Early life stages ofClarias gariepinuswere found to be less sensitive to acute dieldrin toxicity than were those of Nile tilapia,Oreochromis niloticus; 96-h LC50values for 37-day-old fry were 11.7 and 4.95 μg liter−1, respectively. The growth ofC. gariepinusfry was unaffected by 30 days of exposure to 2.4 μg liter−1dieldrin under static conditions with water renewal every 96 h, whereas growth ofO. niloticusfry was significantly reduced. AdultC. gariepinusexposed to dieldrin for 30 days, with water changes every 96 h, rapidly absorbed dieldrin from aqueous solution. Dieldrin concentration was measured just before water changes and from an initial concentration of 4.0 μg liter−1, stabilized after 12 days at about 0.075 μg liter−1, indicating that a balance between uptake and excretion and metabolism had been achieved. Dieldrin accumulated in the tissues during these exposures, especially in the liver, where after 30 days the bioconcentration factor relative to initial concentration was about 900. Chronic exposure ofC. gariepinusto dieldrin had no effect on blood hematocrit and hemoglobin, but appeared to slow the growth of catfish and had a clear negative effect on the reproductive potential of mature females.

Environmental risk assessment of chemicals depends on the production of toxicity data for surrogate species of mammals, birds, and fish and on making comparisons between these and estimated or predicted environmental concentrations of the chemicals. This paper gives an overview of biomarker assays and strategies that might be used as alternatives, that is, to replace, reduce, or refine currently used ecotoxicity tests that cause suffering to vertebrates. In the present context a biomarker is a biologic response to an environmental chemical at the individual level or below which demonstrates a departure from normal status. Of immediate interest and relevance are nondestructive assays that provide a measure of toxic effect in vertebrate species and that can be used in both laboratory and parallel field studies. A major shortcoming of this approach is that such assays are currently only available for a limited number of chemicals, primarily when the mode of action is known. Nondestructive assays can be performed on blood, skin, excreta, and eggs of birds, fish, reptiles, and amphibians. An interesting recent development is the use of vertebrate cell cultures, including transgenic cell lines that have been developed specifically for toxicity testing. The ultimate concern in ecotoxicology is the effects of chemicals at the level of populations and above. Current risk assessment practices do not address this problem. The development of biomarker strategies could be part of a movement toward more ecologic end points in the safety evaluation of chemicals, which would effect a reduction in animal tests that cause suffering.

A semi-natural field study was carried out to assess the likelihood of a potentiation of toxicity between the ergosterol biosynthesis inhibiting (EBI) fungicide, prochloraz, and the organophosphorus (OP) insecticide, malathion, in the red-legged partridge (Alectoris rufa). Groups of partridges kept in four large grassland enclosures were exposed to either prochloraz-treated or control wheat for 7 days after which two of the enclosures were sprayed with malathion whilst the remaining two were sham-sprayed. Cytochrome P-450, aldrin epoxidase and 7-ethoxyresorufin-O-deethylase (EROD) activities were found to be significantly higher in the group exposed to prochloraz alone compared to controls, suggesting that induction of the hepatic microsomal monooxygenase system had occurred by ingestion of prochloraz-treated wheat. However, the level of induction produced was not sufficient to cause a potentiation of malathion toxicity. There was evidence for induction of several forms of P-450 recognised by antibodies raised against 1A1, 2C6 and 4A1 in the prochloraz-exposed partridges.

Biochemical biomarkers measure the exposure of organisms to environmental chemicals. They can also provide measures of toxic effect, e.g. where they are based on molecular mechanisms which underly toxicity. Ideally, biomarkers should be sensitive, specific, simple to use and suitable for the assay of material obtained by non-destructive sampling procedures (e.g. of blood). Recently, there has been encouraging progress in the development of several different types of biomarker assays: (1) The measurement of inhibition of serum ‘B’ esterases to monitor exposure of birds to organophosphorus insecticides. (2) The measurement of DNA damage caused by aromatic hydrocarbons. DNA adduct formation has been studied using the 32P-postlabelling technique. Several other techniques are currently under investigation. (3) The measurement of disturbances to the transthyretin-retinol binding protein complex caused by a metabolite of 3,4,3′,4′,tetrachlorobiphenyl. (4) The measurement of precursors of clotting proteins in blood following the inhibition of the Vitamin K cycle by anticoagulant rodenticides. Of these examples, the first is only a biomarker of exposure but the remaining three examples are, in principle, biomarkers of toxic effect since they all represent measures of molecular mechanisms which underly toxicity. Biochemical biomarkers have considerable potential for measuring effects of chemicals under field conditions — especially where carefully selected combinations of them are used.

Pretreatment of hybrid red-legged partridges (Alectoris rufa cross) with a single oral dose of 167 mg/kg malathion resulted in a marked potentiation of the toxicity of the carbamate insecticide, carbaryl, given 1 hr after malathion. Oral administration of 200 mg/kg carbaryl was lethal to 4 out of 12 malathion-pretreated birds, and a further 6 showed symptoms of cholinesterase poisoning. Birds given either malathion or carbaryl alone showed no visible signs of toxicity. The inhibition of serum butyrylcholinesterase activity was found to be high (75-91%) in both corn oil- and malathion-pretreated birds at 1 and 3 hr after dosing with carbaryl, with no significant difference between them. However, malathion and carbaryl given in combination produced an 88% depression of brain acetylcholinesterase activity compared to a 56% depression in corn oil-pretreated birds given carbaryl; this difference was highly significant. Malathion alone produced a small nonsignificant 5% elevation of activity. Concentrations of carbaryl in the serum and brain were found to be 2- and 7.5-fold greater, respectively, in birds pretreated with malathion compared to those given corn oil. In vitro metabolism of [14C]carbaryl in the presence of NADPH from control birds resulted in the formation of two metabolites (46% of the carbaryl was metabolized), whereas metabolism of carbaryl by microsomes from birds pretreated with malathion was substantially less: only 3% of the carbaryl was metabolized. The inactivation of cytochrome P450 by reactive sulfur liberated during the oxidative desulfuration of malathion is thought to be responsible for these differences in carbaryl metabolism. The potentiation of carbaryl toxicity by malathion was attributed to an inhibition of metabolic detoxication, due largely or entirely to the inhibition of certain forms of cytochrome P450. Other types of pesticides metabolized by cytochrome P450s, such as pyrethroids and organophosphorus insecticides not requiring activation for toxicity, may, like carbaryl, be potentiated following inhibition.

The toxicokinetic interactions between the ergosterol-biosynthesis-inhibiting (EBI) fungicides prochloraz, propiconazole, and penconazole and the organophosphorus (OP) insecticides dimethoate, chlorpyrifos, and diazinon have been studied in the hybrid red-legged partridge. The inhibition of serum butyrylcholinesterase (BuChE) activity provided a useful biochemical indicator of the generation of the toxic oxon metabolites of these OP insecticides. Birds pretreated with 180 mg/kg prochloraz tended to show a greater inhibition of serum BuChE activity at 1, 4, and 24 h following oral exposure to either of the OPs dimethoate (3 mg/kg) or chlorpyrifos (9 mg/kg) compared to birds pretreated with corn oil. Prochloraz-pretreated birds also showed a tendency toward an increased inhibition at 24 h following dosing with the OP diazinon (4.3 mg/kg) compared to corn oil controls. In the case of dimethoate, the inhibition of serum BuChE activity was significantly greater in treated birds than controls at 24 h. Birds pretreated with the EBI fungicide propiconazole (200 mg/kg) showed a similar inhibition of serum BuChE activity to those pretreated with corn oil following administration of 167 mg/kg malathion. Pretreatment with the EBI fungicide penconazole (200 mg/kg) produced significantly greater depression of serum BuChE activity at 1, 4, and 24 h after dosing with malathion, when compared to corn oil controls. The tendency toward increased inhibition of serum BuChE activity by each of the OPs in prochloraz-pretreated birds was attributed to an increased activation of the compound to its active oxon form as a consequence of induction of microsomal monooxygenases by prochloraz.

Recent laboratory studies with hybrid red-legged partridges have shown that the interaction of certain combinations of agricultural pesticides can lead to an enhancement of toxicity. In view of this finding, captive feral pigeons (Columba livia) and European starlings (Sturnus vulgaris), in addition to hybrid red-legged partridges (Alectoris rufa cross), were used in a laboratory study to extend investigations of the enhancement of toxicity of the organophosphorus (OP) insecticide malathion in birds predosed with the ergosterol-biosynthesis-inhibiting (EBI) fungicide, prochloraz. Following pretreatment with either 90 or 180 mg/kg prochloraz, both pigeons and partridges showed significantly greater inhibition of serum butyrylcholinesterase (BuChE) activity (1.4- to 2. 1-fold increase for pigeons and 1.3- to 3.8-fold increase for partridges) when dosed with malathion than control birds; this difference was evident at some or all of the blood-sampling time points. On the other hand, starlings given either 180 or 300 mg/kg prochloraz showed no difference from controls in serum BuChE inhibition after dosing with malathion. The enhanced inhibition of serum BuChE in the pigeon and the partridge after malathion dosing was attributed to the increased activation of malathion to malaoxon following induction of the hepatic monooxygenase system by prochloraz. Serum BuChE inhibition may be useful in studying the interactive effects of OPs and EBI fungicides in the field.

The "biomarker" came into common usage, the inhibition of cholinesterases was recognized as a characteristic indicator of poisoning by organophosphorus and carbamate compounds. In the 1940s Schrader synthesized the first organophosphorus insecticide to have widespread use, parathion; and in the late 1950s the first commercial carbamate insecticide, carbaryl, was synthesized. The relatively low persistence of organophosphorus and carbamate pesticides, compared to organochlorine pesticides, together with their effectiveness has led to wide usage of these compounds. Serum cholinesterases are routinely used to monitor exposure of spray operators to organophosphorus and carbamate compounds, and these methods have been further developed to study the effects of these compounds on wildlife. Esterases which are inhibited by organophosphates are classified as B esterases. On the basis of studies with purified enzymes, two main classes of B esterases have been established: cholinesterases and carboxylesterases.

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.

Butyrylcholinesterase (EC 3.1.1.8) (BChE) was purified from pigeon serum to electrophoretic homogeneity by a four-step procedure involving blue sepharose CL-6B chromatography, ion exchange chromatography, procainamide affinity chromatography and gel filtration. An overall 2789-fold purification was achieved, with a final specific activity of 61.35 mumol/min/mg. The purified enzyme separated into two peaks when filtered through a column of Sephacryl S-300, a smaller peak containing the tetrameric form of BChE (C4) and a larger peak containing the monomeric form of BChE (C1). Native polyacrylamide gel electrophoresis (PAGE) of both peaks revealed single protein bands which coincided with esterase activity, with approximate M(r) values of 84,000 and 340,000, respectively. The C1 monomer represented 85-90% of the activity found in the pigeon serum. It is not clear whether this polymorphism of BChE in vertebrates contributes to the wider inter-individual variations observed in xenobiotics elimination kinetics and in the response to the pharmacological and toxic effects of pesticides. PAGE of the monomeric form of the enzyme in the presence of sodium dodecyl sulphate showed only one protein band with a M(r) of 84,000, while that of the tetrameric form revealed two bands, a major protein band (84,000) and a minor band (170,000), representing the monomer and the dimer of the dissociated tetrameric BChE enzyme under reducing conditions. Highly specific polyclonal antibodies were raised in rabbits against the purified enzyme. These antibodies cross-reacted with other avian BChEs, a criterion which make them useful for the immunopurification of other BChEs from different species as well as for biomonitoring and toxicological studies on the role of esterases as an indicator of avian exposure to organophosphorous pesticides.

Preliminary studies indicate that 3-butylthio-1,1,1-trifluoropropan-2-one (BTFP) is a potent, competitive inhibitor of pigeon serum alpha-naphthyl acetate esterase (alpha-NAE); I50, 9.5 nm.The synthesis of an affinity resin with 3-(4-mercaptobutylthio)-1,1,1-trifluoropropan-2-one (MBTFP) as the ligand is reported; alpha-NAE from serum bound strongly to this. To remove protein from the resin, washing with 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) and subsequent boiling in aqueous sodium dodecylsulfate (SDS) were employed. Recovered protein run on SDS-PAGE showed a single major protein band at 58-60 kDa which agrees well with previous determinations of the molecular weights of carboxylesterases.

The most compelling reason for using biomarkers is that they can give information on the effect of pollutants rather than mere quantification of the levels present. The most critical aspect of environmental assessment is for society to decide how much damage it is prepared to tolerate. Once this is decided it is possible to devise tests to enforce these decisions. Suites of biomarkers have the potential to play an important role in environmental assessment.

Biochemical responses of animals to environmental chemicals (biochemical biomarkers) can give measures of exposure, and sometimes also toxic effect. They are particularly valuable where they can be used to measure the toxic effects of chemicals in the field, employing non-destructive sampling methods. Measurements of exposure are useful in the case of non-persistent chemicals (e.g. organophosphorus, carbamate, or pyrethroid insecticides) which are difficult or impossible to detect by chemical analysis. They can also be useful to provide an integrated measure of the level of exposure to a group of related chemicals. Biochemical biomarkers are likely to provide a measure of toxic effect, where they are based upon a molecular mechanism which underlies toxicity. A widely-used biochemical biomarker is cholinesterase depression, which may involve destructive sampling (brain acetylcholinesterase) or non-destructive sampling (serum butyrylcholinesterase). For genotoxic chemicals, techniques which measure DNA damage (e.g. detection of DNA adducts) provide a powerful tool in measuring environmental effects. The detection of biochemical changes caused by anticoagulant rodenticides (e.g. abnormal levels of clotting proteins in blood) provides another example of this approach. In general, the development of simple, sensitive, and specific assays that are 'user-friendly' would open the way for much wider use of biochemical biomarkers in environmental monitoring.

Starlings (Sturnus vulgaris) were dosed with corn oil, demeton-S-methyl (S-[2-(ethyl thio)ethyl]O,O dimethyl phosphorothioate) or triazophos (O,O-diethyl-O-(1-phenyl-1H-1,2,4-triazol-3-yl)phosphorothioate). Brain and serum esterase activities were measured up to 24 h after dosing. A dose response relationship was observed in serum cholinesterase and carboxylesterase activities following dosing with both pesticides. An increase in serum carboxylesterase activity was observed following exposure to a low dose of demeton-S-methyl, whereas inhibition was observed at higher doses. These results highlighted the need for the development of a method for monitoring exposure which takes into account both release and inhibition of the enzyme.

Resistance to cis-cypermethrin was examined in a range of laboratory and field strains of Heliothis virescens (F.). The PEG87 laboratory strain and the DuPont field strain which was reared in the laboratory for a number of generations were both highly resistant to cypermethrin whilst the other field strains were generally less tolerant. In all but the PEG87 strain, larvae were more resistant in the first instar than in the third instar. Resistance in the field strains was especially weak in larger larvae. Use of the synergist piperonyl butoxide (PBO) indicated that the PEG87 laboratory strain and the DuPont strain possessed a resistance mechanism based on enhanced monooxygenase activity and that this was particularly strongly expressed in the PEG87 strain. The mechanism was absent from the Snook, Hearne and Itta field strains examined. A proportion of individuals from the DuPont, PEG87 and all the field strains displayed nerve insensitivity to pyrethroid action. The DuPont strain was comparatively homogeneous with respect to nerve insensitivity with a high proportion of the insects being resistant. It is concluded that nerve insensitivity is widely distributed in the field strains and confers considerable tolerance to first instar insects. The monooxygenase resistance likewise confers high tolerance to first instar insects. Larvae with a combination of both major mechanisms probably possess high tolerance to the pyrethroid throughout larval life.

Conference Article| August 01 1991 Enzymes and resistance to insecticides in Heliothis virescens Alan R. McCaffery; Alan R. McCaffery 1School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Colin H. Walker; Colin H. Walker 1School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Stephen E. Clarke; Stephen E. Clarke 1School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar K. Seung Lee K. Seung Lee 1School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 2AJ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Received: April 22 1991 Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1991 Biochemical Society1991 Biochem Soc Trans (1991) 19 (3): 762–767. https://doi.org/10.1042/bst0190762 Article history Received: April 22 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation Alan R. McCaffery, Colin H. Walker, Stephen E. Clarke, K. Seung Lee; Enzymes and resistance to insecticides in Heliothis virescens. Biochem Soc Trans 1 August 1991; 19 (3): 762–767. doi: https://doi.org/10.1042/bst0190762 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: Cl2CA, trans-cyclopropanecarboxylic acid, PBO, piperonyl butoxide This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| August 01 1991 Toxicity of pesticides to birds; the enzymic factor C. H. Walker; C. H. Walker 1Department of Biochemistry and Physiology, University of Reading, Reading RG6 2AJ Search for other works by this author on: This Site PubMed Google Scholar C. J. Brealey; C. J. Brealey *Wellcome Research Laboratories, Berkhamsted, Herts. HP4 2QE Search for other works by this author on: This Site PubMed Google Scholar M. I. MacKness; M. I. MacKness †Department of Medicine, University of Manchester, Manchester Royal Infirmary, Manchester M13 9WL, U.K. Search for other works by this author on: This Site PubMed Google Scholar G. Johnston G. Johnston 1Department of Biochemistry and Physiology, University of Reading, Reading RG6 2AJ Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (3): 741–745. https://doi.org/10.1042/bst0190741 Article history Received: April 12 1991 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation C. H. Walker, C. J. Brealey, M. I. MacKness, G. Johnston; Toxicity of pesticides to birds; the enzymic factor. Biochem Soc Trans 1 August 1991; 19 (3): 741–745. doi: https://doi.org/10.1042/bst0190741 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: PTH, phosphoric triester hydrolase, HMO, hepatic microsomal mono-oxygenase This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

Brain and serum esterase activities were measured in starlings (Sturnus vulgaris) dosed with corn oil, demeton-S-methyl (S-[2-(ethyl thio)ethyl]O,O-dimethyl phosphorothioate), chlorpyrifos (O,O-diethylO-(3,5,6-trichloro-2-pyridinyl) phosphorothioate) or triazophos (O,O-diethylO-(1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate). Activity was assayed before and after (1) storage at 20°C and (2) chemical reactivation by pyridine-2-aldoxime methiodide (P2AM). During storage, significant spontaneous reactivation occurred in samples from birds dosed with chlorpyrifos or triazophos but not in the birds dosed with demeton-S-methyl or corn oil. The degree of chemical reactivation of samples was dependent on the compound administered, the type of esterase and the time after dosing. Brain acetylcholinesterase appeared to be more readily reactivated than serum esterases by P2AM. Following dosing with the diethyl organophosphorus compounds, spontaneous or chemical reactivation sometimes resulted in levels of esterase activity above those measured before dosing. This indicates that release and/orde novo synthesis had occurred. It is important to take into account spontaneous reactivation during storage when assessing the inhibition of esterase activity.

Serum cholinesterase (BChE) and carboxylesterase (CbE) activities were investigated in ten species of birds. Multiple forms of serum BChE and CbE were also separated by chromatofocusing. Higher CbE activity and a wider range of CbE and BChE forms were present in the sera of omnivorous/herbivorous birds than carnivores. Omnivores/herbivores studied were the starling, house sparrow, tree sparrow, pigeon, partridge and magpie. Serum CbE activities of these species ranged from 0.46 to 2.93-mu-mol/min/mL with 2-6 forms separated by chromatofocusing. 0-6 forms of BChE were separated by the same method. The serum CbE activities of the little owl, tawny owl, barn owl and razorbill ranged from 0.19 to 0.58-mu-moles/min/mL with 0-2 forms separated by chromatofocusing. No ChE forms were present within the pH gradient. These results may be significant in contributing to the understanding of the selective toxicity of organophosphorus and carbamate pesticides.