BaP-DNA Adduct Formation Research Articles

Effect of dietary restriction on partial hepatectomy-induced liver regeneration of aged F344 rats.

Fourteen weeks-old male F344 rats maintained on a reduced caloric diet (60% of ad libitum (AL) food consumption) for 6 weeks or for 14 months did not affect the hepatic cell proliferation in terms of % S phase population, determined by evaluation of DNA synthesis in hepatocytes isolated from either young (5 months) or aged (18 months) rats. However, hepatic basal cellular DNA synthesis estimated by [3H]thymidine incorporation was reduced through acute dietary restriction (DR) in young rats, but increased in aged animals after 14 months restriction. Partial hepatectomy (PH) on aged rats stimulated hepatocyte regeneration and restored some aging-associated biochemical functions, such as drug metabolizing enzyme-dependent xenobiotic metabolic activation which was determined by measuring the formation of carcinogen-DNA adducts. Forty-eight hours after partial hepatectomy, the % of S phase population and the basal nuclear DNA synthesis of hepatocytes isolated from the partial hepatectomized DR-rats were 4- and 2.8-fold, respectively, greater than those of hepatocytes from AL-animals. DR reduced aflatoxin B1 (AFB1) metabolizing enzyme activity and decreased the AFB1-DNA adduct formation in young rats treated with AFB1. In aged AL-rats, the formation of AFB1-DNA adducts diminished to the same level as that of DR-groups and probably was due to the faster decline of drug metabolizing enzymes in aging AL-rats. However, 48 h after PH, the metabolic activation of AFB1 was restored in AL- and DR-groups which resulted in the increase of AFB1-DNA binding by 4.2 and 1.9-fold, respectively. During the liver regeneration of old PH-rats, DR inhibited the AFB1-DNA adduct formation after the PH-rats received a single dose of AFB1. DR increased benzo[a]pyrene (BaP) metabolic activation in both young and aged rats. Aging also decreased BaP-DNA adduct formation in both DR and AL-rats. The increase of BaP-DNA adduct formation in PH-groups was attributed to the restoration of BaP-metabolizing enzyme activity during liver regeneration. The PH-stimulated BaP-DNA adduct formation in AL- and DR-rats was 3.4- and 2.0-fold greater than control aged rats. Our results indicated that the stimulation of PH-induced liver regeneration by DR in aged animals may be attributed to the retardation of aging by DR and the retention of more active biochemical and enzymological functions in old DR-animals.

Effect of Caloric Restriction on the Metabolism of Benzo[a]pyrene (BaP) and the Formation of BaP-DNA Adducts in Male Fischer 344 Rats

Abstract The effect of caloric restriction (CR) on activities of xenobiotic metabolizing enzymes results in alterations in the metabolic activation of chemical carcinogens, with a resultant impact on DNA-carcinogen adduct formation. Using benzo[a]pyrene (BaP) as a model carcinogen, we have studied the effect of CR on the metabolic activation of BaP, in terms of both BaP metabolism and BaP-DNA adduct formation. Male Fischer 344 rats fed CR diets (60% of the food consumption of ad libitum−fed rats) showed higher activities of BaP metabolizing enzymes resulting in increases of BaP metabolism in vitro and BaP-DNA adduct formation in vivo. The results of the study of the effect of CR on the in vitro metabolism of BaP showed that CR increased the total BaP metabolism, as well as BaP-t-9, 10-diol and BaP-t-4, 5-diol formation. However, BaP-t-7, 8-diol, a proximate carcinogenic metabolite of BaP, was decreased by liver microsomes from CR-rats. Our results indicate that the effect of CR on metabolic activation of ...

Relationship between benzo(a)pyrene-DNA adducts and somatic mutation and recombination in Drosophila melanogaster.

The evaluation of the relationship between the dose to DNA of a mutagen/carcinogen and in vivo somatic cell mutagenesis may provide information on the mechanisms leading to induced mutational events. This can be achieved, for example, by coupling test systems that permit the detection of somatic mutation and recombination on the basis of phenotypic changes in cuticular structures of Drosophila melanogaster, with methods for the quantitation of carcinogen-DNA adducts such as the 32P-postlabeling technique. In this article, we evaluate the quantitative relationship between BaP-DNA adduct formation, determined by 32P-postlabeling, and the induction of mutant cells in the wing marker version of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. The total single clones in the trans-heterozygous mwh/flr3 flies show a linear relationship with the BaP-DNA adduct levels, suggesting a single hit mechanism for the genetic damage giving rise to this type of clones. In contrast, the twin clones (which are of recombinational origin) display a linear-quadratic relationship with the adduct levels, suggesting that multiple hits may be involved in generating these clones. The total single clones in the mwh/TM3, Ser flies (in which mitotic recombination is suppressed) show a logarithmic relationship with the adduct levels. The discussion of these data in terms of the pathways that may be involved in the repair of the BaP-DNA adducts leads to the suggestion that in Drosophila melanogaster the repair of Bap metabolite-DNA adducts in somatic cells may proceed, in large part, via post-replicative recombinational repair.

Ellagic Acid Inhibits DNA Binding of Benzo(a)pyrene Activated by Different Modes.

The possible mechanisms of inhibition of benzo(a)pyrene (BaP) binding to DNA in vivo and in vitro by ellagic acid (EA) were investigated. In the in vitro studies BaP was activated by cytochrome P-450-dependent mixed function oxidase system, ascorbate-dependent lipid peroxidation, and peroxidation of linoleic acid. Reactions were performed in the dark at 37°C for 20 to 30min in buffered aqueous solutions with 2mg DNA and 40nmol tritiated BaP. The levels of BaP-DNA adducts were determined by assay of the radioactivity. BaP-DNA adduct levels ranged from 146 to 170fmol/mg DNA in ascorbate-and NADPH-dependent reactions and the level was 8.25±0.425pmol/mg DNA in the hematin-mediated linoleic acid reaction. Addition of EA to the reaction mixtures resulted in a significant inhibition in BaP-DNA adduct formation, depending upon the concentration of EA; e.g., 500μM EA resulted in 42 to 53% inhibition of binding in the three systems of carcinogen activation. Similarly, EA feeding to male NMRI mice at a concentration of 12μg/ml drinking water significantly decreased the levels of carcinogen-DNA adducts in the lungs. Reactions performed in vitro, in which DNA was preincubated overnight at 4°C with EA, revealed a decrease of 20% in adduct formation. MDA formation during the process of lipid peroxidation, stimulated by NADPH and ascorbate in the liver microsomes of mouse and by hematin in linoleic acid, was taken as an index for the free radical reactions. Addition of EA to in vitro systems inhibited the MDA formation with an IC50 (concentration for 50% inhibition) of 480, 400, and 400μM for NADPH, ascorbate, and hematin-mediated free radical reactions, respectively.

In vitro inhibition of DNA adduct formation by hindered hydroquinols and quinones.

A series of thirty-one chemicals (substituted phenols, hydroquinols, quinones and quinoneimines) were investigated as in vitro inhibitors of DNA-benzo[a]pyrene adducts formation. In order to establish the mechanisms by which such compounds may elicit a protective effect at DNA level, the influence of these compounds upon benzo[a]pyrene (BaP) activation by 3-methyl-cholanthrene-stimulated microsomes was also studied. The data obtained suggest that the tested compounds act mainly (but not only) by BaP metabolism inhibition. Several substituted hydroquinols (e.g. 2-methyl-6-tert-butyl-hydroquinol, 2,6-di-isopropyl-hydroquinol, 2,3-dimethyl-hydroquinol) and quinones (e.g. 2-methyl-6-tert-butyl-1,4-benzoquinone, 2,6-di-isopropyl-1,4-benzoquinone, 2,3-di-methyl-1,4-benzoquinone) were found to be powerful inhibitors of both BaP metabolism and of BaP-DNA adduct formation (> 95%) at a BaP:inhibitor ratio of 1:10. This effect is significantly stronger than that exerted by the 'classical' BHA or other phenolic antioxidants in similar experimental conditions. Generally, quinones were more active than their corresponding hydroquinols, suggesting that the effectiveness of hydroquinols to prevent DNA adduct formation may be due to their oxidation to quinonic forms.

Inhibition of microsome-mediated binding of benzo[ a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [ 3 H]benzo[a] pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72–89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5–1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzoflavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.

Preparation of microgram quantities of BaP-DNA adducts using isolated rat hepatocytes in vitro

The analysis of carcinogen-DNA adducts generally requires the preparation (by chemical or biological means) of DNA adduct standards, in amounts sufficient for chemical characterization. We have established conditions for the in vitro biological preparation of microgram quantities of DNA adducts derived from benzo[a]pyrene (BaP), fluoranthene and 7,12-dimethylbenzanthracene, using isolated rat hepatocytes. The metabolic activation of 180 microM BaP by isolated rat hepatocytes in a calf-thymus-DNA (CT-DNA)-supplemented medium resulted in the formation of 2.9 micrograms of BaP adducted to 56.7 mg of DNA. The average level of binding in this experiment was 148 +/- 8 pmol BaP bound/1 mg DNA, which compares favorably to the 10-30 pmol BAP/1 mg DNA which is typical of mouse skin adducts in vivo. In another experiment, BaP-DNA adduct formation in calf-thymus DNA added to hepatocyte incubations was further increased to 327 +/- 27 pmol/mg DNA, by physical shearing of the DNA prior to the incubation. The HPLC profile of the BaP adducts produced using hepatocytes plus CT-DNA is virtually indistinguishable from that produced by tumor-initiating doses of BaP applied to mouse skin in vivo, and the major DNA adduct formed by the hepatocytes co-elutes with the (+)-anti-diol-epoxide adduct of deoxyguanosine. Similar experiments using fluoranthene and 7,12-dimethylbenzanthracene also resulted in substantial DNA adduct formation; however, incubations using dibenz[a,h]anthracene did not. These results indicate that isolated rat hepatocytes in vitro can be useful for the preparation of DNA adducts of a number of polycyclic aromatic hydrocarbons, in quantities sufficient for chemical characterization.

Effect of sulfite on the covalent reaction of benzo(a)pyrene metabolites with DNA

Sulfur dioxide (SO2) potentiates the carcinogenicity of polycyclic aromatic hydrocarbons. To investigate the mechanism of SO2 cocarcinogenesis, the effect of sulfite, the hydrated form of SO2, on the covalent reaction of benzo[a]pyrene (BaP) metabolites with DNA in vitro was measured. [14C]BaP was incubated with rat lung or liver post-mitochondrial supernatant (S9), an NADPH generating system, calf thymus DNA and sodium sulfite (0-20 mM). In the presence of lung S9, covalent reaction increased linearly from 0.66 to 1.20 pmol BaP metabolites per mg DNA with increasing sulfite concentrations. Addition of sulfite to rat liver S9 also increased BaP-DNA adduct formation with BaP-DNA adducts increasing from 80 to 120 pmol per mg DNA. Sulfite altered the amount and pattern of BaP metabolites formed by either lung or liver enzyme preparations. BaP was metabolized more extensively and the amount of water soluble BaP metabolites formed increased significantly with sulfite present. With lung S9, the amount of BaP-tetrols, diols, and phenols increased slightly. With liver S9, diol and phenol formation was significantly lower while tetrol formation was unchanged. Incubation of rat lung S9 with sulfite resulted in formation of glutathione S-sulfonate (GSSO3H), a known inhibitor of glutathione S-transferases mediating the conjugation of glutathione (GSH) and BaP epoxides. Our results suggest that sulfite may, by altering the overall metabolic activation and detoxication of BaP, or by reacting directly with DNA, subsequently affect the covalent reaction of BaP metabolites with DNA. These are offered as possible mechanisms to explain the cocarcinogenic effect of SO2.