| Abstract: | We have demonstrated that incubation of rat liver microsomes with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) leads to formation of a 2-nitrosofluorene-membrane lipid adduct. This adduct exists as a nitroxyl free radical, termed N-O-LAF, in its oxidized state. When microsomes were incubated with the sulfhydryl binding agent, rho-hydroxymercuribenzoate, a larger amount of N-OL-LAF formed. We interpret this as a slowdown in the rate of endogenous chemical reduction of carcinogen-membrane lipid adduct. In this paper we present evidence that N-OH-AAF is deacetylated by a microsomal enzyme to form N-hydroxy-2-aminofluorene and this is then oxidized to 2-nitrosofluorene which adds covalently to membrane lipid double bonds to form N-O-LAF. Various antioxidants, peroxidase inhibitors, and P450 substrates and inhibitors were ineffective in altering the amount of N-O-LAF formed from N-OH-AAF; but two esterase inhibitors, dietyl-rho-nitrophenylphosphate and alpha-toluene-sulfonyl fluoride, prevented N-O-LAF formation. Of the following purified enzymes tested: porcine liver carboxyl esterase, pepsin, chymotrypsin, cathepsin D, ficin, papain, leucine aminopeptidase, Naja naja phospholipase, acetylcholinesterase (type I), trypsin (type I and V) and epoxide hydrase; only carboxyl esterase was effective in deacetylating N-OH-AAF. |
