L.I.Medved's Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine Kyiv, Ukraine
Abstract. Objective. To study the incidence and peculiarities of intrahepatic cholestasis (IHC) syndrome in patients with toxic liver damage in acute and chronic intoxications with pesticides and to substantiate rational methods of diagnosis and treatment.
Material and methods. The incidence has been analysed with the description of IHC syndrome peculiarities in acute and remote periods in 238patients with acute poisoning with pesticides: 162 — herbicides based on 2,4-dichlorophenoxyacetic acid (2,4-D), 62 — phosphororganic pesticides (POP), 14 — synthetic pyrethroids (SP) and 70 — with chronic intoxication with pesticides (CIP). Clinical-instrumental, biochemical and statistical methods of the study have been used.
Results and conclusions. Along with neurological disorders, the toxic liver damage was observed in 35,8 % of cases of acute poisoning with 2,4-D-based herbicides, in 51,6 % of cases — with POP poisoning, in 64,2 % of cases — with SP poisoning and in 84,2 % of cases — with CIP. IHC syndrome was detected in 22,8 % of cases in patients with pesticide intoxication against toxic hepatitis and in 18,0 % of cases — without signs of hepatitis. The therapeutic efficacy of ursodeoxycholic acid has been established when it is included in the combined therapy of patients with IHC upon intoxication with pesticides.
Key words: pesticides, acute and chronic intoxications, toxic hepatitis, intrahepatic cholestasis syndrome, ursodeoxycholic acid.
The liver is the central body responsible for biotransformation and excretion of medicinal products, pesticides and other xenobiotics. In turn, this important function of the liver may lead to the development of hepatotoxicity, which is often accompanied with hepatic insufficiency [1–6]. A number of studies have established that cholestatic and mixed — cholestatic and hepatocellular liver injury — are the two most severe manifestations of pathology caused by exposure to xenobiotics [1–9]. Chemical substances released during the biotransformation by the liver in the bile often cause the formation of intrahepatic cholestasis (IHC), especially in individuals with individual genetic characteristics of the body[1–6, 8–11]. IHC is based on the dysfunction of the mechanisms of bile synthesis, secretion and outflow, which develops in the absence of obstruction of the bile ducts against the background of lesions in any area — from the basolateral membrane of the hepatocyte or cholangiocyte to the terminal sections of the intrahepatic bile ducts [1–3, 10–14].
Cholestasis in the absence of obstruction is an intra-hepatic process and is caused by impaired bile synthesis or transport in hepatocytes or in the canalicular system (or by combining these mechanisms) [1–8, 32, 36]. Bile synthesis and secretion are vital processes. A large number of toxic exogenous and endogenous compounds are released from the body with bile (bilirubin, cholesterol, metabolites of medicinal products, xenobiotics, etc.). Such bile components as bile acids, emulsifying edible fat, participate in digestion and absorption of fat-soluble vitamins, stimulate pancreatic secretion, motor function of the gallbladder and intestine, provide sterility of bile and duodenal contents [4–7]. Bile synthesis and secretion consist of the following stages: 1) capture of a number of its components (bile acids, bilirubin, cholesterol, etc.) from the blood at the level of the basolateral sinusoidal membrane of hepatocytes; 2) metabolism and synthesis of new constituents of bile and their transport in the cytoplasm of hepatocytes; from the sinusoidal to the biliary pole; 3) secretion of bile elements through the canalicular (biliary) membrane of hepatocytes into the bile canaliculi [4–8]. Through them, bile enters the extralobular bile ducts, which join together, create regional bile ducts, and then a common bile duct. IHC can develop at the level of the hepatocyte or intrahepatic bile ducts. In accordance with this, the following IHC variants are identified: intralobular cholestasis caused by hepatocyte damage (hepatocellular mainly affecting membranes or transport proteins), as well as canaliculi damage (canalicular mainly affecting cholangiocyte membranes or their transport proteins) and extralobular (ductular) caused by damage of intrahepatic bile ducts [4, 6, 8, 32, 36]. IHC aetiology is multifactorial. Thus, hepatocellular and canalicular cholestasis may be due to viral, toxic, including pesticidal, alcoholic, drug-induced liver injury, metabolic disorders (benign IHC, obstetric cholestasis, cystic fibrosis, α-antitrypsin deficiency and others. Estralobular cholestasis often develops in primary biliary cirrhosis, primary sclerosing cholangitis [4, 8, 25, 28, 32], biliary atresia, Caroli disease, and others [4, 6, 8, 32, 36]. The mechanism of interglobular cholestasis development may be due to the reduction in basolateral or canalicular membrane fluidity, inhibition of Na+/K+-ATPase and other membrane enzymes, their translocation from biliary to the sinusoidal pole of the hepatocyte and cytoskeletal hepatocyte damage, impaired integrity of canaliculi and their functions. It should be noted that in most cases, there is a combination of several factors.
Various factors (toxicochemical, metabolic, autoimmune, genetic, infectious agents, etc.) contribute to both IHC formation and its progression [1–4, 9–20]. Many authors believe that whatever aetiologic factor may cause damage to the liver with IHC, its development is always facilitated or predisposed by certain congenital or acquired under exposure to chemical or other agents mutations of genes regulating oxidation, biotransformation, transportation and excretion of metabolites, xenobiotics, as well as the structure, secretion and outflow of the bile [2–4, 10–19].
It is known that hepatocytes are highly polarised cells with various sinusoidal, lateral and apical membrane domains. Fat-soluble chemicals with a molecular weight of ≈ 500 Daltons and above selectively diffuse into the hepatocyte and cholangiocyte through sinusoidal membrane domains. Although some xenobiotics diffuse directly through the cell membrane, most of them require active or lightweight transporters (phase 0 biotransformation) [4, 15, 16, 20, 21]. Binding to cytosolic proteins is accompanied by oxidation and biotransformation of xenobiotics – phases I and II, which leads to more water-soluble metabolites as well. Phase I biotransformation includes oxidation, hydroxylation and other reactions mediated by the cytochrome P-450 (CYP) system, CYP 3A4 in particular. The activity of P-450 cytochrome system varies widely among individuals and depends on the functional activity of nuclear xenoreceptors: pregnane (PXR), androstane (CAR), arylhydrocarbon (AhR), and a number of hormonal nuclear receptors [4, 22, 23, 24]. The reactions of phase II biotransformation of xenobiotics include esterification reactions under the influence of a number of enzymes and conjugation with thiols, sulphates, glucuronic acid, amino acids, glutathione, etc., which usually increases water solubility and reduces the toxicity of xenobiotics. Often, during the biotransformation of xenobiotics, more toxic compounds are formed, in particular, in the biotransformation of many phosphororganic pesticides (POP) [4, 22]. In this regard, chemically induced IHC may develop under conditions of congenital or xenobiotic-induced dysfunction of nuclear receptors and their signalling pathways, inadequate expression of the enzymes of biotransformation phase II, with a decrease in the activity of antioxidants, in particular, glutathione, superoxide dismutase, etc. [4–12, 31]. A special role in IHC formation is played by congenital or acquired under exposure to xenobiotics mutations of genes that regulate the expression of intra- and extracellular transporters of metabolites of chemicals [4, 6, 15–24]. Some of these transport proteins mediate sinusoidal absorption and biliary excretion of bile acids and other bile components, as well as metabolites of xenobiotics through the basolateral membrane. These are, first of all, organic cationic transporters-1 (OCT1), organic anionic transporters-2 (OAT 2), a family of organic anionic transport polypeptides — OATPs, and taurocholate cotransporters — NTCP and family of nucleoside transporters — CNT 1 and 2, ENT1 and 2, etc. [4, 7]. In addition, IHC is formed by mutations of the genes of the canalicular membrane transporters — multidrug resistance-associated proteins — MDR1, MDR2, MDR3, MRP2, MRP3, MRP4 and Canalicularbile – salt – exportpump, BSEP, etc. [4, 6, 7, 15–24, 31]. It is shown that a low level of BSEP expression is considered one of the main risk factors for cholestasis when exposed to medicinal products and other xenobiotics, as well as intrahepatic cholestasis in pregnant women [4, 7]. If most of the medicinal products and xenobiotics are diffused through the basolateral membrane directly or with transport proteins without membrane Na+- / K+- ATPase, the transport of metabolites through the canalicular membrane is always ATP-mediated [4]. The role of transport protein dysfunction in the formation of IHC syndrome is shown in Fig. 1 [4].
Fig. 1. A hepatocyte couplet, illustrating the location of large protein transporters, which determine bile synthesis and secretion and metabolites of xenobiotics. ATP-dependent transporters are dark circles. (Na+ = sodium, BA- = bile acids, OA- = organic anion, OC+ = organic cation, PC = phosphatidylcholine, BA-G = bile acid glucuronides, BA-S = bile sulphates), GSH = glutathione [4].
Under the influence of xenobiotics and other aetiological factors of IHC in hepatocytes and especially in hepatocellular membranes, the biochemical processes are disrupted, the phospholipids content decreases, the activity of Na+- K+- ATPase and other transfer proteins decreases, which breaks the permeability of membranes, as well as the capture and excretion of metabolites and various components of bile. At the same time, cell stocks of thiols, sulphates (glutathione, taurine, etc.) are reduced, which are the main detoxification and antioxidant substances, and their deficiency causes cytolysis of hepatocytes in cholestasis of any aetiology [4, 5, 6, 14–21].
IHC induced by medicinal products, pesticides or other xenobiotics may be present with asymptomatic toxic liver lesions, where the only clinical manifestation is an increase in alkaline phosphatase (AF) or an increase in the level of alkaline phosphatase and gamma-glutamyltranspeptidase (GGTP) [4], but more often it is an increase in the level of alkaline phosphatase and bile acids (BA) in the blood serum [4, 8, 9]. The effects of chemicals are often accompanied by mixed hepatocellular and cholestatic damage with the deterioration of the tubular bile outflow, leading to IHC formation, with the initial site of damage locating at different levels of bile synthesis, secretion and outflow [4, 9, 10, 36]. IHC under the influence of medicinal products or other various chemicals is observed in 20–30 % of cases with a toxic liver injury caused by them [4–12, 14] or in the absence of signs of toxic hepatitis [4].
A wide variety of frequently used medicinal products, including non-steroidal anti-inflammatory drugs, antihypertensive, antidiabetic, hypolipidemic, psychotropic, anticonvulsant agents, in addition, many antibiotics and other antibacterial agents, as well as alcohol, pesticides and other xenobiotics, often affect not only hepatocytes but also cholangiocytes, resulting in xenobiotics-induced cholangiopathy and IHC formation [1–8, 10–14, 29–30, 36]. The delayed IHC development after toxic effects is described [32]. Cholestatic reactions are usually not restored after the cessation of exposure to a medicinal product or another xenobiotic, since the recovery and regeneration of cholangiocytes is slower than those of hepatocytes, and therefore the recovery of the secretory and excretory functions of bile usually occurs later than other functions of hepatocytes [1, 6, 13–16].
Under the influence of xenobiotics, the development of the following clinical forms of IHC is distinguished: 1) acute, induced by medicinal products or other xenobiotics, intrahepatic cholestasis without hepatitis; 2) acute cholestasis with hepatitis; 3) acute cholestasis with isolated bile duct damage and chronic, xenobiotic-induced cholangiopathy [1, 2, 4, 9–11, 36].
In the literature, there are no clear criteria for assessing IHC severity, which adequately characterises the degree of severity of impaired bile synthesis, secretion and outflow. Serum bilirubin level [25], in particular, unconjugated bilirubin level [7] may serve as a possible criterion for severity evaluation. However, later it was shown that bilirubin is not a specific marker of cholestasis in general and IHC in particular, increasing with enzyme defects, cytolysis, liver failure and a number of other pathological processes, but its increase may accompany IHC [26, 27, 28]. A number of authors refer bilirubin to cholestasis markers [2, 5, 25]. It is noted that IHC may not be accompanied by hyperbilirubinaemia [25–28]. It is believed that the degree of severity of impaired bile synthesis, secretion and outflow is most adequately characterised by the increased content of bile acids in the blood [6, 26, 28–30, 36]. At the same time, an increase in the serum concentration of bile acids makes it possible to evaluate the interaction between their absorption in the intestine and capture in the liver and is the most characteristic sign of IHC. It is noted that the highest level of bile acids in the blood serum is recorded in IHC with viral alcoholic hepatitis (79.5 ± 23.7 μmol/L), slightly lower with primary biliary cirrhosis, primary sclerosing cholangitis, viral liver diseases, drug hepatitis, etc., whereas in individuals with chronic liver disease without IHC syndrome their serum level is very low (on the average, 5.4 ± 1.8 μmol/L) [28, 36]. The authors showed that the level of bile acids in the blood correlates with the frequency of detection and intensity of jaundice, pruritus of the skin, weakness, severity in the right hypochondrium, and in viral diseases of the liver and primary biliary cirrhosis — with the level of bilirubin as well. The interrelation of levels of bile acids with levels of cholestasis enzymes — alkaline phosphatase, GGTP, and also leucine aminopeptidase (LAP) is demonstrated [28, 36]. It is noted that IHC syndrome not only aggravates the patient’s condition and the disease outcome, but also necessitates a significant increase in treatment volumes.
Purpose of the study. To identify the frequency of various IHC forms in patients with toxic liver injury in acute and chronic intoxications with pesticides and to assess the efficacy of ursodeoxycholic acid in the treatment complex.
Material and methods. The prevalence of IHC and its individual clinical forms and variants of the course was studied in 236 patients-agricultural workers with acute poisoning with pesticides: 162 — herbicides based on 2,4-dichlorophenoxyacetic acid (2,4-D), 60 — phosphororganic pesticides (POP), 14 — synthetic pyrethroids (SP) and 70 patients with chronic intoxication with pesticides (CIP), conditioned by prolonged occupational exposure to a complex of pesticides. IHC prevalence was studied both in patients with toxic hepatitis, which was diagnosed with the obligatory presence of cytolytic syndrome with an increase in alanine aminotransferase (ALT) levels, less frequently with simultaneous increase of aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGTP), and in patients without toxic hepatitis with an increase in the blood of at least one of cholestasis enzymes (AP, GGTP) or bile acids, as well as their combination. As a control, 30 practically healthy agricultural workers were examined, who did not come into contact with pesticides and other toxic substances in the process of work. All cases of acute poisoning with pesticides developed in agricultural workers due to gross violations of hygienic regulations for their use. The age of the patients with acute pesticide poisoning ranged from 28 to 58 years (mean — 38.2 ± 0.9 years); of patients with CIP — from 32 to 57 years (mean — 48.3 ± 0.6). The mean age of the control group was 37.8 ± 2.2 years.
The object of this study was toxic lesions of the hepatobiliary system in agricultural workers with acute and chronic intoxication with pesticides. The subject is IHC syndrome, its clinical forms and variants of its progression with the improvement of diagnostic and treatment methods.
In the examination of patients, as in the previous work [9], general clinical methods were used: examination, questionnaire, study of medical records (outpatient card data, sanitary and hygienic characteristics of working conditions, industrial accident report, as well as data of toxicological studies on the content of pesticides in the work area air and victims’ blood), anthropometric data, abdominal ultrasound examination, as well as biochemical studies. To eliminate the viral aetiology of hepatitis, serological markers of viral hepatitis were determined and PCR was performed to detect the virus DNA. Haemochromatosis, Wilson disease, etc. were also excluded. When assessing the functional state of the liver, the activity of ALT, AST, APP, GGTP, bilirubin (BR), albumin (A), globulin (Gl), bile acids (BA), cholesterol (CS), fibrinogen, prothrombin, C-reactive protein (СRP) and thymol test parameters were determined with the help of standard test procedures [37, 38]. Taking into account that intoxication with POP, 2,4-D-based herbicides and SP is accompanied with activation of processes of lipid peroxidation, the state of oxidative stress based on the level of malonic dialdehyde (MDA) in the blood was evaluated by its interaction with thiobarbituric acid [38]. To assess the severity of metabolic endotoxicosis, the content of medium molecular weight peptides (MMWP) at 254 nm and 280 nm was determined [39].
Statistical processing was carried out using parametric statistics methods using standard programs, taking into account the basic principles of application of statistical methods in clinical trials [41]. All examinations were carried out with the consent of patients with adherence to ethical standards.
Results and their discussion. In all examined patients with the acute and chronic intoxication of pesticides, neurological disorders predominated in the clinical picture: toxic encephalopathy, asthenovegetative syndrome, less often in combination with vegetative sensory polyneuropathy of the extremities (as reported in the previous study) [9]. With neurological disorders, the toxic liver injury was frequently diagnosed (according to ICD-10), which was recorded as acute toxic hepatopathy for acute poisoning, and chronic toxic hepatitis for chronic poisoning. All patients with toxic liver injury complained of aching pains or discomfort in the right hypochondrium, bitter taste in the mouth, and occasional nausea. In patients with acute or chronic toxic hepatitis, the levels of ALT, less often AST, GGTP, were elevated in the blood serum mainly in the first days after poisoning, less often in 2–3 weeks or 1–2 months.
In acute poisoning with 2,4-D-based herbicides, the toxic liver injury was detected in 58 of 162 examined (35.8 %); in acute poisoning with POP — in 32 of 62 (51,6 %); in acute poisoning with SP — in 9 of 14 (64,2 %). Most often chronic toxic hepatitis was detected with chronic intoxication with pesticides — in 59 of 70 patients (84.2 %). (Fig. 2).
Fig. 2. Frequency of detection of toxic liver injury in patients with acute and chronic intoxication with pesticides.
Key: 2,4-D (1st bar) — dichlorophenoxyacetic acid;
POP (2nd bar) — phosphororganic pesticides;
SP (3rd bar) — synthetic pyrethroids; CIP (4th bar) — chronic intoxication with pesticides.
An increase in IHC biochemical parameters (AP, BA, GGTP, and bilirubin) was observed in patients with pesticide intoxication both with toxic hepatitis syndrome and in patients with intoxication without toxic hepatitis syndrome (Table 1). A concomitant increase in AP and BA levels was observed more often, less often their elevated level was combined with an increase in GGTP or bilirubin. In patients with acute poisoning with 2,4-D-based herbicides, IHC was detected in 10 of 58 patients with toxic hepatitis (17.3 %) and in 15.4 % of cases in patients with 2,4-D poisoning without acute toxic hepatitis. IHC was observed almost with the same frequency both in patients with hepatitis and without hepatitis in acute poisoning with POP, SP and chronic intoxications with pesticides (Table 1), reaching 27 % of cases with CIP. However, it should be noted that in patients with hepatitis, in most cases, concomitant increase in the levels of AP, BA and GGTP was observed, whereas in patients without hepatitis signs in 60–70 % of cases there was an increase only in AP levels, indicating IHC canalicular form as the predominant part of alkaline phosphatase in the liver is produced by canalicular cholangiocytes [4]. In 30–40 % of cases in patients without hepatitis, elevated AP levels or concomitant elevated levels of AP and BA were combined with an increase in GGTP level and only in some cases with an increase in the level of total bilirubin. The mean level of IHC biochemical parameters in patients without signs of toxic hepatitis was almost 1.5–2 times lower than in patients with toxic hepatitis (Table 2).
Table 1. Frequency of detection of intrahepatic cholestasis (IHC) syndrome in acute and chronic intoxications with pesticides
The highest mean level of AP was observed in patients with toxic hepatitis in acute poisoning with POP (238.4 ± 5.2 U/L) and chronic intoxication with pesticides (224.6 ± 4.2 U/L), as well as BA mean level — 62.6 ± 4.2 and 68.3 ± 4.2 μmol/L, respectively, which indicates that IHC formation is caused by the damage of both hepatocytes and canalicular cholangiocytes.
It is established that in patients with cytolytic syndrome indicating the presence of hepatitis was combined with IHC, a higher level of oxidative stress was observed. Thus, the level of malonic dialdehyde in patients with POP poisoning in combination with these syndromes was on the average 8.36 ± 0.26 μmol/L (in the control group — 2.22 ± 0.09 μmol/L, p < 0.05), whereas with cytolytic syndrome without IHC its level averaged 5.13 ± 0.36 μmol/L (p < 0.05). In chronic intoxication with pesticides, the average level of malonic dialdehyde in patients in combination with IHC and toxic hepatitis was 7.24 ± 0.28 μmol/L, while with cytolytic syndrome without IHC it was 5.44 ± 0.29 μmol/L (p < 0.05). A similar pattern was also found in the evaluation of endotoxicosis. In patients with toxic hepatitis with IHC, MMWP level was significantly higher than with hepatitis without IHC. Thus, in patients with poisoning with POP and CIP with IHC and hepatitis, the average MMWP level at 254 nm was 0.288 ± 0.03 and 0.286 ± 0.03, respectively, and at 280 nm — 0.296 ± 0.04 and 0.326 ± 0,02, respectively, whereas in patients with hepatitis without IHC, endotoxicosis parameters were significantly lower.
To optimise IHC treatment in patients with intoxication with pesticides both with signs of toxic hepatitis, and in patients with IHC without toxic hepatitis, the combination detoxification therapy together with carbon enterosorption, Rheosorbilact infusions, antidotal therapy for POP poisoning included UDCA infusions at a dose of 13–15 mg/kg/day for 2–3 weeks, considering that ursodeoxycholic acid has a choleretic, detoxifying, antioxidant, immunomodulatory, anti-inflammatory and anti-fibrogenic action. It is found that in patients with IHC without toxic hepatitis, the use of ursodeoxycholic acid contributed to the normalisation of cholestasis parameters in 2–3 weeks, whereas in patients with IHC and toxic hepatitis, only reduced levels of cholestasis parameters were observed. In connection with this, long-term administration of Ursofalk per OS was continued at a dose of 10 mg/kg for 1–2 months, contributing to IHC resolution or a significant decrease in its intensity. If the levels of GGTP and bilirubin were restored with IHC in 2–3 weeks, the levels of AP and BA in this group of patients were restored much later — in 1–2 months, and in some cases — in 6–12 months.
Therefore, in acute and chronic intoxications with pesticides, both with toxic hepatitis and without hepatitis, intrahepatic cholestasis syndrome develops in 20–30 % of cases, which is characterised mostly by an increase in AP and BA levels. In patients with IHC, more severe oxidative stress and endotoxicosis are observed. The use of infusions of ursodeoxycholic acid in the combination detoxification therapy promotes rapid regression of cholestasis in patients without toxic hepatitis. Combined forms of toxic hepatitis and IHC require a long-term use of Ursofalk since IHC regression in the patients of this group is observed only in 6–12 months.
The role of elective dysfunction or polymorphism of nuclear receptors and their signalling pathways or expression disorders of various transport proteins in IHC formation with intoxications with pesticides requires further study.
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