Phenobarbital Pretreatment Research Articles

Histopathological Studies On The Liver Of Rats Exposed With Toluene And Trichloroethylene Pre-Treated With Phenobarbital

In rats, the effect of phenobarbital Pre-treatment on the toxicity of a variety of halogenated organic solvent has been investigated. The current findings show that Phenobarbital pre- treatment significantly reduces the toxicity of toluene and trichloroethylene. Our observations Show that toluene is more hepatotoxic than trichloroethylene. It causes peculiar ultra-structural changes i.e. involution of nuclear membrane and irregular arrangement of mitochondrial cristae. However, using single high- dose levels of these solvents, a potentiating impact of Phenobarbital on the toxicity of other organic solvents such as methanol, xylene, and methylene chloridecould not be demonstrated. Because barbiturates are commonly used by industrial workers, it’s crucial to be aware of the possibility of synergistic effect from inhaling of organic solvents, as well as of potentiation associated with others.

The anticonvulsant and neuroprotective effects of the Tanacetum Polycephalum hydroalcoholic extract against the experimental pentylenetetrazol‐induced model of the seizures in the male Wistar rats

AbstractBackgroundThe genus Tanacetum is widespread in Europe and Western Asia. It is an important traditional plant which has been used in the treatment of many illnesses.AimIn the present study, the effect of the hydroalcoholic extract of Tanacetum polycephalum (henceforth TP) on the animal model of seizure was studied.MethodsIn this experimental study, 42 male Wistar rats weighing 220‐250 g were randomly selected and were divided to experimental and control groups (six rats per group). The experimental groups were treated by intraperitoneal (ip) single injection of 150, 300, 450, 600, and 750 mg kg−1 of hydroalcoholic extracts of TP. The control negative group received normal saline (0.9%) and the control positive group received phenobarbital (30 mg kg−1, ip) pretreatment. 30 minutes after the treatments, the seizure behaviors were evaluated by pentylenetetrazol (PTZ; 70 mg kg−1, ip) challenge. In addition, after experiment, the rats were put to death and brain was removed for the histological study.ResultsThe ANOVA test was showed that compared to control group, all the TP doses delayed the initiation and duration of the tonic, clonic, and tonic‐clonic seizures and significantly reduced the tonic and clonic seizures (P < 0.001). Furthermore, the administration of all five doses of the extract significantly prevented the production of the dark neurons (P < 0.001) in different areas of the hippocampus compared to PTZ group.ConclusionsWe can conclude that the TP extract has beneficial effects on the prevention of the PTZ‐induced seizure.

Persistent alterations in seizure susceptibility, drug responsiveness and comorbidities associated with chemical kindling after neonatal exposure to an organophosphate

Developmental exposure to organophosphates (OPs), at doses that do not cause cholinergic crisis, induces profound and lasting alterations in different neurotransmitter systems, which contribute to several behavioral outcomes. The present work examines whether neonatal exposure to low dose of chlorpyrifos (CPF), a widely used OP insecticide, alters the general excitability of the adult brain, its responsiveness to drugs with antiepileptic properties, the process of chemical kindling and the kindling-induced behavioral outcomes. Neonatal rats were exposed to daily doses of CPF (1 mg/kg) or dimethyl sulfoxide (DMSO, vehicle) on postnatal days (PND) 1–4. On PND 60, a subgroup of animals from both CPF and DMSO groups were injected with additive doses of pentylenetetrazole (PTZ) to evaluate the latency time to the first seizure, the threshold of PTZ-induced convulsion, and to determine the anticonvulsive action of phenobarbital (20 mg/kg), ethosuximide (100 mg/kg) and scopolamine (0.6 mg/kg) when used as pretreatment. Rats in the other subgroups were kindled by repeated intraperitoneal injections of an initially subconvulsive dose of PTZ (37.5 mg/kg) at 48-h intervals for 4 weeks. Kindled rats were then subjected to radial arm maze, sweet taste preference and forced swim test. Neonatal exposure to CPF shortened the latency time to the first seizure after pretreatment with scopolamine in female rats and decreased the threshold for PTZ-induced clonic convulsions after phenobarbital pretreatment in male rats. Neonatal CPF exposure also decreased the rate of kindling progression in female rats during early stages of PTZ kindling. On the other hand, CPF exposure sex-selectively reduced the number of working memory errors after kindling only in male rats. Drug challenge with MK-801 induced more impairment in the working memory of female kindled rats, indicating more dependence of working memory on NMDA receptor activity in these animals. Female kindled rats from CPF exposed group also showed longer time of immobility in forced swim test, showing an increase in the depressive-like behavior. This difference was also observed in the second session of forced swim test, after treating with fluoxetine, a selective inhibitor of serotonin reuptake. The recent finding, together with lack of difference in the sweet taste preference, suggests that mechanism beyond the reduction of serotonergic activity underlie the increased depressive-like behavior in this animals. To our knowledge, this is the first report describing the potential contribution of developmental exposure to an OP in susceptibility to antiepileptic drug resistance and alteration of seizure-induced behavioral deficits.

Interaction between 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro

The compound 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4‑DCPB and evaluate the effects of 3,4‑DCPB on the activities of CYP450 enzymes. 3,4‑DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4‑DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4‑DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of β-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB−), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4‑hydroxy‑mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6‑hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4‑DCPB (16 μg/mL, 59 μM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB− and PB+) and CYP3A4 (PB+) were not inhibited by 3,4‑DCPB in RLMs. In conclusion, the present study showed that 3,4‑DCPB was metabolized by multiple CYP450 enzymes. 3,4‑DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4‑DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

The antiepileptic and neuroprotective effect of the Buxus hyrcana Pojark hydroethanolic extract against the pentylentetrazol induced model of the seizures in the male rats

Aims: The genus Buxus grows up widespread in Europe and Western Asia. It is an important traditional plant that has been used in the treatment of many illnesses. In the present study, the effect of hydroethanolic extract of Buxus hyrcana Pojark (BHP) on the animal model of seizure was studied.Materials and Methods: In this experimental study, 42 male Wistar rats weighing 220–250 g were randomly selected and were divided into experimental and control groups (six rats per group). The experimental groups were treated by the intraperitoneal (i.p.) single injection of 150, 300, 450, 600 and 750 mg kg−1 of hydroalcoholic extracts of BHP. The control negative group received normal saline (0.9%) and the control positive group received phenobarbital (30 mg kg−1, i.p.) pre-treatment. Thirty minutes after the treatments, the seizure behaviors were evaluated by the pentylenetetrazole (PTZ) (70 mg kg−1, i.p.) challenge. In addition, after the experiment, the rats were put to death and their brains were removed for the histological study.Results: The ANOVA demonstrated that compared to the control group, all the BHP doses delayed the initiation and duration of the tonic, colonic and tonic–colonic seizures and significantly reduced the tonic and colonic seizures (p < 0.001). Furthermore, the administration of all five doses of the extract significantly prevented the production of the dark neurons (p < 0.001) in different areas of the hippocampus compared to PTZ group.Conclusion: We can conclude that the BHP extract has beneficial effects for the prevention of the PTZ induced seizure.

The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1.

Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation.

Early life status epilepticus and stress have distinct and sex-specific effects on learning, subsequent seizure outcomes, including anticonvulsant response to phenobarbital.

Neonatal status epilepticus (SE) is often associated with adverse cognitive and epilepsy outcomes. We investigate the effects of three episodes of kainic acid-induced SE (3KA-SE) and maternal separation in immature rats on subsequent learning, seizure susceptibility, and consequences, and the anticonvulsant effects of phenobarbital, according to sex, type, and age at early life (EL) event. 3KA-SE or maternal separation was induced on postnatal days (PN) 4-6 or 14-16. Rats were tested on Barnes maze (PN16-19), or lithium-pilocarpine SE (PN19) or flurothyl seizures (PN32). The anticonvulsant effects of phenobarbital (20 or 40 mg/kg/rat, intraperitoneally) pretreatment were tested on flurothyl seizures. FluoroJadeB staining assessed hippocampal injury. 3KA-SE or separation on PN4-6 caused more transient learning delays in males and did not alter lithium-pilocarpine SE latencies, but aggravated its outcomes in females. Anticonvulsant effects of phenobarbital were preserved and potentiated in specific groups depending on sex, type, and age at EL event. Early life 3KA-SE and maternal separation cause more but transient cognitive deficits in males but aggravate the consequences of subsequent lithium-pilocarpine SE in females. In contrast, on flurothyl seizures, EL events showed either beneficial or no effect, depending on gender, type, and age at EL events.

Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes

Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2′,7′-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C12), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes.

Influence of Phenobarbital Pretreatment on Toxicity of Calotropis procera Latex in Nubian Goats

Influence of Phenobarbital Pretreatment on Toxicity of Calotropis procera Latex in Nubian Goats

N-Nitrosodiethylamine genotoxicity in primary rat hepatocytes: Effects of cytochrome P450 induction by phenobarbital

Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21–105μg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21–21μg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ∼6-fold and ∼2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105μg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.

A possible mechanism for the decrease in serum thyroxine level by phenobarbital in rodents

Effects of phenobarbital (PB) on the levels of serum thyroid hormones such as total thyroxine (T 4) and triiodothyronine were examined in male mice, hamsters, rats, and guinea pigs. One day after the final administration of PB (80 mg/kg, intraperitoneal, once daily for 4 days), significant decreases in the levels of the serum total T 4 and free T 4 occurred in mice, hamsters, and rats, while a significant decrease in the level of serum triiodothyronine was observed in hamsters and rats among the animals examined. In addition, a significant decrease in the level of serum thyroid-stimulating hormone was observed in only hamsters among the rodents examined. Significant increases in the level and activity of hepatic T 4-UDP-glucuronosyltransferase (UGT1A) after the PB administration occurred in mice, hamsters, and rats, while the increase in the amount of biliary [ 125I]T 4-glucuronide after an intravenous injection of [ 125I]T 4 to the PB-pretreated animals occurred only in rats. In mice, rats, and hamsters, but not guinea pigs, PB pretreatment promoted the clearance of [ 125I]T 4 from the serum, led to a significant increase in the steady-state distribution volumes of [ 125I]T 4, and raised the concentration ratio (Kp value) of the liver to serum and the liver distribution of [ 125I]T 4. The present findings indicate that the PB-mediated decreases in the serum T 4 level in mice, hamsters, and rats, but not guinea pigs, occur mainly through an increase in the accumulation level of T 4 in the liver.

Effects of Exposure Period on the Developmental Toxicity of 2-Bromopropane in Sprague-Dawley Rats

Recently we reported that 2-bromopropane (2-BP) has maternal toxicity, embryotoxicity, and teratogenicity in Sprague-Dawley rats. The aims of this study are to examine the potential effects of 2-BP administration on pregnant dams and embryo-fetal development, and to investigate the effects of metabolic activation induced by phenobarbital (PB) on developmental toxicities of 2-BP. Pregnant rats received 1000 mg/kg/day subcutaneous 2-BP injections on gestational days (GD) 6 through 10 (Group II and Group IIII) or 11 through 15 (Group IV). Pregnant rats in Group III received an intraperitoneal PB injection once daily at 80 mg/kg/day on GD 3 through 5 for induction of the liver metabolic enzyme system. Control rats received vehicle injections only on GD 6 through 15. All dams underwent caesarean sections on GD 20 and their fetuses were examined for external, visceral, and skeletal abnormalities. Significant adverse effects on pregnant dams and embryo-fetal development were observed in all the treatment groups, and the maternal and embryo-fetal effects of 2-BP observed in Group II were higher than those seen in Group IV. Conversely, maternal and embryo-fetal developmental toxicities observed in Group III were comparable to those seen in Group II. These results suggest that the potential effects of 2-BP on pregnant dams and embryo-fetal development are more likely in the first half of organogenesis (days 6~10 of pregnancy) than in the second half and that the metabolic activation induced by PB pre-treatment did not modify the developmental toxic effects of 2-BP in rats.

Effect of Phenobarbital on the Pharmacokinetics of Rutaecarpine and its Metabolite in Rats

To investigate the possible interaction between rutaecarpine and phenobarbital in rats, phenobarbital in saline at 80 mg/kg was given ip to male SD rats for 3 consecutive days. Saline was given to control animals. One day after phenobarbital pre-treatment, rutaecarpine at 16 mg/kg was administered through penile vein. Blood was collected and analyzed by using HPLC. The pharmacokinetic parameters were determined with the non-compartmental model. Pre-treatment with phenobarbital significantly altered the pharmacokinetic profiles of rutaecarpine and its metabolite, 10-hydroxyrutaecarpine. The AUC of rutaecarpine was reduced to approximately 50% of control and the plasma half-life of rutaecarpine was significantly shortened when compared with control. In addition, the Cmax of 10-hydroxyrutaecarpine was increased approximately 160% of control. The AUC and the plasma half-life of 10-hydroxyrutaecarpine were decreased to 76.9% of control and to 82.7 min from 175.9 min, respectively. The results suggested that phenobarbital might accelerate the metabolism of rutaecarpine, thereby changing the pharmacokinetic parameters of rutaecarpine in male SD rats.

Minimal Role of Hepatic Transporters in the Hepatoprotection against LCA-Induced Intrahepatic Cholestasis

The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid-induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse.

Modelo experimental de indução de lesão oxidativa hepática em ratos por halotano

The anesthetic halothane can be reductively metabolized to reactives intermediates that may initiate lipid peroxidation accompanied by hepatic injury. Hypoxia and phenobarbital pretreatment in rats increases metabolism of halothane, the oxidative stress, cause liver antioxidant enzymes changes and tissue damage. We investigated the effect of halothane on hepatic lipid peroxidation and on hepatic histology after increases reductive metabolism of halothane caused by hypoxia and phenobarbital pretreatment. Twenty-five male wistar rats were divided in five equals groups: CO (Control), HO14 (Halothane/Hypoxia), F (fenobarbital alone), O14 (Hypoxia alone) and H (Halothane alone). After 24 hours the rats were killed, their livers removed to determine chemoluminescence, thiobarbituric acid-reactive substances, catalase, superoxide dismutase, and blood samples were taken to determine AST and ALT. The histopathologic evaluation was performed with hematoxylin and eosin staining. Histopathologic scores are presented as 25th-75th percentile/range values and median +/- range. Halothane-hypoxic exposure resulted in a significant changes in the activities of antioxidant enzymes, and induced hepatic lipoperoxidation. Moreover it resulted in histopathologic liver injury as well as significant increase of serum activity of AST and ALT.

Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital

Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T(1/2) and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of (14)C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 approximately 350-fold, but did not increase covalent binding of (14)C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD(100) dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.

Role of Metabolism in Parathion-Induced Hepatotoxicity and Immunotoxicity

The objective of this study was to investigate whether metabolic activation of parathion by cytochrome P-450s (CYPs) was responsible for pesticide-induced hepatotoxicity and immunotoxicity. Initially, to investigate parathion metabolism in vitro, the production of paraoxon and p-nitrophenol, major metabolites of parathion, was determined by high-performance liquid chromatography (HPLC). Subsequently, metabolic fate and CYP enzymes involved in the metabolism of parathion were partially monitored in rat liver microsomes in the presence of the NADPH-generating system. Among others, phenobarbital (PB)-induced microsomes produced the metabolites paraoxon and p-nitrophenol to the greatest extent, indicating the involvement of CYP 2B in parathion metabolism. When female BALB/c mice were treated orally with 1, 4, or 16 mg/kg of parathion in corn oil once, parathion suppressed the antibody response to sheep red blood cells. To further investigate a possible role of metabolic activation by CYP enzymes in parathion-induced toxicity, female BALB/c mice were pretreated intraperitoneally with 40 mg/kg PB for 3 d, followed by a single oral treatment with 16 mg/kg parathion. PB pretreatment produced a decrease in hepatic glutathione content and increases in hepatotoxic paramenters in parathion-treated mice with no changes in the antibody response. In addition, greater p-nitrophenol amounts were produced when mice were pretreated with PB, compared to treatment with parathion alone. These results indicate that parathion-induced hepatotoxicity might be differentiated from immunotoxicity in mice.

Structural Gene Products of the Murine Ah Complex

Antibodies against mouse-liver microsomal cytochromes P1-450 and P-448, two polycyclic aromatic inducible cytochromes, were previously developed [Negishi, M. and Nebert, D.W. (1979) J. Biol. Chem. 254, 11015-11023]. Liver microsomes from 3-methylcholanthrene-treated and phenobarbital-treated and control adult mice and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated adult and fetal mice were examined. Immunoprecipitable radioactivity was measured, following labeling with pyridoxal phosphate/NaB[3H]4 or with 125I-labeled p-aminosulfobenzoic acid/NaNO2 in vitro or with [3H]leucine, [14C]glucosamine, or [32P]O4 in vivo. (a) Induction of cytochrome P1-450 occurs developmentally earlier in gestation than induction of cytochrome P-448 when the mother is treated with polycyclic aromatic compounds. (b) There appears to be a basal form of cytochrome P-448 but no cytochrome P1-450 in control liver microsomes; inducibility of cytochrome P-448 thus ranges between 5--12-fold, whereas that of P1-450 is infinite. (c) Phenobarbital pretreatment induces no detectable P1-450 or P-448. (d) P-448 appears to be either greater in concentration than P1-450 in the membrane or more exposed than P1-450 on the microsomal membrane surface. (e) By the radioimmunoassay methods used, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced P1-450 and P-448 in Ah-nonresponsive mice are indistinguishable from those in Ah-responsive mice; this is true in both the fetus and the adult. (f) Compared with P-448 expression, the expression of P1-450 is more closely associated with 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity, and these two structural gene products are apparently regulated independently. (g) P-448 but not P1-450 appears to be a glycoprotein. These data illustrate further differences between two forms of polycyclic aromatic-inducible P-450 in mouse liver. Neither P1-450 nor P-448 appears to be a phosphoprotein. Neither anti-(P1-450) nor anti-(P-448) precipitates any forms of liver microsomal P-450 from beta-naphthoflavone-treated adult rabbits and, conversely, anti-LM4 (the antibody to rabbit liver microsomal P-450 form 4) does not precipitate any forms of liver microsomal P-450 from 3-methylcholanthrene-treated C57BL/6N mice.

Phenobarbital induction of permethrin detoxification and phenobarbital metabolism in susceptible and resistant strains of the beet armyworm Spodoptera exigua (H�bner)

The permethrin resistant strain (TR-strain) of the beet armyworm, Spodoptera exigua (Hübner), has 92.5-fold resistance to permethrin (at LD50 level) compared to the permethrin susceptible strain (TS-strain). Bioassay involving permethrin mixed with piperonyl butoxide, an inhibitor of microsomal cytochrome P450s, significantly reduced the resistance ratio from 92.5- to 7.9-fold. However, S,S,S-tributylphosphorotrithioate and diethylmaleate which are inhibitors of esterases and glutathione S-transferase, respectively, did not affect the resistance level. These results indicate that the detoxification of permethrin in the TR-strain was primarily due to the cytochrome P450 monooxygenases. LD50 for permethrin was increased to 4.5-fold by the pre-treatment of phenobarbital in the TS-strain. The effect of induction by phenobarbital was almost completely overcome by the piperonyl butoxide treatment. However, it was observed that phenobarbital treatment did not cause any change in the toxicity of permethrin to TR strain. Since this result deviated from the expectation that the metabolism of phenobarbital in the TR-strain should be greater than that in the TS-strain, it was deemed necessary to compare the metabolism of phenobarbital between the TS- and TR-strains. Comparison was made based on the concentration of phenobarbital in the hemolymph and whole body. The results showed no significant difference in phenobarbital treatment between the two strains used in this study suggesting the possibility that the induction system in TS-strain is different from the TR-strain.

Phenobarbital induction of permethrin detoxification and phenobarbital metabolism in susceptible and resistant strains of the beet armyworm Spodoptera exigua (Hübner)

The permethrin resistant strain (TR-strain) of the beet armyworm, Spodoptera exigua (Hübner), has 92.5-fold resistance to permethrin (at LD 50 level) compared to the permethrin susceptible strain (TS-strain). Bioassay involving permethrin mixed with piperonyl butoxide, an inhibitor of microsomal cytochrome P450s, significantly reduced the resistance ratio from 92.5- to 7.9-fold. However, S, S, S-tributylphosphorotrithioate and diethylmaleate which are inhibitors of esterases and glutathione S-transferase, respectively, did not affect the resistance level. These results indicate that the detoxification of permethrin in the TR-strain was primarily due to the cytochrome P450 monooxygenases. LD 50 for permethrin was increased to 4.5-fold by the pre-treatment of phenobarbital in the TS-strain. The effect of induction by phenobarbital was almost completely overcome by the piperonyl butoxide treatment. However, it was observed that phenobarbital treatment did not cause any change in the toxicity of permethrin to TR strain. Since this result deviated from the expectation that the metabolism of phenobarbital in the TR-strain should be greater than that in the TS-strain, it was deemed necessary to compare the metabolism of phenobarbital between the TS- and TR-strains. Comparison was made based on the concentration of phenobarbital in the hemolymph and whole body. The results showed no significant difference in phenobarbital treatment between the two strains used in this study suggesting the possibility that the induction system in TS-strain is different from the TR-strain.