Analysis of Biomarkers of DNA Damage and Mutagenicity in Mice Exposed to Acrylonitrile

Abstract

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in the mouse via unresolved mechanisms. For this report, complementary and previously described methods were used to assess in vivo genotoxicity and/or mutagenicity of ACN in several mouse models, including (i) female mice devoid of cytochrome P450 2E1 (CYP2E1), which yields the epoxide intermediate cyanoethylene oxide (CEO), (ii) male lacZ transgenic mice, and (iii) female (wild-type) B6C3F1 mice. Exposures of wild-type mice and CYP2E1-null mice to ACN at 0, 2.5 (wild-type mice only), 10, 20, or 60 (CYP2E1-null mice only) mg/kg body weight by gavage for 6 weeks (5 days/week) produced no elevations in the frequencies of micronucleated erythrocytes, but induced significant dose-dependent increases in DNA damage, detected by the alkaline (pH >13) Comet assay, in one target tissue (forestomach) and one nontarget tissue (liver) of wild-type mice only. ACN exposures by gavage also caused significant dose-related elevations in the frequencies of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) reporter gene of T-lymphocytes from spleens of wild-type mice; however, Hprt mutant frequencies were significantly increased in CYP2E1-null mice only at a high dose of ACN (60 mg/kg) that is lethal to wild-type mice. Similarly, drinking water exposures of lacZ transgenic mice to 0, 100, 500, or 750 ppm ACN for 4 weeks caused significant dose-dependent elevations in Hprt mutant frequencies in splenic T-cells; however, these ACN exposures did not increase the frequency of lacZ transgene mutations above spontaneous background levels in several tissues from the same animals. Together, the Comet assay and Hprt mutant frequency data from these studies indicate that oxidative metabolism of ACN by CYP2E1 to CEO is central to the induction of the majority of DNA damage and mutations in ACN-exposed mice, but ACN itself also may contribute to the carcinogenic modes of action via mechanisms involving direct and/or indirect DNA reactivity.

Human genetic risk of treatment with antiviral nucleoside analog drugs that induce lethal mutagenesis: the special case of molnupiravir

“…A similar difference in relative sensitivity between the Hprt gene and lacI transgene was observed in Big Blue mice treated with another potent mutagen, N ‐ethyl‐ N 2 nitrosourea (ENU) (Walker et al, 1996), even though the average induced mutant frequencies (i.e., induced mutant frequency = treatment mutant frequency − background mutant frequency) in Hprt and lacI were similar. Additional studies demonstrated the ability to detect significant increases in mutations in the Hprt gene but not the lacI or lacZ genes following treatment of TGR mice with the cytotoxic chemotherapeutic cyclophosphamide (Walker et al, 1999), the direct‐acting alkylating agent ethylene oxide (Walker et al, 1997, 1999), and the epoxide‐forming industrial chemical acrylonitrile (Walker et al, 2020). One of the several possible reasons for these sensitivity differences is that the high baseline for background mutations in TGR mutation assays can make it difficult to resolve spontaneous and induced mutant frequencies and to demonstrate clearly the mutagenic effects of some test compounds in the transgene compared to the Hprt and Pig‐a genes with their relatively low background mutant frequencies (Walker et al, 2009).…”

Acrylonitrile induction of rodent neoplasia: Potential mechanism of action and relevance to humans

“…+, positive; ±, either positive or negative in several assays; -, negative; Fischer 344 rat strain; HID, highest ineffective dose; LEF, lowest effective dose; N/T, not tested; - S9, without metabolic activation; + S9, with metabolic activation; Data from ECHA 2018 3 ; IARC 1999 2 ; Walker et al 2020a,b. 22,23 …”

“…+, positive; ±, either positive or negative in several assays; -, negative; HID, highest ineffective dose; Fischer 344 rat strain; LEF, lowest effective dose; SD, Sprague-Dawley rat strain; Data from ECHA 2018 3 ; IARC 1999 2 ; Walker et al 2020a,b. 22,23 …”

“…No direct production of DNA strand breaks was detected in the target tissues, brain, and Zymbal's gland, of SD and F344 rats and Harderian gland of wild-type B6C3F1 or CYP2E-1-null mice using a conventional comet assay. 23,24 Positive comet assay results were reported in forestomach and liver in wild-type B6C3F1 but not CYP2E-1-null mice, after administration of acrylonitrile at 20 mg/kg by gavage 5 days/week for 6 weeks, indicating the significance of the oxidative pathway in producing DNA damage by acrylonitrile. 23 The authors noted that the nature of DNA damage detected by comet assay was difficult to determine, since the assay does not differentiate between single-strand breaks and excision-repair and alkali-labile sites (including those formed by repair of N-7-guanine DNA adducts).…”

“…While astrocytes accumulated more acrylonitrile compared to microglial cells, they also exhibited higher levels of CYP2E1. 20 Induction of nuclear factor-erythroid 2 related factor 2 (Nrf2), a key transcription factor involved in the 22,23 response to oxidative stress was also observed in rat microglia, but not in astrocytes in rats and mice or glial cells in mice. 19,21 GSH levels were up-regulated in both rat cell types, microglia, and astrocytes, evidencing a protective mechanism against acrylonitrile-associated oxidative stress.…”