Covalent binding of (+)-anti-benzoapyrene 7,8-dihydrodiol 9, 10-epoxide (anti-BPDE) to the N(2)-amino group of deoxyguanine in the oligonucleotides 5'-d(CCTATCGXTATCC) and 5'-d(CCTATm(5)CGXTATCC) (X ...being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPDE-N(2)-dG adduct in single-stranded 13-mer oligonucleotides with 5'-d(m(5)CGT) and 5'-d(m(5)CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respectively) relative to that of the nonmethylated sequences. With the 5'-d(CGC) sequence context, m(5)dC had no significant effect on adduct formation. When the reaction was allowed to proceed in the presence of oligonucleotide duplexes (composed of a 13-mer parent strand and a 9-mer complement), a significant increase in the extent of adduct formation was observed with 5'-d(m(5)CGT)/d(CGA) and 5'-d(m(5)CGA)/d(CGT), but not with 5'-d(CGC)/d(GCG), relative to those of the nonmethylated duplexes. Independent of sequence context, no clear effect of m(5)dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level of diol epoxide binding to the dG target in the 13-mer oligonucleotides is in general higher in single-stranded sequences than in the duplexes. With 5'-d(CGA) and 5'-d(m(5)CGA), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex (composed of a 13-mer parent strand and a full complement) was substantially higher than in the 5'-d(CGT)/d(GCA) sequence context. The stimulating effect of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring system with the nucleobases prior to the nucleophilic addition reaction of the exocyclic amino group. The results from induced circular dichroism studies with the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex are consistent with substantial heterogeneity of adduct conformations, including both external minor groove-localized and intercalated structures.
Covalent binding of (+)-anti-benzoapyrene 7,8-dihydrodiol 9,10-epoxide (anti-BPDE) to the N 2-amino group of deoxyguanine in the oligonucleotides 5‘-d(CCTATCGXTATCC) and 5‘-d(CCTATm5CGXTATCC) (X ...being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPDE−N 2-dG adduct in single-stranded 13-mer oligonucleotides with 5‘-d(m5CGT) and 5‘-d(m5CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respectively) relative to that of the nonmethylated sequences. With the 5‘-d(CGC) sequence context, m5dC had no significant effect on adduct formation. When the reaction was allowed to proceed in the presence of oligonucleotide duplexes (composed of a 13-mer parent strand and a 9-mer complement), a significant increase in the extent of adduct formation was obseved with 5‘-d(m5CGT)/d(CGA) and 5‘-d(m5CGA)/d(CGT), but not with 5‘-d(CGC)/d(GCG), relative to those of the nonmethylated duplexes. Independent of sequence context, no clear effect of m5dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level of diol epoxide binding to the dG target in the 13-mer oligonucleotides is in general higher in single-stranded sequences than in the duplexes. With 5‘-d(CGA) and 5‘-d(m5CGA), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)-trans-anti-BPDE−N 2-dG adduct in the 5‘-d(m5CGT)-containing duplex (composed of a 13-mer parent strand and a full complement) was substantially higher than in the 5‘-d(CGT)/d(GCA) sequence context. The stimulating effect of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring system with the nucleobases prior to the nucleophilic addition reaction of the exocyclic amino group. The results from induced circular dichroism studies with the (+)-trans-anti-BPDE−N 2-dG adduct in the 5‘-d(m5CGT)-containing duplex are consistent with substantial heterogeneity of adduct conformations, including both external minor groove-localized and intercalated structures.
The effect of dietary 2(3)-tert-butyl-4-hydroxy-anisole (BHA) alone and in combination with intraperitoneal injections of 3-methylcholanthrene (MC), on hepatic enzyme activities and benzoapyrene (BP) ...metabolism was compared in male and female NMRI mice. In general, the characteristic induction pattern following dietary BHA administration in female mice could also be seen when male mice were used. The increase in epoxide hydrolase and cytosolic glutathione S-transferase following BHA feeding was however not as pronounced in males as in females. Also, MC treatment appeared to counteract the induction of GST activity to BHA in females but had no such effect in males. Liver microsomes from untreated male mice catalyzed the metabolism of BP less efficiently than did microsomes from females. BHA treatment increased this activity in both sexes to a comparable extent and the overall activity was the same in males and females having received MC. The pattern of BP metabolites was altered following BHA treatment. In general, an increase in BP-4,5-diol and a decrease in 9-OH-BP was observed. This pattern was also noticed when microsomes from MC treated and MC + BHA treated animals were compared. MC treatment alone increased the amount of BP-7,8-diol, the quinones and the phenols. The present report indicates that several factors may contribute to the response to dietary BHA in mice. Whether this has any consequence in regard to this compounds's anticarcinogenic effect remains to be elucidated.
The binding conformations of single anti- and syn-BPDE−N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD) and light absorption ...spectroscopy. The sign of the ICD in single-stranded oligonucleotide adducts correlates with the absolute configuration of the cyclohexyl moiety of the BPDE. Adducts in oligonucleotide duplexes with UV λmax <350 nm exhibiting a significant duplex-induced positive ICD should have a minor groove location as the predominant conformation. Those with UV λmax >350 nm exhibiting either positive or negative contributions to the ICD should have intercalated binding as the predominant conformation. The magnitude of the ICD is dependent on the sequence context of the adducted strand and the particular BPDE−adduct isomer under study. In some cases, the results suggest structural heterogeneity. For instance, the (+)- and the (−)-trans-anti-BPDE−N2-dG adducts in duplexes where a dT flanks the lesion site exhibit weak positive ICD or negative ICD. These results reflect a bimodal conformational adduct distribution with contributions from both externally and internally located adducts. A key observation for the (+)-cis-syn-BPDE−N2-dG complexes in 5‘-d(TGC) and 5‘-d(CGC) sequence contexts is that the near-UV absorption spectra showed distinct bands corresponding to minor groove binding (λmax ≅ 346 nm) as well as intercalative binding (λmax ≅ 354 nm). Evidence for an equilibrium between the different modes of localization is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-cis-syn-BPDE−N2-dG complexes in 5‘-d(TGC) and 5‘-d(CGC) sequence contexts, respectively.
Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzoapyrene-7, 8-dihydrodiol 9,10-epoxide (+)-anti-BPDE by conjugation with ...glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701-1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719-1723; Swedmark and Jenssen (1994) Gene 139, 251-256. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)-or(-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.
Dibenzoa,lpyrene (DBP) is considerably more carcinogenic than benzoapyrene (BP). This fact is most probably related to differences in DNA binding efficiency of their ultimate carcinogenic diol ...epoxide (DE) intermediates, (+)-anti-BPDE in the case of BP and (−)-anti-DBPDE with DBP; differences in structural features of the DNA adducts and differences in DNA adduct recognition; and the subsequent lesion removal by nucleotide excision repair (NER). The complex signaling network involved in adduct recognition, cell cycle check points, and adduct removal is rapidly emerging. In this study we have focused on the effects of (+)-anti-BPDE and (±)-anti-DBPDE on Mdm2, a protein that regulates p53 stabilization/accumulation in an autoregulatory negative feedback loop. The data available so far indicate that both DEs induce accumulation of Mdm2 in HepG2 cells in concentrations without detectable effects on p53 stabilization. However, BPDE was found to be several orders of magnitude more powerful as inducer than the more complex DBPDE. BPDE caused Mdm2 accumulation at low pM concentrations whereas nM concentrations of DBPDE were required for a similar effect. The Mdm2 accumulation induced by each DE was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. This suggests that phosphorylation of Mdm2 is an essential step in accumulation of the protein. Furthermore, available RT-PCR results indicate that the DEs at higher concentrations (1 μM BPDE and 0.1 μM DBPDE) actually lowered the levels of Mdm2 mRNA indicating transcriptional downregulation of Mdm2. As expected, the effect was associated with p53 phosphorylation on serine 15 and, accordingly, stabilization of the protein.
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BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mammalian metabolism of polycyclic aromatic hydrocarbons results in the formation of vicinal diol epoxides (existing as enantiomeric pairs of two diastereomers) considered as important ultimate ...carcinogens if the oxirane ring is located in a bay or fjord region of the parent hydrocarbon. In the present study, individual stereoisomers of the bay region diol epoxides of chrysene, dibenza,h-anthracene and benzoapyrene, as well as of the fjord region diol epoxides of benzocphenanthrene, benzog chrysene and have beenzog incubated with glutathione (GSH) in the presence or absence of human glutathione S-transferase isoenzyme GST A1-1, a class Alpha enzyme. The formation of GSH conjugates was determined and quantified by HPLC. The results demonstrate that the GST A1-1 isoenzyme catalyzes the formation of GSH conjugates of all diol epoxides tested, although a marked variation in catalytic efficiency (>20-fold) was observed. With both bay and fjord region anti-diol epoxides a significant preference for conjugation of the enantiomer with the R configuration at the benzylic position of the oxirane ring was noted. Among the syn diastereomers of the fjord region diol epoxides a similar substrate enantiose-lectivity was noted, i.e. the enantiomer with the corresponding R configuration was again preferentially conjugated. In contrast, for the bay region syn-dioI epoxides this substrate selectivity was reversed, resulting in a preference for the enantiomer with the S configuration. The chemically more reactive syn diastereomers were in general better substrates for GST A1-1 than the corresponding anti diastereomers. However, a comparison between different diol epoxide diastereomers revealed no obvious correlation between chemical reactivity of the compounds and catalytic efficiencies. Furthermore, no significant correlation between diol epoxide lipophilicity and catalytic efficiency was observed. It is suggested that stereochemical factors, including the size and the geometry of the aromatic ring system and the preferred conformation of the diol epoxide, are involved as the major determinant for the rate of catalysis by GST A1-1.
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