DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites.

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates. In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA. In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats. In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide. The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling. The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate. Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose. Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes. The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate. Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate. In liver and heart the adducts were detectable only on the first day after completion of the treatment. The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential.

Genotoxicity of neurotoxic triaryl phosphates: identification of DNA adducts of the ultimate metabolites, saligenin phosphates.

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and neurotoxic metabolite of o-tolyl phosphates. We have investigated the genotoxicity of this saligenin phosphate and the structure of adducts formed by incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with nucleosides and DNA. o-Tolyl phosphate was mutagenic in the Ames test (695 revertants/mumol, Salmonella typhimurium TA 100) only with metabolic activation. 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide, which is a cyclization product similar to those expected from o-tolyl phosphate, was a potent mutagen in bacteria (1452 revertants/mumol, S. typhimurium TA 100) which did not require metabolic activation. Incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with guanosine, deoxycytidine, and deoxyadenosine resulted in formation of guanosine, deoxyuridine, and adenine adducts. These were identified as N-2-(o-hydroxybenzyl)guanosine, N-3-(o-hydroxybenzyl)deoxyuridine, N-1-(o-hydroxybenzyl)adenine, and N-3-(o-hydroxybenzyl)adenine by 1H-NMR spectroscopy, thermospray mass spectrometry, and pH-dependent electronic spectrometry. The deoxyuridine adduct is formed by an alkylation at N-3 of deoxycytidine followed by conversion of the adjacent exocyclic imino group to carbonyl (hydrolytic deamination). The formation of N-2-(o-hydroxybenzyl)-deoxyguanosine, N-3-(o-hydroxybenzyl)deoxyuridine, and N-1-(o-hydroxybenzyl)deoxyadenosine was also demonstrated when 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide was incubated with calf thymus DNA. Adducts formed with nucleosides in calf thymus DNA reacted with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide in vitro were detected by the 32P-postlabeling technique and identified by comparison with synthetic references. DNA adducts are formed by an o-hydroxybenzylation from cyclic phosphoranes derived from o-alkyl-substituted triaryl phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)