“L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine (State Enterprise)”, Kyiv, Ukraine

ABSTRACT. Metolachlor and currently its biological active isomer S–metolachlor is one of the most widely used herbicides in the world. Chronic experiments in rats have found hepatocarcinogenic effect of metolachlor, and epidemiological studies have found positive relationship between enzyme exposure to metolachlor and prevalence of liver cancer. Possibility of the influence of harmful impurities contained in technical products on the detected effects is emphasized.

Objective is to study promotor effect of S–metolachlor generics with different hepatotoxicity in carcinogenesis of liver in rats induced by nitrosodiethylamine (NDEA) and analyse possibility of its realisation in human.

Materials and Methods. Experiments were performed in male Wistar Han rats on hepatocarcinogenesis model “NDEA — hepatectomy”. Two specimens of S–metolachlor generics were studied; and their ratio of S/R enantiomers was 87/13 % with different hepatotoxicity. Substances were administered intragastrically in the doses of 1.5,15 and 150 mg/kg body weight for 8 weeks. Animals of the negative control group received water, and positive control — phenobarbital. Promotor effect was evaluated by the standardised parameters of the total area and number of hepatocyte foci expressing γ-glutamyl transpeptidase (GTP).

Results. No clinical signs of the toxic action of S–metolachlor on the rat body induced to carcinogenesis by NDEA were found. Increase in the number and total area of γ-GTP positive foci in the liver of animals on tumorogenic dose of both specimens of S–metolachlor as well as phenobarbital was found. Mean area of focus in the liver of rats on more toxic specimen was lower. The threshold of promotor action of S–metolachlor on hepatocarcinogenesis has been established at the level of γ 15 mg/kg body weight. Analysis of literature data on the mechanism of hepatotoxic action of metolachlor allowed to make a conclusion aboutphenobarbital-like mechanism of promotor action that is realised through constitutive androstane receptor (CAR). This mechanism is species-specific for rodents; therefore, the results of epidemiological studies on the possibility of liver cancer in human cannot be confirmed experimentally.

Conclusion. Tumorogenic dose of S–metolachlor generics with different degree of hepatotoxicity shows promotor effect in NDEA induced carcinogenesis in rat liver. Hepatotoxicity of S–metolachlor inhibits growth of γ-GTP positive foci. The threshold of hepatocarcinogenesis promotion has been established at the level of γ 15 mg/kg body weight. The mechanism of the observed effect is not relevant for human.

Key Words: S–metolachlor, hepatocarcinogenesis initiated by nitrosodiethylamine, Wistar Han rats, γ-glutamyltranspeptidase.

Metolachlor and currently its biological active isomer S-metolachlor is one of the most widely used herbicides in the world.

By its chemical structure, this substance belongs to the class of chloroacetanilides. They inhibit the synthesis of protein in plants. Racemic metolachlor is a mixture (50/50) of right-handed (R) and left-handed (S) optical isomers. In 1997, the use of S-metolachlor was initiated, and it contained of 88 % S-isomer and 12 % R-isomer. This product was more active as herbicide and, therefore, its drive from the market by racemic metolachlor has been started [1, 3]

Extensive volumes of the use of metolachlor in agriculture have attracted the attention of epidemiologist who studies the influence of pesticides on human health [2, 3]. Epidemiological studies conducted in the US under Agricultural Health Study (AHS) project have studied the relationship between the use of metolachlor and cancer morbidity in farmers of Iowa and North Carolina. No statistically significant relationships with the use of metolachlor in AHS studies by multiple risk factors for certain types of cancer: melanoma, pancreatic cancer and colon or rectal cancer, prostatic cancer were found in applicators. This work has established [2] correlation relationship between the content of nitrates and metolachlor in groundwaters and cancer morbidity in children. However, authors could not differentiate the role of other factors in the development of these tumours. At the same time, a positive relationship was found between the use of metolachlor and hepatic cancer and follicular cellular lymphoma morbidity [3]. Chronic experiments in rats have found a hepatocarcinogenic effect of metolachlor. Detection of a positive relationship between hepatic cancer and farmer exposure to metolachlor is the first report showing that increase in the rate of neoplasms in the rat liver as well as hepatotoxic action to the human hepatic cells in vitro induced by it may be observed in human. Authors of the study underline that obtained results relate to metolachlor only, but no to S-metolachlor [3], referring to possible differences of the toxicological effect of their preparative forms and presence of harmful impurities in the technical products [4].

As opposed to R-metolachlor, the carcinogenicity of S-metolachlor has not been studied in chronic experiments. Upon comparison of the toxicological parameters of both isomers, EPA experts have made a conclusion: by its carcinogenicity, S-metolachlor can be classified as its homologue.

According to EPA classification, metolachlor belongs to possible human carcinogens, group C. Chronic experiments in rats on high dose have found an increase in the rate of hepatic tumours in females and positive tendencies in males. The lack of genotoxicity in both metolachlor isomers allowed assuming promotor mechanism of this effect and establish its safe level [1]. Our previous studies have shown the possibility of induction of pre-tumour conditions in the rat liver by S-metolachlor upon nitrosodiethylamine-induced carcinogenesis [5]. However, the lack of data on the mechanism of hepatocarcinogenic action of metolachlor, the role of impurities in its effect and its relevance for human require additional studies.

Objective of the work was to study the promotor effect of S-metolachlor generics with different hepatotoxicity in carcinogenesis of liver in rats induced by nitrosodiethylamine (NDEA) in rats and analyse the possibility of its realisation in human.

Materials and methods. The experiment was conducted at the Center of Preventive and Regulatory Toxicology — State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety of the Ministry of Health of Ukraine” — in accordance with GLP requirements. The studies correspond to the requirements of the Bioethics Committee on animal welfare, the legislation of Ukraine and international organisations.

The experiment was conducted in accordance with the protocol recommended by N.Ito for detection of hepatocarcinogens with our modifications [6,7]. Male Wistar Han SPF rats were in the “clean” zone of barrier type vivarium under the same conditions in T4 type cages, 5 animals in each. The room was equipped with forced ventilation (12 volumes per hour) with prepared air. Temperature and relative humidity of air were registered on a daily basis, temperature fluctuations were 20–21 ^о С, humidity — 45–46 %. Lightning in the rooms — day-light lamps (12 hours of light, 12 hours of darkness). During the experiment, rats received balanced pelleted feed Altromin (Germany) and decontaminated filtered water.

The experiment included male rats with a body weight of 185 ± 10 g received from the nursery of the Center. After quarantine and randomisation by body weight, animals were divided into the groups. Two control groups — positive and negative control (group 1 and 5) and experimental groups (group 2, 3 and 4), 15 animals per each group. In case of signs of any abnormalities or nonconformity during randomisation, animals were rejected.

After acclimatisation, at day 5 of the experiment all animals received a single injection of nitrosodiethylamine (NDEA), 98 % (Sigma, USA) at a dose of 200 mg/kg. NDEA solution was prepared using a normal saline solution in the concentration of 5 %, and it was administered intraperitoneally.

Two generic specimens of S-metolachlor was expected different hepatotoxicity were studied. The first specimen contained 97.2 % of the basic substance, out of which 87.2 % was for S-enantiomer, and the second specimen — 97.7 % of the basic substance, out of which 86.7 % was for S-enantiomer. Therefore, the ratio of the left- and right-handed enantiomers S/R was 87/13 % that corresponded to the international standards [1, 3]. Administration of substances was initiated 2 weeks after NDEA injection. Experiment with the second substance was initiated a week later, and baseline body weight of rats was 205 ± 15 g.

Animals from the groups 2, 3 and 4 received substance at doses of 1.5, 15 and 150 mg/kg body weight, respectively. Rats from the first group (negative control) received water with OP 10 (0.05 % solution), and from the second group (positive control) — phenobarbital sodium, 99 % (Alkaloida Chemical Company, Hungary) at a dose of 37 mg/kg body weight under the same conditions as in experimental ones. Solutions of S-metolachlor and phenobarbital were prepared on a daily basis (ex tempore) using water, with the addition of OP 10. Solutions were administered intragastrically 5 times per week (exposure from Day 1 to 5, Day 6 and 7 — waiting) in the morning before feeding using the metal probe in the volume acceptable for small laboratory animals [8].

Doses were selected based on the data of toxicological evaluation of metolachlor and OECD recommendation [1, 3, 8]. Dose adjustment was performed every 7 days, considering changes in animal body weight over time during exposure. Three weeks after initiation of the experiment, animals underwent partial hepatectomy by Higgins and Anderson, 1931. Two days after hepatectomy administration of the substance was continued for another 5 weeks. Control of animal condition was performed on a daily basis to detect any deviation associated with exposure to the study substance. The behaviour of animals, their mobility, appetite, condition of the hair, skin mucous membranes were evaluated. Animals were sacrificed 24 hours after the last administration of the study substance. Sacrifice was performed in СО₂ chamber with the observance of the animal welfare rules. Necropsy and gross examination of all internal organs were performed after sacrifice. The absolute and relative weight of the liver was measured to detect hepatotoxic action. Histological examinations were performed if needed.

For histochemical analysis, samples of the liver were taken and used for the preparation of 5–10 µm slices using freezing microtome. After fixation of slices in cold acetone, histochemical reaction to detect γ-glutamyl transpeptidase (GTP) — a marker of the transformed hepatocytes — was performed by Gleaner method [7]. γ-glutamyl transpeptidase (GTP) forms in the hepatic tissue during proliferation — positive foci of hepatocytes. The number and size of these foci are the main criteria in the determination of promotor activity of the study product.

After the reaction, the sizes of detected foci of hepatocytes were statistically processed. The number and area of foci per conventional unit of the liver slice plane using software programs were calculated.

The obtained values of the above parameters were processed using mathematical statistics methods to detect differences between experimental groups of animals and control. Data distribution in the sample was primarily established. In case of normal distribution and homogeneity of variables, parametric paired Student’s t-test for independent samples was used, and in the opposite case — non-parametric Mann — Whitney U-test. Difference between parameters of the control and experimental animals was significant at р ≤ 0.05. Statistical calculations were performed using Excel 2010 package of software programs.

Results and discussion. During the entire period of exposure, experimental groups of animals did not show animal death associated with the administration of a substance, excluding the death of the rats due to postoperative complications after partial hepatectomy.

Behaviour, appearance, motor activity of rats in all groups was unchanged throughout the experiment. Animals ate feed and drank water willingly. Upon comparative evaluation of changes in the growth of male Wistar Han rats over time under conditions of the experiment, no changes in the body weight and its gain were established in experimental animals compared with the negative control. After administration of NDEA, body weight of rats in all groups reduced by 4–7 %. Then, the recovery period and repeated reduction of body weight by 0–4 % after hepatectomy was reported. Administration of substances had no effect on body weight changes and its gain in both experiments. Table 1 provided data on the body weight of animals after the end of exposure to S-metolachlor before sacrifice.

Table 1

The absolute and relative weight of the liver

Notes: ¹ — first experiment; ² — second experiment; ³ — р ≤ 0.05 compared to the negative control.

Body weight of animals in the experimental groups did not statistically differ from the rats of either positive or negative control in both experiments. After sacrifice, external examination of animals in the experimental groups did not find any differences in the appearance of rats exposed to S-metolachlor compared to control.

According to the postmortem examination of rats on S-metolachlor, no gross changes were found in the condition of internal organs and tissues compared to control. Upon comparative analysis of changes in the absolute and relative weight of the liver in experimental rats compared to the negative control in the first experiment, no statistically significant differences were found (Table 1). In the second experiment, rats exposed to a high dose showed an increase in the absolute and relative weight of the liver by 18 and 15 %, respectively. Animals on phenobarbital showed a statistically significant increase in the relative weight of the liver by 12 and 11 %, respectively.

Therefore, products with similar content of S-metolachlor in the study doses did not show general toxic action in NDEA initiated rats. However, upon exposure of rats to the second product in a high dose, the hepatotoxic effect was found.

Multiple studies have shown an increase in the proliferation of cells expressing γ-GTP and other glutamyl transferases of the liver under exposure to hepatocarcinogens [6, 7]. These cells form hyperplastic foci which further are turned to nodules and serve as histochemical markers of carcinogenesis. Small amounts of this enzyme are expressed by cholangiocytes and oval cells in the liver of intact animals. A significant amount of this enzyme if expressed by embryonal hepatocyte, as well as by the tumour cells of the liver [9]. γ-GTP is membrane-bound glycoprotein which active centre is located on the external surface of the cell. Such a location allows decomposition of extracellular glutathione and increase of intracellular content of predictors of its synthesis. Therefore, higher content of glutathione is formed. As is known, glutathione ensures metabolism and detoxification of xenobiotics, NDEA in our case, during which direct carcinogens may develop such as genotoxicants that transform these cells making them resistant to the cytotoxic action of metabolites.

Glutathione provides for not only detoxification of xenobiotics, but also increase in redox potential synthetic processes in these cells contributing to their proliferation. Therefore, conditions for proliferation and selection of clones of transformed cells are created, and this is accompanied by the development of tumour stem cells. Promotors stimulate proliferation of transformed hepatocytes and form foci of their accumulation — foci that further are turned into nodules.

The number and size of hyperplastic γ-GTP positive foci in the liver tissue are the main criteria in the determination of promotor activity of the study compound. Table 2 provides data on the influence of S-metolachlor on the formation and growth of γ-GTP positive foci in hepatic tissue of rats.

Table 2

Area and number of hyperplastic γ-GTP foci in the animal liver by groups

Notes: ¹ — first experiment; ² — second experiment; ³ — M ± SD; ⁴ — median; ⁵ — р ≤ 0.05; ⁶ — р ≤ 0.01 compared to the negative control, Mann — Whitney test.

Upon evaluation of the parameters of γ-GTP active foci in the liver of rats, methods of non-parametric statistics were used, since the sample of the obtained results escaped the law of normal distribution. For a more precise comparison of the parameters with the control, the median of the obtained data was determined along with means.

Analysis of the results of action of the first specimen in terms of formation and growth of γ-GTP positive foci of hepatocytes in the rat liver found a statistically significant increase of their mean total area 4.7 times and 11 times in animals on S-metolachlor at doses of 15 and 150 mg/kg, respectively. Mean the number of foci per cm² in the rat liver of these groups was also increased compared with the control 4.8 and 8.3 times, respectively.

Upon the exposure to the second specimen, a statistically significant increase of the mean total area of γ-GTP foci of hepatocytes 4.8 times was observed only in the liver of rats receiving a high dose. The medium dose also showed an increase in this parameter 1.4 times only (p ≥ 0.05).

Despite differences in the ability to induce foci of γ-GTP expressing hepatocytes in the rat liver in both experiments, it can be concluded that change in the total mean area occurs due to increase in the number of foci. Additional formation of such foci in the rat liver may primarily occur due to mutations in hepatocytes induced by the study agent. However, S-metolachlor has no genotoxic potential [1, 3, 4]. Therefore, it can be expected that an increase in the number of these foci is due to the proliferation of NDEA initiated hepatocytes to the detectable size. We did not find data on the effect of S-metolachlor on the proliferation of hepatocytes in the available literature. However, data are available that metolachlor inhibited in vitro proliferation of HepG2 hepatic cells in human via a change in the cellular cycle. Analysis of the results of flow cytometry of the distribution of cells in the cycle has shown that treatment with metolachlor led to the reduction in the number of cells in the G2/M phase and their accumulation in S-phase. Authors observed an increase in necrosis and apoptosis of hepatocytes [10, 11].

Reduction of synthesis processes in hepatocytes is associated with suppression of energy metabolism by metolachlor [12]. Metolachlor cytotoxicity for rat hepatocytes sharply increases at the background of reduced content of intracellular glutathione [13]. Inhibition of growth of normal hepatocytes establishes the lead for the proliferation of NDEA initiated cells expressing γ-GTP. It is known that clones of such cells for which reduced sensitivity to antiproliferative action of xenobiotics and increased sensitivity to mitogens or regeneration signals is typical, form tumour steam cells. Area of focus may be the parameter of occurrence of such clones in this case. Mean area of focus in animals on the first specimen of the substance at doses of 1.5 and 15 mg/kg did not statistically differ from the control, while its statistically significant increase by 12.8 % was observed for the dose of 150 mg/kg. However, this parameter in the second experiment was virtually unchanged in all groups. To our opinion, this is associated with higher hepatotoxicity of this specimen.

Phenobarbital that was used as a positive control increased mean total area and the number of γ-GTP positive foci in the liver by 5 times. Mean area of the nodule also increased, but by 6.4 % only that is associated with hepatotoxicity of this dose.

The above data shows that generic S-metolachlor with different degree of hepatotoxicity in the tumorigenic doses has a promotor effect on NDEA-initiated hepatocarcinogenesis in rats.

According to a contemporary view, an increase in the proliferative activity of hepatocytes resulted in the formation of GTP foci and further — adenomas and carcinomas is the key event in the mechanism of hepatocarcinogenesis. Stimulation of proliferative activity of hepatocytes is performed through a receptor mechanism that involves the induction of enzymes, induction of peroxisome formation, as well as interaction with oestrogens and statins. The cytotoxic effect just as the influence on apoptosis may be implemented both through the direct action of chemical substances with macromolecules and indirectly — through a receptor mechanism. This effect results in the death of hepatocytes and the development of their compensatory regeneration [14]. Hepatectomy in this model is the factor that enhances hepatocyte proliferation. Therefore, cytotoxic action of metolachlor that would result in hepatocyte death, would cause inhibition of liver regeneration which was not found in our experiments, Table 1. The lack of metolachlor effect on the development of necrosis and apoptosis has been shown in the work [11]. Reduction in the content of intracellular glutathione in hepatocytes increases metolachlor cytotoxicity [13]. GTP expression on the surface of transformed hepatocytes increases the content of intracellular glutathione in them and creates a local advantage for their proliferation over normal hepatocytes in the liver of rats exposed to metolachlor.

At the same time, induction of cytochromes Р-450, Cyp2b 1 and Cyp3а 1 in the rat liver with metolachlor, similar to phenobarbital, as well as an increase in the activity of such enzymes as 7–pentoxyresorufin-O-depenthylase and 7–ethoxyresorufin-O-diethylase [12,13] suggest a receptor mechanism of promotor effect through androstane constitutive androstane receptor (CAR). This mechanism and its species-specificity for rodents have been well established by the example of phenobarbital [14].

Summarising the above, it can be concluded the following: Tumorogenic dose of S-metolachlor generics with different degree of hepatotoxicity shows promotor effect in NDEA initiated carcinogenesis in rat liver. Hepatotoxicity of S-metolachlor inhibits the growth of γ-GTP positive foci. The threshold of hepatocarcinogenesis promotion has been established at the level of ≤ 15 mg/kg body weight. Analysis of the potential mechanism of carcinogenic action of these substances is similar to phenobarbital, and it is implemented through the constitutive androstane receptor (CAR). These data do not confirm the conclusions of the authors of epidemiological studies on the possibility of the development of hepatic cancer in human.

In recent times, the US EPA website contains brief annotated information on the study of the original specimen of S-metolachlor, results of which are in line with our conclusions. Based on these studies, US EPA experts classified S-metolachlor as the substance which mechanism of carcinogenic action is not relevant for human [15].

The work was performed within the scope of scientific and research work at the State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety of the Ministry of Health of Ukraine” on the topic “Scientific justification of the current regulatory requirements to the use of pesticides and agrochemicals: prediction of remote effects of action (carcinogenic, mutagenic, teratogenic activity, reproductive toxicity, chronic intoxication)”, state registration No. 0108U007458.

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Надійшла до редакції 11.06.2019 р.