Cytochrome P450 Monooxygenase Activity Research Articles

Pyriproxyfen Resistance of <I>Bemisia tabaci</I> (Homoptera: Aleyrodidae) Biotype B: Metabolic Mechanism

Juvenile hormone (JH) analog insecticides are relatively nontoxic to vertebrates and provide efficient control of key arthropod pests. One JH analog, pyriproxyfen, has provided over a decade of exceptional management of whiteflies in cotton of the southwestern United States. Thwarting resistance to pyriproxyfen in Bemisia tabaci (Gannadius) (a.k.a. Bemisia argentifolii Bellows and Perring) has been the focus of an integrated resistance management program because this insecticide was first registered for use in Arizona cotton in 1996. Resistance levels have increased slowly in field populations in recent years but have not demonstrably affected field performance of pyriproxyfen. Resistant strains have been isolated and studied in the laboratory to determine the mechanism of resistance and identify optimal strategies for controlling resistant whiteflies. Synergism bioassays showed that resistance in a laboratory-selected strain QC02-R, was partially suppressible with piperonyl butoxid (PBO) and diethyl maleate (DEM) but not with S, S, S-tributyl phosphorotrithioate (DEF). Consistent with the synergism bioassay results, enzymatic assays revealed that the enzyme activities of cytochrome P450 monooxygenases (P450) and glutathione S-transferases (GST) but not esterases were significantly higher in the pyriproxyfen-resistant QC02-R strain than in the susceptible strain. These results indicate that both P450 and GST are involved in whitefly resistance to pyriproxyfen.

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae)

Anopheles funestus, one of the main African malaria vectors, caused a major malaria outbreak in South Africa during 1999/2000, even though South Africa had an effective vector control program in place. The outbreak was due to pyrethroid resistant An. funestus invading KwaZulu/Natal. Increased activity of cytochrome P450 (monooxygenase) was responsible for the pyrethroid resistance in this species. A monooxygenase gene, CYP6P9, was highly overexpressed in the pyrethroid-resistant strain compared with a susceptible strain. Characterization of this gene as well as the redox partners involved in the catalytic cycle of P450s was investigated. The full length of the CYP6P9 sequence was isolated, sequenced and compared between the pyrethroid-resistant and -susceptible strains. Sequence identity between the two strains was 99.3%; the sequence differences were mainly outside of the conserved regions. The functional significance is still unknown, but it is feasible that these variations are associated with differences in insecticide metabolism. A second CYP6 gene (CYP6P13) was also isolated; it shared close similarities with CYP6P9. The putative redox partners, cytochrome b(5) (cyt b(5)) and NADPH-cytochrome P450 reductase (CPR), were isolated from An. funestus (resistant strain) and showed high levels of sequence identity to other insect cyt b(5) and CPRs. Isolation of the coding sequences CYP6P9 and its cognate redox partners enables expression of functional recombinant protein for biochemical and structural analysis.

Cross‐resistance study and biochemical mechanisms of thiamethoxam resistance in B‐biotype Bemisia tabaci (Hemiptera: Aleyrodidae)

B-biotype Bemisia tabaci (Gennadius) has invaded China over the past two decades. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in B. tabaci, a resistant strain was selected in the laboratory. Cross-resistance and the biochemical mechanisms of thiamethoxam resistance were investigated in the present study. A 66.3-fold thiamethoxam-resistant B. tabaci strain (TH-R) was established after selection for 36 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed obvious cross-resistance to imidacloprid (47.3-fold), acetamiprid (35.8-fold), nitenpyram (9.99-fold), abamectin (5.33-fold) and carbosulfan (4.43-fold). No cross-resistance to fipronil, chlorpyrifos or deltamethrin was seen. Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) exhibited significant synergism on thiamethoxam effects in the TH-R strain (3.14- and 2.37-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that cytochrome P450 monooxygenase activities increased 1.21- and 1.68-fold respectively, and carboxylesterase activity increased 2.96-fold in the TH-R strain. However, no difference was observed for glutathione S-transferase between the two strains. B-biotype B. tabaci develops resistance to thiamethoxam. Cytochrome P450 monooxygenase and carboxylesterase appear to be responsible for the resistance. Reasonable resistance management that avoids the use of cross-resistance insecticides may delay the development of resistance to thiamethoxam in this species.

Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, Australia

AbstractFollowing the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two‐ to sixfold when compared with the susceptible strain. Up to a 1.9‐fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S‐transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non‐metabolic mechanisms that were not investigated in the present study.

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies).

BackgroundThe yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood.ResultsThe present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR.ConclusionThese results suggest that the high level of insecticide resistance found in Ae. aegypti mosquitoes from Martinique island is the consequence of both target-site and metabolic based resistance mechanisms. Insecticide resistance levels and associated mechanisms are discussed in relation with the environmental context of Martinique Island. These finding have important implications for dengue vector control in Martinique and emphasizes the need to develop new tools and strategies for maintaining an effective control of Aedes mosquito populations worldwide.

Toxicogenomics Applied to Cultures of Human Hepatocytes Enabled an Identification of Novel Petasites hybridus Extracts for the Treatment of Migraine with Improved Hepatobiliary Safety

Butterbur extracts (Petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive of rare hepatobiliary toxicity. To evaluate its hepatotoxic potential, a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic C(max) levels of 60 ng/ml. Nonetheless, in a 28-day toxicity study at approximately 200-fold of therapeutic doses, induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-month chronic toxicity study, the initial hepatobiliary effects were reproduced, but at the end of the study, liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at > 170-fold of therapeutic C(max) levels, including cytotoxicity (lactate dehydrogenase, MTT, and ATP), transaminase activities (alanine aminotransferase and aspartate aminotransferase), albumin synthesis, urea and testosterone metabolism to assay for cytochrome P450 monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high and well above therapeutic doses, liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport and lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore reemphasizing the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene ( Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC 50) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance ( r 2 = 0.712) compared to the F439W mutation ( r 2 = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced ( r 2 = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.

Characterisation of imidacloprid resistance mechanisms in the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae)

Effective control of the brown planthopper, Nilaparvata lugens Stål, across rice-growing regions of Asia has been seriously compromised over the last 2 years by the appearance of widespread resistance to the neonicotinoid insecticide, imidacloprid. Sequence analysis of the ligand-binding domain of the nicotinic acetylcholine receptor α1 subunit from two field-collected resistant strains (CHN-2 and IND-11) did not reveal the Y151S point mutation previously implicated in conferring target-site resistance in this species. This result was supported by ligand-binding studies with [ 3H]-imidacloprid that showed no significant change in insecticide binding to isolated membranes from susceptible and resistant strains. In contrast, there was an approximate 5-fold increase in the mixed function oxidase activity for the two resistant strains suggesting that imidacloprid metabolism by increased cytochrome P450 monooxygenase activity is the major mechanism of resistance in these strains.

In vitro metabolism of polychlorinated biphenyls and cytochrome P450 monooxygenase activities in dietary-exposed Greenland sledge dogs

The in vitro metabolism of a polychlorinated biphenyl (PCB) mixture was examined using hepatic microsomes of dietary-exposed Greenland sledge dogs ( Canis familiaris) to an organohalogen-rich diet (Greenland minke whale blubber: EXP cohort) or a control diet (pork fat: CON cohort). The associations between in vitro PCB metabolism, activity of oxidative hepatic microsomal cytochrome P450 (CYP) isoenzymes and concentrations of PCBs and hydroxylated metabolites were investigated. The CON dogs exhibited a 2.3-fold higher depletion percentage for the PCB congeners having at least two pairs of vicinal meta-para Cl-unsubstituted carbons (PCB-18 and -33) relative to the EXP dogs. This depletion discrepancy suggests that there exist substrates in liver of the organohalogen-contaminated EXP dogs that can competitively bind and/or interfere with the active sites of CYP isoenzymes, leading to a lower metabolic efficiency for these PCBs. Testosterone (T) hydroxylase activity, determined via the formation of 6β-OH-T, 16α-OH-T, 16β-OH-T and androstenedione, was strongly correlated with the depletion percentages of PCB-18 and -33 in both cohorts. Based on documented hepatic microsomal CYP isoenzyme substrate specificities in canines, present associations suggest that primarily CYP2B/2C and CYP3A were inducible in sledge dogs and responsible for the in vitro metabolism of PCB-18 and -33.

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

BackgroundPermethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.Methodology/Principal FindingsTo determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.ConclusionsThese findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

Effects of Andrographis paniculata extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities after in vivo administration to rats and in vitro in rat and human hepatocyte cultures

The expression of cytochrome P450 (CYP) is regulated by both endogenous factors and foreign compounds including drugs and natural compounds such as herbs. When herbs are co-administrated with a given drug in modern medicine it can lead to drug–herb interaction that can be clinically significant. The ability of Andrographis paniculata extract (APE) and Andrographolide (AND), the most medicinally active phytochemical in the extract, to modulate hepatic CYP expression was examined in vivo in rats and in vitro in rat and human hepatocyte cultures. After in vivo administration, APE at dose levels of 0.5 g/kg/day (i.e. 5 mg/kg/day AND equivalents) and at 2.5 g/kg/day (i.e. 25 mg/kg/day AND equivalents) and AND at dose levels of 5 and 25 mg/kg/day significantly decreased CYP2C11 activity. In primary cultures of rat and human hepatocytes, treatment with AND 50 μM and APE-containing 50 μM AND also resulted in significant decreases in CYP2C expression and activity. In addition, in human hepatocytes, treatment with APE and AND 50 μM resulted in a decrease in CYP3A expression and activity. In conclusion, this study suggests that AND and APE could cause herb–drug interactions in humans through modulation of CYP2C9 and CYP3A4 expression and activities.

Effects of Andrographis paniculata crude extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities

Effects of Andrographis paniculata crude extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities

Effects of the Anticonvulsant, Valproate, on the Expression of Components of the Cytochrome-P-450-Mediated Monooxygenase System and Glutathione S-Transferases

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.

The influence of diet composition on phase I and II biotransformation enzyme induction

The expression of phase I and II biotransformation enzymes was examined with respect to experimental diet composition and with the addition of the bi-functional inducer flavone. Enzymatic activity and mRNA levels of cytochrome P450 monooxygenase (CYP) isoforms (CYP1A1, CYP1A2, CYP2B1/2) and glutathione-S-transferase (GST) isoforms (GSTA, GSTM, and GSTP) were used as indices for the changes in expression. An amino acid based (AA) diet and a semi-purified egg white (EW) diet were designed to include similar levels of nutrients and were compared to a standard laboratory chow (SC) diet. Rats (Sprague-Dawley) and mice (C57BL/6) were used as animal models. Animals were fed one of the three diets for 7 days prior to incorporation of flavone (2%, wt/wt). Diets with or without flavone were next fed for an additional 3 days. Enzymatic activities of the CYPs in mice and GSTs in both mice and rats were determined. In mice, the relative mRNA levels for each of the CYP and GST isoforms were also measured. The increase in phase I and II enzyme expression observed in response to flavone was most dynamic when the AA-based diet was used (often >20-fold for given isoform enzymatic activities and >200-fold for specific mRNAs), followed by the EW diet (10 to 20-fold and 100 to 200-fold, respectively). The SC diet resulted in a higher level of background expression of CYP and GST isoforms and as a consequence the observed fold increases in CYP and GST isoforms (enzymatic and mRNA levels) were substantially less (1 to 10-fold and 1 to 150-fold. respectively), when the SC diet fed group with or without flavone was compared.

Biochemical mechanisms conferring cross-resistance between tebufenozide and abamectin in Plutella xylostella

Experiments have been carried out to confirm the cross-resistance between abamectin and tebufenozide in Plutella xylostella and demonstrate its mechanism. The results showed that the resistant strain of P. xylostella selected by tebufenozide (RF 99.38) really showed high cross-resistance to abamectin (RF 29.25). When this strain was subjected to resistance decaying treatment, breeding without contacting any insecticides, and abamectin resistance selection for 20 generations, the former resulted in decrease of its resistance to both tebufenozide and abamectin to about one third of the original (RF 35.03 and 11.67, respectively), and the later enhanced its resistance to abamectin dramatically (RF 303.77), but not to tebufenozide(RF 50.04). PBO showed high synergism to abamectin (SR 2.11–12.23), and the synergism ratio positively related to the resistance level among different strains. Enzyme analysis also proved that the activity of cytochrome P450 monooxygenase (MFO) was notable enhanced in the strains resistant to both tebufenozide and abamectin (1.71- to 3.01-fold). Based on discussion, it was concluded that tebufenozide selection could resulted in significant cross-resistance of P. xylostella to abamectin. The major mechanism for the cross-resistance should be the enhancement of MFO activity. For resistance management, tebufenozide and abamectin would not recommend for rotational use.

Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity

Detoxification by cytochrome P450 monooxygenases is an important mechanism involved in pesticide resistance in insects and mites. The activity of these enzymes can be induced by a variety of chemicals. The aim of this study was to evaluate the effect of six P450 inducers (phenobarbital, barbital, 3-methylcholanthrene, geraniol, isosafrole, pentamethylbenzene), known to have an inducing activity in insects and mammals, on the O-deethylation activity in the two-spotted spider mite Tetranychus urticae. Treatment with barbital, phenobarbital and geraniol resulted in a dose-dependent increase in activity. Neither 3-methylcholanthrene, isosafrole nor pentamethylbenzene were effective inducers. Time course studies showed that induction by geraniol and barbital started rapidly within a period of 1–4 h after initiation of the treatment, while maximal activity was reached within 4 and 48 h, respectively. In addition, it was shown that induction with xenobiotic compounds can alter the monooxygenase-mediated acaricide tolerance in a susceptible strain of T. urticae. Although barbital induced higher levels of P450 activity, geraniol proved to be a better compound to decrease toxicity of the tested acaricides.

Insecticide resistance in field populations of Laodelphax striatellus Fallén (Homoptera: Delphacidae) in China and its possible mechanisms

Small brown planthoppers, Laodelphax striatellus Fallén, were collected from six provinces in China, and their resistance to common insecticides surveyed. The strains collected from Jiangsu province were found to be the most resistant, and showed high resistance to imidacloprid and moderate resistance to chlorpyrifos, acephate and deltamethrin (resistance factors, RF ranged 66 – 108, 31 – 50, 17 – 24 and 12 – 21, respectively). Another two strains collected from Fujian and Guangdong provinces also displayed moderate or low level resistance to imidacloprid, chlorpyrifos and acephate (RF ranged 27 – 29, 10 – 12, and 9 – 13, respectively). However, the strains collected from Shandong and Yunnan provinces showed little resistance to any of the insecticides tested (RF < 6). The sensitivity of the strain collected from Hebei province (the most sensitive one) was similar to that of the susceptible strains previously reported. In all the strains tested, no obvious resistance to fipronil and carbosulfan was found, although the sensitivity of Jiangsu strains decreased by 5 – 8-fold. Biochemical analysis proved that the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase, as well as the AChE insensitivity, could all contribute to multiple resistance in this pest.

Mechanistic aspects of 4-amino-2,6-dichlorophenol-induced in vitro nephrotoxicity

4-Amino-2,6-dichlorophenol (ADCP) is a potent acute nephrotoxicant in vivo inducing prominent renal corticomedullary necrosis. In vitro, ADCP exposure increases lactate dehydrogenase (LDH) release from rat renal cortical slices at 0.05 mM or greater. The purpose of this study was to examine the ability of antioxidants, cytochrome P450 (CYP) and flavin adenine dinucleotide monooxygenase (FMO) activity modulators, indomethacin, glutathione and inhibitors of glutathione conjugate metabolism to attenuate ADCP cytotoxicity in vitro. Renal cortical slices prepared from untreated male Fischer 344 rats ( N = 4/group) were preincubated at 37 °C under a 100% oxygen atmosphere with an inhibitor or vehicle for 5–30 min. ADCP (0.05–0.5 mM) or vehicle was added and incubations continued for 120 min. At the end of the incubation period, LDH release was measured as an index of nephrotoxicity. ADCP cytotoxicity was partially attenuated by ascorbate (1.0 or 2.0 mM), but not by N, N′-diphenyl- p-phenylenediamine (DPPD), α-tocopherol or deferoxamine. Inhibitors of CYP (metyrapone, piperonyl butoxide and isoniazid) and FMO activity modulators (methimazole, N-octylamine) had no effect on ADCP cytotoxicity. Indomethacin or glutathione 1.0 mM completely and partially blocked ADCP 0.1 and 0.5 mM cytotoxicity, respectively. N-acetylcysteine, AOAA (an inhibitor of cysteine conjugate β-lyase) and probenecid (an organic anion transport inhibitor), but not AT-125 (an inhibitor of γ-glutamyl transferase), partially attenuated ADCP 0.1 mM cytotoxicity. Overall, these results suggest that reactive metabolites may be produced from ADCP primarily via a co-oxidation-mediated mechanism. The difference in the ability of ascorbate and glutathione to attenuate ADCP-induced cytotoxicity in vitro in kidney cells could indicate that alkylation via the reactive benzoquinoneimine metabolite might be responsible for cytotoxicity rather than a free radical-mediated mechanism.

Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD 50) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin- O-deethylase (EROD), 7-pentoxyresorufin- O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA- d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) fromCandida bombicola

Candida bombicola is a yeast with at least two appealing features. The species can grow on alkanes when provided as the sole carbon source, and it produces glycolipids, which have several industrial, cosmetic and pharmaceutical applications. Both metabolic processes require in their pathway the activity of cytochrome P450 monooxygenase. This enzyme needs and gets reducing equivalents from NADPH cytochrome P450 reductase (CPR). The CPR gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes an enzyme of 687 amino acids, which shows homology with known CPRs of other species. The functionality of the gene was proven by heterologous expression in Escherichia coli. The recombinant protein exhibited NADPH-dependent cytochrome c reducing activity. Cloning and characterization of this enzyme is an important step in the study of the cytochrome P450 monooxygenase system of Candida bombicola. The GenBank accession number of the sequence described in this article is EF050789.