Isoform-selective inactivation of human arylamine N-acetyltransferases by reactive metabolites of carcinogenic arylamines.

Human arylamine N-acetyltransferases (NATs) are expressed as two polymorphic isoforms, NAT1 and NAT2, that have toxicologically significant functions in the detoxification of xenobiotic arylamines by N-acetylation and in the bioactivation of N-arylhydroxylamines by O-acetylation. NAT1 also catalyzes the N-acetylation of 4-aminobenzoylglutamic acid, a product of folic acid degradation, and is associated with endogenous functions in embryonic development. On the basis of earlier studies with hamster NAT1, hamster NAT2, and human NAT1, we proposed that human NAT2 would be more susceptible than NAT1 to inactivation by N-arylhydroxamic acid metabolites of arylamines. Kinetic analyses of the inactivation of recombinant NAT1 and NAT2 by the N-arylhydroxamic acid, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), as well as the inactivation of NAT2 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), resulted in second-order inactivation rate constants (k(inact)/K(I)) that were several fold greater for NAT2 than for NAT1. Mass spectrometric analysis showed that inactivation of NAT2 in the presence of the N-arylhydroxamic acids was due to formation of a sulfinamide adduct with Cys68. Treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF revealed that the compounds were less potent inactivators of intracellular NAT activity than the corresponding nitrosoarenes, but unexpectedly, the hydroxamic acids caused a significantly greater loss of NAT1 activity than of NAT2 activity. Nitrosoarenes are the electrophilic products responsible for NAT inactivation upon interaction of the enzymes with N-arylhydroxamic acids, as well as being metabolic products of arylamine oxidation. Treatment of recombinant NAT2 with the nitrosoarenes, 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), caused rapid and irreversible inactivation of the enzyme by sulfinamide adduct formation with Cys68, but the k(inact)/K(I) values for inactivation of recombinant NAT2 and NAT1 did not indicate significant selectivity for either isoform. Also, the IC(50) values for inactivation of HeLa cell cytosolic NAT1 and NAT2 by 4-NO-BP were similar, as were the IC(50) values obtained with 2-NO-F. Treatment of HeLa cells with low concentrations (1-10 microM) of either 4-NO-BP or 2-NO-F resulted in preferential and more rapid loss of NAT1 activity than NAT2 activity. Because of its wide distribution in human tissues and its early expression in developing tissues, the apparent high sensitivity of intracellular NAT1 to inactivation by reactive metabolites of environmental arylamines may have important toxicological consequences.

Activation of the carcinogen N-hydroxy-N-(2-fluorenyl)benzamide via chemical and enzymatic oxidations. Comparison to oxidations of the structural analogue N-hydroxy-N-(2-fluorenyl)acetamide.

Chemical or enzymatic oxidations of the carcinogen N-hydroxy-N-(2- fluorenyl)benzamide (N-OH-2-FBA) were investigated under the conditions facilitating one-electron oxidation or oxidative cleavage of N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA). HPLC methods were developed for separation and quantitation of the above hydroxamic acids and their respective oxidation products. To identify the products of oxidation of N-OH-2-FBA, N-(benzoyloxy)-2-FBA (N-BzO-2-FBA) was synthesized and shown to undergo ortho rearrangement to 1- and 3-BzO-2-FBA. Oxidation of N-OH-2-FBA (4.88 mM) with alkaline K3Fe(CN)6 in benzene was complete and yielded equimolar amounts of 2-nitrosofluorene (2-NOF) and the ester (chiefly N-BzO-2-FBA), indicative of one-electron oxidation to nitroxyl free radical which undergoes bimolecular dismutation. However, one-electron oxidation of N-OH-2-FBA (30 or 10 microM) by horseradish peroxidase/H2O2 at pH 7 or myeloperoxidase/H2O2 at pH 6.5 yielded only approximately 10% as much product as N-OH-2-FAA (30 microM). The addition of 0.1 mM Br- +/- 0.1 M Cl- at pH 4 to 6.5 increased 2-NOF formation in MPO/H2O2-catalyzed oxidations. Simulations of these oxidations with HOCl/Cl- or HOBr/Br- showed that the latter was more efficient, converting N-OH-2-FAA almost completely and less than or equal to 62% of N-OH-2-FBA to 2-NOF. The amounts of the ester (N- and o-BzO-2-FBA), which by itself did not contribute to 2-NOF formation or significant substrate regeneration, indicated that approximately 10% of 2-NOF originated from one-electron oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of bovine serum albumin on the extent of ortho rearrangement of N-(sulfooxy)-2-fluorenylacetamide and of enzymatically activated N-hydroxy-2-fluorenylacetamide and on the binding of reactive esters to nucleic acids.

We have investigated the effect of the bovine serum albumin (BSA)-catalyzed ortho rearrangement of synthetic and enzymatically generated N-(sulfooxy)-2-fluorenylacetamide (NSF) to the O-sulfate esters on the binding of NSF to transfer ribonucleic acid (tRNA) and to deoxyribonucleic acid (DNA). Binding of synthetic NSF to tRNA and DNA decreased approximately 90 and 70%, respectively, in the presence of BSA. Under these conditions, the ortho rearrangement, a minor reaction in the absence of BSA, was nearly quantitative. The decrease of adduct formation to nucleic acids was not attributable to the competitive binding of NSF to BSA. Binding of NSF, generated by cytosolic sulfonation of the arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to tRNA, was diminished approximately 97% in the presence of BSA while the ortho rearrangement of the sulfonated substrate increased from less than 0.5% to approximately 50%. Adduct formation of DNA with N-hydroxy-2-fluorenylacetamide, activated by enzymatic sulfonation, was inhibited approximately 90% in the presence of BSA. In these experiments, the catalytic effect of BSA on the ortho rearrangement of enzymatically sulfonated N-hydroxy-2-fluorenylacetamide was of the same order as observed in the experiments with tRNA. The data obtained on the covalent interaction of DNA with enzymatically activated N-hydroxy-2-fluorenylacetamide indicate that, in addition to NSF, another electrophilic species accounts for binding of activated N-hydroxy-2-fluorenylacetamide to DNA. The data support the view that the reactive electrophile is N-acetoxy-2-fluorenamine, resulting from the N,O-transacetylation of N-hydroxy-2-fluorenylacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH.

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using alpha-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.

Oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide by enzymatically or chemically generated oxidants of chloride and bromide.

The oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA) via one-electron (1e-) oxidation to equimolar 2-nitrosofluorene (2-NOF) and N-acetoxy-2-FAA and via oxidative cleavage to 2-NOF by chemically and myeloperoxidase (MPO)/H2O2 generated oxidants of Cl- and/or Br- were investigated. 2-NOF was determined spectrophotometrically in the reaction mixtures and by HPLC of their extracts; N-acetoxy-2-FAA was determined by HPLC. In the presence of individual or mixed halides at their physiologic concentrations (0.1 M Cl- and/or 0.1 mM Br-) and pH 4-6, MPO/H2O2-catalyzed oxidation of N-OH-2-FAA to 2-NOF via oxidative cleavage was much greater than 1e- oxidation. At the respective pH optima, oxidation was much more rapid with Br- and Br- + Cl- than with Cl-. HOBr or HOCl + Br- oxidized N-OH-2-FAA more rapidly than HOCl, also chiefly via oxidative cleavage. This suggested that, in the presence of MPO/H2O2 + Cl- + Br-, oxidation was due to HOBr from HOCl oxidation of Br- and/or oxidation of Br- by MPO/H2O2. In the presence of taurine (1 or 10 mM), a scavenger of hypohalous acids, MPO/H2O2 catalysis of oxidative cleavage was unaffected with Br-, prevented with Cl-, and partially prevented with Cl- + Br-. These results were linked to N-halotaurine formation since it was found that N-bromotaurine, but not N-chlorotaurine, oxidized N-OH-2-FAA chiefly to 2-NOF. With time N-chlorotaurine and N-bromotaurine appeared to undergo a pH-dependent halide exchange with Br- and Cl-, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

N-glycolylhydroxamic acids: an improved synthetic method and the in situ generation and intramolecular rearrangement of N-acetoxy-N-glycolyl-2-aminofluorene.

A new and improved method for the synthesis of glycolylhydroxamic acids is described. The two-step method involves acylation of arylhydroxylamines with acetoxyacetyl chloride, followed by saponification of the ester bond to give the desired products. The conversion of hydroxamic acids to their thallous salts followed by subsequent acetylation with acetyl chloride to give N-acyloxy esters is described. During the course of this investigation, it was observed that the N-acetoxy ester of an N-glycolylhydroxamic acid was highly unstable and underwent a novel O----O acyl migration. This facile rearrangement reaction was studied for the case of N-acetoxy-N-glycolyl-2-aminofluorene (N-OAc-GAF), which gave N-hydroxy-N-(acetoxyacetyl)-2-aminofluorene (N-OH-AcAAF) as the sole product of this rearrangement. HPLC was used to investigate this reaction and included the assignment of an HPLC peak to be due to N-OAc-GAF. A competition study employed the amide N-glycolyl-2-aminofluorene (GAF) and demonstrated the absence of intermolecular transfer of the N,O-acyl (acetyl) group of N-OAc-GAF. The mechanism for the probable intramolecular rearrangement reaction is presented, along with a consideration of the possible significance it might have for the toxicity of glycolylhydroxamic acids.