Effect of caffeine on the hepatic microsomal mixed function oxidase system during phenobarbital and benzo[a]pyrene treatment in rats

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.     

In vitro activation and resultant binding of benzo[a]pyrene to DNA by microsomes from rats fed corn and menhaden oils

Dietary unsaturated fat is required for maximum induction of the hepatic mixed function oxidases (MFO) responsible for activating carcinogens to forms that may bind covalently to DNA. The aim of this study was to assess the influence of dietary fat type and content on the activities of some enzymes involved in activation and detoxification of the carcinogen benzo[a]pyrene (B[a]P). Modification of these changes by pretreatment with phenobarbital (PB) was also evaluated. Male rats were fed diet devoid of fat or containing 20% corn oil (CO) or 20% menhaden fish oil (MO) for 4 days. PB induced soluble glutathione S-transferase, a detoxifying enzyme, only in rats fed dietary fats. Microsomes from rats fed both types of dietary fat had increased levels of cytochrome P-450 (P-450) and PB induced P-450 only in rats fed these fats. Although ethoxycoumarin O-dealkylase was significantly elevated in the MO group, the induction by PB was not dependent on dietary fat type or level. Dietary fat increased microsome-catalyzed in vitro binding of [3H]-B[a]P to calf thymus DNA, especially in response to PB. Menhaden oil depressed B[a]P hydroxylation and PB treatment depressed this activity to the greatest extent in rats fed this diet. When calculated as B[a]P metabolized per unit of P-450, PB seems to induce a P-450 in fat fed animals having lower affinity and capacity for B[a]P hydroxylation and activation than in rats fed the fat-free diet.     

3H]benzo[a]pyrene utilization in rats following tracheal implant exposure

Open-ended rat tracheal implants (OETI) were exposed to 40 micrograms [3H]benzo[a]pyrene (B[a]P)-gelatin pellets and the 3H activity in the OETI, the host's tissues and excretia was determined 3-96 h after insertion of the pellets. The radioactivity in the OETI reached near peak activity by 3 h, and decreased almost 10-fold by 24 h. Most of the activity was associated with parent B[a]P throughout the 95 h. The 3H activity in the surrounding tissue also was mostly associated with B[a]P, but the 3H activity in the liver, kidney, blood and urine was mostly associated with water-soluble plus conjugated metabolites. In the feces, 68% of the 3H activity was in B[a]P at 3 h, but mostly organic as well as water-soluble plus conjugated metabolites were extracted from it throughout the remaining 96 h. Forty-eight hours after insertion of the B[a]P pellets, the feces contained almost 16% of the total 3H activity. Pre-exposure of the OETI to B[a]P for 4 days before insertion of the [3H]B[a]P pellets stimulated metabolism of B[a]P in the tracheas approximately 2-fold, but had no significant effect on the host tissues.     

Changes in rat lung microsomal lipids after p-xylene: relationship to inhibition of benzo[a]pyrene metabolism

The relationship between p-xylene's effects on microsomal membranes, cytochrome P-450, and benzo[a]pyrene (BaP) metabolism was studied. p-Xylene (1 g/kg, ip, 1 h) inhibited 3-hydroxy BaP (3-OH) formation and decreased arylhydrocarbon hydroxylase (AHH) activity approximately 40% in rat lung microsomes. BaP dihydrodiol and quinone formation were unchanged by p-xylene administration. Cytochrome P-450 was below the limit of detection in lung microsomes from p-xylene-treated rats. Total phospholipid (PL) and phosphatidylcholine (PC) in microsomal membranes were decreased 28% and 17%, respectively. Cholesterol (CL), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were unchanged. The net activity of enzymes involved in the synthesis of PC, phosphatidylethanolamine-N-methyltransferase I and II (PMT I and PMT II), was slightly elevated by p-xylene. PL/CL and PC/PE ratios, indicators of membrane fluidity, were decreased 34% and 13%, respectively, in microsomes from p-xylene-treated rats. Analysis of fluidity by fluorescence polarization showed that the actual fluidity of treated microsomes was slightly decreased (5%) as compared to controls. The decrease in P-450, PL, and PC is considered to contribute to the inhibition of BaP metabolism.     

The effect of toluene on rat lung benzo[a]pyrene metabolism and microsomal membrane lipids

Toluene (1 g/kg, i.p., 1 and 4 h) was shown to decrease total cytochrome P-450 (P450) content in rat lung. At both timepoints, reduction in pulmonary P450 content was associated with a decrease in aryl hydrocarbon hydroxylase (AHH) activity, a detoxication pathway for benzo[a]pyrene (BaP). At 4 h, toluene increased the toxication/detoxication ratios of BaP metabolites in pulmonary microsomes, primarily via inhibition of hydroxy metabolite formation. The structurally analogous solvents p- and m-xylene have been previously shown to produce a similar pattern of MFO changes in rat lung; the inhibition of BaP metabolism was found to be related to alterations in pulmonary microsomal lipids following administration of p- but not m-xylene. In the present study, toluene-induced alterations in MFO parameters were not found to be associated with changes in microsomal lipids. Toluene did not affect either total phospholipid or cholesterol content at either timepoint. Similarly, no changes in speciated phospholipids were observed. Membrane integrity, expressed as conjugated diene formation, also remained unchanged following toluene administration. Toluene did, however, decrease microsomal inner-core membrane fluidity at 4 h but had no effect on membrane leaflet fluidity at either timepoint. These data suggest that the fatty acid composition of microsomal lipids may play a role in the metabolic alterations observed in pulmonary microsomes following toluene administration.     

Effects of aloe, aloesin, or propolis on the pharmacokinetics of benzo[a]pyrene and 3-OH-benzo[a]pyrene in rats

This study was conducted to examine the effects of aloe and aloesin on the weight gain and blood chemistry as well as the pharmacokinetics of benzo[a]pyrene (BaP) and 3-OH-BaP in rats. The rats treated with multiple doses of aloe and aloesin (100 mg/kg every 12 h for 14-19 d) did not show any significant changes in the weight gain and blood biochemical parameters. In addition, the effects of oral treatment with aloe, aloesin, and propolis on the absorption and pharmacokinetics of benzo[a]pyrene (BaP) and its metabolite, 3-OH-BaP, were studied in rats. The treatment with a single oral dose (200 mg/kg) of aloe, aloesin, and propolis did not alter the concentration-time profiles of BaP and 3-OH-BaP after iv and oral administration of BaP. At higher oral doses (500 mg/kg), the biliary excretion of BaP and the urinary excretion of 3-OH-BaP were significantly increased, but the urinary excretion of BaP and the fecal excretion of 3-OH-BaP remained unaltered. Whether high doses of aloe increase the overall elimination of BaP deserves further investigation.     

Acute and subchronic oral toxicities of benzo[a]pyrene in F-344 rats.

We have studied the acute and subchronic oral toxicities of benzo[a]pyrene (BaP) in male and female F-344 rats. Single acute BaP doses of 0, 100, 600, and 1000 mg /kg dissolved in peanut oil were administered by oral gavage. Subchronic doses of 0, 5, 50, and 100 mg/kg/day were administered for 90 days in the animal diet. The major toxicological endpoints examined included animal body weight, selected tissue weights, and histopathological examinations (liver, kidney, stomach, prostate, testes, and ovaries). In addition, we examined blood elements: red blood cells (RBC), white blood cells (WBC), hemoglobin (Hgb), hematocrit (Hct), mean cell volume (MCV), mean cell hematocrit (MCH), and mean cell hemoglobin concentration (MCHC), blood chemistry (ALT, AST, and BUN), and urine chemistry (glucose, bilirubin, specific gravity, pH, protein, urobilinogen, nitrite, occult blood, and leucocytes). In the acute study, WBC were significantly decreased and mean cell-hemoglobin concentration was significantly increased, both in males only. The liver:body weight ratio was significantly increased in males and females (up to 30%). None of the blood chemistry or urine parameters were significantly affected. In the subchronic study, mean body weight was significantly decreased in males only (13%), and the liver:body weight ratio in males was significantly increased. Several of the blood elements were significantly decreased in males and females after 90 days; RBCs (up to 10%), Hct (up to 12%), and Hgb (up to 12%). For blood chemistry parameters (AST, ALT, BUN), only BUN in males was significantly increased in the high dose group (100 mg/kg) at the 90 day time point. The histopathological examination of selected tissues showed significant abnormalities (tubular casts) only in the male kidney, at the 2 highest doses, after 90 days. These studies indicate that the acute and subchronic toxicities of BaP are relatively low, BaP affects specific blood elements and organs, and BaP has a greater effect on males than females. The induction of non-carcinogenic kidney abnormalities in males only may be indicative of renal dysfunction and further substantiates an apparent sex difference in tolerance to BAP:     

A bioactive metabolite of benzo[a]pyrene, benzo[a]pyrene-7,8-dione, selectively alters microsomal Ca2+ transport and ryanodine receptor function

Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca(2+) homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca(2+) transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca(2+) transport across microsomal membranes. B[a]P-7,8-dione induces net Ca(2+) release from actively loaded vesicles in a dose-, time-, and Ca(2+)-dependent manner. In the presence of 5 microM extravesicular Ca(2+), B[a]P-7,8-dione exhibited threshold and EC(50) values of 0.4 and 2 microM, respectively, and a maximal release rate of 2 micromol of Ca(2+) min(-1) mg(-1). The mechanism by which B[a]P-7,8-dione enhanced Ca(2+) efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione (< or = 20 microM) had no measurable effect on initial rates of Ca(2+) accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca(2+)-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca(2+)-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.     

Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.     

Pulmonary retention of [14C]benzo[a]pyrene in rats as influenced by the amount instilled

Studies on the pulmonary retention of benzo[a]pyrene after inhalation have shown that clearance is biphasic, with one component clearing with a half-time greater than 1 day and another with a half-time less than 1 day. In the work reported here we demonstrated that the amount of benzo[a]pyrene instilled in the lungs can affect the rate at which the benzo[a]pyrene is cleared into the blood. Fischer-344 rats were given 16, 90 or 6400 ng of [14C]benzo[a]-pyrene/rat by intratracheal instillation. Rats were sacrificed at various times up to 7 days after instillation. Individual lung lobes and trachea were removed, digested, and analyzed by liquid scintillation spectrometry. At 24 h after instillation the amount of 14C covalently bound to lung macromolecules was determined in some rats. Benzo[a]pyrene equivalents remaining in the lungs was expressed as a percentage of the instilled dose as a function of time. A two-component negative exponential function was fit to the data. With increasing dose (16-6400 ng/rat), an increasing percent (89-99.76%) was cleared with a half-time less than 1 day and a decreasing percent (11.3-0.24%) was cleared with a half-time greater than 1 day, suggesting that the mechanism by which the slower clearances occurred had been saturated at higher doses. At 24 h after instillation, from 1 to 2 pmol of [14C]benzo[a]-pyrene equivalents/lung were covalently bound to lung macromolecules. There was no difference in the amount of covalently bound 14C over the range of instillation doses used, suggesting that a small amount of benzo[a]-pyrene equivalents was bound in the lungs regardless of the amount instilled. These results suggested that linear extrapolation from high dose studies to environmental concentrations might underestimate lung burdens of benzo[a]pyrene.     

Carcinogenic activity of benzo[a]pyrene in a 2 year oral study in Wistar rats

Because of the relatively high human oral exposure to polycyclic aromatic hydrocarbons (PAHs) compared to the inhalation exposure, the known carcinogenicity of this type of compounds and the limited data from oral studies available with polycyclic aromatic hydrocarbons, an oral carcinogenicity study was performed using benzo[a]pyrene (B[a]P) as a PAH representative. Wistar rats, 52 animals per sex and group were exposed daily (5 days a week) to 0, 3, 10 or 30 mg B[a]P/kg bw/day by gavage for 104 weeks and were subject to gross- and histopathology. The main tumours observed were hepatocellular carcinomas and forestomach tumours. Other tumours induced in this study were tumours of the auditory canal, skin and appendages, oral cavity, small intestine, kidney, and soft tissue sarcomas. For hepatocellular carcinomas and forestomach tumours, the BMDL10 were 3 and 1 mg/kg bw/day, respectively. The incidence of altered hepatic foci was increased in the 3mg/kg bw/day group. The increase in liver tumours is considered the most relevant effect for human risk assessment in terms of pathogenesis and sensitivity, and is proposed as the basis for human cancer risk assessment for oral PAH exposure.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [¹⁴C]benzo[a]pyrene (3 μmol/kg) to rabbits, 30% of the ¹⁴C dose appeared in bile and 12% in urine, within six hours.

2. Biliary and urinary metabolites were mainly conjugated; <12% of the ¹⁴C was extractable with ethyl acetate, but after treatment with β-glucuronidase or aryl sulphatase 30–40% became extractable.

3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of ¹⁴C in bile and 24% of ¹⁴C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total <10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total <4%).

4. The proximate carcinogen, the 7,8-diol, was not detected in any extract.

5. After intraduodenal administration of biliary metabolites of [¹⁴C]benzo[a]pyrene (approx. 0·3 μmol), ¹⁴C was excreted in the bile (21% dose) and urine (14%) within 23?h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [14C]benzo[a]pyrene (3 mumol/kg) to rabbits, 30% of the 14C dose appeared in bile and 12% in urine, within six hours. 2. Biliary and urinary metabolites were mainly conjugated; less than 12% of the 14C was extractable with ethyl acetate, but after treatment with beta-glucuronidase or aryl sulphatase 30-40% became extractable. 3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of 14C in bile and 24% of 14C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total less than 10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total less than 4%). 4. The proximate carcinogen, the 7,8-diol, was not detected in any extract. 5. After intraduodenal administration of biliary metabolites of [14C]benzo[a]pyrene (approx. 0 X 3 mumol), 14C was excreted in the bile (21% dose) and urine (14%) within 23 h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

In vitro influence of molybdenum on benzo[a]pyrene metabolism in hepatic and pulmonary rat microsomes

Our study presents the in vitro molybdenum influence on benzo[a]pyrene (BaP) microsomal metabolism. Addition of various concentrations of different molybdenum salts [MoS2, MoCl5, (NH4)6Mo7O24 . 4H2O] to liver and lung microsomal fractions of rats previously treated with 3-methylcholanthrene produces a decrease in the different BaP metabolites assessed by high-performance liquid chromatography (HPLC) analysis. This inhibition varies, depending on the considered metabolite and in relation to both the molybdenum level and the origin of the microsomal suspension. The minimum effective concentration is 0.26 and 0.52 mM Mo from liver and lung, respectively. The inhibitory potencies of the +5 (chloride) and +6 (ammonium heptamolybdate) molybdenum compounds are comparable; that of the sulfide is lower.     

Formation of hemoglobin-benzo[a]pyrene adducts in human erythrocytes incubated with benzo[a]pyrene and hamster embryo cells

Evidence is accumulating that the levels of covalent carcinogen-macromolecule adducts, including adducts with hemoglobin, reflect biologically effective levels of carcinogen exposure. The purposes of the present study were (a) to establish a cellular system for obtaining adducts between intracellular human hemoglobin and metabolites of polycyclic aromatic hydrocarbons (PAH), and (b) to evaluate techniques for chromatographic characterization of the adducts. We showed that hemoglobin-benzo[a]pyrene adducts were formed when human erythrocytes were treated with [3H]benzo[a]pyrene (BP) in the presence of hamster embryo fibroblasts, which are known to be effective for BP metabolism. After lysis of the erythrocytes, noncovalently bound BP and its metabolites were effectively removed from hemoglobin under mild conditions by using hydrophobic interaction and size-exclusion liquid chromatography. Three to five distinct adducts were resolved by reversed-phase and ion-exchange liquid chromatography. As determined by a two-step, reversed-phase liquid chromatographic procedure, trypsin treatment of globin from the cellular system yielded at least three of the four 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy BP tetrols known to arise from mammalian metabolism of BP. This observation is consistent with both (a) the recently described formation of labile carboxyl esters via reaction of BP-7,8-dihydrodiol-9,10-epoxide (BPDE) with hemoglobin and (b) the known formation of both anti- and syn-BPDE in hamster embryo fibroblasts. In addition, high-performance liquid chromatographic analysis demonstrated the presence of other products presumed to be BP-peptide adducts because of their susceptibility to thermolysin treatment.     

Clotrimazole as an inhibitor of benzo[a]pyrene metabolite-DNA adduct formation in vitro and of microsomal mono-oxygenase activity

The fungistatic drug clotrimazole (1-[(o-chlorophenyl)diphenylmethyl]imidazole) in concentrations of 5 or 50 microM completely prevented the formation of benzo[a]pyrene metabolite-DNA adducts in vitro catalyzed by liver microsomes from phenobarbital-or 3-methylcholanthrene-treated rats, respectively. Microsomal 7-ethoxycoumarin de-ethylase and aryl hydrocarbon hydroxylase were effectively inhibited by clotrimazole and three clotrimazole derivatives in all induction states tested, with I50 values down to 7 x 10(-8) M. The mechanism of inhibition was noncompetitive in phenobarbital-stimulated microsomes. Microsomal epoxide hydratase in vitro was enhanced up to 450% by clotrimazole and one of the analogues in concentrations between 5 and 500 microM. Clotrimazole spectrally interacted with reduced cytochrome P-450, exhibiting a double-banded Soret region with peaks at 427 and 446 nm, and partially prevented cytochrome P-450-CO complex formation. When administered in vivo, clotrimazole effectively induced cytochrome P-450 content, mono-oxygenase activity and epoxide hydratase activity in rat liver microsomes. The induction pattern was similar to that obtained with phenobarbital. The analogues were less potent inducers.     

Lung metabolism of benzo[a]pyrene in rats treated with p-xylene and/or ethanol

The metabolism of benzo[a]pyrene (BaP) may be altered by xenobiotic compounds. The effects of p-xylene and ethanol on the lung metabolism of BaP were studied. p-Xylene was administered by ip injection at doses ranging from 0.1 to 1.0 g/kg (1:1 in soybean oil). Ethanol was administered po at 5 g/kg (40% w/v). Rats given p-xylene, ethanol, or p-xylene and ethanol were sacrificed 1 h after treatment. Additional time points of 15 min, 30 min, 4 h, and 24 h after p-xylene (1 g/kg) were examined. 3-Hydroxy-BaP (3-OH) formation was measured fluorometrically as aryl hydrocarbon hydroxylase activity (AHH) in lung microsomes. p-Xylene (1 g/kg) inhibited the formation of 3-OH BaP 40% at 15 min, 27% at 30 min, 43% at 1 h, and 39% at 4 h after treatment. Inhibition of AHH activity was still present 24 h after dosing (41%). AHH activity was inhibited 27% and 46% at 0.5 mg/kg and 1.0 mg/kg p-xylene (1 h), respectively, while the lowest dose (0.1 mg/kg) did not change activity. Analysis of the major metabolites of BaP by high-performance liquid chromatography (HPLC) demonstrated that the formation of 3-OH and 4,5-diol BaP were inhibited 32% and 50%, respectively, in lung microsomes prepared 24 h after a single injection of p-xylene (1 g/kg). None of the other metabolites analyzed were changed by p-xylene. Ethanol had no effect on 3-OH BaP formation during a 1-h treatment. A combined dose of ethanol and p-xylene moderately inhibited 3-OH BaP formation. These findings indicate that BaP detoxication (i.e., 3-OH formation) in rat lung is selectively inhibited by p-xylene but not ethanol. Ethanol appears to modify the inhibitory effect of p-xylene.