Ming W. Chou

Metabolism of the mutagenic environmental pollutant, 6-nitrobenzo[a]pyrene: Metabolic activation via ring oxidation

Abstract Metabolism of 6-nitrobenzo[a]pyrene by rat liver microsomes yielded 1- and 3-hydroxy-6-nitrobenzo[a]pyrene, 6-nitrobenzo[a]pyrene-1,9- and -3,9-hydroquinone and benzo[a]pyrene-3,6-quinone. The monohydroxylated metabolites were more mutagenic than 6-nitrobenzo[a]pyrene in a Salmonella typhimurium / microsome reversion assay. These results indicate that ring hydroxylation is involved in the metabolic activation of this nitro-polycyclic aromatic hydrocarbon.

Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons

Nitrated polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that result from various incomplete combustion processes. We have examined the activity of hepatic microsomal enzymes in rats pretreated with a series of environmentally occurring nitrated PAHs including: 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitrofluoranthene, and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of the compounds increased the cytochrome P-450 content more than 2-fold. 1,8-Dinitropyrene, 6-nitrochrysene, and 1- and 3-nitrobenzo[a]pyrene significantly increased arylhydrocarbon hydroxylase activity 2- to 8-fold higher than solvent-treated controls. The induction of 7-ethoxycoumarin O-deethylase activity paralleled that found with arylhydrocarbon hydroxylase. The maximum induction of aminopyrine N-demethylase was only 1.5-fold, and none of the nitrated PAHs caused significant increases in epoxide hydrase or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. The greatest increase was caused by 1-nitrobenzo[a]pyrene followed by 1,3-dinitropyrene, 3-nitrobenzo[a]pyrene and 6-nitrochrysene. These data suggest that nitrated PAHs may potentiate the effects of subsequent exposures to various chemical carcinogens.

Effect of caloric restriction on the metabolic activation of xenobiotics

The effect of caloric restriction (CR) on xenobiotic metabolizing enzyme activities results in alterations in the metabolic activation of chemical carcinogens, with a resultant impact on DNA-carcinogen adduct formation and DNA repair. Using aflatoxin B1 (AFB1) and benzo[a]pyrene (BP) as model carcinogens, we studied the effect of CR on the metabolic activation of these carcinogens and carcinogen-induced DNA damage and repair in terms of AFB1-DNA and BP-DNA adduct formation and removal. Male Fischer 344 rats fed calorie restricted diets (60% of the food consumption for ad libitum-fed rats) showed a reduction in the metabolic activation of AFB1 and decrease in both the in vitro and in vivo AFB1-DNA adduct formation. However, CR increased the activity of BP metabolizing enzymes resulting in an enhancement of BP-DNA adduct formation. Our results indicate that the effect of CR on metabolic activation of xenobiotics is dependent upon the selected xenobiotic metabolizing enzymes whose activities may be significantly altered by CR, and upon the nature of the chemical carcinogens which exert different structure-activity relationships during the process of chemically induced carcinogenesis.

Effects of aging and caloric restriction on the genotoxicity of four carcinogens in the in vitro rat hepatocyte/DNA repair assay

The effects of aging and chronic caloric restriction (CR) on the genotoxicity of four carcinogens, representing four different classes of chemicals, in the in vitro rat hepatocyte/DNA repair assay were investigated. Hepatocyte cultures were isolated from young, middle-aged, and old male Fischer (F344) rats which were maintained on either an ad libitum (AL) or a CR diet (60% of AL). Hepatocyte cultures from old AL rats, treated with 2-acetylaminofluorene (2-AAF), aflatoxin B1 (AFB1), 7,12-dimethylbenz[a]anthracene (DMBA) and dimethylnitrosamine (DMN), exhibited age-related decreases in DNA repair as compared to young AL rats. By contrast, cultures from young CR rats exhibited significant diet-related decreases in DNA repair with 2-AAF, AFB1, DMBA and DMN, when compared to results from young AL diet-fed rats. Old CR F344 rat derived cultures exhibited no significant age-related dose-dependent decrease in the DNA repair response with any of the chemicals tested. However, in cultures from old CR rats 10.0 microM AFB1 produced an age-related decrease in DNA repair from the response observed in young CR rats. When hepatocytes were isolated from Aroclor 1254-induced rats, increases in DNA repair were observed. These data indicate an age- and diet-related decrease in DNA repair and/or DNA damage and suggest that this decrease is due to a decrease in metabolic activation of these carcinogens to genotoxic species.

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.

Genotoxicity of tacrine in primary hepatocytes isolated from B6C3F1 mice and aged ad libitum and calorie restricted Fischer 344 rats.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine; THA), a reversible centrally acting anticholinesterase, has been shown to be potentially useful for treatment of patients with Alzheimers disease. However, currently available forms of THA may be therapeutically limited by the fact that high doses have resulted in liver and kidney damage. To determine if THA is hepatotoxic via a genotoxic mechanism, we evaluated its ability to induce unscheduled DNA synthesis (UDS) in primary cultures of rodent hepatocytes. Positive dose-dependent increases in UDS were observed in hepatocytes derived from male B6C3F1 mice and from young, middle-aged, old, and old Aroclor-induced (ARO) male F344 rats maintained on either an ad libitum (AL) or a caloric restricted (CR) diet (60% of AL) and exposed to 0.05-1000.0 micrograms/ml of THA. Hepatocytes from old AL rats, treated with THA, exhibited significant age-related decreases in DNA repair compared to young and middle-aged AL rats. By contrast, cultures from CR rats exhibited age- and diet-related decreases in UDS from the AL and young CR animals, respectively. Moreover, ARO-induced old AL- and CR-derived hepatocytes exhibited significant increases in UDS compared to uninduced old AL and CR animals. No cytotoxicity was observed in the uninduced old AL- or any CR-derived hepatocytes. These data indicate that the aged and CR fed animal is less susceptible to the cytotoxic and genotoxic effects of THA; while the younger AL fed and enzyme induced old AL or CR fed animals were more susceptible. The data suggest that THA may be a genotoxic rodent carcinogen. At present, the relationship of these findings to the clinical use of THA are unclear and further study is required.

Effect of caloric restriction on the metabolism of 7-bromobenz[a]anthracene and 7-fluorobenz[a]anthracene by male B6C3F1 mouse liver microsomes: Reduction of metabolic activation pathway

The effect of caloric restriction (CR) on the in vitro metabolism of 7-bromobenz[a]anthracene (7-Br-BA) and 7-fluorobenz[a]anthracene (7-F-BA) by liver microsomes isolated from 5-and 12-month-old male B6C3F1 mice was studied. Mice were fed ad libitum (AL), or starting at 14 weeks of age, received 60% of the calories consumed by control mice. After microsomal incubation, metabolites were separated by HPLC and their structures identified by comparison of their spectra with known standards. The metabolites formed were: trans-3,4-dihydrodiol, trans-5,6-dihydrodiol, trans-8,9-dihydrodiol and trans-10,11-dihydrodiol, 5,6-epoxide, and 4-, 5-, 6-, 8-, and 9-phenolic derivatives. The formation of the 7-Br-BA trans-3,4-dihydrodiol and 7-F-BA trans-3,4-dihydrodiol, the proximate mutagens of 7-Br-BA and 7-F-BA, respectively, was lower in microsomal incubation mixtures derived from the 5-month-old CR mice as compared to those from the AL. These results suggest that CR can diminish the genotoxicity of polycyclic aromatic hydrocarbons by directly altering metabolic activation.

Evidence for the metabolic formation of a vicinal dihydrodiol-epoxide from the potent mutagen 1-nitrobenzo(a)pyrene

Metabolism of 1-nitrobenzo(a)pyrene (1-nitro-BaP) by rat liver microsomes yielded 1-nitro-BaP trans-7,8-dihydrodiol, 1-nitro-BaP trans-9,10-dihydrodiol and 1-nitro-BaP 7,8,9,10-tetrahydrotetrol. Formation of these metabolites suggests that a vicinal 7,8,9,10-dihydrodiol-epoxide is a metabolite of 1-nitro-BaP.

Stereoselective metabolism of 8‐and 9‐fluorobenzo[a]pyrene by rat liver microsomes: Absolute configurations of trans‐dihydrodiol metabolites

Rat liver microsomal metabolism of 8-fluorobenzo[a]pyrene (8-fluoro-BaP) generated 3-hydroxy-8-fluoro-BaP, 8-fluoro-BaP trans-4,5-dihydrodiol, and 8-fluoro-BaP, 3,6-quinone as major products. A minor metabolite of 8-fluoro-BaP was tentatively assigned as 8-fluoro-BaP 9,10-dihydrodiol. Metabolism of 9-fluorobenzo[a]pyrene (9-fluoro-BaP) gave 3-hydroxy-9-fluoro-BaP, 9-fluoro-BaP trans-4,5-dihydrodiol, 9-fluoro-BaP trans-7,8-dihydrodiol, and 9-fluoro-BaP 3,6-quinone. All three dihydrodiol metabolites were optically active. Comparison of the circular dichroism spectra of BaP 4R,5R-dihydrodiol, 6-bromo-BaP 7R,8R-dihydrodiol, and 6-fluoro-BaP 7R,8R-dihydrodiol with those of the respective dihydrodiol metabolites allowed assignments of an R,R absolute configuration to the major enantiomers of the three dihydrodiol metabolites.

Effect of caloric restriction on aflatoxin B1-induced DNA synthesis, AFB1-DNA binding and cell proliferation in Fischer 344 rats.

Young adult male Fischer rats maintained on a reduced calorie diet (60% of ad libitum food consumption) for 6 weeks showed a decrease in the binding of aflatoxin B1 (AFB1) to hepatic or renal nuclear DNA and a reduction of AFB1-induced hepatocellular damage. Repeated dosing of rats with AFB1 resulted in the inhibition of hepatic and renal DNA synthesis measured by [3H]thymidine incorporation. However, the rate of DNA synthesis was greater in ad libitum (AL) rats than in calorically restricted (CR) animals. Three days after AFB1 dosing, the rate of DNA synthesis had recovered to the control level. Cell cycle analyses measured by a flow cytometric method on kidney cells of both AL and CR rats showed that there were no significant changes in cell populations in the S phase between these two groups of rats. AFB1 inhibited the cell proliferation on an average of 33%. The restoration of the cell proliferation in kidney cells was found on the third day after AFB1 dosing. The rate of the regenerative cell proliferation was found to be slightly greater in AL rats than in CR animals. The AFB1-induced regenerative DNA synthesis in both liver and kidney was retarded by CR.