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In Vivo Investigation of Dietary Exposure to 5 Pesticides

6 Discussion

6.1 CNS-symptoms and brain acetylcholinesterase

No dose related changes in clinical appearance were observed in either the dose-response study of chlorpyrifos or the combined exposure study of the five pesticides. Thus, the clinical observation of severe toxic effect on the central nervous system seen in the initial study was not confirmed. The observation that the rats at autopsy in the initial study appeared more sensitive to sound stimuli after 28 days exposure of the mixture of the five pesticides at the NOAEL-level was not seen in the combined exposure study even though group 5 in the combined exposure study represented a similar dose level as the mid-dose group in the initial study.

Two of the pesticides in the mixture are known to affect CNS. Chlorpyrifos acts by inhibiting acetylcholinesterase and signs of intoxication are stimulation of the parasympatic nerve system. Alphacypermethrin acts by changing the sodium channels by increasing the permeability in the nerve membranes, which causes a general stimulation of neurotransmitters. Since the brain acetylcholinesterase was reduced by 8% and 12% in the low and mid-dose group in the initial study, chlorpyrifos was suspected to be the cause of the intoxication. However, the initial study was performed in 1998 and in 2000 the JMPR evaluation considered only statistically significant inhibition of ±20% of brain acetylcholinesterase to represent a clear toxicological effect (39). Preliminary results, from an ongoing experiment in our laboratory, shows that chlorpyrifos dosed subcutaneous once a week in doses up to 30 mg/kg induced inhibition of brain acetylcholinesterase of approximately 60% without any CNS symptoms. Therefore it can be questioned whether an inhibition of brain acetylcholinesterase of 8% and 12 % in the initial study was the cause of the CNS-symptoms seen. It cannot be excluded that alphacypermethrin contributed to the observed effects on CNS in the initial study, since it is known that alphacypermethrin cause increased sensitivity to sounds and causes high stepping and splayed gait (37). However, in the combined exposure study the data from the behavioural tests and the measurement of motor activity did not indicate any substance related change in the function of the nervous system. Another explanation for the lack of CNS-symptoms and inhibition of acetylcholinesterase in the combined exposure study could be the low dose of mancozeb in the diet only between 18-26 % of the intended. A combination effect of mancozeb and chlorpyrifos and/or alphacypermethrin can therefore not be excluded. Finally the lack of CNS symptoms in the combined exposure study could be a consequence of lowering the high dose from five times the NOAEL of all the pesticides in the initial study to two times NOAEL of chlorpyrifos and the level of NOAEL of the other 4 pesticides in the mixture in the combined exposure study.

6.2 Plasma acetylcholinesterase

The data in the combined exposure study did reveal a slight statistically significant reduction in plasma acetylcholinesterase activity in the males of group 2, 5 and 6. This is considered to be without biological significance due to the very low levels. Preliminary results from an ongoing experiment at Institute of Food Safety and Nutrition show that a reduction of approximately 45% in plasma acetylcholinesterase activity is without change in clinical behaviour.

6.3 Plasma pesticide concentration

Plasma pesticide was measured in blood of alphacypermethrin, bromopropylate, carbendazim and chlorpyrifos after one week and after four weeks. The plasma pesticide concentrations found in samples from the first subset of animals euthanized after one week in the combined exposure study seems to be lower than the concentration in the blood samples taken after four weeks. This difference probably reflects the change in steady state levels from one week of exposure to four weeks of exposure. Mancozeb was not measured in plasma. It was planned to develop and validate a method for measuring mancozeb. However, plasma needed to be stabilized in EDTA and unfortunately we only obtained enough blood from few animals to stabilize blood both in heparin and EDTA.

The active metabolite of chlorpyrifos, chlorpyrifos-oxon, was not found in any samples, but the metabolite TCP was found in all samples from group 2-6 in the combined exposure study. The concentrations seemed to be dose dependent. There did not seem to be a combination effect since the levels were similar in group 2 (NOAEL of Chlorpyrifos) and group 5 (NOAEL of chlorpyrifos, bromopropylate, alphacypermethrin, carbendazim, mancozeb). Some reservations have to be taken for the data on chlorpyrifos-oxon, as there apparently were problems with the methods. It seemed like the use of plasma from the group 1 animals in the first subset caused a degradation of the oxon compounds. This could be due to the enzyme paraoxonase, which rapidly degrade chlorpyrifos-oxon (17). However, further investigation is needed.

6.4 Diet pesticide concentration

Pesticide concentrations were measured in the diet of the combined exposure study. Alphacypermethrin, bromopropylate and carbendazim were found at acceptable levels of the intended (60-90%). Chlorpyrifos was found at acceptable levels in group 4-6 (80-135%), but the concentration in group 3 was 300% higher than intended. Mancozeb was only found at levels of 18-26% of the intended. The result from the analysis of the diet was very disappointing, because the low level of mancozeb makes comparison between the initial study and the combined exposure study very difficult. Analysing the diet retrospectively for the intended content of pesticides as done in the combined exposure study is truly a problem when the content deviate op to 300% from the intended. The stability of the pesticides in the diet is another problem some degradation of mancozeb cannot be excluded, as the diet was stored at a room temperature of 16-18^oC.

The reason for dosing the pesticides through the diet was to resample the human situation, where pesticide residues are consumed in the diet through fruit and vegetables. There are advantages and disadvantages by dosing the animals through the diet. One of the advantages of feeding through the diet is that the intake of the compound is continuously dosed to the animals, whereas dosage by gavage is given as a bolus once or twice a day. Disadvantages is that it can only be estimated how much the rat eats of the diet. Two rats were housed together in a cage due to animal welfare. However, one of the rats in a cage is the dominating animal and will eat more than the other rat. When dosing by gavage it is known exactly what the rat gets which is an advantage.

6.5 Organ weight changes

6.5.1 Liver

The relative organ weight of the liver was increased in both sexes in the combination groups (group 3-6) in the combined exposure study. This indicates that the four pesticides rather than chlorpyrifos are responsible for the weight increase. Others have found increase in liver weight at high dose levels for bromopropylate, carbendazim and mancozeb (35;36;38).

Histological examination revealed a mild degree of centrilobular cell hypertrophy in 8 out of 8 males and 3 out of 8 females in group 6 in the combined exposure study. This is a normal histological finding when liver microsomal enzymes like cytochrome-P450 are induced (1;22). The relative liver weight increase in group 6 was between 17 and 20% in females and between 15 to 20% in males. This is in agreement with Amacher et al. (1998), who found that a weight increase of 20 % and above was associated with hypertrophy of the liver. They found a correlation between increased liver weights and microsomal cytocrome-P450 enzyme induction (1). Both bromopropylate and carbendazim are known to induce liver enzymes (35;36). However, despite the indication of liver enzyme induction there was not an increase of the active metabolite chlorpyrifos-oxon in plasma. An increase in liver metabolism of chlorpyrifos might not be the mode of action of the toxicity seen in the initial study. However, liver metabolism cannot be excluded since there apparently were problems with the methods used to measure chlorpyrifos-oxon concentrations in the plasma.

Chlorpyrifos can be both activated by cytochrome P450 enzymes through a desulfuration reaction to form chlorpyrifos-oxon and detoxified through a dearylation reaction (31). Tang et al. found CYP2B6 to have the highest activation (desulfuration) activity, whereas CYP2C19 had the highest detoxification (dearylation) activity and CYP3A4 had a high activity for both activation and detoxification in human liver microsomes (31). One research group suggested CYP1A1 and CYP2B6 as possible metabolic biomarkers for organaphosphorothioate toxicity in humans (3), whereas another group found CYP2D6 to be important in the microsomal bio activation pathway of chlorpyrifos (26). Vodela et al. (1995) found chlorpyrifos to be an inhibitor of hepatic microsomal drug-metabolising enzymes in rats (33). There might be differences between human and rat metabolism of chlorpyrifos. Most of the above mentioned studies were performed in in vitro human liver microsomes. No cytochrome P450 enzymes were measure in the combined exposure study due to the lack of knowledge of which of the enzymes are involved in chlorpyrifos metabolism in rats. Further investigation on which of the cytochrome P450 enzymes that are important in activation and detoxification of chlorpyrifos in the rat liver microsomes is needed.

Differences between male and female human liver microsomes are seen with females possessing greater activity than males (31), this correspond the higher effect seen in vivo in our laboratory in female rats on brain acetylcholinesterase inhibition.

6.5.2 Thyroid

The absolute and relative organ weight of the thyroid gland in the combined exposure study was statistically significantly increased in groups 3-6 in females, absolute weight was significantly increased in group 3, 5 and 6 in males and the relative weight was increased in groups 2-6 in males. The histological examination of the thyroid gland revealed a mild degree of follicular cell hypertrophy in most animals in group 6 of both sexes. This indicates an increased activity in the thyroid gland in the combination group.

Of 240 pesticides screened by the U.S. Environmental Protection Office (EPA) of Pesticide Programs; 15% (37/240) induced effect on the thyroid follicular cells in rodents (13). It is well established that thyroid-stimulating hormone TSH is the main growth factor for thyroid cells maintaining the differentiated state of the thyroid and controlling thyroid hormone secretion (10). The control of the thyroid hormone in the blood is regulated by a negative feedback mechanism involving the hypothalamus, the pituitary and the thyroid gland. The hypothalamus releases thyrotropin-releasing hormone (TRH), which stimulates the pituitary to produce TSH. TSH stimulates the thyroid to produce thyroid hormones. Cells of both the hypothalamus and pituitary respond to levels of circulation thyroid hormone. When levels of thyroid hormones are low, the outputs of TRH and TSH are raised and the thyroid stimulated to increase the output of thyroxin ^(T4⁾ and triiodothyronine ^(T 3^). This feedback loop helps to maintain thyroid hormone homeostasis (13). The thyroid gland is capable of meeting physiological demands for ^T 4 and ^T3 up to a point. However, beyond that point continuous stimulation of the thyroid may lead to hyperplasia and hypertrophy, which eventually can lead to neoplasia (13). Mancozeb and to some extent bromopropylate are the two pesticides in the mixture known to effect the thyroid gland. It is remarkable that the weight increase of the thyroid gland and the histological changes were present in the combined exposure study despite the low content (18-26%) of mancozeb in the diet. Szepvolgyi et al. (1989) found a dose-dependant hyperplasia of the thyroid gland in rats exposed to mancozeb at 113 mg/kg bw/day and above (30). In combined exposure study the weight increase and the histological changes in the thyroid gland indicate that the toxicity of mancozeb may be enhanced by the other four pesticides.

There are many ways by which chemicals can perturb the thyroid-pituitary homeostasis. Inside the thyroid these are: 1) inhibition of the active transport of inorganic iodide into the follicular cells; 2) inhibition of thyroid peroxidase (TPO) that converts inorganic iodide into organic iodide and couples iodinated tyrosyl moieties into thyroid hormone; 3) damage to follicular cells; and 4) inhibition of thyroid hormone release into the blood (13). Outside the thyroid gland these are: a) inhibition of conversion of ^T4 to ^T3 by 5’- monodeiodinase at various sites in the body and b) enhancement of the metabolism and excretion of thyroid hormones by the liver (13;23). Mancozeb has both intrathyroidal and extrathyroidal sites of action. According to the U.S. EPA Office of Pesticide program, mancozeb caused thyroid follicular cell hypertrophy and hyperplasia, increased the thyroid weight, decreased hormone levels of ^T4 and ^T3 and increased levels of TSH^, decreased iodide uptake and thyroid peroxidase activity (13).

The changes in weight and histology of the thyroid gland in the combined exposure study indicate that the toxicity of mancozeb is enhanced. This could be caused partly by increased metabolism of mancozeb to the metabolite ETU or increased metabolism of the thyroid hormones in the liver, which could perturb the thyroid homeostasis by increasing TSH-secretion. Male rats are more sensitive than female rats with regard to effects on thyroid hormones (13), as it was the case in the combined exposure study.

6.5.3 Thymus

Both absolute and relative weight of the thymus was reduced in all combination groups (group 3-6) in the combined exposure study. There was no weight change in the control group and in the chlorpyrifos group (group 2). In addition, the low organ weights were comparable between groups 3 to 6. This could indicate that the four pesticides (alphacypermethrin, bromopropylate, carbendazim and mancozeb) are responsible for this effect in combination at their respective NOAEL levels. There were no recognisable histological changes. The weight reduction of the thymus could be indicative of the beginning of an involution of the thymus which could be stress related (9;19) or caused by toxicity of one or more of the pesticides. The pesticide deltamethrin is, as alphacypermethrin, another member of the pyrethroid family and is known to cause thymus atrophy by apoptosis (6), and cypermethrin can induce alteration in thymocyte distribution in prenatal exposed rats (27). However, mancozeb is to our knowledge the only pesticide in the combination that is known to directly have affected thymus in dogs at dose levels at 1000 ppm or above. In that study a decrease in thymus weight was observed and the histological examination revealed a thymic cortical depletion (35).

6.6 Blood

The haematological data showing reduction in haematocrite (Htc), haemoglobin (Hb) and red blood cells (RBC) indicate that the exposed male animals in groups 3, 5 and 6 of the combined exposure study suffer from a mild degree of anaemia (4-7%). Since no dose related changes were observed in MCV, MCH and MCHC this anaemia is characterised as normocytic. The initial study did not reveal a similar effect, and there is no obvious reason for this discrepancy. However, the mild degree of the anaemia together with the lower number of animals per group in the initial study could partly account for the different result. Both alphacypermethrin, carbendazim and chlorpyrifos are known to cause a reduction in the number of erythrocytes in rats (36-38). In the beagle dog, mancozeb have caused anaemia in two different studies at dose levels of 113 mg/kg bw/day (35). Anaemia may result from a reduced rate of production, increased rate of destruction of red cells or loss of red blood cells from the circulation due to bleeding (34). The blood has a large regenerative capacity and whether a degree of anaemia of 4-7% is an adverse effect can be discussed. However, the mild anaemia observed in male rats in group 3, 5 and 6 in the combined exposure study supports the theory of a combination effect.

In the initial study the number of white blood cells (WBC) was reduced significantly in males in the mid-dose group. In the combined exposure study a significant reduction in WBC was seen in females in the high dose group. In the initial study the number of neutrophils was increased in males in the mid-dose group, but in the combined exposure study there was not a shift in the differential count. Because the changes in WBC were seen in different sexes in the two studies and there is inconsistency in change of differential counts, this effect could be non-specific or considered incidental. However, carbendazim is known to decrease WBC at high dose levels (38), and a combination effect cannot be excluded.

6.7 Risk assessment

In general the combined exposure study showed statistically significant effects on organ weights for the thymus, the thyroid gland and the liver and histological changes in the liver and the thyroid gland, as well a haematological changes. These changes were found at levels of NOAEL of the individual compounds. At NOAEL of the individual compounds none of these changes were present in the literature (35-38). Thus the effects in the combined exposure study could be interpreted as a combined effect. Effects are seen at lower levels when rats are exposed to the five pesticides in combination than when the rats are exposed to the individual pesticides. On basis of the two present combination studies a true NOAEL is not found, which makes risk assessment difficult. Using NOAEL of the individual pesticides it is important with a large safety factor on ADI. This gives a greater safety margin when risk assessment for adverse health effects is evaluated. The suggested method for evaluation of combined effects by assumption of an additive effect (14) and the method used by The Danish Veterinary and Food Administration (25) seems to give some certainty that there is no health hazard by eating food items containing small amounts of more than one pesticide.

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