Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[a]pyrene (BaP), benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11H-benzo[b]fluorene, dibenz[a,h]anthracene, pyrene, benzo[ghi]perylene and benzo[e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer. The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.     

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.     

Genotoxicity testing of six insecticides in two crosses of the Drosophila wing spot test

Among the great variety of genotoxicity assays available, the wing spot test in Drosophila melanogaster has some characteristics that make it very suited for the screening of genotoxic activity, i.e., it is an easy and inexpensive assay using a eukaryotic organism in vivo. One of the most interesting characteristics of the assay is its capacity to detect genotoxic activity of promutagens without the necessity of an exogenous metabolic activation system. In this paper we present results obtained with a recently developed high bioactivation cross of the wing spot test (NORR cross). The positive results obtained with the five well-known procarcinogens 7, 12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, p-dimethylaminoazobenzene, diethylnitrosamine and urethane clearly show that the NORR strains are similar to the other high bioactivation strains previously described, but they lack their methodological disadvantages. We have tested six insecticides, which are characterised by having contradictory results in other genotoxicity tests, using both the standard and the high bioactivation (NORR) cross. The six insecticides analysed are the pyrethroid allethrin, the methylenedioxyphenolic compound piperonyl butoxide, the chlorinated hydrocarbons dieldrin and endrin, and the organophosphates dimethoate and malathion. We obtained negative results for all six compounds. Our results show the suitability of the wing spot test for the evaluation of compounds at the first level of genotoxicity testing.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

New tester strains with improved bioactivation capacity for the Drosophila wing-spot test

In Drosophila melanogaster new tester strains for the somatic mutation and recombination test (SMART) in the wing were constructed with the aim of increasing the metabolic capacity to activate promutagens. Some aspects of the genetic control of the xenobiotics metabolism in Drosophila are already known. In the DDT-resistant strain Oregon R(R) the RI gene at position 65.0 on chromosome 2 is responsible for the high constitutive expression of cytochrome P-450-dependent activities typical for this strain. Therefore, chromosomes 1 and 2 in the original mwh (multiple wing hairs) and flr3 (flare3) tester strains were substituted for chromosomes 1 and 2 from the Oregon R(R) strain. In assays with the model promutagen diethylnitrosamine an increased sensitivity of about 2.5-fold was found for this new set of 'HB' strains ('High Bioactivation (HB) cross').     

Improved high bioactivation cross for the wing somatic mutation and recombination test in Drosophila melanogaster.

The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Fr?lich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibits a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross (ORR; flr3 females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

The genetic regulation of some cytochrome P-450-dependent enzyme activities has been studied in adult Drosophila. Strains having genetically determined high or low enzyme activities were crossed with a marker strain and the metabolism was analyzed in microsomes from hybrids carrying different combinations of chromosomes from the strain under test. High p-nitroanisole (PNA) N-demethylation, biphenyl 3-hydroxylation and an increased amount of a protein with an apparent mol. wt. of 54 000, after SDS-gel electrophoresis of the microsomes in insecticide-resistant Drosophila strains, are inherited as dominant second chromosome traits. A low capacity for benzo[a]pyrene (BP) hydroxylation and 7-ethoxycoumarin O-deethylation in the Hikone R strain is semidominantly inherited in both cases and determined by gene(s) on the third chromosome. A semidominantly inherited high 4-hydroxylation of biphenyl and a high amount of a protein with an apparent mol. wt. of 56 000 in the Oregon R strain are also localized to the second chromosome. The results indicate that several other cytochrome P-450-dependent activities are not regulated by the genes mentioned above. In conclusion, at least three genes regulating the cytochrome P-450 system in Drosophila have been identified.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[a]pyrene (BaP), benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11H-benzo[b]fluorene, dibenz[a,h]anthracene, pyrene, benzo[ghi]perylene and benzo[e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer.The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.     

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated.     

A dual assay for the specific screening of 3-methylcholanthrene- and phenobarbital-like chemical inducers of cytochrome P-450 monooxygenases

We present and evaluate a dual assay, the CYPIA (Cytochrome P-450 induction assay) for the detection and the simultaneous identification of chemicals belonging either to the 3-methylcholanthrene or phenobarbital classes of cytochrome P-450 monooxygenase inducers. These inducers play an important role in the mutagenic activation of chemical compounds as well as in many pharmacological and toxicological events and therefore should be screened by drug and chemical designers. After treatment of male rats or mice by chemicals, the liver preparations (S9) have been used in the Salmonella typhimurium test, to activate either ethidium bromide or cyclophosphamide into mutagenic metabolites. These transformations are specifically catalyzed by cytochrome P-450-dependent monooxygenases induced by 3-methylcholanthrene-like and phenobarbital-like chemical inducers, respectively, The mutagenicity data were strikingly correlated with other methods (production of [³H]benzo[a]pyrene bay-region metabolites, benzphetamine demethylase activity, immunological double-diffusion analysis). Compared to the latter methods, the CYPIA, based on a single and widespread technology, introduces an interesting simplification, and improves the specificity and the sensitivity of the responses.     

Cell-specific metabolic activation of 7,12-dimethylbenz[a]anthracene in rat testis

The binding of metabolites of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) to protein in rat testis seminiferous tubules was studied. Treatment of cultured seminiferous tubule segments with DMBA resulted in very little binding to protein, suggesting that the seminiferous epithelium from rat testis lacks the cytochrome P-450-dependent monooxygenase(s) required for DMBA metabolism. In contrast, Leydig cells from rat testis contain monooxygenase systems which catalyze the metabolism of PAH, such as DMBA. This metabolic activation of DMBA was localized in both mitochondria and microsomes derived from Leydig cells and was decreased by inhibitors of the cytochrome P-450 system and by free radical scavengers, suggesting that the metabolism involved both cytochrome P-450 and free radical-dependent pathways. In the presence of whole Leydig cells or microsomes prepared from Leydig cells, the covalent binding of DMBA metabolites to protein of rat testis seminiferous tubules was increased 5- and 13-fold, respectively. These results suggest that DMBA is metabolized primarily in rat testis Leydig cells and that part of the produced metabolites find their way to the seminiferous epithelium, where they undergo further metabolism producing reactive metabolites, possibly cation radicals and diolepoxides, which interfere with the functions of spermatogonia and spermatocytes by modifying key proteins covalently.     

The homogeneity of rat liver microsomal cytochrome P-448 activity and its role in the activation of benzo[a]pyrene to mutagens

The O-deethylation of ethoxyresorufin and the metabolic activation of benzo[a]pyrene to mutagens were determined in hepatic microsomal preparations from control and induced animals. An excellent direct correlation (r = 0.95) has been observed between ethoxyresorufin O-deethylase and the metabolic activation of benzo[a]pyrene to mutagens when the fraction of cytochromes P-450 present as cytochrome P-448 was altered by the administration of phenobarbitone and 3-methylcholanthrene alone or in combination with 9-hydroxyellipticine. The correlation between these activities was maintained following treatment of animals with Arochlor 1254, benzo[a]pyrene, benzo[e]pyrene, 7,12-dimethylbenzo[a]anthracene,2-anthramine and 2-naphthylamine.     

Rat liver cytochrome P450IA2 synthesized by transfected COS-1 cells efficiently activates food-derived promutagens

A standard calcium phosphate technique was used to obtain transient expression of cDNAs for rat liver cytochrome P450s in COS-1 cells. Cells transfected with a pMT2-based vector expressing P450IA2 cDNA (pMT2-IA2) had high acetanilide-4-hydroxylase activity and very low aryl hydrocarbon hydroxylase (AHH) activity. Cells transfected with a hybrid expression vector, pMT2-IA2/IA1, coding for a P450IA2/IA1 fusion protein (consisting of the amino-terminal region of P450IA2 and the central and carboxy-terminal regions of P450IA1) had high AHH activity. This result and other data indicate that the P450IA2/IA1 fusion protein has the substrate specificity of P450IA1. Extracts of cells transfected with pMT2-IA2 readily converted 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and related food-derived promutagens into mutagenic forms. Extracts of cells transfected with pMT2-IA2/IA1 showed efficient activation of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp P-2). To facilitate comparison of activities of P450s synthesized from cDNA expression vectors, the promutagen activation assays were carried out with limiting enzyme and saturating or nearly saturating substrate concentrations. The transient expression system described here uses a standard expression vector and requires only microgram quantities of cell extract protein for activation of food-derived promutagens such as MeIQ and Trp P-2. It will be useful for identifying P450s active in promutagen activation and for analyzing structure-function relationships of different P450 molecules.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Genotoxic effects of eugenol, isoeugenol and safrole in the wing spot test of Drosophila melanogaster

In the present study, the phenolic compounds eugenol, isoeugenol and safrole were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The Drosophila wing somatic mutation and recombination test (SMART) provides a rapid means to evaluate agents able to induce gene mutations and chromosome aberrations, as well as rearrangements related to mitotic recombination. We applied the SMART in its standard version with normal bioactivation and in its variant with increased cytochrome P450-dependent biotransformation capacity. Eugenol and safrole produced a positive recombinagenic response only in the improved assay, which was related to a high CYP450-dependent activation capacity. This suggests, as previously reported, the involvement of this family of enzymes in the activation of eugenol and safrole rather than in its detoxification. On the contrary, isoeugenol was clearly non-genotoxic at the same millimolar concentrations as used for eugenol in both the crosses. The responsiveness of SMART assays to recombinagenic compounds, as well as the reactive metabolites from eugenol and safrole were considered responsible for the genotoxicity observed.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

Inhibition of stage-specific DNA synthesis in rat spermatogenic cells by polycyclic aromatic hydrocarbons

Changes in the rate of DNA synthesis in spermatogenic cells after treatment of segments of rat seminiferous tubule at defined stages of epithelial cycle with benzo[a]pyrene (BP) or 7,12-methylbenz[a]anthracene (DMBA) were studied. The incorporation of labeled thymidine into DNA was used as a measure of the rate of DNA synthesis. Very little or no inhibition of DNA synthesis at stages V and VIII of the cycle was observed at BP and DMBA concentrations lower than 100 microM. In contrast, in the presence of added mitochondria and/or microsomes from whole rat testis, 20 microM BP or DMBA inhibited DNA synthesis 5% and 80%, respectively. This inhibition of DNA synthesis was prevented by inhibitors of the cytochrome P-450 system and by free radical scavengers. These results suggest that polycyclic aromatic hydrocarbons (PAH) require metabolic activation in order to inhibit DNA replication in seminiferous tubules. The first step of this biotransformation is cytochrome P-450-dependent and occurs in Leydig cells. However, the metabolites produced in this step may be further metabolized to reactive metabolites by peroxidative pathways in the seminiferous tubules; these latter products may affect DNA replication.     

Metabolic activation of cytochrome P-450/P-448 in the yeast Saccharomyces cerevisiae

The strains D6 and JD1 of the yeast Saccharomyces cerevisiae were used to assay the genetic activity of several compounds, benzo[a]pyrene, 15,16-dihydro-11-methyl-cyclopenta[a]phenanthren-17-one, 2-naphthylamine and cyclophosphamide, which require metabolic activation by cytochromes P-450 and P-448 to produce genetically active chemical species. Cells from both strains were harvested from cultures grown in low concentrations of glucose and switched to growth in high glucose containing media. Treatments under these conditions resulted in increased sensitivity of the test systems without the presence of an exogenous S9 mix and the presence of S9 was found not to enhance this sensitivity. The yeasts used under these treatment conditions showed a P-450/P-448 type metabolism.