Mutagenicity Of Benzo[a]pyrene Research Articles

Spices May Prevent Human Cancers

Industrial and domestic use of fossil fuel (coal, gasoline, natural gas), wood burning (for heating, cooking, forest wild fire), and tobacco smoking releases harmful chemicals to human environment. Air pollutants include a mixture of noxious gases such as carbon monoxide, carbon dioxide, nitrous oxide, polycyclic aromatic hydrocarbons (PAHs). PAHs are cancer causing agents are highly toxic and mutagenic/genotoxic. Spices are widely used on a daily basis around the world. Some of the major spices/active principles used in large quantities are turmeric (curcumins), ginger (gingerol, shaogol, zingerone), betel leaf (hydroxy chavicol) and catechu (catechin).With short-term mutagenic assay, these spices extract and their active principles are nonmutagenic. We have tested these compounds against well-known mutagenic/carcinogenic PAHs, benzo(a)pyrene (BaP) and dimethyl(a)benzanthracene (DMBA). Nontoxic doses of extracts of spices/principles, turmeric/curcumins, ginger/gingerol, shaogol, zingerone, betel leaf/hydroxy chavicol and catechu/catechin dose dependently decreased the mutagenicity of BaP and DMBA. The protective activities of these compounds were also confirmed by animal tumor models. Nitrite is a water pollutant that is a precursor of mutagenic/carcinogenic nitrosamines and nitrosoamides. Amines/amides are present in vegetables and cured meat. Human stomach maintains acidic conditions, favors the chemical reaction of nitrite with amines/amides to form mutagenic/carcinogenic nitrosamines/amides. We mimic these conditions and have shown that turmeric and ginger their active principles dose dependently block the formation of nitrosamines/amides. Further the human efficacy of turmeric extract and its principle curcumin was confirmed in cigarette and bidi smokers. Consuming turmeric and curcumin reduced excretion of mutagens in human smokers. Large quantity of active principle are present in these spices, turmeric - curcumin 2%-2.5%, ginger - gingerol, shaogoal, zingerone - 2%-2.5%, betel leaf - hydroxychavicol 5%-10% and catechu - catechu 40%-50%. Human consumption of these spices may play an important role reducing the harmful effects of environmental toxicants, thus reducing cancer and other age related human chronic degenerative diseases.

Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver

It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H 2O 2), sodium azide (NaN 3), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H 2O 2, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN 3 in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST. A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.

Effects of paving asphalt fume exposure on genotoxic and mutagenic activities in the rat lung

Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung. In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied. Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay. To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored. Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo[a]pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029. This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m 3). The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner. In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479±33 mg h/m 3 did not significantly enhance 2-AA mutagenicity with either S. typhimurium YG1024 or YG1029. At a higher total asphalt fume exposure level (1150±63 mg h/m 3), S9 significantly increased the mutagenicity of 2-AA as compared to the control. However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test. These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content.

Evaluation of the mutagenicity and antimutagenicity of forty-two 3-substituted flavones in the Ames test

The mutagenic and antimutagenic activities of forty-two synthetic flavones were assessed by the Ames test. The tested flavones included twenty-three 3-nitroflavones, eighteen 3-aminoflavones and the 3-chloroflavone. The mutagenicity was evaluated with Salmonella typhimurium TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain) with and without metabolic activation (S9 mix). The antimutagenicity of the non mutagenic derivatives was evaluated against 11 known reference mutagens. A total of 39 synthetic flavones were mutagenic. The mutagenic activities ranged from 0.1 rev/nmole (4′-chloro-6-methoxy-3-nitroflavone) to 6240 rev/nmole (4′-methoxy-3,3′-diaminoflavone). Two differences were found between the 3-amino and the 3-nitroflavones: (i) the mutagenicity of the 3-aminoflavones required the presence of the metabolic activation; (ii) the 3-amino derivatives were more mutagenic than their 3-nitro counterparts. Increased mutagenicity, as assessed with strain YG1042, was limited to 17/39 derivatives. The mutagenic activity was induced by the presence of the double bond at the 2,3-position for conjugation of the lone-pair electron with the carbonyl group on the `C' ring. This mutagenicity was modulated by substituents at the 2′-position. Additional mutagenicity was brought by the aminoaromatic and nitroaromatic group reduction by bacterial nitroreductases and by the S9 mix; it was modulated by different substituents on the aromatic rings of the flavones. Three flavones: 3-chloroflavone ( 1C), 4′-hydroxy-3-nitroflavone ( 23N) and 2′,3-diaminoflavone ( 2A) showed antimutagenic properties. Compound 1C was efficient against benzo( a)pyrene (BaP), 2-aminofluorene (2AF), 2-aminoanthracene (2AA), 4-nitroquinoline-1-oxide (4NQO) and 1-methyl-3′-nitro-1-nitrosoguanidine (MNNG). Compound 23N inhibited the mutagenicity of BaP and MNNG. The antimutagenic activity of 2A was limited to MNNG.

Modulation of cytochrome P-450IA1-mediated mutagenicity, DNA binding and metabolism of benzo[ a]pyrene by Chinese medicinal herbs

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. We previously showed that they inhibited mutagenesis, DNA binding and metabolism of benzo[ a]pyrene (BaP) and aflatoxin B 1 (AFB 1) bioactivated by Aroclor 1254-induced rat hepatic S9. The purpose of this study was to investigate the effects of OD and SB on the cytochrome P-450IA1-mediated mutagenicity of BaP in Salmonella typhimurium TA100 using β-naphthoflavone (βNF)-induced rat hepatic S9. We also determined the effects of OD and SB on cytochrome P-450IA1-linked ethoxyresorufin O-deethylase (EROD) activity in βNF-induced hepatic microsomes. In addition, we studied the effects of these two herbs on BaP metabolite binding to calf thymus DNA and using high performance liquid chromatography (HPLC) we investigated the effects of OD and SB on the metabolism of BaP by βNF-induced S9. Our experimental results showed that OD and SB inhibited the mutagenicity of BaP in the presence of either non-induced or βNF-induced S9. SB significantly inhibited BaP binding to DNA. These effects correlated with the inhibition of cytochrome P-450IA1-linked EROD activity in βNF-induced microsomes and with an inhibition of βNF-induced S9 mediated metabolism of [ 3H]BaP as determined by HPLC. These results suggest that OD and SB may possess antimutagenic activity by inhibiting P-450IA-mediated metabolism of BaP.

Microsomal activation of benzo[a]pyrene to mutagens by Alligator mississippiensis in vitro: induction by 3-methylcholanthrene

The metabolism of benzo[a]pyrene (BaP) to mutagens by liver microsomes of the American alligator, Alligator mississippiensis, was studied as a function of induction by pretreatment with 3-methylcholanthrene (3MC). 3MC-pretreatment greatly enhanced the microsomal-mediated oxidation of BaP to mutagens in the umu mutagenicity assay. Analysis of the BaP metabolite profile by reversed-phase HPLC showed that this increase in mutagenicity of BaP corresponded to an increase in the formation of BaP-diols, tetrols and diol epoxide as compared with untreated alligators. SDS-polyacrylamide gel electrophoresis of the microsomal fractions indicated the presence of additional protein bands within the 50kDa molecular weight region in microsomes of 3MC-pretreated alligators. Western blot analysis revealed the presence of a protein band of about 50 kDa that cross-reacted with a polyclonal antibody to rabbit P450 LM4 and a monoclonal antibody to scup P450E. The former recognizes both CYPIA1 and CYPIA2 in liver microsomes from 3MC-treated rats and the latter is believed to be orthologous to these isoforms; thus, our data suggest the 3MC-inducible liver microsomal P450 is the alligator orthologue of one of these isoforms. The results also suggest that 3MC induces P450 in A. mississippiensis with structural and functional similarity to forms present in mammals.

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.

The effects of pretreatment with cytochrome P-450 inducers and preincubation with a cytochrome P-450 effector on the mutagenicity of genotoxic carcinogens mediated by hepatic and renal S9 from two species of marine fish

A series of experiments was designed to characterize the cytochrome P-450-dependent activation of 7 genotoxic carcinogens in the Salmonella preincubation assay by hepatic postmitochondrial fractions (S9) from the oyster toadfish and the Americal eel and by renal S9 from the toadfish. Significant S9-dependent mutagencity was observed for benzo[ a]pyrene (BAP), 2-aminoanthracene (2AA), aflatoxin B1 (AFB1), 7,12-dimethylbenz[ a]anthracene (DBMA) and cyclophosphamide (CP) with hepatic S9 from untreated fish (UI S9) of both species and with renal S9 from untreated toadfish, although renal UI S9 was only marginally effective for activating AFB1. Neither UI S9 from toadfish liver or kidney nor that from eel liver consistently affected the direct mutagenicity of ethylene dibromide (EDB) or substantially activated dimethylnitrosamine (DMN). Pretreatment of toadfish with 3-methylcholanthrene (MC) decreased the mutagenicity of 2AA and increased the mutagenicities of BAP, AFB1 and DMBA, whereas, pretreatment of eels with MC increased the mutagenicities of BAP, 2AA and AFB1. Pretreatment of toadfish with Aroclor 1254 (AC) decreased the mutagenicity of AFB1 and increased the mutagenicity of 2AA, whereas, pretreatment of eels with AC increased the mutagenicities of BAP and DMBA. Pretreatment of toadfish with β-napthoflavone (BNF) effected changes similar to those by pretreatment with MC except that the mutagenicity of AFB1 was not increased. Coincubation with 10 −4 M α-napthoflavone (ANF) decreased the mutagenicity of BAP mediated by toadfish MC and BNF S9 and eel AC S9 and decreased the mutagenicity of AFB1 mediated by toadfish MC and BNF S9 and by eel MC S9. Coincubation with ANF increased the mutagenicity of AFB1 mediated by toadfish and eel AC S9 and increased the mutagenicity of 2AA mediated by eel AC S9. Pretreatment of toadfish with MC, BNF and AC decreased the mutagenicity of 2AA mediated by renal S9 and ANF decreased the mutagenicity of 2AA mediated by renal UI and BNF S9. MC pretreatment of toadfish and eels and BNF pretreatment of toadfish induced BAP monooxygenase activity in hepatic microsomes. ANF (10 −4 M) inhibited the BAP monooxygenase activity of MC microsomes from toadfish and eels and of BNF microsomes from toadfish. The conjugation effectors diethyl maleate and salicylamide alone or combined had little or no effect on the mutagenicities of BAP and 2AA mediated by toadfish and eel UI and MC S9. These results suggest BAP and 2AA are metabolized by different cytochrome P-450-dependent enzymes in both species and that, whereas, MC and BNF pretreatment effect similar changes in cytochrome P-450 populations in toadfish liver, MC and AC pretreatment effect different changes in cytochrome P-450 populations in the livers of either species. The results also suggest that the effects of pretreatment of these marine species with particular cytochrome P-450 inducers on the S9-mediated mutagenicities of particular genotoxic carcinogens can be species-dependent.

A comparison of the effect of selenium on the mutagenicity and metabolism of benzo[ a]pyrene in rat and hamster liver S9 activation systems

When selenium (Na 2SeO 3) was included in the incubation mix containing rat or hamster liver S9 preparations both the metabolism and mutagenicity of benzo[ a]pyrene (BaP) and several of its metabolites were altered. At non-toxic concentrations selenium inhibited the S9 dependent mutagenicity of BaP and number of its metabolites on Salmonella typhimurium strain TA100 as indicated by the number of histidine independent revertants observed. High performance liquid chromatographic analysis of S9 generated metabolites of BaP from rat and hamster liver indicated that selenium caused quantitative differences in the amounts of the metabolic products. In hamster liver S9 differences were reflected in decreased amounts of strongly mutagenic BaP-7,8-dihydrodiol and increased amounts of 4,5- and 9,10-dihydrodiols that were weakly mutagenic to TA100 in that system. In rat liver S9 selenium caused quantitatively similar decreases in BaP-7,8- and 9,10-dihydrodiol and 3-hydroxy-BaP. When used as substrate 3-hydroxy-BaP was the most mutagenic to TA100 in the rat activation system whereas BaP-7,8-dihydrodiol was most mutagenic in the hamster S9 system. Assays that measured the formation of water-soluble conjugates of BaP indicated that selenium did not significantly alter the formation of sulfate ester or glutathione conjugates although a 12–17% reduction of labeled metabolites bound to the glucuronide fraction was observed. Results described in this report suggest that selenium modified the metabolism and hence the mutagenicity of BaP to TA100 by affecting mixed-function oxidase and/or epoxide hydratase activity in both the rat and hamster liver S9 activation systems.