The Myth of the Linear, No-Threshold Dose-Response Relationship for Carcinogens.

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The Myth of the Linear, No-Threshold Dose-Response Relationship for Carcinogens George E. Parris

ABSTRACT: The regulatory standards for low-level chemical and radiation exposure in virtually all developed nations are ultimately tied to cancer risk assessment. The risk assessment methodology that has been used most widely for the last 50 years is based on the hypothesis that the probability of cancer is a linear function of dose with no threshold, i.e., the linear, no-threshold (LNT) dose-response hypothesis. Application of this hypothesis to bioassay and/or epidemiological data frequently projects unacceptable risk from minimal exposures. Regulation of chemical and manufacturing industries to conform to these calculated exposures limits is frequently economically burdensome and leads to the abandonment of otherwise technologically attractive products, processes and applications. Moreover, incorporation of these standards into environmental remediation regulations as clean-up standards has frequently required very expensive alternatives for dealing with legacy contamination issues. Thus, many people have challenged the results of LNT analysis. In general, the regulatory agencies and courts have taken the position that the burden of proof is on the plaintiff in these situation and the plaintiff is put into the position of trying to “prove a negative.” For practical purposes, this burden is impossible to carry. This manuscript takes a hard look at the origins of the LNT hypothesis and concludes that it is a myth. Literally, the LNT hypothesis was created by application of inappropriate assumptions to questionable data. Basically, the LNT theory never should have been accepted as “science” and should be required to

meet a burden-of-proof standard itself in any scientific comparisons. In this manuscript, I will show that the methodology for cancer risk assessment used by regulatory agencies was arbitrarily parsed from the body of scientific literature available circa 1960, because it is simple to apply and inherently over conservative, not because it provided realistic scientific results.

KEY WORDS: Linear, No-threshold, Risk, Assessment, Mutation, Cancer, Muller, USEPA, DNA, Repair Abbreviations: DNA, deoxyribose nucleic acid; eV, electron volt; LNT, linear no-threshold; LET, linear energy transfer; USEPA, United States Environmental Protection Agency XP, Xeroderma Pigmentosum

I. INTRODUCTION “The chairman knew that the linear model would prevail, and so it has.” --Roy E. Albert, 19941 In winning a debate, it is very important who has the burden of proof. As a general rule, the arguments needed to overcome an incumbent position are required to be clear and conclusive. In comparison, the incumbent position will prevail simply by asserting its authority in the absence of clear and conclusive evidence to the contrary. This is the situation that we have been in with respect to the linear, no-threshold (LNT) dose-response model for risk assessment for carcinogens for about 50 years. In this manuscript, I challenge the incumbency of the LNT hypothesis by pointing out that it did not start as a science-based position and has not had its deficiencies corrected. It was an arbitrary political decision, and thus, does not deserve any sort of scientific priority. Superficial support for the theory (e.g., from epidemiology) is fatally flawed because the dose-metrics used are not standard and usually are not consistent with the basis of the LNT hypothesis. In scientific or science-based legal debates, protagonists of the LNT hypothesis should be required to prove their case as decisively as any opposing hypothesis. My approach is to summarize the origins of the LNT dose-response model for

cancer risk assessment and show how it is intertwined with the mutation theory of cancer causation. In particular, I will show that a LNT hypothesis was derived for the dose-response for mutations caused by high energy events (e.g., nuclear decay or photons) and that the hypothesis derived for mutations has arbitrarily been applied to cancer caused by radiation, chemical agents or viruses. While some of the underlying assumptions of the LNT hypothesis are valid for radiationinitiated cancer, it can be easily shown that there was never any scientific basis to apply the LNT hypothesis to risk of cancer caused by chemical carcinogens. In the closing sections, I will discuss how unquestioning belief in the LNT hypothesis has led some epidemiologists to manipulate their data and select dosemetrics to give the illusion that there is a linear relationship between chemical dose and cancer response. A. Theotor Boveri and the nature of cancer By 1900 “cancer” was a defined disease and medical science was seeking a cause and a cure. Boveri, a zoologist, observed abnormal chromosome segregation following failure of cytokinesis in barnacles and noted the abnormal development of the affected organisms. Shortly before his death,2 he proposed that aneuploidy might account for cancer development in higher organisms.3 His hypothesis about the nature of cancer (not necessarily about the cause of cancer) drew attention to the nucleus as the seat of cell differentiation and survived into the 1920s.4 But, the excitement over the discovery of the physical location of genes by the research

group of Thomas Hunt Morgan in 1919-19205, 6 shifted attention from the karyotype of cells to the protein-coding genes themselves. The work of Darwin and Mendel now had a physical model and scientists interested in evolution and the mutations of genes7-9 took center stage in study of cancer, whereas to this point, the nature of the malfunction causing cancer had been a debated hypothesis.10 B. The Virus Hypothesis of Cancer Causation Interestingly, in the medical world of the late 1800s where most diseases were known to be caused by biological agents (broadly called “viruses”), the theory of cancer causation had been initiated on the basis of some very respectable science. Following the work of Louis Pasture (circa 1885) the germ theory of disease greatly influenced the thinking of the medical profession. Bacteria could be isolated and made visible by the microscope, but by the 1900, it was known that some disease-causing agents (viruses) were so small that they passed through porcelain filters (i.e., filterable viruses) and were not visible under any light microscope (i.e., ultramicroscopic). These agents were simply called “filterable viruses” with no idea what they actually were. One of the diseases of interest to immunologists was cancer. In the 1800s, it had been discovered that tumors could be transplanted from one animal to another; and in the early 1900s, Payton Rous was conducting such experiments. Rous discovered a transplantable tumor in fowl.11 His experimentation included

investigations of bacteria and the effects of “cell fragments.” He soon reported that a agent separable from the cells of the tumor could cause the tumor;12 and by 1914, it was found that the agent was a filterable virus.13 In 1925, a paper was published in Lancet by W.E. Gey, which was discussed and elaborated on by Archibald Leitch in the British Medical Journal.14 The hypothesis that emerged from their prematurely circulated work was that all mammalian tumors had a common cause of viruses and that a separable chemical factor was needed to activate the virus to produce a certain type of tumor. Without going into details, they were separating alleged filterable viruses from mammalian tumors and then injecting this liquid along with the alleged activating chemical agent (so far only isolated from Rous’s tumor) into animals and producing Rous’s tumors. Of course, through a series of errors, they had misidentified Rous’s filterable virus as the chemical agent. It did not take long for this hypothesis to be elevated to the status of a theory and circulate widely in the medical profession. 15 For the next few years the virus theory of cancer dominated thinking in the area as other viruses that caused cancer were isolated.16 The cancer virus theory had wide support in the 1930s.17-20 C. Muller and Mutations Because the LNT hypothesis of risk assessment of cancer originated from the work H.J. Muller, it is worth understanding his interests and approach. Many things have been written about Muller’s political and social outlook. Perhaps the

most authentic view of his mind is provided by his autobiographical notes published sympathetically by Sonneborn.21 According to Muller, his life was changed in 1906 by the arguments of his life-long friend Edgar Altenburg who “succeeded in converting me to atheism ....and to the cause of social revolution.” Muller embraced evolution and viewed genetics as the way for humans to control their own evolution.21 This theme is, indeed, the key to understanding Muller’s professional progression and political affiliations. In his early career, he became involved with the U.S. communist movement. When he suffered a mental breakdown and attempted suicide, the note that he intended as his last testament included a donation of $1,000 to the American Communist Party.22 But, he was apparently completely cured of any belief that the Soviet Union was a bastion of true socialism from his years as a visiting researcher there (1933-7). While he, thus, can be regarded as a neutral observer in the ideological “cold war” between the United States and the Soviet Union, his overriding motivation was a belief that humans could and should guide their own evolution and that this might be accomplished by negative and positive eugenics,21 which can only be implemented by government intrusion into the reproductive patterns of individuals. The point that is most relevant here is that his interests and research were on mutations, not on cancer. This is logical considering that at this time cancer was believed to be caused by viruses. Muller saw mutations as the dangerous degradation of the human genome. He did no research on cancer and had very little to say about it.

Muller was not a great experimentalist. In the laboratory of T.H. Morgan, Muller felt slighted because while he developed theories, the experimentalists were the people named on the published papers.23 Because of his interest in mutations and evolution, he attempted to induce artificial mutations into fruit flies, first by heat. These experiments with negative results indicated that non-lethal heat was not sufficient to induce mutations.24 He also experimented with selected toxic chemicals. Ironically, he found that lead acetate and arsenic trioxide (arsenic III) did not cause mutations in Drosophila even after protracted feeding at high doses. He then turned to X-rays, which could be compared qualitatively and on a relative basis; but at the time (1927), X-rays were difficult to generate or control in any absolute quantitative fashion (i.e., wave length or intensity).25 His revelation in 1928 produced a flurry of related papers on radiation-induced mutations26, 27 and speculation among evolutionary scientists that natural radiation was the cause of evolution.28, 29 In his 1946 Nobel lecture, Muller presented a quantitative relationship between radiation dose and mutations: “... the frequency of the gene mutations is directly and simply proportional to the dose of irradiation applied, and this despite the wave-length used, whether X- or gamma- or even beta-rays, and despite the timing of the irradiation. ... They leave, we believe, no escape from the conclusion that

there is no threshold dose, and that the individual mutations result from individual "hits", producing genetic effects in their immediate neighborhood. ...” The linear, no-threshold concept was for mutations caused by the total dose (intensity x duration) of high energy radiation (e.g., gamma rays), which was independent of duration or intensity alone (or wavelength for high-energy radiation).30 However, Muller pointed out that work with UV radiation actually inhibited rearrangements (which can be explained by induction of DNA repair, which was unknown at the time). “... Moreover, as Altenburg (1930, 1935) showed, even the smaller quantum changes induced by ultraviolet exert this effect on the genes. They cause, however, only a relatively small amount of rearrangement of chromosome parts (Muller and Mackenzie, 1939) and, in fact, they also tend to inhibit such rearrangement, as Swanson (1944), followed by Kaufinann and Hollaender (1944 et seq.), has found. ...” Muller went on to mention mutations induced by mustard gases, which were not known to be carcinogenic. Note that Muller is referring to the quantitative work of others (especially, Hanson and Heys26 and Oliver30) and they are all referring to mutations caused by high energy radiation. Recently, these results have been critiqued along with Muller’s

presentation of them.31, 32 The primary issue centers around a conclusion by Oliver30 who wrote: “There is no indication of a threshold below which mutations would not be produced. In so far as this work goes, it therefore indicates that the small amounts of radiation in nature may cause some or all of the natural mutations.” Obviously, Oliver was trying to support the idea that natural radiation was sufficient to provoke variations leading to evolution, 28, 29 which was the hot topic in 1929. At the time of his address (December 12, 1946), Muller was probably also aware of unpublished work by Spencer and Stern33 (received for publication November 15, 1947; according to Spencer and Stern the data were collected between October 1943 and July 1945) that pushed the observed dose down from 400 r (see above; actually as low as 385 r for X-rays33) to 25 r33 and supported the hypothesis of a linear, no-threshold dose-response relationship for mutations caused by X-rays. Given the fact that Muller did not mention the unpublished work of Spencer and Stern that would have supported his position, the recent complaint by Calabrese 31, 32, 34 that Muller did not mention the unpublished results of Caspari and Stern 35 (received for publication November 25, 1947) seems to be an unfair and

incorrect attack on Muller. Caspari and Stern’s work35 was quite different from

the type of experiments that had been reported before as they were studying chronic exposure to gamma rays (higher energy than X-rays). The total dose is the product of dose-rate x time and they were looking at the effects of longer time and lower dose-rates than in the typical X-ray experiments. The dose-rates in the Xray experiments were typically 10 r/min or 22 r/min applied for about 30 minutes whereas Caspari and Stern35 spread this same total dose over 21 days. The idea of “recovery” of genes from mutations (i.e., DNA repair) was purely speculative at the time; nonetheless, Caspari and Stern deduced “...that the dosage which makes recovery possible has to be as low as 1/600 r per minute.” This dose-rate corresponded to 81 ion-pairs per sperm head over 21 days, but grouped in 8 to 17 packets of about 10 simultaneous ionizations (i.e., a gamma ray track through the cell) at intervals of 30 hours.35 To the extent that each ionization event along the photon track represents more energy (translatable into chemical damage) than most chemical molecules are capable of, it is relevant to consider whether a high energy photon is a single “hit” or actually multiple simultaneous “hits” on a cell/nucleus/genome. I will discuss the nature of radiation and what these factors mean to chemical reactions in the following section. Rather than challenging Muller’s objectivity on the issue of use of Stern’s unpublished data in his Nobel lecture, I would point to the two facts (i) clearly there is enough energy released by radioactive decay26 or carried by a high energy photon29 or particles to break chemical bonds and it is not surprising that DNA

could be damaged by these events. However, in some cases the DNA repair mechanisms (unknown in 1929 or 1946) are efficient enough to make simple, infrequent (i.e., sporadic) DNA strand breaks moot. (ii) In Muller’s Nobel presentation he includes reference to work by Altenburg27 showing that mercury atomic emissions can cause mutations, but noting that UV exposure can also inhibit mutations (which can be explained by induced DNA repair ). During the 1940s people began to question the general virus theory of cancer causation. Little real progress had been made in understanding viruses since the experiments of Rous in 1914.13 The theory was poorly supported and people were seeing through the flimsy experiments. However, just as Muller’s work was capturing the attention of the public (1946), mouse mammary tumor virus was isolated in 1948.36-38 The role of viruses as a cause of cancer has been widely discussed since that time and it is still under consideration. D. DNA Damage versus Mutations It should be noted that at this time (pre-1965), there was no conceptual difference between DNA damage (i.e., genotoxicity) and mutations because the repair of DNA was unknown. “DNA damage” refers to an event that occurs in a single cell and my be totally unnoticed, may be repaired, or might be lethal to that cell. On the other hand, the term “mutation” implies that the cell survived and produced viable daughter cells that retained the abnormal DNA that was initially caused by some damaging event. In Muller’s case, he was looking at new fly phenotypes

among the offspring of his radiation-exposed flies. In his Nobel lecture (December 12, 1946) he states “...the lethal mutations greatly outnumbered those with visible effects....” As we will see later, simple DNA damage is not rare but is actually very common in cells and goes unnoticed because the DNA repair mechanisms are very efficient. Most of the mutations (i.e., new phenotypes) that Muller observed were associated with irreparable complex rearrangements of the DNA. E. High Energy Photons and Mutations It is important for the reader to understand the nature of electromagnetic radiation (photons) and its effects on tissue. The fundamental relationships among photon energy (E), wavelength (l), frequency (n), Planck’s constant (h) and the speed of light (c) are captured in this equation:

E = hn = hc(1/l) The energy of photons is typically measured in electron volts (eV) whereas the energy of chemical reactions is typically measured in kilojoules per mole (kJ/mole). A photon will not be absorbed by matter unless its energy exactly matches the energy separation (DE) of two different quantum states (e.g., the energy difference between two molecular orbitals). For reference, the energy needed to excite an electron from one outer orbital of a transition metal complex to a higher energy orbital or to rearrange bonds in long conjugated organic system (e.g., retinaldehyde) is about 1eV (97kJ/mole). This is why we can detect an

array of colored transition metal (e.g., Cr, Fe, Co, Ni) complexes that absorb specific bands from the spectrum of white light (i.e., the spectrum from 350nm to 700nm) and reflect or transmit the rest of the spectrum to our eyes. The minimum energy needed to break a single chemical bond found in biological molecules such as DNA or proteins (e.g., C-H --> C. + H.) is typically about 350kJ/mole (3.6eV). This corresponds to the ultraviolet part of the electromagnetic spectrum ( HO– + lower energy X-ray + H+ + recoil e Since water makes up the bulk of biological tissues, the common result is for the recoil (secondary) electron to react with protons (H+) to formation an additional hydrogen radical (H.) in a process called radiolysis of water. e-(aq) + H-OH --> H– + -OH H– + H2O --> H2 + HO– Notice that in principle two hydroxyl radicals are produced in each scattering event and for photons with energies from 100eV to 100keV, 1 to 4 hydroxyl radicals are typically observed for each scattering event.39, 40 Hydroxyl radicals (HO.) are chemically very reactive and each one soon abstracts a hydrogen atom from another molecule or adds to a multiple bond producing a more stable radical. In the cytoplasm, glutathione (GSH) levels are maintained to scavenge reactive free radicals and produce harmless (less reactive) thiol radicals (GS.) that eventually couple with one another to form disulfides (GS~SG).41, 42 The

range of diffusion of hydroxyl radicals before they are consumed is very short (e.g., 4 nm).43 Thus, most hydroxyl radicals produced in the cell, never penetrate into the nucleus. This is very important because the mitochondria constantly produce reactive oxygen species (ROS, e.g., hydrogen peroxide and hydroxyl radicals) that leak into the cytoplasm even though mitochondria have several very efficient enzymes that neutralize reactive oxygen species (e.g., catalase). catalytic reactions: O2- + 2 H+ --> superoxide dismutase (SOD) --> H2O2 H2O2 --> catalase --> H2O + ½ O2 stoichiometric reactions: GSH or ascorbic acid or vitamin E + ROS --> stable products The conventional interpretation of the effects of high energy radiation, thus, stresses the radiolysis mechanism, which produces a predictable stream of hydroxyl radicals as the mechanism of DNA damage. But, it seems to me that the cells are rather well equipped to deal with a few extra ionization events in the cytoplasm and even if the photon track passes through the nucleus, it is difficult to rationalize massive (un-repairable) DNA damage from the low linear energy transfer achievable by Compton scattering. I want to call your attention, however, the fact that photons with energy below 105eV are absorbed by iron atoms (atomic absorptions involving core electrons) with subsequent release of the entire photon

energy as chemical energy in one spot and in one instant. Absorption by iron is even more probably in the 104 and 103eV range. Thus, soft X-rays (or hard Xrays after degradation to lower energy by Compton scattering) may well be absorbed by mitochondria44, 45 or lysosomes46-49 where iron is concentrated in the cell with the effect of bursting the membranes of these organelles and releasing large quantities of reactive oxygen species that overwhelm the standing concentration of reduced glutathione and other antioxidants that normally protect the cell. The irony here is that gamma-rays are less likely than X-rays to be absorbed by iron because they are less likely to be degraded to low energy by Compton scattering. A tidal wave of hydroxyl radicals (suddenly released from iron-rich organelles) sweeping through the nucleus would overwhelm DNA repair capabilities and have much more chance of forming multiple, closely spaced breaks in DNA associated with rearrangements and irreparable DNA damage, which could lead to mutations. F. Origin of the Mutation Hypothesis of Cancer Causation Shortly after Muller’s discoveries with X-rays were publicized (1928), professor Karl Heinrich Bauer published ‘Die Mutationstheorie der Geschwulstbildung‘(the role of genetic mutations in cancer) in German (cited by Berenblum and Shubik50) and J. Joly (as cited by Hanson28) suggested that natural cosmic rays might cause cancer. Curtis et al. in 193351 published “Is malignancy due to a process analogous to somatic mutation?” which suggest that scientists

were still fairly uncertain of the idea that mutations were linked to cancer; for example, somatic mutations were known to occur in plants and animals without causing cancer. And as noted above, viruses appeared to be a common cause of cancer. As late as 1946, Muller did not mention cancer in his Nobel lecture (see above). Thus, the speculation between 1927 and 1947 regarding cancer and mutations appears to have been very tentative. Nonetheless, the foundation had been lain. The next direction of investigation was opened by Friedewald and Rous,52 who realized that the repeated painting of rabbit ears with coal tar compounds seemed to produce an initial (irreversible) precarcerous state, which was converted into cancer only through repeated skin painting. This idea was formalized by professor Isaac Bernblum, who had been studying the metabolism of polynuclear aromatic hydrocarbon carcinogens and who had discovered that applying croton oil (an inflammatory agent) after exposure of tissue to chemical carcinogens was as effective at producing tumors as continued application of the carcinogen itself.50, 53 He confirmed that a single painting of skin with a carcinogen did not produce detectable cancers even after long observation; but, if the same skin was painted repeatedly with croton oil (containing phorbol esters, 2 times per week for 20 weeks) tumors developed even if there were a lenghty period between the carcinogen application and initiation of the croton oil applications. The following table summarizes their results.53 They also found that repeated application of

croton oil before a single application of the carcinogen had no affect on the number of tumors or latent period. These observations firmly established the idea of an irreversible initiation from a single exposure to a carcinogen followed by a reversible promotion of the initiated cells to the status of (clinically significant) cancer. Berenblum and Shubik (1949)50 attempted to rationalize the phenomena that were known to that time in a paper that included “a re-examination of the somatic cell mutation theory of cancer.” Although they acknowledged that the initiation process was “mutation-like” they were disinclined to endorse the mutation hypothesis of cancer causation because mustard agent (bis-2-chloroethylsulfide) was known to be mutagenic, but were not carcinogenic in their test. In conclusion, they stated, “[t]he accumulating indirect evidence would seem to run counter to the [somatic mutation] theory instead of supporting it.” Recalling that before 1953, the structure of DNA was very poorly understood, it is perhaps understandable that some rather abstract papers were published on the somatic mutation hypothesis. One such paper was authored by Strong,54 “a new theory of mutation and the origin of cancer.” What was new about Strong’s idea was that the mutagenic process was conceptualized in terms of energy dissipated by physical agents or chemical agents against DNA without regard to the mechanism of chemical reaction. The relevant point of Strong’s paper is that the photons being used to produce mutations represented enough energy to change a

large number of chemical bonds (in a very short time), whereas chemical agents represent much less in their potential chemical reactions. It is clear from the work of Burdette (1951),55, 56 Stigler (1952),57 Blum (1953),58 Burdette (1953),59 Fardon (1953),60 Burdette (1954 and 1955)61, 62 and Nordling (1955)63 that this is the period when knowledge of the structure of DNA and Muller’s dose-response hypothesis for mutations were married into the linear, no-threshold hypothesis of cancer causation. However, this was soon attacked by Brues (1958)64 who discussed most of the research to that time and noted the whereas Muller’s mutation data set was linear with respect to total dose (and independent of dose rate and duration of dosing) the experience with cancer seemed to indicate that dose rate (even with high energy radiation) was important to frequency of cancers and latent period. He also points to “...the point mutation in a single cell as the carcinogenic determinant, since that concept is necessary to the linear theory.” In other words, the mutation theory of cancer causation and the linearity of the dose response curve (LNT model) are inseparable.

II. DNA AND DNA REPAIR A. Watson, Crick, Wilkins, Franklin and the Double Helix (1953-1960) The story of the deduction of the structure of DNA as a double helix of complementary nucleotides has been told many times. Although the chromosomes were accepted as the local of the genes there was still debate about the actual chemical format in which the information of heredity was stored. In 1944, Avery

et al.65 ended this debate by demonstrating that DNA (not proteins, sugars or RNA) is the chemical that carries the instructions for heredity. This spurred the young James Watson to focus his research on it. In 1953 and with the insights of others,66 he understood the chemical structure of DNA67 and soon the biological implications were evident.68 Although is was clear by 1950 that rearrangements, misplacements and deletions of parts of the chromosomes were mutations, it was not clear until the early 1960s69-72 that changing the sequence of the base-pairs produced mutations (i.e., when Muller had referred to “point mutations” he was thinking about a specific area of the chromosome as opposed to all of the chromosome). B. Ultraviolet Radiation, Mutations and DNA Repair (1949-2000) While most attention had been given to mutations caused by ionizing radiation (Xrays and gamma rays), Muller had mentioned some puzzling results with ultraviolet light in his 1946 Nobel lecture. In 1952 Altenburg et al.73 followed up their original work (from the 1930s74) observing that the dose-response curve for introduction of mutations by UV light in fruit flies followed an inexplicably irregular dose-response curve. Unlike X-rays, the photons of ultraviolet light do not carry enough energy to kick electrons out of atoms or molecules, but the UV photon energies fall in the range typical of the promotion of electrons from the highest occupied molecular orbitals into unoccupied higher energy molecular orbitals (e.g., p --> p*). These activated molecules then are prone to react with

similar molecules and UV is known to catalyze a variety of rearrangements and couplings of organic molecules similar to the DNA bases. Thus, UV causes mutations in DNA but the mutations are more subtle than extreme rearrangements observed for ionizing radiation. Albert Kelner75, 76 was the first to begin to unravel the biological effects of UV light. Following up on some anecdotal observations that bacteria presumably killed by doses of UV were sometime returned to health in visible light, he designed graphic experiments in which Petri dishes covered with colonies of bacteria were first treated with lethal doses of (253.7 nm) UV and then partially masked from visible light. In the areas protected from visible light by the masks, only about one in a million bacteria (10-6) survived and produced colonies; but in the areas where visible light was allowed to shine on the cultures, about 10% (101) of the bacteria produced colonies. It was observed that this effect was

temperature dependent suggesting that it was chemical in nature. It also was observed that if the exposure to visible light was delayed, survival of the bacteria decreased linearly in time (over 4 orders of magnitude with a 2-hr delay) in the lighted areas. Further examination demonstrated that light at longer than 510 nm was not effective in the saving the bacteria. The wavelength for the recovery visible light was about 400 nm.77, 78 From these experiments, Kelner deduced that there were two mechanisms through which mutations were repaired. The first was a photoreactivation that was fast

and efficient; and the second mechanism worked more slowly but did not require light. When Altenburg et al.73 published their mysterious results in 1952, Perlitsh and Kelner 79 immediately responded with an explanation for the non-linear doseresponse curve. It was clear that Altenburg et al.73 had not controlled the visible light exposure and their results were likely a demonstration of the effects of reversal of mutations by visible light (although Kelner was very gracious in not pointing out their mistake). If we assume that in the absence of repair of mutations, the dose-response for UV exposure would have been essentially linear with no threshold, the Altenburg data demonstrate how DNA repair can produce the effect of an S-shaped curve with a threshold for mutations (i.e., unrepaired DNA damage). Nonetheless, it was also noted that more complex mutations caused by X-rays were not reversed by visible light.77 The nature of the UV-induced mutations was slowly clarified as the photochemistry of DNA bases was studied in the 1960s primarily by Richard B. Setlow.80-82 The mutation was discovered to be composed of thymine dimers of two principal types. With the pyrimidines stacked one on top of the other (face-to-face) in the double helix, photoactivation of one thymine (relatively short 254 nm wavelength is required83) allows it to undergo what is now called a “2+2 cyclo-addition” to an adjacent thymine forming a fourmember (cyclobutane) ring:82 C=C C=C* C---C

DNA / / C=C C=C C---C thymines activated thymine cyclobutane As shown in the diagram, the process is reversible, but thermal (D) rearrangement of the cyclobutane to a different dimer (dimer II) yields a structure that is not readily reversed: cyclobutane -->D (thermal energy)--> C=C-C-C (dimer II) first order t1/2 = 1 hr The effect of visible light (>350 nm) was found to be mediated by photoenzyme (called photolayse) that absorbs energy at longer wavelengths and transfers it to the cyclobutane allowing it to reverse to the unactivated dimers (thus speeding up the reverse reaction shown above) without producing activated thymine:82 C=C C---C DNA Neoplasm -->Cancer -->Metastasis The important point here is that cells that sustain damage to their genomes do not necessarily become mutants even if the DNA is not repaired. Cells have a suicidal capability called apoptosis (programmed cell death), 120, 121 which can prevent critically stressed cells (e.g., irreparable DNA damage) from completing the cell cycle. The machinery that accomplishes this task is complex and highly regulated with many feedback loops because there must be a very fine balance between proliferation and cell death or senescence if tissue is to maintain a stable

state of homostasis. Cells spend most of their time in the G0 phase where the DNA is spread out into fine strands of chromatin not visible to light microscopes and virtually the entire genome is transcribed and manipulated by a variety of proteins. During G0, DNA is damaged by the proteins that routinely manipulate it (e.g., topoisomerases); I compare this to a set of blueprints becoming shopworn in a construction manager’s office. In addition, DNA is also vulnerable to damage from thermal, mechanical, physical (e.g., photons), biological (e.g., viruses) and chemical agents. Contrary to common intuition, most damaging events are probably due to the normal activities inside the cell (not to exogeneous mutagenic agents). During G0, double-strand breaks are repaired by non-homologous end joining (see below), damaged bases and nucleotides are excised and replaced, and singlestrand breaks are ligated. Nonetheless, some damage accumulates, and at some point, the cell moves into the replication cycle (G0 --> G1). Interestingly, accumulation of damage is one factor that provokes entry into the cell cycle122 as an opportunity to (i) restore the genome and produce new duplicate cells, (ii) put the cell into a state of suspended animation (senescence) or (iii) eliminate the cell completely (apoptosis). The cell cycle is driven by transcription factors of the E2F family (especially E2F1, E2F2 and E2F3a).123 These transcription factors are normally sequestered by the protein known as pRb (because it was associated with a rare inherited form

of cancer of the eyes called retinoblastoma).124-126 DNA damage is sensed by a proteins known as ATM (mutated in Ataxia-telangiectasia) and ATR (ATM and Rad3-related), which respond by activation of signaling pathways (which we will develop in more detail below). Damage activated-ATR activates Cyclin, 122 which phosphorylates pRB, 127, 128 causing the release of E2F1, which pushes the cell cycle forward from G0 toward G1. DNA damage -->ATR --> Cyclin --> pRB -->E2F1 --> G0 to G1 to S In the 1940s, Madam Louis-Bar pulled together similar cases of clinical observations and defined a syndrome that became known as Ataxia-telangiectasia (AT). By the 1960s, one facet of the AT syndrome was recognized to be cancer. Examination of the chromosomes in AT-related cancers showed a large number of double-strand breaks and fragmented chromosomes.129 Following the revelations about DNA repair made in the case of Xeroderma Pigmentosum, the first assumption by scientists working on AT was that mutations and cancers in AT patients were caused by failure of DNA repair. However, after it was discovered in 1977 that the frequency of formation of DNA double-strand breaks and the repair of double-strand breaks was the same in cells from AT victims as in normal cells,129 the scientific community was left without a hypothesis. The results of Lehman and Stevens129 were periodically confirmed over the next 10 years and by 1990 the hypothesis was shifting to the idea that perhaps the ATM repairs were less faithful than the normal repairs. But, this idea was quickly forgotten when the

association of the AT gene and its product ATM with a protein called p53 was understood. In 1981 a protein with a mass of 53 kDa was discovered at high concentration in tumor cells.130 For nearly a decade, it was suspected of being an oncogen. Then, in 1989, it was realized that the proteins that were found in high concentration in tumor cells were typically mutant forms of the normal (wild-type) p53 that is found in normal cells.131 Wild-type p53 was subsequently associated with stopping the cell cycle of damaged cells at the G1/S checkpoint.132 In addition, it was soon found (1991) that p53 was instrumental in initiating apoptosis of cancer cells.120, 133, 134 Simultaneously, an anti-apoptotic protein was discovered called Bcl-2.120, 135, 136 Bcl-2 was discovered (1984) in B-cell leukemia (Bcl) where it was over-expressed as a result of a translocation mutation that put its gene under the control of an active promoter.137 These findings created intense interest in the complex interactions of these and other proteins because induction of apoptosis was a promising mode of cancer treatment. ATM began to show up in papers discussing p53 and apoptosis in 1994.138, 139 But, it was not until 1997 that it became clear that ATM140-146 and ATR143, 147, 148 detect damage (or potential damage) to DNA and activate p53 and the cell-cycle checkpoints. Recall that most DNA damage occurs during the G0 phase when the chromatin is exposed to mechanical, thermal and chemical stress and the genes are being actively transcribed. Active repair is continuous during the G0 phase, but

when the cell moves into the cell cycle, there is undoubtedly more work to be done especially with double-strand breaks (some of which may be produced by topoisomerase II (Top2)149-152 associated with the compaction of DNA during the transition from G0 to G1). ATM detects DNA damage (especially double-strand breaks) as the cell enters G1 and signals via Chk2 to activate p53. In turn, p53 activates p21, 138, 153-155 which stops the cell cycle at the G1/S checkpoint. Note that over expression of Myc protein can override the G1/S checkpoint by suppressing p21.156, 157 Failure to suspend the cell cycle at the G1/S checkpoint (or introduction of DNA damage after the G1/S checkpoint158) frustrates most DNA repair and is a major contributor to establishment of mutations, which could lead to a cancer phenotype. We can add more detail to the DNA damage-sensing behavior of ATM and ATR and their relationships with p53. First note that p53 is usually not found in normal cells because it is rapidly marked for destruction with ubiquitin by MDM2. 159, 160 It is perhaps an oversimplification to presume that double-strand breaks

(products of ionizing radiation, mechanical stress and physical stress) are uniquely detected by ATM; while single-strand breaks, base damage, thymine dimers and nucleotide adducts (products of UV radiation and reactions with electrophilic molecules) are uniquely detected by ATR, but there is some justification for this interpretation.161, 162 (For more details see reviews by Ljungman163 and Pluquet and Hainaut.164) ATM phosphorylates p53 on serine 15

directly and it phosphorylates Chk2, which phosphorylates p53 on serine 20. ATM also phosphorylates MDM2. Phosphorylation of p53 on serine 20 and/or phosphorylation of MDM2 blocks MDM2 from adding ubiquitin to p53, and hence, stabilize p53 such that its concentration rapidly builds up in the cell. Note that mutated p53 proteins frequently cannot be ubiquinated by MDM2 and this fact accounts for the observation that certain mutated p53 proteins build up in cancer cells.130 The phosphorylation of p53 on serine 15 activates it to do several important things discussed below. In contrast, ATR activation by less extensive DNA damage, activates Chk1, which phosphorylates p53 on serine 20,161 which builds up p53’s concentration, but denies p53 the tool (i.e., phosphorylated serine 15) needed to activate some of the most important down stream pathways: double-strand breaks (IR) -->ATM --> stabilized p53 armed with phosphorylated serine 15 double-strand breaks (IR) -->ATM --> Chk2 --> stabilized p53 with phosphorylated serine 20

Lesser damage (UV, chemicals) --> ATR --> Chk1 --> stabilized p53 with phosphorylated serine 20

It is relevant that other conditions within the cell also activate p53 in various ways. In particular, hypoxia (lack of oxygen) activates p53 through the ATR-Chk1 pathway shown above.165-169 JNK (c-Jun N-terminal kinase) appears to have a role similar to MDM2 in than it facilitates destruction of p53; thus, when JNK is “activated” and its MDM2-like function is abolished, the levels of p53 increase.164 In addition, JNK phosphorylates p53 on serine 37 both in response to genotoxic and nongenotoxic (metabolic) stresses in the cell. Heat shock

(hyperthermia) also activates p53 by phosphorylation of serine 15 and suppresses degradation of p53.170 In summary, p53 acts as an integrator of various stresses within the cell (genotoxic, metabolic, physical) and directs the cell to (i) slow down until the stress can be resolved (e.g., by pausing the cell cycle while DNA repair can be completed), (ii) sending the cell into indefinite suspended animation (senescence) or (iii) activation the cell death program. I will describe how this happens next. B. p53, p21, Bax and Bcl-2 As discussed above, genotoxic, metabolic and other stresses on the cell are integrated through activation of p53 by phosphorylation. These signals are then executed through two major pathways.171 First p53 that has been phosphorylated on serine 20 activates p21,172 which inhibits Cyclin-dependent kinases (CDKs); thus preventing the progress of the cell cycle past G1 phase.154, 155, 173, 174 Cyclin -->blocked (p21) --> pRB -->E2F1 --> G0 to G1 to S In contrast, phosphorylation of p53 on serine 15 has little if any affect on the interaction of p53 with MDM2 or p21, but it is essential for the transcriptional activity of p53. Phosphorylation of p53 on serine 15 facilitates the interaction of p53 with CBP/p300 and together they initiate transcription of numerous responsive genes. 175-177 Perhaps the three most important genes that are transcriptionally activated by p53(serine15phosphate) are Bax, Puma and

Noxa.177 These genes’ products are pro-apoptotic proteins.178-183 The pro-apoptotic proteins are balanced in the cytoplasm by anti-apoptotic proteins including Bcl-2. For simplicity, this balancing can be though of as the ratio of Bax to Bcl-2. Bcl-2 levels are regulated by processes that attempt to protect the cell from premature apoptosis during times of stress. It is worth mentioning here that invasion of a cell by an internal parasite (e.g., a virus or a bacterium) naturally raises the metabolic and genomic stress levels in cells. The primary defense against these invaders is for the cell to commit suicide using the programmed cell death (i.e., disassembly and recycling of the cell parts) program called apoptosis. Obviously, the viruses have evolved countermeasures to this strategy.184 The two most common approaches to blocking p53-dependent apoptosis are to attack p53 itself and/or raise the stress tolerance of the host cell by forcing expression of Bcl-2 or producing a viral homologue of the host’s Bcl2. It is also important for the virus to continue transcription and replication of the genome by taking over the functions associated with pRB.127, 185 For example, SV40 produces an antibody to p53 that effectively prevents its transcription activity186, 187 and EBV produces a viral Bcl-2 (vBcl-2).188 It turns out that Bcl-2 also has a cell cycle regulating function via p27.189, 190 Apparently, the pathway involves formation of a complex between E2F4 and p130, which suppresses expression of E2F1. Some viruses circumvent p27 by directly activating the E2F1 gene.191, 192 If the p53-dependent apoptotic pathway has not been suppressed, then

activation of E2F1 leads to apoptosis; but if the pathway has been suppressed, transcription and replication of the virus is achieved. It is not necessary here to go into detail about the actual execution of apoptosis. Suffice it to say that when the pro-apoptotic signals out-weight the anti-apoptotic signals, Bax and other pro-apoptotic proteins are freed to attack the mitochondria. These proteins are very similar to bacterial toxins and function by producing pores in the mitochondria through which Cytochrome C193-195 escapes. In turn, Cytochrome C causes activation of caspase 9.193, 194 There is a family of caspases (i.e., cysteine proteases) pro-enzymes that are sequentially triggered in a spreading cascade.196, 197 These enzymes deactivate the proteins of the cell and lead to apoptosis (programmed death). So far, we have summarized p53-dependent apoptosis. Before leaving this topic, it should be noted that the immune system also plays a role in apoptosis. If we assume that the p53 system has been blocked by some combination of events or perhaps there are simply no stress-inducing stimuli, the immune system may still respond if the cell displays abnormal membrane surface proteins, which make the leukocytes suspicious. Engagement of TNF-alpha (either free or membranebound) from the immune cells with the CD95/Fas receptor on the surface of an abnormal cell, results in release of an adaptor molecule inside the target cell, which activates caspase 8. Note that the engagement of Fas by TNF can be avoided or mitigated by the target cell by release of decoy soluble-Fas (sFas)

fragments or the expression of FLIP, which prevents the activation of caspase 8. When it is activated, one of the first things that caspase 8 does is to cleave Bcl-2 (which may be in high concentration if the p53-dependent pathway is blocked) and this converts Bcl-2 into Bax-like fragments that attack the mitochondria.198 From that point on, the caspase cascade is similar to the events in the p53-dependent pathway. Obviously, this is a very complex system. From the standpoint of cancer risk assessment, side-tracking DNA-damaged cells into apoptosis or senescence will reduce the risk of cancer. Suspending the cell cycle alone will give the cell time to repair damaged DNA. These protein-dependent processed can be saturated at high dose-rates. Thus, here again, at low dose-rates, we would expect efficient suppression of mutant cells, which would be come less effective at higher doses (i.e., a non-linear factor). Moreover, we saw examples of actions by viruses to disrupt these protective systems (e.g., suppress p53), which alone or in combination with chemical or radiological exposures could enhance proliferation of cells. C. Repair of DNA Double-strand Brakes with Conservation of Sequence While the p53-dependent mechanisms discussed above have stopped or slowed the cell cycle at the G1/S and or the G2/M checkpoints, the cell is working to clear the damage to DNA. It was pointed out above that the Rad52-catalyzed single-strand annealing is very fast (and may produce dangerous frayed ends of the

double-stranded DNA), but can be blocked by Ku70/80 (unless the damage is so extensive that the standing concentration of these proteins is consumed). Capping the double-strand breaks with Ku70/80 buys the cell time to put an elaborate (but fairly slow) system of repair mechanisms into play.118 Moreover, the end-joining process progresses even if there is no homology in the broken ends or if the ends are blunt (i.e., no extended single strand). This may seem like a dubious advantage relative to single-strand annealing, which involves at least some homology. But, at the cellular level, it is of paramount importance to heal all breaks and only of secondary importance to retain the segment sequences and avoid minor deletions because if a double-strand break exists (anywhere on the chromosome) the part of the chromosome that is not attached to the centromere is likely to be lost in the next cell cycle. Moreover, chromosomal instability and aneuploidy are more dangerous than mutations to multi-cellular organisms.199-202 The Ku70/80 base provides a binding point for a DNA-dependent kinase known as DNA-PKcs and the complex is known as DNA-PK. Among the histones that make up the nucleosomes, is a variant of H2A called H2AX. The relevant feature of H2AX is that it has a carboxy terminal Ser-Gln-Glu (i.e., SQE) motif. Apparently, breakage of the DNA exposes this motif to phosphorylation. The recently activated DNA-PK and ATM (the kinase discussed above) phosphorylate the H2AX histones producing what is know as gamma-H2AX, which is negatively

charged. Phosphorylation of H2AX is greatly accelerated in chromatin regions where the nucleosomes are already acetylated117 (i.e., the positive charge of the nucleosome is partially neutralized). Phosphorylation of the nucleosomes causes electrostatic repulsion between the broken ends of the DNA and loosens the association between the DNA (which is negatively charged) and the nucleosomes.203 Cations (Mg++, K+) move to the DNA to balance the charges. The work of the kinases, especially ATM,204 spreads gamma-H2AX along the double helix for thousands of base pairs. The extent and density of gamma-H2AX is the result of a dynamic process of phosphorylation and dephosphorylation.205 Ubiquitylation of H2A and H2AX histones at double-strand breaks also occurs as part of the end-joining process and this capability may be saturated if the cell is dealing with more than about 40 sites at the same time.206 Now, the broken ends of the dsDNA are brought together in a deliberate sequence of steps. It is relevant that these repair foci can be detected by immunofluorescence of gamma-H2AX, and this is one of the primary ways double-strand-break repair has been studied.207 The important step in minimizing rearrangements, deletions and translocations from this point on is to establish a bridge between nearest-neighbor broken ends as quickly as possibly. The actual processing of the ends and ligation can wait. Because phosphorylation of H2AX tends to make all the broken ends of the double helix negatively charged, they do not naturally approach one another. It appears

that some combination of enzymes and nucleoproteins quickly form among the nearest-neighbor ends and begin bringing them together. Ultimately, new DNA is synthesized to fill the gap and the ends of the DNA are ligated. This leaves and interesting problem, the Ku proteins are still on the DNA like beads on a string. They are subsequently removed by a process involving ubiquination.208, 209 It takes about a half-hour for the repair foci to form and it may take many hours for them to be resolved because they persist even after the DNA strands have been physically joined.210, 211 Assuming there is no shortage of the necessary proteins, the half-life for repair of simple DNA double-strand breaks is approximately 20 to 60 minutes.212, 213 In principle, DNA that makes it through the synthesis phase should look and act like normal DNA. However, double-strand breaks that have never been repaired and errors that may have been introduced may still exist. The final opportunity to correct these mistakes is during mitosis when chromosomes containing errors (deletions) are paired with their sisters (hopefully error-free). In homologous recombination repair, the parity of each chromosome is checked against its sister. When mismatches are found (presumably including deletions that were introduced by non-homologous end-joining), the tendency is to cut the shorter strand (i.e., containing the deletion) and the 5’ ends of the strands at the break are eaten away until sections homologous to the longer sister chromosome appear. Then the exposed overhangs insert into the longer strand of the undamaged sister

chromosome to form D-loops. In this state, the undamaged sister chromosome provides the template for reconstructing the missing part of the damaged chromosome. Once this is accomplished, crossing over may or may not occur. This mode of DNA repair is called homologous recombination (HR) and involves several enzymes to facilitate the complex moves. There are a number of things that must be accomplished in the G2 phase of the cell cycle. In particular, the new strands of DNA must receive the epigenetic imprinting of the parental (template) strand. In addition, the time needed to complete the DNA repair by homologous recombination (and the coincidental crossing over that occurs) must be provided. Thus, the cell is held at the G2/M check point while these things are being completed. The mechanism by which the cell secures the checkpoint involves ATM and ATR. D. The G2/M Checkpoint We left the discussion of double-strand breaks at the G2/M checkpoint. If the G1/S checkpoint has been circumvented by Myc156 and/or double strand breaks persist past the DNA synthesis phase,158 the cell is normally restrained at the G2/M checkpoint. The transition from G2 to mitosis (M) is fairly complex. The cell cycle, which is moved along by Cyclin proteins, has recently been interpreted as being directly regulated by Cyclin D1 and p27.122, 214 Although earlier work suggested that Cyclin C1 was continuously present at high levels through most of

the cell cycle (G1-G2), it is currently believed that Cyclin D1 and p27 are both suppressed during the synthesis of DNA.214 As a matter of fact, Cyclin D1 must be suppressed during the S-phase because it binds the proliferating cell nuclear antigen (PCNA), which is an auxiliary protein for DNA polymerase delta.215 The degradation of Cyclin D1 in the S-phase is caused by phosphorylation of its Thr 286, which is promoted by ATR and/or ATM.122 Once the level of Cyclin D1 is minimized during S-phase, it will stay low through G2 and M and will not be available to help push the cell through the G1/S checkpoint unless it is reestablished by Ras (rat sarcoma protein) during G2. Ras prompts Cyclin D1 transcription during G2 to facilitate escape from the G2 checkpoint of the cell cycle.214 Ras also suppresses p27, which is normally up-regulated during mitosis. Protein p27 binds to Cdk2 and to the Cyclin D/Cdk4 complex. When p27 binds to Cdk2, it inhibits entry into S-phase. Apparently, the increase in Cyclin D/Cdk4, pulls p27 away from Cdk2 facilitating DNA synthesis. The process of building up Cyclin D/Cdk4 and the release of Cdk2 (typically about 3 hrs) determine the length of G1. Thus, the concentration of p27 determines the length of G1. In addition, if Ras levels are inappropriately high (at times other than G2), p27 levels will be low going into G1 and G1 will be too short for complete DNA repair to occur. The cell may either go into apoptosis (if the p53 system is intact) or simply proceed to generate mutant daughters if the p53 system is not

functional. Ras protein is a small GTPase that normally transmits carefully regulated external signals in the form of growth factors to prompt cell proliferation (as required for development). But, mutations in Ras216 (or viral supplemented Ras217) can prompt continuous activation of the proteins down stream from Ras causing uncontrolled proliferation with mutations (hence, the association with sarcomas). E. Promyelocytic Leukemia (PML) Protein Senescence is an alternative to apoptosis when p53 and Rb are functional and the cell cycle is running away (i.e., bypassing checkpoints). It was noticed in some cases where Ras218 or SV40 T-antigen219 was driving proliferation that cells would irreversibly enter a quiescent state. Investigation revealed that promyelocytic leukemia (PML) protein was elevated in these cells. This protein seems to be transcribed or promoted by p53220 and it is degraded after being phosphorylated by CK2.221, 222 PML interacts with p53 and Rb. In particular, it appears to sequester Rb and E2F transcription factors into nuclear bodies.223 In addition, under anaerobic conditions (as found in rapidly growing tumors) PML directly interacts with mTOR, which inhibits HIF-1a and neoangiogenesis.224 These factors both tend to stop the proliferation of cells and block the expansion of tumors. F. Summary of Cell Cycle Regulation

For the most part, the triggering mechanisms for apoptosis and cell-cycle regulation by genotoxic agents were worked out between 1985 and 2000. In the context of the scope of this paper, the introduction of mechanisms for eliminating stressed cells and cells with damaged genomes produces another non-linear process that would stand between DNA damage and cancer.

IV. THE HAYFLICK LIMIT One of the misconceptions that existed in the biological sciences at the time when the linear, no-threshold hypothesis was introduced and widely accepted (19461975) was that somatic cells were routinely immortal. This erroneous belief came from an apparently badly mis-executed experiment with chicken tissue under the direction of the well respected physician Alexis Carrel (1873–1944). On January 17, 1912 Carrel started a culture of cells from embryonic chicken hearts to prove the widely assumed immortality of somatic cells. The experimental design (if carelessly executed) allowed new cells to be added every time nutrients were added to the culture. Apparently, this can explain why the culture persisted for over 20 years. Because of Carrel’s prestige and the fact that his results reinforced the “common knowledge” no one challenged them. It was not until the 1950s and 1960s when efforts were being made to grow human cell cultures that the issue came to a head. Nonetheless, in February 1951 George Gey (18991970) succeeded in growing cells from a human cervical cancer, which he described as “stable.”225 Although this cell line (HeLa) was shown to be

undergoing continuous changes in its karyotype as early as 1959226, the popular notion throughout this period (until aneuploidy was re-associated with cancer,199, 200 circa 2000) was that cancer cells were essentially normal human cells with a

few mutations. Unfortunately, this image in the hands of the children and relatives of the cancer victim (Henrietta Lacks (1920–1951)) led them to believe that the cancer cells were their mother incarnate, when in fact; the cells are nothing more than an evolving species of single-cell organism.200, 202 It was not until Leonard Hayflick began trying to understand aging that the question of cell clone longevity came to the front. About 1960,227 Hayflick became suspicious that stable/normal diploid cells could only go through about 50 doublings before they went extinct.228 But, his observations were not supported by any mechanism and were generally ignored in the cancer field. Subsequently, a hypothesis for incremental counting of cell cycles was introduced in 1994229 and soon Hayflick’s hypothesis was associated with a mechanism of telomer attrition230 that had been known since 1971. Telomers are the end-caps on linear chromosomes that do not contain genes and prevent linear chromosomes from being joined end-to-end. There is an important example of chromosome endjoining in the human chromosome number 2, which was apparently formed from two chromosomes present in our common ancestor with the chimpanzees. In any event, every time a cell cycles and the DNA is replicated, the DNA polymerase enzyme complex falls apart when it runs off the end of the (parent)

template strand. This problem does not exist in circular prokaryote chromosomes. Thus, the end of a linear chromosome is progressively lost in each new generation of daughter cells. After about 50 replications, the functional end of the chromosome starts to erode and is recognized as a double-strand break. Weinberg and co-workers231, 232 were the first apply the technique of introducing the telomerase gene (hTERT) into cells to achieve conversion to tumor cells. However, it is not clear that extending the longevity of cell clones is sufficient in itself to produce cancer clones (with a few other mutations). Two things happen when you extend the life of cell clones in the laboratory (Weinberg’s were over 60 doublings201, 231): (i) the possibility exists that cell-cell fusion or chromosomal instability occurred and caused aneuploidy (Weinberg’s cells changed morphology, but their karyotype was not evaluated)226, 233 and (ii) laboratory contamination (where established cancer cell lines overgrow untransformed cell lines) has proven to be a serious problem in long-term cultures.234, 235 Either one of these events could have compromised Weinberg’s conclusions.

V. MULLER’S RATCHET It is perhaps ironic, that Muller is the source of one of the ideas that undermines the mutation theory of cancer causation. In simple terms, if an asexual cell loses an element of its genetic library (i.e., a gene), its progeny will never get it back.

Every lost piece of genetic information (e.g., by deletion or destructive mutation) is no longer available for service. Carried far enough, the organism must become extinct. It works in only one direction like a ratchet. Muller’s ratchet236, 237 is real and has been demonstrated in a variety of ways including the reduction of the Y-chromosome to its most elementary form,238, 239 and reduction in the capabilities/fitness of bacteria240 and viruses.240-245 Thus, a continuous series of mutations actually lead asexual cell clones to extinction. This observation explains why genotoxic agents are some of the most effective anticancer drugs. On the one hand, ionizing radiation leads normal cells to cancer, but at the same time it is used to kill cancer clones by causing incapacitating mutations. It might be argued that the reason for ionizing radiations effects on cancer clones is not specifically related to causing mutations in the DNA. A more specific DNA-targeted toxin is etopside, which is a specific inhibitor of topoisomerase II. Topoisomerase II is an enzyme that untangles DNA by producing systematic breaks and passing one strand through another followed by ligation of the break.246 Etopside forms complexes with topoisomerase II and DNA that stabilize the cleavage complex and inhibits ligation of the separated ends. Thus, the DNA-topoisomerase-etopside cleavage complex degenerates to a DNA double-strand break.247-249 Etopside causes cancer clones to go extinct by causing a series of mutations that ultimately cause loss of function of the cancer genome as predicted by Muller’s ratchet.

But, you may ask, how can bacteria species survive for billions of years if Muller’s ratchet is killing them off. Well the best escape from the ratchet is exchange of genetic material with another individual. For example, a bacterium can pick up a plasmid or a stray piece of DNA or fuse with a cell (perhaps from an entirely different species) and acquire genetic information needed to evolve and keep going. These para-sexual modes of horizontal gene transfer250-258 make the asexual species viable over long periods of time. In the context of the mutation theory of cancer causation, the probability of a mutation improving the fitness of a somatic cell is very small. Thus, virtually ever mutation in an uncontrolled system will have an adverse effect on the cell/genome. Asexual reproduction leads to extinction, not to cancer.259 In all likelihood, successful cancer clones (i.e., fit parasites) survive by cell-cell fusion which is a form of somatic para-sexual reproduction.202 Endoreduplication260-263 can also mitigate extinction by giving an asexual clone a “spare parts supply” for its genome (like a fault-tolerant computer system), but endoreduplication does not provide the evolutionary possibilities of cell-cell fusion. Endoreduplication is likely to be selected for under conditions of frequent mutations and it represents a commitment to an existing phenotype.261 Cell-cell fusion is an alternative to endoreduplication that facilitates rapid change of phenotype.

VI. THRESHOLD FOR CANCER CAUSED BY MUTATIONS Recall that the linear no-threshold dose-response that Muller proposed was for

mutations, not cancer. The somatic mutation hypothesis of cancer causation was introduced tentatively about the time that Muller was gaining notoriety. From there it grew as an underlying assumption that is consistent with the fact that cancer clones have genomes that are generally very different from their host. That point of view was recently (2011) summarized in a risk assessment publication from the USEPA:264 “Mutagenicity as a mode of action for carcinogenicity is well established and has clear biological plausibility.” But, as we have seen there are several types of mutations. It might be better stated that damage to DNA that leads to inheritable abnormal genomes is well established in cancer, but how abnormal genomes facilitate evolution to clinically significant tumors is still unclear. It is clear that a very large number of errors (i.e., DNA lesions) are introduced into DNA of ever cell every day and repaired.265 In addition to cells in the G0 phase, cells that go through the cell cycle are subject to errors in DNA replication most of which are also repaired.266 Gundry et al.267 have used high-throughput sequencing on single cells to estimate the number of (point) mutations retained by the cell after lesions caused by a direct-acting DNA alkylating agent (N-ethyl-Nnitrosourea, ENU). In the controls, there were approximately 0.1 mutations per million base-pairs after 5 passages of mouse embryonic fibroblasts and 0.4

mutations per million base-pairs in Drosophila S2 cells that had experienced more passages. The cells were treated with 4.3 mM ENU for 30 min, followed by triple washing and culturing for 72 hr. At the end of 72 hr, more than 90% of the cells were viable and “virtually no lesions remained.” Presumably, DNA repair was complete. In the S2 cells, there were now about 3.1 mutations per million base-pairs and in the mouse cells there were about 3.9 mutations per million basepairs.267 This methodology may, in principle, be able to shed light on the doseresponse relationships for mutagens and carcinogens.268 Here, I want to point to a case where we can be rather certain that there is a disconnection between mutations and carcinogenity. It is certain that potassium is essential for cellular life and makes up about 0.2% of the human body. The concentration of potassium (K+) in the in blood plasma is about 4.8 mmol/L and the concentration in cells is about 150 mmol/L.269 The concentration of potassium in the nucleus is even higher270 probably because of its association with DNA.271 The volume of a mammalian nucleus is approximately 10-16 m3 (10-13 L). This means that there are about 1010 potassium nuclei in the nucleus of a cell. The natural abundance of radioactive 40K is 1.18 x 10-4. Thus, there are about 106 radioactive 40K nuclei in each nucleus of every cell. The half-life of 40K is 1.25 x 109 years. So, on the average, we would expect about 10-3 disintegrations of 40K per cell nucleus per year. That may not seem like much, but there are about 1014 cells in the human body. Thus, something like, 1011 cell nuclei have 40K

disintegrations each year (on the order of 103 disintegrations per second in the human body). About 89% of these disintegrations are by emission of a highenergy beta electron (up to 1.33 MeV) and 11% are electron capture events followed by both Auger electrons (200-2000eV) and a gamma ray (1.46 MeV) emissions.269 Thus, 40K provides a wide range of radiation types to cells. Since most people do not die of cancer, it is clear there must be a threshold for cancer even for these major events. Sequencing of human genomes indicates that we all carry numerous loss of function mutations and that every child receives approximately 70 mutations not present in his/her parents.272, 273 With all this happening, how can we state confidently that mutations are the cause of cancers. Much less, how can we say that a single mutation could cause a cancer.

VII. CHEMICAL-SPECIFIC FACTORS Up to this point, the topics covered which cast doubt on the scientific basis for linear, no-threshold dose-response for mutagenicity and carcinogenicity apply to either radiation or chemical exposures, but chemicals have an even greater array of non-linear factors to consider. A. Pharmacodynamics and Enzyme Kinetics Radiation penetrates directly into tissues and cells and the relationship between the dose applied and the dose received at the nucleus is linear unless the tissue is

substantially damaged. On the other hand, chemical substances, such as the wellstudied arsenic(III) oxide, ingested in drinking water must traverse the anatomy of the body (i.e., the mouth, esophagus, stomach, blood vessels, liver, and kidney) before arriving at the tissues where it is believed to cause cancer (i.e., the skin, bladder and lungs). Once at the target cell, the As(III) ion must traverse the cell membrane, cytoplasm, and nuclear membrane before contacting the DNA. Although the proponents of the LNT hypothesis for cancer risk assessment when it was proposed for chemicals (circa 1965) can be excused for not knowing about DNA repair, apoptosis and the Hayflick limit before the LNT hypothesis was applied to cancer risk assessment and regulatory decision-making (circa 1970), it is hard to rationalize why any qualified toxicologist would accepted the nothreshold and linear dose-response concepts for chemicals considering the well understood dose-response behaviors associated with other biological interactions (non-linear pharmacodynamics and Michaelis–Menten enzyme kinetics). Note that this issue has been obscured after the fact by the separation of carcinogens into direct acting (i.e., cause DNA damage without metabolism) and indirect acting (require preliminary metabolism). But, non-linear phenomena apply equally well to either case. having worked within the US Environmental Protection Agency and Food and Drug Administration in the 1970s and 1980s, it was my impression that the people making these decisions held the linear, nothreshold concept as some sort of magical law (with no knowledge of where it

came from). It was a dogma, and any non-toxicologist who dared to question it was sure to be branded as an idiot. It was widely accepted that carcinogens were simply different than non-carcinogens because in principle only one “hit” (i.e., only one molecule making its way to the nucleus) was required to cause cancer. This, of course, is not true. The same pharmacodynamic principles apply to carcinogens (direct and indirect acting genotoxins) as to any other toxin. One of the classic (non-carcinogen) examples of threshold behavior is provided by cyanide (anion). Cyanide is about ten times more toxic that carbon monoxide on a molar basis because carbon monoxide only blocks oxygen transport by hemoglobin depleting oxygen from the blood and producing a blue color in the skin. In contrast, cyanide prevents the utilization of oxygen in the cell and the blood is bright red because it is saturated with oxygen. The cyanide ion forms coordinate complexes with metallo-enzymes (e.g., cytochrome oxidase) in the mitochondria that normally serve to transport electrons in aerobic metabolism. To mitigate this effect, mitochondria also have a family of enzymes called rhodaneses, which react with thiosulfate (S2O32-) to produce persulfides (RS-SH) that react with cyanide (CN-) to form the much-less-toxic thiocynate (SCN-). The protective system can neutralize about 0.017 mg CN/kg.min in the average human. As long as the dose rate stays below this threshold and the supply of thiosulphate holds out, cyanide toxicity is not lethal. But, with an acute dose of cyanide of 200 mg (about 2 mg/kg) the rate of detoxification is exceeded and the dose (i.e., dose

rate) is lethal. Perhaps a more relevant example is provided by the case of “arsenic” (i.e., arsenic(III) oxide). Based on anecdotal evidence, arsenic (used in high oral doses to treat skin diseases) was reported to be a carcinogen in the late 1800s. The idea was attributed primarily to Sir Jonathan Hutchinson (1828 – 1913).274 Today, almost every paper (and there are thousands) that discusses the health effects of arsenic (in its many forms) begins with the statement that “arsenic is a known human carcinogen.” Arsenic is most infamous for its association with skin and bladder cancer; links to other sites have also been made through epidemiological studies (which need to be discussed separately). Two interesting experiments have been conducted by scientist of the USEPA275, 276 using inorganic arsenite (iAs(III)). Kitchin et al.275 used heme oxygenase (HO) induction in tissues of the liver and kidney as an indicator of subclinical effects of iAs(III). They established a linear relationship (r2 = 0.780 in liver and r2 = 0.797 in kidney) for the concentration of iAs(III/V) and HO enzyme induction in the livers and kidneys of rats. So, the induction of heme oxygenase can be used as a probe for the tissue concentration of iAs(III/V). Note that neither mono- nor dimethylated forms of arsenic (i.e., MMA(V) nor DMA(V)) induced HO. This observation was exploited by Kitchen et al. to probe arsenic transport and metabolism in oral (gavage) dosing studies as follows: (i) Doses of 10 micromol/kg (i.e., 0.75 mg/kg) in rats did not induce HO in liver or kidney; (ii)

doses of 30 micromol/kg (i.e., 2.25 mg/kg) induced HO in the liver but not in the kidney; (ii) doses of 100 micromol/kg (i.e., 7.5 mg/kg) or higher induced HO in liver and kidney.275 Since both tissues have similar sensitivity to iAs(III) as shown above, these observations suggest that doses below 10 micromol/kg (0.75 mg/kg) the iAs(III) is detoxified (i.e., methylated and/or oxidized) efficiently (in the liver) and diluted in general circulation. At doses between 30 and 100 micromol/kg (7.5 mg As/kg), a biological response is observed in the liver, but the metabolism in the liver (and other tissues) and dilution in the blood volume keeps the dose of arsenic species to the kidney low (e.g., below 30 micromol/kg diluted in the blood volume). Only at doses over 100 micromol/kg did iAs(III) overwhelm the liver and reach the kidney at substantial concentration. The authors concluded that: "Nonlinear relationships were observed between administered arsenite dose and either liver or kidney iAs concentration. Overall, there was a sublinear relationship between administered arsenite and biological effect in rats." Kenyon et al.276 published a parallel study in mice. Again, in mice the USEPA authors note: “The lowest observed effect levels (LOELs) in this study [in mice] for HO induction are 30 and 100 micromol/kg, respectively, in liver and kidney.”

The authors also showed that inorganic and organic forms of arsenic (i.e., metabolites of iAs(III)) varied from tissue to tissue.276-279 Even very potent carcinogens have shown non-linear dose-response when comparing bladder and liver.280 These results (and many others281-285) are consistent with the well studied Michaelis-Menten kinetics of enzyme (i.e., receptor) and membrane transport reactions. This subject is too mathematical to discuss here, but it can be concluded that most unitary biological processes (catalyzed or affected by specific receptors or enzymes) are inherently non-linear and reach a saturation point beyond which they do not respond to higher concentrations of reactant. It is totally implausible that a linear dose-response curve especially at low doses (i.e., low dose rates) should exist between exposure to chemicals and biological endpoints. Leonor Michaelis (1875-1949) and Maud Menten (1879-1960) published their kinetic equations in 1913. Obviously, (unlike DNA repair, apoptosis, Hayflick limit, Muller’s ratchet, etc. effects that were not know in the early 1970s when the USEPA was adopting the linear, no-threshold dose response model for carcinogens) any qualified toxicologists should have expected that pharmacodynamics would produce non-linear effects on the genotoxicity of chemicals administered orally that caused cancer in remote tissues (e.g., the skin

or bladder). That is why it is very hard to rationalize how Albert makes the following statement in 1994 describing the USEPA logic in the mid-1970s:1 “Cancer is an expression of genetic damage. Mutations are genetic damage. Cancer is therefore caused by mutation. Mutation is linear with radiation dose in micro-organisms. Therefore, linearity for cancer! The difference between chemical carcinogens and ionizing radiation could be waved aside as they both cause genetic damage.” On pharmacodynamic grounds alone, the “waving aside” the differences between chemical carcinogens and high energy radiation is incorrect. As a personal note, I was working in the USEPA Office of Toxic Substances at the time (1976-1977), and as a young chemist, I was baffled how there could be linearity in doseresponse in chemical carcinogens assuming that the agent had to traverse the anatomy and survive the biochemistry of the body to reach the cells that are being effected. But, the toxicologists and regulatory personnel of various stripes carried on with great self-confidence, so I assumed that I was missing some key point. I was not; they were. B. Metabolic Activation, Electrophilic Agents and the Millers While I was struggling to understand the regulatory process at the USEPA, most attention to chemical carcinogens was being given to the work of James and Elizabeth Miller.286

The Miller had been doing metabolism, toxicology and mutation work since the late 1940s. By the mid-1970s, they were legends. As early as 1952, they were interested in adducts of carcinogens with cellular molecules (proteins) as a possible cause of cancer.287 By 1969, they established that many carcinogens were metabolized to an active form that reacted with cellular macromolecules288 and could reasonably be described as the world’s foremost authorities on chemical carcinogens.289 They emphasized that most chemical carcinogens are electrophilic agents that react with nucleophiles in the cell. These include proteins and peptides such as glutathione (GSH) to form readily excretable (polar) adducts as a mode of detoxification. But, compounds that were activated within the cell (inside the defenses of the cytoplasm) tended to be the most potent and tissue specific carcinogens. It is relevant that Miller warned:289 “However, it must be emphasized that the mutagenic activities of these esters [of hydroxylamines and hydroxamide acids] are a consequence of their electrophilic activities and are not necessarily related to their carcinogenic activities.” Miller was keeping an open mind. He also had indications that the DNA adducts were being repaired.290 But, the linkage of chemical carcinogens to mutations was the only message that the USEPA wanted to hear. C. Bruce Ames and Chemical Mutagens

In the early 1970s when the mutation hypothesis of cancer causation and the linear, no-threshold concept were being married by the US Environmental Protection Agency as keystones in the war on cancer,1 Bruce Ames was the man of the hour. Ames had developed a very easily applied test to detect mutations (i.e., the Ames test) using specific strains of Salmonella.291-293 The lure of a cost-effective way to detect mutagens and the then-current belief that the association between mutagens and carcinogens is on the order of 90% was a powerful driver to the acceptance of the relationship in regulatory policy.1 Ames’s early work was very supportive of the idea of identifying carcinogens by their mutagenic characteristics as reflected in the titles of his papers at the time (1972-73), e.g., “Carcinogens are mutagens, their detection and classification.”294-297 It is fair to say that these publications settled the mutagen-carcinogen relationship for chemical substances in many scientific minds, all legal minds and the minds of all laypeople. But, true scientists do not wed themselves to positions that become untenable. First there came an understanding that DNA damage and mutations are not as rare as cancer. 265, 298 Indeed, Saul and Ames265 deduced that there are thousands of DNA damaging events in each cell every day from purely endogenous errors. And, it was soon apparent that the bioassays were producing “too many rodent carcinogens”299, 300 because of the bioassay protocol (purely for statistical reasons1) requiring the use of the “maximum tolerated dose.” The maximum tolerated dose, by definition, stresses cells and causes untimely “cell killing.”

Thus, cell proliferation (mitogenesis) increases, leading to more endogenous mutations and cancer. It follows that the ability of the chemical to produce mutations was irrelevant to the cancerous outcome (which Ames still viewed as being caused by mutations). The “mutagen is a carcinogen / carcinogen is a mutagen” concept that seemed so real in the early 1970s fell apart. The testing protocol was the cause of many positive carcinogen findings and had nothing to do with the dose-response at very low dose-rates.

VIII. EPIDEMIOLOGY AND THE LNT RELATIONSHIP Some readers will argue that in spite of (i) the absence of any connection between Muller’s experimental work and cancer; (ii) the numerous steps in the conversion of a mutated cell into a cancer clone where thresholds and non-linear relationships are expected; and (iii) obvious examples where production of mutations at low dose-rates over a life-time do not produce cancer; the extensive use of linear, no-threshold models to fit experimental carcinogenicity data from bioassays and epidemiological studies proves the validity of the relationship. This argument turns out to be nonsense. A. Muller’s Data were Linear for Total Dose The fundamental discovery that Muller25 made when using high doses of high energy radiation was that the incidence of mutations (not cancer) was directly proportional to the total dose (i.e., not the duration of exposure and not the dose

rate but rather the product of the two factors). Most people at the USEPA never went back to Muller and instead relied on work done by the Atomic Energy Commission1 in developing the USEPA’s risk assessment methodology and policy. It was a legal search for “precedent,” not a scientific search for “truth.” So, the differences between (i) total dose, (ii) dose rate and (iii) duration of dosing were ignored. Ironically, the typical formats for bioassays tend to be a constant daily dose (i.e., a constant dose rate) for a set period of time. Under these conditions the dose rate, duration of dosing and total dose are all directly proportional to one another so that the issue is obscured in bioassays. But, this is not a valid assumption in epidemiological studies. The norm in epidemiology studies is to measure concentration of a chemical constituent in a medium (e.g., drinking water) at some current time. This measurement is at best a surrogate for (current) dose rate (assuming that the exposed individuals consume a specific amount of the medium each day (e.g., 2 L of drinking water per day per person). In the work leading up to the revision of the drinking water standard for “arsenic,” the USEPA301 conducted a study of arsenic exposure and cancer among Mormon families in rural Utah. On the face of it, this should be one of the most ideal populations to study. The subject people avoid many of the most important confounding exposures (smoking, drinking, even caffeinated soft drinks, coffee and tea) and importantly can be tied to specific water sources. Both the people and the water sources had defined histories. The

USEPA’s Dr. Lewis, following previous studies on non-carcinogenic effects, used median drinking water concentrations (which did not vary dramatically from the mean) for each community (which typically included multiple, quality-controlled samples collected over more than 20 years) multiplied by the number of years that individuals had drunk the water as her dose-metric. This data set, thus, in keeping with the total dose concept of Muller, combined the dose-rate (as concentration, ppm) and duration of exposure (in years) to give a realistic impression of total dose (and conceived by Muller). The irony here is that the National Academy of Sciences panel that wrote the review of arsenic risk for the USEPA (1999, 2001) dismissed and disparaged the Lewis study because in its words (National Academy of Sciences 2001 update on Arsenic in Drinking Water, p.5) : “... the subcommittee concluded that the limitations of the Utah study currently preclude its use in a quantitative risk assessment. One limitation was the unconventional method used in that study to characterize exposure. ...” The National Academy of Sciences panel also raised a spurious issue about the control population: “... in contrast to the southwestern Taiwan study where lifestyle differences do not appear to influence relative risk of cancer from arsenic in drinking water, the Utah study used a comparison group with differences in lifestyle

characteristics different from the study population. The study population was composed of individuals with religious prohibitions against smoking, and the unexposed comparison group was the overall population of Utah, where such religious prohibitions are not practiced by all residents.” The USEPA authors (Lewis et al.) responded to the issue about comparison groups302 in the same peer-reviewed journal that their original paper was published301 noting that in 1982, 73% of the residence of Utah were Mormons and Utah has historically been among the states with the lowest incidence of bladder and lung cancer in the US. In the SW Taiwan study, Tseng303 noted that there were three professions among the surveyed rural population (farmers, fishermen and salt producers with carbohydrate-based diets) and Wu304 compared the Taiwan population to the entire world. Tseng also noted that about 10% of his population was constantly away from home. As for the “unconventional” dose-metric used by Lewis et al., I would say that it was unconventional only in the sense that it was done in the most consistent manner associated with the underlying principles associated with the linear, no-threshold model. The dose-metrics that have been conventionally used by epidemiologists in cancer studies appear to be arbitrarily chosen to make the data fit more-or-less into a linear dose-response relationship. In the case that I will present next (the one mentioned by the National Academy of Sciences as superior to the USEPA study), the median concentration (of arsenic) was used as the dose-metric with no

consideration of exposure duration (see below). In other studies “log concentration” or “ln concentration” have been used as the dose-metric. The latter manipulation (i.e., taking the log of dose rate or concentration) can convert an S-shaped curve into a straighter line. It should be obvious that Muller did not observe linear relationships associated with any of these dose-metrics. Why then, would we relate any of these studies to the linear, no-threshold observations that Muller made? Epidemiology is a valid science and an epidemiologist is free to choose any dosemetric to correlate data within the experimental range. The problem comes when anyone attempts to extrapolate data beyond the experimental range. To do this you must have an underlying hypothesis about the shape of the dose-response curve. This hypothesis does not come from the experimental dada, but rather a model of how these processes work (e.g., photon --> mutation). In the cancer arena, epidemiologists appear to believe that they can manipulate the dose-metric any way they wish and then do a linear extrapolation to “zero dose.” Any study that involves linear extrapolation to “zero dose” has no shred of scientific basis unless the “dose” is calculated as total dose (i.e., dose rate (mg/kg.day) x time (days) = total dose). And, I repeat once more, that Muller only considered mutations (not cancer) and only studied high energy photons (not chemicals). B. The SW Taiwan Arsenic in Drinking Water Study Since the prestigious National Academy of Science seems to believe the SW

Taiwan study is acceptable science, I will use it to point out the many ways in which epidemiologists frequently get it wrong (in addition to using irrelevant dose-metrics to make the data fit a model they like). I first became aware of the SW Taiwan study 303 in 1977 when I305 attended a conference on arsenic in the environment and listened to a paper by Dr. W.P. Tseng. Tseng306 had initially been studying “blackfoot disease” (an ascending dry gangrene of the extremities, which is obviously related to some sort of interference with peripheral circulation306309). The working hypothesis in Taiwan at the time was that blackfoot disease

was caused by arsenic in the drinking water, although similar arsenic exposures in other areas do not cause this disease. In fact, the cause of blackfoot disease has been hotly debated,309, 310 and there is strikingly similar animal disease that is caused by a virus.311 During the course of these studies, some 40,000 people had been surveyed for skin lesions and some skin cancers in areas not normally exposed to the sun were observed. Since every one already “knew” that arsenic caused skin cancer,274, 312 the focus of the research shifted to include arseniccaused skin cancers. The large number of people included in this sample potentially provides exceptional statistical power to the study, which has made it a favorite of epidemiologists for that reason. One of the criticisms of the Utah study mentioned above was that it only involved several thousand people. The first problem I have with this study is that arsenic occurs in many chemical forms and the arbitrary use of the word “arsenic” needs clarification. Here is

where the weakness of epidemiologists in chemistry becomes very apparent. For practical purposes, “arsenic” concentration is being operationally defined in this and other studies by the analytical methods used to measure it. There is very little explanation of the analytical methodology used in the SW Taiwan study or the quality control / quality assurance methods applied. The absence of this information would make the data unacceptable by current standards. Nonetheless, we can assume that the methodology used was effective for “total arsenic,” i.e., all arsenic compounds rendered into a form suitable for measurement and we will assume that the analytical measurements were accurate within a few percent at the concentrations measured. Tseng303 compiled the data in what seemed (to me) at the time to be a reasonable (if somewhat arbitrary) manner. In each township, there were a number of wells and Tseng presented a bar-graph of the number of wells of each concentration increment (e.g., 0.05, 0.15, 0.20...) as a function of concentration. The graph (Figure 4 in his paper)303 was roughly normally distributed. My assumption at the time, and for the next 20 years, was that when he characterized a township as “high,” “medium,” or “low” that all the wells in that township fell into only one of these ranges of concentration (i.e., high, medium or low). Amazingly, it is not clear from his paper how Tseng actually compiled the chemistry data. However, his data was apparently the basis for the work by Wu et al.304 Chen et al.313 and the National Academy of Sciences (1999). The method that they followed was to

take the median concentration of the wells (all the wells and no others?) in a township and use that as the basis for characterizing the exposure in the township. The assumption was that all the people in the township were exposed to the same concentration characterized by the median. This would be a fair assumption if all the wells in a township were near the same concentration, if they had all be operating for a similar period of time, and the people drank randomly from all the wells. However, it is clear for Tseng’s paper303 that different people had unique (local) water sources since most of the wells (110) were artesian wells that were being replaced by public water supplies starting in the 1950s. What I discovered when reading the 1999 National Academy of Science’s report on Arsenic in Drinking Water (Addendum to Chapter 10, pp. 308-9) was very disturbing. Many of the townships (villages) had more than one well; and where this was the case, (contrary to my original assumption) the wells were frequently quite different in concentrations. Even more disturbing is the fact that Tseng (1977, p. 111) states: "From the villages surveyed, we examined 142 water samples from 114 wells, of which all but four were artesian." Tseng speaks of 37 villages. When I add up the “villages” in the addendum to the 1999 report, I find 42 villages (as stated by Wu et al., 1989 who indicate that “[t]he arsenic levels in well water determined in 1964-1966 were available in 42 villages of the study area...”).304 However, in the addendum there are a total of

153 chemical analyses identified as “wells.” Thus, in the original report by Tseng (1977) he acknowledges that he had multiple samples from some well and Wu (1989) apparently has even more analytical data (for the same suite of wells?). A forensic look at the analytical data (in the addendum) immediately indicate that someone arbitrarily added zeros (significant figures) to that data so that it would all be three places after the decimal. This is a very bad practice that happens when data are poured into computerized databases. But, what seems particularly odd is the redundancy of certain numbers (e.g., in Village 4-1 we find 0.850 three times, 0.840 twice, 0.810 twice, 0.760 three times, 0.290 twice, 0.270 twice, 0.120 twice and 0.020 twice in 47 “wells”). Even if we only consider the 2 significant figure data, having the same two digits come up randomly three times in essentially 95 (0.02 to 0.97) trials is unlikely (e.g., 1 in 10,000). In short, the data set presumably used by Tseng (1977) and Wu (1989) and the National Academy of Sciences (1999) is very suspicious. This is particularly concerning because the median values were used to establish the exposure for the villages. With data for a village that range over essentially two orders of magnitude, redundant or missing data could completely skew the result. Of course, if there are wells in the villages that are not included in the data set, then we have an additional problem, especially since the median values were used to calculate exposure. On a broader analysis, how do we know that the concentrations determined in

1964-66304 are typical for the entire lifetime of the population. For example, the skin cancers (found in the 1960s) were typically observed in people in their 70s. This means that their life (assuming that they lived in the same place all that time) spanned nominally 1900-1970. A few things happened in the area during that time period (e.g., Russo-Japanese War, Japanese invasion of China, World War II, the rise of Communism in China). Although Tseng indicates that some artesian wells were installed as early as 1900-1910 and Su et al.314 say that the deep artesian wells were not introduced until the 1920s, it is not clear when any individual well was installed. Do they all pump the same amount of water? Has the concentration of arsenic stayed constant in the wells over time? Basically, we know that there are some gigantic holes in this database. The biggest problem I see is that we cannot associate any exposed individual with a particular well and the wells in any township (village) can very from low to high (i.e., the median values for a township appear to mean nothing since individuals likely drank from specific wells and the medians themselves appear to be corrupted by inclusion of multiple sampling events from individual wells as separate wells). The final point I’ll make in the generic critique of epidemiologists is that they tend to focus on one constituent (in this case arsenic) and ignore all other possibilities. Tseng clearly associated both blackfoot disease and skin cancer to “arsenic” (as defined by his analytical method) in drinking water. But, in the case of blackfoot disease, the association with arsenic is considered tenuous.309, 310 If the data do

not hold up for blackfoot disease, why should they hold up for arsenic? It is interesting that although the high-arsenic artesian wells have now been abandoned and replaced with municipal water supplies for a generation (since the mid1970s),314 bladder cancer is reduced but still elevated relative to the rest of Taiwan and lung cancer (which few reports associate with arsenic exposure) was similarly reduced.314 Part of the narrow focus of the epidemiologists here is the indifference to other sources of arsenic. Recall that the rural SW Taiwan population was divided among farmers, fisherman and salt producers (i.e., the evaporation of sea water to produce salt). Sushi is, of course, a staple in Taiwan. Less known is that fact that marine algae are loaded with arsenic in various forms.315-317 Although arsenosugars appear to be relatively non-toxic, substantial concentrations of inorganic arsenic ions are present.318 This exposure is totally ignored and uncontrolled in the drinking water studies from SW Taiwan to date.

IX. IN SUMMARY A. Never a Scientific Basis for LNT Cancer Risk Assessment There has never been a scientific basis for a linear no-threshold dose-response relationship between radiation or chemicals and cancer. Muller’s dramatic “linear, no-threshold” presentation in his Nobel Prize speech was for mutagenicity caused by high energy photons. This idea was progressively comingled by the risk assessment community with the mutation hypothesis of cancer causation until

it became a dogma. The applicability of the LNT hypothesis to radiation was plausible in the 1950s before DNA repair, apoptosis, Hayflick’s limit, and Muller’s ratchet were understood. However, Each of these factors affects the linearity of dose-response of radiation and the USEPA (which has become the leading authority on cancer risk assessment) was particularly negligent when they adopted the LNT concept without even considering pharmacodynamics when it was obvious (and acknowledged) that many carcinogens were activated by metabolism in vivo.1, 300, 319-322 The USEPA has grudgingly moved to a position of considering pharmacodynamics in their mode of action analyses.1, 264, 268, 285 B. Acceptable Risk Determination of acceptable risk levels is not associated with how risks are estimated. But, setting risk targets is another factor in determining the overall impact of risk avoidance strategies. The acceptability of risk should be the type of thing we elect our legislators to determine. It is a social value judgment. In contrast, the calculation of risk that is the subject of this paper should be as scientific as possible although there may be places where judgment will be required to fill in missing data. The target risk for USEPA cancer risk regulation is 10-6 deaths per lifetime of exposure (see for example, target risk criteria in 40CFR300.430(e)(2)(i)(A)(2)). Roy Albert relates a remarkable story about where this commonly used one-in-

one-million lifetime risk criterion came from.1 According to the story, it was the product of a hallway exchange between two USEPA executives and was based on the idea that 10-5 risk of death in mass transportation accidents is apparently acceptable to the general public. It is not clear exactly where this number came from, but this is apparently per trip (e.g., the probability that a trip in public transportation will involve a death). The USEPA reduced that by an order of magnitude to 10-6 and made it the tolerable risk for a life-time of exposure to carcinogens. Moving from per event to life-time was a very important change. Consider the following analysis: People routinely drive their cars (or ride as passengers in cars) for hundreds of thousands of miles in their life-time. Remarkable improvements in automobile safety (e.g., airbags, anti-lock breaks, stability controls) have lowered fatalities in recent years to 1.15 deaths per 100 million vehicle travel miles. That is, roughly 10-8 deaths/mile. If we multiply that by 100,000 miles (likely less than most people travel in cars during their lifetimes), we obtain a lower limit of 10-3 for an individual’s lifetime risk of automobile fatality. If the USEPA regulated automobile transportation, a person would not be allowed to travel more than 100 miles by car in their lifetime! Two other features of these two sources of risk (automobile transportation and exposure to carcinogens) should be considered: (i) Automobile accidents kill indiscriminately by age, whereas, many cancer victims die rather late in life. (ii) The automobile statistics are real (there are actual dead people making up the

statistics, not hypothetical people) whereas cancer risk assessments typically are based on hypothetical exposure scenarios (over a lifetime) and very conservative dose-response relationships.1 It should be shocking that something as fundamental as “acceptable risk” should be determined in a casual conversation among unelected bureaucrats who appear to have not considered the truth or impact of their decision. C. Side Effect of the LNT Hypothesis As noted above, the LNT dose-response hypothesis is tied directly to the mutation theory of cancer causation. Acceptance of the LNT hypothesis reinforces the mutation theory. Most of our regulatory strategies for risk avoidance, our research into cancer causation and cure and our attempts to treat cancer in patients for the last 50 years have been driven by this mind set. Although there have been some incremental improvements mostly associated with elimination of social risks such as smoking, sun tanning and sexual transmitted HPV; these hypotheses have not led to a major breakthrough against a broader range of cancers. Perhaps it is time we should acknowledge that the LNT hypothesis (and to some extent mutation the theory of cancer causation) are not supported by science and look to alternatives.

X. REFERENCES 1. Albert RE. Carcinogen risk assessment in the U.S. Environmental Protection Agency. Crit Rev Toxicol. 1994;24(1):75-85.

2. Goldschmidt R. Theodor Boveri. Science. 1916 Feb 25;43(1104):263-70. 3. Boveri T. Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Harris. J Cell Sci. 2008 Jan;121 Suppl 1:1-84. 4. Metcalf MM. Boveri on cancer. Science. 1926 Nov 19;64(1664):499-500. 5. Morgan TH, Sturtevant AH, Bridge CB. The Evidence for the Linear Order of the Genes. Proc Natl Acad Sci U S A. 1920 Apr;6(4):162-4. 6. Sturtevant AH, Bridges CB, Morgan TH. The Spatial Relations of Genes. Proc Natl Acad Sci U S A. 1919 May;5(5):168-73. 7. de Vries H. The origin of species by mutation. Science. 1902 May 9;15(384):721-9. 8. de Vries H. The principles of the theory of mutation. Science. 1914 Jul 17;40(1020):77-84. 9. Lenay C. Hugo De Vries: from the theory of intracellular pangenesis to the rediscovery of Mendel. C R Acad Sci III. 2000 Dec;323(12):1053-60. 10. Jeffrey EC. The Mutation Myth. Science. 1914 Apr 3;39(1005):488-91. 11. Rous P. A transmissible avian neoplasm (sarcoma of the common fowl). J Exp Med. 1910 Sep 1;12(5):696-705. 12. Rous P. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med. 1911 Apr 1;13(4):397-411. 13. Rous P, Murphy JB. On the causation by filterable agents of three distinct chicken tumors. J Exp Med. 1914 Jan 1;19(1):52-68. 14. Viruses and cancer. Br Med J. 1925 Jul 25;2(3369):174-6. 15. The theory of the cancer virus. Br Med J. 1926 May 22;1(3411):879. 16. Shope RE. A filterable virus causing a tumor-like condition in rabbits and its relationship to virus Myxomatosum. J Exp Med. 1932 Nov 30;56(6):803-22. 17. Chemical agents and viruses in cancer. Br Med J. 1939 Sep 23;2(4107):651-2. 18. Viruses and cancer. Br Med J. 1939 Nov 11;2(4114):959-60. 19. Andrewes CH. Latent Virus Infections and Their Possible Relevance to the Cancer Problem: (Section of Comparative Medicine). Proc R Soc Med. 1939 Dec;33(2):75-86. 20. Rous P, Botsford E. The incidence of cancer in tarred and sheltered mice. J Exp Med. 1932 Jan 31;55(2):247-66. 21. Sonneborn TM. H. J. Muller, crusader for human betterment. Science. 1968 Nov 15;162(3855):772. 22. Paul D. H. J. Muller, communism, and the cold war. Genetics. 1988 Jun;119(2):223-5. 23. Auerbach C. H. J. Muller 1890-1967. Mutat Res. 1968 Mar-Apr;5(2):2017.

24. Muller HJ. The Measurement of gene mutation rate in Drosophila, its high variability, and its dependence upon temperature. Genetics. 1928 May;13(4):279357. 25. Muller HJ. The production of mutations by X-rays. Proc Natl Acad Sci U S A. 1928 Sep;14(9):714-26. 26. Hanson FB, Heys FM. The effects of radium in producing lethal mutations in Drodophila melanogaster. Science. 1928 Aug 3;68(1753):115-6. 27. Altenburg E. The Production of Mutations by Ultra-Violet Light. Science. 1933 Dec 22;78(2034):587. 28. Hanson FB, Heys F. A posssible relation between natural (earth) radiation and gene mutations. Science. 1930 Jan 10;71(1828):43-4. 29. Babcock EB, Collins JL. Does natural inoizing radiation control rate of mutation? Proc Natl Acad Sci U S A. 1929 Aug 15;15(8):623-8. 30. Oliver CP. The effect of varying the duration of X-ray treatment upon the frequency of mutation. Science. 1930 Jan 10;71(1828):44-6. 31. Calabrese EJ. Muller's Nobel lecture on dose-response for ionizing radiation: ideology or science? Arch Toxicol. 2011 Dec;85(12):1495-8. 32. Calabrese EJ. Muller's Nobel Prize Lecture: when ideology prevailed over science. Toxicol Sci. 2012 Mar;126(1):1-4. 33. Spencer WP, Stern C. Experiments to test the validity of the linear rdose/mutation frequency relation in Drosophila at low dosage. Genetics. 1948 Jan;33(1):43-74. 34. Calabrese EJ. Key studies used to support cancer risk assessment questioned. Environ Mol Mutagen. 2011 Oct;52(8):595-606. 35. Caspari E, Stern C. The influence of chronic irradiation with gamma-rays at low dosages on the mutation rate in Drosophila melanogaster. Genetics. 1948 Jan;33(1):75-95. 36. Graff S, Moore DH, et al. Isolation of mouse mammary carcinoma virus. Cancer. 1949 Sep;2(5):755-62. 37. Porter KR, Thompson HP. A particulate body associated with epithelial cells cultured from mammary carcinomas of mice of a milkfactor strain. J Exp Med. 1948 Jul;88(1):15-24. 38. Bittner JJ. Relation of nursing to the extra-chromosomal theory of breast cancer in mice. Am J Cancer. 1939 Jan;35(1):90-7. 39. Fulford J, Bonner P, Goodhead DT, Hill MA, O'Neill P. Experimental determination of the dependence of OH radical yield on photon energy: a comparison with theoretical simulations. J Phys Chem A. 1999;103(51):11345-9. 40. Yamaguchi H, Uchihori Y, Yasuda N, Takada M, Kitamura H. Estimation of yields of OH radicals in water irradiated by ionizing radiation. J Radiat Res

(Tokyo). 2005 Sep;46(3):333-41. 41. Biaglow JE, Varnes ME, Epp ER, Clark EP, Tuttle S, Held KD. Cellular protection against damage by hydroperoxides: role of glutathione. Basic Life Sci. 1988;49:567-73. 42. Moran LK, Gutteridge JM, Quinlan GJ. Thiols in cellular redox signalling and control. Curr Med Chem. 2001 Jun;8(7):763-72. 43. Moiseenko VV, Hamm RN, Waker AJ, Prestwich WV. Calculation of radiation-induced DNA damage from photons and tritium beta-particles. Part I: Model formulation and basic results. Radiat Environ Biophys. 2001 Mar;40(1):23-31. 44. Azzam EI, Jay-Gerin JP, Pain D. Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett. 2012 Dec 31;327(1-2):4860. 45. Levi S, Rovida E. The role of iron in mitochondrial function. Biochim Biophys Acta. 2009 Jul;1790(7):629-36. 46. Kurz T, Eaton JW, Brunk UT. The role of lysosomes in iron metabolism and recycling. Int J Biochem Cell Biol. 2011 Dec;43(12):1686-97. 47. Kurz T, Terman A, Gustafsson B, Brunk UT. Lysosomes in iron metabolism, ageing and apoptosis. Histochem Cell Biol. 2008 Apr;129(4):389406. 48. Persson HL, Kurz T, Eaton JW, Brunk UT. Radiation-induced cell death: importance of lysosomal destabilization. Biochem J. 2005 Aug 1;389(Pt 3):87784. 49. Uchiyama A, Kim JS, Kon K, Jaeschke H, Ikejima K, Watanabe S, Lemasters JJ. Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury. Hepatology. 2008 Nov;48(5):1644-54. 50. Berenblum I, Shubik P. An experimental study of the initiating state of carcinogenesis, and a re-examination of the somatic cell mutation theory of cancer. Br J Cancer. 1949 Mar;3(1):109-18. 51. Curtis MR, Dunning WF, Bullock FD. Is malignancy due to a process analogous to somatic mutation? Science. 1933 Feb 10;77(1989):175-6. 52. Friedewald WF, Rous P. The initiating and promoting elements in tumor production: an analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin J Exp Med. 1944 Aug 1;80(2):101-26. 53. Berenblum I, Shubik P. The role of croton oil applications, associated with a single painting of a carcinogen, in tumour induction of the mouse's skin. Br J Cancer. 1947 Dec;1(4):379-82. 54. Strong LC. A new theory of mutation and the origin of cancer. Yale J Biol

Med. 1949 Mar;21(4):293-9. 55. Burdette WJ. A method for determining mutation rate and tumor incidence simultaneously. Cancer Res. 1951 Jul;11(7):552-4. 56. Burdette WJ. Tumor incidence and lethal mutation rate in a tumor strain of Drosophila treated with formaldehyde. Cancer Res. 1951 Jul;11(7):555-8. 57. Stigler R. [Theory of mutation in cancer]. Langenbecks Arch Klin Chir Ver Dtsch Z Chir. 1952;273:604-7. 58. Blum HF, Alexander J. [Regarding the somatic mutation hypothesis of cancer]. Science. 1953 Aug 14;118(3059):197-8. 59. Burdette WJ. The somatic mutation hypothesis of cancer genesis. Science. 1953 Aug 14;118(3059):196-7. 60. Fardon JC. A reconsideration of the somatic mutation theory of cancer in the light of some recent developments. Science. 1953 Apr 24;117(3043):441-5. 61. Burdette WJ. Somatic mutation and cancer. Acta Unio Int Contra Cancrum. 1954;10(3):97-104. 62. Burdette WJ. The significance of mutation in relation to the origin of tumors: a review. Cancer Res. 1955 May;15(4):201-26. 63. Nordling CO. Evidence regarding the multiple mutation theory of the cancer-inducing mechanism. Acta Genet Stat Med. 1955;5(2):93-104. 64. Brues AM. Critique of the linear theory of carcinogenesis. Science. 1958 Sep 26;128(3326):693-9. 65. Avery OT, Macleod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from a pneumococcus type III. J Exp Med. 1944 Feb 1;79(2):137-58. 66. Maddox B. The double helix and the 'wronged heroine'. Nature. 2003 Jan 23;421(6921):407-8. 67. Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953 Apr 25;171(4356):737-8. 68. Watson JD, Crick FH. Genetical implications of the structure of deoxyribonucleic acid. Nature. 1953 May 30;171(4361):964-7. 69. Lieb M. Deoxyribonucleic acid synthesis and ultraviolet-induced mutation. Biochim Biophys Acta. 1960 Jan 1;37:155-7. 70. Rudner R. Mutation as an error in base pairing. Biochem Biophys Res Commun. 1960 Sep;3:275-80. 71. Rudner R. Mutation as an error in base pairing II. Kinetics of 5bromodeoxyuridine and 2-aminopurine-induced mutagenesis. Z Vererbungsl. 1961;92:361-79. 72. Rudner R. Mutation as an error in base pairing. I. The mutagenicity of base

analogues and their incorporation into the DNA of Salmonella typhimurium. Z Vererbungsl. 1961;92:336-60. 73. Altenburg LS, Altenburg E, Baker RN. Evidence indicating that the mutation rate induced in Drosophila by low doses of ultraviolet light is an exponential function of the dose. Genetics. 1952 Sep;37(5):558-61. 74. Altenburg E. The production of mutations by ultra-violet light Science. 1933 Dec 22;78(2034):587. 75. Kelner A. Photoreactivation of ultraviolet-irradiated Escherichia coli, with special reference to the dose-reduction principle and to ultraviolet-induced mutation. J Bacteriol. 1949 Oct;58(4):511-22. 76. Kelner A. Effect of visible light on the recovery of Streptomyces griseus conidia from ultra-violet irradiation injury. Proc Natl Acad Sci U S A. 1949 Feb;35(2):73-9. 77. Kelner A. Light-Induced Recovery of Microorganisms from Ultraviolet Radiation Injury, with Special Reference to Escherichia Coli. Bull N Y Acad Med. 1950 Mar;26(3):189-99. 78. Kelner A. Action spectra for photoreactivation of ultraviolet-irradiated Escherichia coli and Streptomyces griseus. J Gen Physiol. 1951 Jul;34(6):835-52. 79. Perlitsh M, Kelner A. The reduction by reactivating light of the frequency of phenocopies induced by ultraviolet light in drosophila melanogaster. Science. 1953 Aug 7;118(3058):165-6. 80. Setlow JK. Photorepair of biological systems. Res Prog Org Biol Med Chem. 1972;3 Pt 1:335-55. 81. Setlow RB, Carrier WL, Bollum FJ. Pyrimidine dimers in UV-irradiated poly dI:dC. Proc Natl Acad Sci U S A. 1965 May;53(5):1111-8. 82. Setlow RB, Swenson PA, Carrier WL. Thymine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells Science. 1963 Dec 13;142(3598):1464-6. 83. Wulff DL. Kinetics of thymine photodimerization Biophys J. 1963 Sep;3:355-62. 84. Anastasi C, Buchet FF, Crowe MA, Helliwell M, Raftery J, Sutherland JD. The search for a potentially prebiotic synthesis of nucleotides via arabinose3-phosphate and its cyanamide derivative. Chemistry. 2008;14(8):2375-88. 85. Sutherland BM. The human leukocyte photoreactivating enzyme. Basic Life Sci. 1975;5A:107-13. 86. Liu Z, Guo X, Tan C, Li J, Kao YT, Wang L, Sancar A, Zhong D. Electron tunneling pathways and role of adenine in repair of cyclobutane pyrimidine dimer by DNA photolyase. J Am Chem Soc. 2012 May 16;134(19):8104-14. 87. Schuster RC. Dark repair of ultraviolet injury in E. coli during deprivation

of thymine. Nature. 1964 May 9;202:614-5. 88. Blackburn GM, Davies RJ. The structure of DNA-derived thymine dimer. Biochem Biophys Res Commun. 1966 Mar 22;22(6):704-6. 89. Shuster RC, Boyce RP. The excision of thymine dimer from the DNA of UV-irradiated E. coli 15 T-A-U during thymine deprivation. Biochem Biophys Res Commun. 1964 Jul 27;16(5):489-96. 90. Goss P, Parsons PG. Temperature-sensitive DNA repair of ultraviolet damage in human cell lines. Int J Cancer. 1976 Mar 15;17(3):296-303. 91. Achey P, Billen D. Saturation of dark repair synthesis: accumulation of strand breaks. Biophys J. 1969 May;9(5):647-53. 92. Harm W. Dark repair of photorepairable UV lesions in Escherichia coli. Mutat Res. 1968 Jul-Aug;6(1):25-35. 93. Hill RF. Do dark repair mechanisms for UV-induced primary damage affect spontaneous mutation? Mutat Res. 1968 Nov-Dec;6(3):472-5. 94. Cleaver JE. Repair replication and degradation of bromouracil-substituted DNA in mammalian cells after irradiation with ultraviolet light. Biophys J. 1968 Jul;8(7):775-91. 95. Cleaver JE. Xeroderma pigmentosum: a human disease in which an initial stage of DNA repair is defective. Proc Natl Acad Sci U S A. 1969 Jun;63(2):42835. 96. Cleaver JE. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652-6. 97. Cleaver JE. DNA repair and radiation sensitivity in human (xeroderma pigmentosum) cells. Int J Radiat Biol Relat Stud Phys Chem Med. 1970;18(6):557-65. 98. Setlow RB, Regan JD, German J, Carrier WL. Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. Proc Natl Acad Sci U S A. 1969 Nov;64(3):1035-41. 99. Cleaver JE. Common pathways for ultraviolet skin carcinogenesis in the repair and replication defective groups of xeroderma pigmentosum. J Dermatol Sci. 2000 May;23(1):1-11. 100. Cleaver JE, Bootsma D, Friedberg E. Human diseases with genetically altered DNA repair processes. Genetics. 1975 Jun;79 Suppl:215-25. 101. Giannelli F, Croll PM, Lewin SA. DNA repair synthesis in human heterokaryons formed by normal and UV-sensitive fibroblasts. Exp Cell Res. 1973 Mar 30;78(1):175-85. 102. Paterson MC, Lohman PH, Westerveld A, Sluyter ML. DNA repair in human/embryonic chick heterokaryons. Ability of each species to aid the other in the removal of ultraviolet-induced damage. Biophys J. 1974 Nov;14(11):835-45.

103. Karentz D, Cleaver JE. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells. Mol Cell Biol. 1986 Oct;6(10):3428-32. 104. Painter RB. DNA damage and repair in eukaryotic cells. Genetics. 1974 Sep;78(1):139-48. 105. Lange CS. The organization and repair of mammalian DNA. FEBS Lett. 1974 Aug 25;44(2):153-6. 106. Van Holde KE, Sahasrabuddhe CG, Shaw BR. A model for particulate structure in chromatin. Nucleic Acids Res. 1974 Nov;1(11):1579-86. 107. Lange CS. The repair of DNA double-strand breaks in mammalian cells and the organization of the DNA in their chromosomes. Basic Life Sci. 1975;5B:677-83. 108. Lobban PE, Kaiser AD. Enzymatic end-to end joining of DNA molecules. J Mol Biol. 1973 Aug 15;78(3):453-71. 109. Mezard C, Nicolas A. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae. Mol Cell Biol. 1994 Feb;14(2):127892. 110. Iyer LM, Koonin EV, Aravind L. Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics. 2002 Mar 21;3:8. 111. Davis AP, Symington LS. The Rad52-Rad59 complex interacts with Rad51 and replication protein A. DNA Repair (Amst). 2003 Oct 7;2(10):1127-34. 112. Pannunzio NR, Manthey GM, Liddell LC, Fu BX, Roberts CM, Bailis AM. Rad59 regulates association of Rad52 with DNA double-strand breaks. Microbiologyopen. 2012 Sep;1(3):285-97. 113. Davis AP, Symington LS. The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing. Genetics. 2001 Oct;159(2):515-25. 114. Mortensen UH, Bendixen C, Sunjevaric I, Rothstein R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10729-34. 115. Van Dyck E, Stasiak AZ, Stasiak A, West SC. Binding of double-strand breaks in DNA by human Rad52 protein. Nature. 1999 Apr 22;398(6729):728-31. 116. Kaczmarski W, Khan SA. Lupus autoantigen Ku protein binds HIV-1 TAR RNA in vitro. Biochem Biophys Res Commun. 1993 Oct 29;196(2):935-42. 117. Park EJ, Chan DW, Park JH, Oettinger MA, Kwon J. DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. Nucleic Acids Res. 2003 Dec 1;31(23):6819-27.

118. Pinto M, Prise KM, Michael BD. Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics. Radiat Res. 2005 Jul;164(1):73-85. 119. Mladenov E, Iliakis G. Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways. Mutat Res. 2011 Jun 3;711(1-2):61-72. 120. Sinkovics JG. Programmed cell death (apoptosis): its virological and immunological connections (a review). Acta Microbiol Hung. 1991;38(3-4):32134. 121. Wyllie AH. Apoptosis (the 1992 Frank Rose Memorial Lecture). Br J Cancer. 1993 Feb;67(2):205-8. 122. Hitomi M, Yang K, Stacey AW, Stacey DW. Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression. Mol Cell Biol. 2008 Sep;28(17):5478-93. 123. Engelmann D, Putzer BM. Translating DNA damage into cancer cell deathA roadmap for E2F1 apoptotic signalling and opportunities for new drug combinations to overcome chemoresistance. Drug Resist Updat. 2010 AugOct;13(4-5):119-31. 124. Flemington EK, Speck SH, Kaelin WG, Jr. E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):69148. 125. Slansky JE, Farnham PJ. Introduction to the E2F family: protein structure and gene regulation. Curr Top Microbiol Immunol. 1996;208:1-30. 126. Rubin SM, Gall AL, Zheng N, Pavletich NP. Structure of the Rb Cterminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release. Cell. 2005 Dec 16;123(6):1093-106. 127. Akiyama T, Ohuchi T, Sumida S, Matsumoto K, Toyoshima K. Phosphorylation of the retinoblastoma protein by cdk2. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7900-4. 128. Cobrinik D, Dowdy SF, Hinds PW, Mittnacht S, Weinberg RA. The retinoblastoma protein and the regulation of cell cycling. Trends Biochem Sci. 1992 Aug;17(8):312-5. 129. Lehman AR, Stevens S. The production and repair of double strand breaks in cells from normal humans and from patients with ataxia telangiectasia. Biochim Biophys Acta. 1977 Jan 3;474(1):49-60. 130. Crawford LV, Pim DC, Gurney EG, Goodfellow P, Taylor-Papadimitriou J. Detection of a common feature in several human tumor cell lines--a 53,000dalton protein. Proc Natl Acad Sci U S A. 1981 Jan;78(1):41-5.

131. Finlay CA, Hinds PW, Levine AJ. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083-93. 132. Cox LS, Lane DP. Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage. Bioessays. 1995 Jun;17(6):501-8. 133. Yonish-Rouach E, Grunwald D, Wilder S, Kimchi A, May E, Lawrence JJ, May P, Oren M. p53-mediated cell death: relationship to cell cycle control. Mol Cell Biol. 1993 Mar;13(3):1415-23. 134. Wyllie AH, Carder PJ, Clarke AR, Cripps KJ, Gledhill S, Greaves MF, Griffiths S, Harrison DJ, Hooper ML, Morris RG, et al. Apoptosis in carcinogenesis: the role of p53. Cold Spring Harb Symp Quant Biol. 1994;59:403-9. 135. Hockenbery DM. The bcl-2 oncogene and apoptosis. Semin Immunol. 1992 Dec;4(6):413-20. 136. McDonnell TJ, Marin MC, Hsu B, Brisbay SM, McConnell K, Tu SM, Campbell ML, Rodriguez-Villanueva J. The bcl-2 oncogene: apoptosis and neoplasia. Radiat Res. 1993 Dec;136(3):307-12. 137. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 1984 Nov 30;226(4678):1097-9. 138. Canman CE, Wolff AC, Chen CY, Fornace AJ, Jr., Kastan MB. The p53dependent G1 cell cycle checkpoint pathway and ataxia-telangiectasia. Cancer Res. 1994 Oct 1;54(19):5054-8. 139. Smith ML, Zhan Q, Bae I, Fornace AJ, Jr. Role of retinoblastoma gene product in p53-mediated DNA damage response. Exp Cell Res. 1994 Dec;215(2):386-9. 140. Xie G, Habbersett RC, Jia Y, Peterson SR, Lehnert BE, Bradbury EM, D'Anna JA. Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints. Oncogene. 1998 Feb 12;16(6):721-36. 141. Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATMdependent activation of p53 involves dephosphorylation and association with 143-3 proteins. Nat Genet. 1998 Jun;19(2):175-8. 142. Khanna KK, Keating KE, Kozlov S, Scott S, Gatei M, Hobson K, Taya Y, Gabrielli B, Chan D, Lees-Miller SP, Lavin MF. ATM associates with and phosphorylates p53: mapping the region of interaction. Nat Genet. 1998 Dec;20(4):398-400. 143. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, Taya Y, Prives C, Abraham RT. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 1999 Jan 15;13(2):152-7.

144. Lavin MF. ATM: the product of the gene mutated in ataxia-telangiectasia. Int J Biochem Cell Biol. 1999 Jul;31(7):735-40. 145. Portugal J, Mansilla S, Bataller M. Mechanisms of drug-induced mitotic catastrophe in cancer cells. Curr Pharm Des.16(1):69-78. 146. Westphal CH, Rowan S, Schmaltz C, Elson A, Fisher DE, Leder P. atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity. Nat Genet. 1997 Aug;16(4):397-401. 147. Flaggs G, Plug AW, Dunks KM, Mundt KE, Ford JC, Quiggle MR, Taylor EM, Westphal CH, Ashley T, Hoekstra MF, Carr AM. Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes. Curr Biol. 1997 Dec 1;7(12):977-86. 148. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 1999 Sep 1;59(17):4375-82. 149. Pommier Y, Schwartz RE, Zwelling LA, Kohn KW. Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei. Biochemistry. 1985 Nov 5;24(23):6406-10. 150. Udvardy A, Schedl P, Sander M, Hsieh TS. Topoisomerase II cleavage in chromatin. J Mol Biol. 1986 Sep 20;191(2):231-46. 151. Salceda J, Fernandez X, Roca J. Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. EMBO J. 2006 Jun 7;25(11):257583. 152. Deweese JE, Osheroff N. The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing. Nucleic Acids Res. 2009 Feb;37(3):738-48. 153. Khanna KK, Beamish H, Yan J, Hobson K, Williams R, Dunn I, Lavin MF. Nature of G1/S cell cycle checkpoint defect in ataxia-telangiectasia. Oncogene. 1995 Aug 17;11(4):609-18. 154. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary for the p53mediated G1 arrest in human cancer cells. Cancer Res. 1995 Nov 15;55(22):5187-90. 155. Mauro M, Rego MA, Boisvert RA, Esashi F, Cavallo F, Jasin M, Howlett NG. p21 promotes error-free replication-coupled DNA double-strand break repair. Nucleic Acids Res. 2012 Sep 1;40(17):8348-60. 156. Gartel AL, Ye X, Goufman E, Shianov P, Hay N, Najmabadi F, Tyner AL. Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4510-5. 157. Wong PP, Miranda F, Chan KV, Berlato C, Hurst HC, Scibetta AG. Histone demethylase KDM5B collaborates with TFAP2C and Myc to repress the cell cycle inhibitor p21(cip) (CDKN1A). Mol Cell Biol. 2012 May;32(9):1633-44.

158. Kaufmann WK. Cell cycle checkpoints and DNA repair preserve the stability of the human genome. Cancer Metastasis Rev. 1995 Mar;14(1):31-41. 159. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997 Dec 22;420(1):25-7. 160. Momand J, Wu HH, Dasgupta G. MDM2--master regulator of the p53 tumor suppressor protein. Gene. 2000 Jan 25;242(1-2):15-29. 161. Caspari T. How to activate p53. Curr Biol. 2000 Apr 20;10(8):R315-7. 162. Foray N, Marot D, Gabriel A, Randrianarison V, Carr AM, Perricaudet M, Ashworth A, Jeggo P. A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein. EMBO J. 2003 Jun 2;22(11):2860-71. 163. Ljungman M. Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. Neoplasia. 2000 May-Jun;2(3):208-25. 164. Pluquet O, Hainaut P. Genotoxic and non-genotoxic pathways of p53 induction. Cancer Lett. 2001 Dec 10;174(1):1-15. 165. Germain S, Monnot C, Muller L, Eichmann A. Hypoxia-driven angiogenesis: role of tip cells and extracellular matrix scaffolding. Curr Opin Hematol. May;17(3):245-51. 166. Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ. Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol. 2002 Mar;22(6):1834-43. 167. Hammond EM, Dorie MJ, Giaccia AJ. ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation. J Biol Chem. 2003 Apr 4;278(14):12207-13. 168. Fallone F, Britton S, Nieto L, Salles B, Muller C. ATR controls cellular adaptation to hypoxia through positive regulation of hypoxia-inducible factor 1 (HIF-1) expression. Oncogene. 2012 Oct 22. 169. Lee JH, Jin Y, He G, Zeng SX, Wang YV, Wahl GM, Lu H. Hypoxia activates tumor suppressor p53 by inducing ATR-Chk1 kinase cascade-mediated phosphorylation and consequent 14-3-3gamma inactivation of MDMX protein. J Biol Chem. 2012 Jun 15;287(25):20898-903. 170. Wang C, Chen J. Phosphorylation and hsp90 binding mediate heat shock stabilization of p53. J Biol Chem. 2003 Jan 17;278(3):2066-71. 171. Dumaz N, Milne DM, Jardine LJ, Meek DW. Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover. Biochem J. 2001 Oct 15;359(Pt 2):459-64. 172. Xie S, Wang Q, Wu H, Cogswell J, Lu L, Jhanwar-Uniyal M, Dai W. Reactive oxygen species-induced phosphorylation of p53 on serine 20 is mediated in part by polo-like kinase-3. J Biol Chem. 2001 Sep 28;276(39):36194-9. 173. Shiyanov P, Bagchi S, Adami G, Kokontis J, Hay N, Arroyo M, Morozov

A, Raychaudhuri P. p21 Disrupts the interaction between cdk2 and the E2F-p130 complex. Mol Cell Biol. 1996 Mar;16(3):737-44. 174. O'Connor PM. Mammalian G1 and G2 phase checkpoints. Cancer Surv. 1997;29:151-82. 175. Dumaz N, Meek DW. Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J. 1999 Dec 15;18(24):7002-10. 176. Unger T, Sionov RV, Moallem E, Yee CL, Howley PM, Oren M, Haupt Y. Mutations in serines 15 and 20 of human p53 impair its apoptotic activity. Oncogene. 1999 May 27;18(21):3205-12. 177. Bieging KT, Attardi LD. Deconstructing p53 transcriptional networks in tumor suppression. Trends Cell Biol. 2012 Feb;22(2):97-106. 178. Zhan Q, Fan S, Bae I, Guillouf C, Liebermann DA, O'Connor PM, Fornace AJ, Jr. Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis. Oncogene. 1994 Dec;9(12):3743-51. 179. Grandela C, Pera MF, Grimmond SM, Kolle G, Wolvetang EJ. p53 is required for etoposide-induced apoptosis of human embryonic stem cells. Stem Cell Res. 2007 Nov;1(2):116-28. 180. Wu X, Deng Y. Bax and BH3-domain-only proteins in p53-mediated apoptosis. Front Biosci. 2002 Jan 1;7:d151-6. 181. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis. Trends Mol Med. 2006 Sep;12(9):440-50. 182. Yu Z, Wang H, Zhang L, Tang A, Zhai Q, Wen J, Yao L, Li P. Both p53PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression. Biochem Biophys Res Commun. 2009 Sep 4;386(4):607-11. 183. Korwek Z, Sewastianik T, Bielak-Zmijewska A, Mosieniak G, Alster O, Moreno-Villaneuva M, Burkle A, Sikora E. Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells. DNA Repair (Amst). 2012 Nov 1;11(11):864-73. 184. Gao P, Zheng J. Oncogenic virus-mediated cell fusion: new insights into initiation and progression of oncogenic viruses--related cancers. Cancer Lett. 2011 Apr 1;303(1):1-8. 185. Berkovich E, Ginsberg D. ATM is a target for positive regulation by E2F1. Oncogene. 2003 Jan 16;22(2):161-7. 186. Levine AJ. Tumor suppressor genes. Bioessays. 1990 Feb;12(2):60-6. 187. Levine AJ. The common mechanisms of transformation by the small DNA tumor viruses: The inactivation of tumor suppressor gene products: p53. Virology. 2009 Feb 20;384(2):285-93.

188. Marshall WL, Yim C, Gustafson E, Graf T, Sage DR, Hanify K, Williams L, Fingeroth J, Finberg RW. Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak. J Virol. 1999 Jun;73(6):5181-5. 189. Vairo G, Soos TJ, Upton TM, Zalvide J, DeCaprio JA, Ewen ME, Koff A, Adams JM. Bcl-2 retards cell cycle entry through p27(Kip1), pRB relative p130, and altered E2F regulation. Mol Cell Biol. 2000 Jul;20(13):4745-53. 190. Deng X, Gao F, May WS, Jr. Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS. Blood. 2003 Nov 1;102(9):3179-85. 191. Swenson JJ, Mauser AE, Kaufmann WK, Kenney SC. The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction. J Virol. 1999 Aug;73(8):6540-50. 192. Workman A, Jones C. Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1). Virus Res. 2011 Sep;160(1-2):66-73. 193. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4997-5002. 194. Finucane DM, Bossy-Wetzel E, Waterhouse NJ, Cotter TG, Green DR. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J Biol Chem. 1999 Jan 22;274(4):2225-33. 195. Tatulian SA, Garg P, Nemec KN, Chen B, Khaled AR. Molecular basis for membrane pore formation by Bax protein carboxyl terminus. Biochemistry. 2012 Nov 20;51(46):9406-19. 196. Cohen GM. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326 ( Pt 1):1-16. 197. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998 Aug 28;281(5381):1312-6. 198. Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science. 1997 Dec 12;278(5345):1966-8. 199. Duesberg P, Li R, Rasnick D, Rausch C, Willer A, Kraemer A, Yerganian G, Hehlmann R. Aneuploidy precedes and segregates with chemical carcinogenesis. Cancer Genet Cytogenet. 2000 Jun;119(2):83-93. 200. Duesberg P, Rasnick D. Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskeleton. 2000 Oct;47(2):81-107. 201. Li R, Sonik A, Stindl R, Rasnick D, Duesberg P. Aneuploidy vs. gene mutation hypothesis of cancer: recent study claims mutation but is found to support aneuploidy. Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3236-41.

202. Parris GE. The cell clone ecology hypothesis and the cell fusion model of cancer progression and metastasis: history and experimental support. Med Hypotheses. 2006;66(1):76-83. 203. Bassing CH, Chua KF, Sekiguchi J, Suh H, Whitlow SR, Fleming JC, Monroe BC, Ciccone DN, Yan C, Vlasakova K, Livingston DM, Ferguson DO, Scully R, Alt FW. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):8173-8. 204. Savic V, Yin B, Maas NL, Bredemeyer AL, Carpenter AC, Helmink BA, Yang-Iott KS, Sleckman BP, Bassing CH. Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin. Mol Cell. 2009 May 15;34(3):298310. 205. Chowdhury D, Keogh MC, Ishii H, Peterson CL, Buratowski S, Lieberman J. gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell. 2005 Dec 9;20(5):801-9. 206. Gudjonsson T, Altmeyer M, Savic V, Toledo L, Dinant C, Grofte M, Bartkova J, Poulsen M, Oka Y, Bekker-Jensen S, Mailand N, Neumann B, Heriche JK, Shearer R, Saunders D, Bartek J, Lukas J, Lukas C. TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes. Cell. 2012 Aug 17;150(4):697-709. 207. Kuo LJ, Yang LX. Gamma-H2AX - a novel biomarker for DNA doublestrand breaks. In Vivo. 2008 May-Jun;22(3):305-9. 208. Postow L. Destroying the ring: Freeing DNA from Ku with ubiquitin. FEBS Lett. 2011 Sep 16;585(18):2876-82. 209. Postow L, Ghenoiu C, Woo EM, Krutchinsky AN, Chait BT, Funabiki H. Ku80 removal from DNA through double strand break-induced ubiquitylation. J Cell Biol. 2008 Aug 11;182(3):467-79. 210. Belyaev IY. Radiation-induced DNA repair foci: spatio-temporal aspects of formation, application for assessment of radiosensitivity and biological dosimetry. Mutat Res. 2010 Apr-Jun;704(1-3):132-41. 211. Cao Z, Kuhne WW, Steeb J, Merkley MA, Zhou Y, Janata J, Dynan WS. Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response. Nucleic Acids Res. 2010 Aug;38(14):e144. 212. Long BH, Musial ST, Brattain MG. Single- and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide. Cancer Res. 1985 Jul;45(7):3106-12. 213. de Campos-Nebel M, Larripa I, Gonzalez-Cid M. Topoisomerase II-

mediated DNA damage is differently repaired during the cell cycle by nonhomologous end joining and homologous recombination. PLoS One. 2010;5(9). 214. Stacey DW. Three Observations That Have Changed Our Understanding of Cyclin D1 and p27 in Cell Cycle Control. Genes Cancer. 2010 Dec;1(12):118999. 215. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 28;326(6112):515-7. 216. Richter A, Murua Escobar H, Gunther K, Soller JT, Winkler S, Nolte I, Bullerdiek J. RAS gene hot-spot mutations in canine neoplasias. J Hered. 2005;96(7):764-5. 217. Furth ME, Davis LJ, Fleurdelys B, Scolnick EM. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294-304. 218. Ferbeyre G, de Stanchina E, Querido E, Baptiste N, Prives C, Lowe SW. PML is induced by oncogenic ras and promotes premature senescence. Genes Dev. 2000 Aug 15;14(16):2015-27. 219. Shay JW, Pereira-Smith OM, Wright WE. A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res. 1991 Sep;196(1):33-9. 220. Bourdeau V, Baudry D, Ferbeyre G. PML links aberrant cytokine signaling and oncogenic stress to cellular senescence. Front Biosci. 2009;14:475-85. 221. Lallemand-Breitenbach V, de The H. CK2 and PML: regulating the regulator. Cell. 2006 Jul 28;126(2):244-5. 222. Scaglioni PP, Yung TM, Choi S, Baldini C, Konstantinidou G, Pandolfi PP. CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor. Mol Cell Biochem. 2008 Sep;316(1-2):149-54. 223. Vernier M, Bourdeau V, Gaumont-Leclerc MF, Moiseeva O, Begin V, Saad F, Mes-Masson AM, Ferbeyre G. Regulation of E2Fs and senescence by PML nuclear bodies. Genes Dev. 2011 Jan 1;25(1):41-50. 224. Bernardi R, Guernah I, Jin D, Grisendi S, Alimonti A, Teruya-Feldstein J, Cordon-Cardo C, Simon MC, Rafii S, Pandolfi PP. PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR. Nature. 2006 Aug 17;442(7104):779-85. 225. Scherer WF, Syverton JT, Gey GO. Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med. 1953 May;97(5):695-710. 226. Vogt M. A Study of the Relationship between Karyotype and Phenotype in Clones Lines of Strain Hela. Genetics. 1959 Nov;44(6):1257-70.

227. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961 Dec;25:585-621. 228. Hayflick L. Antecedents of cell aging research. Exp Gerontol. 1989;24(56):355-65. 229. Naveilhan P, Baudet C, Jabbour W, Wion D. A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts. Mech Ageing Dev. 1994 Sep;75(3):205-13. 230. Olovnikov AM. Telomeres, telomerase, and aging: origin of the theory. Exp Gerontol. 1996 Jul-Aug;31(4):443-8. 231. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. Nature. 1999 Jul 29;400(6743):464-8. 232. Hahn WC, Stewart SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M, Weinberg RA. Inhibition of telomerase limits the growth of human cancer cells. Nat Med. 1999 Oct;5(10):1164-70. 233. Hughes DT. Cytogenetical polymorphism and evolution in mammalian somatic cell populations in vivo and vitro. Nature. 1968 Feb 10;217(5128):51823. 234. Torsvik A, Rosland GV, Svendsen A, Molven A, Immervoll H, McCormack E, Lonning PE, Primon M, Sobala E, Tonn JC, Goldbrunner R, Schichor C, Mysliwietz J, Lah TT, Motaln H, Knappskog S, Bjerkvig R. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field on track - letter. Cancer Res. 2010 Aug 1;70(15):6393-6. 235. de la Fuente R, Bernad A, Garcia-Castro J, Martin MC, Cigudosa JC. Retraction: Spontaneous human adult stem cell transformation. Cancer Res. 2010 Aug 15;70(16):6682. 236. Baverstock K. Muller's ratchet hypothesis. Lancet. 2004 Oct 28;364(9441):1213. 237. Carlson EA. H.J. Muller's contributions to mutation research. Mutat Res. 2013 Jan-Mar;752(1):1-5. 238. Charlesworth B, Charlesworth D. The degeneration of Y chromosomes. Philos Trans R Soc Lond B Biol Sci. 2000 Nov 29;355(1403):1563-72. 239. Engelstadter J. Muller's ratchet and the degeneration of Y chromosomes: a simulation study. Genetics. 2008 Oct;180(2):957-67. 240. Andersson DI, Hughes D. Muller's ratchet decreases fitness of a DNAbased microbe. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):906-7. 241. Bergstrom CT, McElhany P, Real LA. Transmission bottlenecks as

determinants of virulence in rapidly evolving pathogens. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5095-100. 242. Chao L. Fitness of RNA virus decreased by Muller's ratchet. Nature. 1990 Nov 29;348(6300):454-5. 243. de la Iglesia F, Elena SF. Fitness declines in Tobacco etch virus upon serial bottleneck transfers. J Virol. 2007 May;81(10):4941-7. 244. Novella IS, Elena SF, Moya A, Domingo E, Holland JJ. Size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness. J Virol. 1995 May;69(5):2869-72. 245. Yuste E, Sanchez-Palomino S, Casado C, Domingo E, Lopez-Galindez C. Drastic fitness loss in human immunodeficiency virus type 1 upon serial bottleneck events. J Virol. 1999 Apr;73(4):2745-51. 246. Wendorff TJ, Schmidt BH, Heslop P, Austin CA, Berger JM. The Structure of DNA-Bound Human Topoisomerase II Alpha: Conformational Mechanisms for Coordinating Inter-Subunit Interactions with DNA Cleavage. J Mol Biol. 2012 Dec 7;424(3-4):109-24. 247. Heisig P. Type II topoisomerases--inhibitors, repair mechanisms and mutations. Mutagenesis. 2009 Nov;24(6):465-9. 248. Smart DJ, Halicka HD, Schmuck G, Traganos F, Darzynkiewicz Z, Williams GM. Assessment of DNA double-strand breaks and gammaH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone. Mutat Res. 2008 May 10;641(1-2):43-7. 249. Bromberg KD, Burgin AB, Osheroff N. A two-drug model for etoposide action against human topoisomerase IIalpha. J Biol Chem. 2003 Feb 28;278(9):7406-12. 250. Babic A, Berkmen MB, Lee CA, Grossman AD. Efficient gene transfer in bacterial cell chains. MBio. 2011;2(2). 251. Canchaya C, Fournous G, Chibani-Chennoufi S, Dillmann ML, Brussow H. Phage as agents of lateral gene transfer. Curr Opin Microbiol. 2003 Aug;6(4):417-24. 252. Chen X, Liang H, Zhang J, Zen K, Zhang CY. Horizontal transfer of microRNAs: molecular mechanisms and clinical applications. Protein Cell. 2012 Jan;3(1):28-37. 253. Dunning Hotopp JC. Horizontal gene transfer between bacteria and animals. Trends Genet. 2011 Apr;27(4):157-63. 254. Dunning Hotopp JC, Clark ME, Oliveira DC, Foster JM, Fischer P, Munoz Torres MC, Giebel JD, Kumar N, Ishmael N, Wang S, Ingram J, Nene RV, Shepard J, Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko BE, Tettelin H, Werren JH. Widespread lateral gene transfer from intracellular bacteria to multicellular

eukaryotes. Science. 2007 Sep 21;317(5845):1753-6. 255. Holmgren L, Bergsmedh A, Spetz AL. Horizontal transfer of DNA by the uptake of apoptotic bodies. Vox Sang. 2002 Aug;83 Suppl 1:305-6. 256. Keeling PJ. Role of horizontal gene transfer in the evolution of photosynthetic eukaryotes and their plastids. Methods Mol Biol. 2009;532:50115. 257. Koonin EV, Wolf YI. Evolution of microbes and viruses: a paradigm shift in evolutionary biology? Front Cell Infect Microbiol. 2012;2:119. 258. Schaack S, Gilbert C, Feschotte C. Promiscuous DNA: horizontal transfer of transposable elements and why it matters for eukaryotic evolution. Trends Ecol Evol. 2010 Sep;25(9):537-46. 259. Parris GE. Chromosomal instability (CIN) leads to clone extinction, not cancer. Med Hypotheses. 2008 Dec;71(6):983. 260. Gagos S, Iliopoulos D, Tseleni-Balafouta S, Agapitos M, Antachopoulos C, Kostakis A, Karayannakos P, Skalkeas G. Cell senescence and a mechanism of clonal evolution leading to continuous cell proliferation, loss of heterozygosity, and tumor heterogeneity: studies on two immortal colon cancer cell lines. Cancer Genet Cytogenet. 1996 Sep;90(2):157-65. 261. Ivanov A, Cragg MS, Erenpreisa J, Emzinsh D, Lukman H, Illidge TM. Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks. J Cell Sci. 2003 Oct 15;116(Pt 20):4095-106. 262. Schwerer MJ, Hemmer J, Kraft K, Maier H, Moller P, Barth TF. Endoreduplication in conjunction with tumor progression in an aneuploid laryngeal squamous cell carcinoma. Virchows Arch. 2003 Jul;443(1):98-103. 263. Wilsker D, Chung JH, Bunz F. Chk1 suppresses bypass of mitosis and tetraploidization in p53-deficient cancer cells. Cell Cycle. 2012 Apr 15;11(8):1564-72. 264. Gehlhaus MW, 3rd, Gift JS, Hogan KA, Kopylev L, Schlosser PM, Kadry AR. Approaches to cancer assessment in EPA's Integrated Risk Information System. Toxicol Appl Pharmacol. 2011 Jul 15;254(2):170-80. 265. Saul RL, Ames BN. Background levels of DNA damage in the population. Basic Life Sci. 1986;38:529-35. 266. Lujan SA, Williams JS, Pursell ZF, Abdulovic-Cui AA, Clark AB, Nick McElhinny SA, Kunkel TA. Mismatch repair balances leading and lagging strand DNA replication fidelity. PLoS Genet. 2012 Oct;8(10):e1003016. 267. Gundry M, Li W, Maqbool SB, Vijg J. Direct, genome-wide assessment of DNA mutations in single cells. Nucleic Acids Res. 2012 Mar;40(5):2032-40. 268. Johnson GE, Doak SH, Griffiths SM, Quick EL, Skibinski DO, Zair ZM, Jenkins GJ. Non-linear dose-response of DNA-reactive genotoxins:

recommendations for data analysis. Mutat Res. 2009 Aug;678(2):95-100. 269. Moore FD, Sastry KS. Intracellular potassium: 40K as a primordial gene irradiator. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3556-9. 270. Paine PL, Pearson TW, Tluczek LJ, Horowitz SB. Nuclear sodium and potassium. Nature. 1981 May 21;291(5812):258-9. 271. Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry. 2008 May 13;47(19):5336-53. 272. Genomes Project C. A map of human genome variation from populationscale sequencing. Nature. 2010 Oct 28;467(7319):1061-73. 273. Keightley PD. Rates and fitness consequences of new mutations in humans. Genetics. 2012 Feb;190(2):295-304. 274. Pye-Smith RJ. Arsenic cancer, with description of a case. Proc R Soc Med. 1913;6(Clin Sect):229-36. 275. Kitchin KT, Del Razo LM, Brown JL, Anderson WL, Kenyon EM. An integrated pharmacokinetic and pharmacodynamic study of arsenite action. 1. Heme oxygenase induction in rats. Teratog Carcinog Mutagen. 1999;19(6):385402. 276. Kenyon EM, Del Razo LM, Hughes MF, Kitchin KT. An integrated pharmacokinetic and pharmacodynamic study of arsenite action 2. Heme oxygenase induction in mice. Toxicology. 2005 Jan 31;206(3):389-401. 277. Hughes MF, Devesa V, Adair BM, Styblo M, Kenyon EM, Thomas DJ. Tissue dosimetry, metabolism and excretion of pentavalent and trivalent monomethylated arsenic in mice after oral administration. Toxicol Appl Pharmacol. 2005 Oct 15;208(2):186-97. 278. Hughes MF, Devesa V, Adair BM, Conklin SD, Creed JT, Styblo M, Kenyon EM, Thomas DJ. Tissue dosimetry, metabolism and excretion of pentavalent and trivalent dimethylated arsenic in mice after oral administration. Toxicol Appl Pharmacol. 2008 Feb 15;227(1):26-35. 279. Cohen SM, Ohnishi T, Arnold LL, Le XC. Arsenic-induced bladder cancer in an animal model. Toxicol Appl Pharmacol. 2007 Aug 1;222(3):258-63. 280. Littlefield NA, Farmer JH, Gaylor DW, Sheldon WG. Effects of dose and time in a long-term, low-dose carcinogenic study. J Environ Pathol Toxicol. 1980;3(3 Spec No):17-34. 281. Belinsky SA, White CM, Devereux TR, Anderson MW. DNA adducts as a dosimeter for risk estimation. Environ Health Perspect. 1987 Dec;76:3-8. 282. Hoel DG, Kaplan NL, Anderson MW. Implication of nonlinear kinetics on risk estimation in carcinogenesis. Science. 1983 Mar 4;219(4588):1032-7. 283. Starr TB, Buck RD. The importance of delivered dose in estimating low-

dose cancer risk from inhalation exposure to formaldehyde. Fundam Appl Toxicol. 1984 Oct;4(5):740-53. 284. Lutz WK. Dose-response relationships in chemical carcinogenesis: from DNA adducts to tumor incidence. Adv Exp Med Biol. 1991;283:151-6. 285. Lutz WK. Dose-response relationships in chemical carcinogenesis: superposition of different mechanisms of action, resulting in linear-nonlinear curves, practical thresholds, J-shapes. Mutat Res. 1998 Sep 20;405(2):117-24. 286. Miller JA, Miller EC. Metabolic activation and reactivity of chemical carcinogens. Mutat Res. 1975 Nov;33(1 Spec No):25-6. 287. Miller EC, Miller JA. In vivo combinations between carcinogens and tissue constituents and their possible role in carcinogenesis. Cancer Res. 1952 Aug;12(8):547-56. 288. Miller JA, Miller EC. The metabolic activation of carcinogenic aromatic amines and amides. Prog Exp Tumor Res. 1969;11:273-301. 289. Miller JA. Carcinogenesis by chemicals: an overview--G. H. A. Clowes memorial lecture. Cancer Res. 1970 Mar;30(3):559-76. 290. Phillips DH, Hanawalt PC, Miller JA, Miller EC. The in vivo formation and repair of DNA adducts from 1'-hydroxysafrole. J Supramol Struct Cell Biochem. 1981;16(1):83-90. 291. Ames BN, Whitfield HJ, Jr. Frameshift mutagenesis in Salmonella. Cold Spring Harb Symp Quant Biol. 1966;31:221-5. 292. Ames BN, Lee FD, Durston WE. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A. 1973 Mar;70(3):782-6. 293. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983 May;113(3-4):173-215. 294. Abad MC, Arni RK, Grella DK, Castellino FJ, Tulinsky A, Geiger JH. The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin. J Mol Biol. 2002 May 10;318(4):1009-17. 295. Ames BN. Carcinogens are mutagens: their detection and classification. Environ Health Perspect. 1973 Dec;6:115-8. 296. Ames BN, Durston WE, Yamasaki E, Lee FD. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2281-5. 297. Ames BN, Gurney EG, Miller JA, Bartsch H. Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc Natl Acad Sci U S A. 1972 Nov;69(11):312832. 298. Storz G, Christman MF, Sies H, Ames BN. Spontaneous mutagenesis and

oxidative damage to DNA in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8917-21. 299. Ames BN, Gold LS. Too many rodent carcinogens: mitogenesis increases mutagenesis. Science. 1990 Aug 31;249(4972):970-1. 300. Ames BN, Gold LS. Chemical carcinogenesis: too many rodent carcinogens. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7772-6. 301. Lewis DR, Southwick JW, Ouellet-Hellstrom R, Rench J, Calderon RL. Drinking water arsenic in Utah: A cohort mortality study. Environ Health Perspect. 1999 May;107(5):359-65. 302. Villanueva C, Kogevinas M. Comments on "Drinking water arsenic in Utah: a cohort mortality study". Environ Health Perspect. 1999 Nov;107(11):A544; author reply A-6. 303. Tseng WP. Effects and dose--response relationships of skin cancer and blackfoot disease with arsenic. Environ Health Perspect. 1977 Aug;19:109-19. 304. Wu MM, Kuo TL, Hwang YH, Chen CJ. Dose-response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol. 1989 Dec;130(6):1123-32. 305. Brinckman FE, Parris GE, Blair WR, Jewett KL, Iverson WP, Bellama JM. Questions concerning environmental mobility of arsenic: needs for a chemical data base and means for speciation of trace organoarsenicals. Environ Health Perspect. 1977 Aug;19:11-24. 306. Tseng WP. Prognosis of blackfoot disease. A 10-year follow-up study. Taiwan Yi Xue Hui Za Zhi. 1970 Jan 28;69(1):1-21. 307. Chen KL, Wu HY. Epidemiologic studies on blackfoot disease. 2. A study of source of drinking water in relation to the disease. J Formos Med Assoc. 1962 Jul 28;61:611-8. 308. Ch'i IC, Blackwell RQ. A controlled retrospective study of Blackfoot disease, an endemic peripheral gangrene disease in Taiwan. Am J Epidemiol. 1968 Jul;88(1):7-24. 309. Tseng CH. Blackfoot disease and arsenic: a never-ending story. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2005;23(1):55-74. 310. Lu FJ. Blackfoot disease: arsenic or humic acid? Lancet. 1990 Jul 14;336(8707):115-6. 311. Loken SD, Norman K, Hirasawa K, Nodwell M, Lester WM, Demetrick DJ. Morbidity in immunosuppressed (SCID/NOD) mice treated with reovirus (dearing 3) as an anti-cancer biotherapeutic. Cancer Biol Ther. 2004 Aug;3(8):734-8. 312. Hutchinson J. SALVARSAN ("606") AND ARSENIC CANCER. Br Med J. 1911 Apr 29;1(2626):976-7.

313. Chen CJ, Chen CW, Wu MM, Kuo TL. Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br J Cancer. 1992 Nov;66(5):888-92. 314. Su CC, Lu JL, Tsai KY, Lian Ie B. Reduction in arsenic intake from water has different impacts on lung cancer and bladder cancer in an arseniasis endemic area in Taiwan. Cancer Causes Control. 2011 Jan;22(1):101-8. 315. Andrewes P, Demarini DM, Funasaka K, Wallace K, Lai VW, Sun H, Cullen WR, Kitchin KT. Do arsenosugars pose a risk to human health? The comparative toxicities of a trivalent and pentavalent arsenosugar. Environ Sci Technol. 2004 Aug 1;38(15):4140-8. 316. Slejkovec Z, Kapolna E, Ipolyi I, van Elteren JT. Arsenosugars and other arsenic compounds in littoral zone algae from the Adriatic Sea. Chemosphere. 2006 May;63(7):1098-105. 317. Nischwitz V, Pergantis SA. Improved arsenic speciation analysis for extracts of commercially available edible marine algae using HPLC-ES-MS/MS. J Agric Food Chem. 2006 Sep 6;54(18):6507-19. 318. Garcia-Salgado S, Quijano MA, Bonilla MM. Arsenic speciation in edible alga samples by microwave-assisted extraction and high performance liquid chromatography coupled to atomic fluorescence spectrometry. Anal Chim Acta. 2012 Feb 10;714:38-46. 319. Albert RE, Train RE, Anderson E. Rationale developed by the Environmental Protection Agency for the assessment of carcinogenic risks. J Natl Cancer Inst. 1977 May;58(5):1537-41. 320. Bull RJ. Cancer risk assessment: importance of identifying mechanisms of action. J Am Water Works Assoc. 1990 Oct;82(10):57-60. 321. Doak SH, Jenkins GJ, Johnson GE, Quick E, Parry EM, Parry JM. Mechanistic influences for mutation induction curves after exposure to DNAreactive carcinogens. Cancer Res. 2007 Apr 15;67(8):3904-11. 322. Moolenaar RJ. Commentary on EPA carcinogen risk assessment guidelines. Regul Toxicol Pharmacol. 1989 Jun;9(3):230-5.