P-nitroanisole O-demethylase Activities Research Articles

Involvement of P450s in the metabolic resistance of Digitaria sanguinalis (L.) Scop. To ALS-inhibiting herbicides

Weed resistance to a range of herbicides has rapidly evolved, often with different mechanisms of action. The resulting uninhibited growth of weeds poses demonstrable threats to crop production and sustainable agriculture. Digitaria sanguinalis (L.) Scop., a troublesome weed in corn and other agricultural fields, has developed resistance to herbicides that inhibiting ALS (Acetolactate Synthase), such as nicosulfuron. Understanding the weed's resistance patterns and mechanisms is crucial. However, little is known of the non-target site resistance (NTSR) mechanisms of D. sanguinalis owing to a lack of relevant genome sequences and other materials. Therefore, in this study, a population of D.sanguinalis presenting multiple resistance was tested and found that its high level of resistance to ALS-inhibiting herbicides was not associated with target-related alterations.Administration of P450 inhibitors reversed the resistance to ALS-inhibiting herbicides. Following the application of ALS-inhibiting herbicides, the activities of NADPH-P450 reductase and p-nitroanisole O-demethylase (PNOD) were notably greater in the resistant population of D. sanguinalis than those in the susceptible population. The results suggested P450 enzyme familyplays a major role in the metabolic resistance mechanism, that increased P450 enzyme activity promote cross-resistance in D. sanguinalis to ALS-inhibiting herbicides. RNA-seq analysis showed that five genes from the P450 family (CYP709B2, CYP714C2, CYP71A1, CYP76C2, and CYP81E8) were upregulated in resistant D. sanguinalis. In conclusion, the upregulation of several P450 genes is responsible for establishing resistance to ALS-inhibiting herbicides in D. sanguinalis.

Biochemical Characterization of Detoxifying Enzymes in Dimethoate-Resistant Strains of Melon Aphid, <i>Aphis gossypii</i>(Hemiptera: Aphididae)

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a highly polyphagous sap sucking pest on wide varieties of crops including cotton and vegetables. It is a notorious vector of many plant viruses that are persistently and non-persistently transmitted. In nature, aphids are regulated by their natural enemies. However, chemical control remains a major management tool even though resistance to insecticides has been documented worldwide. A better understanding of mechanisms by which insecticide resistance occurs and its early detection is desirable to develop effective management strategies. The present investigation was conducted to study the development of resistance to an organophosphate (OP) compound-dimethoate, identify biochemical mechanism(s) involved in resistance and study cross-resistance to imidacloprid in laboratory selected A. gossypii strains in comparison to susceptible strains. Bioassay studies revealed that the LC50 values increased dramatically with dimethoate selection in resistant strains and the resistance ratio (RR) was 270-, 243- and 210-fold greater than that of the susceptible strains by 30th generation. Further, biochemical assays revealed enhanced activities of carboxylesterases (CarE), glutathione S-transferases (GSTs) and cytochrome P450-mediated p-Nitroanisole O-demethylase (PNOD) in resistant strains supporting their role in dimethoate detoxification. This study thus revealed that enhanced activity of detoxifying enzymes viz., CarE, GSTs and PNODs is one of the mechanisms underlying dimethoate resistance in A. gossypii collected from South India. Interestingly, the possibility of negatively correlated cross-resistance to imidacloprid was identified in three OP- resistant strains exhibiting 2.97-, 2.56- and 3.76-fold sensitivity to imidacloprid (a novel neonicotinoid). This indicated that the latter was less affected by the resistance mechanism(s) present.

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification.

Time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic drug-metabolizing enzyme activity in rats.

The time-dependent effects of Klebsiella pneumoniae endotoxin on hepatic cytochrome P450-dependent drug-metabolizing capacity (cytochrome P450 and b5 content, activity of aminopyrine N-demethylase, p-nitroanisole O-demethylase, aniline hydroxylase and benzphetamine N-demethylase) and on the pharmacokinetics of antipyrine have been determined in rats. Measurement of enzyme activity and antipyrine (after intravenous injection of 20 mg kg(-1)) were performed 2, 24 and 96 h after a single intraperitoneal injection of endotoxin (1 mg kg(-1)) and after repeated doses (once daily for 4 days). The contribution of tumour necrosis factor alpha (TNFalpha) to the endotoxin-induced changes was also examined in rats pretreated with granulocyte colony-stimulating factor (G-CSF). The systemic clearance of antipyrine and the activity of hepatic cytochrome P450-dependent drug-metabolizing enzymes were dramatically reduced 24 h after a single injection of endotoxin, but had returned to control levels by 96h. The magnitudes of these decreases in these measurements after repeated doses of endotoxin were similar to those seen 24h after the single dose. The systemic clearance of antipyrine correlated significantly with cytochrome P450 content and aminopyrine N-demethylase activity. In histopathological experiments, moderate hypertrophy of Kupffer cells was observed, with no evidence of severe liver-tissue damage. G-CSF pretreatment suppressed the increased plasma concentrations of TNFalpha produced 2 h after single endotoxin injection, but did not eliminate the endotoxin-induced decrease in the systemic clearance of antipyrine, suggesting that TNFalpha is not the sole component responsible for the reduction of cytochrome P450-mediated drug-metabolizing enzyme activity. These results provide evidence that a single intraperitoneal injection of 1.0 mgkg(-1)K. pneumoniae endotoxin in rats reduces hepatic P450 and b5 levels, and reduces the activity of various cytochrome P450-mediated drug-metabolizing enzymes without causing severe liver-tissue damage. This suggests that the effect of endotoxin on hepatic cytochrome P450-mediated drug-metabolizing isozymes is non-selective.

Resistance selection and biochemical characterization of spinosad resistance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

A Helicoverpa armigera population was collected from Shandong province, China. After 15 generations of selection in the laboratory, the H. armigera strain developed more than 20-fold resistance to spinosad. At LD 50 level, no significant cross-resistance was found between spinosad and chlorpyrifos, methomyl, avermectin and chlorfenapyr except for fenvalerate with a low cross-resistance of 2.4-fold. However, LD 99 values of fenvalerate against the parental and resistant strains were not different significantly. After inhibitors were used, spinosad resistance could be partially suppressed by piperonylbutoxide (PBO) and triphenylphosphate (TPP), but not by diethylmaleate (DEM). Activities of p-nitroanisole O-demethylase (ODM) developed to 8.26-fold compared with the parental strain, but no obvious changes were found in activities of carboxyl esterase (CarE) and glutathione -S-transferase (GST). The results indicated that resistance to spinosad in the cotton bollworm might be associated with an increase in cytochrome P450 monooxygenase.

Effects of spinosad on Helicoverpa armigera (Lepidoptera: Noctuidae) from China: tolerance status, synergism and enzymatic responses

Spinosad is increasingly used in pest management programmes, and resistance to it has been detected in recent years. However, there is no report on the susceptibilities of field populations of Helicoverpa armigera (Hübner) from China. Furthermore, the impact of spinosad on metabolic enzymes in this pest remains unknown. Four populations of H. armigera from different locations in China displayed less than 6.5-fold difference in LC(50) to spinosad, the highest being in the Xinjiang population, followed by Xiajin, Taian and Hubei populations, while there was no significant difference at LC(99) level among the four populations. The toxicity of spinosad could be synergised by piperonyl butoxide (PBO) and triphenylphosphate (TPP), but not by diethyl maleate (DEM). Spinosad exposure for 48 h significantly increased the activities of p-nitroanisole O-demethylase (ODM), while no significant changes in glutathione-S-transferase (GST) and carboxyl esterase (CarE) were observed. Field populations of H. armigera from China displayed marginally different susceptibilities to spinosad and had a relatively low LC(50). Cytochrome P450 monooxygenase might be involved in the metabolism of, and hence resistance to, spinosad in this pest in China.

Comparative effects of dimethylsulfoxide on metabolism and toxicity of carbon tetrachloride and dichloromethane

The effects of dimethylsulfoxide (DMSO) on the metabolism and toxicity of chlorinated methanes were examined. Male mice were treated with DMSO (1, 2.5 or 5 ml kg(-1), i.p.) prior to challenge with dichloromethane (CH(2)Cl(2)) or carbon tetrachloride (CCl(4)). Blood carboxyhemoglobin elevation resulting from metabolic conversion of CH(2)Cl(2) to carbon monoxide was inhibited dose-dependently by DMSO pretreatment. The elevation of serum aspartate aminotransferase, alanine aminotransferase and sorbitol dehydrogenase activities induced by CCl(4) (0.1 mmol kg(-1)) was not changed in mice pretreated with DMSO at 1 ml kg(-1), but depressed significantly at a greater dose of DMSO. However, DMSO failed to alter the hepatotoxicity of CCl(4) injected at a dose of 0.2 mmol kg(-1). DMSO induced the microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities as early as 2 h following the treatment. Microsomal disposition of CH(2)Cl(2) and CCl(4) was measured using a vial equilibration technique. The disappearance of CH(2)Cl(2) was inhibited competitively by addition of DMSO. But DMSO did not affect the metabolic degradation of CCl(4). The results indicate that DMSO has multiple effects on metabolism and toxicity of xenobiotics. DMSO induces the hepatic metabolizing activity mediated by CYP2E1, but the presence of this solvent in the enzyme site may inhibit directly the enzymatic interaction with a substrate. The toxicological significance of DMSO-induced effects on such an interaction may be variable depending on the properties of each substrate. The invulnerability of CCl(4) metabolism to the effects of DMSO appears to be related to its high affinity for the lipophilic CYP enzyme site.

Contrasting Changes in Phase I and Phase II Metabolism of Acetaminophen in Male Mice Pretreated with Carbon Tetrachloride

Effect of carbon tetrachloride (CCl(4)) pretreatment on the biotransformation and elimination of acetaminophen were examined in male mice. A 24 hr initial dose of CCl(4) (0.05 ml/kg, intraperitioneally) reduced the induction of hepatotoxicity resulting from acetaminophen treatment (350 mg/kg, intraperitoneally) as determined by changes in serum alanine and aspartate aminotransferase, and sorbitol dehydrogenase activities. Acetaminophen and the major metabolites in plasma were monitored for 12 hr following acetaminophen treatment. CCl(4) pretreatment decreased the plasma concentrations of acetaminophen-cysteine and acetaminophen-mercapturate, but acetaminophen-glucuronide and acetaminophen-sulfate were increased significantly. The elimination of the parent drug from plasma was not affected by CCl(4). In urine collected for 24 hr, the concentrations of acetaminophen-sulfate and acetaminophen-glucuronide were increased by 84% and 33%, respectively, whilst acetaminophen-cysteine and acetaminophen-mercapturate were reduced to approximately one third of control. Expression of cytochrome P450 (CYP) isozymes was determined using antibodies of 2E1 and 1A2 as probes. CYP2E1 and 1A2 expressions were decreased significantly by CCl(4). Likewise, CCl(4) treatment reduced the microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities to less than one third of control. The results indicate that, although CCl(4) reduces the generation of thioether conjugates of acetaminophen by decreasing the CYP activities, inhibition of the oxidative metabolism of acetaminophen is counterbalanced by the enhancement of conjugate formation via the glucuronide and sulfate pathways, resulting in elimination of the drug at a rate equivalent to that in normal mice. It is suggested that liver injury in patients may not warrant a mandatory reduction of drug doses extensively inactivated via phase II reactions.

Induction of the cytochrome P450 activity by plant allelochemicals in the cotton bollworm, Helicoverpa armigera (Hübner)

The p-nitroanisole O-demethylase activity of P450s and expression of CYP6B6 mRNA induced by two plant secondary substances, 2-tridecanone and quercetin, were investigated in cotton bollworm, Helicoverpa armigera (Hübner). The O-demethylase activity was higher in the fat body than that in the midgut of the sixth-instar larvae fed on the artificial diets mixed with these allelochemicals. The similar results were obtained when induced by the combination of 2-tridecanone and quercetin at different concentrations. When the concentration of inducers, mixture of 2-tridecanone and quercetin, was 0.5 mg/ml in the diet, the maximum O-demethylase activities of P450 in the midgut and fat body were 2.6 and 3.88 times higher than those in the relevant control, respectively. Specific primers were designed according to the CYP6B6 and an actin gene of cotton bollworm and used to determine the expression level of CYP6B6 mRNA in the midgut and fat body by a reverse transcription-polymerase chain reaction (RT-PCR) method. The expression levels of CYP6B6 mRNA were obviously correlated with the concentrations of 2-tridecanone, but no correlation with the concentrations of quercetin. When induced with the mixture of 2-tridecanone and quercetin at a concentration of 0.5 mg/ml in the diet, the maximum expression amount of CYP6B6 mRNA in the midgut and fat body was induced, which was 1.74 and 1.8 times compared with the corresponding control, respectively. It suggests that the cytochrome P450 has an important role in the metabolism of plant secondary substances in cotton bollworm.

Influence of chloramphenicol on rat hepatic microsomal components and biomarkers of oxidative stress: protective role of antioxidants.

The effects of chloramphenicol and antioxidant vitamins on in vivo and in vitro indices of microsomal drug oxidizing system were examined in rats. Chloramphenicol at doses of 28 mg/kg, 57 mg/kg and 86 mg/kg body weight administered for 10 consecutive days resulted in a dose-dependent decrease in body weight, liver weight, relative liver weight and protein content compared to control. Chloramphenicol treatment also resulted in a dose-dependent decrease in microsomal phospholipid and a significant increase in cholesterol content causing an increase in cholesterol/phospholipid molar ratio. The drug produced a significant reduction in the activity of aniline hydroxylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and ethoxyresorufin O- deethylase. Activity of ethoxyresorufin O-deethylase was little affected by the drug. Chloramphenicol ranging from 10-4-10-6 M similarly produced a concentration dependent inhibition in the activities of the enzymes. Kinetic studies revealed that chloramphenicol inhibited the enzymes non-competitively. alpha-Tocopherol, beta-carotene and ascorbic acid decreased the chloramphenicol inhibition of the enzymes within the range of 70 to 81%, 45-63% and 55 to 75% respectively. Also, the antioxidant vitamins attenuated the chloramphenicol-induced formation of malondialdehyde by 60%, 53% and 56% and lipid hydroperoxide by 60%, 54%, and 54% respectively The results indicate that the effect of chloramphenicol on cytochrome P450 drug oxidizing enzyme components is related to the action of the drug presumably, via free radical mechanism and that co-administration with antioxidant vitamins can attenuate its toxic action.

Effect of three structurally related antimalarial drugs on liver microsomal components and lipid peroxidation in rats.

Changes in microsomal drug oxidizing enzymes, microsomal lipids, hepatic glutathione (GSH), glutathione S-trans-ferase (GST) and malondialdehyde (MDA) formation following administration of rats with therapeutic doses of three structurally related antimalarial drugs, amodiaquine (AQ), mefloquine (MQ) and halofantrine (HF) were investigated. There was a significant decrease in the activities of aniline hydroxylase, p-nitroanisole O-demethylase and pentoxyresorufin O-dealkylase in AQ, MQ and HF treated rats. AQ elicited the greatest effect with 50, 37 and 67% reductions in the activities of aniline hydroxylase, p-nitroanisole O-demethylase and pentoxyresorufin O-dealkylase, respectively. All the drugs prolonged hexobarbital-sleeping time to varying extents. The three drugs increased significantly the cholesterol per phospholipid ratio. AQ, MQ and HF decreased significantly the GSH level, GST activity and increased the formation of MDA. The results indicate that the alterations in hepatic microsomal components and lipid peroxidation caused by the antimalarials are related to the structural differences in the compounds.

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.

Auto-induction of E5110 metabolism, a novel non-steroidal anti-inflammatory drug, during toxicokinetic studies in beagle dogs.

Following single-dose intravenous and oral studies to determine the absolute bioavailability of E5110 in beagle dogs, repeated dose pharmacokinetic studies were conducted as toxicokinetics in a subacute toxicity test. E5110 was administered orally once a day for 91 days at doses of 0, 1, 10, 50 and 250 mg/kg/day to dogs. On the first day during repeated administration, the Cmax and AUC values of E5110 in females were lower than those for males, and it seems that this may be due to a sex-related difference in the activity of cytochrome P450 (CYP) 3A. During repeated administration of E5110, the plasma levels of E5110 on day 15 and day 91 were markedly lower than those on the first day. On day 91, the AUCs for E5110 were 55.9%, 38.8%, 10.9% and 7.8% in males, and 76.2%, 80.3%, 10.5% and 11.9% in females on the first day, for doses of 1, 10, 50 and 250 mg/kg/day, respectively. Liver microsomes prepared after the last dose of E5110 showed increased activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase, and aminopyrine N-demethylase, at doses of 1 mg/kg/day or more. A dose-dependent increase in P450 content was also observed. Furthermore, the capacity for 5-hydroxylate E5110 was increased, and Western blot analysis indicated an induction of CYP2B and 3A; therefore CYP3A may contribute to a main metabolic pathway of E5110. These results suggested that the decrease in plasma concentrations of E5110 that were observed during repeated administration represents a typical case of auto-induction of the phenobarbital type.

Hepatic drug metabolizing enzyme induction and serum triacylglycerol elevation in rats treated with chlordiazepoxide, griseofulvin, rifampicin and phenytoin

Five days intraperitoneal administration of rats with chlordiazepoxide (0.4 mg/kg), griseofulvin (7 mg/kg), rifampicin (8.6 mg/kg), phenytoin (4.3 mg/kg) and phenobarbitone (1.4 mg/kg; an established inducer of microsomal enzymes) caused a significant increase in serum triacylglycerol ( P<0.001) and the activities of aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase ( P<0.001). Aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase activities were increased 1.48-, 1.15- and 1.47-fold, respectively, in chlordiazepoxide-treated rats, 1.65-, 1.20- and 1.38-fold in griseofulvin-treated rats, 1.74-, 1.36- and 1.44-fold in rifampicin-treated rats, 1.56-, 1.29- and 1.62-fold in phenytoin-treated rats and 2.26-, 1.72- and 1.93-fold in phenobarbitone-treated rats. Chlordiazepoxide, griseofulvin, rifampicin, phenytoin and phenobarbitone increased the activity of cytosolic phosphatidate phosphohydrolase by 52, 58, 67, 73 and 82%, respectively, while the drugs elicited 50, 60, 60, 73 and 87% increases in the activity of the microsomal phosphatidate phosphohydrolase. Similarly, chlordiazepoxide, griseofulvin, rifampicin, phenytoin and phenobarbitone elicited 2.4-, 2.39-, 2.34-, 1.69- and 3.75-fold increases in serum triacylglycerol concentrations. The correlations between serum triacylglycerol concentrations and the activities of aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase were significant in all treatment groups ( r=0.83, r=0.92 and r=0.87, respectively, n=30, P<0.001). Our results suggest that induction of hepatic enzymes by the administered drugs may lead to hypertriglyceridaemia as an adverse effect, possibly by inducing the activity of regulatory enzymes in the biosynthesis of triglyceride.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg −1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg −1 day −1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one’s appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

P3B65 - Increase in renal metabolizing enzyme activities induced by heapatic damage

1. The drug-metabolizing activities (aminopyrine N-demethylase and p-nitroanisole O-demethylase activities) have been measured at monthly intervals throughout the year in liver microsomes of male and female Japanese bullfrogs, Rana catesbeiana.2. The aminopyrine n-demethylase activity based on cytochrome P-450 of both sexes was significantly higher in May–July (spring-summer) than in August–October (summer-autumn).3. On the contrary, the p-nitroanisole O-demethylase activity based on cytochrome P-450 of males was significantly higher in July–October (summer-autumn) than in May–June (spring). However, there was no seasonal changes in the O- demethylase activity in females.4. There were significant differences between males and females in the n-demethylase activity in August–October and in the O -demethylase activity in May–June (or July–October).

Early hepatic changes induced in rats by two hepatocarcinogenic organohalogen pesticides: bromopropylate and DDT.

The aim of the present studies was to describe the effect of two organohalogen pesticides: DDT and bromopropylate, on early changes in rat liver, proposed in the literature to be useful endpoints in screening of non-genotoxic hepatocarcinogens and/or liver tumor promoters. We investigated the effects on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and cytochrome CYP2B1-dependent monooxygenase induction. The histological and cytochemical changes in the liver were also recorded. Male Wistar rats received bromopropylate in one, three or five daily oral doses of 125, 250, and 500 mg/kg body wt. day-1. DDT was applied as one, three, and five daily oral doses of 24 mg/kg body wt. day-1 (this dose is close to the mean hepatocarcinogenic dose in male Wistar rats: 34.1 mg/kg body wt. day-1). In the case of both pesticides the early effects observed consisted of hepatomegaly accompanied by an increase in the p-nitroanisole O-demethylase activity and hepatocyte proliferation. Hepatocyte proliferation was elevated during the total experimental period. Vacuolated cytoplasm and evident focal necrosis may suggest that the maximal increase in hepatocyte proliferation, preceding hepatomegaly, is at least partly related to a regenerative liver response to pesticides. In addition to the above-mentioned early changes, the present findings provide new evidence for the occurrence of dose-dependent abnormal mitoses (and c-mitoses) in the hepatocytes of the bromopropylate and DDT treated rats.

Postmortem changes in drug-metabolizing enzymes of rat liver microsome

Postmortem changes in the drug-metabolizing enzymes in rat liver microsome were studied. Parameters investigated were: microsomal protein, NADPH-cytochrome P-450 reductase activity, NADH-cytochrome b 5 reductase activity, cytochrome b 5 content, cytochrome P-450 content, aminopyrine N-demethylase activity, aniline p-hydroxylase activity, p-nitroanisole O-demethylase activity, uridine diphosphate (UDP)-glucuronyl transferase activity and glutathione S-transferase activity. Nearly all the parameters based on microsomal protein decreased during autolysis and the time-dependent decrement ratios of the parameters changed by various amounts. Cytochrome b 5 content decreased more rapidly than that of other components. By 36 h post mortem, levels of cytochrome b 5 were not detectable. By 48 h post mortem, NADPH-cytochrome P-450 reductase activity decreased to 91%, NADH-cytochrome b 5 reductase activity decreased to 94%, and cytochrome P-450 content decreased to 92% of relative activities. By 48 h post mortem, aminopyrine N-demethylase activity decreased to 87%, aniline p-hydroxylase activity decreased to 98% and p-nitroanisole O-demethylase activity decreased to 75% of relative activities. The activity of p-nitroanisole O-demethylase appeared to be more stable than that of aminopyrine N-demethylase or aniline p-hydroxylase. These results demonstrate that there are multiple forms of isozymes of the cytochrome P-450-linked monooxygenase system. Hepatic transferases showed decrease patterns different to those of monooxygenases, so UDP-glucuronyl transferase activity of ∼32% of relative activity was detected at 48 h post mortem. Thus, UDP-glucuronyl transferase activity appeared to be more stable than the cytochrome P-450-linked monooxygenases. These results show that these activities and components would be useful as markers of postmortem time. The causes of the variety of instability of these enzyme systems are discussed.

Effect of E-5110, a novel non-steroidal anti-inflammatory drug, on trimethadione metabolism as an indicator of hepatic drug-oxidizing capacity in beagle dog.

1. We examined the effects of N-methoxy-3-(3,5-di-tert-butyl-4- hydroxybenzylidene pyrrolidin-2-one (E-5110), a novel non-steroidal anti-inflammatory drug, on the pharmacokinetics of trimethadione (TMO) and characterized the P450 isozymes involved in the metabolism of TMO in beagle dog. 2. In the E-5110-treated dog (50 mg/kg/day for 7 days: oral) the plasma half-life (t1/2) and the area under the curve (AUC) of TMO (4 mg/kg, i.v.) in vivo were decreased, and total body clearance (CL) was increased; the apparent volume of distribution (Vd) was relatively unchanged. 3. Contents of P450 and b5, and the activity of p-nitroanisole O-demethylase and benzphetamine N-demethylase in vitro were significantly increased compared with controls by repeated E-5110 treatment in dog. 4. Contents of CYP2B and 3A were increased by E-5110 pretreatment in dog. 5. TMO N-demethylation was inhibited by the anti-CYP2B and 3A IgG fractions in liver microsomes obtained from the E-5110-treated dog. 6. Results of both the in vivo and in vitro studies of the effects of E-5110 treatment in dog on TMO indicate that these effects may be attributed to the induction of CYP2B and 3A.

Dual effects of a novel thienodiazepine platelet-activating factor antagonist, on drug-oxidizing enzymes in beagle dog.

1. We have examined the effects of (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8, 11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3, 2-f][1, 2, 4]triazolo[4, 3-a][1, 4]diazepine (E-6123), a novel thienodiazepine platelet-activating factor antagonist, on drug-oxidizing capacity in beagle dog, using antipyrine (AP) and trimethadione (TMO) as two model substrates. 2. The plasma half-life (t1/2) and area under the curve (AUC) of AP (0.5 mg/kg, i.v. injection) increased in a dose-dependent manner after a single oral dose of E-6123 (0.2, 1 or 10 mg/kg), whereas the total body clearance (Cl) of AP was decreased, and the apparent volume of distribution (Vd) was unchanged. 3. The pharmacokinetic parameters (t1/2, Cl and AUC) of the metabolism of TMO (4 mg/kg, i.v.) after repeated oral administration of E-6123 (10 mg/kg for 7 days) were not significantly changed in comparison with findings in control dog. The ratio of dimethadione (DMO), being the only TMO metabolite, to TMO in plasma after i.v. administration of TMO in E-6123-treated dog was increased only 5 and 15 min after the final dose, but was not changed at other sampling times (0.5, 1, 2 4, 6, 8 and 12 h). 4. The content of b5, the activity of p-nitroanisole O-demethylase and benzphetamine N-demethylase were significantly increased, compared with controls, by repeated E-6123 treatment. However, aniline hydroxylase activity was not significantly changed. 5. Content of P450 2B was significantly increased in E-6123 treated dog, while that of 3A was not.(ABSTRACT TRUNCATED AT 250 WORDS)