Carcinogenic Hydrocarbons Research Articles

Predicting toxicity through a computer automated structure evaluation program.

The computer automated structure evaluation program (CASE) has been extended to perform automatic quantitative structure-activity relationships (QSAR). Applications include the carcinogenicity of polycyclic aromatic hydrocarbons and of N-nitrosamines. Agreement with experiment is satisfactory.

Predicting Toxicity through a Computer Automated Structure Evaluation Program

The computer automated structure evaluation program (CASE) has been extended to perform automatic quantitative structure-activity relationships (QSAR). Applications include the carcinogenicity of polycyclic aromatic hydrocarbons and of N-nitrosamines. Agreement with experiment is satisfactory.

Evidence for the existence of isozymes of choline kinase and their selective induction in 3-methylcholanthrene- or carbon tetrachloride-treated rat liver

New evidence is provided that rat liver choline kinase exists in several distinct forms (choline kinases I, II and III) which differ in isoelectric point, molecular size and antigenicity against anti-rat kidney choline kinase IgG. Remarkable and selective induction of the choline kinase II and choline kinase III forms of choline kinase was caused similarly by the administration of polycyclic aromatic hydrocarbon carcinogen, 3-methylcholanthrene or hepatotoxic carbon tetrachloride (CCl 4). The immunochemical approach further indicated that the elevation in the activity of choline kinase in the 3-methylcholanthrene- or CCl 4-treated rat liver was not accompanied by a parallel increase in the amount of choline kinase II enzyme protein, compatible with the induction of either a small amount of new enzyme protein(s) with very high specific activity or another enzyme which might catalyze post-translational modification of choline kinase.

Development of an affinity chromatography resin for the purification of carcinogen binding proteins from mouse liver

Pyrene, a structural analog of benzo[a]pyrene, is an effective competing ligand for high affinity carcinogen binding proteins in mouse liver. A pyrenederivatized Sepharose gel was prepared for affinity chromatography purification of these proteins, and adsorbs all detectable [ 3H]B[a]P-binding activity from hepatic cytosol with the adsorption of less than 1% of total protein. Specific carcinogen binding activity is recovered from pyrene-derivatized Sepharose columns with the enrichment of a 33 kDa polypeptide. This chromatograpy resin represents a major step in the isolation of these unusual receptor-like binding proteins for aromatic hydrocarbon carcinogens.

Oxidative metabolism of 7-methylbenz[ a]anthracene, 12-methylbenz[ a]anthracene and 7,12-dimethylbenz[ a]anthracene by rat liver cytosol

Earlier studies from this laboratory demonstrated that benz[ a]anthracene (BA), 7-methylbenz[ a]anthracene (7-MBA) and 12-methylbenz[ a]anthracene (12-MBA) undergo a bio-alkylation substitution reaction in the mesoanthracenic position(s) or L-region leading to the biosynthesis of the potent carcinogen 7,12-dimethylbenz[ a]anthracene (7,12-DMBA). These results support the hypothesis that for most, if not all, unsubstituted polycyclic aromatic hydrocarbon carcinogens, the chemical or biochemical introduction of an alkyl group in the meso-anthracenic position(s) or L-region is a structural requirement for strong carcinogenic activity. Here we report that the L-region methyl derivatives 7-MBA, 12-MBA and 7,12-DMBA are oxidized to hydroxymethyl derivatives by a rat liver cytosol preparation without any apparent oxidation of the ring positions.

The genotoxicity, metabolism and carcinogenicity of nitrated polycyclic aromatic hydrocarbons

(1985). The genotoxicity, metabolism and carcinogenicity of nitrated polycyclic aromatic hydrocarbons. Journal of Environmental Science and Health. Part C: Environmental Carcinogenesis Reviews: Vol. 3, No. 2, pp. 221-272.

Individual variation in patterns of benzo[ a]pyrene metabolism in the marine fish scup ( Stenotomus chrysops)

The metabolism at specific sites on carcinogenic hydrocarbons such as benzo[ a]pyrene (BP) is responsible for activation to the ultimate mutagens and carcinogens, and patterns of metabolism can thus influence the biological effect of such compounds. Marine fish are known to efficiently metabolize BP at the benzo-ring, forming high percentages of the 9,10-dihydrodiol (DHD) and 7,8-DHD, the latter including the penultimate carcinogen. 1,2 Hydrocarbon-induced cytochrome P-450 in fish is responsible for initiating metabolism on the benzo-ring, but epoxide hydrolase (EH) activity is required for DHD formation. 3,4 Both factors could influence formation of the DHD leading to the ultimate carcinogenic diol-epoxide. In the present study, patterns of BP metabolism were evaluated in a number of individual scup Stenotomus chrysops sampled from local Woods Hole waters, and a correlation is described between variation in the DHD formation and EH activity in these feral fish.

The metabolism of the carcinogen 7-methylbenz[c]acridine by hepatocytes isolated from untreated and induced rats.

The metabolic fate of the carcinogenic aza-aromatic hydrocarbon 7-methyl[7-(14)C]benz[c]acridine (14C-7MBAC) was studied in hepatocytes freshly isolated from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated rats. 14C-7MBAC (4-200 microM) was metabolized in a concentration-dependent manner; Michaelis-Menten kinetics were not followed. Using 100 microM 14C-7MBAC, the bulk of the ethyl acetate-extractable metabolites were found in the incubation medium; about 50% of the total metabolites were not extractable into ethyl acetate. The nature of the water-soluble metabolites was examined by enzyme hydrolysis of glucuronides and sulphates, and by glutathione-depletion experiments. Organo-extractable metabolites were examined by reverse-phase h.p.l.c. and quantified by co-chromatography with standards. pretreatment of the rats with mixed-function oxidase inducers, phenobarbital and 3-methylcholanthrene resulted in 2.85- and 5.70-fold increases, respectively, in total metabolism of 14C-7MBAC. Major metabolites for all three hepatocyte preparations co-chromatographed with 7-hydroxymethylbenz[c]acridine, trans-5,6-dihydro-5,6-dihydroxy-7-methylbenz[c]acridine and trans-8,9-dihydro-8,9-dihydroxy-7-methylbenz[c]acridine.

Investigation of absorption, metabolism kinetics and dna-binding of intratracheally administered benzo[ A]-pyrene in the isolated, perfused rat lung : A comparative study between microcrystalline and particulate adsorbed benzo[ A]pyrene

Benzo[a]pyrene (B[a]P) adsorbed onto urban air particles (UAP) or in microcrystalline form (MCr) was administered intratracheally to the isolated perfused lung in doses of 100 and 1.5 micrograms. The appearance rate constant calculated for B[a]P release to the perfusate buffer was significantly lower for B[a]P administered adsorbed onto UAP (0.007 +/- 0.002 min-1) compared to the microcrystalline preparation (0.051 +/- 0.030 min-1). A classical two-compartmental model fitted well to the elimination of B[a]P from the perfusate buffer, after administration in solution to the buffer reservoir; C = 24 e-0.05t + 14 e-0.01t (pmol/ml). The concentration of polar metabolites in the perfusion buffer, at the end of experiments was approx. 9-fold higher for lungs administered the microcrystalline preparation compared to UAP at 1.5 microgram doses. At the 100 microgram dose level, the difference between preparations was only 2-fold, the data indicating that enzyme saturation might be important at the high dose level. With regard to the metabolite pattern, adsorption of B[a]P onto urban air particles caused a relative increase in the formation of B[a]P-9,10-dihydrodiol, whereas the relative formation rate for phenols was decreased. The absolute levels of B[a]P metabolites covalently bound to DNA was significantly higher in lungs given the MCr preparation compared to the UAP. When calculated as the amount metabolites bound, in relation to the total amount polar metabolites at the end of perfusion, however, the UAP preparation was significantly more efficient to enhance the production of DNA binding metabolites; 2.62 +/- 0.59 X 10(-5) vs. 1.33 +/- 0.21 X 10(-5) (pmol covalently DNA-bound metabolites/mg DNA/pmol metabolites formed). The results indicate that urban air particles may exert a cocarcinogenic effect with polynuclear aromatic hydrocarbons by increasing the pulmonary residence time for the carcinogenic hydrocarbon and/or alter the metabolite pattern in a way that enhances the covalent binding of metabolites to DNA.

Induction of immunotoxicity by polycyclic hydrocarbons: role of the Ah locus.

We have employed the plaque forming cell (PFC) response to sheep erythrocytes as well as lymphocyte proliferation to study the induction of immunotoxicity in AHH-inducible (Ah Locus positive, C57BL/6N; B6C3F1) and AHH non-inducible (Ah Locus negative, DBA2/N) mice following administration of polycyclic aromatic hydrocarbons. When two potent carcinogenic polycyclic hydrocarbons which induce AHH activity, 3-methylcholanthrene (MCA) or 1,2,5,6-dibenzanthracene [DB(a,h)A] were administered IP, immunotoxicity was observed in both AHH-inducible and AHH non-inducible animals. However, the AHH-inducible animals appeared to be more sensitive, and substantial suppression of a PFC response toxicity could be induced with doses as low as 14 mg/kg methylcholanthrene. While suppression of a mitogen response required a dose of 43-125 mg/kg. Administration of the weak carcinogen 1,2,3,4-dibenzanthracene [DB(a,c)A], IP, which similarly induces AHH activity in inducible animals, failed to induce immunotoxicity in either C57B1/6N or DBA/2N mice. In contrast to the results obtained following IP administration, when MCA was administered repeatedly (4X) via an intragastric (IG) route we observed striking immunosuppression of a PFC response in Ah locus negative (DBA/2) animals but minimal effects in Ah locus positive animals (C57B1/6). We finally observed that a single IP dose of MCA (125 mg/kg) to Ah locus positive animals substantially inhibited Natural Killer Cell activity but had more limited effects on the ability of an animal to reject a challenge by an immunogenic syngeneic fibrosarcoma.

Carcinogenesis by polycyclic aromatic hydrocarbons: a multilinear regression on new type PMO indexes

A new MCS model of chemical carcinogenicity of polycyclic aromatic hydrocarbons is presented. M stands for metabolism at the M region, C for carbocation formation, and S for size and solubility. Two perturbational molecular orbitals (MO) indices are introduced and discussed together with the size criterion. Thereby, perturbational molecular orbital method is extended similar to the omega-method in order to differentiate between carbocations and radicals. A three-variable linear regression on carcinogenic potency yields a multiple correlation coefficient r = 0.961 for a representative sample of 26 polycyclic aromatic hydrocarbons. The deviations are within the confidence limits of the experimental Iball index. Predictions are given for some hydrocabons whose carcinogenic potencies are yet unknown.

Biosynthesis of the potent carcinogen 7,12-dimethylbenz[ a]anthracene

Earlier studies from this laboratory of the metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) and of benzo[ a]pyrene as well as studies of mutagenic and carcinogenic activity of some of the metabolic products led to the concept that a necessary first step in carcinogenesis by most alkyl substituted polycyclic hydrocarbons is biotransformation to a meso-anthracenic hydroxyalkyl metabolite [5], whereas most hydrocarbons lacking alkyl substituents undergo a bio-alkylation substitution reaction in the mesoanthracenic positions(s) or L-region as a necessary first step in carcinogenesis [6]. According to this unified hypothesis, all strong polycyclic hydrocarbon carcinogens must either, themselves, bear a meso-anthracenic alkyl substituent or else undergo a bio-alkylation substitution reaction in vitro and in vivo. Here we report that the weak carcinogen benz[ a]anthracene undergoes meso-anthracenic methylation by S-adenosyl- l-methionine (SAM), in the presence of a rat liver cytosol preparation, in vitro, to form DMBA and presumably the moderately active carcinogens 7-methylbenz[ a]anthracene and 12-methylbenz[ a]anthracene. These compounds are substrates for further L-region methylation to form the strong carcinogen, DMBA.

The discrepancy between in vivo and in vitro experiments with polycyclic aromatic hydrocarbon(PAH) carcinogens: a hypothetical explanation

The hypothesis suggested in this paper is an attempt to explain a discrepancy between in vivo and in vitro polycyclic aromatic hydrocarbons (PAH)-carcinogenesis experiments, described frequently in the literature. Whereas, in pretreated animals, a higher level of induced PAH-metabolizing enzymes reduces PAH-carcinogenicity, in the tissues or homogenates from pretreated animals the induced PAH-metabolizing enzymes increase the carcinogenic effects of PAH. In our model, both the pretreatment or route of administration should cause a frameshift in the alternation of active and inactive metabolites in the compartments. In pretreated animals the carcinogenicity of PAH administered per os is reduced, because the PAH metabolism is completed to tetrol, the ultimate inactive metabolite, before it reaches the target tissue. The hypothesis explains the discrepancy and makes predictions which can be tested experimentally.

Aryl Hydrocarbon Hydroxylase, Epoxide Hydrolase, and Benzo[a]pyrene Metabolism in Human Epidermis: Comparative Studies in Normal Subjects and Patients with Psoriasis

Prior studies have shown that human skin possesses a cytochrome P-450-dependent microsomal enzyme that is capable of metabolizing drugs and polycyclic aromatic hydrocarbon (PAH) carcinogens. This study characterized benzo[a]pyrene (BP) metabolism in human epidermis of normal and psoriatic individuals. The basal level of the cytochrome P-450-dependent microsomal enzyme aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH) were measured in freshly keratomed epidermis from 12 normal individuals and from uninvolved skin sites of 12 patients with psoriasis. The induction response of AHH following the in vitro addition of the PAH benz[a]anthracene (BA) was also assessed. The basal activity (mean +/- SE) of AHH in normal epidermis was 62.1 +/- 5.6 units (fmol 3-hydroxybenzo[a]pyrene, 3-OH-BP/min/mg protein) whereas the activity in uninvolved skin of psoriatic individuals was 62.9 +/- 5.1 units (NS), Epoxide hydrolase activity was 25.1 +/- 1.1 (pmol BP 4,5-diol/min/mg protein) unites in normal epidermis and 24.8 +/- 2.1 units in epidermis from patients with psoriasis (NS). Following addition of BA (100 microM), in vitro, AHH activity in normal epidermis increased by a mean value of 165% whereas activity in nonlesional epidermis of psoriatic individuals increased 320%. Kinetic studies in normal epidermis revealed that AHH reaction was linear up to 60 min and to 50 micrograms protein, had a pH optimum of 7.4, and the Km for BP was 0.62 microM. High-performance liquid chromatography (HPLC) confirmed that the pattern of metabolism of BP was quite similar in epidermal microsomes prepared from normal and psoriatic individuals, insofar as the formation of diols, phenols, and quinones was concerned. These studies indicate that human epidermis is capable of metabolizing BP and that there is no significant difference between normal individuals and patients with psoriasis insofar as basal AHH activity or total BP metabolism is concerned. Furthermore, the epidermal enzyme system in patients with psoriasis has a greater responsiveness to environmental PAH than does that of normal individuals.

Synthesis and 1H and 13C NMR spectra of 7-fluoro-, 9-fluoro- and 11-fluoro-benzo[b]fluoranthenes

Abstract We have synthesized 7-fluoro-, 9-fluoro- and 11-fluoro- benzo[b]fluoranthene as part of a study designed to locate the molecular sites involved in the metabolic activation of the carcinogenic parent hydrocarbon. Analysis of the proton NMR spectra reveals the effect of fluorine substitution to be almost entirely localized in the substituted ring, the principal exception being a strong deshielding of opposing protons in sterically crowded sites ( i.e. , peri or pseudo-bay regions).

Metabolism of benzo[a]pyrene by cultured human hepatocytes from multiple donors

Primary hepatocyte cultures from six human donors were established and their abilities to metabolize the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) were examined. Cells from each donor were plated at similar densities (1 X 10(7) cells/100 mm dish). All cultures metabolized B[a]P to a significant extent (24-35 nmol in 24 h) and h.p.l.c. profiles of the organic solvent-soluble and glucuronidated B[a]P metabolites were obtained for all donors. The predominant extracellular organic solvent-soluble B[a]P metabolites were the 9,10- and 7,8-dihydrodiols, 9-hydroxy-B[a]P, and a mixture of tetrols, but the general ratios of these metabolites varied widely among the cells from different donors. In contrast, profiles were highly reproducible in cells from the same donor treated with B[a]P at either 8 or 24 h after initial plating. There was less variability in the amounts of specific B[a]P metabolites conjugated to glucuronic acid by cells from various donors. This variability could not be correlated with cell viability or overall levels of B[a]P metabolism. In addition, B[a]P metabolism by fresh and cryopreserved hepatocytes from the same donor was compared. While there was only a small reduction in the level of total B[a]P metabolism after cell freezing, there was a 3- to 5-fold increase in production of B[a]P-7,8-dihydrodiol, found both in the extracellular medium and as glucuronic acid conjugates, by the cryopreserved cells tested.

Human lymphocytes treated with r-7, t-8-dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene require low-density lipoproteins for DNA excision repair

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA—carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which are depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.

Ellagic acid: a potent naturally occurring inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation and covalent binding to DNA in cultured BALB/C mouse keratinocytes.

The metabolism of [3H]benzo[a]pyrene (BP) by cultured primary keratinocytes prepared from BALB/C mouse epidermis was found to be largely inhibited by the dietary plant phenol, ellagic acid. Varying concentrations of ellagic acid added to the keratinocyte cultures resulted in a dose-dependent inhibition of the cytochrome P-450-dependent monooxygenases aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECD). The major organic solvent-extractable metabolites found intracellularly in the cultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP-7,8-diol) and 3-hydroxybenzo[a]pyrene (3-OH-BP), although small amounts of 9-hydroxybenzo[a]pyrene, quinones and trans-9,10-dihydro-9,10-dihydroxybenzo[a]-pyrene (BP-9,10-diol) were also present. The major organic solvent-extractable metabolites found in the extracellular culture medium were BP-7,8-diol and BP-9,10-diol, with smaller quantities of unconjugated phenols and quinones. The major intracellular and extracellular water-soluble metabolites of BP were conjugated with glucuronide (primarily 3-OH-BP and several BP-quinones), and to a lesser extent with sulfate (primarily BP-7,8-diol). Both intracellular and extracellular metabolism of organic solvent-extractable and water-soluble conjugates was significantly inhibited by ellagic acid in a dose-dependent manner. The intracellular enzyme-mediated binding of BP to mouse keratinocyte DNA was also largely inhibited in a dose-dependent fashion by ellagic acid. Our results indicate that cultured primary mouse keratinocytes offer a useful model system for studying factors affecting the metabolic activation and detoxification of polycyclic aromatic hydrocarbon carcinogens in the epidermis, and that polyphenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer that results from exposure to these environmental chemicals.

Chemical carcinogen-mediated decreases in DNA 5-methylcytosine content of BALB/3T3 cells.

The genomic level of DNA cytosine methylation was significantly diminished in dividing BALB/3T3 A31 CL1-13 cells treated with several aromatic hydrocarbon carcinogens. Benzo[a]pyrene-induced decreases in DNA 5-methylcytosine levels were concentration dependent over the range of 0.1 to 1.0 microgram/ml when determined at the end of the 16 h treatment period. The enzymatic methylation of DNA cytosine residues was the most sensitive to inhibition 48 h after treatment as concentrations of benzo[a]pyrene as low as 0.033 micrograms/ml initiated significant reductions in 5-methylcytosine levels. This inhibition of DNA cytosine methylation may be mediated by alkylation of the DNA. Treatment of hemimethylated DNAs with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene (anti-BPDE), (+/-)-r,7-t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE), and (+/-)-benzo[a]pyrene-4,5-epoxide inhibited the transfer of methyl moieties from [3H]S-adenosylmethionine to cytosine residues in the undermethylated strand in the presence of mouse spleen methyltransferase activity. The syn isomer of BPDE was the most potent in this action while the parent compound, benzo[a]pyrene, did not significantly decrease the methyl accepting abilities of treated DNAs. All chemical carcinogens that were tested and are known to transform BALB/3T3 cells initiated significant reductions in 5-methylcytosine formation by 48 h post-treatment. However, concentrations of some hydrocarbons which do not transform these cells 5- to 50-fold above effective concentrations of the transforming carcinogens also provided significant reductions in DNA cytosine methylation. Thus the inhibition of DNA methylation may be an important step in the initiation of oncogenic transformation of BALB/3T3 cells, but decreases in DNA 5-methylcytosine levels alone cannot account for the onset of this multi-step process.

Enhancement of DNA binding, mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[ a]pyrene (BP) metabolism and activation and, to a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[ a]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).