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.     

Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats

The daily i.t. administration of benzo[a]pyrene (BP) to Sprague-Dawley rats, for 3 consecutive days, did not cause any toxicity or clastogenicity in bone marrow cells, as evaluated by monitoring the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes. However, BP produced a considerable enhancement of binucleated and micronucleated pulmonary alveolar macrophages, as well as a significant increase in polymorphonucleates recovered by bronchoalveolar lavage. These effects were prevented by administering the thiol N-acetylcysteine (NAC) by gavage 5 h before each BP instillation. In addition, the i.t. treatment with BP resulted in the formation of BP diolepoxide (BPDE)-DNA adducts in lungs and liver, as assessed by synchronous fluorescence spectrophotometry, with fluorescence peaks of similar magnitude in the 2 tissues. Pretreatment with NAC by gavage completely prevented BPDE adducts to liver DNA and significantly decreased those to lung DNA.     

The irreversible binding of benzo[a]pyrene to rat liver macromolecules in vivo and in vitro: effects of agents that influence benzo[a]pyrene metabolism

The present study was carried out to determine the effects of agents that influence benzo[a]pyrene (BP) metabolism in vitro on the irreversible binding of BP to rat hepatic macromolecules in vivo. The irreversible binding of [3H]BP was found to be both dose and time dependent after its intraperitoneal administration to male Wistar rats. The SKF 525-A, at doses of 50 and 75 mg/kg, ip 3 h before BP, decreased the level of binding from control by 31 and 34%, respectively. At 35 mg/kg, SKF-525-A had no effect. Diethyl maleate (0.6 mL/kg, ip) and cysteine (150 mg/kg, ip), 30 and 5 min before BP, respectively, did not alter the binding of BP from control. Oral methadone treatment, previously shown to increase selectively epoxide hydrase activity in male Wistar rats, also failed to alter the amount of BP bound to hepatic macromolecules. 3-Methylcholanthrene (20 mg/kg per day, ip, for 2 days) administered 24 h before BP, decreased the level of binding from control by 30%. Parallel in vitro studies were carried out with the various agents used in vivo.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Method for estimation of benzo[a]pyrene DNA adducts.

Polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (B(a)P) are known carcinogens/mutagens. These compounds may be metabolized by the P450 mixed function monooxygenase to more nucleophilic compounds which may form adducts to the cellular macromolecules, e.g., DNA, RNA, and proteins. We have used synchronous fluorescence scanning for the assay of DNA adduct formation. In our earlier work with in vitro exposed human lymphocytes we estimated the adduct formation (femtomoles B(a)P per microgram DNA) to be higher than that estimated by other workers. We suggested that this difference may be related to the DNA isolation method used. In order to elucidate these differences we compared DNA adduct formation in human lymphocytes where DNA was isolated by the two different methods, i.e., using phenol extraction or the Gene Clean method. The data demonstrate that the phenol extraction procedure gives a yield of adducts per microgram DNA lower than that obtained by the Gene Clean method. The principle of the Gene Clean method for DNA isolation is protein denaturation by means of NaI followed by catching of DNA by absorption on silica particles. In contrast, the phenol extraction method is based upon phenol-mediated denaturation of proteins in the cell lysate leaving the hydrophilic nucleotides in the aqueous phase. However, during adduct formation more lipophilic adducts derived from DNA may redistribute between the aqueous phase and the phenol phase. In support of this theory we found higher adduct concentration per microgram DNA by the Gene Clean method 40 to 60 times than that found by the phenol method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of activated oxygen species in benzo[a]pyrene:DNA adduct formation in vitro.

The role of several activated oxygen species in the oxidation and binding of B[a]P to calf thymus DNA in vitro was investigated. B[a]P was reacted with calf thymus DNA in the presence and absence of scavengers of active oxygen species. Reactions were performed in the dark at 37 degrees C for 30 min in a buffered aqueous solution with 250 micrograms of calf thymus DNA. The levels of B[a]P:DNA adducts formed were determined using the 32P-postlabeling assay. B[a]P:DNA adduct levels ranged from 1.5-2.6 and 0.25 pmol adducts/mg DNA in reactions with 120 or 12 nmol of B[a]P, respectively. The addition of scavengers of reactive oxygen species to reaction mixtures resulted in a considerable decrease in the levels of DNA adducts formed in comparison to control reactions. Reactions performed with 500 units catalase or 100 units superoxide dismutase significantly inhibited DNA adduct formation. In these reactions adduct levels were 32 and 48% of control levels, respectively. The addition of both catalase and superoxide dismutase to reactions inhibited adduct formation by 95% relative to control reactions. A decrease in adduct levels was also observed when reactions were performed with citrate-Fe3+ chelate, a scavenger of superoxide. In reactions with 50 mM mannitol and 50 mM sodium benzoate, both of which are hydroxyl radical scavengers, adduct formation was significantly inhibited with adduct levels being 30 and 51% of control values, respectively. Adduct levels were decreased to 26% of control values in reactions with 10 mM 2,5-dimethylfuran, a scavenger of singlet oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Analysis of benzo[a]pyrene deactivation mechanisms in rats

The experimental data on the effects of a widespread carcinogen, benzo[a]pyrene (BP), on individual reactions of rats were treated using mathematical-statistical methods. The individual reactions were analyzed in dependence of doses and modes of administration (single or chronic). The analysis revealed a statistically significant correlation between life span and urinary content of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-BP) in rats treated with BP. The calculated regression equations revealed that the individual sensitivity to carcinogen in case of the BP single administration to rats is mainly determined by efficiency of excretion of the BP active forms out of the organism, whereas after chronic BP administration it is determined by mechanisms of enzymatic deactivation of BP.     

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 DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Epimerization of benzo[a]pyrene-tetrols after acid hydrolysis, implications for determination of benzo[a]pyrene adducts in protein and DNA

Determination of benzo[a]pyrene-DNA or protein adducts with high performance liquid chromatography (HPLC) after acid hydrolysis at high temperature (90 degrees C) enables four isomers of benzo[a]pyrene tetrahydrotetrol to be identified and quantitated. We have investigated the effect of acid treatment of benzo[a]pyrene-tetrahydrotetrol isomers using HPLC and nuclear magnetic resonance spectroscopy (NMR) analysis. By HPLC, we found reversible epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and of (+/-)-benzo[a]pyrene-r-7,t-8,c-9, t-10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,c-9, 10-tetrahydrotetrol, but no interconversion between the two isomer groups. After acid hydrolysis, we found an equilibrium of 87% (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol and 9% (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and 68% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol and 20% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. Minor amounts of two unknown compounds with similar chromatographic characteristics were also found. We have established a NMR method for determination of underivatized (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol confirming the epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10- tetrahydrotetrol. (+/-)-Benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol was treated with aqueous hydrochloric acid in tetrahydro- furan-d8 to give (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol at 57 degrees C while observing the 1H NMR resonances at 500 MHz. Gradient-selected correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to confirm the assignments of the aliphatic hydrogens in the product (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-terahydrotetrol. Thus, when analyzing benzo[a]pyrene-DNA or protein adducts by cleaving the adducts with acid hydrolysis, the only ratio of biological significance is between (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol, due to interconversion (epimerization) at C-10.     

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.     

Protective effect of curcumin and chlorophyllin against DNA mutation induced by cyclophosphamide or benzo[a]pyrene

The current study was carried out to evaluate the potency of curcumin and chlorophyllin as natural antioxidants to reduce the oxidative stress markers induced by cyclophosphamide (CP) and benzo[a]pyrene [B(a)P] which were used as free radical inducers. For this purpose, 126 male albino rats were used. The animals were assigned into 4 main groups: negative control group; oxidant-treated group (subdivided into two subgroups: cyclophosphamide-treated group and benzo[a]pyrene-treated group); curcumin-treated group; and chlorophyllin-treated group. Liver samples were collected after two days post the oxidant inoculation and at the end of the experimental period (10 weeks). These samples were examined for determination of liver microsomal malondialdehyde (MDA), DNA fragmentation, restriction fragment length polymorphism (RFLP) and 8-hydroxy deoxyguanosine (8-OHdG) concentration. Both CP and B(a)P caused increments in DNA fragmentation percentages, liver microsomal MDA, concentration of 8-OHdG and induced point mutation. Treatment of rats with either curcumin or chlorophyllin revealed lower DNA fragmentation percentages, liver microsomal MDA concentration, concentration of 8-OHdG and prevented induction of mutations, i.e., reversed the oxidative stress induced by CP and B(a)P and proved that they were capable of protecting rats against the oxidative damage evoked by these oxidants.     

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

The effect of norharman on the metabolism of benzo[a]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[a]pyrene

The effect of norharman on the metabolism of benzo[a]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones.Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[a]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[a]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[a]pyrene on Salmonella typhimurium.