Colin H. Walker

A esterases and their role in regulating the toxicity of organophosphates.

Esterases which can hydrolyse organophosphates without being inhibited by them are termed “A” esterases. Using paraoxon and pirimiphos-methyl oxon as substrates, high “A” esterase activity is found in the liver and plasma or serum of a range of mammalian species. In a study of serum “A” esterases of sheep and humans, over 80% of the activity separated into the high density lipoprotein (HDL) fraction following ultracentrifugation. When HDL fractions from sheep serum were run on Sepharose gel columns, most of the paraoxonase activity separated as a single peak of estimated molecular weight 360000, which corresponds to that of HDL2 of humans.During the course of purification of “A” esterases by three different column procedures, contrasting esterase elution profiles were obtained with organophosphate and pyrethroid substrates. This was strong evidence for the existence of multiple forms of HDL “A” esterases.Levels of “A” esterase activity in plasma and liver of birds were much lower than those of mammals. This appears to be the main reason why birds are much more susceptible than mammals to organophosphates such as pirimiphos-methyl and diazinon which form active oxons that are good substrates for mammalian “A” esterases.No “A” esterase was detected in strains of rust red flour beetle (Tribolium castaneum) which were resistant to organophosphates. Similar observations have been made with strains of other insects resistant to organophosphates, raising the question to what extent esterases of this type are present in insects.

Control enzyme levels in the plasma, brain and liver from wild birds and mammals in Britain

Brain from 47 avian and 17 mammalian species and the liver from 19 avian and 7 mammalian species has been examined for acetyl cholinesterase and nitrophenyl acetate esterase activities. Plasma from 27 avian and 7 mammalian species has been examined for acetyl cholinesterase, cholinesterase, nitrophenyl acetate esterase, glutamate, oxaloacetate transaminase, glutamate pyruvate transaminase, glutamate dehydrogenase, sorbitol dehydrogenase and lactate dehydrogenase activities. The studies have revealed that variations in enzyme activities occur between species but that there are discernible trends within families. The results indicate that comprehensive control enzyme data is necessary in order to assess the effects of exposure to agricultural chemicals in wildlife.

Comparative metabolism of a dieldrin analogue: Hepatic microsomal systems as models for metabolism in the whole animal

Abstract The metabolism of the liposoluble dieldrin analogue HCE † was studied in vivo and in vitro in the rat, rabbit, pigeon and Japanese quail. Comparisons were made between the metabolites formed by liver microsomal preparations and those released from conjugates in the bile and urine. Under both in vitro and in vivo conditions, metabolism was predominantly oxidative in all species with the formation of a major primary metabolite and the progressive conversion of this to two secondary metabolites. The pattern of oxidative metabolism after 30 min of microsomal incubation was broadly similar to that found in bile after 2 hr collection and in urine after 24 hr collection. A minor metabolic route involved hydration of HCE to a trans -diol by microsomal epoxide hydratase and this was found in all species except the pigeon both in vivo and in vitro . There were certain discrepancies between in vitro and in vivo studies with regard to the formation of minor metabolites and to the quantity of trans -diol formed relative to other metabolites. In the case of the rat, the rate of oxidative transformation by microsomes was much higher than the rate of excretion of the metabolites (both figures being expressed in terms of g liver), suggesting that the excretion rate was not limited by the metabolic rate.

Novel protein targets for organophosphorus compounds

Inhibition of tritiated di-isopropyl phosphorofluoridate labelling by a range of organophopshorus compounds was used to screen for novel OP-reactive targets in rat-brain homogenates. Analysis of target proteins was conducted by SDS/PAGE and detection of tritiated proteins using a thin layer chromatography (TLC) linear analyser. Two major sites of 3H-DFP labelling were found with relative molecular masses of 30 and 85 kDa. Rates of reaction of these labelling sites with a range of OP compounds were compared to that of acetylcholinesterase. The 30 kDa band was found to be more sensitive to paraoxon, dichlorvos and diazoxon than acetylcholinesterase. The 85 kDa band was found to be more sensitive to dichlorvos and diazoxon than acetylcholinesterase. Neither labelling band reacted with chlorfenvinphos or demeton-s-methyl at significant rates.

Evidence for an enhanced metabolism of cypermethrin by a monooxygenase in a pyrethroid-resistant strain of the tobacco budworm (Heliothis virescens F.)

Abstract A pyrethroid-resistant strain (PEG87) and a susceptible strain (BRC) of Heliothis virescens were topically dosed at third instar with trans -(1-[ 14 C]cyclopropyl)cypermethrin in acetone. Penetration studies revealed that trans -cypermethrin was absorbed at a significantly faster rate in the susceptible strain as compared to the resistant strain over a 48-hr period. The times for 50% penetration were 11 and 30 hr for the BRC and PEG87 larvae, respectively. Studies on the metabolism of trans -cypermethrin indicated an increased rate of excretion of radioactivity in PEG87 larvae as compared to BRC larvae. The major metabolic product in both strains was polar-conjugated material; trans -cyclopropane carboxylic acid (Cl 2 CA), a product associated with ester bond cleavage, appeared at later time points. The rate of excretion of conjugate by PEG87 larvae exceeded that of BRC larvae; at 12 hr, PEG87 excreted 20 times more conjugate than larvae of the BRC strain. Radioactivity was detected in the haemolymph of both strains. In the PEG87 strain, radioactivity was associated with two metabolites, hydroxylated trans -cypermethrin and Cl 2 CA, the products of oxidative and esterase attack, respectively. Hydroxylated trans -cypermethrin was not identified in the haemolymph of the BRC strain. Two to four hours after dosing, internal concentrations of 14 C activity were two- to fourfold greater in BRC larvae as compared to PEG87 larvae. In the BRC strain, trans -cypermethrin was the most abundant radiolabelled component, while conjugated material represented the highest percentage of radiolabel within the PEG87 larvae. Pretreatment with piperonyl butoxide resulted in a reduction in the excretion of total radioactivity and conjugate from the PEG87 larvae. The absence of hydroxylated cypermethrin and an increase in the level of parent material in those larvae pretreated with piperonyl butoxide substantiates the importance of the monooxygenase system in the detoxification of cypermethrin in the resistant strain.

Polychlorinated biphenyls in fish-eating sea birds—molecular features and metabolic interpretations

Abstract Polychlorinated biphenyls (PCBs) are an important group of environmental pollutants, there being a total of 209 theoretical congeners. Residue analysis of the adipose tissue of five species of fish-eating sea birds from British and Irish coastal waters revealed the presence of up to 60 different congeners. By GC-MS, GC-MSD and high resolution capillary GC-ECD using authentic standards, it was possible to identify and quantify 40 different congeners. Despite the large number of PCB congeners identified only 10 accounted for > 80% of the total PCBs. A PCB congener was identified accounting for 5% of the total PCBs (2,3′,4,4′,5′,6-hexachlorobiphenyl) [168] not usually reported in biological samples. Comparison of the molecular structure for the persistent PCB congeners revealed the lack of meta-para unsubstituted adjacent carbon atoms. It has been shown that meta-para unsubstituted adjacent carbon atoms facilitate the metabolism of PCBs and it is hypothesised that the formation of hydroxy derivatives may depend upon such a requirement.

The classification of esterases which hydrolyse organophosphates: Recent developments

In the IUB classification of 1984, enzymes which hydrolyse paraoxon and other organophosphorous triesters were included in the category of arylesterases--enzymes which hydrolyse phenylacetate (EC 3.1.1.2). With the discovery that some forms of paraoxonase do not hydrolyse phenylacetate, a new entry was made in the revised classification of 1989, Aryldialkylphosphatase (EC 3.1.8.1) under phosphoric triester hydrolases (EC 3.1.8), to distinguish these enzymes from arylesterases. Also some enzymes that hydrolyse phenylacetate do not hydrolyse paraoxon, whereas other enzymes do. Additionally, there is growing evidence for the existence of a number of enzymes which hydrolyse P-F or P-CN bonds of organophosphorous diesters e.g., the nerve gases tabun and soman. These enzymes are in effect organophosphorous acid anhydrolases, and it has been proposed that the earlier entry of (EC 3.8.2.1) now be deleted, and a new entry diisoprophylfluorophosphatase (EC 3.1.8.2) put in its place. Within this category, there is evidence of several enzymes showing different substrate specificities, and different requirements for divalent cations as cofactors, which presents further problems of classification and nomenclature.

Enhancement of malathion toxicity to the hybrid red-legged partridge following exposure to prochloraz

Abstract The agricultural fungicide, prochloraz, which inhibits ergosterol biosynthesis in fungi, was shown to be a potent inducer of liver enzymes in the hybrid red-legged partridge. Pretreatment with a single oral dose of 180 mg/kg resulted in a dramatic potentiation of the toxicity of the organophosphorous insecticide malathion: three out of four prochloraz-pretreated birds died within 10 min from acute poisoning (82–100% inhibition of brain cholinesterase activity) following intraperitoneal administration of 3.3 or 16.7 mg/kg malathion, whereas no significant inhibition of serum cholinesterase activity was seen in control birds 1 hr after administration of the insecticide. Prochloraz pretreatment also resulted in a significant increase in susceptibility to low oral doses of malathion. In vitro metabolism of [ 14 C]malathion by microsomes from control birds resulted largely in the formation of the malathion monoacid, whereas the anticholinesterase, malaoxon, was the main metabolite generated by microsomes from prochloraz-pretreated birds. The dramatic amplification of malathion toxicity following induction with prochloraz is attributed to an increased activation of malathion to its active metabolite malaoxon by one or more induced monooxygenase forms; inhibition of “B”-esterase activity by malaoxon may contribute to this amplification of toxicity.

The metabolism of dieldrin and two of its analogues: The relationship between rates of microsomal metabolism and rates of excretion of metabolites in the male rat

Abstract Malerats receiving re-entrant bile duct cannulae were given [ 14 C]-dieldrin (HEOD) and [ 14 C]-HEOM (15 mg/kg of each) by intraperitoneal injection. Bile collections were made without anaesthesia and nearly all 14 C excretion occurred by this route. Maximal 14 C excretion rates were observed between 20 and 40 min after dosing and these were much lower for dieldrin (3.17 nmoles/kg body wt/min) than for HCE or HEOM (204 and 298 nmoles/kg body wt/min respectively). Induction of liver enzymes by treatment with sodium phenobarbitone caused a significant increase in 14 C excretion in the case of dieldrin (threefold) but not in the case of HCE. The rates of 14 C excretion in vivo were compared with the rates of metabolism in vitro by liver microsomes for each compound. The pattern of primary metabolism observed in vivo was similar to that found in vitro for both HCE and HEOM. Whereas HCE was metabolised predominantly by microsomal monooxygenase attack, both monooxygenase and epoxide hydratase were important in the degradation of HEOM. The rate of microsomal metabolism of HCE and HEOM by non-induced rat liver microsomes was over 100 times greater than that reported for dieldrin using induced rat liver microsomes. Using values expressed in terms of unit body weight, the maximal rates of microsomal metabolism of HCE and HEOM were 5–6 times greater than the corresponding maximal rates of biliary excretion of their metabolites in non-induced rats. HCE concentrations were measured in liver microsomes from non-induced rats dosed with the chemical. The rates of hydroxylation by liver microsomes at these concentrations were of the same order as the corresponding rates of biliary excretion for the hydroxylated metabolites. The rate of microsomal hydroxylation of HCE increased approximately seventeen-fold in terms of liver weight, after phenobarbitone induction. These results suggest that the rate of metabolism can limit the rate of excretion of dieldrin metabolites in the male rat. With HCE and HEOM, however, the maximum metabolic rate is much faster and does not apparently influence the excretion rate. There may be a ‘threshold metabolic rate’ for these highly liposoluble compounds below which the rate of excretion is limited by the rate of metabolism.

Esterase activity in homogenates of three strains of the rust red flour beetle Tribolium castaneum (Herbst)

Abstract 1. Esterase activity was measured in two malathion-resistant and one malathion-susceptible strain of the flour beetle Tribolium castaneum . 2. No A-esterase activity was found in homogenates of any strain. 3. The resistant Kano C strain showed only half of the esterase activity of the other two strains towards α-naphthylacetate, p -nitrophenylacetate and phenylacetate. The significance of this finding in relation to the resistance mechanism of the Kano C strain is discussed. 4. Some properties of the enzymes hydrolysing phenylacetate in insect homogenates and sheep serum are compared.