P450 Monooxygenase Activity Research Articles

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.

Relative Contribution of Monooxygenase and Esterase to Pyrethroid Resistance in <I>Triatoma infestans</I> (Hemiptera: Reduviidae) from Argentina and Bolivia

Recently, high resistance to pyrethroid insecticides has been associated with ineffective field treatments against Triatoma infestans (Klug) (Hemiptera: Reduviidae) in northern Argentina. Samples were collected from two areas in Argentina (Salta and La Rioja) and one are in Bolivia (Yacuiba), and they were subjected to toxicological and biochemical assays. All populations were resistant to deltamethrin, but they showed different profiles to nonpyrethroid insecticides. The Salta population showed high resistance ratios (RRs) to deltamethrin and only slight differences in the susceptibility to fenitrothion and fipronil compared with the reference strain. Otherwise, the La Rioja population showed a lower RR to deltamethrin and no resistance to fenitrothion or fipronil. Finally, the Yacuiba population had high a RR to deltamethrin, but it was susceptibility to fenitrothion and fipronil. In several cases, deltamethrin-resistant populations had higher susceptibility to bendiocarb than the reference strain. Measured activity of P450 monooxygenase in individual insects (based on ethoxycoumarine-O-deethylase), tended to be higher in the deltamethrin-resistant populations, but the differences were not statistically significant. Activity of specific esterases determined by the hydrolysis of 7-coumaryl permethrate demonstrated an increase in the percentage of insects with higher esterase activity in the Salta and La Rioja populations. Unexpectedly, the Yacuiba population showed lower pyrethroid esterase activity than the reference strain. The different pyrethroid resistance patterns found in T. infestans from three geographical regions within Argentina and in Bolivia suggests that enzyme-based pyrethroid resistance in this species has multiple origins. Nevertheless, because nerve insensitivity (related to the presence of the kdr gene) is also an important mechanism related to pyrethroid resistance, further studies on the kdr gene should be carried to clarify the relative contribution of each pyrethroid-associated mechanism in deltamethrin-resistant populations of T.

Relative Contribution of Monooxygenase and Esterase to Pyrethroid Resistance in Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia

Recently, high resistance to pyrethroid insecticides has been associated with ineffective field treatments against Triatoma infestans (Klug) (Hemiptera: Reduviidae) in northern Argentina. Samples were collected from two areas in Argentina (Salta and La Rioja) and one are in Bolivia (Yacuiba), and they were subjected to toxicological and biochemical assays. All populations were resistant to deltamethrin, but they showed different profiles to nonpyrethroid insecticides. The Salta population showed high resistance ratios (RRs) to deltamethrin and only slight differences in the susceptibility to fenitrothion and fipronil compared with the reference strain. Otherwise, the La Rioja population showed a lower RR to deltamethrin and no resistance to fenitrothion or fipronil. Finally, the Yacuiba population had high a RR to deltamethrin, but it was susceptibility to fenitrothion and fipronil. In several cases, deltamethrin-resistant populations had higher susceptibility to bendiocarb than the reference strain. Measured activity of P450 monooxygenase in individual insects (based on ethoxycoumarine-O-deethylase), tended to be higher in the deltamethrin-resistant populations, but the differences were not statistically significant. Activity of specific esterases determined by the hydrolysis of 7-coumaryl permethrate demonstrated an increase in the percentage of insects with higher esterase activity in the Salta and La Rioja populations. Unexpectedly, the Yacuiba population showed lower pyrethroid esterase activity than the reference strain. The different pyrethroid resistance patterns found in T. infestans from three geographical regions within Argentina and in Bolivia suggests that enzyme-based pyrethroid resistance in this species has multiple origins. Nevertheless, because nerve insensitivity (related to the presence of the kdr gene) is also an important mechanism related to pyrethroid resistance, further studies on the kdr gene should be carried to clarify the relative contribution of each pyrethroid-associated mechanism in deltamethrin-resistant populations of T. infestans.

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.

Monitoring the operational impact of insecticide usage for malaria control on Anopheles funestus from Mozambique

BackgroundIndoor residual spraying (IRS) has again become popular for malaria control in Africa. This combined with the affirmation by WHO that DDT is appropriate for use in the absence of longer lasting insecticide formulations in some malaria endemic settings, has resulted in an increase in IRS with DDT as a major malaria vector control intervention in Africa. DDT was re-introduced into Mozambique's IRS programme in 2005 and is increasingly becoming the main insecticide used for malaria vector control in Mozambique. The selection of DDT as the insecticide of choice in Mozambique is evidence-based, taking account of the susceptibility of Anopheles funestus to all available insecticide choices, as well as operational costs of spraying.Previously lambda cyhalothrin had replaced DDT in Mozambique in 1993. However, resistance appeared quickly to this insecticide and, in 2000, the pyrethroid was phased out and the carbamate bendiocarb introduced. Low level resistance was detected by biochemical assay to bendiocarb in 1999 in both An. funestus and Anopheles arabiensis, although this was not evident in WHO bioassays of the same population.MethodsSentinel sites were established and monitored for insecticide resistance using WHO bioassays. These assays were conducted on 1–3 day old F1 offspring of field collected adult caught An. funestus females to determine levels of insecticide resistance in the malaria vector population. WHO biochemical assays were carried out to determine the frequency of insecticide resistance genes within the same population.ResultsIn surveys conducted between 2002 and 2006, low levels of bendiocarb resistance were detected in An. funestus, populations using WHO bioassays. This is probably due to significantly elevated levels of Acetylcholinesterase levels found in the same populations. Pyrethroid resistance was also detected in populations and linked to elevated levels of p450 monooxygenase activity. One site had shown reduction in pyrethroid resistance since the base line in 1999.

Reconstitution of monooxygenase activity of membrane cytochromes P450 in vitro and detergents

Reconstitution of monooxygenase activity of membrane cytochromes P450 in vitro and detergents

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.

Cross‐resistance and biochemical mechanisms of abamectin resistance in the B‐type Bemisia tabaci

Abstract: To understand the risk of resistance and the possible mechanisms of resistance to abamectin in B‐type Bemisia tabaci (Gennadius) better, a resistant strain of B. tabaci was selected in the laboratory and cross‐resistance pattern and resistance mechanisms to abamectin were investigated. The NJ‐Abm strain of B. tabaci was derived from a field population (NJ) collected in Nanjing, China in 2002 with 18 generations of selection with abamectin in the laboratory. Compared with the unselected NJ strain, the selected NJ‐Abm strain developed 14.5‐fold resistance to abamectin and showed significant cross‐resistance to emamectin benzoate (4.4‐fold) and imidacloprid (3.4‐fold), but no cross‐resistance to fipronil. The oxidase inhibitor piperonyl butoxide (PBO) and glutathione S‐transferase inhibitor diethyl maleate (DEM) produced significant synergism on abamectin in the NJ‐Abm strain (with synergistic ratios of 3.9‐ and 4.1‐fold respectively); however, the esterase inhibitor triphenyl phosphate (TPP) did not act synergistically with abamectin. Biochemical analysis confirmed that P450 monooxygenase activity and glutathione S‐transferase activity of the NJ‐Abm strain were elevated to 2.1‐ and 2.0‐fold, respectively, compared with that of the NJ strain. This indicated that enhanced metabolism mediated by P450 monooxygenase and glutathione S‐transferase is likely to be involved in abamectin resistance and cross‐resistance to imidacloprid and emamectin benzoate in the NJ‐Abm strain.

Broad substrate Cytochrome P450 monooxygenase activity in the cells of Aspergillus terreus MTCC 6324

Cytochrome P450 (CYP) monooxygenase activities with different category of substrates namely, alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents, and steroids were detected in the cells of Aspergillus terreus. High CYP specific activity was observed when methanol (5.6 ± 0.017 U mg −1), acetone (7.76 ± 0.02 U mg −1), dimethylsulphoxide (DMSO) (9.70 ± 0.005 U mg −1), n-hexadecane (4.39 ± 0.02 U mg −1), or n-octadecane (4.23 ± 0.01 U mg −1) were used as substrates. Significant CYP specific activity was also detected when naphthalene (3.80 ± 0.002 U mg −1) was used as substrate. The CYP catalysis of n-hexadecane had followed both terminal and sub terminal oxidations. The activity was localized in the cytosol of n-hexadecane grown cells, while, it was apparently distributed in light mitochondrial fraction and microsomal fraction of glucose grown cells. The substrate specificities of CYP present in all the locations were similar irrespective of the substrates used for the growth. Heme staining of the microsomal fraction containing CYP and other proteins in SDS–PAGE showed single heme protein band with corresponding molecular weight of 110 kDa.

Expression, microsomal and mitochondrial activities of cytochrome P450 enzymes in brain regions from control and phenobarbital-treated rabbits

Expression and monooxygenase activity of various cytochrome P450 (CYP) enzymes along with constitutive androstane (CAR) and the pregnane X (PXR) receptors were investigated in the brain of control and phenobarbital-treated rabbits (80 mg/kg for 4 days). RT-PCR analysis, using specific primers, demonstrated that in control rabbits mRNAs of CYP 2A10, 2B4/5 and 3A6 were expressed, though to a different extent, in the liver, as well as in brain cortex, midbrain, cerebellum, striatum, hippocampus and hypothalamus, whilst CYP2A11 and 4B1 were not expressed in the hypothalamus. CAR was expressed in liver and all the brain regions examined, whereas the PXR was expressed only in liver and cortex. Real time RT-PCR analysis demonstrated that in vivo treatment with phenobarbital, in contrast with what happened in liver, did not induce the expression of CYP 2B4/5 mRNA in cortex, midbrain and cerebellum. NADPH cytochrome c reductase and some other enzymatic activities markers of CYP 2A, 2B, 3A and 4B activities were studied in liver microsomes as well as in microsomes and mitochondria of brain cortex, midbrain and cerebellum of control and phenobarbital-treated rabbits. In contrast to what was observed in liver, phenobarbital treatment did not induce the aforementioned monooxygenase activities in brain. However, we cannot exclude that a longer phenobarbital treatment may lead to a significant induction of CYP activities in brain. These findings indicated that brain CYPs, despite the presence of CAR, were resistant to phenobarbital induction, indicating a possible different regulation of these enzymes between brain and liver.

Sublethal Effects of Three Pesticides on Activities of Selected Target and Detoxification Enzymes in the Aquatic Midge, Chironomus tentans (Diptera: Chironomidae)

Sublethal effects of three pesticides including atrazine (triazine herbicide), DDT (organochlorinated insecticide), and chlorpyrifos (organophosphate insecticide) on acetylcholinesterase (AChE), general esterase (GE), glutathione S-transferase (GST), and cytochrome P450 monooxygenase (P450) activities were evaluated in the aquatic midge Chironomus tentans. Exposures of midges to atrazine at 30 and 150 micrograms per liter (microg/L) for 20 d (i.e., from the first- to fourth-instar larvae) enhanced P450 O-deethylation activity by 12.5- and 15.5-fold, respectively, but did not significantly change AChE, GST, and GE activities. Similar exposures to DDT at 0.01 and 0.05 microg/L did not significantly affect AChE, GE, and P450 activities; however, DDT at 0.05 microg/L enhanced GST activity toward the substrate 1-chloro-2, 4-dinitrobenzene by 33.6%. Exposures of midges to chlorpyrifos at 0.10 microg/L for 20 d reduced AChE activity by 59.8%, and GE activities toward the substrates alpha-naphthyl acetate and beta-naphthyl acetate by 30.7 and 48.8%, respectively. The reduced GE activities appear to be due to the inhibition of several esterases, particularly the one with a slow migration, by chlorpyrifos as demonstrated by non-denaturing polyacrylamide gel electrophoresis. Furthermore, exposure of midges to chlorpyrifos at 0.10 microg/L for 20 d enhanced the P450 O-deethylation activity by 3.3-fold although no significant effect was observed at 0.02 microg/L for the same enzyme. These results provide insights into the sublethal effects of these commonly detected pesticides in aquatic environments on important enzymes in aquatic midges.

Long lasting effects of prenatal exposure to deltamethrin on cerebral and hepatic cytochrome P450s and behavioral activity in rat offspring

Prenatal exposure to different doses (0.25, or 0.5 or 1.0 mg/kg corresponding to 1/320th or 1/160th or 1/80th of LD 50) of deltamethrin to the pregnant Wistar rats from gestation day 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 cytochrome P450 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 indicated that increase in cytochrome P450 activity may lead to the accumulation of deltamethrin and its metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic cytochrome P450s was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.5 and 1.0 mg/kg) of deltamethrin and up to 9 weeks at the highest dose (1.0 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 weeks of age, have further indicated that due to the reduced activity of the cytochrome P450s during the ontogeny, the pyrethroid or its metabolites accumulating in the brain may not be cleared from the brain, thereby leading to the persistence in the increase in the expression of cerebral and hepatic cytochrome P450s in the offspring postnatally up to 9 weeks. The data suggests that low dose prenatal exposure to pyrethroids has the potential to produce long lasting effects on the expression of xenobiotic metabolizing cytochrome P450s in brain and liver of the offspring.

Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China

The mechanisms for multiple resistances had been studied with two field resistant strains and the selected susceptible and resistant strains of Spodoptera litura (Fabricius). Bioassay revealed that the two field strains were both with high resistance to pyrethroids (RR: 63–530), low to medium resistance to organophosphates and carbamates, AChE targeted insecticides (RR: 5.7–26), and no resistance to fipronil (RR: 2.0–2.2). Selection with deltamethrin in laboratory could obviously enhance the resistance of this pest to both pyrethroids and AChE targeted insecticides. Synergism test, enzyme analysis and target comparison proved that the pyrethroid resistance in this pest associated only with the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase. However the resistance to the AChE targeted insecticides depended on the target insensitivity and also the enhanced activity of MFO and esterase. Thus, the cross-resistance between pyrethroids and the AChE targeted insecticides was thought to be resulted from the enhanced activity of MFO and esterase.

Biochemical analysis of a chlorfenapyr‐selected resistant strain of Tetranychus urticae Koch

Tetranychus urticae Koch has recently developed resistance to chlorfenapyr in Australia and Japan, but no attempt has yet been made to describe the biochemical mechanisms involved in chlorfenapyr resistance. In this study a laboratory-selected chlorfenapyr-resistant strain was investigated. Resistance to chlorfenapyr was associated with a strong increase in esterase activity and P450 mono-oxygenase (MO) activity but a decrease in 3,3',5,5'-tetramethylbenzidine (TMBZ) peroxidation activity. Differences in esterase activities between susceptible and resistant strains increased with increasing carbon number of the aliphatic side-chain of the nitrophenol substrate. A 4.4-fold increase in the O-deethylation of 7-ethoxy-4-trifluoromethyl coumarin (7-EFC) mediated by P450 MOs was detected. Remarkably, the resistant strain showed only half of the total TMBZ peroxidation activity found in the susceptible strain. The activity of these enzymes was further determined on different crosses and back-crosses of both strains. Results indicated that activities correlated with chlorfenapyr susceptibility and could be considered as biochemical markers. Esterase isozymes of both strains and their crosses were separated with isoelectric focusing (IEF) and visualised after activity staining. It was clear that two distinct zones of enhanced esterase activity were present in the chlorfenapyr-resistant strain (EST 11, pI = 4.88 and EST 16, pI = 4.71). EST 11 was identified with inhibitors as a carboxylesterase. The relative presence and intensity of these esterase zones changed in the different crosses and could be seen as a marker for chlorfenapyr resistance. Glutathione-S-transferase and glucose-6-phosphate dehydrogenase activities were not significantly different between strains. A twofold decrease in TMBZ peroxidase activity in the resistant strain could reflect decreased activation of chlorfenapyr. On the basis of these results the involvement of P450 MOs and esterases in the activation and detoxification of chlorfenapyr in T. urticae is challenged and discussed.