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

Environ and Mol Mutagen

Dinesdurage

et al. 2015

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The Big Blue® (BB) in vivo mutation assay uses transgenic rodents to measure treatment-induced mutations in virtually any tissue. The BB assay can be conducted in rats or mice and is ideal for investigating tissue-specific mutagenic mode of action of tumor induction. Some tissues such as oral mucosa have not been thoroughly studied. Due to the small quantity and cartilaginous nature of oral cavity tissues, development of special prosection and DNA isolation methods was required to permit robust analysis of mutations in these tissues. Improved surgical methods permitted collection of adequate and reproducible quantities of tissue (∼45 mg gingiva/buccal and ∼30 mg gingiva/palate). Optimized DNA isolation methods included use of liquid nitrogen pulverization, homogenization, nuclei pelleting, digestion, and phenol/chloroform extraction, to yield sufficient quantities of DNA from these tissues. In preliminary optimization work, mutant frequency (MF) in tongue and gingiva was increased in rats exposed to the promutagen, benzo[a]pyrene, and the direct mutagen, N-ethyl-N-nitrosourea. The oral cavity carcinogen, 4-nitroquinoline-1-oxide (4-NQO; 10 ppm in drinking water; 28 days), was qualified as a positive control for mutagenesis in oral tissues since it caused significant increases in cII MFs in gingiva/palate (50.2-fold) and gingiva/buccal tissues (21.3-fold), but not in liver or bone marrow (0.9- and 1.4-fold, respectively). These results are consistent with the observation that 4-NQO primarily induces tumors in oral cavity. Results also demonstrate the utility of the BB rat mutation assay and optimized methods for investigation of oral cavity mutagenicity, and by extension, analysis of other small and cartilaginous tissues.

Section: Resultssupporting

confidence: 91%

A robust method for assessing chemically induced mutagenic effects in the oral cavity of transgenic Big Blue® rats

Young

Environ and Mol Mutagen

Dinesdurage

et al. 2015

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“…The general lack of transcriptional changes in the oral mucosa is entirely consistent with previous histopathology data indicating no non‐neoplastic or pre‐neoplastic changes in the oral mucosa of rats or mice [NTP, ; NTP, ; Thompson et al, ; Thompson et al, ]. Likewise, exposure to 180 ppm Cr(VI) did not increase cII transgene mutant frequency in TgF344 rats [Thompson et al, ]. To date, the only dose‐dependent changes in the oral mucosa in response to Cr(VI) were decreases in GSH and increases in GSSG levels in rats, which were not apparent in mice [Thompson et al, ; Thompson et al, ].…”

Section: Discussionsupporting

confidence: 86%

“…Using the OECD 28 + 3 exposure protocol (i.e., 28 days of exposure followed by necropsy on day 31), the oral mutagenic carcinogen 4‐nitroquinoline‐1‐oxide (4NQO) increased mutant frequency (MF) 20‐ to 50‐fold in the oral mucosa, but not in liver or bone marrow [Young et al, ]. A subsequent study from the same laboratory again found that 4NQO increased MF in the oral mucosa, whereas exposure to 180 ppm Cr(VI) did not increase MF in the oral mucosa of TgF344 rats [Thompson et al, ].…”

Section: Introductionmentioning

confidence: 99%

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

Environ and Mol Mutagen

Rager

Suh

et al. 2016

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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.

“…Toxicogenomic analyses indicate minimal, if any, gene expression changes in the oral mucosa of F344 rats or B6C3F1 mice exposed to ≤180 ppm Cr(VI) for 7 or 90 days (Thompson et al, 2016). Exposure to 180 ppm Cr(VI) for 28 days did not increase mutant frequency in oral tissue of Big Blue® F344 rats (Thompson, Young, et al, 2015). Taken together, these data indicate that Cr(VI) elicits minimal, if any, direct cellular responses in the oral mucosa of rats or mice.…”

Section: Resultsmentioning

confidence: 99%

“…To inform the MOA and risk assessment of oral Cr(VI) exposure, a series of studies were conducted beginning with an overall proposed MOA (Thompson, Haws, Harris, Gatto, & Proctor, 2011), and subsequent 90‐day toxicity studies (Thompson, Proctor, et al, 2011; Thompson et al, 2012), transcriptomic analyses (Kopec et al, 2012; Kopec, Thompson, Kim, Forgacs, & Zacharewski, 2012; Rager et al, 2017), genotoxicity studies (O'Brien et al, 2013; Thompson, Seiter, et al, 2015; Thompson, Wolf, et al, 2015; Thompson, Young, et al, 2015; Thompson et al, 2017), as well as ex vivo gastric reduction studies and pharmacokinetic modeling (De Flora et al, 2016; Kirman et al, 2012; Kirman et al, 2013; Kirman et al, 2016; Proctor et al, 2012). Other genotoxicity studies were conducted in response to early drafts of the NTP 2‐year cancer bioassay (De Flora et al, 2006; De Flora et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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

J of Applied Toxicology

Kirman²,

Hays³

et al. 2017

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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.