1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 [1]. Usually, it is the combinations of all remaining traces of pesticides and other pollutants that cause toxic effects [2] acting as synergists, agonists or antagonists. There is a growing evidence of various mutual actions of common pesticide residues from designed toxicological experiments [3]. In the countries of the European Union, in recent years, significant traces of imazalil, cypermethrin and carbendazim have frequently been documented in food plants ( 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 ( [4]. The extent of exposure to imazalil in everyday consumption is also documented by detection of this pesticide in some commercial soft drinks [5]. This compound is used as a drug (enilconazole) [6]. It has a potential of disturbing hepatocyte homeostasis [7]. 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 [6]. 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 [8]. 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 [6]. 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 [15].

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 [16]. 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 [17]. 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References


Experiments were carried out according to the guidelines on animal experiments legally prescribed in Croatia (Law on the Welfare of Animals, NN# 19, 1999) and in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86–123, ethical standards in Directive 86/609/EEC and OECD 407 guidelines for subchronic (28 days) toxicity testing in rodents [18]. The experimental protocol was approved by the bioethical committee of the Faculty Science, Zagreb. Inbred Swiss mice 60 ± 5 days, from the mouse colony of the Faculty of Science, University of Zagreb, were used. The animals were maintained on a formulated commercial pellet diet (Pliva, d.d, Croatia) and water was provided ad libitum. The animals were maintained in a 12-hr light/dark cycle at 60% humidity.

Pesticide application and treatment.

Within each group, housed according to treatment and sex (N = 10, five ♀ + five ♂), the mice received low doses close to NOEL doses for rodents of imazalil, cypermethrin (10 mg/kg) and carbendazim (20 mg/kg) and combinations of the same doses as with individual pesticides (imazalil, 10 mg/kg + cypermethrin, 10 mg/kg; imazalil, 10 mg/kg + carbendazim, 20 mg/kg; carbendazim, 20 mg/kg + imazalil, 10 mg/kg) per os repeatedly every 48 hr. A total of 70 mice were used in the study with respect to the RRR concept. According to the treatment, the groups were tagged as follows: I. control, II. Imazalil (im), III. Cypermethrin (cyp), IV. Carbendazim (car), V. Imazalil + Cypermethrin (im + cyp), VI. Imazalil + Carbendazim (im + car), and VII. Cypermethrin + Carbendazim (cyp + car). All three pesticides were of tech. grade 95% obtained from pesticide manufacturer Chromos d.d., Zagreb. They were administered as a corn oil suspension in a volume of 0.2 ml per mouse. The combinations of pesticides were not mixed before administration; they were rather given by separate gauges as individual pesticide in corn oil suspensions in a volume of 0.1 ml of pesticide A + 0.1 ml pesticide of pesticide B per mouse. Control group (N = 10, five ♀ + five ♂) received 0.2 ml of corn oil following the same schedule. Daily, during 28 days, the animals received doses of imazalil, cypermethrin and carbendazim per os, prepared as a corn oil suspension in a volume of 0.2 ml per animal. The control group received the same volume of corn oil. Animal weight was recorded before each treatment, and calculation of the weight gain (table 1) was made as the difference between mice body weight at first and last day of the experiment. To avoid false changes influenced by excessive blood loss caused by frequent sampling, the whole blood samples were collected 24 hr from the last day of the treatment. Blood was collected by intracardiac puncture described in EMPReSS, standard operating procedure [19]. Serum was frozen at −80°C until processed to enzyme assay protocols, within the subsequent 3 days. Control group data were within the normal reference ranges at the end of the experiment, and they were the reference point for comparison with the treated groups [16,20]. The whole experiment was repeated twice, and the statistical analysis showed no difference between the first and the second experimental set-up.

Table 1.  Changes in body and liver weight and their relation through the hepatosomatic index in mice after 28 days of treatment with imazalil, cypermethrine, carbendazime and their combinations expressed as mean ± S.D. and median.
Group (m + f)Δ Weight gain (g)Liver weight (g)Hepatosomatic indexess (×10−3)
  1. aWithin columns, means with superscript (letter) are significantly different from control (p ≤ 0.05).

I. Control+2.04 ± 0.81.12 ± 0.144.04 ± 6.3
II. Imazalil (im)+0.28 ± 1.3a1.13 ± 0.244.90 ± 5.3
III. Cypermethrin (cyp)+1.27 ± 2.41.10 ± 0.144.71 ± 6.2
IV. Carbendazim (car)−0.57 ± 2.6a1.03 ± 0.144.03 ± 6.4
V. Im + cyp−0.68 ± 2.7a1.16 ± 0.248.82 ± 10.5
VI. Im + car−0.22 ± 2.3a1.14 ± 0.146.31 ± 6.0
VII. Car + cyp−1.00 ± 2.3a0.92 ± 0.237.21 ± 7.5a

Histological analysis.

Liver tissue from all animals was collected after the animals were killed and then fixed in buffered 10% formaldehyde PH 7.0 (phosphate puffer). After 48 hr, the fixed tissue was paraffin embedded. Paraffin blocks were cut serially in 10–15 6-μm slices and stained with hemalum and eosin stains as described before.

The biochemistry assays.

The experiment and biochemistry analysis were conducted according to the recommendations of the International Federation of Clinical Chemistry (IFCC) methods in enzymology and were performed with commercial kits (Sigma-Aldrich, St. Louis, MO, USA) on a Hitachi 717 automatic analyser (Hitachi, Chiyoda, Tokyo, Japan). All analysed parameters were measured from non-haemolysed blood serum and liver homogenate at room temperature. Briefly, the activity of lactate dehydrogenase in plasma (LDH-P) (E.C. was measured under 340 nm by pyruvate to lactate continuous turnover reaction measurement reaction. Alkaline phosphatase (ALP) (E.C. was measured at 405 nm with 4-nitrophenilphosphate as a substrate. Aspartate transferase (AST) (E.C. and alanine transferase (ALT) (E.C. were measured at 340 nm by a previously described method. Creatinine concentrations were measured with the method of formation of alkaline picrate complex at 492 nm. Total protein concentration was measured by the method of Lowry.

Statistical analysis.

Statistical analyses were performed using Statistica 8.0 software (Stat-Soft, Tulsa, OK, USA). Each sample was characterized by enzyme activity level considering the group means (±standard deviation of the mean) and median. The unit of measurement was the animal. Multiple comparisons between control and treated groups were performed by means of anova on log-transformed data in order to normalize the distribution and to equalize the variances. Post-hoc analysis of differences was conducted by the Scheffé test to establish the between-group differences. Level of statistical significance was p ≤ 0.05. In the correlation analysis of enzymatic activity, only significant (p ≤ 0.05) r-values found after the correlation analysis are shown in table 3 [21].


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Weight gain, liver weight and liver indices.

The ratio of weight gain, liver weight and hepatosomatic index at the end of the experimental period are presented in table 1. The treated groups had significantly lower (p ≤ 0.05) weight gain than controls except in the group treated with cypermethrin. The weight gain was positive in the control and in the groups treated with imazalil and cypermethrin. However, in the groups treated with carbendazim and combinations of pesticides, weight gain was slightly negative. Liver weight remained unchanged in all treated groups except in the group treated with the combination of cabendazim + cypermethrin. These animals had slightly lighter but not significantly lower liver weight. This group was the only one with prominently lower and statistically significant average hepatosomatic index (p ≤ 0.05). All other groups did not have hepatosomatic index difference than the control group except for the combination of imazalil + cypermethrin.

Biochemical markers in serum.

Exposure to imazalil, cypermethrin and carbendazim and their combinations did not affect the total protein concentration in serum (table 2).

Table 2.  Changes of biochemical cytotoxicity markers in serum of male and female mice after 28 days of treatment with imazalil, cypermethrine, carbendazime and their combinations as mean ± S.D. and median.
Group (m + f)TP (g/l)LDH (U/l)AST (U/l)ALT (U/l)ALP (U/l)CK (U/l)Creatinin (μM)Amylase (U/l)
  1. a,b,c,d,e,f,gWithin column means with different superscripts (letters) are significantly different (p ≤ 0.05). Different letters represent different treatment groups, respectively, starting from a control group; NSWithin column means are not significantly different from the control group.

I. Control56.6 ± 6.9771.2 ± 107.1126.8 ± 38.644.9 ± 13.662.2 ± 21.51781.8 ± 939.329.9 ± 3.42153.6 ± 855.4
II. Imazalil (im)58.4 ± 4.1NS930.6 ± 180.0acde123.5 ± 40.4NSfd49.8 ± 12.7acdefg77.3 ± 14.0adefg3222.8 ± 1365.5acedefg29.9 ± 3.0NSg2872.5 ± 1129.8acdefg
III. Cypermethrin (cyp)58.3 ± 4.5NS1032.8 ± 323.5abdefg135.3 ± 55.0abefg46.0 ± 11.1NSbg72.4 ± 13.9adefg2454.6 ± 1413.9abefg30.6 ± 3.3NSg2550.9 ± 770.4acdefg
IV. Carbendazim (car)55.8 ± 3.9NS812.2 ± 206.0NSbceg142.5 ± 56.6abcfg40.9 ± 8.3NSbg67.0 ± 14.5NSbc2921.6 ± 1365.5abefg29.4 ± 3.1NSg2274.5 ± 579.9NSbcg
V. Im + cyp54.4 ± 5.1NS780.7 ± 167.4NSbcdeg120.3 ± 54.3NScdf43.1 ± 11.2NSbg62.8 ± 19.3NSbc1720.8 ± 1203.2NSbcdfg28.0 ± 2.7NSg2121.7 ± 598.5NSbcg
VI. Im + car58.2 ± 6.1NS923.4 ± 110.1acde103.0 ± 25.9abcdeg41.8 ± 29.2NSbg69.6 ± 16.1abce2099.1 ± 1126abcde30.4 ± 4.2NSg2312.5 ± 410.9NSbeg
VII. Car + cyp55.7 ± 5.8NS924.8 ± 335.7acde121.3 ± 30.7NScdf28.4 ± 16.4abcdef65.6 ± 26.2NSbcf2168.4 ± 1383.8abcde26.4 ± 3.1abcdef1800.0 ± 566.3abcdef

LDH activities in serum (table 2) were significantly (p ≤ 0.05) higher in animals treated with imazalil, cypermethrin and in the combinations containing carbendazim, compared with the control group. In all other treated groups, serum LDH activity did not differ significantly from the control group.

From three individually applied pesticides, cypermethrin and carbendazim significantly (p ≤ 0.05) elevated serum AST activity (table 2) compared with the controls. Carbendazim caused the highest elevation of AST activity in comparison with the control and other treatment groups. Combinations of imazalil + carbendazim caused significant (p ≤ 0.05) decrease in serum AST activity.

ALT activity was elevated (p ≤ 0.05) in serum of animals treated with imazalil (table 2). A pronounced decrease (p ≤ 0.05) especially of ALT activity was recorded in serum of the group treated with carbendazim + cypermethrin.

ALP activity was significantly higher (p ≤ 0.05) in serum of groups exposed to imazalil and cypermethrin and combinations of imazalil + carbendazim compared with control (table 2).

In imazalil-treated animals, serum creatine kinase activity (table 2) was the highest of all groups (p ≤ 0.05), but it was also elevated (p ≤ 0.05) in all other groups except in the one treated with imazalil + cypermethrin in comparison with control.

Serum creatine concentration (table 2) was not different between treated groups and the control except in the group receiving the combination of carbendazim + cypermethrin where it was lower (p ≤ 0.05) than in any other group.

Amylase activity in serum (table 2) was elevated (p ≤ 0.05) in the imazalil and cypermethrin group and lowered (p ≤ 0.05) in the carbendazim + cypermethrin group. In all other groups, amylase activity remained similar to the control group. Within each treatment group, men and women had the same overall pattern of increase or decrease in serum enzyme activity typical for each treatment group (table 2). The presented standard deviations clarifies the range of enzyme activity (for the species) in which both sexes are included. Similarly, the pattern of induced histological damage depended on exposure to tested chemicals and was also similar in both sexes.

Liver histology.

Liver tissue of unexposed, control animals is presented in fig. 1 together with liver sections from animals treated with individual pesticides. The liver of animals treated with three combinations of pesticides is presented in fig. 2. Imazalil caused changes in heterochromatin and rather swollen appearance of hepatocyte nuclei (fig. 1). Even though imazalil did not disturb the overall architecture of the lobe, a sporadic cell death could be seen in areas close to central vein, but not as pronounced as in the other treatment groups. On the contrary, cypermethrin (fig. 1) caused noticeable sporadic focal necrosis which was not present in the imazalil-treated animals. Changes in heterochromatin caused by cypermethrin were similar as in the imazalil-treated groups. In animals exposed to carbendazim (fig. 1), heterochromatin had similar appearance as in the other two individually applied pesticides, without necrotic changes as in cypermethrin, but with more pronounced sporadic cell death of individual cells around the central lobule area than in the imazalil-treated animals. In hepatocytes of animals treated with a combination of imazalil and cypermethrin (fig. 2), heterochromatin was condensed and the borders of liver lobule were lost because of cell death. However, this combination did not cause highly pronounced necrotic changes that were detected in animals treated with combinations containing carbendazim (fig. 2). The described patterns were seen in all animals belonging to a specific treatment group regardless of sex within each treatment group.


Figure 1.  Liver tissue of animals exposed to the individual pesticides, imazalil, cypermethrin and carbendazim, compared with liver of unexposed animals. I. Control (Magnifications A: 100× B: 400×) Liver of unexposed animals characterized by normal architecture of the liver lobe normally condensed heterochromatin. II. Imazalil (Magnifications A: 100× B: 400×) Liver of animals treated with imazalil is characterized by normal architecture of the liver lobe with slight indicies of ischaemic processes across tissue, but without zonal or focal ischaemic processes. In imazalil-treated animals, heterochromatin and nuclei have swollen appearance (lightning arrow), and cells around blood vessel have disturbed membrane integrity. III. Cypermethrin (Magnifications A: 400× B: 400×) Liver of animals treated with cypermethrin has visible zonal and focal necrosis in liver tissue (arrow). In (A), cells surrounding the central vein are dying, but also patches of necrotic cells are found in distal areas of the liver lobe, which is shown in (B). IV. Carbendazim (Magnification A: 100× B: 400×) Liver of animals treated with carbendazim has disturbed lobe archiceture caused by death of cells and visible changes in heterochromatin of nuclei compared with the control hepatocytes (lightning arrow). Kupffer cells are numerous.

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Figure 2.  Liver tissue of animals exposed to combinations of imazalil, cypermethrin and carbendazim. V. Imazalil + cypermethrin (Magnifications A: 100× B: 400×) Liver of animals treated with the combination of imazalil + cypermethrin shows nuclear changes in heterochromatin (lightning arrow), but although a sporadic cell death is noted, there are no pronounced necrotic processes as in the other two combinations. VI. Imazalil + carbendazim (Magnifications A: 100× B: 400×) Liver of animals treated with the combination of imazalil + carbendazim, pronounced ischaemic necrosis spreading from the centre of the lobe (arrow). VII. Carbendazim + cypermethrin (Magnifications A: 100× B: 400×) Liver of animals treated with the combination of cypermethrin + carbendazim has very pronounced necrosis (arrow) and cell death higher than in the other treatment groups.

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Correlation analysis of relations between serum enzymes in treated and non-treated animals.

Correlation coefficients (r-values) that were found to be significant in untreated animals and their corresponding changes after exposure are shown in table 3. The columns contain the dependent variables of the control group and show the loss of correlation between the activity levels because of poisoning. In the control group of animals, the high, positive correlation dependence (p ≤ 0.05) was noted between the activity of AST, ALT and LDH, and a negative correlation between ALT and ALP. Correlation of ALT:LDH in the imazalil + carbendazim group remained similar to control. Similarly, AST:LDH correlation because of exposure to cypermethrin became more pronounced than in the control group. Owing to the rise in serum LDH and unchanged AST, this particular correlation changed from mild to high because of slope change towards r = 1. Besides the two very significantly positive correlations in the groups treated with cypermethrin and combinations of imazalil + cypermethrin between AST:LDH and ALT:LDH in all other groups, the correlation ties became very low and insignificant.

Table 3.  Changes of correlations between biochemical cytotoxicity markers in serum of male and female mice after 28 days of treatment with imazalil, cypermethrine, carbendazime and their combinations.
Correlated enzyme activitiesCorrelations within serum
AST versus LDHALT versus LDHALT versus AlP
  1. All measured parameters were tested for the correlations between activities in each dose group, but only significant links between the enzymes (*p ≤ 0.05) are presented as underlined values. Columns represent the significant correlations estimated in control animals that were lost because of pesticide treatment.

I. Control0.738*0.879*−0.802*
II. Imazalil (im)0.3120.5580.213
III. Cypermethrin (cyp)0.928*0.5060.496
IV. Carbendazim (car)0.486−0.3780.082
V. Im + cyp0.2620.2250.077
VI. Im + car0.3390.993*−0.542*
VII. Car + cyp0.0290.5090.054


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 [22]. 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 [23]. 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 [27]. 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 [40]. 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. [2].

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.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are indebted to all employees at the Department of Animal Physiology, who showed great persistence during the laboratory experiments. The work was a part of projects supported by the Ministry of Science, Education and Sports of the Republic of Croatia, no. 119-0000000-1255.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Insitoris L, Siroki O, Undeger U, Desi I, Nagymantenji I. Immunotoxicological effects of repeated combined exposure by cypermethrin and the heavy metals lead and cadmium in rats. Int J Immunopharmacol 1999;21:73543.
  • 2
    Jacobsen H, Ostergaard G, Lam HR, Poulsen ME, Frandsen H, Ladefoged O et al. Repeated dose 28-day oral toxicity study in Wistar rats with a mixture of five pesticides often found as residues in food: alphacypermethrin bromopropylate, carbendazim, chlorpyrifos and mancozeb. Food Chem Toxicol 2004;42:126977.
  • 3
    Groten JP. Mixtures and interactions. Food Chem Toxicol 2000;38:S6571.
  • 4
    Naoki Y, Yumi A, Tomofumi M, Takao M. Rapid determination of five post-harvest fungicides and metabolite in citrus fruits by liquid chromatography/time-of-flight mass spectrometry with atmospheric pressure photoionization. Food Control 2010;21:2126.
  • 5
    Garcia-Reyes JF, Gilbert-Lopez B, Molina-Diaz A, Fernandez-Alba AR. Determination of pesticide residues in fruit-based soft drinks. Anal Chem 2008;80:896674.
  • 6
    Kamrin MA, Montgomery JH. Agrochemical and Pesticide Desk Reference. Interactive CD. Chapman & Hall CRC net BASE, London, 2000.
  • 7
    Van Der Heiden E, Bechoux N, Sergent T, Ribonnet L, Schneider YJ, Muller M et al. Imazalil is an aryl hydrocarbon receptor (AhR) antagonist in AhR-dependent reporter human and rat hepatoma cells. Toxicol Lett 2007;172:S2034.
  • 8
    Bouwman H, Sereda B, Meinhardt HM. Simultanious presence of DDT and pyrethroid residues in human breast milk from amalaria endemic area in South Africa. Environ Pollut 2006;144:90217.
  • 9
    Al-Hamdani NMH, Yajurvedi HN. Cypermethrin reversibly alters sperm count without altering fertility in mice. Ecotoxicol Environ Saf 2010;73:10927.
  • 10
    Elbetieha A, Da`as SI, Khamas W, Darmani H. Evaluation of toxic potential of cypermethrin pesticide on some reproductive and fertility parameters in male rats. Arch Environ Contam Toxicol 2001;41:5228.
  • 11
    Kumar S, Gutam AK, Agarawal KR, Shah BA, Saiyad HN. Determination of sperm shape abnormality and clastogenic potential of cypermethrin. J Environ Biol 2004;25:18790.
  • 12
    Larsen SB, Giwercman A, Spanò M, Bonde JP. A longitudinal study of semen quality in pesticide spraying Danish farmers. The ASCLEPIOS study group. Reprod Toxicol 1998;12:5819.
  • 13
    Muthuviveganandavel V, Muthuraman P, Muthu S, Srikumar KJ. Toxic effects of carbendazim at low dose levels in male rats. J Toxicol Sci 2008;33:2530.
  • 14
    Sangeetha R. Activity of superoxide dismutase and catalase in fenugreek (Trigonella foenum-graecum) in response to carbendazim. Indian J Pharm Sci 2010;72:1168.
  • 15
    Yenjerla M, Cox C, Wilson L, Jordan MA. Carbendazim inhibits cancer cell proliferation by suppressing microtubule dynamics. J Pharmacol Exp Ther 2009;328:3908.
  • 16
    James RW. The relevance of clinical pathology to toxicology studies. Comp Haematol Int 1993;3:1905.
  • 17
    Rej R. Aminotransferases in disease. Clin Lab Med 1989;9:66787.
  • 18
    OECD. Organization of economic cooperation and developments guideline for the testing of chemicals. Repeated dose 28-day oral toxicity study in rodents. OECD Guidelines 1995;407:18.
  • 19
    Green E, Georgios V, Koutos G, Heena V, Blake A, Weekes J et al. EMPReSS-European mouse phenotyping resource for standardized screens. Bioinformatics 2005;21:293041.
  • 20
    Matsuzawa T, Nomura M, Unno T. Clinical pathology reference ranges of laboratory animals. Working group II, nonclinical safety evaluation subcommittee of the Japan pharmaceutical manufacturers association. J Vet Med Sci 1993;55:35162.
  • 21
    Zar JH. Biostatistical Analysis, 4th edn. Prentice-Hall Int, London, 1999;931.
  • 22
    Đikić D, Mojsović-Ćuić A, Čupor I, Benković V, Horvat-Knežević A, Lisičić D et al. Carbendazim combined with imazalil or cypermethrin potentiate DNA damage in hepatocytes of mice. Hum Exp Toxicol 2011; Doi: 10.1177/0960327111417910. [E-pub ahead of print].
  • 23
    Manna S, Bhattacharyya D, Mandal TK, Das S. Repeated dose toxicity of alpha-cypermethrin in rats. J Vet Sci 2004;5:2415.
  • 24
    Kaneko JJ, Harvey JW, Bruss ML. Clinical Biochemistry of Domestic Animals. Academic Press Inc., Elsevier Science and Technology, Int., 1997;916.
  • 25
    Edge K, Chinoy H, Cooper R. Serum alanine aminotransferase elevations correlate with serum creatine phosphokinase in myiositis. Rheumatology 2006;45:4878.
  • 26
    Antonelli AC, Torres GAS, Soares PC, Mori CS, Sucupira MCA, Ortolani EL. Ammonia poisoning causes muscular but not liver damage in cattle. Arq Bras Med Vet Zootec 2007;59:813.
  • 27
    Nakagawa Y, Moore G. Cytotoxic effects of postharvest fungicides phenylphenol, thiobendazole and imazalil on isolated rat hepatocytes. Life Sci 1995;57:143340.
  • 28
    Jagvinder K, Sandhu HS, Kaur J. Subacute oral toxicity of cypermethrin and deltamethrine in buffalo calves. Indian J Anim Sci 2001;71:11502.
  • 29
    Khan A, Faridi HAM, Ali M, Khan M, Siddique M, Hussain I et al. Effects of cypermethrin on some clinico-hemato-biochemical and pathological parameters in male dwarf goats (Capra hircus). Exp Toxicol Pathol 2009;61:15160.
  • 30
    Yousef MI, El-Demerdash FM, Kamel KI, Al-Salhen KS. Changes in some hematological and biochemial indicies of rabbits induced by isoflavonon and cypermethrin. Toxicology 2003;189:22334.
  • 31
    Sergent T, Dupont I, Jassogne C, Ribonnet L, van der Heiden E, Scippo ML et al. CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides. Toxicol Lett 2009;184:15968.
  • 32
    Almli B, Egaas S, Christiansen A, Eklo O, Lode O, Källqvist T. Effects of three fungicides alone and in combination on gluthatione S-transferase (GST) and cytochrome P-450 (CYP1A1) in the liver and gill of brown trout (Salmo trutta). Mar Environ Res 2002;54:23740.
  • 33
    Maurice M, Picahrd L, Daujat M, Fabre I, Joyeux H, Domerque J et al. Effects of imadazole derivates on cytochrome P450 from human hepatocytes in primary culture. FASEB J 1992;6:7528.
  • 34
    Navas JM, Chana A, Herradon B, Segner H. Induction of cytochrome P450 1A (CYP1A) by clotrimazole a non-planar aromatic compound. Computational studies on structural features of cltrimazole and related imadazole derivates. Life Sci 2004;76:699714.
  • 35
    Rodrigues AD, Lewis DF, Ioannides C, Parke DV. Spectral and kinetic studies of imidazole anti-fungal agents with microsomal cytochromes P-450. Xenobiotica 1987;17:131527.
  • 36
    Ronis MJ, Ingelman-Sundberg M, Badger T. Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail (Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs). Biochem Pharmacol 1994;48:195365.
  • 37
    Sanderson JT, Boerma J, Lansbergen GW, van den Berg M. Induction and inhibition of aromatase (CYP19) activity by various class of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmcol 2002;182:4454.
  • 38
    Sun G, Thai SF, Tully DB, Lambert GR, Goetz AK, Wolf DC. Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver. Toxicol Lett 2005;155:27787.
  • 39
    Vingaard AM, Hnida C, Breinholt V, Larsen JC. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol In Vitro 2000;14:22734.
  • 40
    Nagano K, Umeda Y, Saito M, Nishizawa T, Ikawa N et al. Thirteen-week inhalation toxicity of carbon tetrachloride in rats and mice. J Occup Health 2007;49:24959.