Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Genotoxic and Epigenotoxic Alterations in the Lung and Liver of Mice Induced by Acrylamide: A 28 Day Drinking Water Study.

Acrylamide has been classified as a "Group 2A carcinogen" (probably carcinogenic to humans) by the International Agency for Research on Cancer. The carcinogenicity of acrylamide is attributed to its well-recognized genotoxicity. In the present study, we investigated the effect of acrylamide on epigenetic alterations in mice. Female B6C3F1 mice received acrylamide in drinking water for 28 days, at doses previously used in a 2 year cancer bioassay (0, 0.0875, 0.175, 0.35, and 0.70 mM), and the genotoxic and epigenetic effects were investigated in lungs, a target organ for acrylamide carcinogenicity, and livers, a nontarget organ. Acrylamide exposure resulted in a dose-dependent formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine and N3-(2-carbamoyl-2-hydroxyethyl)adenine in liver and lung DNA. In contrast, the profiles of global epigenetic alterations differed between the two tissues. In the lungs, acrylamide exposure resulted in a decrease of histone H4 lysine 20 trimethylation (H4K20me3), a common epigenetic feature of human cancer, while in the livers, there was increased acetylation of histone H3 lysine 27 (H3K27ac), a gene transcription activating mark. Treatment with 0.70 mM acrylamide also resulted in substantial alterations in the DNA methylation and whole transcriptome in the lungs and livers; however, there were substantial differences in the trends of DNA methylation and gene expression changes between the two tissues. Analysis of differentially expressed genes showed a marked up-regulation of genes and activation of the gene transcription regulation pathway in livers, but not lungs. This corresponded to increased histone H3K27ac and DNA hypomethylation in livers, in contrast to hypermethylation and transcription silencing in lungs. Our results demonstrate that acrylamide induced global epigenetic alterations independent of its genotoxic effects, suggesting that epigenetic events may determine the organ-specific carcinogenicity of acrylamide. Additionally this study provides strong support for the importance of epigenetic alterations, in addition to genotoxic events, in the mechanism of carcinogenesis induced by genotoxic chemical carcinogens.

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup.

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to <1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations. Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24hour period in 10 adult male volunteers following ingestion of 30µg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t1/2=0.45h) and elimination of the administered dose was complete 24h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43nM at 1.6h after administration and represented <0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29h vs 0.45h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (<1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies.

Evaluating the toxicokinetics of some metabolites of a C6 polyfluorinated compound, 6:2 fluorotelomer alcohol in pregnant and nonpregnant rats after oral exposure to the parent compound.

The 6:2 fluorotelomer alcohol (6:2 FTOH) is a common impurity in per- and polyfluoroalkyl substances (PFASs) used in many applications. Our previous toxicokinetic (TK) evaluation of 6:2 FTOH calculated times to steady state (tss) of one of its metabolites, 5:3 fluorotelomer carboxylic acid (5:3A), in the plasma and tissues of up to a year after oral exposure to rats. Our current work further elucidated the TK of 5:3A and other metabolites of 6:2 FTOH in pregnant and nonpregnant rats after repeated oral exposure and examined the role of renal transporters in the biopersistence of 5:3A. The tss values for 5:3A in serum and tissues of adult nonpregnant animals ranged from 150 days to over a year. 4:3 fluorotelomer carboxylic acid (4:3A) was an additional potentially-biopersistent metabolite. 5:3A was the major metabolite of 6:2 FTOH in serum of pregnant dams and fetuses at each time interval. 5:3A was not a substrate for renal transporters in a human kidney cell line in vitro, indicating that renal reuptake of 5:3A is unlikely contribute to its biopersistence. Further research is needed to identify the underlying processes and evaluate the impact of these 6:2 FTOH metabolites on human health.

Mechanistic insights into the cytotoxicity and genotoxicity induced by glycidamide in human mammary cells.

Acrylamide (AA) is a well-known industrial chemical classified as a probable human carcinogen. Benign and malignant tumours at different sites, including the mammary gland, have been reported in rodents exposed to AA. This xenobiotic is also formed in many carbohydrate-rich foods prepared at high temperatures. For this reason, AA is an issue of concern in terms of human cancer risk. The epoxide glycidamide (GA) is thought to be the ultimate genotoxic AA metabolite. Despite extensive experimental and epidemiological data focused on AA-induced breast cancer, there is still lack of information on the deleterious effects induced by GA in mammary cells. The work reported here addresses the characterisation and modulation of cytotoxicity, generation of reactive oxygen species, formation of micronuclei (MN) and quantification of specific GA-DNA adducts in human MCF10A epithelial cells exposed to GA. The results show that GA significantly induces MN, impairs cell proliferation kinetics and decreases cell viability at high concentrations by mechanisms not involving oxidative stress. KU55933, an inhibitor of ataxia telangiectasia mutated kinase, enhanced the cytotoxicity of GA (P < 0.05), supporting a role of this enzyme in regulating the repair of GA-induced DNA lesions. Moreover, even at low GA levels, N7-GA-Gua adducts were generated in a linear dose-response manner in MCF10A cells. These results confirm that human mammary cells are susceptible to GA toxicity and reinforce the need for additional studies to clarify the potential correlation between dietary AA exposure and breast cancer risk in human populations.

Benzo[a]pyrene (BP) DNA adduct formation in DNA repair-deficient p53 haploinsufficient [Xpa(-/-)p53(+/-)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days.

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.

Pharmacokinetics and derivation of an anticancer dosing regimen for PAC-1, a preferential small molecule activator of procaspase-3, in healthy dogs.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Quantification of deuterated bisphenol A in serum, tissues, and excreta from adult Sprague-Dawley rats using liquid chromatography with tandem mass spectrometry.

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products, epoxy resin-based food can liners, and paper products. The presence of BPA in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure and is problematic because of the potential for endocrine disruption. The ubiquity of environmental BPA in common laboratory supplies used for sample collection, storage, and analysis greatly increases the likelihood of false positive determinations, particularly at trace levels. The current study validated using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in conjunction with deuterated BPA as the dosing material to circumvent contamination for high sensitivity quantifications in rat serum, tissues, urine, and feces. The methods described provided measurements of both estrogen receptor-active aglycone and metabolically deactivated conjugated forms of BPA, a distinction that is critical to assessing toxicological potential. The adequacy of the described methodology was substantiated by its utility in analyzing samples from rats treated orally with a 100 µg/kg body weight dose of d6-BPA. These results emphasize the challenges inherent in measuring BPA in biological samples and how employing stable isotope labeled dosing can facilitate pharmacokinetic studies needed to understand BPA metabolism and disposition. Such studies conducted in experimental animal models, in conjunction with properly validated human biomonitoring data, will be the basis for PBPK modeling of BPA in environmentally exposed humans.

Reduction by, ligand exchange among, and covalent binding to glutathione and cellular thiols link metabolism and disposition of dietary arsenic species with toxicity.

Arsenic (As) is a common contaminant in the earth's crust and widely distributed in food and drinking water. As exposures have been associated with human disease, including cancer, diabetes, lung and cardiovascular disorders, and there is accumulating evidence that early life exposures are important in the etiology. Mode-of-action analysis includes a critical role for metabolic activation of As species to reactive trivalent intermediates that disrupt cellular regulatory systems by covalent binding to thiol groups. The central role of glutathione (GSH) in the chemical reactions of metabolism and disposition of arsenic species was investigated here. The chemical kinetics were measured for reactions in which GSH is a ligand for trivalent As complex formation, a reductant for pentavalent As species, and a participant in ligand exchange reactions with other biological As-thiol complexes. The diverse reactions of GSH with As species demonstrate prominent roles in: (1) metabolic activation via reduction; (2) transport from tissues that are the primary sources of reactive trivalent As intermediates following ingestion (intestine and liver) to downstream target organs (e.g., lung, kidney, and bladder); and (3) oxidation to the terminal metabolite, dimethylarsinic acid (DMAV), which is excreted. Studies of As metabolism and disposition emphasize the link between metabolic activation vs. excretion of As (i.e., internal dosimetry of reactive species) and the disruption of critical cellular thiol-based regulatory processes that define the dose-response characteristics of disease in human epidemiological studies and animal models and underpin risk assessment.

In vivo localization and postmortem stability of benzo[a]pyrene-DNA adducts.

DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) play a critical role in the etiology of gastrointestinal tract cancers in humans and other species orally exposed to PAHs. Yet, the precise localization of PAH-DNA adducts in the gastrointestinal tract, and the long-term postmortem PAH-DNA adduct stability are unknown. To address these issues, the following experiment was performed. Mice were injected intraperitoneally with the PAH carcinogen benzo[a]pyrene (BP) and euthanized at 24 h. Tissues were harvested either at euthanasia (0 time), or after 4, 8, 12, 24, 48, and 168 hr (7 days) of storage at 4°C. Portions of mouse tissues were formalin-fixed, paraffin-embedded, and immunohistochemically (IHC) evaluated by incubation with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum and H-scoring. The remaining tissues were frozen, and DNA was extracted and assayed for the r7,t8,t9-trihydroxy-c-10-(N 2 -deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct using two quantitative assays, the BPDE-DNA chemiluminescence immunoassay (CIA), and high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ES-MS/MS). By IHC, which required intact nuclei, BPdG adducts were visualized in forestomach basal cells, which included gastric stem cells, for up to 7 days. In proximal small intestine villus epithelium BPdG adducts were visualized for up to 12 hr. By BPDE-DNA CIA and HPLC-ES-MS/MS, both of which used DNA for analysis and correlated well (P= 0.0001), BPdG adducts were unchanged in small intestine, forestomach, and lung stored at 4°C for up to 7 days postmortem. In addition to localization of BPdG adducts, this study reveals the feasibility of examining PAH-DNA adduct formation in wildlife species living in colder climates. Environ. Mol. Mutagen. 61:216-223, 2020. © 2019 Wiley Periodicals, Inc.

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide.

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F1/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1-8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F1/Tk(+/+) mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F1/Tk(+/-) mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7-(2-carbamoyl-2-hydroxyethyl)guanine, the major glycidamide-DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide approximately 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1-8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1-8 are due to another mechanism.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women II: Total estrogenicity calculations accounting for competitive protein and receptor binding and potency.

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4-99.2% (median: 97.3%), ER-ß, 82.7-97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women.

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene.

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.

Tumorigenicity and genotoxicity of an environmental pollutant 2,7-dinitrofluorene after systemic administration at a low dose level to female rats.

Environmental pollution with nitroaromatic compounds may pose health hazards. We have examined the tumorigenicity in female Sprague-Dawley rats of 2,7-dinitrofluorene (2,7-diNF) and 9-oxo-2,7-diNF administered by intraperitoneal (i.p.) and oral routes at 10 micromol/kg body weight, 3 times per week for 4 weeks. After i.p. treatment, the estimated median latency for the combined malignant and benign mammary tumors was decreased in 2,7-diNF- (p = 0.003) or 9-oxo-2,7-diNF-treated (p = 0.007), relative to vehicle-treated rats (42 or 64 vs. 80 weeks, respectively), whereas after oral dosing, there were no significant differences. At 90 weeks, the malignant mammary tumor incidence in 2,7-diNF-, 9-oxo-2,7-diNF- and vehicle-i.p. treated rats was 44 (p = 0.02 vs. vehicle-treated), 25 and 6%, respectively. Liver and mammary gland DNA was analyzed by HPLC combined with electrospray tandem mass spectrometry for the presence of a deoxyguanosine (dG-2,7-diNF) adduct and a deoxyadenosine (dA-2,7-diNF) adduct derived from 2,7-diNF, and a deoxyguanosine (dG-9-oxo-2,7-diNF) adduct derived from 9-oxo-2,7-diNF. Both dG-2,7-diNF and dA-2,7-diNF were detected in DNA of 2,7-diNF-treated rats, whereas only very low levels of dG-9-oxo-2,7-diNF were detected in DNA of 9-oxo-2,7-diNF-treated rats. After i.p. treatment, the dA-2,7-diNF level was higher (p < 0.01) in the mammary gland than liver (13.6 vs. 7.8 adducts/10(8) nucleotides). In the mammary gland, the dG-2,7-diNF level was higher (p < 0.05) after i.p. than oral dosing and also higher (p < 0.05) than in the liver (36 vs. 8.6 and vs. 9.1 adducts/10(8) nucleotides, respectively). The preferential display of carcinogenicity and genotoxicity in the mammary gland by low doses of 2,7-diNF signifies its potential relevance for environmental breast cancer.

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells.

In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/Tk(+/-) mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GA-derived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P=0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.

Metabolism and disposition of arsenic species from controlled oral dosing with sodium arsenite in adult female CD-1 mice. I. Pilot study to determine dosing, analytical measurements, and sampling strategies.

Arsenic (As) is ubiquitous in the earth's crust, with typical dietary intake in developed countries <1 µg/kg bw/d, and atypical groundwater exposures in developing countries approaching 50 µg/kg bw/d. Arsenic exposures are linked with human diseases and doses of toxicological concern are similar to typical dietary intake estimates. The methylation of arsenite by arsenite-3-methyltransferase (As3MT) promotes the clearance of arsenic as pentavalent species, but also generates reactive trivalent intermediates. This study measured inorganic arsenic and its metabolites in pentavalent and trivalent states in blood, tissues, and excreta after oral administration of arsenite (50-200 µg/kg bw). While liver was a major site for clearance of arsenite and formation of methylated species, it also had extensive binding of trivalent intermediates; however, thiol exchange and oxidation reactions of trivalent arsenic were facile since dimethylarsinic acid (DMAV) was the predominant species in blood and urine. Consistent evidence was observed for a non-linear relationship between doses above 50 µg/kg bw and levels of bound trivalent As metabolites. The abundance of protein-bound trivalent arsenic within target tissues should correlate with disruption of critical cellular processes, which rely on defined interactions of thiol functional groups, and could provide dose-response relationships from animal models for human risk assessment.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women I: Serum levels, variability and the basis for urinary biomonitoring of serum estrogenicity.

Biomonitoring of human exposure to estrogens most frequently focuses on environmental and dietary estrogens, and infrequently includes measures of exposure to potent endogenous estrogens present in serum. Pregnancy is a developmentally sensitive period during which "added" serum estrogenicity exceeding normal intra-individual daily variability may be of particular relevance. We made repeated measurements of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), daidzein (DDZ), genistein (GEN) and bisphenol A (BPA) in thirty pregnant women using ultra-performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS) and electrospray ionization (ESI). Serum E1, E2, and E3 concentrations varied significantly (coefficients of variation 9-10%) with broad ranges across the cohort: 1.61-85.1 nM, 9.09-69.7 nM, and 1.5-36.3 nM respectively. BPA (undetected, estimated from total exposure), DDZ and GEN concentrations were 1-5 orders of magnitude lower. The 24-h urinary elimination profiles of endogenous estrogens were each strongly correlated with their corresponding serum concentrations (Pearson's Correlation Coefficients of 0.83 (E1), 0.84 (E2) and 0.94 (E3)). A multivariate regression analysis produced equations for estimating serum concentrations of E1, E2, E3, E4, GEN and DDZ from urinary elimination rates and gestation period, an important step towards non-invasive biomonitoring for assessment of "added" estrogenicity during pregnancy.

Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated at minimally cytotoxic concentrations of (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene for 72 h and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene generated distinct gene-expression profiles, whereas B[a]P had only a slight effect on gene expression. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was minimal. Among the carcinogen-altered genes, transferrin (TF) emerged as sensitive marker for an initial screening of chemicals for their potential liver carcinogenicity. Potential liver carcinogens (i.e., chemicals causing altered TF gene expression) could then be subjected to gene-expression analyses to differentiate genotoxic from non-genotoxic liver carcinogens. This approach may substantially enhance the identification and assessment of potential liver carcinogens.

Acrylamide Production in Autoclaved Rodent Feed.

Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only considerthe beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures andpressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemicalchanges to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide weresignificantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urineacrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, whichcorrelate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutionsthat autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and thetype of studies in which these autoclaved diets are used.

Effect of N,N-didesmethyltamoxifen upon DNA adduct formation by tamoxifen and alpha-hydroxytamoxifen.

Tamoxifen undergoes sequential metabolism to N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Whereas N-desmethyltamoxifen is a major metabolite in humans, nonhuman primates, and rats, appreciable concentrations of N,N-didesmethyltamoxifen are formed in humans and nonhuman primates but not in rats. This difference in the extent of N,N-didesmethyltamoxifen formation may be important because it has been proposed that N,N-didesmethyltamoxifen inhibits the cytochrome P450 (CYP)-catalyzed alpha-hydroxylation of tamoxifen and resultant tamoxifen-DNA adduct formation. To test this hypothesis directly, we compared the extent of tamoxifen-DNA adduct formation in rats co-administered 27micromol N,N-didesmethyltamoxifen per kg body weight and either 27micromol tamoxifen per kg body weight or 27micromol alpha-hydroxytamoxifen per kg body weight daily for 7days. Female Sprague-Dawley rats treated with N,N-didesmethyltamoxifen had a 44% decrease (p >0.05) in CYP 3A2 content (the CYP isoform responsible for tamoxifen alpha-hydroxylation), an 18% decrease (p =0.010) in CYP 3A activity, and higher blood levels of tamoxifen and N-desmethyltamoxifen compared to rats treated with solvent. Total tamoxifen-DNA adduct levels were 4.1-fold higher (p <0.001) in rats given alpha-hydroxytamoxifen as compared to tamoxifen. N,N-Didesmethyltamoxifen treatment caused a 1.2-fold increase in total tamoxifen-DNA adduct levels with both tamoxifen and alpha-hydroxytamoxifen, a difference that was not significant. These results indicate that, with this experimental model, N,N-didesmethyltamoxifen does not impair the metabolism of tamoxifen to a reactive electrophile.