Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

Comparative enzyme activities and cytochrome P-450 levels of some rat tissues with respect to their metabolism of several pesticides

Cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in the postmitochondrial centrifugal fraction, microsomes, and supernatant of rat liver, lungs, kidneys, and testes. Liver microsomes contained the highest P-450 levels and A-esterase activity. B-esterase activity was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in rat lung and lowest in the liver (per mg protein). Glutathione S-aryl transferase activity was highest in the liver. The in vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed many differences. Carbaryl metabolism was greater in the liver microsomal fractions than in any other preparation. 1-Naphthol was the major metabolite in all tissue fractions. Although very little metabolism of phosphamidon occurred in the rat, metabolism in the rat liver postmitochondrial fraction was slightly higher with respect to the production of metabolites than in the supernatant and microsomes combined. Chlorotoluron was not metabolized by any of the tissue fractions of the rat. At least a low level of activity toward some compounds was observed in all tissues, but this study confirmed that the liver was the most active metabolizing tissue as well as having the highest levels of enzymatic activity usually associated with pesticide metabolism.     

The detection and characterization of malathion resistance in field populations of Anopheles culicifacies B in Sri Lanka

Malathion resistance was first detected in Sri Lankan Anopheles culicifacies in limited regions of the island in 1982. The frequency of resistance has been increasing slowly since then, but is not yet high enough to be considered an operational problem. Malathion toxicity is synergised in the resistant population by triphenyl phosphate, and metabolism studies suggest the involvement of a carboxylesterase enzyme. The spread of general esterase activity in individuals in an area of the island where resistance is present is wider than that in a totally malathion-susceptible area. However, the frequency of individuals with high esterase activity does not correlate well with resistance in the two field populations studied in detail. This suggests that a qualitative rather than a quantitative change in esterase activity may be involved in this resistance. Extrapolation from similar qualitatively changed carboxylesterases in other anophelines leads us to predict that the resistance in A. culicifacies will be malathion specific and inherited as a single semidominant characteristic.     

Species differences in the in vitro inhibition of brain acetylcholinesterase and carboxylesterase by mipafox,paraoxon, and soman

Acute intraperitoneal toxicity of mipafox, paraoxon, and soman was highest in chicken, followed by rat, and lowest in frog. Species differences in organophosphorus toxicity were not related to differences in the specific activities of either acetylcholinesterase or carboxylesterase in brain. The sensitivity to inhibition of brain acetylcholinesterase in vitro by the organophosphorus compounds was closely related to the susceptibility of the species to acute organophosphorus poisoning. Both the acute toxicity and the sensitivity of brain acetylcholinesterase to inhibition in vitro by organophosphorus compounds in all the species were in the following order of increasing activity: mipafox < paraoxon < soman. The sensitivity of brain carboxylesterase to inhibition by the organophosphorus compounds were less than that of acetylcholinesterase and it could not be related to species susceptibility to acute organophosphorus toxicity. Paraoxon-insensitive phenyl valerate hydrolase in chicken brain was more sensitive to inhibition by mipafox and soman compared to rat; in chicken the sensitivity of paraoxon-insensitive phenyl valerate hydrolase to inhibition by soman was 9000 times more than that by mipafox, while in rat it was 100 times more. Frog brain had no paraoxon-insensitive phenyl valerate hydrolase activity. No evidence of age dependence was noticed in the specific activities of brain acetylcholinesterase, carboxylesterase, neurotoxic esterase, and paraoxon-insensitive phenyl valerate hydrolase or in the sensitivity of these enzymes to in vitro inhibition by organophosphorus compounds in both rats and chickens.     

Organophosphorus poisoning: A comparative study of the toxicity of chlorfenvinphos [2-chloro-1- (2′, 4′, -dichlorophenyl) vinyl diethyl phosphate] to the pigeon,the pheasant and the Japanese Quail

The acute oral toxicity of chlorfenvinphos [2-chloro-1-(2′, 4′-dichlorophenyl)vinyl diethyl phosphate] was measured in pigeon (Columba livia), pheasant (Phasianus colchicus), and quail (Coturnix coturnix japonica) and the compound was shown to be particularly toxic to pigeons. Additionally, all three species were fed chlorfenvinphos at 100 ppm in their diet for two or four weeks and esterase measurements were made by conventional and electrophoretic methods on extracts of liver, kidney and brain. The conventionally measured esterase inhibition correlated well with the acute oral toxicity figures. A more detailed study of the histochemically stained electrophoregams showed some discrepancies compared with conventional methods but offered a possible explanation of the inter-species toxicity difference in that it revealed differential inhibition of some brain iso-esterases in pigeon but not in pheasant or quail.     

华北大黑鳃金龟Holotrichia oblita中肠羧酸酯酶基因 HoCL1的克隆与序列分析

利用粉纹夜蛾(Trichoplusia ni)围食膜蛋白多克隆抗体,从已构建的华北大黑鳃金龟 Holotrichia oblita 中肠cDNA表达文库中筛选得到1个编码羧酸酯酶的cDNA克隆 HoCL1 ,其开放阅读框(ORF)长1 599 bp,编码532个氨基酸,推导的蛋白质分子质量为59.5 kDa,等电点(p I)为4.5。 HoCL1蛋白具有羧酸酯酶的保守结构域:1个二硫键形成的位点和1个丝氨酸活性中心,三联体催化活性中心位于Ser207、Asp333和His422上,不含有氮联糖基化位点和氧联糖基化位点,只含有3 个半胱氨酸残基。依据氨基酸序列同源性分析和保守结构域分析,HoCL1属于B类酯酶,与赤拟谷盗 Tribolium castaneum 羧酸酯酶相似性最高,为35.2%。通过与其他昆虫羧酸酯酶序列比对及构建系统发育树,发现HoCL1羧基端的氨基酸序列保守性低,但靠近N端的活性中心处的氨基酸序列则高度保守,可与赤拟谷盗、异色瓢虫 Harmonia axyridis 羧酸酯酶聚类在一起。羧酸酯酶 HoCL1 基因的克隆鉴定为进一步研究该基因在华北大黑鳃金龟体内的表达及功能奠定了基础。     

Biochemical Characterization of Carboxylesterase in the Small Brown Planthopper Laodelphax striatellus (Fallén)

Several forms of carboxylesterase were observed in a malathion-resistant small brown planthopper, Laodelphax striatellus Fallén, by isoelectrofocusing. To study the mechanisms of increased esterase activity, esterases were purified and their biochemical characteristics were investigated in five active esterase isozymes of two resistant strains. These esterases have polymorphic characteristics and their molecular weights ranged from 66 to 70 kDa, due to variations in glycosylation. The pI values of these esterases ranged from 5.3 to 4.7. These esterases were immunologically related and NH₂-terminal amino acids were identical in all isozymes regardless of pI or molecular weight. No differences have been found in kinetic parameters (K_m and V_max) to α-naphthylacetate and specific activity toward α-naphthylacetate and malathion as a substrate in all isozymes. Resistant individuals showed high ali- and malathion carboxylesterase activities and these enhancements were caused by quantitative differences of carboxylesterases with several different pI.     

Metabolism of juvenile hormone I by microsomal oxidase,esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

House fly (Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with ³H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly (Sarcophaga bullata Parker), and blow fly [Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.     

Detoxication capacity in the honey bee,Apis mellifera L

Various detoxifying enzymes, including microsomal oxidases, glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, were assayed in adult worker bees (Apis mellifera L.) using midguts as the enzyme source. A cell-free system was used for all enzyme assays, except that microsomal oxidases required intact midgut because of the inhibitor encountered. Midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes. All enzymes studied were active, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity. Sublethal exposure of honey bees to various insecticides had no effect on these enzyme activities, with the exception of permethrin which significantly stimulated the glutathione S-transferase, and malathion, which significantly inhibited the α-naphthylacetate esterase and carboxylesterase.     

Metabolic chemistry of pyrethroid insecticides

This review of studies on 20 pyrethroids with nine different acid moieties and ten different alcohol moieties reveals a diversity of functional groups undergoing metabolism in mammals, insects, other organisms, and microsomal esterase and oxidase systems. Seventy-nine metabolites are identified from the cis-isomer of permethrin and transpermethrin but fewer from other pyrethroids examined in less detail. The sites and rates of metabolic attack on each pyrethroid depend on the organism or system. Metabolism of pyrethoids by esterase and oxidase action usually limits their toxicity to mammals more than to insects, thereby conferring useful selective toxicity properties.     

Selective inhibition of separate esterases in rat and mouse liver microsomes hydrolyzing malathion,trans-permethrin,and cis-permethrin

Separate esterase activities of rat and mouse liver microsomes hydrolyzing malathion, trans-permethrin, and cis-permethrin were differentiated on the basis of their sensitivities to inhibition by paraoxon and α-naphthyl N-propylcarbamate (NPC). In rat liver microsomes, the malathionhydrolyzing activity was more sensitive to both inhibitors and showed a different time course of NPC inhibition than the activities hydrolyzing the permethrin isomers. Paraoxon completely inhibited trans-permethrin hydrolysis, but only partially inhibited that of cis-permethrin. The paraoxonsensitive trans- and cis-permethrin-hydrolyzing activities were not differentially inhibited, but separate inhibition curves were obtained for the inhibition of trans- and cis-permethrin hydrolysis by NPC. The mouse liver esterase activity hydrolyzing trans-permethrin showed a similar paraoxon sensitivity to that of rat liver, but that the paraoxon-sensitive portion of the cis-permethrinhydrolyzing activity was 5.5-fold less sensitive to paraoxon than the corresponding rat liver activity and was clearly differentiated from the mouse liver trans-permethrin-hydrolyzing activity. The mouse liver malathion-hydrolyzing activity was 100-fold less sensitive to paraoxon and 14-fold less sensitive to NPC than the corresponding rat liver activity. Rat and mouse liver esterase activities hydrolyzed trans- and cis-permethrin at similar rates under standard assay conditions, but mouse liver esterases were 10-fold less active in hydrolyzing malathion. The higher specific activity of rat liver malathion-hydrolyzing esterases resulted from the greater apparent affinity and maximum velocity for malathion hydrolysis. These results demonstrate that the hydrolysis of malathion, trans-permethrin, and cis-permethrin by rat and mouse liver microsomal preparations involves several esterases with differing substrate specificities and inhibitor sensitivities.     

Fipronil metabolism,oxidative sulfone formation and toxicity among organophosphate‐ and carbamate‐resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide‐resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil + the mono‐oxygenase inhibitor piperonyl butoxide, and fipronil's oxidative sulfone metabolite in two populations (one resistant with elevated mono‐oxygenase activity). Fipronil and fipronil‐sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [¹⁴C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub‐cellular protein fractions (microsomal and cytosolic), and glutathione‐agarose purified glutathione‐S‐transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione‐S‐transferase (MW = 27 kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione‐S‐transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. © 2000 Society of Chemical Industry     

Subcellular distribution of malathion,phenthoate, and diethylsuccinate carboxylesterases in rat lungs

The subcellular distribution of malathion, phenthoate, and diethylsuccinate carboxylesterases was determined in the lungs of male Sprague-Dawley rats and in rats administered lung toxic doses of bromobenzene and paraquat. In control with the former two substrates, the highest activity was encountered in the 6500g and 12,000g pellets. In addition, significant activity was found in the 100,000g supernatant. These fractions hydrolyzed diethylsuccinate very slowly, and the major diethylsuccinate carboxylesterase activity was recovered in the 100,000g pellet. The bromobenzene treatment had no effect on these carboxylesterases; however, the paraquat treatment significantly decreased the phenthoate and diethylsuccinate carboxylesterases in the 100,000g pellet without altering the activity in the other fractions. The present study suggested that the subcellular distribution of malathion and phenthoate carboxylesterases is different from that in liver. The present study revealed that, in the lungs, the highest total activity for malathion and phenthoate carboxylesterases was found in the 100,000g supernatant, in comparison with liver, where the 100,000g pellet contains a much higher activity of these enzymes. The decrease in specific activities of diethylsuccinate and phenthoate carboxylesterases following the treatment with a pneumotoxicant may serve as an indicator of lung damage.     

Studies on the chiral isomers of fonofos and fonofos oxon: II. In vitro metabolism

The in vitro metabolism of the chiral isomers of fonofos and fonofos oxon in the presence of mouse liver mixed-function oxidase and serum esterase was investigated. The metabolism of ³⁵S-labeled phenyl-(S)_P-fonofos mediated by mixed-function oxidase took place stereoselectively, resulting predominantly in (R)_P-fonofos oxon. Similarly, (R)_P-fonofos was converted to (S)_P-oxon. In each case, however, a significant amount of racemization occurred. Other products were diphenyl disulfide and diphenyl disulfide oxide. In addition to stereospecificity, the oxidative metabolism of (R)_P-fonofos proceeded at a rate faster than that of (S)_P-fonofos. Stereoselective rate differences also were observed in mouse or rat serum-catalzyed degradation of the fonofos oxon enantiomers, the (S)_P isomer being degraded about twofold faster than its enantiomer. The differences in toxicities of the isomers of fonofos and fonofos oxon were consistent with the in vitro metabolism data.     

Organophosphorus poisoning: A comparative study of the toxicity of carbophenothion to the Canada goose,the pigeon and the Japanese quail

The acute single dose oral toxicity of carbophenothion (S-4-chlorophenylthiomethyl OO-diethyl phosphorodithioate) has been determined in Canada geese (Branta canadensis), pigeons (Columba livia) and Japanese quail (Coturnix coturnix japonica). At post mortem examination gross pathological changes were observed in Canada geese and pigeons and esterase levels were determined by conventional and electrophoretic methods on extracts of liver and brain from these two species. Carbophenothion residue levels were determined in liver, brain and gizzard contents from the geese and pigeons. The overall pattern of results suggests that esterase inhibition may not be the dominant factor in carbophenothion poisoning in geese. It is suggested that a brain carbophenothion residue level of 1 part/10⁶ is indicative of death by poisoning in geese.     

农药混剂的选择毒性研究

稻瘟净或异稻瘟净与乐果或马拉松混用,对抗性黑尾叶蝉有明显的增效作用,对小白鼠急性口服毒性也有所增加。小白鼠口服稻瘟净或异稻瘟净后,鼠肝中水解乐果和N—甲基正己酰胺的羧酰胺酶活力明显下降;水解醋酸α-萘酯的羧酸酯酶活力也被抑制。羊肝微粒体的羧酰胺酶在体外分别被稻瘟净、异稻瘟净、磷酸三苯酯、磷酸三甲苯酯、苯硫磷、氧乐果和西维因强烈抑制,它们的I_(50)值为1.3×10~(-8)—4.0×10~(-5)克分子浓度。稻瘟净、异稻瘟净、磷酸三苯酯和西维因分别是羊肝羧酰胺酶和羧酸酯酶的竞争性抑制剂,它们的K_i值在4.8×10~(-8)—1.0×10~(-7)克分子浓度之间。这些结果表明稻瘟净、异稻瘟净等增加乐果和马拉松对哺乳动物的毒性的机制,可能主要是由于它们抑制了哺乳动物体内正常解毒乐果和马拉松的水解酶。文中讨论了这些农药混剂的选择毒性问题。     

Mode of action of naphthalic anhydride as a safener for the herbicide AC 263222 in maize

In hydroponic experiments, seed-dressing with the herbicide safener 1,8-naphthalic anhydride (NA), significantly enhanced the tolerance of maize, (Zea mays L., cv. Monarque) to the imidazolinone herbicide, AC 263222, (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-5-methylnicotinic acid). Uptake, distribution and metabolism studies where [¹⁴C]AC 263222 was applied through the roots of hydroponically grown maize plants showed that NA treatment reduced the translocation of radiolabel from root to shoot tissue and accelerated the degradation of this herbicide to a hydroxylated metabolite. Reductions in the lipophilicity and, therefore, mobility of this compound following hydroxylation may account for NA-induced retention of radiolabel in the root system. Hydroxylation of AC 263222 suggested that NA may stimulate the activity of enzymes involved in oxidative herbicide metabolism, such as the cytochrome P₄₅₀ mono-oxygenases. In agreement with this theory, the cytochrome P₄₅₀ inhibitor, 1-aminobenzotriazole (ABT), synergized AC 263222 activity and inhibited its hyroxylation in vivo. NA seed-dressing enhanced the total cytochrome P₄₅₀ and b₅ content of microsomes prepared from etiolated maize shoots. Isolated microsomes catalyzed AC 263222 hydroxylation in vitro. This activity possessed the characteristics of a cytochrome P₄₅₀ mono-oxygenase, being NADPH-dependent and susceptible to inhibition by ABT. Activity was stimulated four-fold following NA seed treatment. Differential NA enhancement of AC 263222 hydroxylase and the cytochrome P₄₅₀-dependent cinnamic acid-4-hydroxylase (CA4H) activity, suggested that separate P₄₅₀ isozymes were responsible for each activity. These results indicate that the protective effects of NA result from enhancement of AC 263222 hydroxylation and concomitant reduction in herbicide translocation. This may be attributed to the stimulation of a microsomal cytochrome P₄₅₀ system. © 1998 SCI.     

Metabolic interactions of agrochemicals in humans

Agrochemicals and other xenobiotics are metabolized by xenobiotic-metabolizing enzymes (XMEs) to products that may be more or less toxic than the parent chemical. In this regard, phase-I XMEs such as cytochrome P450s (CYPs) are of primary importance. Interactions at the level of metabolism may take place via either inhibition or induction of XMEs. Such interactions have often been investigated, in vitro, in experimental animals, using subcellular fractions such as liver microsomes, but seldom in humans or at the level of individual XME isoforms. The authors have been investigating the metabolism of a number of agrochemicals by human liver microsomes and recombinant CYP isoforms and have recently embarked on studies of the induction of XMEs in human hepatocytes. The insecticides chlorpyrifos, carbaryl, carbofuran and fipronil, as well as the repellant DEET, are all extensively metabolized by human liver microsomes and, although a number of CYP isoforms may be involved, CYP2B6 and CYP3A4 are usually the most important. Permethrin is hydrolyzed by esterase(s) present in both human liver microsomes and cytosol. A number of metabolic interactions have been observed. Chlorpyrifos and other phosphorothioates are potent inhibitors of the CYP-dependent metabolism of both endogenous substrates, such as testosterone and estradiol, and exogenous substrates, such as carbaryl, presumably as a result of the interaction of highly reactive sulfur, released during the oxidative desulfuration reaction, with the heme iron of CYP. The hydrolysis of permethrin in human liver can be inhibited by chlorpyrifos oxon and by carbaryl. Fipronil can inhibit testosterone metabolism by CYP3A4 and is an effective inducer of CYP isoforms in human hepatocytes.     

The effects of salinity and temperature on the in vitro metabolism of the organophosphorus insecticide fenitrothion by the blue crab,Callinectes sapidus

The in vitro metabolism of fenitrothion [O,O-dimethyl-O-(3-methyl-4-nitrophenyl)phosphorothioate] by subcellular fractions prepared from the hepatopancrease of blue crabs, Callinectes sapidus, which had been acclimated to either 22°C, 34‰ (parts per thousand); 22°C, 17‰; or 17°C, 34‰ seawater was investigated. In the microsomal fraction, fenitrothion was metabolized to fenitrooxon and 3-methyl-4-nitrophenol. Fenitrothion was metabolized to desmethyl fenitrothion in the cytosolic fraction. The rates of formation of the detoxification products, 3-methyl-4-nitrophenol and desmethyl fenitrothion, were greater in subcellular fractions prepared from crabs which had been acclimated to the lower salinity seawater. The rate of formation of the more toxic metabolite fenitrooxon was greater in the microsomal fraction prepared from crabs which had been acclimated to higher salinity water. All three of these metabolites were formed at considerably faster rates in subcellular fractions from crabs acclimated to and incubated at 22 than at 17°C. These results suggest that enzyme activity contributes to the increased in vivo toxicity of fenitrothion to blue crabs at elevated salinities and temperatures. Also, the observed differences in the rate of formation of the oxon have a greater effect on toxicity than differences in the rate of formation of 3-methyl-4-nitrophenol and desmethyl fenitrothion.