Metabolic activation of benzo[a]pyrene in human skin maintained in short-term organ culture

The metabolic activation of benzo[a]pyrene (BP) was examined in six samples of human skin after topical application of the hydrocarbon to the skin in short-term organ culture. The results show that all of the samples were capable of metabolizing BP to water-soluble products and to ether-soluble products that included the 4,5-, 7,8- and 9,10-dihydrodiols and a product which had chromatographic properties identical with those of authentic trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (BP-11,12-diol). The major BP-deoxyribonucleoside adduct detected in each skin sample appeared to be formed from the reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BP-7,8-diol 9,10-oxide) with deoxyguanosine residues in DNA.     

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane.

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol x min(-1) x pmol(-1) CYP1A1 of the enantiomeric promutagens (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1.     

Some properties of vicinal diol-epoxides derived from benzo(a)anthracene and benzo(a)pyrene

The alkylating properties of pairs of syn- and anti-isomers of 2 diol-epoxides derived from benzo(a)pyrene (BP) and of 1 derived from benz(a)anthracene (BA) have been investigated. Of the anti-diol-epoxides, anti-BP 7,8-diol-9,10-oxide was the most reactive compound towards DNA, towards sodium p-nitrothiophenolate in a non-aqueous solvent system, and towards 4-(p-nitrobenzyl)pyridine in aqueous solution; anti-BP 9,10,-diol-7,8-oxide was of intermediate reactivity and anti-BA 8,9-diol-10,11-oxide was least reactive. The syn-diol-epoxides gave unsatisfactory results with DNA and 4-(p-nitrobenzyl)pyridine because of their rapid solvolysis in aqueous solution, but with sodium p-nitrothiophenolate showed the order of reactivity syn-BP 7,8-diol-9,10-oxide greater than syn-BA 8,9-diol-10,11-oxide greater than syn-BP 9,10-diol-7,8-oxide. The products of the reaction between diol-epoxides and nucleic acids were examined by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC) and the diol-epoxides were shown to react principally with the guanosine and adenosine moieties of RNA.     

Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.     

HPLC analysis of benzo[a]pyrene-albumin adducts in benzo[a]pyrene exposed rats. Detection of cis-tetrols arising from hydrolysis of adducts of anti- and syn-BPDE-III with proteins

Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

A new type of biological chemiluminescence: The microsomal chemiluminescence of benzo[a]pyrene arises from the diol epoxide product of the 7,8-dihydrodiol

The chemiluminescence, CL, accompanying the metabolism of the carcinogen benzo[a]pyrene, BP, by the aryl hydrocarbon hydroxylase system is a new type of low intensity biological chemiluminescence. It is the result of spontaneous oxygenation of a specific reactive metabolic intermediate; not inhibitable by superoxide dismutase or catalase. The reactive metabolite is the strongly mutagenic 7,8-dihydrodiol-9,10-epoxide, produced enzymatically from the 7,8-dihydrodiol precursor. Hydroxylation of benzo[a]pyrene at the 3 position does not lead to chemiluminescent emission; the CL quantum yields of BP and 3-OH-BP are the same. The CL quantum yields of microsomal metabolism of (?) 7,8-diol-BP and the racemic 7,8-diol-BP are identical. The kinetics of CL of the latter show a much faster initial reaction rate, correlating with the greater reactivity of diol epoxide I formed from (+) 7,8-diol-BP. CL may therefore be used to follow the pathways and the rates of production of the mutagenic diol epoxides of BP.     

Absolute configuration of trans-7,8-dihydroxy-7,8-dihydro-7-methylbenzo[a]pyrene enantiomer and the unusual quasidiequatorial conformation of the diacetate and dimenthoxyacetate derivatives

The enantiomers of trans-7,8-dihydroxy-7,8-dihydro-7-methylbenzo[a]pyrene (7-MBaP 7,8-dihydrodiol) and of trans-7,8-dihydroxy-7,8,9,10-tetrahydro-7-methylbenzo[a]pyrene (7-MBaP 7,8-tetrahydrodiol) were directly resolved by high-performance liquid chromatography (HPLC) using a commercially available column packed with an (R)-N-(3,5-dinitrobenzoyl)-phenylglycine derivative of gamma-aminopropylsilanized silica. The absolute configurations of the resolved enantiomers were determined by the exciton chirality method. Circular dichroism (CD) spectral analysis of the quasidiequatorial benzo[a]pyrene 7R,8R-dihydrodiol enantiomer and its diacetate and dimenthoxyacetate derivatives indicated conformational changes were induced upon derivatization. However, the characteristic CD Cotton effects of the quasidiequatorial 7-MBaP 7,8-dihydrodiol and its diacetate and dimenthoxyacetate derivatives were similar indicating that the conformation of 7-MBaP trans-7,8-dihydrodiol was not altered upon derivatization. Proton nuclear magnetic resonance (NMR) spectral analyses confirmed that 7-MBaP 7,8-dihydrodiol, its diacetate and dimenthoxyacetate derivatives all have quasidiequatorial conformations. The results indicate that the methyl substituent of 7-MBaP 7,8-dihydrodiol maintains a quasiaxial position regardless of the size of the acyl derivatives linked to the hydroxyl groups.     

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     

Differential nucleotide excision repair susceptibility of bulky DNA adducts in different sequence contexts: hierarchies of recognition signals

The structural origin underlying differential nucleotide excision repair (NER) susceptibilities of bulky DNA lesions remains a challenging problem. We investigated the 10S (+)-trans-anti-[BP]-N(2)-2'-deoxyguanosine (G*) adduct in double-stranded DNA. This adduct arises from the reaction, in vitro and in vivo, of a major genotoxic metabolite of benzo[a]pyrene (BP), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, with the exocyclic amino group of guanine. Removal of this lesion by the NER apparatus in cell-free extracts has been found to depend on the base sequence context in which the lesion is embedded, providing an excellent opportunity for elucidating the properties of the damaged DNA duplexes that favor NER. While the BP ring system is in the B-DNA minor groove, 5' directed along the modified strand, there are orientational distinctions that are sequence dependent and are governed by flanking amino groups [Nucleic Acids Res.35 (2007), 1555-1568]. To elucidate sequence-governed NER susceptibility, we conducted molecular dynamics simulations for the 5'-...CG*GC..., 5'-...CGG*C..., and 5'-...TCG*CT... adduct-containing duplexes. We also investigated the 5'-...CG*IC... and 5'-...CIG*C... sequences, which contain "I" (2'-deoxyinosine), with hydrogen replacing the amino group in 2'-deoxyguanosine, to further characterize the structural and dynamic roles of the flanking amino groups in the damaged duplexes. Our results pinpoint explicit roles for the amino groups in tandem GG sequences on the efficiency of NER and suggest a hierarchy of destabilizing structural features that differentially facilitate NER of the BP lesion in the sequence contexts investigated. Furthermore, combinations of several locally destabilizing features in the hierarchy, consistent with a multipartite model, may provide a relatively strong recognition signal.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

Absolute configuration of cis-5,6-dihydrodiol enantiomers derived from helical conformers of 1,12-dimethylbenz[a]anthracene

1,12-Dimethylbenz[a]anthracene (1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBA with osmium tetroxide in pyridine in low yield (less than or equal to 3%) and was purified by sequential use of reversed-phase and normal-phase HPLC. Two pairs of 1,12-DMBA cis-5,6-dihydrodiol enantiomers, derived from P (right-handed helix) and M (left-handed helix) conformers, were eluted as a single chromatographic peak on both reversed-phase and normal-phase HPLC. However, these four enantiomers were resolved by sequential use of two chiral stationary phase (CSP) HPLC columns. CSP (Pirkle type I) columns were packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine, which is ionically bonded to gamma-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6-dihydrodiol enantiomers of 4-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene with known absolute stereochemistry.     

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.     

Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.

Carcinogenic benzo[a]pyrene (BP) is generally considered to show genotoxicity by forming DNA adducts of its metabolite, BP-7,8-diol-9,10-epoxide. We investigated oxidative DNA damage and its sequence specificity induced by BP-7,8-dione, another metabolite of BP, using (32)P-5'-end-labeled DNA. Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of 5'-TG-3' sequence and at poly(C) sequences, in DNA incubated with BP-7,8-dione in the presence of NADH and Cu(II), whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. BP-7,8-dione strongly damaged the G and C of the ACG sequence complementary to codon 273 of the p53 gene. Catalase and a Cu(I)-specific chelator attenuated the DNA damage, indicating the involvement of H(2)O(2) and Cu(I). BP-7,8-dione with NADH and Cu(II) also increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation. We conclude that oxidative DNA damage, especially double base lesions, may participate in the expression of carcinogenicity of BP in addition to DNA adduct formation.     

The effect of manganese and ferric ions on the in vitro formation of dihydrodihydroxy metabolites of benzo(a)pyrene

The effect of ferric and manganese ions on the in vitro metabolism of benzo(a)pyrene (BP) to dihydrodihydroxy (diol) metabolites by rat liver microsomal preparations was studied. Of the 3 diols separated by high-pressure liquid chromatography (HPLC) and called diols 1, 2 and 3 in order of elution, diol 1 was identified by its U.V. spectrum as the 9,10-diol; diols 2 and 3 have not yet been identified positively but are probably the 4,5- and 7,8-diols respectively. Higher concentrations of both metals altered the diol profile; 10 and 50 mumol Fe3+ per incubation caused the disappearance of diols 1 and 2 and an increase in diol 3; 10 mumol Mn2+ caused a significant decrease in diol 2 while 50 mumol reduced diol 2 to a negligible amount and inhibited the formation of diol 1; both concentrations caused a relative increase in diol 3. If the tentative identification of diol 3 as the 7,8-diol is correct, manganese and ferric ions could be significant in the metabolism of BP to the active metabolite, the 7,8-diol-9,10-epoxide.     

Action of some retinol derivatives and their provitamins on microsome-catalyzed formation of benzo[a]pyrene-DNA adduct

Several vitamin A compounds have been tested for their ability to suppress formation of DNA adduct by the carcinogen benzo[a]pyrene (B[a]P) in an in vitro reaction catalyzed by rat liver microsomes. Retinol, retinal, 3-dehydroretinol and 3-hydroxyretinol were found to be effective inhibitors of adduct formation. Certain carotenoids that are precursors of these retinoids also displayed considerable inhibitory capacity. Carotenoids and the 3-substituted retinoids appeared to modulate the DNA adduct formation exclusively through their action on microsomal enzymes, since an effective inhibition in each case was observed on the formation of B[a]P-7,8-diol, a proximate carcinogenic metabolite of B[a]P. Unsubstituted retinoids, on the other hand, had marginal effect on enzymes but were found effective in accelerating inactivation of B[a]P-7,8-diol-9,10-epoxide, the ultimate carcinogenic metabolite that binds to DNA.     

A comparison of the intercalative binding of non-reactive benzo[a]pyrene metabolites and metabolite model compounds to DNA

The reversible DNA physical binding of a series of non-reactive metabolites and metabolite model compounds derived from benzo[a]pyrene (BP) has been examined in UV absorption and in fluorescence emission and fluorescence lifetime studies. Members of this series have steric and pi electronic properties similar to the highly carcinogenic metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and the less potent metabolite 4,5-epoxy-4,5-dihydrobenzo(a)pyrene (4,5-BPE). The molecules examined are trans-7,8-dihydroxy-7,8-dihydrobenzo[a]-pyrene (7,8-di(OH)H2BP), 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (tetrol) 7,8,9,10-tetrahydrobenzo[a]pyrene (7,8,9,10-H4BP), pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (4,5-di(OH)H2BP) and 4,5-dihydrobenzo[a]pyrene (4,5-H2BP). In 15% methanol at 23 degrees C the intercalation binding constants of the molecules studied lie in the range 0.79-6.1 X 10(3) M-1. Of all the molecules examined the proximate carcinogen 7,8-di(OH)-H2BP is the best intercalating agent. The proximate carcinogen has a binding constant which in UV absorption studies is found to be 2.8-6.0 times greater than that of the other hydroxylated metabolites. Intercalation is the major mode of binding for 7,8-di(OH)H2BP and accounts for more than 95% of the total binding. Details concerning the specific role of physical bonding in BP carcinogenesis remain to be elucidated. However, the present studies demonstrate that the reversible binding constants for BP metabolites are of the same magnitude as reversible binding constants which arise from naturally occurring base-base hydrogen bonding and pi stacking interactions in DNA. Furthermore, previous autoradiographic studies indicate that in human skin fibroblasts incubated in BP, pooling of the unmetabolized hydrocarbons occurs at the nucleus. The high affinity of 7,8-di(OH)H2BP for DNA may play a role in similarly elevating in vivo nuclear concentrations of the non-reactive proximate carcinogen.     

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.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.     

Stereoselective aspects of the metabolic activation of benzo[a]pyrene by human skin in vitro

Benzo[a]pyrene (BP) is activated within tissues in both a regio- and a stereoselective manner and, since human skin is sensitive to tumour induction by polycyclic aromatic hydrocarbons (PAH), the steroselective metabolism of BP in this tissue has been investigated. Samples of skin from eleven individuals were treated with [3H]BP in short-term organ culture. Two samples were also treated with mixtures of [14C](+)- and (-)-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) in varying proportions. Following application of [3H] BP, more 7,8-dihydrodiol was recovered from the skin itself than from the culture fluid in ten cases; no 7.8-dihydrodiol was detected in extracts from the eleventh. The 7,8-dihydrodiol metabolite was extracted predominantly (range 74-greater than 99%) as the (-)-enantiomer in nine of these ten patients, although proportionately more (+)-enantiomer was recovered from the culture fluid than from the skin in each case. The relative proportions of [3H]BP tetrols derived from syn- and anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE) detected in these extracts was more variable. When skin samples were treated with [14C]BP-7,8-dihydrodiol, more anti- than syn-BPDE-derived tetrols were extracted, irrespective of the optical purity of the dihydrodiol applied. These findings provide evidence for interindividual variations in the stereoselective metabolism of BP, which may be of some importance in determining individual susceptibility to PAH-induced skin carcinogenesis.