Microsomal Cytochrome P-450 Content Research Articles

Phenobarbital, β-Naphthoflavone, Clofibrate, and Pregnenolone-16α-Carbonitrile Do Not Affect Hepatic Thyroid Hormone UDP-Glucuronosyl Transferase Activity, and Thyroid Gland Function in Mice

The effects of representative liver enzyme inducers such as clofibrate (CLO), phenobarbital (PB), pregnenolone-16α-carbonitrile (PCN), and β-naphthoflavone (NF) on hepatic microsomal thyroxin (T4)- UDP-glucuronosyl tranferase (UGT) and triiodothyronine (T3)- UGT activities and thyroid function were evaluated in OF-1 male mice after a 14-day po administration. CLO, PB, and PCN induced histological liver hypertrophy, increases in liver weights, in microsomal protein and cytochrome P450 contents as well as increases in specific UGT activities. Despite this, no significant changes in T4-UGT and T3-UGT activities occurred after treatment by any of these compounds. Furthermore, no significant changes in serum T4and T3levels were observed and thyroid histology was not affected. NF treatment induced microvacuolation of hepatocytes but did not affect any of the other tested parameters. The results show that, in contrast to the widely described effects in rats, liver enzyme inducers do not affect hepatic thyroid hormone metabolism and thyroid function in mice, suggesting that this species should be less sensitive to thyroid tumor promotion by hepatic microsomal enzyme inducers than rats.

Effect of benzene exposure on hematology and hepatic drug metabolic enzymes in ethanol administrated rats

There are few reports regarding the effects of benzene and ethanol being administered simultaneously. In our experiments with 4 groups (controls, ethanol, benzene and ethanol plus benzene) Wistar male rats were treated with ethanol (20%) for 5 weeks, and then exposed to benzene (0.26 g/kg) for 5 days per week for 3 weeks. We also investigated the effects of benzene on the body weight, organ weight, peripheral hematology and hepatic drug metabolizing enzymes in the ethanol administrated rats. The liver weight increased significantly, but spleen weight decreased significantly in the benzene exposed group. Hematological examination showed a decrease of leukocyte in the two groups of benzene and ethanol plus benzene in comparison with the controls, but an effect promoted by ethanol was not found. Serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) values were not significantly different in the exposure groups when compared with the controls. The contents of microsomal cytochrome P450 in all the exposed groups showed a tendency to increase, but they were not significantly different in comparison with the controls. On the other hand, hepatic glutathione S-transferase activity in the exposed groups increased significantly.

Effect of Oxidized Fat on Performance and Some Physiological Responses in Broiler Chickens.

The purpose of the present experiments was to determine influences of feeding or injection of oxidized fat on growth performance and some physiological functions in broiler chickens. Feeding of oxidized fat (10 meq peroxide value (POV)/kg diet) for 14 days depressed growth rate, feed utilization, plasma alpha-tocopherol and ascorbic acid concentrations, but increased 2-thiobarbituric acid reactants (TBARS) in plasma and ratio of heterophile to lymphocyte in blood (HL ratio). Feeding oxidized fat reduced microsomal cytochrome P-450 content in liver and primary antibody production to Brucella abortus, but did not affect microsomal cytochrome b5 content in liver and primary antibody production to sheep blood red cell. Oxidized fat injection (1megPOV/day/100g BW) into breast muscle for 14 days did not affect growth performance and TBARS, but influenced plasma alpha-tocopherol concentration and HL ratio as observed in the experiments 1 and 3. The results suggested that oxidized fat feeding or injection resulted in occurrence of oxidative stresses, and that feeding oxidized fat is a convenient way to reproduce oxidative stresses rather than by the injection under the present experimental condition. In addition, feeding oxidized fat extensively affects wide ranges of physiological functions in chicks as well.

Effect of inhibition of ornithine decarboxylase activity in a model of acute hepatocellular necrosis.

The effect of blockade of the enzyme ornithine decarboxylase by difluoromethylornithine (DFMO) on hepatocellular necrosis and survival in rats treated with thioacetamide (TAA) was investigated. In one experiment, the effect of DFMO on survival of rats with TAA-induced acute hepatocellular necrosis was determined. In another experiment, blood and liver specimens were obtained from DFMO or saline-treated rats 24 h after the administration of TAA for determinations of serum alanine aminotransferase (ALT) and liver content of polyamines and microsomal cytochrome P-450 and for assessment of hepatic histology. Liver polyamines were determined by reversed-phase HPLC and microsomal cytochrome P-450 content by dithionite-difference spectroscopy of CO-treated homogenates. The severity of hepatocellular necrosis was scored blindly. TAA-treated rats that received DFMO survived longer than saline-treated controls (P < 0.01). Serum ALT and liver putrescine concentrations were lower and the histological severity of acute hepatocellular necrosis was less in DFMO-treated rats with TAA-induced hepatocellular necrosis than in saline-treated controls (P < 0.05, P < 0.01 and P < 0.05, respectively). Total cytochrome P-450 levels were similar in DFMO and saline-treated rats with TAA-induced hepatocellular necrosis. DFMO increases survival in TAA-induced fulminant hepatic failure by decreasing the severity of acute hepatocellular necrosis. The beneficial effects of DFMO do not appear to be mediated by its effects on polyamine metabolism, but may be attributable to an effect of DFMO on thioacetamide metabolism or on an alternative pathway of ornithine metabolism.

Metabolic Fate of NKH477. (2). Blood Concentration, Distribution, Excretion and Effects on Drug-Metabolizing Enzyme System in Rats after Repeated Intravenous Administration.

The blood concentration, distribution and excretion of radioactivity were investigated in male rats after daily intravenous administration of 14C-NKH477 for 21 days. The effects of NKH477 on hepatic drug metabolizing enzyme system were also investigated in male rats. 1. The radioactivity in the blood at 24 hr after daily administration rose as number of dose increased, the elimination of radioactivity from the blood after the last dosing was slower than that after a single dosing. 2. In most tissues, the radioactivity accumulated during the period of repeated administration, especially in the spleen, blood, kidney and mesenteric lymph node that rose markedly. Disappearance of radioactivity from most tissues after the last dosing was slower compared with that after a single dosing. 3. Within 168 hr after a repeated administration for 21 days, urinary and fecal excretion amounted to 7.8% and 88.9% of cumulative dose, respectively. The ratio of excretion between urine and feces was approximately the same as after a single dosing. 4. Repeated administration of NKH477 at doses of 0.3 and 1.0 mg/kg/day for 7 days had no effect on liver weight, microsomal cytochrome P-450 contents and hepatic drug-metabolizing enzyme system.

Inhibition of Testicular Steroid Metabolism by Administration of 1-Aminobenzotriazole to Rats

Effects of 1-aminobenzotriazole (ABT) on testicular steroid metabolism were evaluated in rats. Administration of ABT to adult male rats caused dose-dependent decreases in testicular microsomal and mitochondrial cytochrome P450 concentrations. Significant losses of P450 occurred within 8 h of ABT treatment. Accompanying the declines in testicular P450 content were decreases in microsomal 17α-hydroxylase and mitochondrial cholesterol sidechain cleavage activities. Incubation of testicular microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on rates of steroid metabolism. By contrast, incubation of hepatic microsomes with ABT under the same conditions decreased P450 levels and xenobiotic-metabolizing activity. The results indicate that ABT in vivo causes inactivation of steroidogenic P450 isozymes in the testis, but the mechanism of inactivation differs from that on xenobiotic-metabolizing isozymes.

EFFECT OF HYPERTHYROIDISM ON THE IN VITRO METABOLISM AND COVALENT BINDING OF 1,1-DICHLOROETHYLENE IN RAT LIVER MICROSOMES

Hyperthyroidism potentiates the in vivo hepatotoxicity of 1,1-dichloroethylene (DCE) in rats, with a concomitant increase in [14C]-DCE covalent binding. The enhanced injury produced in hyperthyroid livers by DCE could be due to alterations in either the bioactivation or detoxication phases of DCE metabolism. Previous in vitro studies suggested that hyperthyroidism did not potentiate DCE hepatotoxicity by increasing DCE oxidation to intermediates which were able to covalently bind. Several factors, however, that could contribute to the magnitude of DCE bioactivation or covalent binding were not examined. Our objectives were to characterize the effects of hyperthyroidism in male Sprague-Dawley rats on: (1) covalent binding of [14C]-DCE to microsomes and other subcellular fractions, (2) microsomal mixed-function oxidase (MFO) and glutathione S-transferase (GST) activities, and (3) inactivation of microsomal enzyme activities by presumptive DCE reactive intermediates. Hyperthyroid (HYPERT) and euthyroid (EUT) rats received 3 sc injections of thyroxine (100 µg/100 g) or vehicle, respectively, at 48-h intervals; microsomes and other subcellular fractions were isolated from HYPERT and EUT livers 24 h after the last injection. [l4C]-DCE-derived covalent binding was consistently greater in EUT than HYPERT microsomes. The absence of NADH, and the addition of low concentrations (0.1 and 0.5 mM), but not higher concentrations (>l mM), of glutathione (CSH) diminished covalent binding to a greater extent in HYPERT than EUT microsomes. Covalent binding in mitochondrial, nuclear, and cytosolic fractions of EUT and HYPERT livers was equivalent. Regression analysis of covalent binding to liver cell fractions from both EUT and HYPERT rats showed a significant correlation with P-450 content. Hyperthyroidism decreased microsomal, but not mitochondrial, cytochrome P-450 content, and MFO activities for 7-ethoxycoumarin and benzphetamine were similarly decreased. Hyperthyroidism also diminished microsomal CST activity, and altered CST kinetics for both CSH and 1-chloro-2,4-dinitrobenzene (CDNB). The magnitude of inactivation of MFO and CST activities in the presence of DCE (presumably by DCE reactive intermediates) was comparable between EUT and HYPERT microsomes. When covalent binding was standardized to cytochrome P-450 concentrations in microsomes and mitochondria, HYPERT fractions exhibited slightly greater covalent binding than EUT fractions, suggesting that hyperthyroidism does not reduce the capacity of P-450 hemoproteins to bioactivate DCE. Thus, potentiation of DCE hepatotoxicity by hyperthyroidism may be predominantly due to diminished Phase II constituents, and major increases in reactive intermediate/conjugates that covalently bind to and impair critical cellular molecules.

Comparison of the Hepatic and Renal Effects of 1,4-Dichlorobenzene in the Rat and Mouse

The effects of 1,4-dichlorobenzene (DCB) have been compared in male F344 rats given 0 (corn oil control), 25, 75, 150, and 300 mg/kg DCB and male B6C3F1 mice given 0 (corn oil control), 300, and 600 mg/kg DCB by daily oral gavage five days per week for 1, 4, and 13 weeks. The two highest rat and both mouse dose levels were the same as those employed in a NTP bioassay, where DCB produced kidney tumors in male rats and liver tumors in mice. DCB produced significant dose-related increases in relative liver weight in both the rat and the mouse which was associated with, respectively, mild and marked centrilobular hypertrophy. Administration of DCB also produced a sustained induction of microsomal cytochrome P450 content and 7-pentoxyresorufin O-depentylase activity in both species. Western immunoblotting studies demonstrated that DCB induced CYP2B isoenzyme(s) in both rat and mouse liver microsomes. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2'-deoxyuridine in study Weeks 0-1, 3-4, and 12-13. In the rat hepatocyte labeling index values were only increased in animals given 300 mg/kg DCB for 1 week, whereas hepatocyte labeling index values were significantly increased in mice given 300 and 600 mg/kg DCB for 1 and 4 weeks. DCB treatment produced significant increases in rat renal P1/P2 proximal tubule cell labeling index values at all time points, whereas little effect was observed in mouse kidney. The observed species difference in DCB-induced liver tumor formation may reflect the greater sensitivity of the mouse to tumor promotion by a CYP2B inducer. For the kidney, the present data provides further evidence that while DCB-induced alpha2U-globulin nephropathy is associated with a sustained stimulation of cell replication in male rat renal proximal tubule cells, this effect is not observed in the male mouse.

Role of 3,4-Dichlorophenyl Methyl Sulfone, a Metabolite ofo-Dichlorobenzene, in the Changes in Hepatic Microsomal Drug-Metabolizing Enzymes Caused byo-Dichlorobenzene Administration in Rats

2,3- and 3,4-Dichlorophenyl methyl sulfoxides and 2,3- and 3,4-dichlorophenyl methyl sulfones (2,3- and 3,4-DCPSO2Mes) were detected in the urine of rats administeredo-dichlorobenzene (o-DCB). After administration ofo-DCB to rats, swift decreases were observed in the concentrations ofo-DCB in blood, liver, and kidneys, whereas 3,4-DCPSO2Me appeared in blood, liver, kidneys, and adipose tissue. The concentrations of 3,4-DCPSO2Me in the blood and three tissues reached maxima at 24 hr. Both aminopyrineN-demethylase and aniline hydroxylase activities and cytochrome P450 content of hepatic microsomes decreased 24 hr after administration ofo-DCB. In contrast, 3,4-DCPSO2Me increased the activities of these enzymes and cytochrome P450 and b5contents in rat liver microsomes. In both antibiotic-pretreated and bile duct-cannulated rats dosed witho-DCB, the concentrations of 2,3- and 3,4-DCPSO2Mes in blood, liver, kidneys, and adipose tissue were dramatically reduced. These findings suggest that the process of formation of methylsulfonyl metabolites ofo-DCB involves biliary secretion of DCPSO2Mes and/or their precursors which will be subjected to metabolism by intestinal microflora. In antibiotic-pretreated rats, the inhibitory effects ofo-DCB administration on the activities of aminopyrine- and aniline-metabolizing enzymes and the contents of cytochromes P450 and b5in hepatic microsomes were greater than those observed in the intact rats. In bile duct-cannulated rats, the decrease in aminopyrineN-demethylase activity after administration ofo-DCB was greater than that observed in the intact rats. These findings suggest that the apparent inhibition of drug-metabolizing enzymes byo-DCB is the result of simultaneous contrary effects, namely, the inductive effect of 3,4-DCPSO2Me and the stronger inhibitory effect of an unknown factor(s) on drug-metabolizing enzymes.

Dose-related increase in liver heme catabolism during rabbit aflatoxicosis

Aflatoxin B1 (AFB1) has been reported to decrease microsomal hepatic cytochrome P450 (P450) content and increase both total plasma bilirubin concentration and liver heme oxygenase activity. The purposes of this study were to determine whether liver hemoproteins contents and heme catabolizing enzymes were affected by the mycotoxin and whether these alterations were linked to hyperbilirubinemia. Male New Zealand rabbits were divided into three groups of five animals, each receiving for 5 days either arabic gum as vehicle or AFB1 at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments affected neither cytochrome b5 content nor NADPH-cytochrome reductase activity. A linear dose-dependent decrease in cytochrome P450 content and increases in both heme oxygenase and biliverdin reductase activities were observed. Bilirubin UDP-glucuronyltransferase activity was dramatically decreased at both doses, whereas cholestasis occurred only at 0.10 mg/kg. An exponential dose-dependent increase in plasma bilirubin concentration was also observed. Both the simultaneous exponential increase in bilirubinemia associated to a reduced bilirubin UDP-glucuronyltransferase activity and the absence of cholestasis at 0.05 mg/kg, suggested that the hyperbilirubinemia is more probably related to an increased heme catabolism than to an altered bile duct permeability.

Effect of piperonyl butoxide on cell replication and xenobiotic metabolism in the livers of CD-1 mice and F344 rats.

Male CD- 1 mice were fed diets containing 0 (control), 10, 30, 100, and 300 mg/kg/day piperonyl butoxide (PBO) and 0.05% sodium phenobarbital (NaPB) and male F344 rats were fed diets containing 0 (control), 100, 550, 1050, and 1850 mg/kg/day PBO and 0.5% NaPB for periods of 7 and 42 days. In both species PBO and NaPB increased relative liver weight and whereas PBO produced a midzonal (mouse) or periportal/midzonal (rat) hypertrophy, NaPB produced a centrilobular hypertrophy. In the rat, individual cell necrosis was also observed at 42 days after high doses of PBO. Replicative DNA synthesis, assessed as the hepatocyte labeling index following implantation of 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during Study Days 0-7 and 35-42, was increased in mice given 300 mg/kg/day PBO and NaPB for 7 days and in rats given 550 and 1050 mg/kg/day PBO and NaPB for 7 days and 1050 mg/kg/day PBO for 42 days. While PBO had no effect on body weights in mice, the body weights of rats given 550, 1050, and 1850 mg/kg/day PBO for 42 days were reduced to 92, 89, and 70% of control, respectively. PBO induced microsomal cytochrome P450 content and mixed function oxidase activities in the mouse and rat, although the effects were less marked than those produced by NaPB. In summary, this data demonstrates that PBO can produce liver enlargement in the mouse and the rat which is associated with induction of xenobiotic metabolism, hypertrophy, and hyperplasia. The hepatic effects of PBO in the mouse were similar to but less marked than those produced by NaPB. In the rat high doses of PBO were hepatotoxic and resulted in a marked reduction in body weight. Thus while the reported formation of eosinophilic nodules in mouse liver by PBO may occur by a mechanism(s) similar to that of NaPB and other nongenotoxic enzyme inducers, the reported tumor formation in rats at greater than the maximum tolerated dose is most likely associated with marked enzyme induction in conjunction with a regenerative hyperplasia resulting from PBO-induced hepatotoxicity.

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.

Influence of amphotericin B treatment duration of hepatic microsomal enzyme function in rats.

The influence of amphotericin B on various cytochrome P450-dependent mixed-function oxidases, antipyrine clearance and glucose-6-phosphatase was investigated in rats treated daily with deoxycholate amphotericin B (3 mg/kg body weight, intravenously) either for 1 or 4 days. Enzyme activity was measured ex vivo in hepatic microsomes. Following amphotericin B plus deoxycholate application for day 1, ethoxycoumarin-O-deethylase activity decrease significantly whereas microsomal cytochrome P450 concentration, cytochrome c reductase activity, antipyrine clearance and glucose-6-phosphatase activity did not change significantly. In contrast, following application of amphotericin B plus deoxycholate for 4 days the cytochrome P450 concentration was reduced by 50% (p < 0.05) as well as ethoxycoumarin-O-deethylase activity, antipyrine clearance and glucose-6-phosphatase activity: ethoxycoumarin-O-deethylase 232 +/- 68 pmol/mg/min, control 442 +/- 99 pmol/mg/min (p < 0.01); antipyrine clearance 0.56 +/- 0.21 ml/min, control 0.96 +/- 0.18 ml/min (p < 0.01), and glucose-6-phosphatase 193 +/- 28 mU/mg, control 351 +/- 95 mU/mg (p < 0.05). Cytochrome c reductase activity did not decrease significantly. Besides an increase in cytochrome c reductase activity, sodium deoxycholate (a vehicle of amphotericin B) alone induced no significant changes. Microsomal protein related to liver wet weight was significantly reduced by 50% (p < 0.01) only in animals treated for 4 days with amphotericin B plus deoxycholate. The results show that a 1-day treatment of rats with amphotericin B decreases ethoxycoumarin-O-deethylase activity, whereas the hepatic microsomal cytochrome P450 content, cytochrome c reductase and glucose-6-phosphatase activity did not change. Amphotericin B given for 4 days significantly decreases hepatic microsomal enzyme function. The inhibitory effect of amphotericin B on hepatic cytochrome P450 may be due to inhibition of hepatic protein synthesis.

Biochemical responses to sediment-associated contaminants in brown bullhead (Ameriurus nebulosus) from the Niagara River ecosystem

Brown bullhead (Ameriurus nebulosus) were collected from three sites in the Niagara River ecosystem in June and September of 1991, and sediment samples from these sites were obtained in July 1991. The sites were located in the Buffalo River, the Niagara River adjacent to the Love Canal dump site, and in Black Creek, a Canadian tributary of the Niagara River which served as a reference site. Sediment samples from these sites contained measurable concentrations of various polycyclic aromatic hydrocarbons (PAHs) and chlorinated hydrocarbons (CHs). However, the Buffalo River and Love Canal samples were significantly more contaminated than those from Black Creek. Moreover, Buffalo River samples contained greater PAH concentrations than samples from the Love Canal, while the reverse was observed for CHs. Bile and liver of bullhead were used for the following analyses: fluorescent aromatic compounds in bile, a measure of exposure to PAHs, microsomal cytochrome P450 (CYP) and P450IA (CYP1A) contents and ethoxyresorufin-O-deethylase (EROD) activities, total glutathione (TH-GSH) concentrations, concentrations of 8- oxodeoxyguanosine (8-oxo-dG), and concentrations of hydrophobic DNA adducts (as measured by 32P-postlabelling). Additionally, a laboratory experiment was performed to examine CYP1A-associated responses in bullhead exposed to the model inducer, β- naphthoflavone (BNF). Results from the laboratory induction study were generally consistent with those observed in the field study, but the field study results suggested induction of CYP1A in bullhead from the reference site (Black Creek). For both field collections, fish from the Buffalo River displayed the greatest concentrations of fluorescent compounds in bile and hepatic DNA adducts, whilst fish from the Love Canal site displayed the greatest microsomal CYP1A concentrations and EROD activities. TH- GSH concentrations were significantly greater in Buffalo River fish versus Black Creek only for the June sampling. No statistically significant differences in 8-oxo-dG concentrations in bullhead hepatic DNA were observed among the sites at either sampling date. The different patterns in biochemical responses observed were consistent with sediment chemistries, and these results suggest that exposure of feral teleosts to different suites of bioavailable contaminants can be associated with expression of a characteristic array of biochemical responses

Effect of gossypol on hepatic and serum gamma-glutamyltransferase activity in rats.

A single intraperitoneal dose (25 mg/kg) of gossypol given to male Sprague-Dawley rats caused marked changes in the activity of the hepatic and serum gamma-glutamyltransferase (GGT) and microsomal monooxygenases. The GGT activity in liver homogenate, S-9 supernatant fraction and microsomes was significantly depressed; however, the level of serum GGT was elevated. While the hepatic glutathione concentration was not greatly changed, the aminopyrine N-demethylase activity and microsomal cytochrome P450 content of the liver were significantly decreased in the treated rats. At necropsy, the livers of the treated rats appeared generally pale with distinct pinpoint foci. Histopathological examination of the liver showed degenerative changes and coagulative necrosis. The results indicate that gossypol is a strong hepatotoxic agent which can produce severe hepatic damage.

Microsomal and Peroxisomal Fatty Acid Oxidation in Liver of Rats With Bile Duct Ligation and Two-Thirds Hepatectomy

Microsomal cytochrome P450 and peroxisomal activity were studied in liver of rats 7 days after two-thirds hepatectomy or bile duct ligation (BDL). Both surgical models decreased the hepatic microsomal cytochrome P450 content, but only cholestasis, produced by BDL, decreased the microsomal metabolism of lauric acid and aminopyrine, peroxisomal fatty acid β-oxidation and catalase activity. The microsomal and peroxisomal activities responded in a coordinate way to cholestasis and two-thirds hepatectomy. These results suggest a cause-effect relationship between the microsomal cytochrome P450 and peroxisomal activity.

Effect of Piperonyl Butoxide on Cell Replication and Xenobiotic Metabolism in the Livers of CD-1 Mice and F344 Rats

Male CD- 1 mice were fed diets containing 0 (control), 10, 30, 100, and 300 mg/kg/day piperonyl butoxide (PBO) and 0.05% sodium phenobarbital (NaPB) and male F344 rats were fed diets containing 0 (control), 100, 550, 1050, and 1850 mg/kg/day PBO and 0.5% NaPB for periods of 7 and 42 days. In both species PBO and NaPB increased relative liver weight and whereas PBO produced a midzonal (mouse) or periportal/midzonal (rat) hypertrophy, NaPB produced a centrilobular hypertrophy. In the rat, individual cell necrosis was also observed at 42 days after high doses of PBO. Replicative DNA synthesis, assessed as the hepatocyte labeling index following implantation of 7-day osmotic pumps containing 5-bromo-2′-deoxyuridine during Study Days 0–7 and 35–42, was increased in mice given 300 mg/kg/day PBO and NaPB for 7 days and in rats given 550 and 1050 mg/kg/day PBO and NaPB for 7 days and 1050 mg/kg/day PBO for 42 days. While PBO had no effect on body weights in mice, the body weights of rats given 550, 1050, and 1850 mg/kg/day PBO for 42 days were reduced to 92, 89, and 70% of control, respectively. PBO induced microsomal cytochrome P450 content and mixed function oxidase activities in the mouse and rat, although the effects were less marked than those produced by NaPB. In summary, this data demonstrates that PBO can produce liver enlargement in the mouse and the rat which is associated with induction of xenobiotic metabolism, hypertrophy, and hyperplasia. The hepatic effects of PBO in the mouse were similar to but less marked than those produced by NaPB. In the rat high doses of PBO were hepatotoxic and resulted in a marked reduction in body weight Thus while the reported formation of eosinophilic nodules in mouse liver by PBO may occur by a mechanism(s) similar to that of NaPB and other nongenotoxic enzyme inducers, the reported tumor formation in rats at greater than the maximum tolerated dose is most likely associated with marked enzyme induction in conjunction with a regenerative hyperplasia resulting from PBO-induced hepatotoxicity.

Induction and Inhibition of Mouse Cytochrome P-450 2B Enzymes by Musk Xylene

Musk xylene (MX) (1,3,5-trinitro-2-t-butylxylene) is a nitromusk perfume ingredient that although uniformly negative in a battery of genotoxicity tests, produces a high incidence of liver tumors in mice. The purpose of this work was to characterize the profile and dose–response relationship of microsomal enzyme induction following exposure to MX. MX was dosed by gavage to male B6C3F1 mice for 7 days at 0, 1, 5, 10, 20, 50, 100, and 200 mg/kg after which microsomes were prepared. At 200 mg/kg, MX increased liver weight by about 65% and increased microsomal cytochrome P-450 content 2-fold over control. MX increased microsomal activity forO-dealkylation of 7-ethoxy and 7-methoxyresorufin 4- and 2-fold, respectively, and increased theN-demethylation of erythromycin approximately 2-fold. These results were generally consistent with increased CYP1A1, 1A2, and 3A protein levels determined by Western blotting. In contrast, whereas no increase inO-dealkylation of 7-pentoxyresorufin (PROD) was observed, MX treatment increased CYP2B protein levels about 25-fold over control at 200 mg/kg. Furthermore, a single dosage of MX (200 mg/kg) increased Cyp2b-10 mRNA to a maximal level and with a time course similar to phenobarbital (PB). To study inhibition of CYP2B enzymesin vivo,mice were treated with PB (0.05% in drinking water for 5 days), then given a single dosage of corn oil or MX (200 mg/kg) at 2 or 18 hr before necropsy. PB treatment increased PROD activity 25-fold, and at 2 hr after MX treatment (associated with peak plasma levels of MX), there was no change in the PB-induced PROD activity. However, at 18 hr, MX treatment decreased PROD activity by 90%. Despite thein vivoinhibition,in vitrostudies indicated that MX did not cause mechanism-based inactivation of CYP2B enzymes. The potential for nitroreduction of MX (catalyzed by anaerobic intestinal bacteria) to contribute to the inhibition of CYP2B enzyme activity was evaluated in a separate group of PB-induced mice that were dosed orally with a regimen of broad spectrum antibiotics (neomycin, tetracyline, and bacitracin) to reduce gut flora prior to administration of MX. In these animals, MX (200 mg/kg) did not inhibit PB-induced PROD activity. In summary, MX treatment produced general hepatic changes consistent with induction of CYP2B enzymes in mice and caused a large increase in CYP2B protein and mRNA levels. These data indicate that MX is a PB-like inducer of cytochrome P-450 enzymes and may cause liver tumors in a manner analogous to PB. However, no increase in CYP2B enzyme activity was observed, suggesting that MX or metabolites of MX also inhibit this enzyme. When the intestinal flora was eliminated by antibiotic treatment, MX no longer inhibited the CYP2B enzyme, indicating that anaerobic bacteria are capable of metabolizing MX, and suggesting that amine metabolites formed by nitroreduction are involved in the inhibition of mouse CYP2B enzymes.

Chronic cannabinoid, CP-55,940, administration alters biotransformation in the rat

The objective of this study was to investigate the effects of single and repeated administration of CP-55,940 {(−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]- trans-4-(3-hydroxypropyl)cyclohexandol)} on behavior, energy metabolism and biotransformation. Single intraperitoneal administration to male Sprague-Dawley rats of CP-55,940 (0.4 mg/kg), induced a behavioural response characterized by ‘splayed hind limbs’, antinociception, hypothermia and a decrease in locomotor activity. Brain and liver mitochondria of the CP-55,940-treated rats exhibited an increase in respiration and no changes in ADP O and citrate synthase specific activity. Repeated intraperitoneal administration of CP-55,940 (0.4 mg/kg, 11 days) induced behavioural tolerance, disappearance of the increase in the mitochondrial oxygen consumption as well as an increase in the monooxygenase activities and the content of liver microsomal cytochrome P450. Some hepatic metabolizing enzymes of the cytosolic glutathione-centre system were also affected. Previous studies had indicated that the tolerance after chronic administration of CP-55,940 could be due to down-regulation of brain cannabinoid receptors. The present findings demonstrate that the behavioural tolerance occurs together with modified biotransformation activities.

Cytochrome P-450 and free radical generation in rat liver microsomes under the influence of prostaglandin E1.

There is evidence to suggest that the mechanism of antioxidant effect of prostaglandin E1 (PGE1) is due to decrease of radical species generation by cytochrome P-450 in rat liver microsomes. Chronic alcohol intoxication increased NADPH oxidation, cytochrome P-450 content and NADPH-stimulated chemoluminiscence of microsomes. Ethanol also raised superoxide dismutase (SOD) activity in microsomes. PGE1 decreased cytochrome P-450 content, normalized NADPH oxidation, NADPH-induced chemoluminiscence and SOD activity in the liver of alcohol-treated rats. PGE developed the similar effect after microsomal induction by both acetone combined with starvation and phenobarbital normalizing all the above parameters. Therefore, PGE1 affects on both, ethanol-inducible IIE1 and phenobarbital-inducible IIB1 isoforms.