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Abstract: Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10 mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg + cy, 10 mg/kg; im, 10 mg/kg + car, 20 mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy > im > car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.
The toxic potential of different pesticides is usually established for each compound individually to extrapolate risk estimation to human beings by exposure to food-borne traces or residues. In reality, exposure to a single pesticide via food or water residues is rare . Usually, it is the combinations of all remaining traces of pesticides and other pollutants that cause toxic effects  acting as synergists, agonists or antagonists. There is a growing evidence of various mutual actions of common pesticide residues from designed toxicological experiments . In the countries of the European Union, in recent years, significant traces of imazalil, cypermethrin and carbendazim have frequently been documented in food plants (https://webgate.ec.europa.eu/rasffwindow/portal/index.cfm?event=searchResultList/2011). Imazalil or (+)-1-(2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl)-1H--imidazole (CAS No., 73790-28-0, 35554-44-0) is a widely used imidazole-antifungal pesticide. Traces of this pesticide are mainly found in citrus fruits and sporadically detected in other fruits and vegetables in significant concentrations (https://webgate.ec.europa.eu/rasffwindow/portal/index.cfm?event=searchResultList/2011) . The extent of exposure to imazalil in everyday consumption is also documented by detection of this pesticide in some commercial soft drinks . This compound is used as a drug (enilconazole) . It has a potential of disturbing hepatocyte homeostasis . Cypermethrin or (RS)-α-cyano-3 phenoxybenzyl-(1RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2 dimethylcyclopropane carboxylate (CAS No., 52315-07-8) is the most worldwide used type II pyrethroid insecticide in agriculture, home pest control, protection of foodstuff and disease vector control . It is highly accumulative, and one of the best examples of this is that traces of it were found alongside dichlorodiphenyltrichloroethane (DDT) in breast milk in endemic areas of South Africa . The toxicity of cypermethrin is well studied in fruit fly, fish, rats and mice and is reported to cause neurotoxicity and endocrine disruption [9–12]. Carbendazim or methyl benzimidazol-2-ylcarbamate (CAS No., 10605-21-7) is a systemic broad-spectrum fungicide controlling a wide range of pathogens . It is also used as a preservative in paint, papermaking and in the leather industry and further used as a preservative of fruits. It is known that carbendazim may cause endocrine disruption and oxidative stress [13,14]. It has also been studied as a pharmacological compound .
In spite of the number of scientific papers on imazalil, cypermethrin and carbendazim in the last years, the majority of experiments describing imazalil, cypermethrin and carbendazim toxicity were conducted in vitro or ex vivo. To the best of our efforts, no publications exploring toxic effects of combined exposure to those three pesticides were found. To expand the existing limited knowledge of physiological changes in vivo, concerning the hepatotoxicity that leads to misbalance of metabolism, the aim was to detect subtle physiological changes in metabolic pathways caused by mixtures of imazalil, cypermethrin and carbendazim allied with histological analysis of liver tissue of poisoned animals . Detection of enzyme activity in serum by their catalytic activity as a reporter of tissue damage is one of the cornerstones of physiology; thus, analysis was carried out by measuring biochemical parameters in poisoned animals and correlated simultaneously to changes in tissue . We simulated combined subchronic exposure that occurs in human beings and animals consuming food contaminated with residues of imazalil, cypermethrin and carbendazim. Two of the analysed pesticides, imazalil and cypermethrin, are potential therapeutic agents; thus, it is especially important to examine every potential aspect of their toxicology. Given results might serve as a risk estimation model and directional guideline to further toxicology evaluation of poisoning of human beings and animals by combined exposure to these three widely used pesticides.
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The study presents results of mutual action of combinations of imazalil, cypermethrin and carbendazim, three frequently found pesticide residues in fruits and vegetables. Existing reports on imazalil, cypermethrin and carbendazim in similar dose/time applications refer to dose-dependent hepatotoxicity of those compounds when applied alone. Previously, we have shown the DNA-damaging effects of singly applied pesticides and their combinations in hepatocytes . Thus, an experiment was designed to analyse the major serum biochemical biomarkers and hepatotoxicity in situ. The aim was to direct further research towards target organs or physiological systems. The results essentially need to demonstrate whether there is a synergistic, potentiative or antagonistic interaction between these pesticides in repeated low doses. The results showed that changes in serum biochemical markers were different and specific in all treated groups, and they indicate that combinations containing carbendazim were highly hepatotoxic.
In all exposed groups, correlation changes in the ratios of enzyme activities indicate effects leading to the disappearance of all significant correlations that existed in the control group of animals.
Cypermethrin had a similar pattern of disturbance in serum activity of all assessed parameters as imazalil. The single major difference was that cypermethrin raised AST activity, while ALT activity remained unchanged compared with control, whereas imazalil had the opposite effect on the same variables. A histological analysis showed a possible explanation of this phenomenon. In liver treated with cypermethrin, there were dispersed necrotic patches found in the tissue that was not found in imazalil-treated livers and carbendazime-treated livers (fig. 1). AST is known to be a cytosolic and mitochondrial enzyme; therefore, elevation in serum in cypermethrin-treated animals might be a result of more pronounced cell damage and leakage of inner cellular enzymes as cypermethrin is a known lipophilic molecule that can easily pass through the cell lipid bilayer and obstruct its integrity . Interestingly, when compared with the first two individually applied pesticides, carbendazim, which was almost double in administered concentration, did not cause severe changes as cypermethrin and imazalil that indicates its different pattern of toxicity. From the overall pattern of enzyme activities and histological findings, it might be concluded that carbendazim in this experiment had milder toxic influence than the other two pesticides. However, in almost all groups, the elevated creatine kinase (CK) activity was present. Elevated CK activity together with higher LDH activity indicates that there was damage to other peripheral tissues as well. Usually, LDH and CK indicate muscle damage. CK elevation is usually indicative of myositis or myocardial damage. Creatine kinase is a catalyst of ATP renewal in peripheral tissues under anaerobic conditions, and LDH in peripheral tissues turns pyruvate into lactate to compensate ATP generation under diminished oxidative phosphorylation. Thus, elevation in those two enzymes also points that there was a hypoxic condition because of pesticide treatment, especially in the cypermethrin and imazalil group and to a lesser extent in the carbendazim group [24–26]. Impaired respiration related to oxidative phosphorylation and rapid depletion of cellular ATP was previously documented for imazalil . Nevertheless, it is obvious that there was an obstruction of transaminase cycles, alanine cycle and Cori cycle (transfer of lactate to glucose which) that occurred in individual pesticides with graded toxic potential. All those cycles are usually a marker of hypoxic states in peripheral tissue with high energetic demands, such as brain or muscle. CK elevation supports this thesis. Based on serum enzyme activity, moderate subtle obstruction of metabolic pathways appeared to have caused unequal metabolic homeostasis of the whole body. However, considering the low dose of individual pesticides applied here, toxic effects are at the starting point towards more systemic obstruction which would be achieved if the doses were higher or exposure longer. Besides metabolic disequilibrium, elevated amylase activity by cypermethrin and imazalil not only points to pancreatic malfunction, but together with all previously analysed enzymes points that the major effects were causing systemic metabolic misbalance of equilibrium of nutrients between liver and catabolic tissues. Although data on imazalil toxicity are scarce, there is evidence from dog and mice experiments that imazalil has rapid absorption and elimination rate, while cypermethrin persists longer in the body. Thus, it may be argued that the slightly different toxicokinetic properties between the two might be responsible for small differences in enzymatic changes and differences in hepatotoxic potential, especially if we are aware that both imazalil and cypermethrin were administered under the same conditions (time/doses/animals). Similar findings with approximately the same doses and time of exposure were found in other vertebrates [28–32]. Consequently, from these results, we established a graded array of toxic potential characteristic for each individual pesticide (cyp > im > car).
When this assortment has been established, it would be interesting to analyse in what way these three pesticides interact. Contrary to findings for individual pesticides, combinations of pesticides caused a decrease in enzymatic activity of assorted liver-bound enzymes.
However, the least extent of damage was noted in the combination of two pesticides that individually caused the highest elevation of enzymes. Histological findings followed this pattern. These apparently antagonizing effects might be explained in a few possible ways.
Decreased activity in serum enzymes, noted in combinations, must be viewed as the dynamic toxic pathological processes in time linked to enzyme production and clearance equilibrium. Thus, the fall of activity may be caused by inhibition of biochemical enzymatic synthesis on a transcriptional level, higher clearance rate (possible damage to kidney but also higher ALP activity as a consequence of active elimination processes through the bile by the enterohepatic pathway) and inhibition of activity caused by pH change or even more specific mechanisms. Similarly, this effect might also be due to inhibition or induction of monooxygenase enzymes or other specific and non-specific biotransformational pathways. This is a very feasible explanation of differences in (decreased) enzyme activities between the combination groups, as it is known that imazalil induces CYP 1A1 and inhibits CYP 3A4 and generally acts as an inhibitor of biotransformational enzymes. Carbendazim inhibits CYP 2D6, and for cypermethrin, it is known that it has little effect on at least eight different cypenzimes in the living organism [31–39]. In fact, the histological finding proves that the combinations of pesticides until the 28th day probably killed cells that contribute to the total serum activity sooner than individual pesticides evoked necrotic changes. The elevated activities that were observed in individual pesticides must have occurred in combinations of pesticides earlier than the day of analysis, and the extent of damage to the cells was more severe than in individual pesticides after the experimental period, and thus, the total enzymatic activity in serum diminished below that of the control animals.
From the results, we conclude that mild toxic pathological obstruction of liver functions, consequently followed by higher leakage of enzymes to cytoplasm, did not stop the progress of the maintenance of homeostasis, accomplished through increased metabolic rate and through induced metabolic pathways in groups treated with individual pesticides.
In groups receiving a combination of pesticides, there was an additive effect that until 28th day caused more severe systemic and hepatic poisoning than in individual pesticides. Serum enzyme profiles strongly suggest that there is some sort of liver necrosis in the hepatic tissue, and histological findings confirm centrilobular and focal necrosis (Piecemeal necrosis) as described in known hepatotoxic agents . This is especially evident in the carbendazim + cypermethrin group that had the most negative weight gain and lowest liver indices and a decrease in serum enzyme activity of at least three measured parameters. This is in concordance with the report of Jacobson et al. .
Overall systemic pathological effects even in these groups were still not recognizable on the systemic level of the whole animal, and the serum enzyme pattern was merely a beginning of total pathological processes which would be even more pronounced if the experiment lasted longer or the doses were higher. Even though the tissue damage observed by histology was considerable, total protein concentration in serum was not affected by individual pesticides or their combinations. As the liver is a major centre of serum protein synthesis, unchanged circulating concentrations support the thesis that even under such condition liver remained functional.
We should bear in mind that the applied doses were at the reported acute NOEL level for the mice, and had we applied them acutely; the toxic effects would have been observable simply on the molecular level of biotransformation/elimination, or they would not have been observable at all. It is necessary to consider this when applying the results to extrapolation of reference doses (RfD, ADI) of these pesticides in food items. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have additive effects. Similarly, as is the case with the poisoned animals in this study, human beings would not develop any systemic poisoning observable at the level of the whole organism. Slight biochemical changes in biomarkers probably would not revile the ongoing hepatotoxic processes either. Finally, the results represent an important addition to exploring low-dose toxicity in combinations of imazalil, cypermethrin and carbendazim, analogous to the ones found in food, and advice that allows residual concentrations may not cause toxic effects, but certain combinations of different pesticides in similar doses may have hazardous consequences. It is evident from this work that repeated low doses, especially the combination of carbendazim with cypermethrin had additive cytotoxic effects on tissue and physiology of metabolic pathways.