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Background: Naphthalene is a polycyclic aromatic hydrocarbon that has been associated with health effects, including cancer. As the state of the science on naphthalene toxicity continues to evolve, updated toxicity reference value(s) may be required to support human health risk assessment. Objectives: We present a systematic evidence map of studies that could be used to derive toxicity reference value(s) for naphthalene. Methods: Human and animal health effect studies and physiologically based pharmacokinetic (PBPK) models were identified from a literature search based on populations, exposures, comparators, and outcomes (PECO) criteria. Human and animal studies meeting PECO criteria were refined to a smaller subset considered most informative for deriving chronic reference value(s), which are preferred for assessing risk to the general public. This subset was evaluated for risk of bias and sensitivity, and the suitability of each study for dose–response analysis was qualitatively assessed. Lowest observed adverse effect levels (LOAELs) were extracted and summarized. Other potentially relevant studies (e.g., mechanistic and toxicokinetic studies) were tracked as supplemental information but not evaluated further. Existing reference values for naphthalene are also summarized. Results: We identified 26 epidemiology studies and 16 animal studies that were considered most informative for further analysis. Eleven PBPK models were identified. The available epidemiology studies generally had significant risk of bias and/or sensitivity concerns and were mostly found to have low suitability for dose–response analysis due to the nature of the exposure measurements. The animal studies had fewer risk of bias and sensitivity concerns and were mostly found to be suitable for dose–response analysis. Conclusion: Although both epidemiological and animal studies of naphthalene provide weight of evidence for hazard identification, the available animal studies appear more suitable for reference value derivation. PBPK models and mechanistic and toxicokinetic data can be applied to extrapolate these animal data to humans, considering mode of action and interspecies metabolic differences. https://doi.org/10.1289/EHP7381

Background The environmental health community needs transparent, methodologically rigorous, and rapid approaches for updating human health risk assessments. These assessments often contain reference values for cancer and/or noncancer effects. Increasingly, the use of systematic review methods are preferred when developing these assessments. Systematic evidence maps are a type of analysis that has the potential to be very helpful in the update process, especially when combined with machine-learning software advances designed to expedite the process of conducting a review. Objectives To evaluate the applicability of evidence mapping to determine whether new evidence is likely to result in a change to an existing health reference value, using inhalation exposure to the air pollutant acrolein as a case example. Methods New literature published since the 2008 California Environmental Protection Agency’s Office of Environmental Health Hazard Assessment (OEHHA) Reference Exposure Level (REL) for acrolein was assessed. Systematic review methods were used to search the literature and screening included the use of machine-learning software. The Populations, Exposures, Comparators and Outcomes (PECO) criteria were kept broad to identify studies that characterized acute and chronic exposure and could be informative for hazard characterization. Studies that met the PECO criteria after full-text review were briefly summarized before their suitability for chronic point of departure (POD) derivation and calculation of a reference value was considered. Studies considered potentially suitable underwent a targeted evaluation to determine their suitability for use in dose–response analysis. Results Over 15,000 studies were identified from scientific databases. Both machine-learning and manual screening processes were used to identify 60 studies considered PECO-relevant after full-text review. Most of these PECO-relevant studies were short-term exposure animal studies (acute or less than 1 month of exposure) and considered less suitable for deriving a chronic reference value when compared to the subchronic study in rats used in the 2008 OEHHA assessment. Thirteen epidemiological studies were identified but had limitations in the exposure assessment that made them less suitable for dose–response compared to the subchronic rat study. Among the 13 studies, there were four controlled trial studies that have the potential to be informative for future acute reference value derivation. Thus, the 2008 subchronic rat study used by OEHHA appears to still be the most appropriate study for chronic reference value derivation. In addition, advances in dosimetric modeling for gases, including new evidence pertinent to acrolein, could be considered when updating existing acrolein toxicity values. Conclusions Evidence mapping is a very useful tool to assess the need for updating an assessment based on understanding the potential impact of new studies on revising an existing health reference value. In this case example, the focus was to identify studies suitable for chronic exposure dose–response analysis, while also identifying studies that may be important to consider for acute exposure scenarios, hazard identification, or for future research. This allows the evidence map to be a useful resource for a range of decision-making contexts. Specialized systematic review software increased the efficiency of the process in terms of human resources and time to conduct the analysis.

In support of the Integrated Risk Information System (IRIS), the U.S. Environmental Protection Agency (EPA) completed an evaluation of the inhalation carcinogenicity of ethylene oxide (EtO) in December 2016. This article reviews key findings and scientific issues regarding the carcinogenicity of EtO in EPA’s Carcinogenicity Assessment. EPA’s assessment critically reviewed and characterized epidemiologic, laboratory animal, and mechanistic studies pertaining to the human carcinogenicity of EtO, and addressed some key scientific issues such as the analysis of mechanistic data as part of the cancer hazard evaluation and to inform the quantitative risk assessment. The weight of evidence from the epidemiologic, laboratory animal, and mechanistic studies supports a conclusion that EtO is carcinogenic in humans, with the strongest human evidence linking EtO exposure to lymphoid and breast cancers. Analyses of the mechanistic data establish a key role for genotoxicity and mutagenicity in EtO-induced carcinogenicity and reveal little evidence supporting other mode-of-action hypotheses. In conclusion, EtO was found to be carcinogenic to humans by inhalation, posing a potential human health hazard for lymphoid and breast cancers.

Knowledge of the ontogeny of Phase I and Phase II metabolizing enzymes could be used to inform children’s vulnerability based upon likely differences in internal dose from xenobiotic exposure. This can provide a qualitative assessment of toxicokinetic variability and uncertainty pertinent to early lifestages and can help scope a more quantitative physiologically-based toxicokinetic (PBTK) assessment. Although much is known about the ontogeny of metabolizing systems, this is not commonly used in the scoping and problem formulation stage of human health risk evaluation. We propose a framework for introducing this information into problem formulation which combines the data on enzyme ontogeny and chemical-specific toxicokinetics to explore potential child/adult differences in internal dose and whether such differences may be important factors in risk evaluation. The framework is illustrated with 5 case study chemicals including some which are data rich and provide proof of concept, while others are data poor. Case studies for toluene and chlorpyrifos indicate potentially important child/adult toxicokinetic differences while scoping for acetaminophen suggests enzyme ontogeny unlikely to increase early-life risks. Scoping for trichloroethylene and aromatic amines indicates numerous ways that enzyme ontogeny may affect internal dose which necessitates further evaluation. PBTK modeling is a critical and feasible next step to further evaluate child-adult differences in internal dose for a number of these chemicals.

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200μg/kg rat/day) for 4 weeks and receiving either vehicle or CDDO-Im (three times weekly), one week prior to and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared to a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N7-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P positive foci increased substantially (0% to 13.8%) over the 4 weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development.

The use of genetically modified (GM) mice to assess carcinogenicity is playing an increasingly important role in the safety evaluation of chemicals. While progress has been made in developing and evaluating mouse models such as the Trp53⁺/⁻, Tg.AC and the rasH2, the suitability of these models as replacements for the conventional rodent cancer bioassay and for assessing human health risks remains uncertain. The objective of this research was to evaluate the use of accelerated cancer bioassays with GM mice for assessing the potential health risks associated with exposure to carcinogenic agents. We compared the published results from the GM bioassays to those obtained in the National Toxicology Program's conventional chronic mouse bioassay for their potential use in risk assessment. Our analysis indicates that the GM models are less efficient in detecting carcinogenic agents but more consistent in identifying non-carcinogenic agents. We identified several issues of concern related to the design of the accelerated bioassays (e.g., sample size, study duration, genetic stability and reproducibility) as well as pathway-dependency of effects, and different carcinogenic mechanisms operable in GM and non-GM mice. The use of the GM models for dose-response assessment is particularly problematic as these models are, at times, much more or less sensitive than the conventional rodent cancer bioassays. Thus, the existing GM mouse models may be useful for hazard identification, but will be of limited use for dose-response assessment. Hence, caution should be exercised when using GM mouse models to assess the carcinogenic risks of chemicals.

This chapter is focused on the reproductive and developmental toxicity of selected commonly used volatile solvents and gases to which inhalation is a primary route of exposure in humans. Limited data are available regarding the human reproductive and developmental toxicity of carbon tetrachloride, styrene, gasoline, and kerosene and jet fuels. Indirect or direct exposure to tetrachloroethylene has been associated with risks of pregnancy outcomes in women and reproductive dysfunctions in men. Toluene and benzene have been associated with adverse pregnancy outcomes in experimental animals. Formaldehyde, a toxic gas, has been associated with adverse pregnancy outcomes in occupationally exposed women as well as developmental defects in some epidemiological studies. As most of the human studies involve exposure to mixtures of solvents or gases, it is important to gather information on exposure to a single agent and better understand the composition of the mixtures to evaluate the potential for reproductive and developmental toxicity risks to a particular chemical. In general, very limited data exist to characterize reproductive and developmental toxicity risks following exposure to solvents.

Over the past three decades, a formal risk assessment process has been developed to provide consistent and transparent methods for the assessment of potential human health risks from exposure to environmental chemicals. Given a focus on risk to human health, epidemiological studies that identify associations between exposure to environmental chemicals and adverse health effects in humans have the potential to provide critically important information to this process. For many chemicals, however, available epidemiology studies have been found to have limited utility in informing human health risk assessments. In order to investigate this paradox, we have used several case examples to explore the utility of various types of epidemiological data in informing key elements of the risk assessment process (hazard identification, exposure-response assessment, and exposure assessment). Examples from the epidemiologic literature on environmental chemicals are used to illustrate the issues that arise in using available studies for various types of chemical risk assessments. The case examples illustrate several advantages in using epidemiology data, but also identify a number of barriers to its use, frequently related to limitations in exposure assessment. The examples also highlight ways in which the utility of both toxicology and epidemiology data can be enhanced by considering the data in combination, and integrating the results across study categories. Recent scientific developments offer hope for improving the utility of both types of data, and thus enhancing the reliability of future risk assessment efforts.

This study examined the potential to minimize the cytotoxic or genotoxic effects that dimethylsulfoxide (DMSO), when used as solvent, has on the in vitro interleukin-8 (IL-8) release, mammalian cell cytotoxicity and micronuclei formation, and bacterial mutagenesis induced by cigarette smoke wet total particulate matter (WTPM). The use of DMSO as a solvent vehicle for test articles of limited water solubility is widely applied in in vitro assays due to its moderate toxicity to test organisms and its excellent solvent properties for both polar and non-polar compounds, such as WTPM. A significant DMSO dose-dependent depletion in the IL-8 release was observed, with or without the addition of WTPM, at concentrations spanning those typically employed in in vitro assays. DMSO at 3.6% reduced cell viability 40-50%. Overall, DMSO at final concentrations of 0.5% and 4.0% resulted in about 50% and 90% depletion of final IL-8 levels, respectively. DMSO-induced cytotoxicity was evident only at concentrations of 1.5% or more, a concentration higher than that typically employed in such testing. The WTPM-induced cytotoxicity was equivalent at low ranges of DMSO concentrations. DMSO concentrations of 3.6% or higher resulted in an increase of cytotoxicity by 20-25%. DMSO alone did not give rise to bacterial mutagenicity at doses from 0% to 3.9%; however, WTPM exposure with increasing levels of DMSO resulted in increased toxicity of the WTPM at doses of DMSO greater than 6.9%, as indicated by lower revertant counts. This effect suggests that for Ames assay analysis of WTPM collected in DMSO, the level of DMSO should be minimized to prevent lower revertant counts due to DMSO-induced toxicity. DMSO alone gave a dose-dependent increase in the background micronuclei percentage, with a statistically significant increase at 4%. In the presence of WTPM, DMSO at 3% concentration resulted in a significantly higher micro-nucleus frequency, suggesting a possible clastogenic role of DMSO or potential cell permeability differences. In conclusion, this study should provide guidance for the range of DMSO concentrations when used as a solvent for WTPM-mediated in vitro toxicological assays and therefore help in properly designing and conducting in vitro studies that utilize DMSO as the extraction solvent of choice.

Metabolomics is a technology for identifying and quantifying numerous biochemicals across metabolic pathways. Using this approach, we explored changes in biochemical profiles of human alveolar epithelial carcinoma (A549) cells following in vitro exposure to mainstream whole smoke (WS) aerosol as well as to wet total particulate matter (WTPM) or gas/vapor phase (GVP), the two constituent phases of WS from 2R4F Kentucky reference cigarettes. A549 cells were exposed to WTPM or GVP (expressed as WTPM mass equivalent GVP volumes) at 0, 5, 25, or 50 microg/mL or to WS from zero, two, four, and six cigarettes for 1 or 24 h. Cell pellets were analyzed for perturbations in biochemical profiles, with named biochemicals measured, analyzed, and reported in a heat map format, along with biochemical and physiological interpretations (mSelect, Metabolon Inc.). Both WTPM and GVP exposures likely decreased glycolysis (based on decreased glycolytic intermediaries) and increased oxidative stress and cell damage. Alterations in the Krebs cycle and the urea cycle were unique to WTPM exposure, while induction of hexosamines and alterations in lipid metabolism were unique to GVP exposure. WS altered glutathione (GSH) levels, enhanced polyamine and pantothenate levels, likely increased beta-oxidation of fatty acids, and increased phospholipid degradation marked by an increase in phosphoethanolamine. GSH, glutamine, and pantothenate showed the most significant changes with cigarette smoke exposure in A549 cells based on principal component analysis. Many of the changed biochemicals were previously reported to be altered by cigarette exposure, but the global metabolomic approach offers the advantage of observing changes to hundreds of biochemicals in a single experiment and the possibility for new discoveries. The metabolomic approach may thus be used as a screening tool to evaluate conventional and novel tobacco products offering the potential to reduce risks of smoking.

Polycyclic aromatic hydrocarbons (PAHs) are known to be activated by the cytochrome P450 (P450) 1 family. However, the precise role of individual P4501 family members in PAH bioactivation remains to be fully elucidated. We therefore investigated the formation of PAH-DNA adducts in the epidermis of Cyp1a2(-/-), Cyp1b1(-/-), and Ahr(-/-) knockout mice. A panel of different PAHs was used, ranging in carcinogenic potency. Mice were treated topically on the dorsal skin with the following tritium-labeled PAHs: dibenzo[a,l]pyre-ne (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]P). At 24 h after treatment, mice (two male and two female mice per group) were sacrificed, and epidermal DNA was isolated and hydrolyzed with DNase I; subsequently, DNA adducts were quantitated by liquid scintillation counting. In the DB[a,l]P-treated mice, levels of DNA adducts were significantly lower in Cyp1a2(-/-) and Cyp1b1(-/-) mice by 57 and 46%, respectively, as compared to wild-type (WT) mice (C57BL/6 background). The levels of DB[a,l]P DNA adducts formed in Ahr(-/-) mice were 26% lower, but this was not statistically significant. The levels of DMBA-DNA adducts in Cyp1a2(-/-) mice were not different than the WT mice but were significantly lower in Cyp1b1(-/-) and Ahr(-/-) mice by 64 and 52%, respectively. DMBA-DNA adduct samples were further analyzed by HPLC following further digestion to deoxyribonucleosides. HPLC analysis of individual DMBA-DNA adducts revealed differences in the ratio of syn-DMBA-diol epoxide- to anti-DMBA-diol epoxide-derived adducts in the Ahr(-/-) and Cyp1b1(-/-) mice. The ratio of syn-/anti-derived adducts in WT mice was 0.49. This ratio was 0.23 in the Cyp1b1(-/-) mice and 0.87 in the Ahr(-/-) mice. In contrast to the results with DB[a,l]P and DMBA, the levels of B[a]P-, DB[a,h]A-, B[g]C-, and B[c]P-DNA adducts were significantly lower in Ahr(-/-) mice by 73, 75, 50, and 81%, respectively, as compared to WT mice but were not significantly lower in the Cyp1a2(-/-) or Cyp1b1(-/-) mice. Collectively, these and other results support a role for both P4501A1 and P4501B1 in the bioactivation of DMBA; P4501A2, P4501B1, and possibly P4501A1 in the bioactivation of DB[a,l]P; and P4501A1 in the bioactivation of B[a]P, DB[a,h]A, B[g]C, and B[c]P in mouse epidermis. Furthermore, in the metabolic activation of DMBA in mouse epidermis, P4501B1 shows a preference for the formation of syn-DMBA-diol epoxide adducts, whereas P4501A1 shows a preference for the formation of anti-DMBA-diol epoxide adducts.

The current study was designed to evaluate the effects of oral administration of the citrus coumarin, isopimpinellin, on skin tumor initiation by topically applied benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). To evaluate the effects of orally administered isopimpinellin on skin tumor initiation by B[a]P and DMBA, its effects on DNA adduct formation were first evaluated. Female SENCAR mice were pre-treated twice with corn oil, or isopimpinellin (70 mg/kg body wt per os) at 24 h and 2 h prior to topical treatment with B[a]P or DMBA. Another citrus coumarin, imperatorin, was also included in these experiments for comparison. Orally administered isopimpinellin and imperatorin significantly inhibited B[a]P-DNA adduct formation by 37 and 26%, respectively. Imperatorin also blocked DMBA-DNA adduct formation by 43%. In a second dose-response study, orally administered isopimpinellin (35, 70 and 150 mg/kg) blocked DMBA-DNA adduct formation by 23, 56 and 69%, respectively. For the tumor study, mice were pretreated orally with corn oil or isopimpinellin at 24 and 2 h prior to initiation with DMBA, and 2 weeks later promotion began with 12-O-tetradecanoylphorbol-13-acetate (TPA). Isopimpinellin significantly reduced the mean number of papillomas per mouse by 49, 73 and 78% compared to corn oil controls at 30, 70 and 150 mg/kg body wt, respectively. Orally administered isopimpinellin also significantly reduced the percentage of mice with papillomas at the highest dose tested (150 mg/kg). The effectiveness of isopimpinellin given topically over a broad dose range against DMBA tumor initiation was also evaluated for comparison. As part of this study, several parameters of systemic toxicity were evaluated following oral dosing with isopimpinellin and imperatorin. Mice were treated orally with corn oil, isopimpinellin or imperatorin (35, 70 and 150 mg/kg body wt per os) once daily for four consecutive days, killed at 24 h after the last dose, and livers, lungs, and kidneys evaluated histologically. In addition, urinary parameters of nephrotoxicity, blood parameters of liver and kidney function, and thrombin clotting time were assayed. No significant changes in blood clotting, or renal or hepatic function were observed. There was, however, a significant increase in liver wt accompanied by cytoplasmic vacuolation of hepatocytes. There were no histopathological changes in lungs or kidneys. Overall, these data indicate that isopimpinellin (and imperatorin) have chemopreventive effects when administered orally on skin tumor initiation by DMBA.

The current study was designed to determine the mechanistic basis for differences in the effects of naturally occurring furanocoumarins on skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA). Female SENCAR mice were pretreated topically with bergamottin, imperatorin, or isopimpinellin (100-3200 nmol), 7,8-benzoflavone (7,8-BF, 5-40 nmol, a known inhibitor of DMBA skin carcinogenesis in mice), or acetone (vehicle control) 5 min prior to topical treatment with DMBA (10 nmol). Imperatorin, isopimpinellin, and 7,8-BF, but not bergamottin, significantly blocked total DMBA-DNA adduct formation. HPLC analysis of DNA adducts revealed that bergamottin preferentially inhibited formation of anti-DMBA diol-epoxide (DMBADE) derived DNA adducts, imperatorin, and isopimpinellin inhibited both anti- and syn- derived adducts, whereas 7,8-BF showed some selectivity for reduction of syn-DMBADE-DNA adducts. Mouse embryo fibroblast C3H/10T1/2 (10T1/2) cells, and mouse hepatoma-derived 1c1c7 (Hepa-1) cells, which preferentially express P450 1b1 and P450 1a1, respectively, were co-incubated with 2 microM bergamottin, imperatorin, isopimpinellin, and 7,8-BF, and with DMBA (2 microM). Hepa-1 cells (P450 1a1) formed mainly anti-DMBADE-DNA adducts. In contrast, 10T1/2 cells (P450 1b1) formed mainly syn-DMBADE-DNA adducts. Bergamottin inhibited DMBA metabolism to DMBA-3,4-diol and blocked DNA adduct formation in Hepa-1 cells, but had little effect in 10T1/2 cells. In contrast, 7,8-BF completely blocked DMBA metabolism and DNA adduct formation in 10T1/2 cells, but had little effect in Hepa-1 cells. Imperatorin and isopimpinellin inhibited DMBA bioactivation in both cell lines. These results indicate that bergamottin is a more selective inhibitor of P450 1a1 and overall a less effective inhibitor of the metabolic activation of DMBA in mouse epidermis. In contrast, imperatorin, isopimpinellin, and especially 7,8-BF, which block metabolic activation of DMBA in mouse epidermis, appear more selective for P450 1b1. On the basis of our studies using 10T1/2 cells and Hepa-1 cells, it appears that P450 1a1 is primarily responsible for converting DMBA-3,4-diol to anti-DMBADE, whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to syn-DMBADE. These data demonstrate the role of P450 1a1 and 1b1 in the metabolic activation of DMBA in mouse epidermis and provide a mechanistic explanation for the differential effects of naturally occurring furanocoumarins (and 7,8-BF) on polycyclic aromatic hydrocarbon skin carcinogenesis.

Our previous study has shown that chronic exposure to tamoxifen (TAM) induced formation of high levels of DNA adducts in the liver, the target tissue of TAM-induced carcinogenesis in rats. One of the major DNA adducts (spot 1), as detected by 32P-postlabeling, accounted for 53% of the total adducts. To characterize this major adduct, the current study has compared spot 1 with two previously identified TAM-DNA adducts, i.e. alpha-TAM-N2-deoxyguanine (alpha-TAM-N2-dG) and alpha-N-desmethyl TAM-N2-deoxyguanine (alpha-N-dmTAM-N2-dG) by various rechromatography methods. It was found that spot 1 was further resolved into two fractions during rechromatography analysis, one fraction co-migrated with the alpha-TAM-N2-dG and the other fraction co-migrated with the alpha-N-dmTAM-N2-dG. These findings have demonstrated that chronic exposure to tamoxifen induced the same major DNA adducts, i.e. alpha-TAM-N2-dG and alpha-N-dmTAM-N2-dG as those detected in acutely exposed rats.

The purpose of this study was to examine the level of smoking-related aromatic DNA adducts and oxidative DNA damage in current smokers from a lung cancer case-control study in African Americans and Mexican Americans. In addition, mutagen sensitivity (bleomycin-induced chromatid breaks), a marker of genetic susceptibility, was assessed in these patients and correlated with the level of DNA damage. Lymphocyte DNA from cases and age-, sex-, and ethnicity-matched controls was analyzed for aromatic DNA adducts (43 cases and 47 controls) and the level of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) was determined in 46 cases and 48 controls using 32P-postlabeling. Overall, lung cancer cases had significantly (P

I-compounds are endogenous bulky DNA modifications which are detected by nuclease P1-enhanced 32P-post-labeling in tissue DNA of animals not knowingly exposed to carcinogens. Their profiles and levels depend inter alia on animal age, species, strain, tissue, gender, diet and exposure to chemicals such as cytochrome P450 inducers and carcinogens. Due to lack of sufficient material obtainable from in vivo sources, chemical structures of I-compounds and their parent normal bases have not yet been identified. In this report we provide 32P-post-labeling and chromatographic evidence that two prominent I-compounds, herein called C1 and C2, which occur at relatively high levels in pig liver DNA are guanine derivatives. This result was obtained by showing that both compounds, isolated from 32P-post-labeling thin-layer maps, were chemically unstable, i.e. they could be readily hydrolyzed to 32P-post-labeled deoxyguanosine 3',5'-bisphosphate by heating in water. C1 appeared particularly labile, undergoing hydrolysis during thin-layer chromatography at pH 3.3 without heating. Several other I-compounds and adducts, as well as the four normal DNA nucleotides, were, however, highly resistant to hydrolysis under the conditions used here. The possible significance of these findings will be briefly discussed.

The majority of mercury (Hg) exposure in the US population is from consumption of fish contaminated with methylmercury (MeHg). Since inorganic Hg is the predominant form excreted in the feces and urine, hepatic biotransformation is a critical step in its normal clearance. This study was set to test the hypothesis that compromised liver function is associated with body burden of Hg as indirectly reflected by Hg sampled in blood and urine. From the National Health and Nutrition Examination Survey (NHANES, 2003–2008), 3769 adults aged 20 years and above were selected for analysis. Hepatic function was inferred from the three standard serum liver-related enzyme activities, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyltransferase (GGT). Multivariate regression models were used to examine the associations of interest. Although urinary Hg was significantly correlated with serum Hg, the blood–urinary Hg relationship was influenced by liver function, which is also a function of demographic and lifestyle factors (e.g., gender). Although the results were only marginally significant for examined enzymes (p = 0.06–0.08), urinary Hg tended to be lower among subjects with elevated liver enzymes, as compared to those with normal enzyme measurements. Conversely, MeHg generally represents a higher fraction of the total circulating Hg among those with elevated liver enzyme levels, especially among participants with elevations in all three enzymes (p = 0.01). In conclusion, this population-based study identified an association between liver function, serum Hg and urinary Hg. Urinalysis may not be the optimal approach to monitor Hg elimination toxicokinetics or Hg exposure, since the majority of Hg excretion is fecal and the fidelity of urinary excretion may depend on healthy liver function. Future prospective studies are warranted to expand these findings. Keywords: Liver, Mercury, Methyl mercury, Blood, Urine, Toxicokinetics

Genetic toxicology encompasses the study of a wide variety of DNA damage endpoints, including, but not limited to mutagenicity, clastogenicity, and aneugenicity. A wide variety of in silico, in vitro, and in vivo assays are used to predict the three common types of genotoxic damage, through qualitative and quantitative measurement of genotoxic biomarkers. The main emphasis of this chapter is placed on providing a general overview of currently available genotoxic biomarkers. It is beyond the scope of this chapter to discuss carcinogenesis and relevant predictors/assays. Here we discuss the genotoxicity assays that can be used to monitor and measure biomarkers of DNA damage. In addition, the mechanisms of different types of DNA damage, and the molecular bases of genetic variability and susceptibility are also elaborated on with examples.

Systems biology is a holistic approach to study complex interactions in biological systems in their entirety. It could be applied to the field of toxicology for studying the cellular perturbations occurring due to exposure to xenobiotics (e.g., drugs, chemicals) and/or other stressors. These xenobiotic-induced alterations are monitored using high-throughput tools or approaches, such as phenomics, epigenomics, and toxicogenomics and the obtained data are then integrated with computational tools such as bioinformatics. Overall, systems biology approaches could be used effectively in identifying biomarkers of toxicant exposure and understanding the molecular mechanisms of toxicity pathways. To emphasize the utility of these tools, a case study with the well-studied toxicant benzene is presented.

I-compounds are indigenously appearing covalent DNA modifications that can be detected by 32P-postlabeling assay in tissues of normal animals without known exposure to any carcinogens or toxins. Although these compounds have not been structurally identified, indirect evidence from earlier work suggested the possibility of involvement of molecular fragments derived from lipid peroxides. Diquat is a herbicide that stimulates lipid peroxidation and massive intrahepatic oxidant stress through redox cycling-mediated generation of reactive oxygen species. In the present study, we examined the effects of diquat on hepatic I-compounds of male Fischer-344 rats. Two groups of rats, approximately 14 weeks and 8 weeks old, were given a hepatotoxic dose (0.1 mmol/kg) of diquat or equal volumes of saline, i.p. Two and 6 h later plasma alanine aminotransferase (ALT) activities were measured and hepatic DNA I-compound levels were examined by nuclease P1-enhanced 32P-postlabeling. Elevated ALT activities were observed in some animals in both groups, at both time points, but considerable inter-animal variation was seen. A total of 15–16 I-compound fractions were measured in control and in diquat-treated animals, but no extra spots indicative of treatment-induced adducts were detected. Despite the qualitative similarities, the quantities of individual I-compounds were markedly decreased at 2 h in diquat-treated animals of both age groups. In 14 week old rats the hepatic I-compound contents were decreased at 2 h by 22–59%, which was statstically significant (ANOVA, P < 0.05) for all of the 9 polar I-compound fractions and none of the non-polar fractions. Eleven I-spots from this group showed significant negative linear correlations (P < 0.05) with ALT values. In 8 week old rats treated with diquat a 22–43% depletion in I-compound contents was statistically significant for 4 of the 7 non-polar and 2 of the 8 polar adduct fractions, but there was no significant correlation of I-compound contents with ALT values at the 2 h time point. By 6 h most of the I-spot levels had returned to normal or above normal values in both groups of animals. While most I-spots from 14 week old rats did not correlate with ALT levels at 6 h, two I-spots displayed positive correlations in the 8 week group. Overall, the susceptibility to diquat-associated DNA alterations appeared to differ with age. Decreases in hepatic I-compound contents of diquat-treated Fischer-344 rats and the absence of additional I-compounds in these animals were not consistent with the hypothesis that I-compounds are derived from lipid peroxide fragments. However, the rapid but transient decrease in I-compound levels is notable because of similar trends previously observed during a number of procarcinogenic experimental manipulations.

Mutagenic agents have long been inferred to act through low-dose linear, nonthreshold processes. However, there is debate about this assumption, with various studies interpreting datasets as showing thresholds for DNA damage and mutation. We have applied rigorous statistical analyses to investigate the shape of dose-response relationships for a series of in vitro and in vivo genotoxicity studies using potassium bromate (KBrO(3) ), a water ozonation byproduct that is bioactivated to a reactive species causing oxidative damage to DNA. We analyzed studies of KBrO(3) genotoxicity where no-effect/threshold levels were reported as well as other representative datasets. In all cases, the data were consistent with low-dose linear models. In the majority of cases, the data were fit either by a linear (straight line) model or a model which was linear at low doses and showed a saturation-like downward curvature at high doses. Other datasets with apparent upward curvature were still adequately represented by models that were linear at low dose. Sensitivity analysis of datasets showing upward curvature revealed that both low-dose linear and nonlinear models provide adequate fits. Additionally, a simple biochemical model of selected key processes in bromate-induced DNA damage was developed and illustrated a situation where response for early primary events suggested an apparent threshold while downstream events were linear. Overall, the statistical analyses of DNA damage and mutations induced by KBrO(3) are consistent with a low-dose linear response and do not provide convincing evidence for the presence of a threshold.

Exposure to xenobiotics induces complex biochemical responses in mammalian cells resulting in several perturbations in cellular toxicity pathways. Within the context of systems biology, such biochemical perturbations can be studied individually using “omics” approaches such as toxicogenomics, transcriptomics, proteomics and metabolomics (Heijne et al., 2005). The objective of this chapter is to examine how the metabolomics approach can be used in identifying the risk posed by environmental chemicals to human health using selective examples of organ toxicity. Metabolomics is a medium-to-high throughput technique employing predominantly mass spectrometry (MS) and nuclear magnetic resonance (NMR) technology (Roux et al., 2011) for the identification and characterization of endogenous metabolites of low molecular weight (

The 32P-postlabeling assay has been used widely in carcinogen-DNA adduct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphorylates the 5'-OH groups of adducted nucleotides in the presence of [gamma-32P]ATP. However, as an exception to this property, PNK has been reported to phosphorylate non-adducted carcinogen metabolites, such as tetrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphorylates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [32P]phosphate from [gamma-32P]ATP to rat bile components or purified bile acids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absence of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100,000-fold less efficient than labeling of 2'-deoxyadenosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, was resistant to phosphorylation. On polyethyleneimine-cellulose TLC maps, 32P-labeled rat bile extract gave an array of non-polar radioactive spots which resembled carcinogen-DNA adducts, while 32P-labeled purified bile acids each gave a single spot. These 32P-labeled products liberated 32Pi upon incubation with prostatic acid phosphatases. Two of the radioactive spots obtained from rat bile were identified as phosphorylated taurocholic and taurodeoxycholic acids by co-chromatography with 32P-labeled standards. These findings demonstrate for the first time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination with bile acids and possibly other bulky/hydrophobic alcohols when analyzing DNA samples by 32P-postlabeling.

Background and Aims: Formaldehyde is ubiquitous in ambient and indoor air. Human exposure primarily results from off-gassing of processed wood products and as a combustion by-product. Evidence of c...

Publisher Summary This chapter discusses the reproductive and developmental toxicity of several commonly used solvents that are easily volatilized so that inhalation is a primary route of exposure. In both sexes, infertility can occur as a result of toxic exposures. With regard to developmental toxicity, there is an increased risk of small-for-gestational-age live born babies has been observed in women exposed to CCl4 during their second or third trimester of pregnancy. PCE has been associated with increased risks of spontaneous abortions, time to pregnancy and decrease in fertility in women who were directly employed or whose spouses were working in the dry-cleaning industry. Men working in these facilities have shown abnormal sperm morphology and other reproductive dysfunctions. Occupational exposure studies have examined the reproductive and developmental toxicity of styrene; these studies did not find statistically significant alterations in the occurrence of stillbirths, infant deaths, malformations or birth weight. Toluene has been associated with spontaneous abortions in women with no developmental effects observed. The most common reproductive symptoms of benzene exposure in women include menstrual disorders and in men, a decrease in sperm count, motility and concentration as well as decreased semen volume. No well-conducted reproductive and developmental toxicity studies exist to evaluate human health risk of acute or chronic exposure to kerosene and jet fuels. Developmental outcomes of formaldehyde include fetal loss, structural alterations, growth retardation, and delays in functional development. Formaldehyde has been associated with spontaneous abortions and increased time to pregnancy in occupationally exposed women.

Both experimental animals and humans exhibit complex cellular responses upon exposure to xenobiotics and may undergo similar types of metabolic changes leading to adverse outcomes. Exposure to xenobiotics results in perturbation of many cellular events (e.g. oxidative stress, lipid peroxidation, inflammation, genotoxicity, cytotoxicity, etc.), and during this process biochemicals (endogenous metabolites) of a given metabolic pathway are increased, decreased or unaffected. Metabolomics is an emerging medium to high-throughput technology that can automatically identify, quantify and characterize hundreds to thousands of low molecular weight biochemicals simultaneously, using targeted or global analytical approaches, yielding a metabolic fingerprint and understanding of biochemical pathway perturbations. Herein, we illustrate how metabolomics can be utilized to explore the mechanisms of action of xenobiotics which affect different ‘key events’ contributing to different mode(s) of action. The extensively studied hepatotoxicant carbon tetrachloride (CCl4) is specifically described.

32P-postlabeling analysis recently revealed that in addition to 5-methylcytosine, mammalian DNA contains covalently modified nucleotides of unknown structures and functions termed I-compounds whose levels increase with age. I-compound levels, in addition, depend on species, strain, sex, tissue, and diet and are generally lowered by carcinogen exposure. As shown here, levels of several non-polar I-compounds in liver DNA of untreated male C3H mice were elevated 2 to 8.5 times at 1800 h and 2400 h as compared to 0600 h and 1200 h, while polar I-compounds and persistent carcinogen-DNA adducts induced by safrole were unaffected by time of day. In liver DNA of male F-344 rats 4 non-polar I-compounds and 4 polar I-compounds showed significant circadian rhythm at 2000 h compared to 0800 h. This novel circadian variation of DNA structure implies mechanisms precisely regulating I-compound levels in vivo and may conceivably be linked to diurnal differences of DNA synthesis and gene expression.

Cadmium is a transition metal and an ubiquitous environmental and industrial pollutant. Laboratory animal studies and epidemiological studies have shown that exposure to cadmium is associated with various organ toxicities and carcinogenic effects. Several national and international regulatory agencies have classified cadmium compounds as carcinogenic to humans. In 2012, International Agency for Research on Cancer (IARC) reviewed the relevant information on cadmium exposure and concluded that there is sufficient evidence in humans for the carcinogenicity of cadmium and its compounds. However, the underlying molecular mechanism(s), especially for the carcinogenicity, are unknown. The mechanisms proposed for carcinogenicity include aberrant gene expression, induction of oxidative DNA damage, stimulation of cell proliferation, malignant cell transformation, inhibition of DNA damage repair, inhibition of apoptosis and epigenetic mechanisms including alteration of DNA methylation. Studies have shown that due to the slow elimination, cadmium is a cumulative toxicant and past exposures could result in toxic effect of the residual metal. To better understand the mechanisms of cadmium carcinogenicity we have reviewed in vitro experimental and in vivo animal studies focused on the proposed mechanisms of carcinogenicity as well as epidemiological evidence on carcinogenicity in lung and kidney following cadmium exposure. Disclaimer: The views expressed are those of the authors and do not necessarily represent the views and/or policies of the U.S. EPA Citation Format: Raghu G. Nath, BR Sonawane, SV Vulimiri, YS Lin. Mechanisms of cadmium carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2015-2738

Egypt shows a parallel increase in premenopausal breast cancer and environmental pollution. The purpose of this study is to explore a possible relationship between oxidative DNA damage, urinary estrogen metabolites and breast cancer in Egyptian premenopausal women. We conducted a pilot study of Egyptian breast cancer involving 29 cases and 32 controls and analysed lymphocyte DNA levels of 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG), a measure of oxidative DNA damage using high performance liquid chromatography with electro-chemical detection (HPLC-ECD) method. We analysed levels of urinary estrogen metabolites, 2-hydroxyestrone (2-OHE) and 16alpha-hydroxyestrone (16alpha-OHE) by an enzyme immuno assay. We also collected residential, occupational, and reproductive histories of all study subjects. We detected, in all subjects, exceptionally high levels of 8-oxo-dG and thus oxidative DNA damage, the levels (mean 8-oxo-dG/10(5) dG+/-SD) were significantly (P0.01) higher in breast cancer cases (139.4+/-78.4) than in controls (60.9+/-51.5). Urinary 2-OHE and 16alpha-OHE or their ratio was not significantly different between cases and controls. However, 8-oxo-dG levels were positively correlated (P0.05) with 2-OHE and 16alpha-OHE from cases while controls showed a negative correlation (P0.05). Urban residence (Odds Ratio [OR] 3.1; Confidence interval [CI], 1.1-9.3), infertility (OR [9.8]; CI [1.1-89.7]), age (OR [2.6]; CI [1.4-4.6]) and 8-oxo-dG (OR 5.8; CI 1.9-17.5) levels were found to be significant predictors of breast cancer. Our finding of exceptionally high levels of 8-oxo-dG, a common result of oxidative DNA damage, warrant future studies on a larger population of premenopausal women in Egypt with consideration of other susceptibility markers and dietary characteristics.

Phenolics, common plant constituents, form up an important part of human diet and are considered potential chemopreventive agents. In the present study, structurally diverse phenolics, such as tannic acid, protocatechuic acid, chlorogenic acid and resveratrol, were investigated for their inhibitory effects on covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA in vitro and the suppression of oxidative burst in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human polymorphonuclear neutrophils (PMNs). 32P-postlabeling analysis of DNA incubated with DMBA in the presence of 3-methylcholanthrene (3-MC)-induced microsomes produced three major adducts derived from anti-, syn- and anti-dihydrodiol epoxides through reactions with dGuo and dAdo, respectively. Phenolic compounds at the concentration of 150 microM reduced the levels of all DMBA-DNA adducts by 55-98%. The most dramatic effect was observed in case of tannic acid, which completely inhibited the formation of DMBA-dAdo adducts. Chlorogenic acid was the least effective inhibitor of DMBA-DNA adducts formation particularly syn-DMBADE-dAdo (20%). Human neutrophils showed a significant dose-related decrease of TPA-induced chemiluminescence after pretreatment with phenolic compounds. The most effective inhibitors were tannic acid and resveratrol with IC(50)=5.19 and 5.76 microM, respectively. These results suggest that the suppression of reactive oxygen species (ROS) and carcinogen-DNA adducts formation may be important for anticarcinogenic activity of the examined phenolics.

A bulky DNA adduct (Spot 1) was previously detected in normal adjacent breast tissues of 41% (36/87) of women with breast cancer and in none (0/29) of the noncancer controls by (32)P-postlabeling. To characterize this adduct, it was chromatographically compared with DNA adduct profiles generated in several in vitro and in vivo experimental systems. First, MCF-7 cells were exposed to a number of chemical carcinogens, that is, benzo[a]pyrene (B[a]P), 4-OH-B[a]P, 9-OH-B[a]P, 11-OH-B[a]P, B[a]P-trans-4,5-dihydrodiol, 1-nitropyrene, 6-nitrochrysene, dibenzo[a,l]pyrene, benzo[c]phenanthrene, dibenzo[a,h]anthracene, 3-methylcholanthrene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Spot 1 was detected as a minor adduct in cells treated with B[a]P but not other compounds. Second, to determine whether Spot 1 is derived from lipid peroxidation products or estrogen metabolites, it was compared with adduct profiles of cells or DNAs exposed to 17beta-estradiol, 4-hydroxy estradiol, 4-hydroxynonenal, or oxidized oat oil. Spot 1 was not detectable in these samples. In addition, Spot 1 did not comigrate with the 1,N(2)-ethenodeoxyguanosine adduct standard. Third, to explore the mechanism of Spot 1 formation, it was compared with adduct profiles detected in DNA or mononucleotides reacted with BPDE, 1-OH-7,8-dihydrodiol of B[a]P, and 3-OH-7,8-dihydrodiol of B[a]P as well as in rats orally treated with B[a]P. Spot 1 comigrated with a minor adduct in BPDE-treated DNA during anion exchange rechromatography but these two adducts were separated by partition chromatography. Spot 1 also behaved in a manner that was very similar to that of the polar B[a]P adducts detected in rat liver, but the two adducts were separated by HPLC. Fourth, Spot 1 was compared with CD1 mice exposed to 7H-benzo[c]fluorene (B[c]F). Spot 1 from some patients comigrated with a major adduct induced by B[c]F. Finally, we found that the presence of Spot 1 in human breast tissues was not related to smoking status but, rather, with CYP1A1 MspI polymorphism. The CYP1A1 mutant carriers had a significantly higher frequency of this adduct than did the wild-type genotypes. Furthermore, individuals with Spot 1 had a significantly higher staining intensity for BPDE-PAH adducts in their tissue sections than those without it. These results demonstrate that this major bulky DNA adduct detected in human breast tissues is related to PAH exposure.

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.

The formation of DNA adducts in mouse epidermis has been examined following topical application of dibenz[a,j]anthracene (DB[a,j]A) and its metabolites, i.e., DB[a,j]A-3,4-diol, DB[a,j]A-3,4-10, 11-bis-diol, DB[a,j]A-3,4-8,9-bis-diol, 10-OH-DB[a,j]A-3,4-diol, or 11-OH-DB[a,j]A-3,4-diol, using a 32P-postlabeling assay. At initiating doses (400-1600 nmol), DB[a,j]A produced at least 23 DNA adduct spots, including four less polar (derived from the bay-region syn- and anti-diol-epoxides) and 19 highly polar DNA adducts. DB[a, j]A-3,4-diol produced 13 DNA adduct spots, four less polar and nine highly polar DNA adducts, and DB[a,j]A-3,4-10,11-bis-diol produced nine highly polar DNA adducts. Eight and seven of the highly polar DNA adducts generated by DB[a,j]A-3,4-diol and DB[a,j]A-3,4-10, 11-bis-diol, respectively, migrated in the chromatography system like the highly polar DNA adducts produced by the parent compound. Sufficient amounts of radioactivity were associated with highly polar adduct spots 11, 13, and 22 to confirm their chromatogaphic identity in DNA samples from DB[a,j]A-, DB[a,j]A-3,4-diol-, and DB[a, j]A-3,4-10,11-bis-diol-treated mice. 10-OH-DB[a,j]A-3,4-diol and 11-OH-DB[a,j]A-3,4-diol did not produce any highly polar DNA adducts that could be detected under our experimental conditions. At an initiating dose of 400 nmol, DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a, j]A-3,4-10,11-bis-diol produced 22.4 +/- 13.0, 15.6 +/- 10.1, and 5. 5 +/- 0.3 (mean +/- SD) adducts/10(9) nucleotides, of which 77, 65, and 100%, respectively, represented highly polar DNA adducts. At the same dose of 400 nmol per mouse, DB[a,j]A and its 3,4-diol were able to initiate papillomas in SENCAR mouse skin (3.08 +/- 1.89 and 3.48 +/- 2.72 papillomas per mouse, respectively, after 16 weeks of promotion with 12-O-tetradecanoyl phorbol 13-acetate), while the 3, 4-10,11-bis-diol of DB[a,j]A was inactive as a tumor initiator. A quantitative correlation (r = 0.935; p = 0.0196) between levels of less polar DNA adducts and tumor-initiating activity of DB[a,j]A, DB[a,j]A-3,4-diol, and anti-DB[a,j]ADE was observed. This study demonstrates that the highly polar DNA adducts formed from DB[a,j]A in mouse epidermis arise primarily from the DB[a,j]A-3,4-10, 11-bis-diol. However, the contribution of this metabolite to the tumor-initiating activity of DB[a,j]A appears to be small.

The development of short-term or accelerated cancer bioassays using genetically modified (GM) animals that exhibit high sensitivity to chemically induced cancers and that act through mode(s) of action relevant to humans continues to be an important focus of research. However, to date only a few GM mouse models such as the Trp53+/−, the Tg.AC and the rasH2 models have effectively been evaluated to substitute for the two-year rodent cancer bioassays. Whether these models provide an acceptable replacement for the conventional rodent cancer bioassay for assessing human cancer risks is the subject of ongoing debate. The objective of this study was to evaluate the current status of the use of GM mice for accelerated cancer bioassays in assessing the potential human health risks associated with exposure to carcinogenic agents. We compared the published data from the GM bioassays with results obtained using the National Toxicology Program's conventional 2-year mouse cancer bioassay presently used in human cancer risk assessment. The results demonstrate a moderate success for these GM models to distinguish carcinogens from non-carcinogens. Although the GM models are less efficient in detecting rodent carcinogens, they are more consistent in identifying non-carcinogens. Our analysis also shows that there continue to be concerns about the assay design and protocols used for the GM models, including issues related to sample size, study duration, genetic instability, and reproducibility. Additional issues of concern include specific toxicity pathway-dependent effects, and differences in the potential carcinogenic mechanisms operating in GM and non-GM animals, topics that still need to be investigated. In addition, the dose-responses of the GM models can vary substantially from those seen in conventional cancer bioassays. Overall, we conclude that the existing GM mouse models are of value for hazard identification, but are of limited use for dose-response analysis with currently used study designs. Hence, until these models are thoroughly validated using more robust study designs and the dose-response issues adequately addressed, caution should be exercised when using the current GM models to assess the carcinogenic risks of chemicals to humans. Disclaimer: The views expressed in this abstract are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency or the National Institute of Environmental Health Sciences. Citation Format: David A. Eastmond, Suryanarayana V. Vulimiri, John E. French, Babasaheb Sonawane. An evaluation of the use of genetically modified mouse models in human cancer risk assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1561. doi:10.1158/1538-7445.AM2013-1561