Derivation of oral cancer slope factors for hexavalent chromium informed by pharmacokinetic models and in vivo genotoxicity data.

Hexavalent chromium [Cr(VI)] is present in drinking water from natural and anthropogenic sources at approximately 1 ppb. Several regulatory bodies have recently developed threshold-based safety criteria for Cr(VI) of 30-100 ppb based on evidence that small intestine tumors in mice following exposure to ≥20,000 ppb are the result of a non-mutagenic mode of action (MOA). In contrast, U.S. EPA has recently concluded that Cr(VI) acts through a mutagenic MOA based, in part, on scoring numerous in vivo genotoxicity studies as having low confidence; and therefore derived a cancer slope factor (CSF) of 0.5 (mg/kg-day)-1, equivalent to ∼0.07 ppb. Herein, we demonstrate how physiologically based pharmacokinetic (PBPK) models and intestinal segment-specific tumor incidence data can form a robust dataset supporting derivation of alternative CSF values that equate to Cr(VI) concentrations ranging from below background to concentrations similar to those derived using threshold approaches-depending on benchmark response level and risk tolerance. Additionally, we highlight weaknesses in the rationale EPA used to discount critical in vivo genotoxicity studies. While the data support a non-genotoxic MOA, these alternative toxicity criteria require only PBPK models, robust tumor data, and fair interpretation of published in vivo genotoxicity data for Cr(VI).

Predictive modeling of biological responses in the rat liver using in vitro Tox21 bioactivity: Benefits from high-throughput toxicokinetics.

Computational methods are needed to more efficiently leverage data from in vitro cell-based models to predict what occurs within whole body systems after chemical insults. This study set out to test the hypothesis that in vitro high-throughput screening (HTS) data can more effectively predict in vivo biological responses when chemical disposition and toxicokinetic (TK) modeling are employed. In vitro HTS data from the Tox21 consortium were analyzed in concert with chemical disposition modeling to derive nominal, aqueous, and intracellular estimates of concentrations eliciting 50% maximal activity. In vivo biological responses were captured using rat liver transcriptomic data from the DrugMatrix and TG-Gates databases and evaluated for pathway enrichment. In vivo dosing data were translated to equivalent body concentrations using HTTK modeling. Random forest models were then trained and tested to predict in vivo pathway-level activity across 221 chemicals using in vitro bioactivity data and physicochemical properties as predictor variables, incorporating methods to address imbalanced training data resulting from high instances of inactivity. Model performance was quantified using the area under the receiver operator characteristic curve (AUC-ROC) and compared across pathways for different combinations of predictor variables. All models that included toxicokinetics were found to outperform those that excluded toxicokinetics. Biological interpretation of the model features revealed that rather than a direct mapping of in vitro assays to in vivo pathways, unexpected combinations of multiple in vitro assays predicted in vivo pathway-level activities. To demonstrate the utility of these findings, the highest-performing model was leveraged to make new predictions of in vivo biological responses across all biological pathways for remaining chemicals tested in Tox21 with adequate data coverage (n = 6617). These results demonstrate that, when chemical disposition and toxicokinetics are carefully considered, in vitro HT screening data can be used to effectively predict in vivo biological responses to chemicals.

An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: a case study with hexavalent chromium, captan, and folpet.

Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.

Exposure to environmentally-relevant concentrations of hexavalent chromium does not induce ovarian toxicity in mice.

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water (≥250 ppm) is reported to decrease ovarian follicle counts and increase follicular atresia in mice. To assess effects at lower concentrations, herein we exposed B6C3F1 mice to 0.1-150 ppm Cr(VI) in drinking water for 90 days in a GLP-compliant study. Ovarian follicular counts, differentiation, and degeneration were assessed from every 10th serial section (up to 14 sections per ovary). Ovarian follicular counts, differentiation, and rate of atresia were not altered in any exposure group. Gross and microscopic changes were not apparent in any of the evaluated reproductive or glandular organs. The no observable adverse effect level (NOAEL) for follicular effects was 150 ppm. In addition to these findings, published Cr(VI) studies examining follicles were scored using two methods for assessing study quality for use in risk assessment-including the Toxic Substance Control Act (TSCA) scoring method. Both methods revealed that studies reporting adverse effects on follicles generally received low scores. Overall, the current study indicates no/low potential for Cr(VI) to induce follicular toxicity in mice below 150 ppm Cr(VI) in drinking water (17.7 mg/kg bodyweight).

Comparison of Gene Expression Responses in the Small Intestine of Mice Following Exposure to 3 Carcinogens Using the S1500+ Gene Set Informs a Potential Common Adverse Outcome Pathway.

Carcinogenesis of the small intestine is rare in humans and rodents. Oral exposure to hexavalent chromium (Cr(VI)) and the fungicides captan and folpet induce intestinal carcinogenesis in mice. Previously (Toxicol Pathol. 330:48-52), we showed that B6C3F1 mice exposed to carcinogenic concentrations of Cr(VI), captan, or folpet for 28 days exhibited similar histopathological responses including villus enterocyte cytotoxicity and regenerative crypt epithelial hyperplasia. Herein, we analyze transcriptomic responses from formalin-fixed, paraffin-embedded duodenal sections from the aforementioned study. TempO-Seq technology and the S1500+ gene set were used to analyze transcription responses. Transcriptional responses were similar between all 3 agents; gene-level comparison identified 126/546 (23%) differentially expressed genes altered in the same direction, with a total of 25 upregulated pathways. These changes were related to cellular metabolism, stress, inflammatory/immune cell response, and cell proliferation, including upregulation in hypoxia inducible factor 1 (HIF-1) and activator protein 1 (AP1) signaling pathways, which have also been shown to be related to intestinal injury and angiogenesis/carcinogenesis. The similar molecular-, cellular-, and tissue-level changes induced by these 3 carcinogens can be informative for the development of an adverse outcome pathway for intestinal cancer.

Identifying Attributes That Influence In Vitro-to-In Vivo Concordance by Comparing In Vitro Tox21 Bioactivity Versus In Vivo DrugMatrix Transcriptomic Responses Across 130 Chemicals.

Recent efforts aimed at integrating in vitro high-throughput screening (HTS) data into chemical toxicity assessments are necessitating increased understanding of concordance between chemical-induced responses observed in vitro versus in vivo. This investigation set out to (1) measure concordance between in vitro HTS data and transcriptomic responses observed in vivo, focusing on the liver, and (2) identify attributes that can influence concordance. Signal response profiles from 130 substances were compared between in vitro data produced through Tox21 and liver transcriptomic data through DrugMatrix, collected from rats exposed to a chemical for ≤5 days. A global in vitro-to-in vivo comparative analysis based on pathway-level responses resulted in an overall average percent agreement of 79%, ranging on a per-chemical basis between 41% and 100%. Whereas concordance amongst inactive chemicals was high (89%), concordance amongst chemicals showing in vitro activity was only 13%, suggesting that follow-up in vivo and/or orthogonal in vitro assays would improve interpretations of in vitro activity. Attributes identified to influence concordance included experimental design attributes (eg, cell type), target pathways, and physicochemical properties (eg, logP). The attribute that most consistently increased concordance was dose applicability, evaluated by filtering for experimental doses administered to rats that were within 10-fold of those related to likely bioactivity, derived using Tox21 data and high-throughput toxicokinetic modeling. Together, findings suggest that in vitro screening approaches to predict in vivo toxicity are viable particularly when certain attributes are considered, including whether activity versus inactivity is observed, experimental design, chemical properties, and dose applicability.

Integration of mechanistic and pharmacokinetic information to derive oral reference dose and margin-of-exposure values for hexavalent chromium.

The current US Environmental Protection Agency (EPA) reference dose (RfD) for oral exposure to chromium, 0.003 mg kg-1  day-1 , is based on a no-observable-adverse-effect-level from a 1958 bioassay of rats exposed to ≤25 ppm hexavalent chromium [Cr(VI)] in drinking water. EPA characterizes the confidence in this RfD as "low." A more recent cancer bioassay indicates that Cr(VI) in drinking water is carcinogenic to mice at ≥30 ppm. To assess whether the existing RfD is health protective, neoplastic and non-neoplastic lesions from the 2 year cancer bioassay were modeled in a three-step process. First, a rodent physiological-based pharmacokinetic (PBPK) model was used to estimate internal dose metrics relevant to each lesion. Second, benchmark dose modeling was conducted on each lesion using the internal dose metrics. Third, a human PBPK model was used to estimate the daily mg kg-1 dose that would produce the same internal dose metric in both normal and susceptible humans. Mechanistic research into the mode of action for Cr(VI)-induced intestinal tumors in mice supports a threshold mechanism involving intestinal wounding and chronic regenerative hyperplasia. As such, an RfD was developed using incidence data for the precursor lesion diffuse epithelial hyperplasia. This RfD was compared to RfDs for other non-cancer endpoints; all RfD values ranged 0.003-0.02 mg kg-1  day-1 . The lowest of these values is identical to EPA's existing RfD value. Although the RfD value remains 0.003 mg kg-1  day-1 , the confidence is greatly improved due to the use of a 2-year bioassay, mechanistic data, PBPK models and benchmark dose modeling.

Comparison of Toxicity and Recovery in the Duodenum of B6C3F1 Mice Following Treatment with Intestinal Carcinogens Captan, Folpet, and Hexavalent Chromium.

High concentrations of hexavalent chromium (Cr(VI)), captan, and folpet induce duodenal tumors in mice. Using standardized tissue collection procedures and diagnostic criteria, we compared the duodenal histopathology in B6C3F1 mice following exposure to these 3 carcinogens to determine whether they share similar histopathological characteristics. B6C3F1 mice ( n = 20 per group) were exposed to 180 ppm Cr(VI) in drinking water, 12,000 ppm captan in feed, or 16,000 ppm folpet in feed for 28 days. After 28 days of exposure, villous enterocyte hypertrophy and mild crypt epithelial hyperplasia were observed in all exposed mice. In a subset of mice allowed to recover for 28 days, duodenal samples were generally indistinguishable from those of unexposed mice. Changes in the villi and lack of observable damage to the crypt compartment suggest that toxicity was mediated in the villi, which is consistent with earlier studies on each chemical. These findings indicate that structurally diverse agents can induce similar (and reversible) phenotypic changes in the duodenum. These intestinal carcinogens likely converge on common pathways involving irritation and wounding of the villi leading to crypt regenerative hyperplasia that, under protracted high-dose exposure scenarios, increases the risk of spontaneous mutation and tumorigenesis.

Ten factors for considering the mode of action of Cr(VI)-induced gastrointestinal tumors in rodents.

The determination of whether a chemical induces a specific cancer through a mutagenic or non-mutagenic mode of action (MOA) plays an important role in choosing between linear and nonlinear low-dose extrapolation to derive toxicity criteria. There is no formal framework from the U.S. EPA for determining whether environmental chemicals act through a mutagenic or non-mutagenic MOA; consequently, most such determinations are made on an ad hoc basis. Eastmond [Mutat Res 751 (2012)] recently conducted a systematic investigation of MOA determinations by U.S. and international regulatory agencies and organizations, and identified ten major factors that influence them, including toxicokinetics, in vivo genotoxicity in target organs, data quality, and evidence for alternative MOAs. We have used these ten factors to evaluate mutagenic vs. non-mutagenic MOA for gastrointestinal tumors induced by oral exposure to hexavalent chromium [Cr(VI)]. We also highlight similarities between Cr(VI) and other intestinal carcinogens previously determined to have non-genotoxic MOAs. Based on these analyses, we conclude that the MOA for Cr(VI) induced gastrointestinal tumors is non-mutagenic and that threshold risk assessment approaches are appropriate.

Assessment of the mutagenic potential of hexavalent chromium in the duodenum of big blue® rats.

A cancer bioassay on hexavalent chromium Cr(VI) in drinking water reported increased incidences of duodenal tumors in B6C3F1 mice at exposures of 30-180ppm, and oral cavity tumors in F344 rats at 180ppm. A subsequent transgenic rodent (TGR) in vivo mutation assay in Big Blue® TgF344 rats found that exposure to 180ppm Cr(VI) in drinking water for 28days did not increase cII transgene mutant frequency (MF) in the oral cavity (Thompson et al., 2015). Herein, we extend our analysis to the duodenum of these same TgF344 rats. At study termination, duodenum chromium levels were below either the limit of detection or quantification in control rats, but were 24.6±3.8µg/g in Cr(VI)-treated rats. The MF in control (23.2×10-6) and Cr(VI)-treated rats (22.7×10-6) were nearly identical. In contrast, the MF in the duodenum of rats exposed to 1-ethyl-1-nitrosourea for six days (study days 1, 2, 3, 12, 19, 26) increased 24-fold to 557×10-6. These findings indicate that mutagenicity is unlikely an early initiating event in Cr(VI)-induced intestinal carcinogenesis.

High-Throughput Screening Data Interpretation in the Context of In Vivo Transcriptomic Responses to Oral Cr(VI) Exposure.

The toxicity of hexavalent chromium [Cr(VI)] in drinking water has been studied extensively, and available in vivo and in vitro studies provide a robust dataset for application of advanced toxicological tools to inform the mode of action (MOA). This study aimed to contribute to the understanding of Cr(VI) MOA by evaluating high-throughput screening (HTS) data and other in vitro data relevant to Cr(VI), and comparing these findings to robust in vivo data, including transcriptomic profiles in target tissues. Evaluation of Tox21 HTS data for Cr(VI) identified 11 active assay endpoints relevant to the Ten Key Characteristics of Carcinogens (TKCCs) that have been proposed by other investigators. Four of these endpoints were related to TP53 (tumor protein 53) activation mapping to genotoxicity (KCC#2), and four were related to cell death/proliferation (KCC#10). HTS results were consistent with other in vitro data from the Comparative Toxicogenomics Database. In vitro responses were compared to in vivo transcriptomic responses in the most sensitive target tissue, the duodenum, of mice exposed to ≤ 180 ppm Cr(VI) for 7 and 90 days. Pathways that were altered both in vitro and in vivo included those relevant to cell death/proliferation. In contrast, pathways relevant to p53/DNA damage were identified in vitro but not in vivo. Benchmark dose modeling and phenotypic anchoring of in vivo transcriptomic responses strengthened the finding that Cr(VI) causes cell stress/injury followed by proliferation in the mouse duodenum at high doses. These findings contribute to the body of evidence supporting a non-mutagenic MOA for Cr(VI)-induced intestinal cancer.

Estimating serum concentrations of dioxin-like compounds in the U.S. population effective 2005-2006 and 2007-2008: A multiple imputation and trending approach incorporating NHANES pooled sample data.

Dioxin-like compounds (DLCs) are monitored in the U.S. population using data collected with the National Health and Nutrition Examination Survey (NHANES). Until recently, participants' serum samples have been analyzed individually, and summary statistics defining reference ranges by age, gender, and race/ethnicity have served as the background by which other biomonitoring data can be evaluated. In the most recent NHANES DLC data, 2005-2006 and 2007-2008, participants' sera have been physically pooled prior to laboratory analysis, introducing major challenges to their utility as a reference population: variability among individuals and relations with covariates are lost, and individual design effects cannot be applied. Further, the substantial drop in limits of detection (LODs) in pooled sample biennials prevents reliable comparisons to individual data, and has complicated estimates of change over time. In this study, we address the drawbacks introduced by pooled samples by generating U.S. population reference ranges based on individual-level data adjusted to 2005-2006 and 2007-2008 levels. Using publicly available data, multiple imputation (MI) generated four NHANES biennials (2001-2008) of individual DLC data; we then trended the change over time in each DLC by demographic stratum. NHANES 2003-2004 individuals were adjusted by the trended change over time. Population estimates of toxic equivalency (TEQ) concentrations were calculated using traditional MI survey analysis methods and reference tables provided for 2005-2006 and 2007-2008 by age, race, and gender. Demographic differences in TEQ concentrations and trended change are reported, e.g. TEQ continues to drop in young adults aged 20-39, but distributions appear stable in older adults 60+; Mexican Americans have consistently lowest dioxins, furans, and PCBs, with non-Hispanic Blacks dropping to the same levels as non-Hispanic Whites in dioxins and PCBs and significantly below non-Hispanic Whites in furans by 2007-2008. Additionally, the ratio of 95th percentile to mean in DLC distributions was found to vary by age, between dioxins, furans, and PCBs, and across mean, making a simple ratio approach impractical for describing population concentrations using pooled samples. We discuss the practical implications of the pooled sample method, the performance of this trending solution in the context of other methods, and expected effects of distribution assumptions on variability and TEQ estimates, particularly in largely undetected congeners. These updated reference populations of individuals, along with information on trending, provide a common and valid basis for interpreting other individually sampled biomonitoring data.

Transcriptomic responses in the oral cavity of F344 rats and B6C3F1 mice following exposure to Cr(VI): Implications for risk assessment.

Exposure to hexavalent chromium [Cr(VI)] in drinking water was previously reported to increase oral tumor incidence in F344 rats. To investigate the mode of action for these tumors, transcriptomic profiles in oral mucosa samples of F344 rats and B6C3F1 mice were analyzed following exposure to 0.1-180 ppm Cr(VI) for 7 or 90 days. In rats, genome-wide microarray analyses identified no significantly differentially expressed genes (DEGs) at either time point. In mice, 14 and 1 DEGs were respectively identified after 7 and 90 days of exposure. Therefore, relaxed statistical criteria were employed to identify potential DEGs (pDEGs), followed by high-throughput benchmark dose modeling to identify responsive pDEGs for pathway enrichment analysis. This identified 288 and 168 pDEGs in the rat oral mucosa, of which only 20 and 7 showed evidence of dose-response. No significant pathway enrichment was obtained with either pDEG or dose-responsive pDEG lists. Similar results were obtained in mice. These analyses indicate a negligible transcriptional response in the oral mucosa of both species. Comparison of the total number of gene changes in the oral mucosa of rats and mice with responses in the duodenum of animals from the same study demonstrated remarkable dose-response concordance across tissues and species as a function of tissue chromium concentration. The low chromium levels in the oral mucosa and negligible transcript response are consistent with an absence of tissue lesions. These findings are used to compare the merits of linear and nonlinear approaches for deriving toxicity criteria based on the oral tumors in rats. Environ. Mol. Mutagen. 57:706-716, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Reduction of hexavalent chromium by fasted and fed human gastric fluid. II. Ex vivo gastric reduction modeling.

To extend previous models of hexavalent chromium [Cr(VI)] reduction by gastric fluid (GF), ex vivo experiments were conducted to address data gaps and limitations identified with respect to (1) GF dilution in the model; (2) reduction of Cr(VI) in fed human GF samples; (3) the number of Cr(VI) reduction pools present in human GF under fed, fasted, and proton pump inhibitor (PPI)-use conditions; and (4) an appropriate form for the pH-dependence of Cr(VI) reduction rate constants. Rates and capacities of Cr(VI) reduction were characterized in gastric contents from fed and fasted volunteers, and from fasted pre-operative patients treated with PPIs. Reduction capacities were first estimated over a 4-h reduction period. Once reduction capacity was established, a dual-spike approach was used in speciated isotope dilution mass spectrometry analyses to characterize the concentration-dependence of the 2nd order reduction rate constants. These data, when combined with previously collected data, were well described by a three-pool model (pool 1 = fast reaction with low capacity; pool 2 = slow reaction with higher capacity; pool 3 = very slow reaction with higher capacity) using pH-dependent rate constants characterized by a piecewise, log-linear relationship. These data indicate that human gastric samples, like those collected from rats and mice, contain multiple pools of reducing agents, and low concentrations of Cr(VI) (<0.7 mg/L) are reduced more rapidly than high concentrations. The data and revised modeling results herein provide improved characterization of Cr(VI) gastric reduction kinetics, critical for Cr(VI) pharmacokinetic modeling and human health risk assessment.

Comparison of in vivo genotoxic and carcinogenic potency to augment mode of action analysis: Case study with hexavalent chromium.

Recent analyses-highlighted by the International Workshops on Genotoxicity Testing Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment-have identified a correlation between (log) estimates of a carcinogen's in vivo genotoxic potency and in vivo carcinogenic potency in typical laboratory animal models, even when the underlying data have not been matched for tissue, species, or strain. Such a correlation could have important implications for risk assessment, including informing the mode of action (MOA) of specific carcinogens. When in vivo genotoxic potency is weak relative to carcinogenic potency, MOAs other than genotoxicity (e.g., endocrine disruption or regenerative hyperplasia) may be operational. Herein, we review recent in vivo genotoxicity and carcinogenicity data for hexavalent chromium (Cr(VI)), following oral ingestion, in relevant tissues and species in the context of the aforementioned correlation. Potency estimates were generated using benchmark doses, or no-observable-adverse-effect-levels when data were not amenable to dose-response modeling. While the ratio between log values for carcinogenic and genotoxic potency was ≥1 for many compounds, the ratios for several Cr(VI) datasets (including in target tissue) were less than unity. In fact, the ratios for Cr(VI) clustered closely with ratios for chloroform and diethanolamine, two chemicals posited to have non-genotoxic MOAs. These findings suggest that genotoxicity may not play a major role in the cancers observed in rodents following exposure to high concentrations of Cr(VI) in drinking water-a finding consistent with recent MOA and adverse outcome pathway (AOP) analyses concerning Cr(VI). This semi-quantitative analysis, therefore, may be useful to augment traditional MOA and AOP analyses. More case examples will be needed to further explore the general applicability and validity of this approach for human health risk assessment.

Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy.

Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

Assessment of the mutagenic potential of Cr(VI) in the oral mucosa of Big Blue® transgenic F344 rats.

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water was associated with an increased incidence of oral tumors in F344 rats in a 2-year cancer bioassay conducted by the National Toxicology Program. These tumors primarily occurred at 180 ppm Cr(VI) and appeared to originate from the gingival mucosa surrounding the upper molar teeth. To investigate whether these tumors could have resulted from a mutagenic mode of action (MOA), a transgenic mutation assay based on OECD Test Guideline 488 was conducted in Big Blue(®) TgF344 rats. The mutagenic oral carcinogen 4-nitroquinoline-1-oxide (4-NQO) served as a positive control. Mutant frequency was measured in the inner gingiva with adjacent palate, and outer gingiva with adjacent buccal tissue. Exposure to 10 ppm 4-NQO in drinking water for 28 days increased mutant frequency in the cII transgene significantly, from 39.1 ± 7.5 × 10(-6) to 688 ± 250 × 10(-6) in the gingival/buccal region, and from 49.8 ± 17.8 × 10(-6) to 1818 ± 362 × 10(-6) in the gingival/palate region. Exposure to 180 ppm Cr(VI) in drinking water for 28 days did not significantly increase the mutant frequency in the gingival/buccal (44.4 ± 25.4 × 10(-6)) or the gingival/palate (57.8 ± 9.1 × 10(-6)) regions relative to controls. These data indicate that high (∼180,000 times expected human exposure), tumorigenic concentrations of Cr(VI) did not significantly increase mutations in the gingival epithelium, and suggest that Cr(VI) does not act by a mutagenic MOA in the rat oral cavity.

Synchrotron-based imaging of chromium and γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water.

Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum--the latter evidenced by lengthening of the crypt compartment by ∼2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. These findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.