In vivo roles of conjugation with glutathione and O6-alkylguanine DNA-alkyltransferase in the mutagenicity of the bis-electrophiles 1,2-dibromoethane and 1,2,3,4-diepoxybutane in mice

Abstract

Several studies with bacteria and in vitro mammalian systems have provided evidence of the roles of two thiol-based conjugation systems, glutathione (GSH) transferase and O(6)-alkylguanine DNA-alkyltransferase (AGT), in the bioactivation of the bis-electrophiles 1,2-dibromoethane and 1,2,3,4-diepoxybutane (DEB), the latter an oxidation product of 1,3-butadiene. The in vivo relevance of these conjugation reactions to biological activity in mammals has not been addressed, particularly with DEB. In this work, we used transgenic Big Blue mice, utilizing the cII gene, to examine the effects of manipulation of conjugation pathways on liver mutations arising from dibromoethane and DEB in vivo. Treatment of the mice with butathionine sulfoxime (BSO) prior to dibromoethane lowered hepatic GSH levels, dibromoethane-GSH DNA adduct levels (N(7)-guanyl), and the cII mutation frequency. Administration of O(6)-benzylguanine (O(6)-BzGua), an inhibitor of AGT, did not change the mutation frequency. Depletion of GSH (BSO) and AGT (O(6)-BzGua) lowered the mutation frequency induced by DEB, and BSO lowered the levels of GSH-DEB N(7)-guanyl and N(6)-adenyl DNA adducts. Our results provide evidence that the GSH conjugation pathway is a major in vivo factor in dibromoethane genotoxicity; both GSH conjugation and AGT conjugation are major factors in the genotoxicity of DEB. The latter findings are considered to be relevant to the carcinogenicity of 1,3-butadiene.

Figure 1

Quantitative analysis of DNA adducts in livers of Big Blue® transgenic mice 6 h after treatment with dibromoethane (DBE), BSO/dibromoethane, or O⁶-BzGua/dibromoethane (A) and DEB, BSO/DEB, or O⁶-BzGua/DEB (B). O⁶-BzGua (80 mg/kg, ip) was administered 1 h prior to treatment with dibromoethane (30 mg/kg, ip) or DEB (25 mg/kg, ip), and BSO (8 mg/kg, ip) was administered 2 h prior to treatment with dibromoethane (30 mg/kg, ip) or DEB (25 mg/kg, ip).

Figure 2

cII mutant frequencies in liver of Big Blue® transgenic mice at 6 (A, B) and 24 h (C, D) after treatment with vehicle, dibromoethane (DBE), BSO/dibromoethane, or O⁶-BzGua/dibromoethane (A, C) and vehicle, DEB, BSO/DEB, or O⁶-BzGua/DEB (B, D). O⁶-BzGua (80 mg/kg, ip) was administered 1 h prior to treatment with dibromoethane (30 mg/kg, ip) or DEB (25 mg/kg, ip) and BSO (8 mg/kg, ip) was administered 2 h prior to treatment with dibromoethane (30 mg/kg, ip) or DEB (25 mg/kg, ip). The statistical significance values were from compareisons with the cII mutant frequencies induced by dibromoethane or DEB and evaluated using Student’s t-test (shown in figure). In Part A, the relative mutations frequencies (compared to the highest value, with dibromethane treatment set at 100%) were 15% for vehicle, 42% for +BSO, and 88% for +O⁶-BzGua. In Part B, the relative mutations frequencies (compared to the highest value, with dibromethane treatment set at 100%) were 20% for vehicle, 43% for +BSO, and 88% for +O⁶-BzGua. In Part C, the relative mutations frequencies (compared to the highest value, with DEB treatment set at 100%) were 32% for vehicle, 57% for +BSO, and 67% for +O⁶-BzGua. In Part D, the relative mutations frequencies (compared to the highest value, with DEB treatment set at 100%) were 49% for vehicle, 68% for +BSO, and 80% for +O⁶-BzGua.

Figure 3

Relative independent mutations induced by vehicle (A), dibromoethane (DBE) (B), BSO/dibromoethane (C), or O⁶-BzGua/dibromoethane (D) in liver of Big Blue® transgenic mice. O⁶-BzGua (80 mg/kg, ip) was administered 1 h prior to treatment with dibromoethane (30 mg/kg, ip) and BSO (8 mg/kg, ip) was administered 2 h prior to treatment with dibromoethane (30 mg/kg, ip). Transition mutations (■); transversion mutations (□); frameshifts and others (diagonal shading). The P values are for comparison of the GC to AT transversions between Parts A and B (P <0.001) and between Parts B and C (P <0.001).

Figure 4

Relative independent mutations induced by vehicle (A), DEB (B), BSO/DEB (C), or O⁶-BzGua/DEB (D) in livers of Big Blue® transgenic mice. O⁶-BzGua (80 mg/kg, ip) was administered 1 h prior to treatment with DEB (25 mg/kg, ip) and BSO (8 mg/kg, ip) was administered 2 h prior to treatment with DEB (25 mg/kg, ip). Transition mutations (■); transversion mutations (□); frameshifts and others (diagonal shading). The P values are for comparison of the AT to GC transversions between Parts A and B (P <0.0001), between Parts B ad C ((P <0.02) and between Parts B and C (P <0.05).

Scheme 1. GSH (A, B) and AGT (C, D) Conjugation Pathways for Activation of Dibromoethane (A, C) and DEB (B,D)

For the identities of the other DNA adducts of dibromoethane (GSH), DEB (GSH), and dibromoethane (AGT) see the indicated references.

Scheme 2. DNA Adducts from Reaction of Oxidized Products of 1,3-Butadiene

See the references.^– (Known stereoisomers of several of the adducts are not considered here.)