Metabolic disorders, effects of obese genes and imbalance of fatty tissue hormones in patients after acute and chronic intoxication with pesticides

  • Authors: N.M. Bubalo, G.M. Balan
  • UDC: 615.91:056.52:615.27
  • DOI: 10.33273/2663-4570-2018-82-83-2-3-51-70
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State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine

Abstract. The objective is to study the incidence of metabolic disorders and effects of obese genes in patients after acute and chronic intoxication with pesticides and to justify methods for evaluating their severity to optimize differentiated therapy and prevention. Material and methods. In 104 agricultural workers after acute poisoning with 2,4-D based herbicides, organophosphorus pesticides and synthetic pyrethroids, and 66 patients with chronic intoxication with pesticides in the initial period and a year later, parameters of oxidative stress, carbohydrate and fat metabolism were studied, depending on the development of toxic liver damage syndrome. In patients after acute poisoning with 2,4-D-based herbicides, imbalance of fatty tissue hormones — leptin, resistin, adiponectin and TNF-α — has also been studied.
Conclusion. Dynamic observation has made it possible to establish that in those who have had acute and chronic intoxication with pesticides with toxic liver damage syndrome, metabolic disorders and effects of obese genes develop as the progression of hepatosteatosis develops. The increased level of fatty tissue hormones in the blood — leptin, resistin and TNF-α, at the background of a slight decrease in adiponectin levels in patients who have had poisoning with 2,4-D-based herbicides, allows predicting an increased risk of a progressive course of steatohepatosis and obesity, prevention of which is based on the long-term use of metformin and statins.
Key words: metabolic disorders, effects of obese genes, imbalance of fatty tissue, acute and chronic intoxications, pesticides.

Metabolic syndrome (MS) or X syndrome, according to the conclusion of the American Heart Association [1], is a complex of symptoms characterized by dyslipidaemia, insulin resistance, abdominal obesity, hyperglycaemia and hypertension, as well as this is a risk factor for the development of non-alcoholic fatty liver disease (NAFLD), cardiovascular diseases, type 2 diabetes mellitus, stroke, chronic renal conditions and cancer [2, 3]. The Japanese programme for MS diagnostics includes similar criteria [8, 9]. One of the main MS components is dyslipidaemia. According to the definition of Ukrainian Heart Association, dyslipidaemia should be defined as impairment of function and/or composition of blood lipids and lipoproteins arising from various causes and capable of inducing manifestations of MS, metabolic diseases and atherosclerotic process, both independently and in combination with other risk factors [5].

Over the past decades, metabolic disorders and obesity became pandemic all over the world, not only among adults but also among children, as well as domestic and wild animals. Just over the past 30 years, obesity among the adult population in the US has risen from 13 % to 35 % [6]. The growing incidence of MS is one of the global health problems, and not only of developed countries [6]. Such situation is associated by many researchers with the effects of environmental factors — endocrine disruptors-obesogens stimulating adipogenesis, dyslipidaemia causing metabolic changes, obesity contributing to the development of type 2 diabetes [9–17]. Endocrine disruptors (EDs) are pollutants of the environment and food products that have a damaging effect on the metabolic systems of the body, interfering with the functioning of the endocrine system by interacting with intracellular nuclear receptors (NRs) of sex hormones, hormones of thyroid gland, adrenal glands, fatty tissue, and others. [10, 11, 12]. It has been noted that the use of molecular imaging technologies in cell culture to identify the spectra of damaging effects by the evaluation of NRs reactivity has made it possible to detect a variety of disorders in the signalling pathways of NRs under the action of EDs at the cellular, extracellular and epigenetic levels. EDs are a wide class of structurally diverse compounds capable of modulating both the secretion and structure of hormones, as well as the polymorphism of NRs and endogenous hormonal signalling pathways [11, 18–21]. These chemical substances include industrial environmental pollutants, waste, pesticides, pharmaceuticals, phytopreparations, consumer products, plastics, etc. [9–17]. EDs are very different in chemical structure, in the mechanism of action on the endocrine system, namely endocrine glands [24–25], hormones [18, 19, 25], hormonal NRs [11, 20–25], especially oestrogen nuclear receptor (ER), androgenic (AR), NRs of the family of peroxisome proliferation activators (PPARs), NRs of thyroid gland (TR), liver (LXR), etc. [11, 8, 19, 20–25]. Furthermore, EDs impairs both the function of NRs and gene expression, their polymorphism and also cause dysfunction of all signalling pathways of NRs [10, 11, 22, 20–25, 56]. The following endocrine or metabolic disruptors are the most studied: organic compounds of tin — organotins [26, 37], compounds of arsenium [27], cadmium [10–13, 25], phthalates [10–12, 29], parabens [10–13, 34], various organochlorines, including polychlorinated and polybrominated biphenyls, furans, dioxins, as well as chlorine-containing pesticides (DDT, DDE, hexachlor, etc.) [11, 30–36, 55] that cause endocrine and metabolic disorders not only in adults, but also in children, adolescents, domestic and wild animals, even at the level of low doses [33]. For example, endocrine effects of pesticides DDE and HCB in adolescents 14–15 years of age were assessed by hormone content, sexual maturation, and metabolic disorders. The average concentrations of DDE and HCB were 30.7 and 36.5 ng/L, respectively [55]. A positive correlation of HCB exposure with sexual maturation and content of testosterone, aromatase in boys, as well as with content of thyroxine, thyroid stimulating hormone and metabolic disorders in both sexes was established. In girls, the effect of DDE and HCB was negatively correlated with impaired sexual development and positively correlated with BMI [55]. These pesticides, as well as organophosphates (diazinon, parathion, malathion, thiophos, metaphos, etc.) [11, 30, 35, 36], carbamates, synthetic pyrethroids [11, 30, 43], herbicides (atrazine, 2,4-D, etc.) [30, 39, 40, 41], fipronil [42], etc. are associated with an increased risk of metabolic disorders. Many pesticides stipulate an increased risk of impaired carbohydrate and lipid metabolism [30–47], development of obesity [9–17, 44–46], non-alcoholic fatty liver disease with predominance of hepatosteatosis [11, 43, 48–50], type 2 diabetes mellitus [33–35, 51–53]. It should be noted that endocrine disruptor effects, including metabolic and obese genes, are caused by low doses of environmental pollutants, including pesticides. Their dysmetabolic effects and effects of obese genes have been demonstrated both in experimental in vivo and in vitro models, and in conducting epidemiological studies [11, 12–25, 47–55]. For example, organochlorine and organophosphorus pesticides are found in the human body at very low levels due to occupational and environmental exposure, but their biological effects are dangerous because they interact with a variety of enzymes, proteins, receptors and transcription factors, disrupt the metabolism of glucose and lipids, cause formation of metabolic disorders, hepatosteatosis, obesity and more frequent development of type 2 diabetes mellitus. These processes are caused by the interaction of these pesticides with a number of nuclear receptors: especially PPARγ, PPARs, LXR, TR, altering gene expression involved in the metabolism of lipids and glucose, as well as with oestrogen (ER) and androgenic (AR) NRs modulating gene expression involved in both metabolic processes and ensuring the functioning of the reproductive system and reproduction, and also interacting with xenoreceptors regulating the processes of xenobiotic biotransformation. It has been shown that these EDs disrupt metabolic and transcription processes at the cellular and molecular level, as well as at the epigenetic level [7, 16–18, 20, 24, 28, 30, 33, 35–38, 98].

For example, chronic exposure (50 days) to fungicide-triazole paclobutrazol causes a dose-dependent disturbance of lipid metabolism in the liver with an increase in the level of triglycerides, total cholesterol, free fatty acids, increasing mRNA expression of lipid and glucose metabolism and expression of the regulatory genes of PPARγ, LXRα, AR NRs, as well as activating the synthesis of a number of enzymes — acetyl-CoA carboxylase 1, fatty acid-binding protein 4, and stearoyl-CoA desaturase [99] that also indicates that this ED causes metabolic disorders through a variety of NRs signalling pathways.

More severe metabolic disorders cause a combined exposure to pesticides. For example, study of the cumulative toxicity of the fungicide mancozeb and neonicotinoid insecticide imidacloprid [100] showed that their effect on mice during lactation increases the risk of weight gain in offspring induced by hypothyroidism, hyperprolactinaemia and dyslipidaemia due to interaction with TR, PPARγ and other signalling lines, which indicates the inclusion of many signalling pathways in the mechanisms of metabolic dysregulation. The same signalling pathways include low doses of phenylpyrazole insecticide — fipronil, activating the key regulators of lipogenesis and differentiation of adipocytes, but to a greater extent this process under the action of this pesticide is stipulated by AMPK-mediated pathway [96].

The majority of researchers note that many EDs, including pesticides, acting on experimental animals or the human body, cause not only lipid metabolism but also carbohydrate metabolism disturbances, in particular, in the formation of insulin resistance, development of hyperglycaemia and type 2 diabetes mellitus or their combination [57–69]. For example, the formation of disturbances in glucose homeostasis with the development of oxidative stress in the liver and pancreas is observed upon intoxication with dimethoate [59]. The development of hyperglycaemia followed by an increase in the level of glycogen phosphorylase and phosphoenolpyruvate carboxylase in rat liver has been also described with the subchronic action of malathion, which also indicates impairment of glucose homeostasis [60]. The relationship between the content of DDT, DDE, HCB, trans-nonachlor, oxychlordane, heptachlor, β-HCH, mirex, aldrin, dieldrin, chlordane, alachlor, pentachlorophenol, parathion, phorate, phonophos, trichlorophone, cyanazine or 2,4-5T in those exposed to low doses of these pesticides under occupational or environmental impact with the development of metabolic disorders and type 2 diabetes mellitus has been established [11, 30, 64, 69, 74, 75, 99, 100]. It has been established that some EDs have specific mechanisms of action, causing only obesity, diabetes or hepatosteatosis, while others affect many aspects of metabolism leading to metabolic syndrome and combined development of these diseases [11, 30]. EDs, including pesticides, not only cause metabolic disorders with dyslipidaemia, formation of fatty hepatosis and obesity, but many of them, especially organic tin compounds, phthalates, organochlorine pesticides, etc., cause their inheritance in a transgenerational manner — development of metabolic disorders and obesity in the next generation [11, 30, 37, 57, 64, 66].

In recent years, the term metabolic toxicity has emerged, which is defined along with carcinogenicity, reproductive toxicity and neurotoxicity in assessing the degree of toxicity of pesticides and other xenobiotics [30]. Among pesticides, metabolic toxicity has already been determined in 12–26 % of cases [30, 33, 61, 64, 75, 99, 100]. In their structure, 90 % are insecticides (chlorine, organophosphorus), 5 % — herbicides, and 5 % — fungicides [30].

Endocrine disrupter effects of 2,4-D herbicide have not been sufficiently studied.

When studying the effect of herbicide 2,4-D (amine salt) on the hormonal status of experimental animals under acute exposure in doses of 1/2 LD50, 1/10 LD50 and 1/20 LD50, and subacute exposure (20 days) in total doses 1/20 LD50, 1/10 LD50 and LD50, daily multidirectional changes in the content of iodinated thyroid hormones have been detected that indicates a thyrotoxic effect of 2,4-D in organs and tissues [71]. The authors found a dose-dependent decrease in the concentration of the hormone T3 and an increase in T4 with an acute exposure to 2,4-D, and an increase in T3 and decrease in T4 with subacute exposure, and they associate these shifts with thyroid damage and an impaired consumption of T4 in peripheral tissues. A decrease in the blood insulin level was noted with acute exposure to medium and high doses of 2,4-D, whereas in the subacute experiment, blood insulin content in animals increased significantly. At the same time, the concentration of testosterone in the blood decreased [71]. Unfortunately, the authors used fairly high doses of herbicide 2,4-D, which caused endocrine disruptor effects both in the acute and subacute experiment. However, it is much more important to study the effect of low doses of this herbicide on human under certain occupational and environmental conditions.

At the same time, establishment of the morphological aspects of the reaction of thyroid stimulating hormones of anterior pituitary gland under the chronic action of relatively low 2,4-D (30 mg/kg) doses revealed the development of dystrophic destructive processes in the cytoplasm of thyroid stimulating hormones and haemomicrocirculation disorders in the structure of anterior pituitary gland [72]. This indicates a potential endocrine-damaging effect of herbicide 2,4-D. Experimental studies also demonstrated membrane-toxicity effects of 2,4-D, which allowed establishing that 2,4-D interacts predominantly with polar groups of membrane lipids, which is due to their anionic properties [96].

The main mechanism of the 2,4-D toxic effect on warm-blooded animals involves partial separation of oxidation and phosphorylation processes, which leads to disturbances in energy metabolism with dissociation of dehydrogenases isoenzymes, accumulation of lactic acid and development of energy deficiency [24, 75]. In turns, the bioenergetic imbalance is accompanied by a disturbance of cell membrane resistance and ion channel functions, electrical potentials of muscle and nerve cells. Watt B.E. et al. [73] believe that one of the main directions of the toxic effect of 2,4-D are disorders of the metabolism of acetyl-CoA cycle of tricarboxylic acids and β-oxidation of fatty acids. This process may be associated with a decrease in the activity of PPARα and PPARβ nuclear receptors, which contribute to the regulation of fatty acid oxidation and mitochondrial lipid oxidation, leading to the development of metabolic disorders, formation of hepatosteatosis, and appearance of obesity.

An increase in the body weight of rats exposed to non-toxic doses of the herbicide 2,4-D was revealed [74]. In rats fed 2,4-DA herbicide in a concentration of 0.015 mg/L (MAC — 0.5 mg/L) with drinking water within 6 weeks detected a body weight gain in comparison with the control group in as little as a week, which was significantly increased in 6 weeks as well. At the same time, there was an approximately 1.5-fold increase in the weight of epididymal fat (4.8 g in the control group and 6.4 g in the experimental group), which to a certain extent indicates the effect of obese genes of low doses of the herbicide 2,4-DA, mechanisms of which require further study.

Metabolic effects of herbicide 2,4-D in concentrations of 100 nM, 10 μM and 1 mM after 50 hours of exposure in rats have been identified [41]. The authors described glucose dysmetabolism, increased intracellular lactate content, and a decrease in the lactate/alanine ratio as an indicator of intracellular energy balance disturbances, more pronounced when exposed to low doses — 10 μM and 1 mM. Herbicide 2,4-D caused dyslipidaemia, lipid dysmetabolism in the liver against the background of induction of oxidative stress and activation of lipid peroxidation in rats [75].

Given the suspicion of the endocrine disruptor potential of herbicide 2,4-D, in 2009, the US Environmental Protection Agency (EPA) included it in the 1st screening program. The program included investigation of the potential endocrine activity of 2,4-D using in vitro tests to evaluate the interaction with oestrogen, androgenic receptors and the degree of inhibition of aromatase activity [К.К.Соаdy et al., 76]. The authors did not find convincing data on the interaction of 2,4-D with oestrogen, androgenic receptors and steroidogenic system. It should be assumed that other mechanisms (possibly receptors of PPARs family, fatty tissue hormones, etc.) are involved in the formation of metabolic disorders of glucose and lipids detected earlier in experimental animals under exposure to 2,4-D [41, 73, 74, 75].

To assess metabolic shifts, it is common to investigate lipid and carbohydrate dysmetabolism. However, in recent years it has been proved that fatty tissue is a powerful endocrine organ that synthesizes various hormones (adipokines) involved in the formation of metabolic disorders, inflammation, immune processes, etc. [77–95], giving a special role to leptin, adiponectin, resistin and tumour necrosis factor-α (TNF-α) in the formation of metabolic disorders [77–90].

Leptin is the first described and most studied adipokine. In human, leptin is synthesised by cells of white and brown fatty tissue, skeletal muscles, stomach, placenta. Leptin is considered a “voice” of fatty tissue. It acts on hunger and satiation centres in the hypothalamus and controls body weight by lowering the synthesis and release of neuropeptide Y, causing hunger [77, 88, 89], reducing food intake and increasing energy consumption. In case of abdominal obesity, the level of leptin sharply increases as a compensation due to its increased production in white fat tissue and the development of leptin resistance [77–92]. It is believed that in case of obesity, the resistance of the hypothalamus receptors to the central action of leptin is formed, leading to hyperleptinaemia [77, 89, 92]. In turn, prolonged hyperleptinaemia inhibits insulin mRNA expression resulting in hyperglycaemia and development of diabetes mellitus [77, 91].

Some authors have noted an increase in blood leptin level as insulin resistance increases, which also leads to the progression of the metabolic syndrome, obesity and development of diabetes mellitus [77, 78, 89, 91]. Hyperleptinaemia is considered a predictor of atherosclerotic vascular disease, arterial hypertension, metabolic syndrome, myocardial infarction and stroke [89, 91, 92].

Fatty tissue hormone adiponectin is also produced by fat tissue and has the opposite effect. This adipokine has an anti-inflammatory effect, suppressing the activity of the transcription factor — nuclear factor-kappa B (NF-kB) in macrophages, monocytes, endothelial cells, and also in hepatocytes. Adiponectin inhibits the activity of liver enzymes involved in gluconeogenesis, which helps to lower the level of glucose in the liver. It also facilitates the transport of glucose to the muscles, activates oxidation of fatty acids and increases tissue sensitivity to insulin [77, 80, 87–89, 91]. The content of adiponectin is sharply reduced in metabolic syndrome, obesity, and especially in diabetes mellitus [88–91]. Its reduction may be a biomarker of these conditions and perhaps characterises their progression.

In turn, resistin is considered as insulin resistance hormone, it affects fat metabolism on the principle of feedback: on the one hand, its level increases with differentiation of adipocytes, on the other — it inhibits adipogenesis [88–91]. An increased level of resistin is associated with the development of metabolic disorders, dyslipidaemia and insulin resistance [77, 89, 91, 93, 94]. Resistin is an antagonist of the anti-inflammatory adipokine — adiponectin, therefore it exerts a pro-inflammatory effect [93–94]. There is an idea that resistin stimulates the expression of pro-inflammatory cytokines, in particular, TNF-α [94].

TNF-α is secreted by adipocytes and macrophages and plays an important role in the development of inflammation and insulin resistance by reducing the tyrosine phosphorylation of the insulin receptor and the substrate of the type 1 insulin receptor in muscle and abdominal fatty tissue [82–85, 89]. The level of TNF-α increases in the blood and abdominal fatty tissues in metabolic syndrome, obesity and diabetes [82–85, 89, 95].

Investigation of the level of leptin, adiponectin, resistin and TNF-α in the blood in patients after acute poisoning with 2,4-D herbicide with metabolic disorders and obesity, determination of informative value and relationship with dyslipoproteinaemia, BMI, and dependence on hepatosteatosis is of special scientific interest.

Thus, the metabolic toxicity of 2,4-D herbicide has not been sufficiently studied. However, given its wide application in agriculture, reports about acute poisoning, especially when hygiene regulations are violated during its use, assessment of the potential risk of development of metabolic effects and obese genes effects in individuals who have experienced acute poisoning with this herbicide is of scientific interest. The risk of metabolic effect and obese genes effects in individuals who have experienced acute poisoning with organophosphorus pesticides, synthetic pyrethroids, and those with chronic intoxication due to the effect of a complex of pesticides should be studied for their rational correction and prevention.

Study objective was to study the incidence of metabolic disorders and effects of obese genes in patients after acute and chronic intoxication with pesticides and to justify methods for evaluating their severity to optimise differentiated therapy and prevention.

Materials and methods. To study the incidence and peculiarities of formation of metabolic effect and obese genes effects, 108 patients with acute pesticide poisoning were examined over time: 56 — with herbicides based on 2,4-D amine salt, 40 — with OPC, 12 — with SP, and 66 patients — with chronic pesticide intoxication (CPI) due to the long-term occupational exposure to a complex of pesticides. As a control, 30 virtually healthy agricultural workers with no history of intoxication with pesticides were examined.

All cases of acute poisoning with 2,4-D, OPC and SP occurred in agricultural workers due to gross violations of hygienic regulations for their use. Occupational composition of patients with acute 2,4-D poisoning was represented by beet growers-fielders, OPC — vine-growers and gardeners. Among 12 cases of SP poisoning — 8 gardeners and 4 handymen. Age of patients with acute pesticide poisoning ranged from 36 to 58 years (mean — 46.2 ± 0.06 years), and patients with CPI — from 40 to 57 years (mean — 48.8 ± 0.6). The mean age of the subjects from the control group is 47.8 ± 2.2 years. All examined persons with acute poisoning were women. Among patients with CPI were 36 men and 30 women.  The occupational structure of 66 patients with CPI was represented by 16 heads and workers of chemical pesticide stores (24.2 %), 20 gardeners and grape growers (30.3 %), 25 machine operators (37.8 %), and 5 disinfectors (7.7 %). The work experience of patients with acute poisoning and control group averaged 24.6 ± 0.08 years, and CPI — 20.8 ± 0.09 years. In all 40 patients with OPC poisoning, it arose when exposed to dimethoate. Out of 12 cases of acute SP poisoning, in 9 patients — acute poisoning with “Decis” (active substance is deltamethrin), and in 3 — with sumicidin (fenvalerate).

The object of this study is an investigation of metabolic disorders and obese genes effects (overweight and obesity) in patients who have experienced acute and chronic intoxication with pesticides. Subject is the analysis of anthropometric parameters, lipid and carbohydrate metabolism disorders, imbalance of fatty tissue hormones (leptin, adiponectin, resistin, and TNF-α).

The general clinical methods were used to examine the patients: examination, questioning, study of medical documentation (outpatient record data, sanitary and hygienic characteristics of working conditions, industrial accident report, toxicological studies on the content of pesticides in the working zone air and in the blood of victims), anthropometric data, abdominal ultrasound with elastography, as well as biochemical tests. Anthropometric examinations were conducted using standard methods: measuring the waist circumference (WC) and calculating the body mass index (BMI) according to the formula:

BMI  =  (body weight in kg) / (height m2).

To assess the lipid metabolism in patients with acute and chronic intoxication with pesticides, the level of total cholesterol (TH), high density lipoprotein cholesterol (HDL cholesterol), triglycerides (TG), which were measured in serum using Cormay (Poland) reagents, were measured by the colorimetric enzymatic method on a biochemical analyser. The content of low-density lipoprotein cholesterol (LDL cholesterol) was calculated by W. T. Friedewald:

LDL-C  =  TH – (HDL-C + TG / 2.22).

In order to study the carbohydrate metabolism, blood glucose was measured by a glucose oxidative method using Human (Germany) reagents; ELISA in the serum was used to measure the insulin content (Insulin ELISA DPG Instruments GmbH, Germany). To measure insulin resistance, HOMA-IR (Homeostasis Model Assessment of Insulin Resistance) index was calculated using the formula:

HOMA-IR = fasting blood glucose (mmol/L) × fasting blood insulin (µU/mL) / 22.5.

The level of dyslipoproteinaemia in 8–10 years in patients who had experienced acute 2,4-D poisoning, was measured at DILA medical laboratory, Kyiv. The content of the fatty tissue hormones in the blood serum was measured by ELISA — rider Anthos 2020 (Austria). Leptin, resistin and adiponectin were measured using Demeditec diagnostics (Germany) reagents. TNF-α in blood serum was measured using Human TNF-α Platinum Bioseins reagents, Bender Med-sistems (Austria) in the laboratory of the Institute.

In assessing the functional state of the liver in the clinic of Institute, activity of alanine (ALT), asparagic (AST) aminotransferase, alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GTTP), bilirubin (B), albumin (A), bile acids (BA), fibrinogen, prothrombin, C-reactive protein (C-RP), malonic dialdehyde (MDA) and thymol test values were measured using harmonised standard methods [96].

Statistical processing was carried out using parametric statistics methods with standard programs taking into account the basic principles of their application in clinical trials [97]. All examinations were carried out with the consent of the patients and consistent with ethical standards.

Results and discussion. In the cynical picture of patients with acute poisoning with pesticides and chronic intoxication with pesticides, the following neurologic disorders prevailed: asteno-vegetative syndrome (AVS), toxic encephalopathy (TE), vegetative-sensory polyneuropathy of the upper and lower extremities (VSP). Biochemical and ultrasonographic methods made it possible to detect toxic liver damage in a number of patients with acute and more often chronic intoxications (according to ICD-10). According to the medical documents and medical history, examined patients had no liver conditions before pesticide poisoning. Toxic liver damage in acute exogenous intoxications is more often manifested in the acute period as toxic hepatopathy, in chronic — toxic hepatitis [101–102]. Neurological disorders prevailed in all examined patients: in 60–85% of cases, AVS was diagnosed, rarely (14–40 %) — TE and in most of the examined, especially with chronic pesticide intoxication (CPI), VSP of upper and lower extremities was established (Table 1). Toxic liver damage with a predominance of cytolytic syndrome was defined in 46.4 % of cases in patients with acute 2,4-D-based herbicide poisoning, in 55 % of cases — poisoning with OPC, in 66.6 % of cases — poisoning with SP and in 84.8 % of cases — in patients with CPI (Table 1).

The main clinical symptoms of toxic liver damage were nausea, sometimes vomiting, abdominal distension, discomfort in the right upper quadrant of the abdomen, an increase in the anteroposterior size of the right hepatic lobe, increased liver density, and an increase in the activity of liver enzymes and parameters indicative of the development of cytolytic syndrome, intrahepatic cholestasis syndrome and hepatodepressive syndrome, the evaluation criteria of which we have described in previous works [104, 105]. Just in the first 10–14 days after acute pesticide poisoning in patients with toxic hepatopathy, an increase in the density of the liver structure was found out during ultrasonography with elastography, which increased during an examination in a year. If in healthy individuals the ultrasound attenuation factor for elastography of the liver averaged 1.2 ± 0.02 dB/cm, then in patients with 2,4-D poisoning it increased in 20-30 days to 2.02 ± 0.09 dB/cm that according to M.Sasso et al. [103] corresponds to mild hepatosteatosis. In patients with OPC poisoning, ultrasound attenuation factor increased on average to 2.42 ± 0.08 dB/cm, and for chronic hepatitis in patients with CPI — up to 2.66 ± 0.06 dB/cm (p < 0.05) that which according to the criteria of M.Sasso et al. [103] corresponds to moderate steatosis. Criteria for diagnostics in examined patients with toxic liver damage and hepatosteatosis with pesticide intoxications, as well as the incidence and severity of liver damage syndromes and the rationale for differentiated therapy, are detailed in our previous studies [104, 105]. The incidence of clinical syndromes in patients with pesticide intoxications, who were studied for metabolic and obese gene effects over time, is presented in Table 1.

Table 1. Incidence of clinical syndromes in patients experienced pesticide intoxication.



To assess the incidence of metabolic disorders and obese genes effects, the patients were divided into two groups: group I — patients without toxic liver damage, group II — with toxic liver damage, with the formation of hepatosteatosis. Such a conditional allocation of patients was performed taking into account the fact that metabolic disorders, obese genes effects and the formation of hepatosteatosis are considered to be an interrelated process [11, 43, 44–46].

Analysing the anthropometric parameters over the time of observation, a tendency to an increase in the BMI and waist circumference (WC) in patients who have had pesticide intoxication with the toxic liver damage syndrome (group II) was revealed. Assessment of BMI was performed according to WHO recommendations: 18.5–25 kg/m² — normal range, 25–30 — excess body weight (pre-obesity), 30–35 — obesity of the degree I, 35–40 — obesity of the degree II, 36 and more — obesity of the degree III (morbid). Waist circumference in women up to 88 cm is considered normal.

Time course of the average parameters of BMI and WC in the patients examined in a year in comparison with the baseline is presented in Table 2. The table shows that the baseline level of BMI and WC in patients with poisoning without liver damage, although slightly higher than normal, indicating an excess body weight, but at the same time does not significantly differ from parameters in the comparison group (p > 0.05). In a year after the examination, there is a certain tendency to increase of these parameters in this group of patients, but the mean levels are insignificantly different from the baseline (p > 0.05). At the same time, individuals with acute 2,4-D-based herbicide and OPC poisoning with a syndrome of toxic liver damage and hepatosteatosis, show more significant increase in BMI and WC in comparison with the baseline, especially with the comparison group over time of observation in a year (Table 2, р < 0.05), and this corresponds to obesity of degree I. In patients with CPI without a toxic hepatitis syndrome, both baseline and observational anthropometric parameters in a year were slightly higher than in the comparison group. In patients with CPI with toxic hepatitis and hepatosteatosis, both baseline and in one year of observation, these parameters were significantly higher than in the comparison group (p < 0.05) and corresponded to obesity of degree II–III. Increase in BMI and WC values (reflecting the appearance of predominantly abdominal and probably visceral obesity) to a certain extent indicates the appearance of metabolic disorders and obese genes effects of pesticides (2,4-D, OPC, and pesticide complex in CPI).

Table 2. Time course of anthropometric parameters in patients experienced acute and chronic intoxications (М±m, numerator — baseline period, denominator — in a year).


Note: *  – with р < 0.05 compared to the baseline,
          ** – with р < 0.05 compared to the comparison group.


The severity of hepatosteatosis, revealed by ultrasonography with elastography, was assessed by ultrasound attenuation factor according to M.Sasso et al. [103]. While healthy subjects in the control group had ultrasound attenuation factor averaged 1.26 ± 0.06 dB/cm, in individuals of the comparison group who have had acute poisoning without toxic liver damage, it differed insignificantly (p  0.05), then in patients who have had acute poisoning with toxic liver damage, it was significantly higher: with 2,4-D poisoning - 2.29 ± 0.12 dB/cm (p < 0.05), OPC poisoning - 2.32 ± 0.14 (p < 0.05) that according to M.Sasso et al. corresponded to mild steatosis, and in cases of CPI with toxic hepatitis — 2.58 ± 0.16 dB that corresponded to moderate steatosis.

The study of biochemical parameters in patients with acute 2,4-D-based herbicide, OPC and SP poisoning without toxic liver damage syndrome in acute period (Table 3) revealed only a significant increase in the level of MDA (p < 0.05), which indicates the formation of oxidative stress, however observation over time in a year revealed a normalisation of its level (p > 0.05). The level of transaminases, parameters of carbohydrate and lipid metabolism in patients of this group in the acute period and in a year did not differ significantly from the level of the control group (p > 0.05). In patients with CPI without toxic hepatitis, both in the baseline period and in a year of observation, only a significant increase in the level of MDA (p < 0.05) was detected, the remaining studied parameters of carbohydrate and lipid metabolism both in the baseline period and in a year of observation did not significantly differ from the level of the control group (p > 0.05), although there was a tendency to increase in the level of insulin resistance, total cholesterol and LDL-C (Table 3).

Table 3. Main biochemical parameters in patients experienced pesticide intoxications (numerator — baseline, denominator — in a year), depending on the presence of toxic liver damage syndrome (М±m).


Note: *  – with р < 0.05 compared to the control group,
          ** – with р < 0.05 compared to the baseline.


In turn, biochemical tests in patients with acute pesticide poisoning with toxic liver damage syndrome revealed an elevated level of ALT in the acute period (p < 0.05), the average level of which almost normalised in a year. The level of ALT in patients with CPI with toxic hepatitis syndrome was significantly increased both in the baseline period of observation and in a year (p < 0.05, Table 3). In patients with acute 2,4-D, OPC poisoning and CPI, a significant increase in the GGTP level was observed (p < 0.05). There was also a more significant increase in the average level of MDA, especially in CPI patients (p < 0.05). Regularly, transient hyperglycaemia was observed in a number of examined patients, but the average blood glucose level did not differ significantly from the level in the comparison group (p > 0.05). The average level of blood insulin, although tended to increase in patients with toxic liver damage syndrome and especially in CPI patients, but no significant changes were observed (p > 0.05). At the same time, there was an increase in insulin resistance in patients who have had acute pesticide poisoning with toxic liver damage, in a year of observation, which was positively correlated with severity of hepatosteatosis (r = 0.68) and MDA level (r = 0.72). The increase in insulin resistance (HOMA-IR) was in patients with CPI with toxic hepatitis both in the baseline period and in a year (p < 0.05), except for CPI patients. Among the parameters of lipid metabolism, there was a significant increase in the level of total cholesterol (TC) and TG in patients with acute 2,4-D and OPC poisoning with toxic liver damage in a year of observation (p < 0.05), as well as in CPI patients with toxic hepatitis syndrome, both in the baseline period and in a year (p < 0.05). The tendency towards the decrease in the level of HDL cholesterol and increase in the level of LDL cholesterol was reported in patients with toxic liver damage, however, there were no significant changes (p > 0.05). Such parameters as mean level of MDA, HOMA-IR (level of insulin resistance), as well as the level of TC and TG, positively correlated with BMI and WC, as well as with the severity of hepatosteatosis in the examined patients with liver damage (r = 0.52–0.78) , which indicates that these parameters of metabolic disorders are the predictors of the formation and progression of overweight and obesity. As is well known, products of lipid peroxidation damage membranes of liver cells and mitochondria, increase the intake and synthesis of free fatty acids (FFA) in hepatocytes, promote the development of insulin resistance. In turn, insulin resistance also causes increased intake of FFA in the liver, enhances their excessive synthesis, and also reduces the rate of β-oxidation of FFA in mitochondria of hepatocytes, increases the synthesis and secretion of LDL and VLDL, which leads to the formation and progression of hepatosteatosis and obesity [43, 48, 50, 75].

For the prevent the progression of metabolic and obese genes effects, along with a decrease in the caloric intake of food, normalisation of diet and increased motor activity, 500 mg metformin twice daily is recommended to these patients for a month twice a year with a constant administration of statins. Metformin in obesity reduces the synthesis of cholesterol and triglycerides, enhances lipolysis in both liver and adipose tissue, and inhibits synthesis of glucose in the liver and helps to reduce insulin resistance. Statins, in turn, reduce the level of TC, TG and contribute to the normalisation of dyslipoproteinaemia, their use reduces the risk of cardiovascular complications.

To study the time course of metabolic disorders and obese genes effects in patients after acute poisoning with pesticides, 23 patients who have had acute 2,4-D-based herbicide poisoning were examined after 8–10 years in a remote period. Of these, 14 patients in the baseline period were found to have toxic hepatopathy in the structure of clinical syndromes with the formation of mild to moderate hepatosteatosis (group 1), and in 9 cases liver damage was not diagnosed (group 2). The third group consisted of 9 virtually healthy individuals. All subjects underwent an anthropometric status assessment (BMI, WC), peculiarities of dyslipoproteinaemia and content of the main hormones of fatty tissue — leptin, resistin, adiponectin and TNF-α in the blood. Measurement of these adipokines is preferred due to the fact that leptin, resistin, adiponectin and TNF-α play a key role in the development of metabolic disorders, the formation of insulin resistance, hepatosteatosis and obesity [77–95].

Anthropometric and biochemical tests in patients after acute 2,4-D-based herbicide poisoning in the remote period made it possible to reveal that in 14 of 23 patients who had toxic liver damage during the acute poisoning in the structure of clinical syndromes with the formation of mild to moderate hepatosteatosis, there was a significant increase in the body weight and WC (Table 4).  BMI significantly increased to an average of 42.75 ± 2.39 (P < 0.05), and WC to an average of 116.9 ± 5.95 (P < 0.05) that corresponds to the obesity of degree 2, namely of abdominal type. There was also an increase in dyslipoproteinaemia — a significant increase in TC, TG, LDL, and atherogenic index (Table 4, P < 0.05). At the same time, significant changes in anthropometric and biochemical parameters were not observed in patients of group 2, who experienced acute poisoning without toxic liver damage and formation of hepatosteatosis (P > 0.05).

Table 4. Anthropometric and biochemical parameters in patients experienced acute 2,4-d-based herbicide poisoning in remote period (M±m).


NOTE: *with P < 0.05 compared to the control group,
             **with P < 0.05 compared to group 2.


A similar tendency was observed when studying the levels of fatty tissue hormones in the blood (Table 5). In patients, who experienced acute 2,4-D-based herbicide poisoning with toxic liver damage with the formation of hepatosteatosis, a significant increase in the level of leptin, resistin to WTNF-α at the background of a slight decrease in the level of adiponectin (P > 0.05, Table 5).

Investigation of the relationship between the levels of these adipokines and anthropometric parameters revealed a direct positive correlation of leptin levels with BMI, WC, the content of TC and TG (r = 0.72; 0.64; 0.62 and 0.76, respectively). Levels of resistin and TNF-α are also in direct correlation with these parameters (r = 0.59–0.68). The level of adiponectin tended to decrease, however, the average level differed insignificantly from the comparison group (P > 0.05).

Table 5. Content of fatty tissue hormones in patients experienced acute 2,4-d-based herbicide poisoning in remote period.


NOTE: *with P < 0.05 compared to the control group
             **with P < 0.05 compared to group 2.


Conducted studies confirm published data on the key role of these adipokines in the development of metabolic disorders with the formation of hepatosteatosis and the effects of the obese gene [76–95]. Our data suggest that increased secretion of leptin, resistin and TNF-α not only maintain metabolic disorders and obese genes effects but also contribute to their progression, which is apparently due to their biological role in the body.

It is known that the synthesis of leptin is regulated by the OB gene — obesity gene. Normally, leptin promotes energy consumption and catabolism of fats and carbohydrates [77–78]. However, selective leptin resistance and compensatory hyperleptinaemia develop under toxic effects, activation of lipid peroxidation processes, and an increase in the tone of the sympathetic nervous system. This increases the activating effect of leptin on insulin metabolism and suppresses insulin resistance — the transcription of preproinsulin gene, and the excretion of insulin, as well as the activity of insulin receptors in tissues,  are suppressed.

Lipid peroxidation products, metabolic disorders caused by ED, also increase the secretion of resistin and tumour necrosis factor- (TNF-α), which in turn are one of the main factors contributing to the progression of insulin resistance, activation of adipogenesis, development of obesity and type 2 diabetes mellitus [82–86]. TNF-α and resistin have a pro-inflammatory effect, support smouldering inflammation in fatty tissue and liver, causing an imbalance of fatty tissue hormones and progression of hepatosteatosis [82–86]. It can not be ruled out that an increased level of resistin in the examined patients of the group 1 also supports smouldering inflammation, hepatosteatosis and obesity. In healthy individuals, fatty tissue hormone adiponectin promotes inhibition of lipid peroxidation, insulin resistance, and stimulates secretion of anti-inflammatory, anti-obese genes and anti-diabetic factors. However, lipid peroxidation products, hyperleptinaemia, increased secretion of resistin and TNF-α dramatically suppress synthesis of adiponectin, which contributes to the progression of hepatosteatosis and obesity [80, 81, 86–92]. Among the examined patients of group 1, in 7 out of 14 patients, it was significantly reduced, but the average level did not differ significantly from the comparison group.

Thus, the conducted dynamic studies on the assessment of the incidence and severity of metabolic disorders and obese genes effects in patients, who experienced acute and chronic intoxication with pesticides allow the following conclusions:

1. In patients with acute 2,4-D-based herbicide, SP and OPC poisoning with a syndrome of toxic liver damage and especially in patients with chronic pesticide intoxication with toxic hepatitis syndrome, just in the baseline period there are elevated levels of MDA (p < 0.05), and dyslipoproteinaemia, which increase over time in a year of observation with a simultaneous increase in TC and triglycerides (p < 0.05), insulin resistance (p < 0.05) and overweight, which indicates the formation of metabolic disorders and obese genes effects.

2. Individual monitoring of the increase in levels of MDA, triglycerides and insulin resistance — predictors of the formation of overweight, with timely correction of them in patients who have experienced intoxication with pesticides, will make a significant contribution to preventing the development of metabolic syndrome, steatohepatosis and obesity.

3. The increased level of fatty tissue hormones in the blood — leptin, resistin and TNF-α (р < 0.05), at the background of a moderate decrease in adiponectin levels in patients who have had poisoning with 2,4-D-based herbicides, in a remote period allows predicting an increased risk of a progressive course of steatohepatosis and obesity, for prevention of which long-term administration of metformin and statins is recommended along with reduced caloric content of food and increased motor activity.



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Надійшла до редакції 17.07.2018