Michael D. Corbett

The uptake and in vivo metabolism of the organophosphate insecticide fenitrothion by the blue crab, Callinectes sapidus☆

Callinectes were exposed to [14C]fenitrothion at a level of 5.2 micrograms/liter in either 22 degrees C, 34 ppt; 22 degrees C, 17 ppt; or 17 degrees C, 34 ppt seawater. Uptake from the water, as measured by a decrease in fenitrothion, and the distribution of radioactivity throughout the Callinectes were determined. The nature of radiolabeled metabolites in the water and hepatopancreas was also determined. Fenitrothion was absorbed more rapidly from the water at the higher salinity and temperature. Radioactivity was detected in all the organs assayed by 24 hr postexposure, though the levels increased in most tissues throughout the experiment. The highest concentrations of radioactivity were found in the hepatopancreas and stomach. The metabolites which were detected in the water and liver indicate that Callinectes metabolize fenitrothion by oxidation of the phosphorothioate to a phosphate to yield fenitrooxon. The presence of aminofenitrothion and 3-methyl-4-aminophenol shows that reduction of the nitro group to an amino group also occurs. The isolation of desmethyl forms of fenitrooxon and fenitrothion as well as 3-methyl-4-nitrophenol indicates that hydrolysis of both the P-O-aryl and P-O-alkyl bonds occurred. Glycoside and sulfate conjugates of both phenols were inferred in the hepatopancreas. Higher levels of fenitrooxon and lower levels of desmethyl fenitrothion were detected in the 34 ppt seawater than in the 17 ppt seawater. The 5 degrees C differential had no significant effect on the nature and concentrations of metabolites detected in the seawater.

Reduction potentials in relation to physiological activities of benzenoid and heterocyclic nitroso compounds: Comparison with the nitro precursors

Abstract Reduction potentials were determine for various physiologically active benzenoid and heterocyclic nitroso compounds, namely, substituted nitrosobenzenes, 1-nitrosopyrene, and 1-methyl-2-nitrosoimidazole. The values, favorable for biological activity, ranging from 0.2 to −0.2 V, increased in acidic medium. These potentials were appreciably higher than those for the corresponding nitro parents. In most cases, the nitroso form was more biologically active than the nitro counterpart. Catalytic electron transfer processes may play a role in vivo , along with other actions, in the observed responses from the nitroso category.

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.

The effects of temperature, salinity and a simulated tidal cycle on the toxicity of fenitrothion to Callinectes sapidus

The 96 hr LC50 for Callinectes sapidus exposed to fenitrothion at 22 degrees C and a salinity of 34 ppt (parts per thousand) was estimated to be 8.6 micrograms/l with a 95% confidence interval of 7.4-9.9 micrograms/l. Acute toxicity was shown to increase with increasing temperature as well as increasing salinity. Exposure of Callinectes to a simulated tidal cycle of 17 ppt salinity change at 6 hr intervals increased the acute toxicity of fenitrothion to Callinectes. The autotomization response in Callinectes was shown to be affected at subacute exposure levels as low as 0.1 microgram/l.

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.

The effects of salinity and temperature on the in vitro metabolism of the organophosphorus insecticide fenitrothion by the blue crab, Callinectes sapidus

Abstract The in vitro metabolism of fenitrothion [ O,O -dimethyl- O -(3-methyl-4-nitrophenyl)phosphorothioate] by subcellular fractions prepared from the hepatopancrease of blue crabs, Callinectes sapidus , which had been acclimated to either 22°C, 34‰ (parts per thousand); 22°C, 17‰; or 17°C, 34‰ seawater was investigated. In the microsomal fraction, fenitrothion was metabolized to fenitrooxon and 3-methyl-4-nitrophenol. Fenitrothion was metabolized to desmethyl fenitrothion in the cytosolic fraction. The rates of formation of the detoxification products, 3-methyl-4-nitrophenol and desmethyl fenitrothion, were greater in subcellular fractions prepared from crabs which had been acclimated to the lower salinity seawater. The rate of formation of the more toxic metabolite fenitrooxon was greater in the microsomal fraction prepared from crabs which had been acclimated to higher salinity water. All three of these metabolites were formed at considerably faster rates in subcellular fractions from crabs acclimated to and incubated at 22 than at 17°C. These results suggest that enzyme activity contributes to the increased in vivo toxicity of fenitrothion to blue crabs at elevated salinities and temperatures. Also, the observed differences in the rate of formation of the oxon have a greater effect on toxicity than differences in the rate of formation of 3-methyl-4-nitrophenol and desmethyl fenitrothion.

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.