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 for the roles of two thiol-based conjugation systems, glutathione (GSH) transferase and O6-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 the present 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 adducts (N7-guanyl), and cII mutation frequency. Administration of O6-benzylguanine (O6-BzGua), an inhibitor of AGT, did not change the mutation frequency. Depletion of GSH (BSO) and AGT (O6-BzGua) both lowered the mutation frequency induced by DEB, and BSO lowered the levels of GSH-DEB N7-guanyl and N6-adenyl DNA adducts. Our results provide evidence that the GSH conjugation pathway is a major in vivo factor in dibromoethane genotoxicity; both GSH and AGT conjugation are major factors in the genotoxicity of DEB. The latter findings are considered to be of relevance to the carcinogenicity of 1,3-butadiene.

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

“…, N 6 -oxopropenyl-dA, N 6 -carboxymethyl-dA, N 6 -(2-hydroxy-3-butan-1,4-diyl)-dA) have not reported any mutagenic events ( 79 , 80 , 81 ). In the case of mutations arising from EDB, most are G to A transitions, and the numbers of A mutations we have observed in Escherichia coli ( 51 , 82 ) yeast ( 83 ) or mouse liver ( 75 ) are too small, relative to background, to accurately assign a prominent base-pair substitution. However, hPol κ inserted C opposite to an N 6 -A dihydroxybutyl GSH adduct formed from diepoxybutane ( 70 ).…”

“…EDB induces AGT-DNA cross-links in cellular environments ( Fig. 1 ) ( 48 , 49 , 50 , 51 , 75 ). The possibility exists that proteolytic degradation of AGT-DNA cross-links occurs under cellular conditions by DNA-dependent proteases as well as the proteasome.…”

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation

“…Although GS-DNA adduct provides an immediate protection against the toxic effect of PN, it could be mutagenic, as observed with 1,2,3,4diepoxybutane and ethylene dibromide. 31,32 Accordingly, to test the mutagenic potential of the GS-DNA adduct reported here, we performed the Ames test in a TA100 histidinedependent strain of Salmonella typhimurium bacteria. The number of revertants growing in the absence of histidine was counted and corrected with a degraded PN treatment control.…”

Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases

“…17, 18 Mutations of a transgene in mouse liver were attenuated by an inhibitor of GSH synthesis, indicating that these adducts strongly contribute to DBE genotoxicity in vivo . 19 …”

“…17,18 Mutations of a transgene in the mouse liver were attenuated by an inhibitor of GSH synthesis, indicating that these adducts strongly contribute to DBE genotoxicity in vivo. 19 A similar chemical mechanism is involved in the DBE modification of the DNA repair protein O 6 -alkylguanine DNA alkyltransferase (AGT, MGMT) 20−25 and other proteins that become cross-linked to DNA. 26,27 After calf thymus DNA was treated with DBE in the presence of AGT, we identified adducts at the N7, N1, N2, and O6 positions of dG 24,28 and an N 6 -dA adduct, which had not been seen in earlier work with GSH.…”