Marcel Amichot

Inducibility of the Drosophila melanogaster cytochrome P450 gene, CYP6A2, by phenobarbital in insecticide susceptible or resistant strains

The importance of cytochrome P450s in the biology of cells or organisms is clearly established. While numerous studies concern vertebrates, little is known about invertebrates cytochrome P450s. In this paper, we have focused on CYP6A2 gene expression in Drosophila melanogaster. We show the expression of this cytochrome P450 gene in the Canton(s) strain (wild type) to be under the control of phenobarbital. In adults treated with phenobarbital, this gene is transcribed in the midgut, the pericuticular fat bodies and the Malpighian tubules. The induction factor is 15. In the RDDTR strain of Drosophila melanogaster, which is resistant to the insecticide DDT, this gene is constitutively overexpressed in the same tissues (overexpression factor is 6 relative to untreated Canton(s) flies). Phenobarbital is not as effective on RDDTR (induction factor is 2.5 relative to untreated RDDTR flies) as on wild type strains.

Purification and characterization of a carboxylesterase involved in malathion-specific resistance from Tribolium castaneum (Coleoptera: Tenebrionidae)

Specific resistance to malathion in a strain of Tribolium castaneum is due to a 44-fold increase in malathion carboxylesterase (MCE) activity relative to a susceptible strain, whereas non-specific esterase levels are slightly lower. Unlike the overproduced esterase of some mosquito and aphid species, MCE in Tribolium castaneum accounts for only a small fraction (0.033-0.045%) of the total extractable protein respectively in resistant and susceptible strains. The enzyme was purified to apparent homogeneity from these two strains and has a similar molecular weight of 62,000. However, preparative isoelectricfocusing indicated that resistant insects possess one MCE with pI of 7.3, while susceptible insects possess a MCE with a pI of 6.6. Purified MCE from both populations had different K(m) and V(m) values for hydrolysis of malathion as well as for alpha-naphthyl acetate. The kinetic analysis suggests that MCE of resistant insects hydrolyses malathion faster than the purified carboxylesterase from susceptible beetles and that this enzyme has greater affinity for malathion than for naphthyl esters. Malathion-specific resistance is due to the presence of a qualitatively different esterase in the resistant strain.

Induction of cytochrome P450 activities in Drosophila melanogaster strains susceptible or resistant to insecticides.

We analysed Drosophila melanogaster cytochrome P450s (P450) through the measurements of four enzymatic activities: ethoxycoumarin-O-deethylase, ethoxyresorufin-O-deethylase, lauric acid hydroxylation, and testosterone hydroxylation. We did these measurements in two Drosophila strains: one is susceptible to insecticides (Cantons) and the other is resistant to insecticides by enhanced P450 activities (RDDTR). In addition, we also treated the flies with eight chemicals (beta-naphtoflavone, benzo-alpha-pyrene, 3-methylcholanthrene, phenobarbital, aminopyrine, rifampicin, prochloraz, and clofibrate) known to induces genes from the families CYP1, CYP2, CYP3, CYP4, and CYP6. Metabolisation of all the substrates by P450 from flies microsomes was observed. The chemicals had different effects on these activities, ranging from induction to inhibition. The effects of these chemicals varied with the strains as most of them were ineffective on the RDDTR strain. The results showed that P450-dependent activities are numerous in Drosophila. Regulation features of these activities are complex. The availability of mutant strains as RDDTR should allow fundamental studies of P450 in insects.

Target modification as a molecular mechanism of pyrethroid resistance in Drosophila melanogaster

Abstract “Knock down” resistance (kdr) to the pyrethroid deltamethrin was investigated using susceptible (Tubingen) and resistant (TubingenDDT) strains of Drosophila melanogaster. Toxicological and pharmacological data show that resistance involves a modification of the affinity of the insecticide for its receptor site on the voltage-dependent sodium channel. Genetic studies indicate that the kdr factor is linked to the second chromosome where one sodium channel gene, sch, is located. Cloning and sequencing the alleles of this gene from both strains revealed a single substitution that may be responsible for the loss of toxicity of deltamethrin in the resistant strain.

Tissue-specific induction and inactivation of cytochrome P450 catalysing lauric acid hydroxylation in the sea bass, Dicentrarchus labrax.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

Comparative ability to detoxify alder leaf litter in field larval mosquito collections.

The larvicidal effects of polyphenols from dietary alder leaf litter were investigated in different field collections of three detritivorous Aedes taxa (Ae. detritus, Ae. cataphylla, Ae. rusticus) and compared to the cytochrome P450 monooxygenase, glutathione S-transferase, and esterase activities. Larvae from polyphenol-rich habitats had a higher tolerance for polyphenols and higher midgut cytochrome P450 and esterase activities than larvae from polyphenol-poor habitats. Furthermore, the role of P450 enzymes in the mechanism of resistance to alder polyphenols was suggested by the synergistic effect in vivo of piperonyl butoxide in the resistant Ae. rusticus. This confirms the importance of polyphenols to larval mosquito performance, and provides evidence for the importance of specific detoxification mechanisms for tolerance to dietary polyphenols. Arch.

Molecular cloning of a CYP1A cDNA from the teleost fish Dicentrarchus labrax

A cDNA encoding for cytochrome P450 1A has been cloned in the marine teleost fish Dicentrarchus labrax. This fish, common in the Mediterranean, was chosen since it is considered a good sentinel species. Moreover, biomarkers of exposure to organic contaminants (such as EROD) are often measured in this species and make it possible to evaluate the quality of waters. For cloning purposes, RNAs were extracted from the liver of benzo[a]pyrene (BaP)-treated animals and used as template in degenerate RT-PCR. The cDNA product was cloned and used for the design of highly stringent primers that were utilized in Rapid Amplification of cDNA Ends (RACE) PCR. The cloned cDNA hybridizes with a 2.7 kb mRNA which is induced by treatment of the fish with BaP, a classical CYP1A inducer. The closest sequences found in data banks belong to fish CYP1A.

Cloning and detection of insecticide resistance genes

Insecticides act in impairing the function of molecular targets, usually proteins, which are crucial for life, and most resistances develop through the selection of mutations which either modify the target so that it remains at least partially functional, or increase the rate of insecticide degradation so that less toxicant reaches the target. Other resistance mechanisms include behavioural avoidance of the insecticide, decreased penetration and increased excretion; the first two mechanisms have been described in insects.

Modeling impact of parathion and its metabolite paraoxon on the nematode Caenorhabditis elegans in soil

Parathion is an insecticide of a group of highly toxic organophosphorous compounds. In vivo, it is activated to the toxic metabolite paraoxon. Laboratory experiments have shown that a single relationship between the variable (concentration x time of application) and the percentage of paralyzed nematodes is relevant. Aqueous (0.01 M CaCl2) extracts from soil that had received a dose of parathion as used in practice during an incubation experiment had no effect on nematodes, because sorption and biodegradation of the pesticide decreased the pesticide concentration in the soluble phase. To predict the toxicological effects of parathion and paraoxon on nematodes under various soil conditions during a simulation period of 20 d, we used a model predicting the concentrations of parathion and paraoxon over time in the soil liquid phase. In this model, sorption and biodegradation of both parathion and paraoxon were taken into account, and the results indicated that sorption effects were dominant and determined the differential toxicological risks between soils. Variable effects were predicted for short times (typically <5 d), and critical toxicological conditions were predicted for longer duration (typically >10-15 d), in all cases.

Transcription analysis of the para gene by in situ hybridization and immunological characterization of its expression product in wild-type and mutant strains of Drosophila

In Drosophila, the para gene has been shown to encode a functional voltage-dependent sodium channel. We used a cDNA clone to study the distribution of its transcripts by in situ mRNA hybridization on adult fly sections. These transcripts are found in cortical regions of the central nervous system and in the eyes. On immunoblots, antibodies raised against expression products of part of the gene recognize a polypeptide of M(r) approximately 270,000 in head membranes. Immunolocalization experiments indicate that anti-para antibodies bind to cortical regions of the brain and give heavy signals in the eyes. Immunohistochemistry was also performed on napts and seits1, two mutant Drosophila strains known to be defective in sodium channel activity. Only napts flies displayed a decrease in the expression of the para protein.