Evaluation of conventional and non-conventional biomarkers of liver toxicity in greenhouse workers occupationally exposed to pesticides

Highlights

• Pesticides have the potential to cause liver toxicity.

• Greenhouse workers exposed to pesticides and non-exposed controls were assessed.

• Biomarkers of hepatocellular and biliary system impairment were measured in serum.

• Increased AST and decreased ALP, OCT and ARG1 were observed in greenhouse workers.

• Results failed to show unambiguous evidence of pesticide-related hepatotoxicity.

Abstract

The liver plays a prominent role in maintenance of homeostasis and is the major organ for xenobiotic metabolism, including pesticides. Conventional liver function tests are widely used to assess hepatocellular and biliary system dysfunction by measuring serum levels of aminotransferases (ALT, AST) and cholestasis enzymes (alkaline phosphatase –ALP– and γ-glutamyl transferase –GGT–), respectively. Although these tests are not entirely specific for liver damage, their specificity increases when measured concurrently, but still have limited usefulness to predict early liver dysfunction. Hence, non-conventional biomarkers may have a better performance for the early detection of biochemical hepatotoxicity with a greater specificity and sensitivity. A cross-sectional study with a follow-up component was conducted on 175 greenhouse workers regularly exposed to pesticides under integrated production system, and 91 controls living in the same geographical area. All individuals were evaluated for conventional (ALT, AST, ALP, GGT) and non-conventional biomarkers of hepatotoxicity (ornithine transcarbamylase (–OTC–), Arginase-1 –ARG1– and glutathione S-transferase alpha –GSTα–) over two periods of the same crop season, one of high pesticide exposure and other of low exposure. A slight increase in AST was observed in greenhouse workers relative to controls, suggestive of subtle hepatocellular toxicity. Although ALP, ARG1 and GST-α levels were decreased in greenhouse workers, this might be related to a potential homeostatic mechanism that regulates their expression. Altogether, these findings do not represent unambiguous evidence of liver dysfunction (e.g., hepatocellular or biliary system impairment) but may be the result of the low-toxicity pesticides used by greenhouse workers.

Introduction

Pesticides have been increasingly used worldwide as they play an important role in reducing crop losses caused by pests. Greenhouses constitute a productive system with special conditions of humidity, temperature and ventilation, which increase the crop production. However, such conditions contribute to a greater occurrence of pests and therefore there is a need to rely on the use of pesticides to prevent yield losses. Intensive agriculture production systems thus represent an optimal context to study the potential adverse effects on human health from long-term exposure to pesticides, which greatly increases the risk of developing clinical conditions (Gangemi et al., 2016). Nonetheless, overt clinical manifestations are preceded by earlier biochemical changes in target organs that can be quantitatively assessed by using biomarkers of organ-specific toxic response. This approach allows preventive measures to be implemented.

The liver plays a prominent role in the maintenance of homeostasis and is the major site for xenobiotic metabolism. However, as a result of phase I microsomal oxidation reactions, many chemical agents undergo bioactivation instead detoxification, resulting in toxic metabolites and generation of reactive intermediate species capable of inducing liver damage (Hernández et al., 2019; Pearson and Patel, 2016; Poljšak and Fink, 2014). Those toxic metabolites can interact with intracellular macromolecules and eventually produce hepatocellular necrosis (Hernández et al., 2019). However, genetic polymorphisms in genes encoding phase I xenobiotic metabolising enzymes may influence the metabolisation and toxicity of pesticides and their metabolites (Docea et al., 2017, Gómez-Martín et al., 2015). Conventional or traditional liver biomarkers are widely used to assess impairment of the liver function as they provide information on hepatocellular damage (aminotransferases, formerly called transaminases, i.e. alanine aminotransferase –ALT– and aspartate aminotransferase –AST–) and on the biliary system (alkaline phosphatase (ALP) and γ-glutamyl transferase –GGT–) (Gil and Hernández, 2009). However, the sensitivity of these biomarkers is limited for measuring subclinical alterations, and they also show reduced specificity for liver injury as they can be present in other organs. For that reason, non-conventional biomarkers (e.g., ornithine transcarbamylase –OTC–, Arginase-1 –ARG1– and glutathione S-transferase alpha –GSTα–) can be used to increase the sensitivity and specificity and to improve the performance for an early detection of hepatocellular damage (Bailey et al., 2012; Campion et al., 2013; Ozer et al., 2008).

The increase of AST and ALT in serum indicates damage to periportal hepatocytes and is usually associated to high intake of some drugs (Limdi and Hyde, 2003). AST is released into the blood when there is an increase in the permeability of the hepatocyte (or skeletal muscle cell) membrane or actual cell necrosis. When a hepatocellular injury occurs, ALT leaks into the extracellular space and later into the bloodstream, where it shows a slow clearance rate, with a half-life of approximately 42 h (Ozer et al., 2008). The magnitude of ALT elevation is usually greater than that of AST when both are elevated due to hepatocellular injury because of the longer half-life of ALT and the greater fraction of AST that is bound to the mitochondria (Aulbach and Amuzie, 2017). Furthermore, the specificity of ALT for liver damage is greater than that of AST since it is found in significant lower quantities in other tissues (Yang et al., 2014).

There are more specific biomarkers of toxic liver damage affecting centrilobular hepatocytes, such as glutathione-S-transferase alpha (GSTα). GST represent a superfamily of multifunctional proteins serving as phase II detoxification enzymes of xenobiotics that afford cell protection against toxic metabolites or reactive intermediates by facilitating their inactivation and elimination through the urine (Dessì et al., 2012). Since GSTα is a cytosolic enzyme also expressed in the epithelial cells of the proximal tubule, it may also reflect a functional impairment of proximal tubular cells. However, in this case the enzyme is usually measured in urine instead of serum (Hernández et al., 2019).

Urea cycle enzymes have also been used as non-conventional biomarkers for periportal hepatocytes damage. Urea cycle is the primary metabolic pathway for the disposal of ammonia produced by the degradation of amino acids, which consists of two mitochondrial and four cytoplasmic reactions that occurs exclusively in the liver (Moedas et al., 2017). This metabolic process ultimately results in urea, a waste product that is excreted by the urine. OTC is a mitochondrial enzyme located exclusively in periportal hepatocytes where it catalyzes the reaction of carbamoyl phosphate (the first storage form of ammonia) and ornithine (the end product of the extraction of urea from arginine) to produce citrulline (Ishikawa et al., 2003). Although OTC is also located in the small intestine, the intestinal enzyme may be released into the alimentary canal and not into the circulation, so increased serum levels of OTC are considered as a useful liver-specific diagnostic marker of damage to the periportal hepatocytes. Although OTC has been considered a potential biomarker of severity of liver disease (Ceriotti, 1983; Ohnishi et al., 2017), it has seldom be used as biomarker of hepatotoxicity following exposure to chemicals or other stressors. Arginase I (ARG1) is another enzyme of the urea cycle mostly expressed in the cytosol of hepatocytes. ARG1 is an enzyme responsible for the breakdown of arginine to urea and ornithine. While urea is further excreted by the urine, ornithine is transported back to the mitochondria to begin a new urea cycle. Since ARG1 is highly specific for liver, this enzyme is considered as one of the most specific biomarkers for liver damage as compared to other enzymes (Ozer et al., 2008).

In relation to cholestatic hepatotoxicity, this liver condition is characterized by raised serum levels of ALP and GGT out of proportion to elevation of aminotransferase enzymes as a result of impaired bile secretion (Chen et al., 2018; Memon et al., 2016; Siddique and Kowdley, 2012). ALP is a high molecular weight enzyme responsible for removing phosphate groups from biological molecules. While its elevation in serum may be due to cholestasis, other possible causes include bone disease, skeletal muscle alterations and consumption of certain drugs (Limdi and Hyde, 2003, Yang et al., 2014). In order to ensure that the alteration of serum ALP is the result of a dysfunction of the biliary system, serum GGT levels should concurrently be measured (Yang et al., 2014). GGT is an enzyme synthesized in the liver and located on the outer surface of the plasma membrane where it plays an important role in the homeostasis of reduced glutathione by hydrolyzing it into γ-glutamyl-cysteine and cysteinylglycine (Lee and Jacobs, 2009).

This study sought to assess the potential hepatotoxic damage in greenhouse workers exposed to pesticides by measuring serum levels of conventional (ALT, AST, ALP, and GGT) and non-conventional (OTC, ARG1 and GSTα) biomarkers of liver dysfunction as surrogate indicators of subclinical liver toxicity. The combination of these two sets of biomarkers adds value by improving specificity and sensitivity to traditional biomarkers alone for assessing chemical-induced liver injury. Thereby, the integration of all these biomarkers provides better information to assess potential hepatotoxic risk in workers exposed to pesticides.