The kinetics of benzo(a)pyrene anti-7,8-dihydrodiol 9, 10-epoxide formation from benzo(a)pyrene and regulatory membrane effects

r-7,c-10,t-8,t-9-Tetrahydroxybenzo(a)pyrene (7,10/8,9-tetrol), which is the principal hydrolysis product of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-diol-epoxide), was resolved and measured by HPLC in organic extracts of incubations which contained induced rat liver microsomes and BP. Kinetic analyses showed that: (a) following a 5- to 7-min lag period, anti-diol-epoxide formation was linear, and (b) levels of anti-diol-epoxide formed were highly dependent upon the starting BP concentration. anti-Diol-epoxide production increased at starting BP concentrations of 0–12 μm and decreased in incubations containing 12–25 μm BP. However, between 25 and 100 μm BP, anti-diol-epoxide formation was stable at a level representing 65% of the peak production which occurred at a starting BP concentration of 12 μm. BP oxidation was competitively inhibited by (?)-trans-BP-7,8-dihydrodiol and about five times less effectively by the (+)-trans-BP-7,8-dihydrodiol. The inability of a severalfold excess of BP (25–100 μm) to totally inhibit BP-7,8-dihydrodiol oxidation was explained by the presence of a microsomal substrate compartment which was saturated at only 6–8 μm BP, the remaining BP present as aggregates in the aqueous compartment. Purification of microsomes by Sepharose 2B gel filtration after reaction with [³H]BP also indicated that BP-7,8-dihydrodiol was preferentially concentrated in the microsome compartment leading to a net increase in the ratio of BP-7,8-dihydrodiol to BP in the microsomal compartment, which favored BP-7,8-dihydrodiol oxidation to yield the biologically active anti-diol-epoxide.     

The interaction of platinum II compounds with bacteriophages T7 and R17

The inactivation of the DNA-containing bacteriophage T7 and of the RNA-containing bacteriophage R17 by dichloro-ethylenediamine Pt II and by cis- and trans-dichloro-diammine Pt II has been studied under a variety of experimental conditions. It has been found that the mean lethal concentrations vary for T7 from 0.007 to 0.5 μg/ml and for R17 from 0.09 to 1.1 μg/ml depending upon which compound is used and the manner in which the experiment is performed. Experiments with the ¹⁴C-labelled ethylenediamine derivative have shown that inactivation is primarily related to the extent of reaction of the compound with the bacteriophage and that variations in inactivation observed under different experimental conditions simply reflect differences in the kinetics of the binding reactions. Quantitative comparisons have shown that at the mean lethal dose 1.5 molecules of platinum compound are bound to each R17 bacteriophage and 5 molecules to each T7.96% of the compound bound to R17 was bound to RNA and 76% of the compound bound to T7 was bound to the DNA.     

Oxidation of benzo(a)pyrene and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene by horseradish peroxidase-H2O2 intermediate: fluorometric study

The capacity of oxidation of benzo(a)pyrene (BP) and its analog to be oxidized by peroxidases in several tissues has been studied. The kinetics of the horseradish peroxidase (HRP) oxidation of BP and 7,8-dihydro-7,8-dihydroxy benzo(a)pyrene (BP-7,8-diol) were examined. Effective ratios of H2O2 and HRP for catalytic oxidation were 13.74 for BP and 4.58 for BP-7,8-diol. The maximum ratio was approximately 90 for both hydrogen donors (BP and BP-7,8-diol) to the ES complex. The maximum ratio of oxidized BP and BP-7,8-diol to HRP was 5.7. Ks values for H2O2 were 1.68 and 6.35 microM for BP and BP-7,8-diol, respectively. The mean values of the rate constants, k5, for the oxidation of BP and BP-7,8-diol were 0.56 X 10(5) M-1 sec-1 and 4.1 X 10(5) M-1 sec-1, respectively, at low concentrations. At low concentrations a Hill plot of the oxidation of BP showed a negative value (nH = 0.5) and at high concentrations nH = 1.0. On the other hand, that of BP-7,8-diol showed positive cooperativeness (nH = 1.8). These oxidation reactions caused substrate (donor) inhibition at high concentrations. The inhibition constants, KA', were 9.8 and 5.65 microM for BP and BP-7,8-diol, respectively. The reactivity of the oxidation of BP-7,8-diol was five to six times larger than that of BP.     

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Residue R216 and catalytic efficiency of a murine class alpha glutathione S-transferase toward benzo[a]pyrene 7(R),8(S)-diol 9(S), 10(R)-epoxide

Murine class alpha glutathione S-transferase A1-1 (mGSTA1-1), unlike mammalian class alpha GSTs, is the most efficient in the glutathione (GSH) conjugation of the ultimate carcinogenic metabolite of benzo[a]pyrene, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] [Hu, X., Srivastava, S. K., Xia, H., Awasthi, Y. C., and Singh, S. V. (1996) J. Biol. Chem. 271, 32684-32688]. Here, we report the crystal structures of mGSTA1-1 in complex with GSH and with the GSH conjugate of (+)-anti-BPDE (GSBpd) at 1.9 and 2.0 A resolution, respectively. Both crystals belong to monoclinic space group C2 with one dimer in the asymmetric unit. The structures reveal that, within one subunit, the GSH moiety interacts with residues Y8, R14, K44, Q53, V54, Q66, and T67, whereas the hydrophobic moiety of GSBpd interacts with the side chains of F9, R14, M207, A215, R216, F219, and I221. In addition, the GSH moiety interacts with D100 and R130 from the other subunit across the dimer interface. The structural comparison between mGSTA1-1.GSH and mGSTA1-1.GSBpd reveals significant conformational differences. The movement of helix alpha9 brings the residues on the helix into direct interaction with the product. Most noticeable are the positional displacement and conformational change of R216, one of the residues located in helix alpha9. The side chain of R216, which points away from the H-site in the mGSTA1-1.GSH complex, probes into the active site and becomes parallel with the aromatic ring system of GSBpd. Moreover, the guanidinium group of R216 shifts approximately 8 A and forms a strong hydrogen bond with the C8 hydroxyl group of GSBpd, suggesting that the electrostatic assistance provided by the guanidinium group facilitates the ring-opening reaction of (+)-anti-BPDE. The structure of mGSTA1-1. GSBpd is also compared with those of hGSTP1-1[V104,A113].GSBpd, hGSPA1-1.S-benzylglutathione, and mGSTA4-4. 4-S-glutathionyl-5-pentyltetrahydrofuran-2-ol. The comparison provides further evidence that supports the functional roles of R216 and helix alpha9. The lack of mobility of helix alpha9 and/or the lack of electrostatic assistance from R216 may be responsible for the relatively lower activity of hGSTA1-1, mGSTA4-4, and hGSTP1-1 toward (+)-anti-BPDE.     

Induction,modification and inhibition of rat liver microsomal benzo(a)pyrene hydroxylase; Correlation with the S-9-mediated mutagenicity of benzo(a)pyrene

The effects of various pretreatments $\text{in vivo}$ (3MC, PB, 2 and 4FAA) and of various inhibitors $\text{in vitro}$ (7,8 BF, SKF525A and MN ^R) on the activity of rat liver microsomal BP hydroxylase were analyzed and correlated with the S-9 mediated mutagenicity of BP. 3MC is the only treatment which both induces and modifies the hydroxylase activity; it also specifically increases the enzyme mediated mutagenicity. Miconazole ^R which inhibits all the tested microsomal preparations, also reduces the mutagenicity mediated by all the S-9 preparations whereas the inhibitory effects of 7,8 BF and SKF525A are limited respectively to enzyme preparations from 3MC induced and control or PB treated rats.     

The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

Metabolic fate of glutathione conjugate of benzo[a]pyrene-(7R,8S)-diol (9S,10R)-epoxide in human liver.

Benzo[a]pyrene-(7R,8S)-diol (9S,10R)-epoxide [(+)-anti-BPDE] is believed to be the activated form of the widely spread environmental pollutant benzo[a]pyrene. Glutathione (GSH) S-transferase (GST)-catalyzed conjugation of (+)-anti-BPDE with GSH is an important mechanism in its cellular detoxification. Here, we report that the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is a potent inhibitor (K(i) 15 microM) of class Mu human GST isoenzyme, which, among human liver GSTs, is a highly efficient detoxifier of (+)-anti-BPDE. Thus, the inhibition of GST activity by (-)-anti-BPD-SG may hinder GSH conjugation of (+)-anti-BPDE, unless the conjugate is metabolized and/or eliminated. The results of the present study show that gamma-glutamyltranspeptidase (gamma-GT) can metabolize (-)-anti-BPD-SG at a rate of about 0.29 nmol/min/mg protein. Our studies also show that (-)-anti-BPD-SG is transported across the human canalicular liver plasma membrane (cLPM) in an ATP-dependent manner at a rate of about 0.33 nmol/min/mg protein. The ATP-dependent transport of (-)-anti-[(3)H]BPD-SG across human cLPM follows Michaelis-Menten kinetics (K(m) 84 microM; V(max) 0.33 nmol/min/mg). In conclusion, the results of the present study suggest that both gamma-GT-mediated metabolism and ATP-dependent canalicular transport may be important steps in overall detoxification of (+)-anti-BPDE in the human liver.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

Metabolism of benzo[a]pyrene and 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a pyrene in lung and liver of newborn mice

Although the newborn mouse has been extensively used to test the tumorigenic activities of polynuclear aromatic hydrocarbons and their diol epoxide metabolites, no information is available on their metabolism in the newborn mouse in vivo. Therefore, we have investigated the metabolism and distribution of [3H]benzo[a]pyrene ([3H]BaP) and (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene ([3H]BPDE) in liver and lung of mice given i.p. injections of these compounds on their 1st, 8th and 15th days of life. In lung, identified metabolites of [3H]BaP included diols, quinones, and phenols. Their levels were higher on the 1st day compared to the 8th and 15th days of life. The pattern of organic extractable metabolites detected in mouse liver was different from that in lung, being dominated by unidentified polar metabolites, the levels of which increased with age. Levels of [3H]BPDE in liver and lung were measured by trapping with 2-mercaptoethanol. It was demonstrated that [3H]BPDE rapidly reaches the lung after i.p. injections. The half-lives of [3H]BPDE in lung and liver were similar to those observed in vitro. The results are discussed with respect to the known tumorigenic activities of BaP and BPDE in newborn mice and in mouse skin.     

Benzo(a)pyrene 7,8-dihydrodiol-9,10-oxide modification of DNA: Relation to chromatin structure and reconstitution

Purified duck reticulocyte DNA was incubated in vitro with a 7,8-dihydrodiol-9,10-oxide derivative of benzo(a)pyrene (BPDE). The carcinogen-modified DNA was somewhat more susceptible to partial digestion by the single strand specific endonuclease S₁ than unmodified DNA, suggesting slight denaturation of the helix at sites of modification. Chromatin was reconstituted in vitro utilizing this carcinogen-modified DNA and unmodified-chromatin associated proteins. This reconstituted chromatin showed the same kinetics and extent of digestion by Staphylococcal nuclease, and similar nucleosome profiles on sucrose density gradient centrifugation, as those obtained with native chromatin or chromatin reconstituted with unmodified DNA. Moreover, polyacrylamide gel electrophoresis of DNA fragments obtained from nuclease digests gel electrophoresis of DNA fragments obtained from nuclease digests of the reconstituted chromatins suggested that the chromatin containing carcinogen-modified DNA had the same subnucleosome structure as that reconstituted with unmodified DNA. In a separate set of studies intact duck reticulocyte chromatin was reacted directly with BPDE. Nuclease digestion studies indicated that 65% of the carcinogen was bound to the ‘open’ regions of chromatin, and 35% to ‘closed’ regions.These results indicate that although covalent binding of a benzo(a)pyrene (BP) derivative to DNA produces local distortions in conformation of the helix, this modification does not appear to interfere with the ability of the DNA to associate with histones to form nucleosome structures. In addition, although DNA in the open regions of chromatin is more susceptible to reaction with the BP derivative, there is appreciable reaction with the DNA associated with histones.     

Modification of glutathione levels in C3H/10T1/2 cells and its relationship to benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide-induced cytotoxicity

Levels of reduced glutathione (GSH) in C3H/10T1/2 cells were selectively altered to determine what quantitative role GSH transferase-catalyzed conjugation plays in regulating the cytotoxic effects of benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene, anti-diol epoxide). A 65% decrease in 10T1/2 cell GSH content from 0.16 mM (control cell GSH concentration) to 0.06 mM was accompanied by a 46% decrease in the anti-diol epoxide LD80; a 98% increase in GSH content resulted in a 44% increase in anti-diol epoxide LD80. This nonlinear relationship between changes in cellular GSH concentration and anti-diol epoxide LD80 was directly relatable to the nonlinear change in the rate of anti-diol epoxide conjugation which was catalyzed by 10T1/2 cell GSH transferases. Purified 10T1/2 cell cytosol catalyzed the GSH conjugation of anti-diol epoxide to yield a GSH conjugation product with a distinct UV absorbance spectrum; the apparent GSH Km for this cell cytosol-catalyzed reaction was 0.20 mM. Variations in the cellular GSH concentration around the GSH Km resulted in a nonlinear change in the amount of anti-diol epoxide-GSH conjugate formed, and a reciprocal change in the amount of free anti-diol epoxide available for cytotoxic alkylation events. These results clarify in quantitative, biochemical terms how GSH transferase-catalyzed conjugation can regulate the level of an electrophilic carcinogen metabolite in a biological system.     

The microsomal metabolism of benzo(a) pyrene phenols.

Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region. Based on the chromatographic retention time, absorbance, and fluorescence spectra, the two major products of 9-hydroxybenzo(a)pyrene metabolism may be diphenols. The existence of spectral intermediates which resemble phenols rather than quinones during the steady-state metabolism of 3-hydroxybenzo(a)pyrene strongly indicates that either the major product is a diphenol which slowly oxidizes to yield 3,6-quinone and/or that an active quinone reductase exists in liver microsomes.