Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver

1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups.

2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only.

3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase.

4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane.

5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups.

6. Lindane induced periportal necrosis with haemorrhagic foci in all groups.

7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction.     

Prolonged phenobarbital pretreatment abolishes the early oxidative stress component induced in the liver by acute lindane intoxication

Lindane administration to rats (60 mg/kg b.w.) led to an enhancement in the oxidative stress status of the liver at 4 h after treatment, characterized by increases in hepatic thiobarbituric acid reactants (TBARS) formation and chemiluminescence, reduced glutathione (GSH) depletion, and diminution in the biliary content and release of GSH. These changes were observed in the absence of changes in either microsomal functions (cytochrome P450 content, NADPH-dependent superoxide radical production, and NADPH-cytochrome P450 reductase or NADPH oxidase activities) or in oxidative stress-related enzymatic activities (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and glutathione-S-transferases), over control values. Phenobarbital (PB) administration (0.1% in drinking water; 15 days) elicited an enhancement in liver microsomal functions, lipid peroxidation, and GSH content, without changes in oxidative stress-related enzymatic activities, except for the elevation in those of glutathione reductase and glutathione-S-transferase, compared to control rats. Lindane given to PB-pretreated rats did not alter liver microsomal functions, lipid peroxidation, glutathione status, or oxidative stress-related enzymatic activities, as compared to PB-pretreated animals. In addition, lindane induced periportal necrosis with hemorrhagic foci in untreated rats, but not in PB-pretreated animals. It is concluded that the early oxidative stress response of the liver to lindane and hepatic injury are suppressed by PB pretreatment via induction of microsomal enzymes in all zones of the hepatic acinus. reserved.     

Regulation of rat liver epoxide hydratase activity by phenobarbital and 3-methylcholanthrene.

Phenobarbital-pretreatment of rats increased liver microsomal epoxide hydratase activity 2.6-fold over controls even after elimination of inherent latency problems. However. 3-methylcholanthrene pretreatment of rats does not alter the levels of hepatic epoxide hydratase activity. Epoxide hydratase was purified from control rats or rats pretreated with phenobarbital or 3-methylcholanthrene. The enzymes isolated from all three sources appear to be very similar in size, immunological activity and specific activity. These experiments strongly suggest that phenobarbital stimulates epoxide hydratase activity by selectively increasing microsomal content of a single form of the enzyme. The possible existence of multiple forms of epoxide hydratase is discussed.     

Effect of phenobarbital or 3-methylcholanthrene pretreatment on carbon tetrachloride-induced lipid peroxidation in rat liver.

The enhancement of carbon tetrachloride hepatotoxicity following phenobarbital pretreatment is associated with an increase in lipid peroxidation in vivo when CCl₄ is administered orally or by inhalation. However, pretreatment with 3-methylcholanthrene did not increase in vivo lipid peroxidation following CCl₄ administration by the oral or inhalation route. CCl₄ stimulated lipid peroxidation, as determined by malonaldehyde formation in vitro, and was increased by phenobarbital pretreatment but not by 3-methylcholanthrene pretreatment. These data support a relationship between microsomal drug metabolizing activity and alterations in hepatic injury and lipid peroxidation following CCl₄ administration.     

Three forms of trichloroethylene-metabolizing enzymes in rat liver induced by ethanol, phenobarbital, and 3-methylcholanthrene

In vitro metabolism of trichloroethylene (TRI) and trichloroethanol (TCE) was investigated using liver microsomes from control and ethanol-, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated rats. At least three forms of enzymes were involved in TRI metabolism. One was a low-Km type normally existing in microsomes from control rats. The ethanol-inducible enzyme was found to be catalytically identical to this low-Km isozyme. Another was a high-Km type which was induced exclusively by PB, and a third was an MC-inducible isozyme with a Km value between those of ethanol- and PB-inducible isozymes. Although MC treatment did not affect the rate of TRI metabolism in vitro, both ethanol and PB treatment markedly enhanced the metabolism. Ethanol-induced enhancement was different from PB-induced enhancement in that ethanol enhanced the metabolism predominantly at low substrate concentrations, whereas PB did so at high concentrations. In addition, TRI metabolism with enzymes from ethanol-treated rats was inhibited by the substrate itself at high concentrations. MC treatment of rats had little or no influence on the rate of TCE metabolism in vitro, whereas both ethanol and PB enhanced the microsomal conversion of TCE to chloral hydrate. As in the case of TRI metabolism, ethanol induced a microsomal TCE-metabolizing enzyme of low Km, whereas PB preferentially induced an enzyme of high Km.     

Effect of phenobarbital and 3-methylcholanthrene on biphenyl hydroxylations and fluidity of rat liver microsomal membranes

The metabolism of biphenyl by rat liver microsomes after administration of phenobarbital and 3-methylcholanthrene was studied. Phenobarbital increased the activity of biphenyl-4-hydroxylase and 3- methylcholanthrene increased the activity of both biphenyl-4-hydroxylase and biphenyl-2-hydroxylase as compared to non-treated (control) rats. Phenobarbital increased the lipid fluidity while 3-methylcholanthrene increased the lipid rigidity of microsomal membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius plots of [ro/r)-1]-1 indicated that the lipid phase separation of the control membrane at 22.1 +/- 1.1 degrees was reduced in phenobarbital treated (14.5 +/- 0.8 degrees) and increased in 3-methylcholanthrene treated rats (32.7 +/- 2.2 degrees). Arrhenius plots of biphenyl-4-hydroxylase and biphenyl-2-hydroxylase activities exhibited a break point at 21.8 +/- 1.1 degrees and 32.1 +/- 2.1 degrees, respectively, suggesting differences in the interactions of the enzymes with their annular lipids. It is suggested that biphenyl-4-hydroxylase requires a liquid state of its lipid microenvironment to be fully active, while biphenyl-2-hydroxylase a gel state of its lipid microenvironment. These studies provide a basis for postulating that a "non-genomic" mechanism of phenobarbital and 3-methylcholanthrene induces cytochrome P-450 dependent monoxygenases.     

Effects of phenobarbital and 3-methylcholanthrene administration on glutathione-S-epoxide transferase activity in rat liver

Glutathione-S-epoxide transferase activity in rat liver was studied with three epoxide substrates. The specific activities in the high-speed liver supernatant fraction for 3MC 11, 12 oxide, styrene oxide and 3,3,3-trichloro-1,2 epoxy propane were 5.7, 86.4 and 165 nmoles/5 min/mg protein, respectively. Phenobarbital (75 mg/kg body wt for 3 days) or 3-methylcholanthrene (20 mg/kg body wt for 2 days) administration to rats resulted in a 40–60% increase in enzyme activity. β-napthoflavone administration on the other hand was without any effect on glutathione-S-epoxide transferase activity.     

Effect of phenobarbital and spironolactone treatment on the oxidative metabolism of antipyrine by rat liver microsomes.

The effects of pretreating rats with the inducers, phenobarbital or spironolactone, on the formation rate of the three major oxidative metabolites of antipyrine in vitro by hepatic microsomal fractions have been investigated. Both inducers reduced the rate of 3-methylhydroxylation of antipyrine by approximately 50%. In contrast, N-demethylation and 4-hydroxylation were enhanced 1.7-fold and 3.4-fold, respectively, in case of phenobarbital induction and 1.4-fold and 2.6-fold, respectively, following spironolactone treatment. To elucidate the role of some cytochrome P450 isoenzymes in the production of the three major metabolites of antipyrine, the effects of form selective enzyme inhibitors on antipyrine oxidation were also studied. Troleandomycin did not alter 3-methylhydroxylation but reduced both N-demethylation and 4-hydroxylation of antipyrine in microsomes from induced rat liver. Cimetidine and chloramphenicol decreased the rate of formation of all three metabolites in microsomes from induced and uninduced animal livers as well. Chloramphenicol seemed to be the most potent inhibitor of in vitro antipyrine oxidation. Alpha-methyldopa significantly enhanced the rate of formation of 4-hydroxyantipyrine and slightly reduced the rate of N-demethylation and 3-methylhydroxylation. According to the data obtained with microsomes from uninduced rat livers, the formation of the three major metabolites of antipyrine is extensively mediated by CYP2C11/C6. In microsomes from induced animal liver, CYP2B and CYP3A may contribute to both N-demethylation and 4-hydroxylation of antipyrine.     

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene

The responsiveness of the hepatic supernatant NAD⁺-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley.A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged (non-responsive) in all rats after treatment with PB while it increased (responsive) 5- to 19-fold in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.     

Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene induction of microsomal drug-metabolizing enzymes of the rat

The effect of riboflavin deficiency on the induction of hepatic microsomal enzymes by phenobarbital and 3-methylcholanthrene has been investigated. A decrease in microsomal flavin levels of 56 per cent was associated with a decrease in NADPH cytochrome c reductase (52 per cent), azoreductase (71 per cent) and benzpyrene hydroxylase (74 per cent). Microsomal cytochrome P-450 content and aminopyrine demethylase were not significantly affected by flavin deficiency. Phenobarbital or 3-methylcholanthrene pretreatment did not affect hepatic microsomal flavin levels in normal or deficient animals. In flavin-deficient animals, phenobarbital pretreatment significantly increased cytochrome c reductase, cytochrome P-450 content, aminopyrine demethylase and azoreductase. Thus the carbon monoxide-sensitive pathway (cytochrome P-450 mediated) of azoreductase was essentially unaffected by flavin deficiency. In deficient animals, the carbon monoxide-insensitive microsomal azoreductase pathway (non-cytochrome P-450 mediated) normally induced by 3-methylcholanthrene was unaffected. Thus, induction of azoreductase by 3-methylcholanthrene was found to be flavin dependent. However, 3-methylcholanthrene did increase cytochrome P-450 content and benzpyrene hydroxylase in flavin-deficient animals. The induction of benzpyrene hydroxylase by 3-methylcholanthrene increased with increasing microsomal flavin content. Part of the mechanism of azoreductase induction by 3-methylcholanthrene was due to an induced change in the structure or composition of microsomal flavoprotein. This interpretation is supported by the findings that: (1) induction by 3-methylcholanthrene in riboflavin-deficient rats required a minimal flavin level, (2) increased enzyme activity was not compensated by an increase in microsomal flavin and (3) induction by 3-methylcholanthrene augmented FMN-stimulation of microsomal azoreductase in vitro.     

Comparison of hepatic drug-metabolizing enzymes induced by 3-methylcholanthrene and phenobarbital between pre- and postnatal rats

Effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) on the hepatic drug-metabolizing enzyme system in fetal liver of rats were investigated. Intraperitoneal administration of 3MC (25 mg/kg, 72 and 48 hr before death) to pregnant rats significantly increased hexobarbital (HB) and aminopyrine (AM)-metabolizing activities in fetuses on the 21st day of gestation to 148.0 and 150.6% of control fetuses, respectively. In contrast, HB and AM-metabolizing activities in 4-day-old neonates and mothers were decreased by administration of 3MC on the 21st day of gestation. Benzo[a]pyrene (BP)-metabolizing activity, NADPH-cytochrome c reductase activity, and cytochrome P-450 content in 3MC-treated fetuses were significantly increased to 2143.6, 137.6, and 323.8% of the control, respectively. Following 3MC administration, the maximum absorption of the cytochrome P-450-CO difference spectra in liver microsomes of fetuses was observed at 449-450 nm. The induction profile following 3MC administration in the fetal livers was different from that in the neonatal and the maternal livers. On the other hand, intraperitoneal administration of PB (60 mg/kg, 72, 48, and 24 hr before death) significantly increased HB, AM, and BP-metabolizing activities in fetal livers to 263.7, 231.0, and 151.2% of the respective controls. The profile induced by PB in the fetal livers was similar to that in maternal livers. These results suggest that HB and AM-metabolizing enzymes in fetal livers treated with 3MC or PB possess the capacity to be induced, and the responsiveness of the drug-metabolizing enzyme system to 3MC during the prenatal stage may differ from the postnatal stage.     

Effects of rubratoxin B on the hepatic monooxygenase system in phenobarbital and 3-methylcholanthrene induced male mice

Alterations in the hepatic microsomal monooxygenase system and in the concentrations of rubratoxin B in urine and feces were examined in male mice pretreated with corn oil, phenobarbital or 3-methylcholanthrene (3MC) and then given a single i.p. dose of rubratoxin B (1 mg/2.5 ml propylene glycol/kg). Twenty-four hours later the following parameters were determined: hepatic cytochrome P-450 content, enzyme activities of NADPH-cytochrome c reductase, NADPH-dependent dehydrogenase, aniline hydroxylase and ethylmorphine N-demethylase, and hapatic microsomal protein and reduced glutathione levels. Excretion of rubratoxin B in urine and feces also was determined. Rubratoxin B reduced the elevated cytochrome P-450 (136%, 134%) and protein (128%, 112%) to control values in animals pretreated with phenobarbital or 3MC, respectively; whereas, in the corn oil pretreated group, the mycotoxin reduced cytochrome P-450 by 38%. Aniline hydroxylase activity was reduced 31% or more in all pretreated animals. Rubratoxin B did not affect ethylmorphine N-demethylase activity in mice pretreated with phenobarbital; however, the enzyme activity was decreased significantly in the 3MC group. Rubratoxin B reduced the hepatic glutathione level in animals receiving 3MC (33%) or corn oil (22%). More rubratoxin B was detected in the urine than in the feces regardless of pretreatment. Only trace amounts of toxin were detected in the feces of animals from the 3MC group. These data suggest a greater effect of rubratoxin B in the 3MC pretreated mice than in the phenobarbital animals.     

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K_m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V_max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V_max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V_max using BOX was enhanced after TCDD administration only. The K_m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K_m using STOX, while it increased the K_m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.     

Cholestasis as an in vivo model for analysis of the induction of liver microsomal monooxygenases by sodium phenobarbital and 3-methylcholanthrene.

Cholestasis was used as a model of sharp decrease in the amounts of substrate-binding and catalytic centres of cytochrome P-450 for sodium phenobarbital and 3-methylcholanthrene. Based on this model, the induction effects of sodium phenobarbital and 3-methylcholanthrene on rat liver microsomal monooxygenases were analyzed. Under conditions excluding the primary binding and metabolism of the inducer by monooxygenases, sodium phenobarbital retains its capacity for induction. By contrast, 3-methylcholanthrene exerted no inducing effects under the same conditions as confirmed by the lack of increase of cytochrome P-448 content. From the data obtained it is suggested that in mechanism of sodium phenobarbital induction of liver microsomal monooxygenases the activation of protein synthesis is affected by the inducer itself. As for 3-methylcholanthrene, it is assumed that the synthesis of specific protein (cytochrome P-448) could be initiated by the microsomal metabolites of this inducer.     

Alcohol-induced oxidative stress in rat liver

1. Livers from rats treated acutely with ethanol showed increased chemiluminescence, malondialdehyde production, and diene formation. Previous administration of (+)-cyanidanol-3 completely abolished acute ethanol-induced chemiluminescence.

2. Rats fed alcohol liquid diets for 3 weeks showed significant increases in microsomal and mitochondrial malondialdehyde formation, and in microsomal H₂O₂ and O₂^? generation.

3. Rats fed a solid basal diet plus ethanol solution for 12 weeks also showed increased microsomal production of O₂^? and increased content of microsomal cytochrome P-450. Hydroperoxide-induced chemiluminescence was higher in homogenates, mitochondria and microsomes from ethanol-treated rats than from controls. Vitamins E and A were more effective inhibitors of hydroperoxide-stimulated chemiluminescence in liver homogenates from ethanol-treated rats than from control animals.

4. Results are consistent with peroxidative stress leading to increased lipid peroxidation in liver of rats fed ethanol both acutely and after long-term dosing.