Aroclor 1254-treated Rats Research Articles

Effects of quercetin on Aroclor 1254-induced expression of CYP450 and cytokines in pregnant rats

The present study aimed to investigate the protective effect of quercetin on polychlorinated biphenyls (PCB)-induced liver and embryo damage in pregnant Sprague–Dawley rats. Pregnant rats were divided into five groups, and then were orally gavaged daily with peanut oil (vehicle) or a commercial PCB mixture (Aroclor 1254) — with or without co-treatment with 75, 150, or 300 mg/kg quercetin — on gestation days (GD) 4–7. At GD 9, all rats were euthanized, and their blood, liver, and uterus were collected. Expressions of CYP450 mRNA and protein in liver, cytokines (IFNγ, IL-2, IL-4, and IL-6) and IFNγ/IL-4 ratios in liver and sera, liver morphology, and the status of implanted embryos were analyzed. The results showed Aroclor 1254 treatment alone caused hepatic cord damage (i.e. cell disorganization, swelling, decreased cytoplasm, vacuolization), and that quercetin co-treatment appeared to mitigate this damage. Similarly, levels of CYP1A1 and CYP2B1 mRNA in livers of Aroclor 1254-only-treated rats were significantly higher than those in rats co-treated with quercetin. Hepatic and sera levels of IFNγ, IL-2, IL-6, and IFNγ/IL-4 ratios, and the ratio of delayed-development embryos, all increased in Aroclor 1254-treated rats, but were relatively decreased as a result of quercetin co-treatments. IL-4 levels were decreased by Aroclor 1254 and tended to increase back to normal when quercetin was used. The results indicated that quercetin imparted a protective effect against Aroclor 1254-induced toxicity in pregnant rats, in part, by modulating levels of important pro-inflammatory cytokines and reducing induced CYP1A1 and CYP2B1 expression.

Impact of selenium status on Aroclor 1254-induced DNA damage in sperm and different tissues of rats

Aroclor 1254 is a commercial mixture of polychlorinated biphenyls (PCBs), which are widespread environmental pollutants. It is used as non-flammable heat transfer agent and plasticizer. Animal studies have reported that Aroclor 1254 exerted toxic effects in different organs and systems. Although the evidences are limited, it seems reasonable that Aroclor 1254 may have a potential for similar adverse effects in humans. Selenium (Se) is a trace element and an important component of cellular antioxidant defense. This study was designed to investigate the effects of different Se status on the genotoxicity of Aroclor 1254 in sperm and different organs of Sprague-Dawley rats using Comet assay. Se deficiency (SeD) was generated by feeding 3-week old Sprague-Dawley rats with <0.05 Se mg/kg diet for 5 weeks. Se supplementation groups (SeS) were fed with 1 mg Se/kg diet. Aroclor 1254-treated rats received 10 mg/kg dose by gavage during the last 15 d of feeding period. SeD increased DNA damage in all of the organs as well as in lymphocytes and sperm. Aroclor 1254 treatment caused pronounced changes in liver, kidney and brain cells along with marked increases in lymphocytes and sperm. Se supplementation provided full or partial protection decreases in Aroclor 1254-induced DNA damage in sperm and all of tissues. Se deficiency aggravated the toxicity by increasing DNA damage caused by Aroclor 1254. Further studies should be performed to clarify the mechanism(s) underlying the protective role of Se status against Aroclor 1254 genotoxicity.

Exposure to Isothiocyanates Suppresses Urinary Mutagenicity in Rats Treated With Heterocyclic Amine IQ: Lack of Association With CYP1 Activity

The principal objectives of this study were to evaluate whether exposure of rats to low doses of isothiocyanates modulates the overall metabolism of heterocyclic amine 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), as exemplified by urinary mutagenicity, a food carcinogen, and to relate any modifications in metabolism to changes in CYP1 and glutathione S-transferase activities. Animals were exposed to isothiocyanates either for 2 wk (long-term) or 1 day (short-term), and all animals were then treated with a single oral dose of IQ, and urine was collected daily for 3 days; animals continued to receive the isothiocyanates during this period. Urinary mutagenic activity was determined using the Ames mutagenicity assay in the presence of an activation system from Aroclor 1254-treated rats. At the end of the study, animals were killed and hepatic methoxy- and ethoxyresorufin dealkylations were determined as well as glutathione S-transferase activity. All isothiocyanates studied, namely sulforaphane, erucin, and phenethyl isothiocyanate, decreased urinary mutagenic activity, implying enhanced IQ metabolism, but only after long-term intake. Changes in mutagenic activity were not related to changes of any of the enzyme activities determined. It is concluded that long-term intake of isothiocyanates may stimulate the metabolism of IQ, but this effect is not linked to changes in hepatic CYP1A2 and glutathione S-transferase activities.

Stereoselective Metabolism of the Environmental Mammary Carcinogen 6-Nitrochrysene to trans-1,2-Dihydroxy-1,2-dihydro-6-nitrochrysene by Aroclor 1254-Treated Rat Liver Microsomes and Their Comparative Mutation Profiles in a lacI Mammary Epithelial Cell Line

The environmental pollutant 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. Our previous studies have shown that 6-NC is metabolized to trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (1,2-DHD-6-NC) in rats and in several in vitro systems, including human breast tissue, and the latter is the proximate carcinogenic form in the rat mammary gland. Because optically active enantiomers of numerous polynuclear aromatic hydrocarbon (PAH) metabolites including chrysene have different biological activities, we hypothesized that the stereochemical course of 6-NC metabolism might play a significant role in the carcinogenic/mutagenic activities of the parent 6-NC. The goal of this study is to evaluate the effect of stereochemistry on the mutagenicity of 1,2-DHD-6-NC using the cII gene of lacI mammary epithelial cells in vitro. Resolution of (+/-)-1,2-DHD-6-NC was obtained by either nonchiral or chiral stationary phase HPLC methods. We determined that the ratio of (-)-[R,R]- and (+)-[S,S]-1,2-DHD-6-NC formed in the metabolism of 6-NC by rat liver microsomes is 88:12. The mutation fractions and mutation spectra of [R,R] and [S,S]-enantiomers were examined. Our results showed that the [R,R]-isomer is a significantly (p < 0.01) more potent mutagen than the [S,S]-isomer. The major types of mutation induced by the [R,R]-enantiomer are AT > GC, AT > TA, and GC > TA substitutions, and these are similar to those obtained from 6-NC in vivo in the mammary glands of rats treated with 6-NC. The mutation spectra of the [S,S]-isomer were similar to the [R,R]-isomer, but a higher percentage of AT > GC substitutions in the [R,R]-isomer was noted. On the basis of the results of the present study, we hypothesize that [R,R]-1,2-DHD-6-NC is the proximate carcinogen of 6-NC in the rat mammary gland in vivo and will test this hypothesis in a future study.

Global Gene Expression Analysis of Human Bronchial Epithelial Cells Treated with Tobacco Condensates

Gene expression patterns were assessed in normal human bronchial epithelial (NHBE) cells exposed to cigarette smoke condensates (CSC) from commercial cigarettes in order to develop a better understanding of the genomic impact of tobacco exposure, and to define biomarkers that can potentially discriminate tobacco-related effects and outcomes in a clinical setting.NHBE cells were treated with CSCs from two American brands for up to 12 hours in the presence of S9 microsomal fraction from Aroclor 1254-treated rats. High-density oligonucleotide microarrays coupled with a novel statistical analysis that relies on statistical significance levels rather than arbitrary fold-change differences was used to identify genes that undergo expression alterations upon treatment. Expression patterns of approximately 3700 genes were altered after CSC treatments. While a majority of these genes were affected by both CSCs, each condensate also affected a unique subset of ~1000 genes. An unexpected finding was that S9, required for metabolizing procarcinogens in CSCs to carcinogenic metabolites, also altered the expression of approximately 1700 genes. Exposure of NHBE cells to different CSCs alters the expression of a large set of genes that affect a common set of biological pathways including those relevant to carcinogenesis. Identification of CSC-affected genes and underlying biological processes may generate an atlas of molecular events that includes biomarkers of tobacco exposure and disease status in smokers. Finally, the finding that S9 affects the expression of a number of genes may have implications for various toxicogenetic assays currently used by regulatory agencies to evaluate harmful effects in exposed humans.

COMPARATIVE METABOLISM OF PHENANTHRO[3,4-B]THIOPHENE, PHENANTHRO[4,3-B]THIOPHENE AND THEIR CARBON ANALOG BENZO[C]PHENANTHRENE BY RAT LIVER MICROSOMES

Phenanthro[3,4-b]thiophene (P[3,4-b]T) and phenanthro[4,3-b]thiophene (P[4,3-b]T) are thiasters of weakly mutagenic benzo[c]phenanthrene (B[c]P). These polycyclic sulfur heterocycles (thia-PAHs) represent a group of chemicals which have been identified in cigarette smoke. P[3,4-b]T is a potent mutagen in Salmonella typhimurium strain TA100 in the presence of rat liver S9, whereas its isosteric isomer P[4,3-b]T is a nonmutagenic compound. In order to understand the mechanism underlying the differences in the mutagenic activity of P[3,4-b]T and P[4,3-b]T, we have investigated the metabolism of P[3,4-b]T, P[4,3-b]T, and their carbon analogue B[c]P by rat liver microsomes. The liver microsomes from rats treated with Aroclor 1254 metabolized P[3,4-b]T, P[3,4-b]T, and B[c]P at a rate nearly 7- to 9-fold greater than of the control liver microsomes. High-performance liquid chromatography (HPLC) analysis of the metabolites formed showed that B[c]P was metabolized almost exclusively to its dihydrodiols which comprised predominantly K-region diol as noted in the previous studies. Our preliminary studies on the metabolism of P[3,4-b]T, P[4,3-b]T and B[c]P by liver microsomes from control and Aroclor 1254-treated rats have shown a significant reduction in the formation of 6,7-diol (K-region diol) and 8,9-diol (diol with a bay-region double bond) of the two thia-PAHs compared to the formation of analogous 5,6-diol (K-region diol) and 3,4-diol (diol with a bay-region double bond) from B[c]P. Both P[3,4-b]T and P[4,3b]T produced a major, relatively nonpolar metabolite(s) (80–96% of total metabolites). These studies indicate that the highly mutagenic P[3,4-b]T is not metabolized to dihydrodiol with a bay-region double bond to any greater extent than the weakly or nonmutagenic B[c]P or P[4,3-b]T, suggesting that the metabolite(s) other than P[3,4-b]T8,9-diol is likely to be involved in the mutagenicity of P[3,4-b]T.

467 Induction of dominant lethal mutations in male mice by perezone

L5178Y/TK+/− cells were treated with either N-acetyl-2-aminofluorene (AAF), 2-aminoanthracene (2A), benzanthracene (BA), benzo[a]pyrene (BP), 3-methylcholanthrene (3MCA), or dimethylnitrosamine (DMN) in the presence of varied concentrations (2.5–10% v/v) of liver S9 (9000 × g) postmitochondrial fraction from Aroclor 1254-dosed male CD rats. Consistent S9 concentration-dependent decreases in trifluorothymidine-resistant (TFTr) mutant induction were noted following 3 h exposure of L5178Y/TK+/− cells to either 2.5 μg/ml BP, 50 or 67 μg/ml AAF, 0.96 μg/ml 2A, 7.35 μg/ml BA, or 5.4 μg/ml 3MCA. The exception was DMN which yielded a moderate S9 concentration-dependent increase in TFTr mutant induction when L5178Y/TK+/− cells were treated for 3 h with 74.1 μg/ml DMN. Depending upon the promutagen being tested, these results suggest two different metabolic events: (1) activation via non-induced enzymes or other factors whose activities are totally S9 volume-dependent (DMN), and (2) deactivation via induced detoxification pathways, a sequence favored by increasing S9 concentration (all others). Utilization of a single “standard” (10%) concentration of liver S9 from Aroclor 1254-treated rats did not appear to provide optimal metabolic activation for 5 of these 6 promutagens.

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test.

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.

Induction of nuclear transcription factors, cytochrome P450 monooxygenases, and glutathione S-transferase alpha gene expression in Aroclor 1254-treated rat hepatocyte cultures

Aroclor 1254 is a complex mixture of polychlorinated biphenyls and is well known for its potency to induce drug-metabolising enzymes, but little is known about its ability to modulate gene expression of transcription factors, which code for proteins that bind to the regulatory elements of DNA and facilitate transcriptional activation. We therefore investigated the gene expression of the liver-specific transcription factors CCAAT/enhancer-binding protein α (c/EBPα), hepatic nuclear factor (HNF) 1 and 4, and major cytochrome P450 (CYP) isozymes in addition to glutathione S-transferase alpha 2 (GSTA-2) in cultures of primary rat hepatocytes. We found highly significant and dose-dependent increases of c/EBPα (up to 62-fold), HNF-1 (up to 7-fold), HNF-4 (up to 8-fold), and 50- and 4-fold inductions of GSTA-2 and CYP monooxygenases, respectively. Based on the ethoxyresorufin- O-deethylase assay, the gene expression and enzyme activity for CYP1A1 were in good agreement, but for other CYP isozymes similar correlations could not be obtained. In conclusion, the simultaneous induction of liver-specific TFs and of several detoxifying enzymes may point to a coordinate genomic response in cultures of rat hepatocytes upon treatment with Aroclor 1254.

Caffeine metabolism by microsomal and cDNA-expressed cytochrome P450 1A from scup ( Stenotomus chrysops)

Consequences of CYP1A induction depend in part on the substrates metabolized. We are employing model compounds to determine the substrate specificity of teleost CYP1As, and to assess functional similarities to both CYP1A1 and CYP1A2, vis-à-vis the structural relationships among these proteins. Caffeine (CA; 1,3,7-trimethylxanthine) is a natural alkaloid that is N-demethylated predominantly by CYP1A2 in mammals, to paraxanthine (PX; N-3-demethylation) and theobromine (TB; N-1-demethylation). Theophylline (TP; N-7-demethylation) and 1,3,7-trimethyluric acid (TMU; 8-hydroxylation) are formed largely by other catalysts. BNF-induced scup liver microsomes oxidized CA primarily to PX (173 pmol/min/mg) and TB (50 pmol/min/mg). TP and TMU formation was not detected. Polyclonal and the 1A1-specific monoclonal (1-12-3) antibodies to scup CYP1A inhibited CA metabolism by 80-90%. Chinese hamster V79 cells stably expressing scup CYP1A metabolized CA to PX (major) and TB (minor). The CA metabolite profile was remarkably similar to that formed by V79 cells expressing human CYP1A2. Liver microsomes from Aroclor-treated rats oxidized CA to PX, TB, TP and TMU, all at rates between 25 and 60 pmol/min/mg. CA N-demethylations by rat liver microsomes were inhibited about 40%, and 8-hydroxylation (TMU) was inhibited 90% by Mab 1-12-3, indicating involvement of rat CYP1A1. The results establish that teleost CYP1As metabolize caffeine, with regiospecificity apparently more similar to CYP1A2s than to CYP1A1s. [Support: EPA R-823890-01 and Sea Grant NA46RG0470, R/P-60.]

Differential modulation of the genotoxicity of food carcinogens by naturally occurring monomeric and dimeric polyphenolics

Naturally occurring dimeric polyphenolics and their gallate esters were isolated from grape seeds, almond fruits, and apple skin, and their ability to modulate the mutagenicity of food carcinogens was studied in the Ames test, and compared to that of the monomeric green tea flavonols, (+)-catechin and (-)-epicatechin. Neither the monomeric nor the dimeric polyphenols and their galloylated derivatives influenced the mutagenic activity elicited by the indirectly acting food carcinogens benzo[a]pyrene and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), in the presence of a hepatic activation system derived from Aroclor 1254-treated rats; the only exception was the B7 dimer, which, at concentrations above 1 microM, suppressed the mutagenicity of IQ. None of the polyphenolics modulated the mutagenic activity elicited by the directly acting carcinogen N'-methyl-N'-nitro-nitrosoguanidine (MNNG). In contrast, all the dimeric polyphenols and the galloylated metabolites, at concentrations over 1 microM, potentiated the mutagenic activity induced by the indirectly acting carcinogen N-nitrosopyrrolidine, in the presence of an activation system derived from isoniazid-treated rats. In conclusion, dimeric polyphenols and galloylated derivatives of plant origin are unlikely to influence the initiation stage of the carcinogenicity of chemicals through mechanisms that involve inhibition of their cytochrome P450-mediated bioactivation or scavenging of the reactive, genotoxic intermediates.

Consumption of tea modulates the urinary excretion of mutagens in rats treated with IQ. Role of caffeine

The present study was undertaken to investigate whether the consumption of green tea and black tea influences the excretion of mutagens and promutagens in rats treated orally with the food carcinogen 2-amino-3-methylimidazo[4,5- f]quinoline (IQ). Rats were maintained on aqueous extracts (2.5%, w/v) of green tea, black tea or decaffeinated black tea as their sole drinking liquid. After 4 weeks, the animals received, by gastric intubation, a single dose of IQ (5 mg/kg), and urine was collected for 48 h. Direct and indirect mutagenicity, in the presence of an activation system derived from Aroclor 1254-treated rats, was determined in the urine samples using the Ames mutagenicity assay. Consumption of green tea and black tea, but not of decaffeinated black tea, markedly decreased the urinary excretion of mutagens and promutagens. In a further study, supplementation of decaffeinated black tea with caffeine suppressed the excretion of mutagens and promutagens in the urine of rats pretreated with IQ. It is concluded that both green tea and black tea modulate the bioactivation and metabolism of IQ, and that caffeine is largely responsible for this effect.

Microsomal deacetylation of ring-hydroxylated 2-(acetylamino)fluorene isomers: effect of ring position and molecular mechanics considerations.

Metabolism of arylamides such as 2-(acetylamino)fluorene to mutagenic products is catalyzed by various liver microsomal and cytosolic enzymes. Deacylation is believed to be a deactivating pathway, and the activity of the microsomal deacetylase toward N-hydroxy-2-AAF is exceedingly greater than toward the parent 2-AAF. Another deactivating pathway is cytochrome P450-catalyzed ring hydroxylation. We have studied the effect of ring hydroxyl substitution on the activity of the liver microsomal deacetylase from Aroclor 1254-treated rats in vitro. The deacetylase activity was generally decreased toward ring-hydroxylated derivatives in the order of 2AAF approximately 1-OH-AAF > 3-OH-AAF > 7-OH-AAF > 5-OH-AAF approximately 9-OH-AAF. The difference in activity between 2-AAF and 5-OH- and 9-OH-AAF was about eightfold. Molecular mechanics calculations reveal that structural and geometrical parameters are more important than the energies associated with the different isomers. We show that the greater the distance of the hydroxyl group on the fluorenyl ring structure from the acetylamino group, the slower the rate of deacetylation. The difference in reactivity between the 1-hydroxy-2-AAF and the other hydroxy-2-AAF isomers is due to the lack of planarity of the 1-hydroxy isomer as compared to the essentially planar configuration of the other isomers. The relative contribution of microsomal ring hydroxylation and deacetylation to detoxification of arylamides remains to be established.

The substrate specificity of the rat hepatic cytosolic arylamine oxidase catalyzing the bioactivation of aromatic amines

The ability of the cytosolic arylamine oxidase to convert structurally diverse aromatic and heterocyclic amines to mutagens in the Ames test was investigated using hepatic cytosol from Aroclor 1254-treated rats as the activation system. Using this system, only amino compounds containing at least three fused aromatic rings elicited a strong mutagenic effect; heterocyclic amines failed to exhibit mutagenicity. In contrast, when Aroclor 1254-induced rat hepatic microsomal preparations served as the activation system, monocyclic, bicyclic as well as larger amino compounds induced a clear mutagenic response; moreover, heterocyclic amines were potent mutagens. Nitrosamines displayed mutagenicity in the presence of only the microsomal activation system. In the presence of the cytosol, the mutagenic response of aromatic amines was much lower in the bacterial strain TA98-1,8-DNP 6, which is deficient in O-acetyltransferase activity, compared to the normal TA98 strain. This finding implies that the cytosolic activation of aromatic amines involves N-hydroxylation.

Synthesis of fjord region tetraols and their use in hepatic biotransformation studies of dihydrodiols of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene.

Metabolic activation of the racemic benzo[c]chrysene-trans-9,10-, benzo[g]chrysene-trans-11,12- and dibenzo[a,l]pyrene-trans-11,12-dihydrodiols to fjord region syn- and anti-dihydrodiol epoxides by microsomes of Aroclor 1254-treated Sprague-Dawley rats has been examined. Since the fjord region dihydrodiol epoxides were hydrolytically unstable under the experimental conditions, their enzymatic formation was determined by analyzing the tetraols as their products of acidic hydrolysis upon addition of perchloric acid. The various stereoisomeric tetraols formed were separated by HPLC and identified by co-chromatography with authentic tetraols, which had been prepared by acidic hydrolysis of synthetically available syn- and anti-dihydrodiol epoxides and characterized by NMR and UV spectroscopy. Under standardized conditions the acidic hydrolysis of syn-dihydrodiol epoxides of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene resulted in the formation of two tetraols with cis/trans ratios of 81:19, 77:23 and 80:20, respectively, whereas the anti-dihydrodiol epoxides underwent almost exclusively trans hydrolysis. The proportion of the stereoisomeric tetraols obtained from microsomal incubations indicates that all three dihydrodiols are predominantly oxidized at the adjacent olefinic double bond to the anti-diastereomers of the corresponding fjord region dihydrodiol epoxides accounting for 4-35% of the ethyl acetate-extractable metabolites. To allow quantitative assessment of the metabolites 3H-labeled trans-dihydrodiols were synthesized by reduction of the corresponding o-quinones with sodium borotritide. Metabolic conversion of benzo[c]chrysene-trans-9,10- and dibenzo[a,l]pyrene-trans-11,12-dihydrodiol by rat liver microsomes were in a similar low range during the first 10 min of incubation (6.2 +/- 1.2 and 3.4 +/- 1.0 nmol substrate/nmol cytochrome P450/10 min, respectively), whereas the conversion of benzo[g]chrysene-trans-11,12-dihydrodiol was much higher (20.6 +/- 2.2 nmol substrate/nmol cytochrome P450/10 min). Given the strong intrinsic mutagenic and carcinogenic activity of the fjord region dihydrodiol epoxides, our data indicate that their formation, even at a relatively low level, may contribute significantly to the biological activity of the parent hydrocarbons.

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling.

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.

Metabolic Activation of the (+)-S,S- and (−)-R,R-Enantiomers of trans-11,12-Dihydroxy-11,12-dihydrodibenzo[a,l]pyrene: Stereoselectivity, DNA Adduct Formation, and Mutagenicity in Chinese Hamster V79 Cells

Polycyclic aromatic hydrocarbons require metabolic activation in order to exert their biological activity initiated by DNA binding. The metabolic pathway leading to bay or fjord region dihydrodiol epoxides as ultimate mutagenic and/or carcinogenic metabolites is thought to play a dominant role. For dibenzo[a,l]pyrene, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon, the formation of the fjord region syn- and/or anti-11,12-dihydrodiol 13,-14-epoxide (DB[a,l]PDE) diastereomers has been found to be the principal metabolic activation pathway in cell cultures leading to DNA adducts. In order to further elucidate the stereoselectivity involved in this activation pathway via the formation of the trans-11,12-dihydrodiol, we have synthesized the enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]-pyrene and investigated their biotransformation in rodents. Incubations with liver microsomes of Sprague-Dawley rats and CD-1 mice pretreated with Aroclor 1254 revealed that the enzymatic conversion to the fjord region DB[a,l]PDE strongly depends on the absolute configuration of the 11,12-dihydrodiol enantiomers. While oxidation at the 13,14-position of the (+)-(11S,12S)-dihydrodiol is limited to a small extent, the (-)-11R,12R-enantiomer is metabolized to its fjord region dihydrodiol epoxides in considerably higher amounts. Moreover, this substrate is transformed with high stereoselectivity to the corresponding (-)-anti-dihydrodiol epoxide by liver microsomes of Aroclor 1254-treated rodents. The metabolism results were in good accordance with the extent of stable adduct formation in calf thymus DNA as investigated by the 32P-postlabeling technique and with the mutagenicity in Chinese hamster V79 cells of the two enantiomeric 11,12-dihydrodiols mediated by hepatic postmitochondrial preparations of Aroclor 1254-treated rats. The results indicate that both genotoxic events occurred predominantly by the stereoselective activation of the (-)-(11R,12R)-dihydrodiol to the (-)-anti-DB[a,l]PDE with R,S,S,R-configuration.

Correlation of the Extent of Fjord-Region Oxidation with DNA Binding and Mutagenicity of the Enantiomeric 11,12-Dihydrodiols of Dibenzo[a,l]pyrene

Abstract In vitro studies on the hepatic biotransformation of the enantiomeric trans-11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) using microsomal fractions of animals pretreated with Aroclor 1254 revealed that the formation of fjord-region dihydrodiol epoxides strongly depends on the absolute configuration of the substrate. Both the (-)-11R,12R- and the (+)-11S,12S-enantiomer are converted diastereoselectively to the (-)- and (+)-anti-dihydrodiol epoxide, respectively, by either rat or mouse liver microsomes. Fjord-region oxidation occurs to greatest extent on incubation of the (-)-11R,12R-dihydrodiol with preparations from rats. This finding is in line with the differences seen for the two enantiomers on the total DNA binding under identical activation conditions as well as on the mutagenic activity in Chinese hamster V79 cells using the postmitochondrial hepatic fraction of Aroclor 1254-treated rats as metabolizing system.

Use of Precision-Cut Liver Slices to Evaluate Species Differences in 2-Acetylaminofluorene-Induced Unscheduled DNA Synthesis

Precision-cut liver slices were prepared from male Sprague–Dawley rats (pretreated with or without Aroclor 1254), male Dunkin–Hartley guinea pigs, male cynomolgus monkeys, and humans. Liver slices were cultured for 24 hr using a dynamic organ culture system in medium containing [3H]thymidine and 2-acetylaminofluorene (2-AAF), aflatoxin B1(AFB1), or 6-aminochrysene (6-AC). The liver slices were then harvested and processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). All three genotoxins induced UDS in liver slices from untreated and Aroclor 1254-treated rats. In human liver slices 2-AAF produced a concentration-dependent induction of UDS and at the highest concentration examined 2-AAF also induced UDS in guinea pig liver slices. However, 2-AAF did not induce UDS in cynomolgus monkey liver slices, although both AFB1and 6-AC induced UDS in liver slices from this species as well as from guinea pigs and humans. The inability of 2-AAF to induce UDS in cynomolgus monkey liver slices appears to be at least partially due to the absence of hepatic CYP1A2 in this species. Precision-cut liver slices appear to be a useful alternative to primary hepatocyte cultures for studies of xenobiotic-induced genotoxicity employing the UDS technique. As shown by this study they may also be employed to evaluate species differences in xenobiotic-induced genotoxicity.

Neonatal Polychlorinated Biphenyl Treatment Increases Adult Testis Size and Sperm Production in the Rat

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants which decrease serum thyroxine (T4) concentrations. We have previously demonstrated that neonatal hypothyroidism in the rat increases Sertoli cell numbers, adult testis weight, and daily sperm production (DSP). The aim of this study was to determine if neonatal PCB treatment increases adult testis weight and DSP. Treated rats received either Aroclor 1242 or 1254 (0.4–3.2 mg/day), from birth to Day 25 by daily injection; some treated litters also received T4replacement. Controls received vehicle alone. Tritiated thymidine autoradiography was used to assess Sertoli cell proliferation in 15-day control and Aroclor-treated rats. Serum T4was measured at 25, 45, and 135 days of age, and serum testosterone, testis weight, DSP, and testicular histology were examined at 135 days. Both Aroclor 1242 and 1254 suppressed serum T4concentrations; Aroclor 1254 was more potent and long lasting. Testis weight was increased 22 and 13% in rats that received the 1.6 and 3.2 mg/day Aroclor 1242 doses, respectively, while the 0.4 mg/day dose did not produce significant increases. Aroclor 1254 produced significant increases in testis weight of 13 and 23% at the 0.4 and 1.6 mg/day doses, respectively. The 1.6 mg/day Aroclor 1242 and the 0.4 and 1.6 mg/day Aroclor 1254 doses increased DSP by 27, 11, and 42%, respectively; other treatments did not produce significant increases. At 15 days of age, Sertoli cell proliferation was greater in treated rats than in controls. T4replacement decreased or eliminated the increased testis weight and DSP seen in Aroclor-treated rats. The highest dose of Aroclor 1242 and both doses of Aroclor 1254 decreased adult body weight, while other treatments did not. These results indicate that neonatal PCB treatment increases adult testis weight and DSP in rats. PCBs produce this effect primarily by inducing hypothyroidism, which leads to increased Sertoli cell proliferation, testis weight, and DSP. Thus PCBs, despite inhibitory effects on adult reproductive organs, can paradoxically stimulate increases in adult testis weight and DSP when administered neonatally. These data emphasize the pleiotropic nature of PCB effects and the susceptibility of the developing reproductive system to environmental factors.