Recent discoveries on the historical foundations of cancer risk assessment: Shedding light on the limits of LNT.

Within the past three years there has been a spate of historical discoveries by our research team on various different facets of the historical foundations of cancer risk assessment. This series of discoveries was stimulated by the creation of a 22-episode documentary of the historical foundations of cancer risk assessment by the US Health Physics Society and the need to provide documentation. This process yielded nearly two dozen distinct historical findings which have been published in numerous papers in the peer-reviewed literature. These discoveries are itemized and summarized in the present paper, along with the significance of each discovery within the historical context of ionizing radiation research and cancer risk assessment.

Newly discovered letter: why Muller failed to cite the negative mouse mutation findings of Snell, preserving his chances to receive the Nobel Prize.

A recently acquired letter between Hermann Muller and his wife (March 21, 1933) reveals that Muller had learned that he had been nominated for the Nobel Prize in 1932 with about 1/3 of the total votes being supportive. Muller was hopeful that over time sufficient votes would lead to receiving the award. The knowledge of Muller on this matter and its timing provide a likely explanation why Muller never cited the negative mouse mutation findings of George Snell, performed under Muller's direction during that time period. This action of Muller, along with the failure of Snell to promote his discovery, greatly reduced the chances that those findings would complicate his attempt to garner support for his LNT single-hit model and its application to hereditary and cancer risk assessment. It also helped Muller achieve the Nobel Prize, allowing him the necessary international visibility to promote his ideologically driven ionizing radiation-related LNT-based paradigm.

First report of X-ray induced somatic mutation by Muller's department chair fails to support Muller's linearity hypothesis.

This paper reevaluates the first report of X-ray-induced somatic gene mutations. It was undertaken by John Patterson, Department Chair of Hermann Muller, using the same biological model, methods and equipment of Muller. Patterson reported X-ray induced mutation frequencies for X-chromosome-linked (sex-linked) recessive gene mutations in somatic cells of Drosophila melanogaster that resulted in color changes in the ommatidia of the eyes. Results were based on color changes detected in both male and female offspring irradiated while in egg, larval or pupal stages and for unirradiated controls. Patterson claimed that the observed dose response displayed linearity, with a clear implication that the linear response extended to background exposure levels of unirradiated controls. This reanalysis disputes Patterson's interpretation, showing that the dose response in the low-dose zone strongly supported a threshold model. The doses in the experiment, which were not clearly presented, were so high that it would preclude the assumption that the experiment provided any information of relevance to radiation exposures of humans at low doses, or even at high doses delivered at low-dose rates. Induced phenotypical changes that occurred at the higher doses, especially in female offspring, overwhelmingly resulted from X-ray-induced chromosome breaks instead of point mutations as initially expected by Patterson. The Patterson findings and linearity interpretations were an important contributory factor in the acceptance of the linear non-threshold (LNT) model during the formative time of concept consolidation. It is rather shocking now to see that the actual data provided no support for the LNT model.

Muller and mutations: mouse study of George Snell (a postdoc of Muller) fails to confirm Muller's fruit fly findings, and Muller fails to cite Snell's findings.

In 1931, Hermann J. Muller's postdoctoral student, George D. Snell (Nobel Prize recipient--1980) initiated research to replicate with mice Muller's X-ray-induced mutational findings with fruit flies. Snell failed to induce the two types of mutations of interest, based on fly data (sex-linked lethals/recessive visible mutations) even though the study was well designed, used large doses of X-rays, and was published in Genetics. These findings were never cited by Muller, and the Snell paper (Snell, Genetics 20:545-567, 1935) did not cite the 1927 Muller paper (Muller, Science 66:84, 1927). This situation raises questions concerning how Snell wrote the paper (e.g., ignoring the significance of not providing support for Muller's findings in a mammal). The question may be raised whether professional pressures were placed upon Snell to downplay the significance of his findings, which could have negatively impacted the career of Muller and the LNT theory. While Muller would receive worldwide attention, and receive the Nobel Prize in 1946 "for the discovery that mutations can be induced by X-rays," Snell's negative mutation data were almost entirely ignored by his contemporary and subsequent radiation genetics/mutation researchers. This raises questions concerning how the apparent lack of interest in Snell's negative findings helped Muller professionally, including his success in using his fruit fly data to influence hereditary and cancer risk assessment and to obtain the Nobel Prize.

Muller's genetic load/species extinction hypothesis.

The genetic load hypothesis of Hermann Muller raised the profound question of possible species extinction, even for humans, following a prolonged accumulation of recessive genes due to ionizing radiation exposure within the population. Two major mouse radiation research teams in the United States provided the most extensive tests of Muller's hypothesis. One group continued its study for more than two decades, over 82 consecutive generations, approximating 2500 human years. Even though Muller had stressed for decades his fear of species-threatening effects, no significant effects were observed for related factors such as reproductive fitness and longevity. Yet, the paper presenting the data of the 82-generation negative study has only been cited five times in 45 years. Altogether numerous laboratories worldwide collected vast amounts of data on mice, rats, and swine in an unsuccessful attempt to see if there was convincing evidence to support the genetic load theory and claims that species might deteriorate or be rendered extinct. This paper re-examines Muller's genetic load hypothesis with a new evaluation of how that hypothesis was tested and the significance of the findings, with most of those studies being completed before the BEIR I Committee Report in 1972. That committee briefly discussed the available evidence, mostly ignoring those results as they proceeded to make hereditary risk estimates both for (1) the first generation after a radiation exposure and (2) for the time, in the distant future, when a hypothetical genetic equilibrium would be reached. Their estimates assumed accumulation of harmful mutations and a linear no-threshold dose response extending all of the way down to a single ionization. More recent data on induction by ionizing radiation of dominant mutations that affect the skeletons of mice provide further robust supporting evidence that the generationally cumulative and LNT-based assumptions underpinning Muller's genetic load hypothesis are not correct.

Muller misled the Pugwash Conference on radiation risks.

The Pugwash Conferences have been a highly visible attempt to create profoundly important discussions on matters related to global safety and security at the highest levels, starting in 1957 at the height of the Cold War. This paper assesses, for the first time, the formal comments offered at this first Pugwash Conference by the Nobel Prize-winning radiation geneticist, Hermann J. Muller, on the effects of ionizing radiation on the human genome. This analysis shows that the presentation by Muller was highly biased and contained scientific errors and misrepresentations of the scientific record that resulted in seriously misleading the attendees. The presentation of Muller at Pugwash served to promote, on a very visible global scale, continued misrepresentations of the state of the science and had a significant impact on policies and practices internationally and both scientific and personal belief systems concerning the effects of low dose radiation on human health. These misrepresentations would come to affect the adoption and use of nuclear technologies and the science of radiological and chemical carcinogen health risk assessment, ultimately having a profound effect on global environmental health.

Comet assay and hormesis.

The paper provides the first assessment of the occurrence of hormetic dose responses using the Comet assay, a genotoxic assay. Using a priori evaluative criteria based on the Hormetic Database on peer-reviewed comet assay experimental findings, numerous examples of hormetic dose responses were obtained. These responses occurred in a large and diverse range of cell types and for agents from a broad range of chemical classes. Limited attempts were made to estimate the frequency of hormesis within comet assay experimental studies using a priori entry and evaluative criteria, with results suggesting a frequency in the 40% range. These findings are important as they show that a wide range of genotoxic chemicals display evidence that is strongly suggestive of hormetic dose responses. These findings have significant implications for study design issues, including the number of doses selected, dose range and spacing. Likewise, the widespread occurrence of hormetic dose responses in this genotoxic assay has important risk assessment implications.

Hermann Muller and his LNT scientific and policy leadership: Private communication reveals uncertainties.

The present paper highlights numerous publications of Hermann J. Muller with a focus on his opinions concerning the validity of the linear no-threshold dose response model for hereditary and cancer risk assessment. These comments reflect a very consistent and powerfully supporting position for the LNT model. However, newly discovered correspondence between Muller and Robley D. Evans reveals that Muller was highly uncertain about the supportive science, and therefore hid his real opinions, deliberately misleading the scientific community and governmental agencies. Of further historical value is that in the correspondence with Evans, Muller proposed what might be the first articulation of an environmentally based Precautionary Principle. These perspectives have remained unknown since Muller requested Evans to keep this letter private.

Background radiation and cancer risks: A major intellectual confrontation within the domain of radiation genetics with multiple converging biological disciplines.

This paper assesses the judgments of leading radiation geneticists and cancer risk assessment scientists from the mid-1950s to mid-1970s that background radiation has a significant effect on human genetic disease and cancer incidence. This assumption was adopted by the National Academy of Sciences (NAS) Biological Effects of Atomic Radiation (BEAR) I Genetics Panel for genetic diseases and subsequently applied to cancer risk assessment by other leading individuals/advisory groups (e.g., International Commission on Radiation Protection-ICRP). These recommendations assumed that a sizeable proportion of human mutations originated from background radiation due to cumulative exposure over prolonged reproductive periods and the linear nature of the dose-response. This paper shows that the assumption that background radiation is a significant cause of spontaneous mutation, genetic diseases, and cancer incidence is not supported by experimental and epidemiological findings, and discredits erroneous risk assessments that improperly influenced the recommendations of national and international advisory committees, risk assessment policies, and beliefs worldwide.

Muller mistakes: The linear no-threshold (LNT) dose response and US EPA's cancer risk assessment policies and practices.

This paper identifies the occurrence of six major conceptual scientific errors of Hermann Muller and describes how these errors led to the creation of the linear no-threshold (LNT) dose response historically used worldwide for cancer risk assessments for chemical carcinogens and ionizing radiation. The paper demonstrates the significant role that Muller played in the environmental movement, affecting risk assessment policies and practices that are in force even now a half century following his death. This paper lends support to contemporary research that shows significant limitations of the LNT model for cancer risk assessment.

How self-interest and deception led to the adoption of the linear non-threshold dose response (LNT) model for cancer risk assessment.

This paper clarifies scientific contributions and deceptive/self-serving decisions of William L. Russell and Liane Russell that led to the adoption of the linear non-threshold (LNT) model for cancer risk assessment by the US EPA. By deliberately failing to report an extremely large cluster of mutations in the control group of their first experiment, and thereby greatly suppressing its mutation rate, the Russells incorrectly claimed that the male mouse was 15-fold more susceptible to ionizing-radiation-induced gene mutations as compared with fruit flies. This self-serving error not only propelled their research program into one of great prominence, but it also promoted the LNT-based doubling dose (DD) concept in radiation genetics/cancer risk assessment, by the US National Academy of Sciences (NAS) Biological Effects of Atomic Radiation (BEAR) I Genetics Panel (1956). The DD concept became a central element in their recommendation that regulatory agencies switch from a threshold to an LNT model. This error occurred because of a decision by W. Russell not to report that a large cluster of control group mutations found in an experiment for which preliminary results were reported in 1951. This failure to report that cluster and similar clusters continued throughout the careers of the Russells, resulting in massive overestimation of low dose radiation risks supporting the LNT. The Russell database and the repeated claim that those data show that there is no threshold dose rate for mutation in irradiated mouse stem-cell spermatogonia, have provided mechanistic validation supporting the epidemiological LNT hypothesis for radiation-induced leukemias and cancers. This reanalysis supports the threshold model for both males and females, thereby rebutting epidemiological extrapolations from the NAS and EPA claiming support for the LNT hypothesis for cancer risk assessment. The implications of the Russell errors/deceptions, how/why they occurred, and their impact upon society are enormous and need to be addressed by scientific/regulatory agencies, affecting regulatory and litigation activities.

Manhattan Project genetic studies: Flawed research discredits LNT recommendations.

This paper reexamines the technical report (∼ one page) of Uphoff and Stern (1949) in Science that was highly relied upon by the US National Academy of Sciences (NAS) Biological Effects of Atomic Radiation (BEAR) I Genetics Panel to support a linearity dose response for radiation risk assessment. The present paper demonstrates that research of Uphoff and Stern (1949) to evaluate whether total dose or dose rate best estimated radiation risks included two variables, thereby precluding the ability to accurately derive a reliable conclusion about this topic. Furthermore, the acute dose selected by Uphoff and Stern was given at a strikingly low dose rate that may have precluded the capacity to adequately test the total dose/dose rate hypothesis, even with a proper study design which also this research did not possess. The issue of total dose and dose rate was much later successfully addressed by Russell et al. (1958) using a murine model, yielding a dose-rate rather than a total dose conclusion. The failure to subject the experimental details of the Uphoff and Stern (1949) study to peer-review and publication in the open literature precluded a rigorous and necessary evaluation, profoundly and improperly impacting the adoption of the linear dose response model.

Ethical challenges of the linear non-threshold (LNT) cancer risk assessment revolution: History, insights, and lessons to be learned.

This paper provides historical review and evaluation of the development, adoption, and advocacy of the linear non-threshold (LNT) dose response model for cancer risk assessment as applied in practices and policies worldwide. It extends previous historical assessments and provides novel insights regarding: 1) how LNT bias became institutionalized in US governmental agencies, 2) how improper editorial practices at the journal Science promoted the adoption of LNT, 3) how a Nobel Prize winning scientist unjustifiably espoused and influenced support for replacing the threshold dose response model with the LNT model, 4) how the cover-up of striking and substantial experimental cancer data by US government scientists reduced support for the threshold dose response model at a critical period of cancer risk assessment policy adoption, and 5) how these events have negatively influenced cancer risk assessment practices and environmental and public health decisions for decades. These findings are presented to illustrate how profound and recognized mistakes, biases and unethical activities, inclusive of frank scientific misconduct, converged, and should motivate regulatory agencies worldwide to critically evaluate any existing policies that apply the LNT model as well as to serve as object lessons for current and future ethical conduct of research, and the provision of ethico-legal education in and across scientific curricula and institutions.

Cover up and cancer risk assessment: Prominent US scientists suppressed evidence to promote adoption of LNT.

This paper reports that William Russell, Oak Ridge National Laboratory (ORNL), conducted a large-scale lifetime study from 1956 to 1959 showing that exposure of young adult male mice to a large dose of acute X-rays had no treatment effects on male and female offspring concerning longevity or the frequency, severity, or age distribution of neoplasms and other diseases. Despite the scientific, societal and crucial timing significance of the study, Russell did not publish the findings for almost 35 years, nor did he inform governmental advisory committees, thereby significantly biasing decisions made during this period which supported the adoption of LNT for risk assessment. Of further significance, Arthur Upton, an ORNL colleague of Russell during this study and later Director of the US National Cancer Institute (NCI), was also fully knowledgeable of this study, its findings and its negative impact on the acceptance of LNT. Upton later worked along with Russell to publish these data (i.e., Cosgrove et al., 1993) to dispute the case-specific claim that children developed cancer because of the radiation exposure of their fathers as workers at the Sellafield nuclear plant. Thus, while Russell's data were available, but were not used to challenge the key radiation and leukemia paper of Edward B. Lewis, (1957) when LNT was being adopted by regulatory agencies, they were used in a major trial in the United Kingdom (UK) for the client (i.e., British Nuclear Fuels Plc) that hired Upton. While the duplicity of Russell's and Upton's actions is striking, the key finding of the present paper is that Russell and Upton intentionally orchestrated and sustained an LNT cover up during the key period of LNT adoption by regulatory agencies, thereby showing an overwhelming bias to enhance the adoption of LNT.

Prescribing in Chronic Severe Hepatic Impairment.

Therapeutic Reviews aim to provide essential independent information for health professionals about drugs used in palliative and hospice care. Additional content is available via www.palliativedrugs.com. The series editors welcome feedback on the articles.

Ombitasvir/paritaprevir/ritonavir plus dasabuvir regimen may be used safely in combination with sirolimus for the treatment of chronic hepatitis C.

The era of direct acting antivirals has revolutionised the management of chronic hepatitis C infection and improved patient outcomes. The optimal management of patients who require liver transplantation remains a matter of ongoing discussion. Treatment in the post-transplantation setting may be complicated by significant drug-drug interactions between antiviral agents and standard immune suppressive treatment regimens. We describe what we believe to be the first reported case of a patient successfully treated for CHC with ombitasvir/paritaprevir/ritonavir plus dasabuvir, while taking sirolimus following liver transplantation.

Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.

PURPOSE: To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS: Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS: The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS: Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.

The influence of dominant lethal mutations on litter size and body weight and the consequent impact on transgenerational carcinogenesis.

The reported untreated mouse control data from the Oak Ridge National Laboratory assessment of dominant damage (ADD) experiments demonstrate a strong negative correlation between body weight at 4, 5, 7, 9, and 11 weeks of age and number in litter at 3 weeks of age. Independently from the above finding, mother mice are also shown to differ substantially as to the mean weights of their litters. Much literature suggests that, as a general rule, (a) heavier mice are more likely to develop spontaneous and induced tumors earlier and (b) caloric restriction decreases body weights and tumor incidences and increases longevity. The above findings make it likely that many experiments that have been interpreted to demonstrate radiation-induced transgenerational carcinogenesis have instead merely illustrated a confounding effect of extensive induced dominant lethality. That is, because of induced dominant lethality, experimental groups contain heavier mice or rats, which accordingly develop more spontaneous tumors at a faster rate than control groups, with the increased tumor rates having nothing to do with induction of dominant tumor mutations. Our findings prompt suggestions as to possible modifications in the analysis and experimental design of such experiments.

An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression.

Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5-E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development.