Formation and removal of benzo[a]pyrene metabolites--DNA adducts in cultured normal human fibroblasts using a microsome-activating system

The rate of removal of DNA adducts of several benzo[a]pyrene metabolites from nuclear DNA was compared by introducing a microsome-activating system in human fibroblast cells. Confluent human fibroblasts were exposed to benzo[a]pyrene in the presence of a microsomal activating system and DNA adducts were formed in the nuclear DNA. The adducts present in DNA were determined after 1 h of incubation and 48 h later. There was no difference in the rate of removal between 7S- and 7R -N2-[10-(7 beta, 8 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine, 7R -N2-[10(7beta, 8 alpha, 9 beta-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine and the covalent adduct of 9-hydroxybenzo[a]pyrene-4,5-epoxide to guanosine. This finding does not agree with the idea that metabolites forming 'persistent DNA adducts' are always responsible for the carcinogenicity of their parent compound.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

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     

DNA--benzo[a]pyrene adducts formed in a Salmonella typhimurium mutagenesis assay system.

The DNA adducts formed in Salmonella typhimurium when bacteria are incubated with radioactive benzo[a]pyrene and liver microsomal enzymes from several sources has been investigated. When enzyme preparations from Aroclor I254 or 3-methylcholanthrene induced C57BL/6N (B6) mice were used to mediate activation, the predominant product was an adduct between the 10 position of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the N-2 position of deoxyguanosine. Similar results were obtained with human liver and with Aroclor-induced rat-liver enzyme preparations. This adduct is also the major DNA product previously found when human tissues or certain rodent cells were incubated with benzo[a]pyrene. On the other hand, when activation of benzo[a]pyrene was mediated by a phenobarbital-induced B6 mouse-liver enzyme preparation, the extent of binding was quite low and the profile of DNA adducts in S. typhimurium DNA was quite different. Thus, under appropriate conditions, the activation and DNA binding of benzo[a]pyrene inthe microsome mediated S. typhimurium mutagenesis assay generally resembles that seen in intact mammalian cells. Caution must be exercised, however, in the choice of microsome-activation systems.     

Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Resolution of optical isomers by high-pressure liquid chromatography: The separation of benzo[a]pyrene trans-diol derivatives

The optical isomers of (±)r-7,t-8-dihydroxy-7,8-dihydrobenzo[a]pyrene and its synthetic precursor (±)r-7,t-8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene were resolved as their di-(−)menthoxyacetates using high-pressure liquid chromatography. Saponification of the resolved diesters yielded the corresponding enantiomers. The specific rotation, CD spectra, and ORD curves are reported. The resolution of these optical isomers permits detailed studies on the enzymatic intermediates and the mechanism of benzo[a]pyrene activation to its carcinogenic form. The method is of general usefulness for the resolution of optical isomers.     

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.     

Probing the microenvironmental of benzo[a]pyrene diol epoxide-DNA adducts by triplet excited state quenching methods

Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25±1°C were investigated: covalent adducts derived from the reaction of (±)-7β,8α-dihydroxy-9α,10α-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) and of (±)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of K_O2^T≈10⁹ M^?1·s^?1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of K_O2^T is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of K_O2^T≈10⁸ M^?1·s^?1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The K_O2^T values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1·10^?4?3·10^?3 M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of K_O2^T for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.     

Non-covalent intercalative binding of 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene to DNA

When the benzo(a)pyrene diol epoxide (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is mixed into a DNA solution, a 10nm red shift in the absorption maximum of BPDE appears at 354nm which is due to a non-covalent intercalation complex. The major reaction pathway at this intercalation site is the hydrolysis of BPDE to its tetraol which is accompanied by a decrease in the absorbance and a shift from 354 to 353nm (the latter is due to intercalated tetraol). The non-covalent binding constants are approximately 8200M^?1 for BPDE and 3300M^?1 for the tetraol at 25°C, pH 7.0. Covalent adduct formation between BPDE and DNA occurs either at another, external binding site, or after some rearrangement of the intercalated BPDE, since covalent adducts display a 345nm absorption maximum (2nm red shift only).     

Induction of mutations by chemical agents at the hypoxanthine-guanine phosphoribosyl transferase locus in human epithelial teratoma cells

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N′-nitro-N-nitrosoguanidine (MNNG); 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 × 10⁴ cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4–6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5′-monophosphate (IMP)/min/μg protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polyclinic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 μM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10⁶ colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.     

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.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

Antioxidative and antitumor promoting effects of [6]-paradol and its homologs

Benzo[a]pyrene diol epoxide, a metabolite of benzo[a]pyrene (BaP), and chlorohydrin, the reaction product of chloride and the epoxide, form in vitro the same trans- and cis-stereoisomeric DNA adducts, but in different proportions. In this study, we asked whether the DNA adduct concentration can be kept the same by applying the appropriate dose of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)and (+/-)-7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-BPDCH) to rodent skin and whether the DNA adducts formed differ only in their trans- and cis-stereoisomerism. Skin from C57Bl6 mice, spontaneous hypertension rats (SHR) and Sprague-Dawley (SD) rats was treated ex vivo immediately after the death of the animals with anti-BPDE and its corresponding bay region chlorohydrin trans-BPDCH and the epidermis was analyzed for DNA adducts 1h after the application. We found that adduct formation at the exocyclic amino groups of deoxyguanosine and deoxyadenosine in epidermal DNA followed a linear dose-response within 6--100 nmol/cm(2) with both chemicals. In order to achieve the same adduct concentration in mouse, spontaneous hypertension rat (SHR), and Sprague-Dawley (SD) rat skin, respectively, a 37-, 23- and 10-fold lower dose of anti-BPDE than of trans-BPDCH had to be applied. The order of 2'-deoxyguanosine (dGuo) adduct concentration with anti-BPDE was similar to what has been reported, but the order with trans-BPDCH was (+)-cis-BPDE-N(2)-dGuo adduct>(+)-trans-BPDE-N(2)-dGuo=(-)-trans-BPDE-N(2)-dGuo>(-)-cis-BPDE-N(2)-dGuo in mouse skin. Irrespective of species or strain, a significantly higher proportion of cis-adducts was obtained after treatment with trans-BPDCH than after treatment with anti-BPDE. Therefore, DNA adduct concentration can be kept the same by applying the appropriate dose of anti-BPDE and trans-BPDCH to rodent skin and the DNA adducts formed differ only in their trans- and cis-stereoisomerism.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N²-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts

The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF). For this purpose NF were prelabeled in their DNA with [14C]-thymidine and treated with [3H]BPDE I. The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine. The adduct composition did not change significantly in 24-h post treatment incubation. Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis. The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p. chromatosomal DNA and in total nuclear DNA. From these data the concentration in nucleosomal linker-DNA was calculated. The initial adduct distribution was non-random and 6.3 times higher in 47 b.p. linker-DNA relative to 145 b.p. core-DNA and 9.2 times higher in 27 b.p. linker-DNA relative to 165 b.p. chromatosomal DNA. Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA. After this early phase the adducts located in 145 b.p. core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period. In contrast, further adduct removal was observed from nucleosomal linker-DNA.     

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.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

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.