Microsome-mediated Binding Research Articles

Flavonoids of Honey and Propolis: Characterization and Effects on Hepatic Drug-Metabolizing Enzymes and Benzo[a]pyrene−DNA Binding in Rats

The influence of dietary sunflower honey, propolis, and a flavonoid extract of propolis was examined on drug-metabolizing enzyme activities in rat liver and on microsome-mediated binding of benzo[a]pyrene to DNA. Characterization of flavonoids present in sunflower honey and propolis was achieved in order to assess the relative effects of different components of honey and propolis. Honey and propolis contained the same major flavonoids, pinocembrin, chrysin, galangin, and pinobanksin. The concentration of flavonoids was higher in propolis. Sunflower honey produced no significant changes on phase I and phase II enzyme activities and no modification of in vitro binding of benzo[a]pyrene to DNA. Propolis treatment produced an increase of ethoxyresorufin deethylase, pentoxyresorufin depentylase, ethoxycoumarin deethylase, glutathione transferase, and epoxide hydrolase activities. A flavonoid extract from propolis slightly enhanced only few enzyme activities, ethoxycoumarin deethylase and epoxide hydrolase. The...

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl by cultured human breast epithelial cell line MCF 10A.

Metabolism and nucleic acid binding of the mammary gland carcinogen N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) was investigated using the human mammary epithelial cell line MCF 10A. Chromatographic analysis of the ethyl acetate extract of the media from cultured MCF 10A after 24 h exposure to N-OH-AABP revealed the formation of two metabolites, 4-aminobiphenyl (ABP) and 4-acetylaminobiphenyl (AABP). Incubation of [3H]N-OH-AABP with calf thymus DNA in the presence of the cytosols or microsomes revealed a binding of 0.21 and 2.36 nmol/mg DNA/mg protein respectively. In contrast to cytosol-mediated binding, the microsome-mediated binding of [3H]N-OH-AABP to DNA was inhibited by paraoxon. Furthermore, exogenous addition of non-labelled N-hydroxy-4-aminobiphenyl (N-OH-ABP) to the incubation mixture blocked the binding of [3H]N-OH-AABP to DNA, suggesting that the metabolic activation process involves inter-molecular transacetylation. Cytosols from MCF 10A also catalyzed acetyl coenzyme A (AcCoA)-dependent binding of [3H]N-OH-ABP to DNA; the amount of binding was 0.51 nmol/mg DNA/mg protein. HPLC of the DNA hydrolysate obtained after incubation of [3H]N-OH-AABP and [3H]N-OH-ABP with the MCF 10A microsomes and cytosols showed N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-ABP) as the primary adduct, based on the mobility of the radioactive peak in comparison with the synthetic standard. 32P-postlabeling of adducted DNA obtained on incubation with N-OH-ABP or N-OH-AABP showed similar adduct profiles, with the major adduct corresponding with the bisphospho derivative of dG-ABP and a minor adduct corresponding with N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-ABP). Additionally, the cellular DNA isolated from MCF 10A following exposure to N-OH-AABP also revealed a major spot corresponding with the dG-ABP derivative. These results suggest that the mammary gland carcinogen N-OH-AABP is activated to reactive electrophilic species in the target human mammary tissues by acetyl transferase(s) enzyme systems.

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.

Microsome-mediated transacetylation and binding of N-hydroxy-4-aminobiphenyl to nucleic acids by hepatic and bladder tissues from dog.

Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes were investigated. HPLC analysis of the ethyl acetate extracts of hepatic microsomal incubates of [3H]ABP in the presence of 4-acetylaminobiphenyl (AABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), or acetyl coenzyme A (AcCoA) as acetyl donors showed the formation of [3H]AABP, suggesting that microsomes catalyze N-acetylation of ABP involving transacetylation. Dog hepatic microsomes also catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of AABP, N-OH-AABP or AcCoA, and the binding was blocked by paraoxon, an inhibitor of microsomal deacetylases. Binding of [3H]N-OH-ABP to DNA was catalyzed also by dog hepatic microsomes, and the extent of binding was 266, 156 and 135 pmol/mg DNA for AABP, N-OH-AABP and AcCoA as acetyl donors respectively. HPLC analyses of the DNA hydrolysates showed that the major adduct formed was N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the adduct in comparison with the synthetic standard. The acetyl adduct N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl was not detected in the DNA hydrolysates. Adduct profiles obtained from 32P-postlabeling of DNA samples from the microsome-mediated binding of [3H]N-OH-ABP showed similarities to the profile obtained previously from the chemical interaction of N-OH-ABP with DNA under acidic conditions, suggesting that the microsome-mediated binding of N-OH-ABP may proceed via formation of aryl nitrenium ions as the ultimate electrophilic species. Microsomes from dog bladder also catalyzed the binding of [3H]N-OH-ABP to RNA and DNA in the presence of AABP, N-OH-AABP or AcCoA as acetyl donors, though the levels of binding were less than those observed with hepatic microsomes. The prevalence of these acetyl transferases in the target organs for ABP and AABP carcinogenesis raises the possibility that metabolic activation of the proximate metabolite N-OH-ABP could occur directly in these tissues and these reactions could play a critical role in the initiation of cancers.

Comparison of acyltransferase-mediated mutagenicity and nucleic acid binding of N-acetoxy-4-acetylaminobiphenyl by hepatic and bladder microsomes from rats and dogs.

Acyltransferase-mediated mutagenic and metabolic activation of N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) by hepatic tissues of rats and dogs were compared. N-OAc-AABP was mutagenic in Salmonella typhimurium TA98 even in the absence of exogenous enzyme(s). However, supplementation with hepatic microsomes from dogs showed a dose-dependent increase in mutagenicity of N-OAc-AABP, whereas under the same conditions, rat microsomes were inactive. Incubation of liver microsomes with RNA showed that 46.4 and 11.2 nmole of [3H]N-OAc-AABP were bound/mg RNA/mg protein with dogs and rats, respectively. The hepatic microsome-mediated binding and mutagenicities of N-OAc-AABP were blocked by paraoxon, suggesting the involvement of deacetylase(s) in the activation process. Analyses of the in vitro incubates of N-OAc-AABP with rat and dog liver microsomes revealed the O-deacetylation product N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) as the major metabolite. The ratios of O-deacetylation of N-O[14C]Ac-AABP versus N-deacetylation of N-OAc-[14C]AABP for hepatic microsomes from dogs and rats were 2.9 and 7.2, respectively. The O- and N-deacetylases are also distributed in bladder tissues and their activities in comparison to the hepatic tissues were lower and amounted to 14.2 and 5.0 nmoles (O/N-deacetylation ratio 2.8) for dogs and 14.8 and 1.7 nmoles per mg protein per min (O/N-ratio of 8.7) for rats. The microsomes from bladder tissues also catalyzed the binding of [3H]N-OAc-AABP to RNA and enhanced its mutagenic response in TA98, both of which were blocked by paraoxon. The occurrence of deacetylase(s) in the target tissues of the bladder carcinogen 4-acetylaminobiphenyl (AABP) suggests that metabolic activation of some of the proximate metabolites could occur within these target organs. Furthermore, since the O-deacetylation product N-OH-AABP is relatively innocuous compared to the N-deacetylation product N-acetoxy-4-aminobiphenyl, these results imply that the refractiveness of rats for 4-aminobiphenyl or AABP-induced bladder carcinogenesis might in part be associated with the higher ratios of microsomal O/N-deacetylase activities. Thus susceptibility to arylamine or arylacetamide-induced liver and bladder carcinogenesis might be influenced by the microsomal deacetylases.

Mechanisms of anti-carcinogenesis by indole-3-carbinol: Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B 1 activation

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B 1 (AFB 1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500–2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM 2 and LM 4b as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HC1) to act as scavengers for the direct alkylating agent AFB 1-8,9-Cl 2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB 1-8,9-Cl 2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB 1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB 1. RXM increased the apparent K m for the AFB 1-DNA binding reaction without changing the associated V max; the apparent K m values at 0, 3.5, 35, and 350 μM RXM were 35, 38, 66, and 86 μM for trout liver microsomes. RXM also inhibited the activation of AFB 1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB 1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB 1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by β-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Stimulation of binding of benzo[ a]pyrene metabolites to DNA by diet-induced peroxidative stress

To investigate the influence of unsaturation of dietary fat on the oxidation of benzo[ a]pyrene-7,8-dihydrodiol to DNA binding products, we fed diets containing 10% by weight of either safflower oil or lard to weanling rats. Compared with the group fed lard, the group fed safflower oil had 2.0- to 2.5-fold higher levels of unstimulated and peroxidation-stimulated activation of benzo[ a]pyrene-7,8-dihydrodiol to DNA-binding metabolites, respectively, in hepatic nuclei. The rats fed safflower oil had a significant 75% higher level of lipid peroxidation as measured by the thiobarbituric acid assay. Rats fed safflower oil also showed 30% greater binding of (−)-benzo[ a]pyrene-7,8-dihydrodiol oxidation products to DNA compared with animals fed lard, following administration of this dihydrodiol enantiomer through the hepatic portal vein. Significant diet-dependent differences were not apparent in DNA binding of the (+)-isomer, or in the tetrol production from either isomer; however, rats fed safflower oil showed a trend towards production of higher levels of anti-benzo[ a]pyrene diol epoxide-derived tetrols. Activities of hepatic nuclear and microsomal aryl hydrocarbon hydroxylase and of cytosolic and microsomal glutathione S-transferases were not significantly affected by diet, nor was the activity of microsome-mediated binding of (+)- or (−)-benzo[ a]pyrene-7,8-dihydrodiol to DNA in vitro. The results indicate that polyunsaturated fat in quantities as low as 10% by weight of the diet is sufficient to increase significantly the extent to which DNA-binding metabolites of benzo[ a]pyrene are produced, and that this increased metabolism is likely to be independent of mixed-function oxidases.

The Different Genotoxicity of P-Dichlorobenzene in Mouse and Rat: Measurement of the in Vivo and in Vitro Covalent Interaction with Nucleic Acids

Twenty-two hours after i.p. injection to male Wistar rats and BALB/c mice para-dichlorobenzene (p-DCB) is bound covalently to DNA from liver, kidney, lung and stomach of mice but not of rats. DNA adducts in mouse liver are repaired in seventy-two hours. The covalent binding index value, calculated on the labelling of mouse liver DNA, classifies p-DCB as a weak initiator with an oncogenic activity lower than that of chlorobenzene. The labelling of RNA and proteins from the different organs of both species is, however, low. In vitro interaction with calf thymus DNA mediated by mouse and rat microsomes from liver and lung did occur. Binding extent was strongly reduced by addition of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH. Cytosolic fractions from kidney and lung were able to induce binding of p-DCB to DNA to a lower extent with respect to microsome-mediated binding. These results indicate that microsomal mixed function oxidase system and microsomal GSH-transferases can be involved in overall activating metabolism whereas cytosolic GSH-transferases play a minor role. This study, which is a part of a structure-activity relationship approach on benzene and its haloderivatives, provides the first evidence of genotoxicity of p-DCB in mammalian cell. It allows to partly explain variations of susceptibility of different species to hepatocarcinogenesis and of hepatotoxicity of different isomers.

Altered hepatic microsomal function and elevated protooncogene expression as residual effects in rats exposed to delta-9-tetrahydrocannabinol

The microsomal activation of the potent hepatocarcinogen aflatoxin B 1 (AFB 1) and the expression of selected protooncogenes were investigated in the livers of rats exposed to Δ 9-tetrahydrocannabinol (THC). At equimolar levels of cytochrome P-450, the microsome-mediated binding of AFB 1 to DNA was significantly lower (56% of the controls) in preparations from drug exposed rats. Hepatic expression of the c-k-ras protooncogene was 3-fold higher in THC exposed animals. These results suggest the possible occurrence of long lasting residual effects in the rats exposed to THC.

Comparative metabolism of (—)benzo[a]pyrene-7,8-dihydrodiol by hepatocytes isolated from two species of bottom-dwelling fish

To investigate the influence of unsaturation of dietary fat on the oxidation of benzo[a]pyrene-7,8-dihydrodiol to DNA binding products, we fed diets containing 10% by weight of either safflower oil or lard to weanling rats. Compared with the group fed lard, the group fed safflower oil had 2.0- to 2.5-fold higher levels of unstimulated and peroxidation-stimulated activation of benzo[a]pyrene-7,8-dihydrodiol to DNA-binding metabolites, respectively, in hepatic nuclei. The rats fed safflower oil had a significant 75% higher level of lipid peroxidation as measured by the thiobarbituric acid assay. Rats fed safflower oil also showed 30% greater binding of (−)-benzo[a]pyrene-7,8-dihydrodiol oxidation products to DNA compared with animals fed lard, following administration of this dihydrodiol enantiomer through the hepatic portal vein. Significant diet-dependent differences were not apparent in DNA binding of the (+)-isomer, or in the tetrol production from either isomer; however, rats fed safflower oil showed a trend towards production of higher levels of anti-benzo[a]pyrene diol epoxide-derived tetrols. Activities of hepatic nuclear and microsomal aryl hydrocarbon hydroxylase and of cytosolic and microsomal glutathione S-transferases were not significantly affected by diet, nor was the activity of microsome-mediated binding of (+)- or (−)-benzo[a]pyrene-7,8-dihydrodiol to DNA in vitro. The results indicate that polyunsaturated fat in quantities as low as 10% by weight of the diet is sufficient to increase significantly the extent to which DNA-binding metabolites of benzo[a]pyrene are produced, and that this increased metabolism is likely to be independent of mixed-function oxidases.

Glutathione plus cytosol- and microsome-mediated binding of 1,2-dichloroethane to polynucleotides

1,2-[1,2- 14C]Dichloroethane was metabolized by rat hepatic microsomes to products that irreversibly bound polynucleotides. The polynucleotides were then enzymatically hydrolyzed and the products separated by a high-performance liquid chromatograph (HPLC) equipped with an ODS or a SCX column. The products of microsome-mediated binding were identified in the HPLC eluate as 1, N 6 -ethenoadenosine to polyadenylic acid, 3, N 4 -ethenocytidine to polycytidylic acid, and two cyclic derivatives to polyguanylic acid. 1,2-[1,2- 14C]Dichloroethane was also metabolized in the presence of a glutathione (GSH)-cytosolic fraction and a polynucleotide. After enzymatic hydrolysis of the polynucleotide, the major peak of radioactivity was eluted from a Sephadex G-25 column in the salt volume which would exclude the presence of a product containing both GSH and a nucleoside. Chromatography by ODS-HPLC of the major peak from Sephadex G-25 indicated the presence of a GSH metabolite of 1,2-dichloroethane that did not contain a nucleoside. A similar hydrophilic peak was obtained for the hydrolysis products of polynucleotides from a glutathione plus cytosol incubation in which the polynucleotide instead of being added prior to the incubation was added after the incubation. The products of the glutathione plus cytosol metabolism of 1,2-[1,2- 14C]dichloroethane appear to be glutathione metabolites that coisolated with the polynucleotides rather than covalently bound adducts. In conclusion, covalently bound adducts were identified for microsomemediated binding of 1,2-dichloroethane to polynucleotides, while no evidence was obtained for glutathione plus cytosol-mediated covalent binding to polynucleotides.

Inhibition of microsome-mediated binding of aflatoxin B 1 to DNA by glutathione S-transferase

Hamster liver microsome-mediated [ 14C]aflatoxin B 1 binding to DNA is inhibited by 105,000 × g liver supernatant fraction. Absolute requirement of glutathione for the inhibitory activity of the dialyzed supernatant and irreversible loss of the inhibitory activity by the heat-treated supernatant indicate involvement of glutathione S-transferase. Concomitant with inhibition of aflatoxin B 1-DNA binding, the formation of an aflatoxin B 1-glutathione conjugate is indicated.

Effect of UDP-glucuronic acid on the microsome-mediated binding of benzo(a)pyrene metabolites to calf thymus DNA

The binding of benzo(a)pyrene (BP) to DNA in the presence of liver microsomal preparations from 3-methylcholanthrene-treated rats was increased 28.5% (P < 0.001) by addition of UDP-glucuronic acid to the incubation mixture at a 100 microM concentration of BP. However this increase was not observed at lower concentrations of BP. Analysis of DNA-hydrolysates by column chromatography on Sephadex LH-20 indicated that this increase was due to greater binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. The amount of bound adducts derived from 9-hydroxybenzo(a)pyrene (9-OH BP) was not significantly affected by UDP-glucuronic acid. Addition of UDP-glucuronic acid to incubation mixtures containing microsomes from untreated rat liver caused only a slight increase in total binding. These findings suggest that UDP-glucuronyl transferase may be involved in the activation of BP.

Rat liver microsome-mediated binding to DNA of 3-amino-1-methyl-5H-pyrido[4,3-b]indole, a potent mutagen isolated from tryptophan pyrolysate.

Rat liver microsome-mediated binding to DNA of 3-amino-1-methyl-5H-pyrido[4,3-b]indole, a potent mutagen isolated from tryptophan pyrolysate.

The role of 9-hydroxybenzo(a)pyrene in the microsome mediated binding of benzo(a)pyrene to DNA.

A study of the liver microsome-mediated binding to added DNA of the phenol metabolites of benzo(a)pyrene (BP-OH) and of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BP-7,8-diol) suggested that as in the case of BP itself the reaction was catalysed by the enzyme aryl hydrocarbon hydroxylase. The addition of glutathione to the microsomal incubation inhibited the binding of BP and BP-OH more than that of BP-7,8-diol. Analysis by LH20 chromatography of the deoxyribonucleoside products from BP-DNA showed greater inhibition by glutathione of formation of the major product believed to result from further metabolism of BP-OH, than of the product arising by metabolism of BP-7,8-diol. The chromatographic behaviour and fluorescence spectrum of this major product were consistent with its derivation from 9-hydroxybenzo(a)pyrene (BP-9-OH) and furthermore suggested that BP-9-OH-4,5-oxide was the derivative whose reaction with DNA yielded this microsome-mediated BP-DNA product.

(+/-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene is an intermediate in the metabolism and binding to DNA of benzo(a)pyrene.

The addition of borate buffer to the aqueous methanol used to elute hydrocarbon-deoxyribonucleoside derivatives from an LH 20 Sephadex column resulted in the separation of the products of reaction with DNA of the stereoisomers, (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy- and (+/-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrenes, i.e., the syn- and anti-benzo(a)pyrene-diolepoxide, respecitvely. By this technique it was shown that the microsome-mediated binding to DNA of benzo(a)pyrene-7,8-dihydrodiol involved exclusively the anti-benzo(a)pyrene-diolepoxide. The benzo(a)pyrene binding to DNA that resulted on exposure of BHK21/C13 cells to this carcinogen was also shown to result predominantly by reaction of the anti-benzo(a)pyrene-diolepoxide. However, in this case other derivatives, including the syn-benzo(a)pyrene diolepoxide, might also be involved.

Rat liver microsome-mediated binding of benzo(a)pyrene metabolites to DNA.

Individual metabolites of benzo (a) pyrene were isolated from rat liver microsomal incubation of the parent hydrocarbon, and subsequently bound to DNA in separate incubations with microsomes. Of the metabolites examined, by far the greatest binding resulted with 7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (BP-7,8-diol); the binding od the benzo (a) pyrene phenols (BP-OH) was about 50% that of the 7,8-diol. Analysis of the hydrocarbon-deoxyribonucleoside derivatives obtained by enzymic degradation of these DNA samples revealed that the binding of BP-7,8-diol was accounted for mainly by a single product identical to one of the product identical to one of the products obtained from DNA with bound benzo(a)pyrene. Furthermore, the microsome-induced binding of BP-OH to DNA yielded mainly a single product identical in chromatographic behaviour to the major product derived from benzo(a)pyrene when bound to DNA during incubation with microsomes.