Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

Illustrative case using the RISK21 roadmap and matrix: prioritization for evaluation of chemicals found in drinking water.

The HESI-led RISK21 effort has developed a framework supporting the use of twenty-first century technology in obtaining and using information for chemical risk assessment. This framework represents a problem formulation-based, exposure-driven, tiered data acquisition approach that leads to an informed decision on human health safety to be made when sufficient evidence is available. It provides a transparent and consistent approach to evaluate information in order to maximize the ability of assessments to inform decisions and to optimize the use of resources. To demonstrate the application of the framework's roadmap and matrix, this case study evaluates a large number of chemicals that could be present in drinking water. The focus is to prioritize which of these should be considered for human health risk as individual contaminants. The example evaluates 20 potential drinking water contaminants, using the tiered RISK21 approach in combination with graphical representation of information at each step, using the RISK21 matrix. Utilizing the framework, 11 of the 20 chemicals were assigned low priority based on available exposure data alone, which demonstrated that exposure was extremely low. The remaining nine chemicals were further evaluated, using refined estimates of toxicity based on readily available data, with three deemed high priority for further evaluation. In the present case study, it was determined that the greatest value of additional information would be from improved exposure models and not from additional hazard characterization.

Computational modeling identifies key gene regulatory interactions underlying phenobarbital-mediated tumor promotion.

Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and ß-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesis.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

A proposed framework for assessing risk from less-than-lifetime exposures to carcinogens.

Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.

Risk assessment of toxaphene and its breakdown products: time for a change?

Technical toxaphene (TT) was last used in commerce in about 1982. Any environmental exposure to toxaphene in this century is to environmentally degraded forms of toxaphene, termed weathered toxaphene. Several hundred chlorinated bornane congeners have been identified in technical toxaphene. The degradation of technical toxaphene to weathered toxaphene can result in various congener mixtures, but the primary mode of degradation is dechlorination. The U.S. Environmental Protection Agency (EPA) presently estimates the risk of exposure to toxaphene by relying upon rat and mouse toxicology studies performed on technical toxaphene. No adjustment is made for the dechlorination of toxaphene in the environment. The European Union (EU), however, has modeled toxaphene risks from eating fish with chlorinated bornane residues through a series of studies on toxaphene degraded by either ultraviolet light, or biodegradation in fish. The EU risk assessment relies upon rat liver studies in vivo and mouse in vitro studies on the inhibition of gap junction intercellular communication (GJIC). This article reviews the current state of knowledge of technical and weathered toxaphene toxicology. We discuss the various current methods and opportunities to advance the risk assessment of weathered toxaphene beyond the existing U.S. EPA assessment of technical toxaphene.

Predicting future human and environmental health challenges: the Health and Environmental Sciences Institute's scientific mapping exercise.

To predict important strategic issues in product safety during the next 10 years, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute initiated a mapping exercise to evaluate which issues are likely to be of societal, scientific, and regulatory importance to regulatory authorities, the HESI membership, and the scientific community at large. Scientists representing government, academia, and industry participated in the exercise. Societal issues identified include sensitive populations, alternative therapies, public education on the precautionary principle, obesity, and aging world populations. Scientific issues identified include cancer testing, children's health, mixtures and co-exposures, sensitive populations, idiosyncratic reactions, "omics" or bioinformatics, and environmental toxicology. Regulatory issues identified include national and regional legislation on chemical safety, exposure inputs, new technologies, transitioning new science into regulations and guidelines, conservative default factors, data quality, and sensitive populations. Because some issues were identified as important in all three areas (e.g. sensitive populations), a comprehensive approach to assessment and management is needed to ensure consideration of societal, scientific, and regulatory implications. The resulting HESI Combined Challenges Map is not intended to offer a universal description of challenges in safety assessment, nor is it intended to address, advocate, or manage the prioritized issues. Rather, the map focuses on and predicts issues likely to be central to the strategic agendas of individual companies and regulatory authorities in the developed world. Many of these issues will become increasingly important in the future in rapidly developing economies, such as India and China. The scientific mapping exercise has particular value to the toxicology community because it represents the contributions of key scientists from around the world from government, academia, and industry.

Correction: Goodbye to the bioassay.

[This corrects the article DOI: 10.1039/C8TX00004B.].

Goodbye to the bioassay.

It is time to say goodbye to the standard two-year rodent bioassay. While a few, primarily genotoxic, compounds which are clearly associated with human cancer test positive in the bioassay, there is no science-based, sound foundation for presuming it provides either a valid broad (across different chemicals) capability for discerning potential human carcinogens or a valid starting point for making human risk assessment decisions. The two basic assumptions underlying the bioassay are: (1) rodent carcinogens are human carcinogens; and (2) results obtained at high doses are indicative of results that will occur at lower, environmentally relevant, doses. Both of these assumptions are not correct. Furthermore, a reevaluation of National Toxicology Program bioassay data has revealed that if the dose group size were increased from 50 to 200 rodents per group the number of bioassays deemed to be positive would increase from approximately 50% to very close to 100%. Thus, under the extreme conditions of the bioassay (e.g., high doses, lifetime exposure and, at times, a non-physiological route of administration) virtually all chemicals tested could be made into rodent carcinogens. In recent years there have been a number of proposals to move away from the standard bioassay. In particular, a recently formulated decision tree (Cohen, 2017), which places an emphasis on dose-response relationships and invites the use of MOA information, provides a sound basis for moving on from the bioassay and towards a rational approach to both identify chemicals which appear to have the potential to cause cancer in humans and take dose-response relationships into consideration in order to place the extent, if any, of the risk they might pose into proper perspective.

Orphan nuclear receptor constitutive active/androstane receptor-mediated alterations in DNA methylation during phenobarbital promotion of liver tumorigenesis.

Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197-7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.

A review of large granular lymphocytic leukemia in Fischer 344 rats as an initial step toward evaluating the implication of the endpoint to human cancer risk assessment.

Large granular lymphocyte leukemia (LGLL) is a common fatal disease in aging F344 rats. The current understanding of rat LGLL and a search for mechanistic data/correlations to human leukemia were examined with the goal of improving evaluation of the LGLL endpoint in cancer bioassays as it relates to human cancer risk assessments. The exact cell of origin of the F344 rat LGLL is not fully resolved, although natural killer (NK) cell characteristics were demonstrated in most, if not all cases. Similarities between rat LGLL and a rare human NK-LGLL exist, invalidating claims of no human counterpart, although the underlying etiopathogenesis may be different. There is insufficient data to establish a mode of action of chemical-induced rat LGLL. Evaluation of the National Toxicology Program database revealed only 34 substances (out of over 500 studied) that were possibly associated with increased incidences of LGLL. Of these, only five produced definitive LGLL effects in both sexes; the remaining 29 produced single sex responses and/or only "equivocal" associations with LGLL. Trends of increasing background/variability in LGLL incidence and its modulation by extraneous factors (e.g., corn oil gavage) are key confounders in interpretation. Given that LGLL is a common tumor in control F344 rats, interpretations of bioassays can be improved by increasing the statistical stringency (e.g., p<0.01 over traditional p<0.05), as an indicator of possible carcinogenic effects, but that alone would be insufficient evidence for declaring treatment-related increases. Thus, it was concluded that the evaluation of possible chemically related increases in rat LGLL utilize a "weight-of-evidence" approach.

Incorporation of an Epigenetic Evaluation into Safety Assessment: What we First Need to Know.

The rapidly evolving field of epigenetic regulation of gene expression is having an impact across the spectrum of biomedical research. Toxicologists have embraced this area as evidenced by their increasing focus on discerning potential epigenetic mechanisms underlying mechanisms by which chemical and physical agents might cause toxicity. It is not surprising that an interest in epigenetic mechanisms of toxicity would lead to a desire to incorporate an epigenetic component into safety assessment. However, premature movement in this direction carries the risk of imposing more confusion than light. This commentary provides an overview of epigenetics, with an emphasis on how the various epigenetic parameters are integrated, as a basis for understanding the complexity behind the desire to include epigenetic evaluations in safety evaluations. Basically, we have much more to learn before turning the goal into a reality. However, considerable progress has been made with regard to using epigenetic profiles as signatures of xenobiotic exposure.

Diethanolamine and phenobarbital produce an altered pattern of methylation in GC-rich regions of DNA in B6C3F1 mouse hepatocytes similar to that resulting from choline deficiency.

DNA methylation is an epigenetic mechanism regulating transcription, which when disrupted, can alter gene expression and contribute to carcinogenesis. Diethanolamine (DEA), a non-genotoxic alkanolamine, produces liver tumors in mice. Studies suggest DEA inhibits choline uptake and causes biochemical changes consistent with choline deficiency (CD). Rodents fed methyl-deficient diets exhibit altered methylation of hepatic DNA and an increase in liver tumors, e.g., CD causes liver tumors in B6C3F1 mice. We hypothesize that DEA-induced CD leads to altered methylation patterns which facilitates tumorigenesis. B6C3F1 hepatocytes in primary culture were grown in the presence of either 4.5 mM DEA, 3 mM Phenobarbital (PB), or CD media for 48 h. These concentrations induced comparable increases in DNA synthesis. PB, a nongenotoxic rodent liver carcinogen known to alter methylation in mouse liver, was included as a positive control. Global, average, DNA methylation status was not affected. The methylation status of GC-rich regions of DNA, which are often associated with promoter regions, were assessed via methylation-sensitive restriction digestion and arbitrarily primed PCR with capillary electrophoretic separation and detection of PCR products. DEA, PB, and CD treatments resulted in 54, 63, and 54 regions of altered methylation (RAMs), respectively, and the majority were hypomethylations. A high proportion of RAMs (72%) were identical when DEA was compared to CD. Similarly, 70% were identical between PB and CD. Altered patterns of methylation in GC-rich regions induced by DEA and PB resemble that of CD and indicate that altered DNA methylation is an epigenetic mechanism involved in the facilitation of mouse liver tumorigenesis.

Phenobarbital induces progressive patterns of GC-rich and gene-specific altered DNA methylation in the liver of tumor-prone B6C3F1 mice.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.

Altered methylation in gene-specific and GC-rich regions of DNA is progressive and nonrandom during promotion of skin tumorigenesis.

Altered DNA methylation, an epigenetic mechanism, likely contributes to tumorigenesis, with an inverse relationship existing between methylation in a promoter region and transcription. Using the SENCAR two-stage mouse skin tumorigenesis model, altered methylation was characterized in precancerous tissue and in tumor tissue. Mouse skin was initiated with 7,12-dimethylbenz[a]anthracene and promoted three times a week with 3, 9, 18, or 27 mg cigarette smoke condensate (CSC) for 4, 8, or 29 weeks; tumors were collected at 29 weeks. In addition, reversibility of changes in methylation was assessed following cessation of the promoting stimulus. DNA was isolated, and GC-rich methylation was assessed quantitatively via methylation-sensitive restriction digestion, arbitrarily primed PCR, and electrophoretic separation of PCR products. Analysis focused on regions of altered methylation (RAMs), which persisted from 4 to 8 weeks and from 8 weeks to tumor tissue. Persistent RAMs (i.e., seen in precancerous tissue and carried forward to tumors) are likely to play a key role in tumorigenesis. Twenty-two CpG sites in the upstream region of the Ha-ras promoter were unmethylated in control skin, 27 mg CSC, and tumor tissue. At two CpG sites closer to the transcriptional start site the incidence of hypomethylation increased with the dose of CSC. Hypomethylation was detected in all tumor samples. Expression of Ha-ras increased with 18 and 27 mg CSC promotion and more so in tumor tissue. These data support our hypothesis that tumor promotion involves instability of the epigenome, providing an environment where changes in the methylation status of specific regions of the genome accumulate progressively and contribute to the clonal expansion of initiated cells that leads to tumor formation.