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

Abstract

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.

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

“…The mechanism of 4‐NQO tumor induction likely involves oxidative stress and direct DNA interaction of 4‐NQO metabolites [Fronza et al, ; Arima et al, ; Kanojia and Vaidya, ; Vitale‐Cross et al, ]. Herein, background and 4‐NQO‐induced mutant frequencies in gingival/buccal and gingival/palate samples were similar to those reported in Young et al []. The consistency of these data increases the confidence that the TGR gingiva assay can detect mutagenic signals from oral carcinogens that act by a mutagenic MOA.…”

“…Statistical analysis of the mutant frequency was conducted as described in Young et al []. In brief, at least 2 packaging reactions were performed for each tissue from each animal and at least 125,000 phage were scored per tissue per animal.…”

“…Statistical analysis of the mutant frequency was conducted as described in Young et al [2015]. In brief, at least 2 packaging reactions Environmental and Molecular Mutagenesis.…”

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

“…However, two recent developments indicate that these oral mucosa transcriptomic data should be revisited. First, the oral mucosa (including the gingiva, palate, and buccal regions) was recently qualified for use in transgenic rodent (TGR) in vivo mutation assays to permit investigation of mutagenic potential in the oral cavity of Big Blue® TgF344 rats [Young et al, ]. 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, ].…”

“…First, the oral mucosa (including the gingiva, palate, and buccal regions) was recently qualified for use in transgenic rodent (TGR) in vivo mutation assays to permit investigation of mutagenic potential in the oral cavity of Big Blue® TgF344 rats [Young et al, ]. 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, ].…”