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In Vivo Investigation of Dietary Exposure to 5 Pesticides

3 The five pesticides mode of action, target organ and possible interactions

Below, metabolism and target organs are described for the five pesticides alphacypermethrin, bromopropylate, carbendazim, chlorpyrifos and mancozeb. Based upon these data, the possible combined effects of the exposure to a mixture of the five pesticides are discussed.

3.1 Alphacypermethrin

Alphacypermethrin is a synthetic pyrethroid insecticide. Alphacypermethrin is rapidly absorbed and excreted via urine and faeces. Alphacypermethrin is metabolised by ester cleavage to give cyclopropanecarboxylic acid and the 3-phenoxybenzene moiety, which is excreted as the sulphate conjugate of 3-(4’hydroxyphenoxy) benzoic acid (7). Cypermethrin is a non-systemic insecticide with contact and stomach action (32). Alphacypermethrin is more biologically active than cypermethrin.

Alphacypermethrin is a neurotoxic compound interacting with the sodium channels in the peripheral and central nervous system (15). Alphacypermethrin causes a long-lasting prolongation of the normally transient increase in sodium permeability of nerve membrane during excitation, resulting in long-lasting trains of repetitive firing.(5). Signs of intoxication with alphacypermethrin in 28 and 90 day oral toxicity studies in rats at dose levels of 40 mg/kg bw and above were salivation, high-stepping and splayed gait, hunched posture, hypersensitivity to stimuli, reduced body weight and food consumption (5). In 90 days oral toxicity study in rats increase in relative liver weight, decrease in haemoglobin (males), decrease in mean corpuscular volume (MCV, males) and decrease in eosinophils (males) was seen (37).

3.2 Bromopropylate

Bromopropylate is an acaricide of the class benzilates. Bromopropylate is metabolised into 8 distinct metabolites, these are excreted in faeces, urine and expired air. There is a pronounced sex difference in metabolite pattern in urine where benzilic acid is a minor fraction in males (10-14%) and a major fraction in females (65-72%). Males excrete 90% trough faeces and 6% in urine, whereas females excrete 55% through faeces and 33% in the urine, a small amount is excreted through CO2 expiration (35).

Bromopropylate induces liver enzymes such as cytochrome-P450, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylases, styrene oxide hydrolase’s, cytosol gluthathione S-transferase, UDP-glucoronyl-transferase and testosterone hydrolase. This causes increase in liver weights at high dose levels as a result of functional overload, this is regarded a reversible adaptation (35).

The critical effects seen in 28 and 90 days oral toxicity studies are reduced body weight, increased liver, kidney and testis weight. In long term oral toxicity studies liver and kidney weights were increased, in addition the weight of the testis and the thyroid gland were increased in male rats. The histological changes in the testis were testicular tubular atrophy and oedema. In the thyroid gland the histological changes were follicular cystic dilatation and hyperplasia (35).

3.3 Carbendazim

Carbendazim is a fungicide of the class benzimidazoles. Carbendazim is readily absorbed after oral exposure in laboratory animals and rapidly metabolised. Carbendazim is metabolised to 5-HBC-S (main metabolite in males rats), 5,6-HOBC-Noxide (main metabolite in female rats), 5,6-DHBC-S and 5,6-DHBC-G the two minor metabolites. Carbendazim is excreted in faeces (25% male and 33-38% in females) and urine (36).

Relative liver weight increased in rats fed above 2000 ppm. Feeding carbendazim to rats at 2000 ppm or more resulted in slight-to-moderate induction of several phase-I drug metabolising enzymes: 7-ethoxycoumarin-O-deethylase, biphenyl-4-hydroxylase, aniline hydroxylase, 4-methoxybiphenyl-N-demethylase and cytochrome-c-reductase. The activities of the phase-II drug enzymes glucoronyl transferase I and II and the gluthathione content were moderately-to-markedly increased at this dose (36).

In 90 days oral toxicity studies in rats an increased liver weight, decreased RBC, decreased WBC, increased Alkaline Phosphatase (ALP) in male, decreased blood urea (males) and increased bilirubin were seen. Histological kidney damage with tubular dilatation and hydropic degeneration at 16 mg/kg, fibrosis and congestion at 32 and 64 mg/kg were seen. In a 90 oral toxicity study in dogs increase in liver and thyroid weight and decrease in heart weight was observed (36). Clinical signs of intoxication after a high single dose are generally non-specific. Tetisticular degeneration has been observed after high single doses (>1000 mg/kg bw) (36).

3.4 Chlorpyrifos

Chlorpyrifos [O, O,-diethyl O-(3,5,6-thrichloro-2pyridinyl) phosphorothiate] is a broad-spectrum organ phosphorus pesticide used as an insecticide. Chlorpyrifos is bio activated by the microsomal cytochrome-P450 system to the active oxon metabolite, which is about three times more potent as an acetylcholinesterase inhibitor than chlorpyrifos itself (18). Most bio activation takes place in the liver, while detoxification takes place in the liver and plasma. The degradation step occurs by conversion directly to 3,5,6-trichloro-2-pyridinol (TCP) and diethyl thiophosphate. The oxon can be deactivated by hydrolysis to diethylphosphate and TCP (18;29). A minor reaction pathway is hydrolysis to monethyl 3,5,6-trichloro-2-pyridyl phosphorothionate. The oxon is further metabolised by hydrolysis catalysed by paraoxonase.

Acute toxicity of chlorpyrifos is caused by acetylcholinesterase inhibition in CNS. Acetylcholine is the neurotransmitter of the parasympatic nervous system. Acetylcholinesterase is the enzyme that cleaves acetylcholin in to acetate and choline. An inhibition of acetylcholinesterase increases the amount of acetylcholin available for the parasympatic nervous system and signs of intoxication are therefore stimulation of the parasympatic nervous system and include salivation, dyspnoea, flaccid paralysis, vomiting, piloerection, exophtalmia and diarrhoea. Female animals are generally more sensitive to acute effects of chlorpyrifos than male animals (38).

The critical effects in 28 and 90 days oral toxicity studies in rats are decreased body weights (bw), increased vacuolation of the adrenal zona fasciculata, decrease in red blood cell count (RBC), increase in platelet count (Plt), reduced serum total protein, albumin and globulin concentrations, decrease in alanine aminotransferase (ALAT) and ALP activity in males, decrease serum glucose concentration and increase in specific urine gravity in females. Other targets are acetylcholinesterase inhibition in plasma, erythrocyte and brain (38).

3.5 Mancozeb

Mancozeb is a dithiocarbamate used as fungicide. Mancozeb is rapidly absorbed, metabolised and excreted in both sexes in rats. Mancozeb is metabolised to ethylene thiourea (ETU), ethylenethiuram monosulfide, EBIS, ethylenethiourea-N-thiocarbamid N-acetyl-ethylenediamine, ethylenediamine, ethylene urea, creatine, allantoin plus six unknown metabolites. ETU is the major metabolite. Mancozeb is distributed to various organs with the highest concentrations in thyroid tissue (35).

In 28 and 90 days oral toxicity studies in rats at 1000 ppm increased liver and thyroid weights and decreased body weights were seen. In female rats increased spleen weights were seen at dose levels of 1000 ppm. Serum Thyroxin (T 4) was decreased in rats at 1000 ppm, TSH increased in rats at 250 ppm and plasma cholinesterase increased at 2576 ppm (35).

In rats histological changes were thyroid follicular cell hyperplasia in 90% of the rats receiving 1000 ppm, in the kidney a yellow-brow pigment in the lumen of the cortical tubules was seen of rats at dose levels above 125 ppm, in the adrenal gland hypertrophy of cells in zona glomerulosa was seen at dose level 1000 ppm and in the liver centrilobular cell hypertrophia was seen in males receiving 1000 ppm (35).

In dogs a decrease in thymus weight and an increase in thyroid weight at dose levels at 1000 ppm or above was seen. By microscopy this was recognized as thymic cortical depletion and thyroid follicular cell hyperplasia. In the blood a reduction in RBC, haemoglobin and haematocrit was seen accompanied by increase in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) at dose levels of 1000 ppm for females and 5000 ppm for males. In another dog study females showed frank anaemia at dose levels of 113 mg/kg bw/ day and a dose-related decrease in RBC. In blood chemistry T3, T4 and ALAT was decreased at 5000 ppm, and cholesterol and bilirubin was increased for females at dose levels at 1000 ppm and in males at dose levels of 5000 ppm (35). Long-term animal studies have shown mancozeb to be a multi-potent carcinogen causing tumours in mammae, zimbal gland,  liver, pancreas, thyroid gland, bone and blood system (2). The metabolite ETU of mancozeb is also a thyroid carcinogen (28).

3.6 Possible interaction between the five pesticides

Main critical effects and ADI for the 5 pesticides are presented in Table 1.

The liver is target organ for four of the five pesticides, these are alphacypermethrin, bromopropylate, carbendazim and mancozeb. Carbendazim and bromopropylate induce liver enzymes like cytochrome-P450 and ethoxycoumarin O-deethylase. It is therefore likely that a combination of all or some of the pesticides will cause increased liver weight and induce liver enzymes. Induction of liver enzymes might increase the metabolism of some of the other pesticides, which might increase or decrease their toxicity.

The kidneys, testis and the thyroid gland are organs commonly affected by bromopropylate, carbendazim and mancozeb. Reduced/increased weights of these organs might be expected as a combination effect.

Blood is affected by chlorpyrifos, carbendazim and mancozeb. RBC is reduced at high doses of chlorpyrifos, carbendazim and mancozeb. A reduction in RBC might be expected as a possible result of interaction between the pesticides.

CNS is the target organ of two of the pesticides chlorpyrifos and alphacypermethrin. Chlorpyrifos acts by inhibiting acetylcholinesterase and signs of intoxication are stimulation of the parasympatic nervous system causing salivation, dyspnoea, flaccid paralysis, vomiting, piloerection, exophthalmia and diarrhoea (38). Alphacypermethrin acts by changing sodium channels by increasing the permeability of sodium in nerve membranes during excitation, resulting in long-lasting trains of repetitive firing. Signs of intoxication with alphacypermethrin are salivation, high-stepping and splayed gait, hunched posture, hypersensitivity to stimuli, reduced body weight and food consumption (5). Thus both alphacypermethrin and chlorpyrifos interfere with the function of the nervous system, however, resulting in different clinical signs.

At dose-levels of NOAEL none of the compounds are expected to have an effect by themselves. However, there might be toxicological effect of the mixture due to interaction of the individual compounds when these chemicals are given simultaneously at dose levels of NOAEL. Expected target organs for combined effects of the five pesticides in the mixture are CNS, blood, liver, kidney, testis and thyroid gland.

Table 1. Critical effects and Acceptable-Daily-intake (ADI) in rats or dogs of the five pesticides chlorpyrifos, alphacypermethrin, bromopropylate, carbendazim and mancozeb. ADI is not always based on 28 and 90 oral toxicity studies.


Click on the table to see: ‘‘Table 1‘‘

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