Indole-3-carbinol protects against covalent binding of benzo[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60-90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63-85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.     

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

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.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.     

Granuloma pouch assay. II. Induction of 6-thioguanine resistance by MNNG and benzo[a]pyrene in vivo

Growth of granulation tissue was initiated with croton oil on the inside of a subcutaneous air pouch, on the back of adult male rats. Two days later, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (BP) were applied directly into the pouch in doses ranging from 0.05 to 1.8 mg and from 0.03 to 0.5 mg, resp. The granulation tissue was excised after 48 h. Isolated single cells were checked for their 6-thioguanine resistance in vitro. The influence of cell density during expression time in vitro, of 6-thioguanine concentration and cell density in selective media on the recovery of mutant cells was investigated. The spontaneous mutation frequency was 0.53 x 10(-5). There was a dose-dependent increase in mutation frequencies with both compounds. The frequency was 5 times as high with MNNG as with BP.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats.

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [3H]benzo[a]pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72-89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5-1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzo-flavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.     

Analysis of benzo[a]pyrene deactivation mechanisms in rats

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

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450-catalyzed stereoselective epoxidation at the K region of benz[a]anthracene and benzo[a]pyrene

The enantiomers of K-region benz[a]anthracene (BA) 5,6-epoxide and benzo[a]pyrene (BP) 4,5-epoxide were resolved by chiral stationary-phase high-performance liquid chromatography (CSP-HPLC). The K-region epoxides formed in the metabolism of BA by liver microsomes from untreated (control), phenobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats were determined by CSP-HPLC to have a 5R,6S/5S,6R enantiomer ratio of 25:75, 21:79, and 4:96, respectively. The K-region 4,5-epoxide formed in the metabolism of BP by the same rat liver microsomal preparations contained a 4R,5S/4S,5R enantiomer ratio of 48:52 (control), 40:60 (PB), and 5:95 (MC), respectively. The results indicate that various cytochrome P-450 isozymes of rat liver exhibit different stereoselective properties in catalyzing the epoxidation reactions at the K region of BA and of BP.     

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

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

Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.     

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

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

Organ-selective induction of cytochrome P-450-dependent activities by indole-3-carbinol-derived products: influence on covalent binding of benzo[a]pyrene to hepatic and pulmonary DNA in the rat.

Indole-3-carbinol (I3C) is a dietary modulator of carcinogenesis that can reduce the level of carcinogen binding to DNA. I3C-derived products are potent inducers of certain cytochrome P-450(CYP)-dependent enzyme activities. To investigate whether the protective effects of I3C against carcinogen damage to DNA are associated with increased activities of CYP1A1 enzymes, we examined the relationship of I3C-mediated organ-specific CYP enzyme induction with total levels of benzo[a]pyrene (BP) binding to hepatic and pulmonary DNA of rats. Oral intubation (PO) of I3C (500 mumol/kg body wt.) in 10% DMSO in corn oil produced after 20 h, increases in ethoxyresorufin O-deethylase (EROD) activities (associated with CYP1A1 isozyme) of 700-fold, 245-fold and 36-fold in small intestine, lungs and liver, respectively, compared with activities in untreated controls. Hepatic aryl hydrocarbon hydroxylase (AHH) activity was increased 4-fold under these conditions. Pentoxyresorufin O-depentylase (PROD) activity (associated with CYP2B isoenzyme) was increased 6-fold in the liver but was unaffected in lung and small intestine. Intraperitoneal injection (IP) of I3C (500 mumol/kg body wt.) produced no significant change in EROD or PROD activities in lung, liver, or small intestine. PO administration of the acid reaction mixture (RXM) of I3C increased hepatic AHH activity (5-fold) and EROD activities in small intestine (650-fold), lung (100-fold) and liver (18-fold). IP administration of RXM (equivalent to 500 mumol I3C/kg body wt.) significantly increased only EROD activity in lung and liver, but did not affect EROD activity in small intestine, AHH activity in liver, or PROD activity in any of the organs examined. Twenty hours after inducer treatment, half of the rats were treated PO with 0.2 mumol [3H]BP in corn oil. Analysis of tissues 5 h after BP administration indicated that compared with untreated controls, administration of I3C and RXM by either route reduced by 30-50% the level of BP binding to hepatic DNA, an effect that was not correlated to CYP1A1 enzyme induction in any of the organs examined. However, PO administration of I3C and RXM produced a 50-70% decrease in carcinogen binding to pulmonary DNA, while IP administration of inducers had no effect on DNA binding in this organ. These results with the lung are consistent with an increased presystemic clearance of BP in the intestine and are discussed in terms of the role of induction of intestinal CYP1A1 activity in the decreased lymphatic and venous transport of unmetabolized BP to the lung.     

Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and ~50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O₂ and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450S were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450_PB-B greatly inhibited the reaction (>90%), while antibodies to P-450_PB-C or P-450_PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9–75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced ~140-fold at this dose and ~4-fold by a dose of phenobarbital as low as 0.9 mg/kg.     

Dietary fat inhibits the intestinal metabolism of the carcinogen benzo[a]pyrene in fish

Following the intestinal absorption of dietary benzo[a]pyrene (BP) by the killifish, this compound becomes incorporated along with dietary triglycerides into membrane-bound fat vacuoles within the intestinal epithelial cell (1985. J. Lipid Res. 26: 428-434). These vacuoles, arising from the smooth endoplasmic reticulum, are important transient structures involved in both the uptake and metabolism of dietary BP and, presumably, other lipophilic toxicants as well. In the present study we used subcellular fractions isolated from the intestines of spot (Leiostomus xanthurus), a teleost fish, to study factors that influence the metabolism of BP in a fat vacuole/microsomal system. Triglyceride-solubilized BP is capable of diffusion from fat vacuoles to microsomal enzymes. Increases in the concentration of fat vacuoles decrease the availability of BP to microsomal BP hydroxylase. The effect of fat vacuoles on the activity of BP hydroxylase becomes more pronounced as the concentration of BP in our test system decreases. Addition of cytosolic glutathione transferases to the fat vacuole/microsomal system enhances the activity of BP hydroxylase. Examination of binding of 3H-labeled BP to killifish (Fundulus heteroclitus) intestinal cytosolic proteins in vivo indicated that a large fraction of the radioactivity was associated within glutathione transferase. These results suggest that dietary fat inhibits metabolism of low levels of BP in the intestine. A consequence of this would be greater exposure of peripheral tissues to dietary carcinogens.     

Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice

PCBs are complete rodent carcinogens and their potent tumor promoting activity has been reported, but their tumor-initiating activity remains controversial. Macromolecular binding of PCB metabolites has been demonstrated in vitro, but this issue remains unclear in vivo. The purpose of this study was to determine the binding affinity of 4-chlorobiphenyl and 3,3',4,4'-tetrachlorobiphenyl to proteins and DNA in vivo. C57/BL6 female mice were treated intraperitoneally with hepatic enzyme inducers (phenobarbital and beta-naphthoflavone) and then with 14C-labelled polychlorinated biphenyls or benzo[a]pyrene. The short-term distribution of labeled compounds into liver, lungs and kidneys and into different sub-cellular fractions of these tissues was assessed and the DNA and proteins from the 700 x g pellet were further purified to assess covalent binding. All compounds were distributed in low amounts into the liver, kidneys and lungs, with the greatest accumulation in the liver, and the lowest in lungs. In all tissues, test compounds were mostly found in cytosols and organellar pellets (10,000 x g), and lower amounts were present in nuclear pellets (700 x g) and microsomes. In lungs and kidneys, only benzo[a]pyrene showed significant covalent binding to proteins. In the liver, protein binding indices were significant for all compounds (P<0.05), but no significant binding of the test compounds to DNA could be demonstrated with this approach. Our results suggest that at the 24 h time point, all compounds were activated to electrophilic intermediates prone to macromolecular binding. Hepatic proteins apparently act as a sink for PCB-derived electrophiles, thus preventing detectable levels of covalent binding to hepatic DNA or to proteins in less metabolically active tissues.     

Ascorbic acid reduces accumulation of [3H]spiperone in mouse striatum in vivo

[3H]Spiperone was administered (20 microCi/kg, 0.0003 mg/kg, sc) to mice. In agreement with other published reports, 2 hr later the accumulation of tritium was three to four times greater in the corpus striatum than in the cerebellum. Ascorbic acid (100, 1000, 2000 mg/kg, ip, 30 min) reduced the 2-hr accumulation in the corpus striatum 16, 42, and 63%, respectively, with only the highest dose producing any significant (18%) reduction in the cerebellum. The effect was still evident in striatum 18 hr after a single dose of 1000 mg/kg. Striatal minces taken from mice treated 1 or 2 hr earlier with ascorbic acid (2000 mg/kg, ip) showed no reduction in [3H]spiperone binding. However, preincubation of striatal minces for 2 hr with ascorbic acid (10(-3) M) produced an 82% reduction in specific binding while not having any effect on nonspecific binding. While it cannot be certain that the reduction of striatal [3H]spiperone concentrations after ascorbic acid in vivo was not a result of some nonspecific alteration in the pharmacokinetics of [3H]spiperone, the in vitro observation strongly suggests that it resulted from an alteration of binding characteristics at the receptor level.