Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

N-benzylimidazole, a high magnitude inducer of rat hepatic cytochrome P-450 exhibiting both polycyclic aromatic hydrocarbon- and phenobarbital-type induction of phase I and phase II drug-metabolizing enzymes

The effects of N-benzylimidazole on hepatic microsomal and cytosolic drug-metabolizing enzymes were compared to the effects produced by phenobarbital, beta-naphthoflavone, a polycyclic aromatic hydrocarbon, and Aroclor 1254, a polychlorinated biphenyl mixture. N-Benzylimidazole was a "high magnitude" inducer of male rat hepatic cytochrome P-450, inducing cytochrome P-450 over 3 times above control. N-Benzylimidazole exhibited mixed type induction of cytochrome P-450, producing both polycyclic aromatic hydrocarbon- and phenobarbital-type induction. There was no evidence of imidazole (isoniazid) type induction characteristics. Microsomes from rats treated with either Aroclor 1254 or N-benzylimidazole showed a common pattern of induction of the cytochrome P-450-dependent properties and glucuronosyltransferase activities, and the electrophoretic profiles of proteins were also similar. Cytosolic glutathione transferase activity was also induced similarly after treatment with the two agents.     

Effect of enzyme induction on Sandimmun (cyclosporin A) biotransformation and hepatotoxicity in cultured rat hepatocytes and in vivo

This study was designed to examine the relationship between the extent of Sandimmun (cyclosporin A, SIM) metabolism and SIM-induced hepatotoxicity both in vivo and in primary cultures of rat hepatocytes. Firstly, SIM (50 mg/kg p.o.) was administered daily to male Wistar rats for 10 days with or without co-administration of Aroclor 1254. SIM-induced hepatotoxicity appeared after 4 days of treatment and was enhanced after 10 administrations of SIM. Total plasma proteins were decreased and hyperbilirubinemia as well as increased levels of plasma bile salts were prominent. Aroclor 1254 stimulated total hepatic cytochrome P-450 3.7-fold, and markedly increased the rate of SIM metabolism and plasma elimination as determined by both HPLC and RIA techniques. However, this induction did not change the degree of SIM-induced hepatotoxicity. Secondly, short-term cultures of hepatocytes obtained from normal rats and from rats pretreated with either Aroclor 1254 or dexamethasone, a specific inducer of the cytochrome P-450 III gene family responsible for the formation of the primary SIM metabolites M1, M17 and M21, were incubated with various concentrations of SIM for up to 17 hr. At 1 microM SIM, both inducers greatly increased the rate of SIM metabolism in vitro, producing, however, different metabolite patterns. In the hepatocyte cultures, SIM inhibited the incorporation of amino acids into proteins. In addition, a small fraction of [3H]-labeled SIM was covalently bound to hepatocellular macromolecules. Although the fraction of covalently bound SIM was markedly increased in cells from dexamethasone-treated rats, the degree of inhibition of hepatocellular protein synthesis was not changed in cells from induced rats. In contrast to SIM-induced nephrotoxicity, these results suggest that increased rates of SIM biotransformation by inducers of drug metabolism are not associated with an attenuation of hepatotoxicity both in vivo and in vitro.     

Differential induction of cytochrome P-450 catalyzed activities by polychlorinated biphenyls and benzo [a]pyrene in B6C3F1 mouse liver and lung

The properties of some constitutive and inducible enzyme activities of liver and lung microsomes were determined in B6C3F1 mice pretreated by either intratracheal (i.t.) administration of benzo[a]pyrene (BaP) or polychlorinated biphenyl (PCBs) mixture (Aroclor 1254), or intraperitoneal (i.p.) administration with Aroclor 1254. After i.p. administration of Aroclor 1254, liver cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH), benzphetamine N-demethylase and nitroreductase activities were increased 2.8-, 2.0-, 2.2-, and 2.0-fold, respectively. Lung cytochrome P-450 content was also increased (1.9-fold) after i.p. administration of Aroclor 1254; AHH and nitroreductase activities, however, were not affected and benzphetamine N-demethylase activity was decreased. Aroclor 1254 administered i.t. did not affect liver cytochrome P-450 content. However, AHH and benzphetamine N-demethylase activities were decreased 1.4- and 1.2-fold, respectively, and nitroreductase activity was increased 1.6-fold. After i.t. administration of Aroclor 1254, lung cytochrome P-450 content and AHH activity were increased 1.4- and 2.2-fold, respectively. Benzphetamine N-demethylase activity was decreased 2.1-fold and nitroreductase activity was not affected. After i.t. administration of BaP, liver 7-ethoxyresorufin O-deethylase and nitroreductase activities were increased 2.2- and 1.5-fold, respectively, and benzphetamine N-demethylase activity was decreased 1.3-fold. Lung AHH and 7-ethoxyresorufin O-deethylase activities were increased 4.3- and 3.1-fold, respectively, and cytochrome P-450 content, benzphetamine N-demethylase and nitroreductase activities were decreased 1.4-, 1.2- and 1.3-fold, respectively, after BaP administration. These data indicate that different cytochrome P-450 isozymes induced in B6C3F1 mice are responsible for monooxygenase and nitroreductase activities, and that the route of administration of chemicals is important in the expression of cytochrome P-450 catalyzed activities.     

Xenobiotic metabolizing enzymes are not restricted to parenchymal cells in rat liver

To characterize the distribution and inducibility of drug metabolizing enzymes within different hepatic cell populations, the activities of aminopyrine N-demethylase, ethoxyresorufin O-deethylase, microsomal epoxide hydrolase and cytosolic glutathione transferase were measured in liver parenchymal, Kupffer, and endothelial cells isolated from untreated rats or rats pretreated with phenobarbital, 3-methylcholanthrene, or Aroclor 1254. Enzyme activities, measurable in all cases, were 2.3- to 5.7-fold higher in parenchymal cells than in Kupffer and endothelial cells. Phenobarbital increased aminopyrine N-demethylase, microsomal epoxide hydrolase, and cytosolic glutathione transferase activities, whereas 3-methylcholanthrene enhanced ethoxyresorufin O-deethylase, epoxide hydrolase, and glutathione transferase activities in the three cell populations. Aroclor 1254 consistently induced each of the enzyme activities in parenchymal, Kupffer, and endothelial cells. Western blot analyses revealed clear differences in the expression of proteins immunologically related to cytochrome P-450 PB-1, and glutathione transferases B and X in parenchymal cells compared with the corresponding Kupffer and endothelial cells. In contrast, only minor differences between the cell types were apparent in the expression of cytochromes P-450 PB-4, P-450 MC1a, P-450 MC1b and microsomal epoxide hydrolase. These studies establish that oxidative and postoxidative drug metabolizing enzymes are not restricted to parenchymal cells: similar but distinguishable complements of these enzymes are also found in Kupffer and endothelial cells.     

Triglyceride accumulation in long-term cultures of adult rat hepatocytes by chronic exposure to Aroclor 1254

The effect of chronic exposure to micromolar concentrations of Aroclor 1254 (Aro) on the hepatic lipid metabolism was studied in long-term cultures of adult rat hepatocytes. Hepatocytes were cocultivated with mitomycin C-treated 3T3 cells and exposed for 2 wk to Aroclor 1254 concentrations ranging from 0.01 to 20 micrograms/ml. The Aro-exposed cultures showed intracytoplasmic lipid droplets and a maximum increase of 55% in the triglyceride (TG) content and of 4.4-fold in the cytochrome P-450 content. Labeling studies with [14C]acetic and [14C]oleic acid showed no changes in the uptake of fatty acid and TG precursors by the Aro-treated cultures; the synthesis of cellular lipids from [14C]acetic acid was slightly inhibited by Aroclor 1254, but that from [14C]oleic acid was increased, specially for TG (37%). The secretion of total lipids and TG was 2.1- and 2.7-fold lower, respectively, in the cultures treated with 20 micrograms/ml of Aroclor 1254, resulting in an increase of 1.9-fold in the intracellular content of TG. The synthesis of cellular proteins labeled with [3H]leucine was unchanged in the Aro-treated cultures, but the secretion of exportable proteins was 1.7-fold lower in the cultures treated with 20 micrograms/ml of Aroclor 1254. Our results showed that long-term exposure to in vivo relevant concentrations of Aroclor 1254 produced morphological and biochemical changes in cultured hepatocytes, like those described in vivo, and intracellular TG accumulation due mostly to impaired secretion of TG by the hepatocytes. Our results also suggest that this culture system could be useful for the screening of toxic agents producing fatty liver and the study of the involved mechanism(s).     

Binding of reactive metabolites of CCl4 to specific microsomal proteins

The covalent binding of [14C]carbon tetrachloride to microsomal proteins in rat liver microsomes under anaerobic conditions was investigated by SDS-polyacrylamide slab gel electrophoresis and fluorography. Most of the labeled proteins were observed in the molecular weight range of 52-61 kDa, indicating that cytochrome P-450 forms (EC 1.14.14.1) were labeled. Protein bands at the position of the NADPH-cytochrome P-450 reductase (78 kDa) (EC 1.6.2.4) and NADH-cytochrome b5 reductase (33 kDa) (EC 1.6.2.2) also showed radioactivity. The fluorographic pattern of the protein labeling was cytochrome P-450-dependent, as was demonstrated by CO and metyrapone inhibition as well as by pretreatment of rats with inducing drugs such as 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Aroclor 1254. Immuno-precipitation with a purified anti-P-450 immunoglobulin against cytochrome P-450 PB-B (52 kDa) of rat liver indicated that this protein contained about 10-20% of the total bound radioactivity in an average ratio of 0.8 mol [14C]CCl4-metabolite/mol cytochrome P-450 PB-B.     

Potentiation of carbon tetrachloride hepatotoxicity in rats by pretreatment with polychlorinated biphenyls.

Pretreatment of male rats with Aroclor 1254 at a dose of 25 mg/kg i.p. for 6 days resulted in potentiation of the hepatotoxicity of inhaled carbon tetrachloride (CCl4) as evidenced by a decrease in liver glucose-6-phosphatase and elevations of serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), isocitrate dehydrogenase, and sorbitol dehydrogenase. Aroclor 1254 alone did not demonstrate hepatotoxicity. Aroclor 1254 administration resulted in large increases in cytochrome c reductase, cytochrome P-450 (448) AND P-Nitroanisole demethylation. Subsequent exposure to CCl4 vapor resulted in over 70% decreases in the latter two parameters. The potentiation was dose-dependent with a dose of 5 mg/kg or higher being effective. Aroclor 1260 administration gave results similar to those of Aroclor 1254, but Aroclor 1221 enhanced CCl4 toxicity to a lesser extent.     

In vivo oxidative damage in rats is associated with barbiturate response but not other cytochrome P450 inducers

Previously published studies have shown that cytochrome P450 (P450) enzyme systems can produce reactive oxygen species and suggest roles of P450s in oxidative stress. However, most of the studies have been done in vitro, and the potential link between P450 induction and in vivo oxidative damage has not been rigorously explored with validated biomarkers. Male Sprague-Dawley rats were pretreated with typical P450 inducers (beta-naphthoflavone, phenobarbital (PB), Aroclor 1254, isoniazid, pregnenolone 16alpha-carbonitrile, and clofibrate) or the general P450 inhibitor 1-aminobenztriazole; induction of P4501A, -2B, -2E, -3A, and -4A subfamily enzymes was confirmed by immunoblotting and the suppression of P450 by 1-aminobenztriazole using spectral analysis. PB and Aroclor 1254 significantly enhanced malondialdehyde and H2O2 generation and NADPH oxidation in vitro and significantly enhanced formation in vivo, in both liver and plasma. Some of the other treatments changed in vitro parameters but none did in vivo. The PB-mediated increases in liver and plasma F2-isoprostanes could be ablated by 1-aminobenztriazole, implicating the PB-induced P450(s) in the F2-isoprostane elevation. The markers of in vivo oxidative stress were influenced mainly by PB and Aroclor 1254, indicative of an oxidative damage response only to barbiturate-type induction and probably related to 2B subfamily enzymes. These studies define the contribution of P450s to oxidative stress in vivo, in that the phenomenon is relatively restricted and most P450s do not contribute substantially.     

Protective role of Lycopene against Aroclor 1254-induced changes on GLUT4 in the skeletal muscles of adult male rat

Aroclor 1254 is the commercial mixture of highly toxic environmental pollutant, polychlorinated biphenyls (PCBs). Being immensely durable, it is extensively used and widely distributed. Studies show that Aroclor 1254 causes a variety of adverse health effects through free radical generation. The present investigation was designed to check the effect of Aroclor 1254 on the glucose transporter protein, GLUT4, which plays a key role in glucose homeostasis. The protective role of lycopene against the adverse effect of Aroclor 1254 was also tested. Group 1 rats received corn oil as vehicle and served as control. Groups 2, 3, and 4 were administered with Aroclor 1254 [2?mg kg^?1 body weight (b.w.) day^?1] intraperitoneally for 30 days. Groups 3 and 4 received lycopene (2 and 4?mg kg^?1 b.w. day^?1, respectively) orally in addition to Aroclor 1254. After 30 days, animals were euthanized and the skeletal muscles were dissected to determine the following parameters: GLUT4 messenger RNA (mRNA), GLUT4 protein (both plasma membrane and cytosolic fractions), and ¹⁴C-2-deoxyglucose uptake. Though there was no change in GLUT4 mRNA and fasting plasma glucose levels, Aroclor 1254 significantly decreased the GLUT4 protein level in both the subcellular fractions of the gracilis and triceps muscles. Most important, ¹⁴C-2-deoxyglucose uptake showed a significant decrease in Aroclor 1254 alone treated rats, and Aroclor 1254 plus 4?mg lycopene supplementation treatment maintained the same at par with control. Thus, Aroclor 1254 has adverse effects on GLUT4 translocation and ¹⁴C-2-deoxyglucose uptake, and lycopene administered along with Aroclor 1254 has a protective role over it.     

Organ specific metabolic activation of five extracts of indoor and outdoor particulate matter

In this study liver and lung homogenates of untreated and Aroclor 1254-pretreated rats (Wistar) and mice (Swiss) were compared for their P-450 content and their capacity to activate extracts of airborne particulate matter, sampled indoors and outdoors. Results show that in addition to liver, lung homogenates of rat (Wistar) and mouse (Swiss) are also able to activate extracts of airborne particulate matter in a comparative way. Uninduced liver and lung homogenates showed only minor differences in activation capacity in the metabolism of airborne particles. In contrast to liver homogenates, Aroclor 1254 pretreatment of test animals did not give strong induction of metabolic activation capacity of lung homogenates. P-450 content was observed in all liver and lung homogenates of mouse and rat and in human lung homogenates. The results obtained in this study suggest that the respiratory tract may be an important site for in vivo bioactivation of respirable particles.     

Structure-activity relationships in the inhibitory effects of ellipticines on benzo(a)pyrene hydroxylase activity and 3-methylcholanthrene mutagenicity

The structural features which determine the ability of ellipticine (5,11dimethyl6Hpyrido[4-3b] carbazole) and its derivatives to interact with cytochrome P-450 and to inhibit rat liver microsomal benzo(a)pyrene hydroxylase as well as to inhibit the mutagenicity of 3-methylcholanthrene have been studied. Spectral interactions studies were carried out with either Aroclor 1254-, 3-methylcholanthrene-or phenobarbital-induced microsomes. Inhibitory activities towards benzo(a)pyrene hydroxylase and 3-methylcholanthrene mutagenicity (Ames test), were determined using Aroclor 1254-induced microsomes. It appears that every ellipticine derivative having significant inhibitory effects on hydroxylation of benzo(a)pyrene or mutagenicity of 3-methylcholanthrene also exhibits a very good affinity for microsomal cytochromes P-450. The accessibility of the pyridinic nitrogen of ellipticine derivatives appears as the most important factor for their binding to cytochromes P-450 and the presence of methyl groups in 5 and 11 positions of ellipticine derivatives is an essential condition for the expression of the inhibitory power. Various substitutions in the A ring of ellipticine appear to be of secondary importance. On the other hand the location of the pyridinic ring and consequently the arrangement of the molecule within the hydrophobic pocket of cytochrome P-450 seems also to play an important role in the inhibitory power since isoellipticines are devoid of such properties. These results should help in the design of particularly efficient inhibitors of drug and carcinogen metabolism.     

Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and chlorinated paraffins (CPs) in rats-testing interactions and mechanisms for thyroid hormone effects

The effects of the polybrominated diphenyl ether (PBDE) congener 2,2'4, 4'-tetrabromodiphenylether (DE-47), and technical preparations of polychlorinated biphenyls (PCBs; Aroclor 1254) and chlorinated paraffins (CPs; Witaclor 171P) on thyroid hormone (TH) levels were examined in rats. To study possible interactive effects, also combinations of the three compounds were used. Thus, female Sprague-Dawley rats, 7 weeks old, were treated with approximately isomolar doses (ca. 30 micromol/kg bw per day) of DE-47 (6.0 mg/kg per day), Aroclor 1254 (4.0 mg/kg per day) and Witaclor 171P (6.8 mg/kg per day), alone or in combinations, daily for 14 days by gastric intubation. DE-47 was also administered in a higher (18 mg/kg per day) and lower (1.0 mg/kg per day) dose. In order to test possible mechanisms behind the TH effects, microsomal enzyme (cytochrome P-450 isozymes and uridine diphosphoglucuronyl transferase-UDPGT) activity (indicating both metabolic activation and/or biliary clearance), ex vivo-binding of 125I-T4 to plasma proteins (suggesting effects on peripheral TH transport) and light microscope morphology of the thyroid gland were studied. The observed degree of TH reduction after Aroclor 1254 and DE-47 exposure corresponded with a decrease in the ex vivo binding of 125I-T4 to the plasma TH-transporter transthyretin (TTR), and with induction of the microsomal phase I enzymes (ethoxy- and methoxy-resorufin dealkylases, EROD and MROD). The phase II enzyme UDPGT was also elevated, but only moderately. The thyroid morphology showed an activation of the epithelia, but no degenerative alternations, that was correlated to exposure to Aroclor 1254. In our model, the observed effects match the hypothesis that the T4 decrease is chiefly due to disturbances in serum transport, caused by binding of in vivo-formed Aroclor 1254 and DE-47 metabolites to TTR. However, decreased plasma TH levels due to increased glucuronidation activity may also be of some importance. The thyroid gland hyperactivity is probably a feed-back consequence of the T4 decrease, in spite of the lack of TSH alterations. In the mixed DE-47 and Witaclor 171P group synergistic effects were indicated on free T4 (FT4) and EROD induction levels, results that may suggest that such effects should be considered in risk assessment of mixtures of persistent organohalogens.     

Neonatal exposure to Aroclor 1254: Effects on adult hepatic testosterone hydroxylase activities

1. The effects of neonatal exposure to Aroclor 1254 (100 μmol/kg) on the metabolism of testosterone by adult male and female rats were determined by comparing their hepatic microsomal testosterone hydroxylase activities at 60, 90 and 120 days after the initial exposure.

2. The most pronounced effects in male rats were observed 90 days after treatment with Aroclor 1254, whereas in female rats the major changes in testosterone hydroxylase activities were observed after 60 days.

3. Ninety-day-old male rats neonatally treated with Aroclor 1254 exhibited decreased basal testosterone 7α-hydroxylase and increased basal testosterone 16α-, 2α- and 15β-hydroxylase activities and androstenedione formation. In addition, the Aroclor 1254-mediated induction of testosterone 7α- and 6α-hydroxylase activities and androstenedione formation was decreased, and that of testosterone 2β- and 15β-hydroxylase activities was increased.

4. Sixty-day-old female rats exposed neonatally to Aroclor 1254 exhibited increased basal testosterone 16α-, 2β-, 6α- and 15β-hydroxylase activities and androstenedione formation, and increased Aroclor 1254-induced metabolism of testosterone at all positions except 16α and 2α.

5. Changes in testosterone hydroxylase activities indicative of permanent damage (or imprinting) in androgen metabolism, i.e. altered activities in 120-day-old animals, were observed only in male rats. These activities included basal testosterone 6β-, 16α- and 2α-hydroxylase activities and androstenedione formation.     

Metabolism of a potential 8-aminoquinoline antileishmanial drug in rat liver microsomes

The metabolism of the 8-aminoquinoline, 8-(6-diethylaminohexylamino)-6-methoxy-lepidine dihydrochloride (WR 6026 X 2HCl), was studied in a rat hepatic microsomal system. The results show that WR 6026 X 2HCl was metabolized into two more polar compounds. The structures of these metabolites as proven by gas chromatography-mass spectrometry, ultraviolet absorption, and high performance liquid chromatography were: 8-(6-ethylaminohexylamino)-6-methoxy-lepidine (metabolite 1) and 8-(6-diethylaminohexylamino)-6-methoxy-4-hydroxymethyl quinoline (metabolite 2). The formation of both metabolites was NADPH dependent and also linearly dependent on incubation time and microsomal protein concentration at 0.24 mM WR 6026 X 2 HCl. Studies on the effects of pretreatment of animals with either phenobarbital or Aroclor 1254 suggest that cytochrome P-450 isozymes catalyzed both N-deethylation and hydroxylation reactions. N-deethylase activity was induced by either pretreatment: however, hydroxylase activity was unaffected by phenobarbital pretreatment and significantly elevated by Aroclor 1254 pretreatment. These results suggest that these two reactions are catalyzed by different cytochrome P-450 isozymes. The formation of these two metabolites in vivo may play an important role in the antileishmanial activity of WR 6026 X 2HCl.     

Cytochromes P-4501A, P-4503A and P-4502B in liver and heart of Mugil capito treated with CYP1A inducers

Hepatic microsomes of Aroclor 1254-treated Mugil capito showed a single protein band detected in immunoblot with monoclonal antibody 1-12-3 to teleost (scup) CYP1A. The hepatic CYP1A like protein was induced with dose dependency after exposure of the fish to β-naphthoflavone (BNF) as well as to Aroclor 1254. The induced mullet hepatic CYP1A protein was confined to a distinct fraction obtained by DE-52 anion exchange chromatography, and its relative content in that fraction increased in fish that were treated with higher doses of inducer. EROD (7-ethoxyresorufin O-deethylase) activity in hepatic microsomes from mullet treated with various doses of BNF correlated significantly (r(2)=0.81502, P<0.01) with CYP1A content. Treatment of the mullet with low dose of Aroclor 1254 (25 mg/kg) induced only traces of CYP1A in liver microsomes (5.1±4.8 mg/kg). However, in mullet treated with the high dose of Aroclor 1254 (100 mg/kg) there was a dramatic induction in CYP1A content (408±275 pmol/mg) and this hemoprotein comprised about 83% of the total P-450 content of liver microsomes. The total level of P-450, although induced in the liver tissue, was not induced in heart tissue of Aroclor 1254 treated mullet. On the other hand, P-4501A was induced in treated mullet to a level that comprised almost all of the cardiac P-450 content. EROD activity in the heart tissue of induced mullet was characterized by low V(max) and high K(m) values (K(m)=2.35 mM, V(max)=39.5 pmol/min per mg) compared to the values recorded for the enzyme from the liver (K(m)=1.0 mM, V(max)=288.0 pmol/min per mg). Cardiac CYP1A with low catalytic activity and repression of CYP-types other then CYP1A in heart of CYP1A induced fish may be part of a mechanism aimed to preserve crucial levels of electron donors and molecular oxygen in cardiac muscle of fish exposed to CYP1A inducers.     

Studies on sex related differences in elimination of theophylline in the rat after pretreatment with phenobarbital, chrysene, Aroclor 1254 or lindane.

Cytochrome P-450 in liver microsomes plays a central role as a drug metabolizing enzyme. The sex-dependent differences in the properties of cytochrome P-450 have been extensively studied using liver microsomes, and marked differences have been found in microsomal metabolism of xenobiotic. Effect of pretreatment with phenobarbital, chrysene, lindane and polychlorinated biphenyls (Aroclor 1254) on the theophylline elimination in male and female rats was studied. Levels of theophylline were measured spectrophotometrically.     

Oxidative biotransformation in primary cultures of chick embryo hepatocytes: induction of cytochrome P-450 and the metabolism of benzo(a)pyrene

Primary cultures of chick embryo hepatocytes are known to maintain their initial level of cytochrome P-450 for a number of days. To explore the possibilities of chick embryo hepatocyte cultures as a tool in drug metabolism, induction profiles of cytochrome P-450 were determined and the metabolism of benzo(a)pyrene as a model substrate was studied.Maximum induction by phenobarbitone and Aroclor 1254 is reached after 21 h and 18 h, respectively, both in the presence and absence of serum. For -naphthoflavone induction is maximal after 31 h in the presence and 43 h in the absence of serum. The levels of P-450 after induction are comparable to those found in vivo in rats: increases of 200% for phenobarbitone, 200% for -naphthoflavone and 210% for Aroclor 1254. Ethoxyresorufin-0-deethylase activities are induced by -naphthoflavone and Aroclor 1254, but as expected only slightly by phenobarbitone. In the absence of serum in the culture medium, for the control as well as the induced cells a plateau of activity is maintained for at least 24 h. In the presence of serum a decline in P-450 levels is observed. Especially in the case of Aroclor, an increase in porphyrin content of 320% of control values is seen at the same time.A number of representative metabolites of benzo(a)pyrene were quantitated during a 4-h incubation. Relative amounts are comparable to those observed with rat liver microsomes. As expected, -naphthoflavone and Aroclor induce the rate of metabolism (by 500% and 400%, respectively, in the absence of serum), but phenobarbitone has no or very little effect.Interestingly, when benzo(a)pyrene is incubated with control or phenobarbitone-induced cells an increase in rate of metabolite formation with time is observed: benzo (a)pyrene seems to induce its own metabolism. The chick embryo hepatocytes thus offer the possibility of studying induction and biotransformation in the same system at the same time, in vitro.     

Experimental hepatic porphyria induced by polychlorinated biphenyls

Aroclor 1254, which consists of a mixture of polychlorinated biphenyls (PCBs) containing 54% chlorine, produced an experimental hepatic porphyria in rats resembling hexachlorobenzene poisoning and human porphyria cutanea tarda. The PCB-induced porphyria is characterized by delayed development, increased excretion of urinary uroporphyrins, accumulation of 8- and 7-carboxyporphyrins in the liver and increased drug-metabolizing capacity of the liver. Cytochrome P-450 and microsomal heme were increased maximally at 1 week, in the absence of an increase in the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid (ALA) synthetase. Induction of ALA synthetase and porphyria occurred later, after 2–7 months' exposure to PCBs. No induction of ALA synthetase could be demonstrated prior to the onset of porphyria. Marked induction of ALA synthetase occurred 5 hr after large single doses of Aroclor 1254; however, the doses required were larger than those used to produce porphyria when administered chronically, and induction appeared to be related to the marked increase in cytochrome P-450 seen 24 hr after administration of the drug.     

In vitro activation of isophosphamide and trophosphamide to metabolites mutagenic for bacteria

The ability of S9 liver fractions from uninduced rats to activate isophosphamide (IP) and trophosphamide (TP) to metabolites mutagenic for bacteria was compared to that of S9 fractions prepared from rats pretreated in vivo with three inducers of hepatic monooxygenase. Pretreatment of rats with phenobarbital (PB) and Aroclor 1254 increased IP and TP mutagenic activation by S9 fractions as compared to control and 3-methylcholanthrene (3-MC)-induced rat liver S9. Furthermore, the effect of mixed-function oxidase inhibitors, such as alpha-naphthoflavone, metyrapone and SKF 525-A on S9-mediated mutagenic activation of IP and TP was investigated. The data obtained suggest the involvement of a PB-inducible form of cytochrome P-450 in the activation of IP and TP to mutagenic species.