Equilibrium binding of four anthracyclines to nucleic acids: thermodynamic properties and sequence selectivity.

The thermodynamic parameters of the interaction of the two anthracyclines 13-dihydrodaunomycin and marcellomycin with calf thymus DNA were examined by equilibrium binding studies. Enthalpy and entropy changes of the binding of both drugs show salt dependence profiles that cannot be rationalized by the polyelectrolyte theory. This feature is common to other anthracycline compounds. The nucleotide sequence binding preferences of daunomycin, adriamycin, 13-dihydrodaunomycin and marcellomycin have been studied by monitoring the degree of protection from cleavage by restriction endonucleases of linearized pBR322. Differential protection of pBR322 DNA against the cleavage of Bgl I and Ava II suggests that these drugs recognize changes in the sequences near the enzyme recognition site. Alterations of the electrophoretic restriction pattern of pBR322 in the presence of anthracyclines are dependent on time and on concentration. These results are discussed in relation to the existence of nucleotide sequences with different affinity for these drugs.     

Covalent binding of benzo[a]pyrenediol epoxides to polynucleotides

Spectroscopic studies on the trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene- (anti-BPDE-) modified synthetic polynucleotide solutions reveal interesting sequence-dependent stereoselective covalent binding of anti-BPDE to DNA. Absorption spectral results indicate that the G.C polymers are much more reactive than the A.T polymers toward this metabolite and the homopolymer suffers higher modification than its corresponding alternating polynucleotide. The covalently attached anti-BPDE exhibits only a 2-3-nm red shift in the guanine-containing polynucleotide and native DNA solutions as opposed to the 8-nm red shift in poly(G) and none in the A.T polymers. Distinct stereoselectivities are exhibited by poly(dG-dC).poly(dG-dC) vs. poly(dG).poly(dC) as suggested by the oppositely signed CD in the pyrene spectral region. Comparison with the syn-BPDE modified polynucleotides reveals some interesting differences with its anti diastereomer. Significant contributions from the intercalated syn-BPDE are apparent in the modified guanine-containing polynucleotides as indicated by the appearance of 10-nm red-shifted shoulders. In contrast to the strong dependence on polynucleotides for anti-BPDE, the rate of hydrolysis of syn-BPDE appears to be insensitive to their presence in the solution. anti-BPDE modification on the 50 microM hexaamminecobalt-induced Z-form poly(dG-dC).poly(dG-dC) is much less extensive than its corresponding B form, possibly the consequence of both structural and ionic strength factors. The spectral characteristics of anti-BPDE bonded to these two forms are distinctly different, with the Z form resembling more closely those of A.T polymers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.     

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.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.     

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.     

Metabolism and binding of benzo[a]pyrene in randomly-proliferating,confluent and s-phase human skin fibroblasts

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-³H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.Abbreviations BaP benzo[a]pyrene - BaP-4,5-diol trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene - BaP-7,8-diol trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene - Bap-9,10-diol trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene - CM complete medium - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - PAH polycyclic aromatic hydrocarbon - PDL population doubling - RP randomly proliferating     

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     

A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins

A method is presented for the quantitative determination of covalent binding of metabolically activated benzo[a]pyrene to microsomal proteins. After incubation of radiolabelled benzo[a]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.     

Sequence selectivity, a test of the nature of the covalent adduct formed between benzo[a]pyrene and DNA

A theoretical study is presented of the energetic and structural properties of covalent adducts of benzo[a]pyrene and a DNA fragment. Energy optimisation is performed with the use of minimiser with constraints and an advanced semiempirical energy formula. Three types of adducts are studied: an external complex with the benzopyrene located in the DNA minor groove and two types of intercalative complexes with the carcinogen situated on the 3' side and 5' side of the covalently bound guanine. For each of the adducts the effects of DNA base sequence are examined. It is shown that the results for the intercalative complex with the carcinogen situated on the 5' side of the modified guanine correlate with the experimentally determined sequence preference.     

The oxidation of benzo[a]pyrene mediated by lipid peroxidation in irradiated synthetic diets

The effect of gamma-irradiation (1000-4000 Gy) on the formation of lipid peroxides and on the oxidation of the environmental carcinogen benzo[a]pyrene (BP) has been studied in mixtures of starch/fat and BP which were used as models for natural foods. When mixtures containing polyunsaturated fats (mackerel oil and cod-liver oil which contain relatively large proportions of C20:5 and C22:6) were exposed to gamma-irradiation, large concentrations of lipid peroxide were formed and a concomitant oxidation of BP to mutagenic and toxic BP quinones took place. The rate of BP oxidation was closely related to the extent of peroxidation of the lipids in the starch mixtures and was dependent on the dose of gamma-irradiation and the presence of air. Mackerel oil also underwent peroxidation during the storage of both irradiated and unirradiated starch/mackerel oil/BP mixtures and this resulted in a significant oxidation of the BP present in these samples. Antioxidants such as vitamin E and BHA inhibited both lipid peroxidation and BP oxidation resulting from gamma-irradiation. These results demonstrate that the species generated during the peroxidation of unsaturated fats in foodstuffs can react with polycyclic aromatic hydrocarbons such as BP and convert them into active mutagenic and toxic products. This has important toxicological implications, particularly as the consumption of polyunsaturated fat in the Western world is increasing and gamma-irradiation may soon be widely used for food sterilization.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Metabolism and covalent binding of benzo[alpha]pyrene in human peripheral lung

Short-term organ cultures of peripheral lung from lung cancer patients metabolise benzo[alpha]pyrene to ethylacetate-soluble metabolites, which covalently bind to tissue macromolecules. The nature and quantities of metabolites formed and the extent of covalent binding are dependent upon the time of incubation, the substrate concentration and interindividual variability in the metabolic activity of the lung. Individuals whose lungs rapidly metabolise the carcinogen exhibit more extensive further metabolism of primary metabolites and higher levels of covalent binding. Certain striking differences in the relative retention in the tissue or release into the extra-cellular medium of different metabolites have been found as illustrated by the observation that the ratio of 7,8-dihydro-7,8-dihydroxybenzo[alpha]-pyrene to 9,10-dihydro-9,10-dihydroxybenzo[alpha]pyrene was always significantly higher in the tissue than in the extracellular medium.     

Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene

The lipid peroxidation (as malondialdehyde, MDA), activities of superoxide dismutase (SOD) and catalase (CAT), and benzo[a]pyrene (BaP) metabolites were investigated in sera and erythrocytes of male Sprague-Dawley rats treated with BaP (20 mg per rat). MDA levels were significantly increased in sera (16.98+/-3.29 nmol/ml serum, P<0.05) 12 h after BaP treatment and persisted up to 96 h (13.80+/-1. 65 nmol/ml serum, P<0.05), but no significant change in NIDA levels was observed in erythrocytes. SOD and CAT activities were significantly increased in erythrocytes shortly after BaP exposure, and they were slightly decreased in sera, indicating an inverse correlation between lipid peroxidation and antioxidant enzyme activity. BaP and BaP-quinones (BaP-1,6-quinone and BaP-3,6-quinone) were measured in sera during the study period. A rapid increase of unmetabolized BaP was observed in sera (41.27+/-4.14 pmol/ml serum) 3 h after BaP treatment, reaching a peak at 6 h (48.56+/-4.62 pmol/ml serum) followed by a sharp decrease. Formation of the BaP-1, 6-quinone and BaP-3,6-quinone started in sera 3 h after BaP treatment, reached a peak at 24 h (7.23+/-1.02 pmol/ml serum) and 12 h (9.20+/-0.98 pmol/ml serum), respectively, and then decreased gradually. The time-dependent pattern of serum lipid peroxidation and the level of erythrocyte antioxidant enzymes were shown to be related to the concentrations of the BaP-quinone metabolites. These results suggest that BaP treatment, probably via the formation of BaP-quinones, oxidatively altered lipids and antioxidant enzymes in the blood, and might be associated with BaP-related vascular toxicity including carcinogenesis.     

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.