Bernadette R. Corbett

The covalent binding of acetaminophen to cellular nucleic acids as the result of the respiratory burst of neutrophils derived from the HL-60 cell line.

After being induced to differentiate into a neutrophilic type, cultures of the leukemic cell line HL-60 were able to cause the bioactivation and nucleic acid binding of acetaminophen upon stimulation of the respiratory burst. This phenomenon was found to simulate the same process as that previously shown with normal human granulocytes. Binding to both DNA and RNA of the cells was determined quantitatively by use of 14C-labeled acetaminophen congeners. Protein binding occurred to about the same extent as did RNA binding. Simultaneous labeling experiments with [ring-14C]- and [14C = O]acetaminophen further showed that the acetaminophen molecule was bound to DNA in an intact manner, while binding to RNA showed about a 50% excess binding of the acetaminophen ring relative to the carbonyl group. Experiments with certain inhibitors showed that catalase and azide ion strongly inhibited DNA binding, while superoxide dismutase had a slight stimulatory effect on binding. These results suggest a significant role for myeloperoxidase in the bioactivation process, which contrasts with the proposed bioactivation mechanism of certain arylamine compounds. A mechanism was proposed for acetaminophen binding to nucleic acids that requires the 1 e- oxidation of this substrate to its phenoxyl radical, although the production of the N-acetyl-p-benzoquinoneimine metabolite, which has been proposed to account for the extensive protein binding known to occur for acetaminophen, might also contribute to such binding. The potential genotoxicity of acetaminophen was considered in view of what might be a unique pathway which can metabolize this chemical to a nucleic acid-binding species.

Hydroxamic acid production and active-site induced Bamberger rearrangement from the action of α-ketoglutarate dehydrogenase on 4-chloronitrosobenzene

The α-ketoglutarate dehydrogenase complex obtained from E. coil has been found to convert 4-chloronitrosobenzene (3) into N-(4-chlorophenyl)succinohydroxamic acid (4) and N-(4-chloro-2-hydroxyphenyl)succinamic acid (5). The conversion of 4-chloronitrosobenzene (3) into these two products is not quantitative and attempts to identify other, significant low-molecular-weight metabolites have been unsuccessful. Partial enzyme-inactivation has been observed during the incubation of 4-chloronitrosobenzene (3) with α-ketoglutarate dehydrogenase. The direct enzymic conversion of the hydroxamic acid (4) into the isomeric product (5) did not occur. These results are interpreted on the basis of a mechanism in which N-(4-chlorophenyl)hydroxylamine (6) is generated at the enzyme active-site by a redox process. Condensation of the active-site bound products would give rise to the hydroxamic acid (4) directly, while a Bamberger-like rearrangement of the active-site bound hydroxylamine (6), followed by condensation of the resulting o-aminophenol, would explain the production of the succinamic acid (5).

A comparison of the HL-60 cell line and human granulocytes to effect the bioactivation of arylamines and related xenobiotics. The binding of 2-aminofluorene to nucleic acids as the result of the respiratory burst.

Studies were made on the ability of the leukemic cell line, HL-60, to substitute for normal human granulocytes in research concerned with the bioactivation of arylamines. The arylamine carcinogen, 2-aminofluorene (2-AF), was used as the model substrate in the form of 2-[9-14C]AF, and was incubated with HL-60 cell cultures, both in the presence and absence of phorbol myristate acetate (PMA) which induces the respiratory burst. The HL-60 cultures were generally employed after having been induced to undergo differentiation to neutrophils by the action of dimethyl sulfoxide (DMSO). Comparisons of the amounts of DNA and RNA binding by 2-AF between HL-60 and normal human granulocyte cultures demonstrated close similarities in the amount and nature of nucleic acid binding by this arylamine substrate. HL-60 cells that had been induced to differentiate to neutrophils to the extent of about 80% showed high levels of the respiratory burst along with extensive covalent binding of 2-[9-14C]AF to cellular nucleic acids. Although normal human granulocytes tended to metabolize 2-AF slightly faster than did highly differentiated HL-60 cells, the extent of nucleic acid binding relative to the amount of 2-AF metabolized was similar. A major difference in the metabolic fate of 2-AF in these cell cultures was the unique ability of HL-60 cultures at all stages of differentiation to effect the slow N-acetylation of 2-AF to give 2-acetylaminofluorene (2-AAF). Extensive analyses of incubation extracts showed that the major differences in apparent metabolites were quantitative. With few exceptions, both activated HL-60 and granulocyte cell cultures produced the same metabolites, most of which remain unidentified. Studies with inhibitors such as catalase, superoxide dismutase and azide ion further suggest that these two related cell cultures metabolize 2-AF in similar manner. The DMSO-differentiated HL-60 culture is proposed as a convenient model with which to investigate the metabolism and bioactivation of arylamines by human granulocytes or pure neutrophils.

Hydroxamic acid production by α-ketoglutarate dehydrogenase. Part 2. Evidence for an electrophilic reaction intermediate at the enzyme active site

The α-ketoglutarate dehydrogenase-catalyzed conversion of 4-chloronitrosobenzene (1) into the hydroxamic acid (3) and the Bamberger-rearrangement product (6) was investigated by use of radio-tracer methods and nucleophilic trapping agents. 14C-Labelled 4-chloronitrosobenzene (1) failed to give any significant incorporation of radiolabel into the protein of the enzyme, or into calf thymus DNA. The production of a third and highly polar metabolite during this reaction was confirmed; however, the structure of this metabolite has not been elucidated. In the presence of high concentrations of halide salts, the product distribution for the enzymic reaction was markedly altered. In the order I– > Br– > Cl–, halides inhibited the production of the rearrangement product (6) and of the unknown polar product. The inhibition of the formation of these products was accompanied by a considerable increase in the amount of hydroxamic acid (3), and by the production of a new metabolite, the structure of which was dependent upon the halide employed in the reaction. In the case of both Br– and Cl–, the new metabolite [(8a) and (8b), respectively] was indicative of the trapping of an enzyme-generated electrophile by halide anion. In the case of I–, the initial trapping of the electrophilic species was followed by a redox process to give 4-chloroaniline (9).

Mutagenicity of the C-nitroso analog of fenitrothion

The chemicals fenitrothion, nitroso fenitrothion, amino fenitrothion and 3-methyl-4-nitrophenol were tested for mutagenicity to Salmonella typhimurium strains TA98 and TA100, both in the presence and absence of rat liver S-9 mix. The strong mutagenicity of nitroso fenitrothion to both strains either in the presence or absence of S-9 mix contrasted with the observation that fenitrothion displayed no mutagenicity in these tester strains. The results suggest that the normal nitroreductases present in TA98 and TA100 cannot metabolize fenitrothion to a mutagenic metabolite. This inability of the tester strains to effect partial nitroreduction results in the failure of this screening system to predict the potential genotoxicity of this pesticide.

HRP-catalyzed bioactivation of carcinogenic hydroxamic acids. The greater reactivity of glycolyl-versus acetyl-derived hydroxamic acids

An analysis of the hydroxamic acid oxidation reaction by H2O2 and horseradish peroxidase (HRP) was made with three pairs of hydroxamic acids. Each pair consisted of the aceto- and glycolhydroxamic acid derivatives from one of three different arylhydroxylamines. The parent arylhydroxylamines were the known carcinogens, N-hydroxy-2-aminofluorene and N-hydroxy-4-aminobiphenyl and the noncarcinogen 4-chlorophenyl-hydroxylamine. All the hydroxamic acids appeared to be converted to products that were expected on the basis of the previously-proposed mechanism of this peroxidative reaction. Each acetohydroxamic acid gave the corresponding nitroso compound and O-acetyl ester of the starting material in approximately equal amounts. The glycolhydroxamic acids gave the corresponding nitroso compound and a relatively unstable product that was proposed, by analogy, to be the O-glycolyl ester of the starting material. A comparison of the initial rates of reaction of each hydroxamic acid pair showed that the glycolhydroxamic acid was much more susceptible to the peroxidation reaction than was the corresponding acetohydroxamic acid. The initial rate of the reaction was also highly dependent upon the nature of the aromatic ring in the order fluorene greater than biphenyl greater than 4-chlorophenyl. The relative degree of HRP-catalyzed covalent binding to DNA of the aceto- and glycolhydroxamic acids in the fluorene series was studied and found to parallel the relative rates of reaction of these substrates in the H2O2/HRP system. It was proposed that glycolhydroxamic acids are likely to be more genotoxic than are acetohydroxamic acids when subjected to peroxidative bioactivation conditions.

Enzymatic generation of N-[4-dimethylamino)phenyl]acetohydroxamic acid by the action of pyruvate decarboxylase on 4-(dimethylamino)nitrosobenzene

Abstract The established ability of pyruvate decarboxylase to catalyze the conversion of nitroso aromatics to hydroxamic acids was utilized to generate the previously unknown hydroxamic acid 2. Although 2 could not be isolated in pure form from enzymatic reactions, evidence for its production is presented in this study. Under the conditions of the enzymatic reaction, 2 undergoes a slower reduction to give the corresponding acetanilide 3, which was isolated and characterized. The isomeric hydroxamic acid 4 was synthesized and its stability compared to that of 2. The much greater reactivity of the hydroxamic acid 2, particularly evidenced by its facile reduction to 3, was explained on the basis of the potential for the formation of the N -acylquinonediimine cation, 9.

Biochemical Studies on the Putative Nitroso Metabolite of Chloramphenicol: A New Model for the Cause of Aplastic Anemia

Chloramphenicol (CAP) is one of the oldest and more potent broad spectrum antibiotics available for the treatment of certain severe infections. CAP is an unusual natural product since it possesses both the nitro functional group and C-Cl bonds, neither of which are particularly common among terrestrial natural products. The necessity of the nitro functional group to the antimicrobial action of CAP has been thoroughly studied (reviewed in ref. 1). Unfortunately, the clinical use of CAP often causes toxic reactions, most notably to the hemopoietic system (2). The most serious of these actions is the rare, but generally fatal condition of aplastic anemia (hypoplastic marrow failure). In 1980 Tunis made the hypothesis that reduced intermediates (nitroso, hydroxylamine) of the nitro group of CAP were responsible for the aplastic anemia associated with CAP (2); however, this hypothesis had actually been proposed by the Weisburgers in 1967 (3). It was the Weisburgers’ hypothesis that prompted us to synthesize CAP-NO, CAP-NHOH and related analogs in 1978 (1), and to investigate the potential toxicity of such putative metabolites to hemopoietic precursor cells (4,5).

Irreversible inhibition of the cytosolic metabolism of N-hydroxy-2-acetylaminofluorene by its glycolyl analog

The glycolyl hydroxamic acid derivative of 2-aminofluorene was found to be a potent inhibitor of its own metabolism and the metabolism of N-hydroxy-2-acetylaminofluorene by rat liver cytosol. The inhibition was irreversible, as well as time and concentration dependent, which indicates a suicide-inhibition type of metabolism. There was a direct correlation between the inhibition of N-hydroxy-2-acetylaminofluorene disappearance and 2-acetylaminofluorene formation. In contrast, both the glycolyl and acetyl hydroxamic acid derivatives were metabolized to a similar extent by enzymes in the microsomal fraction.

The generality of the nitroso-glyoxylate reaction: Conversion ofp-nitronitrosobenzene to the hydroxamic acid

The reaction of glyoxylic acid withp-nitronitrosobenzene (1b) in dilute aqueous solution gave the hydroxamic acid (2b) as the major detectable product. The significance of this observation with respect to the title reaction is discussed.