An in vitro evaluation of the potential toxicities and interactions of carbamate pesticides

Toxic. in Vitro Vol. 3, No. 2, pp. 91-93, 1989 Printed in Great Britain. All rights reserved

088%2333/89 $3.00+0.00 Copyright © 1989 Pergamon Press plc

AN IN VITRO EVALUATION OF T H E POTENTIAL TOXICITIES A N D INTERACTIONS OF CARBAMATE PESTICIDES T. T. IYANIWURA* Laboratory for General Toxicology, National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands (Received 15 February 1988; revisions received 2 September 1988)

Abstract--The inhibition of rat plasma chofinesterase was used to screen the toxicity of carbamate pesticides /n vitro. All three carbamates studied--aldicarb, carbofuran and oxamyl--inhibited the rat cholinesterase in a dose-dependent manner, as determined by the Ellman technique. The concentrations that produced 25 and 50% inhibition of cholinesterase (IC25 and IC~o) were, respectively, 1 x 10-s and 5 x 10-TM for aldicarb, I x 10-9 and 1.5 x 10-TM for carbofuran, 5 x I0 -~t and 5 x 10-sM for oxamyl and 4 x 10-H and 6.5 x 10-sM for a mixture consisting of equal proportions of all three carbamates. In the interaction study, based on the data for the mixture of all three carbamates, and for the individual carbamates, chofinesterase inhibition by aldicarb was found to be potentiated by carbofuran and oxamyl. The effects of carbofuran were also potentiated by aldicarb and oxamyl but the presence of carbofuran and aldicarb was found to reduce cholinesterase inhibition by oxamyl.

INTRODUCTION The traditional method of evaluating the toxicity of pesticides to non-target species is usually based on the independent actions of single compounds. Unusually high toxicity observed with pesticide combinations (Dubois, 1969; Fmwley et al., 1957) has drawn attention to the potential for interaction between pesticides. This consideration is especially important in the case of food crops for man, since food crops are often exposed to a combination of chemicals. Considering organophosphorus and carbamate pesticides, used both for crop protection and in the control of insect vectors of disease, cholinesterase inhibition is their main mode of toxicity both in target and non-target species (Hayes, 1962). Thus the inhibition of plasma cholinesterase activity has been used to determine the extent of human exposure and the toxicity of these chemicals to man (Copplestone, 1980; Izmirova, 1980; Roberts, 1980). In the following studies we attempted to screen, in vitro, for the potential toxicity of three commonly used carbamate pesticides (used on food crops both as contact and systemic pesticides), using cholinesterase inhibition as an index of toxicity. Concentrations of carhamate pesticides that produced 25 and 50% inhibition (IC25 and ICs0) of the cholinesterase enzyme were determined both for individual compounds and for a mixture of aldicarb (2-methyl-2-(methylthio)propionaldehyde O-(methylcarbamoyl) oxime), oxamyl(N',N'-dimethyl-N-[(methylcarbamoyl)oxy]- lthiooxamimidic acid methyl esters) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl ester). We aimed to identify a n / n vitro technique that could be *Present address: Department of Pharmacology and Clinical Pharmacy, Ahmadu BeUo University, Zaria, Nigeria. Abbreviation: AChE ffi acetylcholinesterase. 91

readily used along with in vivo data to assess to mammalian toxicity of this class of pesticides. MATERIALS AND METHODS Fresh plasma was collected in heparinized tubes from male rats approximately 8wk old (Wistar strain, 250-300g, RIVM, Bilthoven, The Netherlands) and cholinesterase enzyme activity was determined within 2 hr of collection. Plasma rather than a purified cholinesterase enzyme preparation was used in the current investigation for the following reasons: the studies are intended to be used in predicting the toxicity of these compounds to man and the standard procedure for monitoring human exposure to these compounds is the depression of plasma cholinesterase enzyme. Levels of cholinesterase activity in the plasma correlate well with the development of the symptoms of intoxication from the cholinesterase inhibitors (Ngatia and Mgeni, 1980; Rosival and Batora, 1980). Moreover, the Ellman technique (Ellmann et al., 1961) which was used for the cholinesterase assays in the current studies has been found to provide a rapid and most reliable assay for cholinesterase activity in the plasma. In addition, Decinorm E, which is a standard plasma enzyme preparation (Sigma Chemical Co., St Louis, MO, USA), was used to validate the activity of the plasma cholinesterase enzyme. Measurement of enzyme activity was made over a l-rain period. Cholinesterase activity was determined colorimetrically (Ellman et al., 1961; George and Abernethyl, 1983; Lewis et al. 1981) using an LKB reaction rate analyser with a flat bed potentiometer recorder (LKB Biochrom Ltd, Cambridge, UK). Acetylthiocholine iodide was used as the substrate. Thus carbamate (reagent grade, CibaGeigy Ltd, Agrochemical Division, Switzerland) solutions were prepared in distilled water and

T. T. IYANIWURA

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analysed for cholinesterase inhibition using the Ellman et al. (1961) technique. Inhibitor (80/~1) was mixed with 400 #1 of a working solution containing 165 mM-NaCI, 16.5 mM-tris(hydroxyl)methylamine (pH 7.8), 0.75 mM-5,5'-dithiobis-2-nitrobenzoate and 100#1 plasma and incubated for I rain at 37°C. The reaction was started by the addition of 125#1 I3 mM-acetylthiocholine iodide and the increase in absorbance (AA) at 410 nm was determined. A blank consisting of working solution excluding plasma and inhibitor was prepared for all experiments. Decinorm E (Sigma; for plasma enzyme activity validation) was treated exactly as the control plasma and then assayed for activity. The activities of carbamates were expressed as percentage inhibition of cholinesterase activity in pesticide-treated plasma compared with activity in non-treated plasma (controls). Thus enzyme activity in control plasma was considered to be 100% (0% inhibition). To determine whether there was any interaction between the carbamates, a mixture was made of the three compounds in equal proportions. The concentrations of the mixture tested ranged from 10 -I° to 10-SM. The volume of each mixture assayed for cholinesterase inhibition was exactly the same as the volume of the pure compounds used as controls (carbamate or inhibitor volume is 11.3% of overall working volume, physiological saline was added to control plasma to correct for volume). All determinations were made within a linear range of plasma cholinesterase activity to ensure that the enzyme remained unsaturated during the assay period. RESULTS AND DISCUSSION

The three pesticides inhibited the cholinesterase enzyme even at very low (nM) concentrations (Table 1). The 1C25 and ICs0 values, shown in Table 2, were obtained from the log dose-response plots (Fig. 1). The control plasma (i.e. untreated or inhibitor-treated plasma; Table 3) was used to determine the extent of cholinesterase inhibition in the samples treated with pesticides. Thus the percentage inhibition of treated samples ~ (A Absorbanceupte / A A b s o r b a n c e p ~ ) x 100. The co-toxicity coefficient (i.e. the extent to which the toxicity of a pesticide in the mixture is influenced by other pesticides also present; Sun and Johnson, 1960) is given by the relationship: c.c. = ICs0 of pesticide/ICs0 of combination, where c.c. > 1 implies that the combination is synergistic, c.c. < 1 implies that the combination is inhibitory and c.c. -- 1 implies that the combination is additive; in cases where one of the compounds does Table 1. The effects of the carbamates aldicarb, carbofuran and oxamyl, and of a mixture of the three compounds, in equal proportions, on rat plasma cholinesterase activity Carbamate conffll (~/)

Cholinesteraee inhibition (%) Aldicarb

Carbofuran

Oxamyl

Mixture

10-'° 10-9 10-s 10-7 10-e 10-s

8.0:1:0.0 15.0±0.0 27.0+2.0 42.0 ± 0.5 73.0 + 4.0 96.0+1.0

15.0±0.0 23.0±3.0 40.0+2.0 50.0 + 3.0 72.0 ± 3.0 98.0±0.0

5.0+0.5 30.0±0.4 46.0-1-0.3 63.0 + 0.0 84.0 ± 0.0 99.0±0.0

26.0+0.3 27.5+0.0 42.0±0.0 58.0 + 0.0 73.0 ± 1.0 95.4+0.0

Values are means ± SD for four determinations.

Table 2. Carbamate concentrations that produce 25 and 50% inhibition of rat plasma cholinesterase (IC:5 and IC5o). The IC:s and ICs0 values were calculated from the doseresponse data for the compounds and the mixture

Carbamate Aldicarb Carbofuran Oxamyl Mixture

IC2s(M) 1.0 x 10-s 1.0 x 10-9 5.0 x 10-I~ 4.0 x 10T M

ICs0(M) 5.0 x 10-7 1.5 x 10-7 5.0 x 10-s 6.5 x 10-s

The mixtures consisted of equal proportions of aldicarb, carbofuran and oxamyl. Table 3. Plasma cholinesterase activity in samples not treated with pesticide Sample

Activity (AA/min)

Blank Decinorm E Plasma

0.020 + 0.001 0.190 +0.002 0.180 ± 0.002

AA = increase in absorbance Values are means ± S D for four determinations.

not show any toxicity at the test concentrations then its contribution to the combustion compound is undefined. The co-toxicity coefficients of the three pesticides, calculated from the ICs0s in Table 3 were 7.69 for aldiearb, 2.31 for carbofuran and 0.77 for oxamyl. The toxicity of aldiearb was potentiated by earbofuran and oxamyl; the combination was also synergistic for carbofuran (c.c. > 1), but was inhibitory for oxamyl (c.c. < 1). In the present investigation we ensured that pesticide or inhibitor concentration remained rate-limiting. For the enzyme-inhibitor interaction, the pesticide concentration range was especially low (10l°-10 -5 M) to ensure that the enzyme system remained unsaturated throughout the measurement period. All measurements were made within a linear range of plasma cholinesterase activity (see the log dose-response plots, Fig. 1). The insecticidal action of earbamates derives mainly from their ability to inhibit acetylcholinesterase (ACHE), although they are capable of inhibiting other esterases such as pseudocholinesterase. The toxic effects derive primarily from the inhibition of the cholinesterase enzyme (Hayes, 1982). The earbamylation of AChE is unstable and the regeneration of the enzyme is relatively rapid when compared with phosphorylated AChE from organophosphates. In this sense carbamates are less toxic than the organophosphates and for all practical considerations carbamates are regarded as reversible inhibitors of the cholinesterase enzyme. Thus it is important, especially in in vitro measurements of AChE-carbamate interactions, that the reaction time is very short otherwise regeneration of the earbamylated enzyme sets in. Once this happens the data cease to be meaningful and any distinct dose-related effect is lost. A 1-min reaction time was found to be adequate in the present study. Although it is impossible on the basis of in vitro data alone to advance adequate explanations for the mechanism of interaction of chemicals (because of the pharrnacodynamics of interactions of drugs with biological systems and the processes of absorption, distribution and elimination), the potentiation of aldicarb and earbofuran toxicity observed in the

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Log corbomote concn( M I Fig. 1. Log dose-response plot showing the effects of the carbamates aldicarb, carbofuran and oxamyl and of a ndxtur¢ of the three compounds, in equal proportions, on the activity of cholinesteras¢ in rat plasma. Values are means + SD for four determinations.

present study is perhaps best explained in terms of competition for binding sites on proteins other than the cholinesterase enzyme. I f these compounds have a strong affinity for such proteins, competition may take place, so that more molecules o f one or both c o m p o u n d s are available to inhibit the target enzyme, ACHE. A similar mechanism has been suggested for potentiation o f organophosphate toxicity (Fleisher et al., 1963). It must be emphasized, however, that a complete elucidation o f the mechanism o f interaction between carbamates would require further studies in vivo.

REFERENCF_S Copplestone J. F. (1980). Methods for field assessment of exposure to pesticides. In Field Worker Exposure During Pesticide Application. Edited by W. F. Tordoir and E. A. H. van Heemstra. pp. 17-19. Elsevier, Amsterdam. Dubois K. P. (1969). Combined effects of pesticides. Can. med. Ass. J. 100, 173. Ellman G. L., Courtney K. D., Andres V. and Featherstone R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88. Fleisher J. H. L., Harris L. W., Prudhomme C. and Bursel J. (1963). Effects of ethyl p-nitrophenyl thionobenzene phosphate (EPN) on the toxicity of isopropyl methyl phosphono-fluoridate (OB). J. Pharmac. exp. Ther. 139, 390. Frawley J. P., Fuyat H. N., Hagen E. C., Blake J, R. and Fitzugh O. L. (1957). Marked potentiation of mammalian toxicity from simultaneous administration of two anti-

cholinesterase compounds. J. Pharmac. exp. Ther. 121, 96. George P. M. and Abernethyl H. M. (1983). Improved Ellman procedure for erythrocyte cholinesterase. Clin. Chem. 29, 365. Hayes W. J., Jr (Editor) (1982). Carbamate pesticides. In Pesticides Studied in Man. pp. 436-462. Williams and Wilkins, Baltimore. Izmirova N. (1980). Method of determination of exposure of agricultural workers to organophosphorus pesticides. In Field Worker Exposure During Pesticide Application. Edited by W. F. Tordoir and E. A. H. van Heemstra. pp. 169-172. Elsevier, Amsterdam. Lewis P. J., Lowing R. K. and Gompertz D. (1981). Automated discrete kinetic method for erythrocyte acetylcholinesterase and plasma cholinesterase. Clin. Chem. 27, 926. Ngatia J. and Mgeni A. Y. (1980). The effects of continuous exposure to organophosphorus and carbamate insecticides on cholinesterase (ChE) levels in humans. In Field Worker Exposure During Pesticide Application. Edited by W. F. Tordoir and E. A. H. van Heemstra. pp. 63-66. Elsevier, Amsterdam. Roberts D. V. (1980). Blood cholinesterase monitoring of workers exposed to organophosphorus pesticides: theory and practice. In Field Worker Exposure During Pesticide Application. Edited by W. F. Tordoir and E. A. H. van Heemstra. pp. 173-176. Elsevier, Amsterdam. Rosival L. and Batora V. (1980). Consequences for exposure of impurities in pesticide formulation. In Field Worker Exposure During Pesticide Application. Edited by W. F. Tordoir and E. A. H. van Heemstra. pp. 173-176. Elsevier, Amsterdam. Sun Y. and Johnson E. R. (1960). Synergistic and antagonistic actions of insecticide-synergist combinations and their mode of action. Agric. Fd Chem. 8, 163-168.