Detection of DNA adducts originating from 1-methoxy-3-indolylmethyl glucosinolate using isotope-dilution UPLC-ESI-MS/MS.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, present at substantial levels in several food crops (e.g., broccoli and cabbage), forms DNA adducts in vitro and is mutagenic to bacterial and mammalian cells after activation by the plant enzyme myrosinase. Moreover, a breakdown product, 1-MIM alcohol, is metabolized to a secondary reactive intermediate by some mammalian sulfotransferases (SULTs). First, we incubated herring-sperm DNA with 1-MIM glucosinolate in the presence of myrosinase. We identified and synthesized the predominant adducts, N(2)-(1-MIM)-dG and N(6)-(1-MIM)-dA, and developed an UPLC-ESI-MS/MS method for their specific detection using isotopic dilution. Second, we demonstrated both DNA adducts in target cells (Salmonella typhimurium TA100 and Chinese hamster V79) of standard mutagenicity tests treated with 1-MIM glucosinolate/myrosinase as well as in 1-MIM alcohol-treated Salmonella and V79 cells engineered for expression of human SULT1A1. Similar excesses of N(2)-(1-MIM)-dG over N(6)-(1-MIM)-dA adducts were found in all cellular models independent of the test compound (1-MIM glucosinolate or alcohol), whereas dA adducts predominated in the cell-free system. Finally, we detected both DNA adducts in colon tissue of a mouse orally treated with 1-MIM glucosinolate. We are going to use this specific and sensitive method for investigating genotoxic risks of food-borne exposure to 1-MIM glucosinolate in animal and human studies.

Synthesis, absolute configuration, and bacterial mutagenicity of the 8 stereoisomeric vicinal diol epoxides at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene.

The synthesis of the 8 possible stereoisomeric diol epoxides (DEs) at the terminal benzo ring of carcinogenic dibenz[a,h]anthracene (DBA) is reported. trans-3,4-Dihydroxy-3,4-dihydro-DBA (1) afforded the 4 bay region DEs: the enantiomeric pairs of the anti diastereomers (+)-3/(-)-3 and of the syn diastereomers (-)-4/(+)-4, respectively. trans-1,2-Dihydroxy-1,2-dihydro-DBA (2) served as precursor of the 4 reverse DEs: the enantiomeric pairs of the anti diastereomers (+)-5/(-)-5 and of the syn diastereomers (-)-6/(+)-6, respectively. The transformation of the olefinic double bond in the enantiomeric trans-dihydrodiols to epoxides was achieved by either (i) oxidation with m-chloroperoxybenzoic acid or (ii) formation of a bromohydrin with N-bromoacetamide/H(2)O followed by dehydrobromination with an anion exchange resin. Because of the pseudodiequatorial conformation of the hydroxyl groups in 1, both reactions proceeded highly stereoselectively, while the stereoselectivity was impaired by the pseudodiaxial conformation of the hydroxyl groups in 2. Diastereomers and racemic compounds were efficiently separated without derivatization by HPLC on achiral or chiral stationary phases, respectively. The absolute configurations of the DEs were deduced from the absolute configuration of 1 and 2 considering the regio- and stereoselectivity of the subsequent reactions and resulted in (+)-(1R,2S,3S,4R)-3/(-)-(1S,2R,3R,4S)-3, (-)-(1S,2R,3S,4R)-4/(+)-(1R,2S,3R,4S)-4, (+)-(1R,2S,3S,4R)-5/(-)-(1S,2R,3R,4S)-5, and (-)-(1R,2S,3R,4S)-6/(+)-(1S,2R,3S,4R)-6. The bacterial mutagenicity of the 8 stereoisomeric DEs was determined in histidine-dependent strains TA98 and TA100 of Salmonella typhimurium in the absence of a metabolizing system. In general, the bay region DEs of DBA were stronger mutagens than the reverse DEs. In strain TA98, the syn diastereomers of bay region DEs were stronger mutagens than their anti isomers, while in the case of reverse DEs the anti diastereomers were more potent than their syn isomers. In strain TA100, all syn diastereomers surpassed the bacterial mutagenicity of their anti isomers. Concerning the bay region DEs of DBA, this corresponds to the situation described for benzo[a]pyrene: of the 4 enantiomeric bay region DEs of DBA and benzo[a]pyrene, the syn diastereomer with [(R,S)-diol (R,S)-epoxide] absolute configuration is the most potent mutagen in both bacterial strains, while the anti isomer with [(S,R)-diol (R,S)-epoxide] configuration is the weakest mutagen.

DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells.

Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (±)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.

1-Methoxy-3-indolylmethyl glucosinolate; a potent genotoxicant in bacterial and mammalian cells: Mechanisms of bioactivation.

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, contained in many Brassica vegetables, is strongly mutagenic in Salmonella typhimurium TA100 when activated by myrosinase. Here, we describe the synthesis and evaluation of two breakdown products, 1-MIM nitrile and 1-MIM alcohol. 1-MIM nitrile was not mutagenic and 1-MIM alcohol showed low direct mutagenicity in TA100, indicating that other breakdown products mediated the mutagenicity of 1-MIM glucosinoate/myrosinase in this strain. However, 1-MIM alcohol was strongly mutagenic to a TA100-derived strain expressing human sulphotransferase SULT1A1. Likewise, 1-MIM glucosinolate (with myrosinase) showed 10 times higher mutagenic activity in TA100-SULT1A1 than in strain TA100. Identical adducts, N(2)-(1-MIM)-dG and N(6)-(1-MIM)-dA, were detected in both strains, but the levels were higher in TA100-hSULT1A1. A similar influence of SULT1A1 was observed in recombinant V79-hSULT1A1 cells compared to parental SULT-deficient Chinese hamster V79 cells. 1-MIM glucosinolate (with myrosinase) as well as 1-MIM alcohol induced sister chromatid exchange in both cell lines, but with clearly higher efficiency in V79-hSULT1A1 cells. Gene mutation assays were conducted at the HPRT locus with 1-MIM alcohol in V79-hSULT1A1 cells, and with 1-MIM glucosinolate/myrosinase in V79 parental cells. In both cases, the result was clearly positive. Thus, 1-MIM glucosinolate is mutagenic in bacterial and mammalian cells via at least two different metabolites.

Structural requirements for mutation formation from polycyclic aromatic hydrocarbon dihydrodiol epoxides in their interaction with food chemopreventive compounds.

Chinese hamster V79 cells were used to investigate the protective effect of four known antimutagens present in food, chlorophyllin (CHL), ellagic acid (EA), epigallocathechingallate (EGCG) and benzylisothiocyanate (BITC), against potent mutagenic polycyclic aromatic hydrocarbon diol epoxides (PAH-DE) derived from benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) known to be deposited on crops from polluted ambient air or formed during food processing. As fjord-region PAH-DE are more toxic and mutagenic than bay-region PAH-DE, we adjusted the concentrations of PAH-DE to induce approximately the same levels of adducts. The studies were performed using an assay indicating toxicity in terms of reduced cell proliferation together with the V79 Hprt assay for monitoring mutant frequencies. CHL significantly increased the survival and showed a protective effect against the mutagenicity of all PAH-DE. A significant protective effect of EA was found towards the mutagenicity of BPDE, DBPDE and BPhDE and with EGCG for BPDE and BPhDE. BITC had a slight positive effect on the mutagenicity of DBADE and BPhDE. Taken together, a novel and unexpected finding was that the antimutagenic activity could differ as much as by a factor of 7 towards four carcinogenic PAH metabolites being relatively similar in structure and genotoxic activity.

Probenecid, an inhibitor of transmembrane organic anion transporters, alters tissue distribution of DNA adducts in 1-hydroxymethylpyrene-treated rats.

1-Methylpyrene (1-MP), an abundant alkylated polycyclic aromatic hydrocarbon, is activated by side-chain hydroxylation to 1-hydroxymethylpyrene (1-HMP) and subsequent sulfo-conjugation to electrophilic 1-sulfooxymethylpyrene (1-SMP). In rats, this activation mainly occurs in liver. 1-SMP may react with hepatic DNA or be exported into the blood circulation to reach other tissues, in particular kidneys. Findings with recombinant cell lines suggest that renal 1-SMP uptake proceeds via organic anion transporters (OATs). Here, we tested the hypothesis that probenecid, a characteristic OAT inhibitor, interferes with kidney damage brought about by 1-SMP formed in rats. 1-HMP was administered intraperitoneally to 30 rats, half of which were co-treated with probenecid. The tissue distribution of DNA adducts was analyzed using (32)P-postlabeling and isotope dilution LC-MS/MS for the detection of the adducts N(2)-(1-methylpyrenyl)-2'-deoxyguanosine and N(6)-(1-methylpyrenyl)-2'-deoxyadenosine. In rats treated solely with 1-HMP, adduct levels in kidney tissue were about 3-fold and 8-fold higher than those in liver and lung, respectively. After co-treatment with probenecid, hepatic and pulmonary adduct levels were 12-fold and 4-fold elevated, respectively, whereas renal adduct levels were slightly lower compared to those of rats receiving 1-HMP alone. Moreover, serum levels of 1-SMP were increased 23-fold in animals pre-treated with probenecid. The differential effects on hepatic and pulmonary adduct levels suggest that not only renal OATs, but also additional anion transporters, e.g. those mediating the hepatic export of 1-SMP into the bile, were inhibited. Thus, transmembrane transport proteins play a crucial role in the distribution of reactive phase II metabolites, and thereby in tissue allocation of DNA adducts.

Conversion of the common food constituent 5-hydroxymethylfurfural into a mutagenic and carcinogenic sulfuric acid ester in the mouse in vivo.

5-Hydroxymethylfurfural (HMF), formed by acid-catalyzed dehydration and in the Maillard reaction from reducing sugars, is found at high levels in numerous foods. It was shown to initiate colon aberrant crypt foci in rats and skin papillomas and hepatocellular adenomas in mice. HMF is inactive in in vitro genotoxicity tests using standard activating systems but is activated to a mutagen by sulfotransferases. The product, 5-sulfoxymethylfurfural (SMF), is a stronger carcinogen than HMF. SMF has not been detected in the biotransfomation experiments conducted on HMF in humans and animals in vivo up to date. Here, we report pharmacokinetic properties of HMF and SMF in FVB/N mice. Sensitive assays for the quantification of HMF and SMF by LC-MS/MS multiple reaction monitoring were devised. SMF, intravenously injected (4.4 micromol/kg body mass), showed first-order elimination kinetics in blood plasma (t(1/2) = 7.9 min). HMF, injected intravenously (793 micromol/kg body mass), demonstrated biphasic kinetics in plasma (t(1/2) = 1.7 and 28 min for the initial and terminal elimination phases, respectively); the volume of distribution of the central compartment corresponded approximately to the total body water. The maximum SMF plasma level was observed at the first sampling time, 2.5 min after HMF administration. On the basis of these kinetic data, it was estimated that between 452 and 551 ppm of the initial HMF dose was converted to SMF and reached the circulation. It is likely that additional SMF reacted with cellular structures at the site of generation and thus is ignored in this balance. Our work supports the hypothesis that HMF-related carcinogenicity may be mediated by its reactive metabolite SMF.

Renal organic anion transporters OAT1 and OAT3 mediate the cellular accumulation of 5-sulfooxymethylfurfural, a reactive, nephrotoxic metabolite of the Maillard product 5-hydroxymethylfurfural.

5-Hydroxymethylfurfural (HMF) is formed when sugars are acidified or heated. It is present at high levels in numerous foods. HMF is inactive in standard genotoxicity tests, but can be metabolized to a chemically reactive intermediate, 5-sulfooxymethylfurfural (SMF), which is mutagenic and carcinogenic. We recently found that direct parental administration of SMF to mice leads to abundant acute necrosis and proteinaceous casts in the proximal tubules as the dominating toxicological effect. Since proximal tubule cells actively mediate the excretion of many organic anions, we hypothesized that transporter-mediated uptake of SMF into the cells could be the reason for this selective organotoxicity. To test this hypothesis, we used human embryonic kidney (HEK293) cells stably expressing human (h) OAT1 or OAT3. SMF was a competitive inhibitor of p-aminohippurate uptake by hOAT1 and estrone sulfate uptake by hOAT3 with K(i) values of 225 microM and 1.5mM, respectively. Moreover, the initial rates of SMF uptake were 5.2- and 3.1-fold higher in cells expressing hOAT1 and hOAT3, respectively, than in control HEK293 cells. Likewise, the sensitivity of hOAT1- and hOAT3-expressing cells to SMF cytotoxicity was significantly higher than that of control cells, and was reduced by addition of probenecid, an inhibitor of OATs. Taken together, these results indicate that OAT1 and OAT3 mediate the uptake of SMF into proximal tubule cells and thereby may be involved in SMF-induced nephrotoxicity.

Time course of hepatic 1-methylpyrene DNA adducts in rats determined by isotope dilution LC-MS/MS and 32P-postlabeling.

The alkylated polycyclic aromatic hydrocarbon 1-methylpyrene is a carcinogen in rodents and has been detected in various environmental matrices and foodstuffs. It is activated metabolically by benzylic hydroxylation to 1-hydroxymethylpyrene followed by sulfoconjugation to yield electrophilic 1-sulfooxymethylpyrene (1-SMP) that is prone to form DNA adducts. An LC-MS/MS method using multiple reaction monitoring (MRM) of fragment ions has been developed for specific detection and quantification of N (2)-(1-methylpyrenyl)-2'-deoxyguanosine (MP-dGuo) and N (6)-(1-methylpyrenyl)-2'-deoxyadenosine (MP-dAdo) formed in DNA in the presence of 1-SMP. DNA samples were spiked with stable isotope internal standards, [ (15)N 5, (13)C 10]MP-dGuo and [ (15)N 5]MP-dAdo, followed by enzymatic digestion to 2'-deoxynucleosides and solid-phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS. The limits of detection were 10 fmol of MP-dGuo and 2 fmol of MP-dAdo or three molecules of MP-dGuo and 0.6 molecules of MP-dAdo per 10 (8) 2'-deoxynucleosides using 100 mug of herring sperm DNA as the sample matrix. The method was validated with herring sperm DNA reacted with 1-SMP in vitro. Hepatic DNA was analyzed from rats that were dosed intraperitoneally with 9.3 mg 1-SMP per kg body weight and killed after various time periods. Levels of MP-dGuo and MP-dAdo in rat liver were found to increase, reaching their maxima at approximately 3 h, and then decrease over time. A good correlation was observed between the results obtained using LC-MS/MS and MRM and those from (32)P-postlabeling. MRM allowed the more precise quantification of specific 1-MP adducts, in addition to a time reduction of the analysis when compared with (32)P-postlabeling.

Detoxification of promutagenic aldehydes derived from methylpyrenes by human aldehyde dehydrogenases ALDH2 and ALDH3A1.

Methylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulpho conjugation to reactive esters, which can induce mutations and tumours. Yet, further oxidation of the alcohol may compete with this toxification. We previously demonstrated that several human alcohol dehydrogenases (ADH1C, 2, 3 and 4) oxidise various benzylic alcohols (derived from alkylated pyrenes) to their aldehydes with high catalytic efficiency. However, all these ADHs also catalysed the reverse reaction, the reduction of the aldehydes to the alcohols, with comparable or higher efficiency. Thus, final detoxification requires elimination of the aldehydes by further biotransformation. We have expressed two human aldehyde dehydrogenases (ALDH2 and 3A1) in bacteria. All pyrene aldehydes studied (1-, 2- and 4-formylpyrene, 1-formyl-6-methylpyrene and 1-formyl-8-methylpyrene) were high-affinity substrates for ALDH2 (K(m)=0.027-0.9 microM) as well as ALDH3A1 (K(m)=0.78-11 microM). Catalytic efficiencies (k(cat)/K(m)) were higher for ALDH2 than ALDH3A1 by a moderate to a very large margin depending on the substrate. Most important, they were also substantially higher than the catalytic efficiencies of the various ADHs for the reduction the aldehydes to the alcohols. These kinetic properties ensure that ALDHs, and particularly ALDH2, can complete the ADH-mediated detoxification.

Oxidation of alcohols and reduction of aldehydes derived from methyl- and dimethylpyrenes by cDNA-expressed human alcohol dehydrogenases.

Some methylated pyrenes can form DNA adducts in rat tissues after benzylic hydroxylation and sulpho conjugation. However, oxidation of the intermediate alcohols to carboxylic acids is an important competing pathway leading to detoxification. We previously showed that co-administration of ethanol or 4-methylpyrazole strongly enhances DNA adduct formation by 1-hydroxymethylpyrene, indicating an involvement of alcohol dehydrogenases (ADHs) in the detoxification. This mechanism may be involved in the observed synergism of smoking and alcohol consumption in certain human cancers. In a preceding study, cDNA-expressed human ADH2 efficiently oxidised 1-, 2- and 4-hydroxymethylpyrene; these reactions were inhibited in the presence of ethanol or 4-methylpyrazole. Here we report that ADH1C, ADH3 and ADH4 also show substantial activity towards these substrates and two further congeners, 1-hydroxymethyl-6-methylpyrene and 1-hydroxymethyl-8-methylpyrene. All four ADH forms also catalysed the reverse reaction, implying that the aldehydes have to be sequestered by other enzymes, such as aldehyde dehydrogenases, for final detoxification. ADH1C and ADH4 activities towards hydroxymethylpyrenes were more strongly inhibited in the presence of ethanol and 4-methylpyrazole than those of ADH2. ADH3 was only inhibited at very high concentrations of the modulators. In conclusions, several human ADHs are capable of detoxifying benzylic alcohols of alkylated polycyclic aromatic hydrocarbons. However, some competing substrates and inhibitors may affect all these redundant detoxification systems, although to various extents.

Efficient oxidation of promutagenic hydroxymethylpyrenes by cDNA-expressed human alcohol dehydrogenase ADH2 and its inhibition by various agents.

Alkylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulphation to electrophilically reactive esters. However, we previously found that the predominant biotransformation route for the hepatocarcinogen 1-hydroxymethylpyrene (1-HMP) in the rat in vivo is the oxidation of the side chain by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases to the carboxylic acid. Inhibition of this pathway by ethanol (competing ADH substrate) or 4-methylpyrazole (ADH inhibitor) led to a dramatic increase in the 1-HMP-induced DNA adduct formation in rat tissues in the preceding study. In order to elucidate the role of individual ADHs in the metabolism of alkylated polycyclic aromatic hydrocarbons, we expressed the various members of the human ADH family in bacteria. Cytosolic preparations from bacteria expressing ADH2 clearly oxidized hydroxymethylpyrene isomers (1-, 2- and 4-HMP) with the highest rate. This form was purified to near homogeneity to perform detailed kinetic analyses. High catalytic efficiencies (V(max)/K(m)) were observed with HMPs. Thus, this value was 10,000-fold higher for 2-HMP than for the reference substrate, ethanol. The corresponding aldehydes were also efficiently reduced by ADH2. 4-Methylpyrazole inhibited the oxidation of the HMP isomers as well as the reverse reaction. Daidzein, cimetidine and the competing substrate ethanol were further compounds that inhibited the ADH2-mediated oxidative detoxification of 1-HMP.

Directing role of organic anion transporters in the excretion of mercapturic acids of alkylated polycyclic aromatic hydrocarbons.

Excretion of mercapturic acids of a xenobiotic is a good indicator for the formation of electrophilic intermediates. However, the route of excretion, urine or feces, is important for usage of a given mercapturic acid as a biomarker in humans. In the present study we investigated the excretion routes of 1-methylpyrenyl mercapturic acid (MPMA) and 1,8-dimethylpyrenyl mercapturic acid (DMPMA) formed from the corresponding benzylic alcohols in rats. Whereas MPMA was primarily excreted in urine (72% of the total urinary and fecal level), DMPMA clearly preferred the fecal route (88%). We then examined interactions of these mercapturic acids with renal basolateral organic anion transporters (OATs) using HEK293 cells stably expressing human OAT1 and OAT3. The uptake rates of MPMA by OAT1- and OAT3-expressing cells were 2.8- and 1.7-fold, respectively, higher than that by control cells. MPMA was a competitive inhibitor of p-aminohippurate uptake by OAT1 and estrone sulfate uptake by OAT3 with K(i) values of 14.5 microM and 1.5 microM, respectively. In contrast, DMPMA was not transported by OAT1 and only modestly transported by OAT3 (1.25-fold over control). Thus, we suspect that the substrate specificities, alone or together with other factors, played a directing role in the excretion of MPMA and DMPMA. Although the mechanistic link requires verification, our results clearly show that a minute structural difference (the presence or absence of an additional methyl group in an alkylated four-ring polycyclic hydrocarbon) can strongly affect the interaction with transporter proteins and direct the excretion route of mercapturic acids.

Formation of estrogenic metabolites of benzo[a]pyrene and chrysene by cytochrome P450 activity and their combined and supra-maximal estrogenic activity.

Metabolism of polycyclic aromatic hydrocarbons (PAHs) has been studied intensively, and potential metabolites with estrogenic activity have been identified previously. However, little attention has been paid to the metabolic pathways in mammalians and to the combined effect of individual metabolites. Several hydroxylated metabolites of benzo[a]pyrene (BaP) and chrysene (CHN) were formed by rat liver microsomal cytochrome P450 (CYP) activity, some of which possess estrogenic activity. All mono- and several dihydroxylated metabolites of BaP and CHN were tested for ER affinity and estrogenic activity in a proliferation assay (E-screen) and in a reporter-gene assay (ER-CALUX). Twelve estrogenic metabolites were identified with EC50 values ranging from 40nM to 0.15mM. The combined effect of a mixture of seven PAH-metabolites was also studied in the ER binding assay. At concentrations that show little activity themselves, their joint action clearly exhibited significant estrogenic activity. BaP itself exhibited estrogenicity in the ER-CALUX assay due to bio-activation into estrogenic metabolites, probably via aryl hydrocarbon receptor (AhR) induced CYP activity. Furthermore, 2-hydroxy-CHN (2-OHCHN) induced supra-maximal (400%) estrogenic effects in the ER-CALUX assay. This effect was entirely ER-mediated, since the response was completely blocked with the ER-antagonist ICI182,780. We showed that 2-OHCHN increased ER-concentration, using ELISA techniques, which may explain the observed supra-maximal effects. Co-treatment with the AhR-antagonist 3',4'-dimethoxyflavone (DMF) enhanced ER-signaling, possibly via blockage of AhR-ER inhibitory cross-talk.

Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases, sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells.

3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites, we developed a panel of Chinese hamster V79MZ-h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 microM 3-NBA, or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the (32)P-postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYP1A2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA.

Detection of inducible nitric oxide synthase and vascular endothelial growth factor in choroidal neovascular membranes.

Vascular endothelial growth factor (VEGF) is among the cytokines which have been implicated in the pathogenesis of choroidal neovascularization secondary to age-related macular degeneration (ARMD). There is, however, evidence that intercellular signaling molecules, such as nitric oxide (NO), are involved in this process. NO is synthesized via the inducible isoform of NO synthase (iNOS), which is expressed after induction by cytokines. In the current study, we investigated whether VEGF and iNOS are coexpressed in choroidal neovascular membranes (n = 7) from patients with ARMD. Immunohistochemistry was performed on cryosections with anti-iNOS and anti-VEGF. Moderate to intense immunostaining for iNOS and VEGF was observed in retinal pigment epithelial cells, macrophages, and in spatial relation to vessel walls. As scored by light microscopy, we found a significant correlation between immunoreactivity for VEGF and iNOS (p < 0.0341) in vascular endothelial cells. Our study supports a significant role for iNOS in the pathogenesis of neovascularization and membrane growth in ARMD. Moreover, our findings suggest a possible relationship between NO and VEGF in the regulation of pathologic angiogenesis in this disease.

Microsomal activation of dibenzo[def,mno]chrysene (anthanthrene), a hexacyclic aromatic hydrocarbon without a bay-region, to mutagenic metabolites.

Metabolically formed dihydrodiol epoxides in the bay-region of polycyclic aromatic hydrocarbons are thought to be responsible for the genotoxic properties of these environmental pollutants. The hexacyclic aromatic hydrocarbon dibenzo[def,mno]chrysene (anthanthrene), although lacking this structural feature, was found to exhibit considerable bacterial mutagenicity in histidine-dependent strains TA97, TA98, TA100, and TA104 of S. typhimurium in the range of 18-40 his(+)-revertant colonies/nmol after metabolic activation with the hepatic postmitochondrial fraction of Sprague-Dawley rats treated with Aroclor 1254. This mutagenic effect amounted to 44-84% of the values determined with benzo[a]pyrene under the same conditions. The specific mutagenicity of anthanthrene in strain TA100 obtained with the cell fraction of untreated animals was 6 his(+)-revertant colonies/nmol and increased 2.7-fold after treatment with phenobarbital and 4.5-fold after treatment with 3-methylcholanthrene. To elucidate the metabolic pathways leading to genotoxic metabolites, the microsomal biotransformation of anthanthrene was investigated. A combination of chromatographic, spectroscopic, and biochemical methods allowed the identification of the trans-4,5-dihydrodiol, 4,5-oxide, 4,5-, 1,6-, 3,6-, and 6,12-quinones, and 1- and 3-phenols. Furthermore, two diphenols derived from the 3-phenol, possibly the 3,6 and 3,9 positional isomers, as well as two phenol dihydrodiols were isolated. Three pathways of microsomal biotransformation of anthanthrene could be distinguished: The K-region metabolites are formed via pathway I dominated by monooxygenases of the P450 1B subfamily. On pathway II the polynuclear quinones of anthanthrene are formed. Pathway III is preferentially catalyzed by monooxygenases of the P450 1A subfamily and leads to the mono- and diphenols of anthanthrene. The K-region oxide and the 3-phenol are the only metabolites of anthanthrene with strong intrinsic mutagenicity, qualifying them as ultimate mutagens or their precursors. From the intrinsic mutagenicity of these two metabolites and their metabolic formation, the maximal mutagenic effect was calculated. This demonstrates the dominating role of pathway III in the mutagenicity of anthanthrene under conditions where it exhibits the strongest bacterial mutagenicity.