Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate β-lyase purified from Peptostreptococcus magnus

Abstract

To determine the role of cysteine conjugate β-lyase (β-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of β-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10- epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified β-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified β-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55°C for 5 min. The molecular weight of β-lyase was 150,000, as determined by fast protein liquid chromatography. S- Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of β-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, β-lyase had little effect on the cysteine conjugate of 1-NP 4,5- oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified β-lyase or xanthine oxidase. The levels of DNA binding were highest in the presence of both enzymes; under these conditions the binding of the cysteine conjugate of 1-NP 4,5-oxide increased 12.8-fold and the binding of the cysteine conjugate of 1-NP 9,10-oxide increased 21.3-fold. Aminooxyacetic acid and allopurinol inhibited the enhancement of the DNA binding of both conjugates. Our results suggest that a 1-NP oxide-Cys is changed to a more genotoxic form by β-lyase-mediated deconjugation and nitroreduction.