Polymorphism of the human GSTP gene may be of importance in PAH-induced carcinogenesis. A higher frequency of GSTP*B (encoding for Val at position 105) genotype among individuals with certain tumours ...has been observed. The altered susceptibility may be associated with a decreased GST-dependent elimination of reactive PAH-intermediates. We have determined the catalytic efficiency of two naturally occurring GSTP1-1 allelic variants with Ile or Val at position 105 towards bay- and fjord-region diol epoxides of chryscne (CDE) and dibenzoa, lpyrene (DBPDE), respectively. With both variants, the bay-region diol epoxides were more efficiently conjugated than the fjord-region analogues. GSTP1-1/Val was about 3-fold more active with (+)-anti-CDE relative to GSTP1-1/Ile. With (+)-syn-CDE, equal activity was observed. GSTP1-1/Val was about 2-fold more efficient than the Ile-variant with (-)-anti-DBPDE. No difference in activity was observed with (+)-syn-DBPDE.
The kinetics of the enzymatic conjugation of glutathione (GSH) with the anti-diastereoisomers of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzoapyrene (BPDE), ...trans-3,4-dihydroxy-1,2-epoxy-1,2,3, 4-tetrahydrobenzaanthracene (BADE) and trans-1,2- dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (CDE) catalyzed by transferase 4-4 from rat liver have been compared. When the concentration of these diol-epoxides was varied (using 2 mM GSH) the apparent Vmax values were 560, 2100 and 1500 nmol/mg/min for (+/-)-anti-BPDE, (+/-)-anti-BADE and (+/-)-anti-CDE, respectively, with corresponding apparent Km values of 11, 125 and 105 microM. The catalytic efficiency of transferase 4-4 in the GSH conjugation of (+/-)-anti-BADE and (+/-)-anti-CDE is thus approximately one-third of (+/-)-anti-BPDE (0.014 and 0.012 s-1 microM-1 respectively versus 0.042 s-1 microM-1). Similar non-linear Lineweaver-Burk plots were obtained with each diol-epoxide when the concentration of GSH was varied, and two apparent Km values of 0.02-0.04 and 0.4-0.9 mM GSH were estimated. The GSH-conjugates formed with the individual enantiomers of the racemic substrates used were resolved by h.p.l.c. The data indicate that with each diol-epoxide transferase 4-4 is highly selective (greater than or equal to 95%) towards the biologically most active (+)-enantiomer.
Covalent adducts of the carcinogenic polycyclic aromatic hydrocarbon (+)-anti-benzoapyrene 7,8-dihydrodiol 9,10-epoxide ((+)-anti-BPDE) in polynucleotides have been studied by fluorescence ...spectroscopy. The pyrenyl chromophores of the BPDE adducts, linked by the C10 atom to the exocyclic nitrogen of guanine, interact in the photoexcited state, as evidenced by excimer fluorescence. Strong BPDE excimer fluorescence is observed in the alternating poly(dGdC)-(dGdC) sequence, whereas it is weak in the homopolymeric poly(dG)-poly(dC) and in calf thymus DNA. No excimer fluorescence is observed for the BPDE adducts in poly(dAdC)-(poly(dGdT) or poly(dAdG)-poly(dCdT). It is concluded that the formation of BPDE excimers in polynucleotides requires binding to guanines on different strands on consecutive basepairs. The experimental results are supported by graphics modeling and energy minimization of BPDE adducts in various oligonucleotide sequences. The results show that the most favorable arrangement for excimer formation of the BPDE-dG adducts is in a 5'(dCdG-BPDE)-5'(dCdG-BPDE) sequence, where the pyrenyl chromophores interact in the minor groove.
The occurrence of inflammatory processes and of cancer in the human respiratory tract is intimately associated. One of the major factors in this is probably the recruitment of and stimulated activity ...of polymorphonuclear leukocytes (PML) in conjunction with the ability of these cells to convert various carcinogens to their ultimate active metabolites. In this study, we demonstrate that nitrite and sulfite, the major dissolution products of the environmental pollutants nitrogen dioxide and sulfur dioxide in water enhance the metabolic activation of trans-7,8-dihydroxy-7,8-dihydrobenzoapyrene (BP-7,8-dihydrodiol), the proximal carcinogen of benzoapyrene, to trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzoapyrene (BPDE) and tetraols, the corresponding hydrolysis products, in human PML prestimulated with 12-O-tetradecanoylphorbol-13-acetate. Nitrite was more efficient than sulfite in stimulating the formation of reactive intermediates of BP-7,8-dihydrodiol in PML that covalently bind to extracellular DNA and, in particular, to intracellular proteins. The mechanism by which sulfite stimulates the metabolism of BP-7,8-dihydrodiol most probably involves the intermediate formation of a sulfur trioxide radical anion (${\rm SO}_{3}^{\overline{\bullet}}$)the subsequent formation of the corresponding sulfur peroxyl radical anion (^{\bullet}{\rm OOSO}{}_{3}^{-}$) in the presence of oxygen. The mechanism underlying the stimulatory action of nitrite is less clear but the major pathway seems to involve myeloperoxidase. These results offer an explanation for the increased incidence of lung cancer in cigarette smokers living in urban areas. The major glutathione transferase (GST) isoenzyme in human PML is GST P1-1, a Pi-class form. The GST activity of PML was found to be inversely correlated with the extent of binding of BP-7,8-dihydrodiol products to exogenous DNA. These results suggest that individuals exhibiting high GST-activity in the PML may be better protected against the type of carcinogenic dealt with in this study.
Human Alpha class glutathione transferases (hGSTs) have been incubated with the ultimate carcinogenic ( m )- anti - and (+)- syn -diol epoxides (DE) of the nonplanar dibenzo a,l pyrene (DBP). ...hGSTA1-1, A2-2, and A3-3 demonstrate activity with both diol epoxides ( R -absolute configuration at the benzylic oxirane carbon) whereas hGSTA4-4 virtually was inactive. (+)- syn -DBPDE was superior as substrate compared to the ( m )- anti enantiomer with hGSTA1-1 as the most efficient enzyme (k cat /K M = 464 mM m 1 s m 1 ) followed by A3-3 and A2-2 (k cat /K M = 190 mM m 1 s m 1 and 30.4 mM m 1 s m 1 , respectively). With ( m )- anti -DBPDE, the k cat /K M values were in general about 10-fold lower. Replacing ( m )- anti -DBPDE with the less complex ( m )- anti -BCDE or the less structurally distorted (+)- anti -BPDE, revealed that GSTA1-1 was 19- and 25-fold less active, respectively. hGSTA1-1 is present in human lung, a primary target tissue for PAH-induced tumors. Considering the great efficiency of this isoform relative to both Pi and Mu-class GSTs toward DBPDE, its presence and extent of expression may play a significant role in protection against this type of highly carcinogenic compounds.
The (-)-anti-diol epoxide of benzocphenanthrene (BPhDE) and (+)-anti-diol epoxide of benzoapyrene (BPDE) were used to site-specifically modify oligonucleotides containing the binding site for the ...transcription factors AP-1 and TFIID, respectively. The modified oligonucleotides were incubated with the appropriate transcription factor and protein-DNA interaction analysed by the electrophoretic mobility shift assay. The available results show that a trans-BPhDE-N
6
-dA adduct located in the AP-1 binding sequence totally inhibits protein-DNA interaction. The presence of a trans-BPDE-N
2
-dG adduct near the TFIID binding site and thus, an expected bending of the helix, has no obvious effect on protein binding or the further assemblance of the transcription initiation complex.
Hepatocytes and liver microsomes isolated from 3-methylcholanthrene-treated rats metabolize benzo(a)pyrene to products that bind to endogenous DNA and exogenously added calf thymus DNA, respectively. ...By using a sensitive fluorescence technique, it has been possible to characterize the major DNA-binding metabolite in hepatocytes as being produced by further metabolism of 9-hydroxybenzo(a)pyrene. In microsomes, two products binding to calf thymus DNA were recovered, a major species formed by activation of 9-hydroxybenzo(a)pyrene and a minor fraction formed by further metabolism of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. The available evidence indicates that the ultimate products responsible for binding to DNA were identical to 9-hydroxybenzo(a)pyrene 4,5-oxide and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide, respectively. Our data further suggest that metabolic activation of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene results in quite different DNA:metabolite complexes. The former product(s) seems to be strongly associated with hydrophobic regions in DNA, whereas the latter metabolite(s) appears to be more exposed to the exterior.
The association kinetics of RecA protein from Escherichia coli to DNA is strongly enhanced if even a minor fraction of DNA bases has been modified by a carcinogenic (+)-anti metabolite of ...benzoapyrene (BPDE). The enhancement is much smaller with the less carcinogenic (-)-anti enantiomer of BPDE suggesting the possibility that the RecA protein binds selectively to the proto-oncogenic target. Most importantly, the binding of RecA to DNA modified with the latter enantiomer is found to give rise to a reorganization of this BPDE adduct from an intercalation site into a minor groove site. This indicates that the binding mechanism of RecA is via intercalation of some amino acid moiety, a discovery that could explain the approximately 50% contour length increase of the DNA within its fibrous complex with RecA.
Sodium sulfite, a hydrolysis product of the environmental pollutant sulfur dioxide increased the activation of (−)-
trans-7,8-dihydroxy-7,8-dihydrobenzo
apyrene (BP-7,8-diol) to the (+)
...anti-enantiomer of
trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo
apyrene (BPDE) in phorbol myristate acetate (PMA)-stimulated human polymorphonuclear leukocytes (PMNs). This effect was potentiated in the presence of DMSO. No significant effect of sulfite on BP7,8-diol activation was observed in resting leukocytes. As revealed by the
32P-postlabelling technique the dominant adduct in both intracellular DNA and to DNA added to the leukocytes was (+)-
anti-BPDE bound to the exocyclic nitrogen of deoxyguanosine. The mechanism underlying the stimulatory effect of sulfite on diol epoxide production and increased DNA-binding probably involves one-electron oxidation of sulfite to a sulfur trioxide radical anion and subsequent reaction with molecular oxygen to form the corresponding peroxyl radical. This step obviously requires PMA-initiated oxidative burst and thus, production of Superoxide radical anions (O
2⨪).
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