Total Microsomal Cytochrome P450 Research Articles

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7–28 days and 8000 ppm metazachlor for 3–28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3–28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5–50 μM) and NaPB (500 μM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5–25 μM metazachlor or 500 μM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver

BackgroundThis study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia–reperfusion (IR) in alcoholic fatty liver. Materials and methodsRats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. ResultsAfter the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. ConclusionsWe suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I2) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005mg/kg Se, and I2 deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T4 (TT4), total T3 (TT3)], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I2 deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I2 and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I2 status.

Effect of Sulfur Dioxide Inhalation on CYP2B1/2 and CYP2E1 in Rat Liver and Lung

Sulfur dioxide (SO2) is a ubiquitous air pollutant, present in low concentrations in the urban air and in higher concentrations in the working environment. In this study, we investigated the effects of inhaled SO2 on the O-dealkylase of pentoxyresorufin (PROD) and p-nitrophenol hydroxylases (p-NP) activities and mRNA levels of CYP2B1/2 and CYP2E1 in the lung and liver of Wistar rats. Male Wistar rats were housed in exposure chambers and treated with 14.11 ± 1.53, 28.36 ± 2.12, and 56.25 ± 4.28 mg /m3 SO2 for 6 h/day for 7 days, while control rats were exposed to filtered air in the same condition. The mRNAs of CYP2B1/2 and -2E1 were analyzed in livers and lungs by using reverse-transcription polymerase chain reaction (RT-PCR). Our results showed that the PROD activities and mRNA of CYP2B1/2 were decreased in livers and lungs of rats exposed to SO2. The p-NP activities and mRNA of CYP2E1 were decreased in lungs but not in livers of rats exposed to SO2. Total liver microsomal cytochrome P-450 (CYP) contents were diminished in SO2-exposed rats. These results lead to two conclusions: (1) SO2 exposure can suppress CYP2B1/2 and CYP2E1 in lungs and CYP2B1/2 in livers of rats, thus modifying the liver and lung toxication/detoxication potential, and (2) the total liver microsomal CYP contents were diminished, although the activity and mRNA expression of CYP2E1 in rat livers were not affected by SO2 exposure.

Transgenic mice with a hypomorphic NADPH-cytochrome P450 reductase gene: effects on development, reproduction, and microsomal cytochrome P450.

A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr(low) allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr(low/low) mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr(low) allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr(low) females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr(low/low) mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.

The impact of oil-derived products on the behaviour and biochemistry of the eleven-armed asteroid Coscinasterias muricata (Echinodermata)

The present study examines the impact of exposure to oil-derived products on the behaviour and physiology of the Australian 11-armed asteroid Coscinasterias muricata.Asteroids were exposed to dilutions of water-accommodated fraction (WAF) of Bass Strait stabilised crude oil, dispersed oil or burnt oil ( n=8) for 4 days whereby, prey-localisation behaviour was examined immediately after exposure, and following 2, 7, and 14 days depuration in clean seawater. The prey-localisation behaviour of asteroids exposed to WAF and dispersed oil was significantly affected though recovery was apparent following 7 and 14 days depuration, respectively. In contrast, there was no significant change in the prey-localisation behaviour of asteroids exposed to burnt oil. Behavioural impacts were correlated with the total petroleum hydrocarbon concentrations (C6–C36) in each exposure solution, WAF (1.8 mg l −1), dispersed oil (3.5 mg l −1) and burnt oil (1.14 mg l −1), respectively. The total microsomal cytochrome P450 content was significantly lower ( P Dunnett test<0.01) in asteroids exposed to dispersed oil than in any other asteroids, whilst asteroid alkaline phosphatase activity was not significantly affected ( P ANOVA=0.11). This study further documents the deleterious impact of dispersed oil to marine organisms and supports further research in the area of in situ burning as a less damaging oil spill response measure towards benthic macro-invertebrates.

The contribution of 2,3,5-trichlorophenyl methyl sulfone, a metabolite of 1,2,4-trichlorobenzene, to the δ-aminolevulinic acid synthetase induction by 1,2,4-trichlorobenzene in rat liver

In the present study, we investigated the contribution of methylsulfonyl metabolite derived from 1,2,4-trichlorobenzene (1,2,4-TCB) on the δ-aminolevulinic acid (ALA) synthetase induction by the parent compound in rats. The time courses of increasing of hepatic microsomal total cytochrome P450 content after a single ip administration of 1,2,4-TCB (1.36 mmol/kg), and 2,3,5- and 2,4,5-trichlorophenyl methyl sulfones (2,3,5- and 2,4,5-TCPSO 2Mes) (50 μmol/kg each) were in parallel with those of increasing of the total heme content in liver microsomes. 1,2,4-TCB significantly increased the heme oxygenase activity, but 2,3,5- and 2,4,5-TCPSO 2Mes did not. On the other hand, 1,2,4-TCB and 2,3,5-TCPSO 2Me markedly enhanced the ALA synthetase activity. No change was observed in this enzyme activity after the administration of 2,4,5-TCPSO 2Me. After the administration of 1,2,4-TCB to the rats treated with dl-buthionine-( S, R)-sulfoximine (BSO) and to the non-BSO-treated rats, the concentrations of both 2,3,5- and 2,4,5-TCPSO 2Mes were significantly lower in liver of the BSO-treated rats than in liver of the non-BSO-treated rats. Additionally, the 1,2,4-TCB did not elevate the ALA synthetase activity in the BSO-treated rats. On the other hand, the administration of 2,3,5-TCPSO 2Me to BSO-treated rats resulted in induction of ALA synthetase. The results strongly suggest that the methyl sulfone derived from 1,2,4-TCB, i.e., 2,3,5-TCPSO 2Me, contributes highly to the induction of the ALA synthetase activity by the parent compound.

High induction of cytochrome p4501a activity without changes in retinoid and thyroid hormone levels in flounder (Platichthys flesus) exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

AbstractOal dosesof 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) were administered twice within 7 d (total concentration 0.01, 0.1, 1, 10, or 100 μg TCDD/kg body weight) to flounder (Platichthys flesus). After 10 d of exposure, flounder were sacrificed, and the effects of TCDD exposure on hepatic microsomal total cytochrome P450 content, 7‐ethoxyresorufin‐O‐deethylase (EROD) activity, and glutathione‐S‐transferase activity were examined. In addition, plasma and hepatic retinoid and plasma thyroid hormone levels were analyzed. Overall a good correlation existed between the dose and liver concentrations of TCDD. However, only 0.75% of the total dose of TCDD was retained in the flounder liver. The TCDD caused a dose‐related, statistically significant induction of the hepatic microsomal total cytochrome P450 content (4.4‐fold; p &lt; 0.001) and associated EROD activity (27‐fold; p &lt; 0.001), without obvious changes in body weight, liver weight, and condition factor. Total glutathione‐S‐transferase activity was not induced in livers of flounder exposed to TCDD. No TCDD‐induced changes in liver retinoid, plasma retinol, and plasma thyroid hormone parameters were observed. These results indicate that the flounder is an aryl hydrocarbon (Ah) receptor‐responsive species with respect to CYP1A induction but not with respect to other mammalian Ah receptor‐associated responses.

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct–ligated male rats: Importance to reduced hepatic content of cytochrome P450 in cholestasis

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased to 32% to 52% of control, but the activities of two male sex-specific CYPs, 2C11 and 3A2, were reduced to a significantly greater extent (P < .05). The microsomal contents of CYP proteins 2C6 and 2E1 were decreased after BDL to 61% and 63% of control, whereas 2C11 and 3A2 proteins were 21% and 45% of control. Corresponding reductions of the messenger RNA (mRNA) species for CYP 2C11 (9% of control) and 3A2 (37%) were detected, whereas there was no reduction of 2C6 mRNA. These findings are consistent with downregulation of the CYP 2C11 and 3A2 genes. Nuclear run-on studies performed 3 days after BDL showed that there was a generalized impairment of gene transcription after BDL, but a disproportionate reduction in transcription of CYPs 2C11 and 3A2. A possible explanation for downregulation of CYP 2C11 and 3A2 was provided by the observation that serum estradiol concentrations were threefold greater in BDL male rats, while serum testosterone was reduced; estradiol is known to downregulate CYPs 2C11 and 3A2. It is concluded that male sex-specific CYP enzymes are decreased to a greater extent than other microsomal proteins in BDL male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: Importance to reduced hepatic content of cytochrome P450 in cholestasis

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased to 32% to 52% of control, but the activities of two male sex-specific CYPs, 2C11 and 3A2, were reduced to a significantly greater extent ( P < .05). The microsomal contents of CYP proteins 2C6 and 2E1 were decreased after BDL to 61% and 63% of control, whereas 2C11 and 3A2 proteins were 21% and 45% of control. Corresponding reductions of the messenger RNA (mRNA) species for CYP 2C11 (9% of control) and 3A2 (37%) were detected, whereas there was no reduction of 2C6 mRNA. These findings are consistent with downregulation of the CYP 2C11 and 3A2 genes. Nuclear run-on studies performed 3 days after BDL showed that there was a generalized impairment of gene transcription after BDL, but a disproportionate reduction in transcription of CYPs 2C11 and 3A2. A possible explanation for downregulation of CYP 2C11 and 3A2 was provided by the observation that serum estradiol concentrations were threefold greater in BDL male rats, while serum testosterone was reduced; estradiol is known to downregulate CYPs 2C11 and 3A2. It is concluded that male sex-specific CYP enzymes are decreased to a greater extent than other microsomal proteins in BDL male rats. It is proposed that the reduced activity of steroid metabolizing enzymes, which occurs after BDL, results in altered serum sex steroid concentrations, which, in turn, lead to decreased transcription of CYP 2C11 and 3A2 genes.

Thiabendazole Is an Inducer of Cytochrome P4501A1 in Cultured Rabbit Hepatocytes

The effect of TBZ (30-100 μM) was investigated on cytochromes P450 of cultured rabbit hepatocytes considered 72 h after plating. At the highest concentrations and without apparent cellular toxicity, the drug provokes a dose-dependent increase in total microsomal cytochrome P450 and a rise in EROD activity which was correlated to a specific increase in P4501A1 level. Northern blot analysis of RNA reveals an increased level of mRNA specific to P4501A1. The transcriptional activation of this isoenzyme is proposed because of the significant inhibition of the above-mentioned increases when actinomycin was added to the culture medium. Data obtained from competition experiments demonstrate that TBZ is not a ligand of Ah receptor. A down-regulation process could explain the slight decrease in both ANOH and P4502E1 level observed in hepatocytes treated with the highest dose of TBZ.

Sex and strain differences in mouse hepatic microsomal coumarin 7-hydroxylase activity

Hepatic microsomal coumarin 7-hydroxylase activity has been determined in male and female mice of strains A/J, AKR, BALB/c, CBA/Ca, C3H/He, C57BL/6J, DBA/2 and 129. In males, coumarin 7-hydroxylase activity was highest in liver microsomes from DBA/2 mice and lowest in BALB/c mice. With female mice enzyme activity was highest in DBA/2 and 129 strains, intermediate in the CBA/Ca strain and comparatively low in the other five strains. Marked sex differences were observed in coumarin 7-hydroxylase activity with enzyme activity in female animals from strains DBA/2, 129 and CBA/Ca being 4.8-, 6.2- and 4.8-fold higher, respectively, than in male mice. In contrast, only minor sex and strain differences in levels of total microsomal cytochrome P-450 were observed. These results demonstrate marked sex and strain differences in mouse hepatic microsomal coumarin 7-hydroxylase activity. Such differences may be due to variations in particular cytochrome P-450 isoenzymes such as CYP2A5, not all of which can be explained by the known allelic difference in the Cyp2a-5 locus.

Fasciola-Hepatica: Liver Microsomal Membrane Functions in Host Rat

Changes in different microsomal membrane functions were measured in the liver of rats 3, 6, or 9 weeks following an oral infection with 20 metacercariae of Fasciola hepatica. The parasitic pathology noted at autopsy was accompanied by increased levels in both plasma aspartate aminotransferase (EC 2.6.1.1) and microsomal γ-glutamyltransferase (EC 2.3.2.2). Heme oxygenase activity of microsomes was significantly decreased by Weeks 3 and 6 postinfection and this decrease correlates with those of total microsomal cytochrome P450 and certain P450-dependent monooxygenase activities, namely, benzphetamine demethylation, ethoxycoumarin deethylation, and benzopyrene hydroxylation. Microsomal epoxide hydrolase (EC 3.3.2.3) was only altered 6 weeks after the infection. During the early stages of the parasitism, there were decreases in both microsomal calcium uptake and calcium ATPphosphohydrolase activity (EC 2.6.1.1), whereas membrane fluidity, estimated by the order parameter S, was lower in the infected rats than that in the controls. These alterations could be related to the already described increase in liver cytosolic calcium or lipid peroxidation which occurs in experimental fascioliasis.

Immunologic and endocrine effects of the flame-retardant pentabromodiphenyl ether (DE-71) in C57BL/6J mice

Polybrominated diphenyl ethers are manufactured for use as flame retardants in commercial plastics and textiles in Europe and North America. These studies investigated the acute and subchronic immunotoxicity and endocrine effects of a commercial pentabromodiphenyl ether mixture, DE-71, in female C57BL/6 mice. Mice were orally exposed to acute single doses of DE-71 of 0, 0.8, 4.0, 20, 100, or 500 mg/kg, or to subchronic daily doses totaling 0, 250, 500, or 1000 mg/kg over a 14 day period. Immunotoxicity was assessed by measuring the plaque-forming cell response to sheep erythrocytes (SRBC) and natural killer cell (NKC) activity (basal and poly I:C stimulated) to YAC-1 target cells. Liver cytochrome P450 content and activities (ethoxyresorufin- o-deethylase (EROD) and pentoxyresorufin- o-deethylase (PROD)) as well as corticosterone (CS) and thyroxine (T4) concentrations were also measured. PROD activity was induced 3–5-fold in mice exposed acutely or subchronically to DE-71 at doses >250 mg/kg. EROD activity and total microsomal cytochrome P450 content were significantly induced only in mice treated subchronically with DE-71; maximum induction of EROD was 3.3-fold. Total serum T4 concentrations were significantly lower in mice treated acutely with DE-71 at all doses except the 100 mg/kg dose. Total and free T4 concentrations were dose-dependently decreased in DE-71-treated mice following subchronic exposure. Plasma CS levels were elevated following subchronic exposure to DE-71. The elevation of CS was correlated with order of capture at necropsy, suggesting an interactive effect of DE-71 and stress. In regard to immunotoxicity, significant suppression of the anti-SRBC response was seen only in mice exposed subchronically to 1000 mg DE-71/kg, an exposure that also resulted in decreased thymus weight. NKC activity was not altered by exposure to DE-71.

Acetaminophen Hepatotoxicity: Influence of Phenobarbital and β-Naphthoflavone Treatment in Obese and Lean Zucker Rats

The effect of phenobarbital (PB) and β-naphthoflavone (β-NF) on acetaminophen (APAP)-induced hepatotoxicity was evaluated in obese and lean Zucker rats. In addition, the consequences of APAP overdose on hepatic CYP2B1/2B2 enzyme activities following PB treatment were assessed. A single oral dose of APAP 3 g/kg (total body weight) was administered to both littermates 24 hr after the last dose of PB or β-NF. Histologic evidence of hepatocellular necrosis and serum hepatic aminotransferase enzymes 48 hr after APAP administration was utilized to evaluate hepatic damage. Hepatic microsomal total cytochrome P450 concentrations, alkoxyresorufin O-dealkylase activities, and 16β-testosterone hydroxylase activities were determined to evaluate the effect of APAP overdose on cytochrome P450 enzyme-substrate activities in the presence and absence of PB treatment. APAP overdose produced lower hepatotoxicity in the obese Zucker rat compared to lean controls. A similar trend was observed in animals treated with PB prior to APAP administration. In contrast, β-NF treatment produced potentiation of APAP toxicity and/or death of both obese and lean Zucker rats. Generally, APAP overdose produced reduction of hepatic cytochrome P450 enzyme-substrate activities. However, obese Zucker rats retained a higher percentage of their pre-APAP-treated enzyme activities which is consistent with the observation that obese Zucker rats are less affected by the hepatotoxic effects of APAP overdosage.

Induction of CYP2B1 and 3A1, and associated monooxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice

Previous studies suggest long-term feeding of tamoxifen { Z-1-[4-(2-dimethylaminoethoxy)phenyl]1, 2-diphenyl-1-butane} to rats gives rise to liver tumours, while mice are resistant. The effects of tamoxifen on cytochrome P450 isoenzymes and associated monooxygenase activities in the livers of female Fischer rats and C57B1/6 and DBA/2 mice have been compared. Total microsomal cytochrome P450 was not induced in the livers of rats given tamoxifen (45 mg/kg daily for 4 days) and was in fact significantly reduced after 3 days treatment. In contrast, there was a 30–60-fold increase in the metabolism of benzyloxy- and pentoxyresorufins to resorufin. Ethoxyresorufin O-deethylase was induced only 2.5-fold. The regio- and stereo-specific hydroxylation of testosterone following tamoxifen pretreatment of rats showed a general time- and dose-dependent induction. 6β- and 16α-hydroxylation of testosterone together with oxidation to androstenedione were increased 2–3-fold while 2β-hydroxylation was induced only marginally, suggesting that tamoxifen produces a mixed pattern of induction with a significant phenobarbitone-like component. No induction of the 2β- or 6β-hydroxylation pathway occurred in either mouse strain. In rats, immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or 3A1 showed that tamoxifen pretreatment resulted in 2–3-fold increases in both CYP2B1, 2B2 and 3A1 proteins, relative to controls. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced proteins. Similar patterns of induction as measured by immunoblotting experiments and testosterone hydroxylation were seen following the administration of structurally related analogues, toremifene and droloxifene (3-hydroxytamoxifen), thought to be noncarcinogenic in the rat. No induction of these monooxygenase activities was seen in C57B1/6 mice and only small increases in benzyloxy- and pentoxyresorunn metabolism were in DBA/2 mice. It is suggested that the induction of cytochrome P450-dependent activities by tamoxifen may result in accelerated liver metabolism of this drug with important implications for the disposition of tamoxifen in vivo and also for its metabolic conversion to genotoxic metabolite(s). The difference in inducibility of cytochrome P450-dependent monooxygenase activities between rats and mice offers a plausible and testable hypothesis that the difference in tamoxifen metabolism between the two species may contribute to their carcinogenic response to tamoxifen.

Inhibition of rat methylbenzylnitrosamine metabolism by dietary zinc and zinc in vitro

Methylbenzylnitrosamine is an esophageal-specific carcinogen in the rat, and the incidence of methylbenzylnitrosamine-induced esophageal carcinoma is increased by dietary zinc deficiency. Methylbenzylnitrosamine requires activation by cytochrome P-450 to be mutagenic; the present study examined the role of dietary zinc deficiency and the in vitro addition of zinc on the cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. Dietary zinc deficiency significantly increased the cytochrome P-450-dependent esophageal and hepatic microsomal metabolism of methylbenzylnitrosamine. These changes occurred without alteration in the specific content of total microsomal cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. These results suggest that physiological levels of zinc may be an endogenous inhibitor of methylbenzylnitrosamine metabolism. Dietary zinc deficiency appears to reduce this inhibition of cytochrome P-450 activity, resulting in an increase in carcinogen activation.

Herbicide-insecticide interaction in maize: Malathion inhibits cytochrome P450-dependent primisulfuron metabolism

Tolerance of maize to sulfonylurea herbicides such as primisulfuron has recently been reported to be impaired by the use of some organophosphorus insecticides. In an effort to elucidate the mechanism of this interaction, the effect of the insecticide, malathion, on the metabolism of primisulfuron was studied in whole plants, in excised leaves, and in a microsomal in vitro system from maize. Foliar application of malathion to 7-day-old plants had no influence on leaf uptake and translocation of primisulfuron, but caused a decrease in the rate of herbicide metabolism. In excised leaves, malathion increased the metabolic half-life of primisulfuron. In microsomal preparations, malathion inhibited cytochrome P450-dependent primisulfuron phenyl- and pyrimidinering hydroxylation. Loss of primisulfuron phenyl-ring hydroxylase activity was time-dependent, saturable with respect to malathion concentration, and attenuated in the absence of NADPH. The kinetic data suggest a mechanism-based cytochrome P450 inactivation by malathion. The oxoanalogue of malathion, malaoxon, did not influence the metabolic half-life of primisulfuron in excised leaves and was a poor inhibitor of microsomal primisulfuron hydroxylation. Neither insecticide had any effect in vitro on total microsomal cytochrome P450 content. From the present results it may be concluded that malathion affects primisulfuron tolerance of maize due to the inhibition of cytochrome P450 monooxygenases involved in herbicide metabolism.

Effects of cytochrome P450 inducers on I-compounds in rat liver and kidney DNA.

I-compounds are covalent DNA modifications presumably derived from endogenous electrophiles. To investigate the possible role of cytochrome P450 in I-compound metabolism, groups of female Sprague-Dawley rats (225-250 g) were treated i.p. with vehicle or cytochrome P450 inducers, i.e. 80 mg/kg phenobarbital (PB), 20 mg/kg 3-methylcholanthrene (MC) or 50 mg/kg pregnenolone-16 alpha-carbonitrile (PCN), once daily for 4 days. DNA synthesis rate was measured via [3H]methylthymidine incorporation. DNA adducts and I-compounds in liver and kidney were analyzed 1 and 8 days after the last treatment. Total liver and kidney microsomal cytochrome P450 content and activities of representative drug-metabolizing enzymes for PB, MC and PCN, i.e. benzphetamine N-demethylase, ethoxycoumarin O-deethylase (ECD) and erythromycin N-demethylase, were also determined in all groups. PCN caused significant depletion of total non-polar I-compounds at 1 day, compared to controls. Levels of several individual I-spots in liver were differentially reduced by each of the three inducers at 1 day. Most I-spots were restored to control levels at 8 days. Kidney I-compounds were not affected by PB or PCN, but MC reduced the level of one non-polar individual I-compound at 1 day. Except for the expected DNA adduct formation from MC, there were no qualitative changes in profiles of postlabeled modified nucleotides. Total cytochrome P450 content in liver microsomes and activities of individual P450 enzymes were significantly increased by treatment with each of the inducers at 1 day. This was, however, not the case at 8 days in PB- and PCN-treated livers. MC-treated rats, on the other hand, displayed elevated levels of liver cytochrome P450 and ECD at 8 days. In kidney, PB and PCN did not elicit induction of P450 and individual enzymes, but MC increased total P450 content and ECD activity at 1 day, and ECD activity alone at 8 days. These results suggest a major role for cytochrome P450 enzymes in the metabolism of I-compounds.