Research Article: Oxidative stress and mitochondrial uncoupling protein 2 expression in hepatic steatosis induced by exposure to xenobiotic DDE and high fat diet in male Wistar rats

Date Published: April 25, 2019

Publisher: Public Library of Science

Author(s): Vincenzo Migliaccio, Rosaria Scudiero, Raffaella Sica, Lillà Lionetti, Rosalba Putti, Partha Mukhopadhyay.


Oxidative stress plays a key role in steatohepatitis induced by both xenobiotic agents and high fat diet (HFD). The present study aimed to evaluate hepatic oxidative stress and anti-oxidant systems response in rats exposed to HFD and/or non-toxic dose of dichlorodiphenyldichloroethylene (DDE), the first metabolite of dichlorodiphenyltrichloroethane. Groups of 8 rats were so treated for 4 weeks: 1- standard diet (N group); 2- standard diet plus DDE (10 mg/kg b.w.) (N+DDE group); 3- HFD (D group); 4- HFD plus DDE (D+DDE group). Oxidative stress was analyzed by determining malondialdehyde as lipid peroxidation product, while the anti-oxidant systems were evaluating by measuring the levels of the principal cytosolic and mitochondrial antioxidant proteins and enzymes, namely superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxidase 1 (GPx1) and uncoupling protein 2 (UCP2) involved in the control of hepatic reactive oxygens species (ROS) accumulation. The results showed malondialdehyde accumulation in livers of all groups, confirming the pro-oxidant effects of both HFD and DDE, but with a greater effect of DDE in absence of HFD. In addition, we found different levels of the analyzed anti-oxidant systems in the different groups. DDE mainly induced UCP2 and SOD2, while HFD mainly induced GPx1. Noteworthy, in the condition of simultaneous exposure to DDE and HFD, the anti-oxidant response was more similar to the one induced by HFD than to the response induced by DDE. Present findings confirmed that both HFD and xenobiotic exposure induced hepatic oxidative stress and showed that the anti-oxidant defense response was not the same in the diverse groups, suggesting that UCP2 induction could be an adaptive response to limit excessive ROS damage, mainly in condition of xenobiotic exposure.

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The liver is the main organ involved in xenobiotic detoxification as well as in dietary lipid metabolism, and hepatic steatosis is the most common pathologic liver responses to both high fat diet (HFD) and chemical exposures [1]. The metabolism disrupting chemical (MDC) hypothesis suggested by Heindel et al., (2017) [2] postulates that environmental chemicals have the ability to promote metabolic changes that can result in obesity, diabetes and/or fatty liver disease. MDC hypothesis provides a framework for the integration of different aetiology of steatohepatitis: alcoholic, non-alcoholic and toxicant-associated steatohepatitis (ASH, NASH, and TASH, respectively). A common mechanism for the etiologically different liver diseases may be found in inflammation and oxidative stress. It is well known that HFD induced hepatic mitochondrial dysfunction and oxidative stress [3,4,5,6]. On the other hand, liver xenobiotic metabolism may increase oxidative stress [7]. Little is known on the effect of simultaneous exposure to xenobiotics and HFD on liver oxidative stress and metabolic disorders. Under physiological conditions, low levels of ROS are essential in many biochemical processes, including intracellular signalling, defence against microorganisms, and cell function. On the contrary, the excessive production of ROS modifies the balance between the oxidants / prooxidants and antioxidants agents, leading to lipid peroxidation and depleting the antioxidant cellular reserves (both enzymatic and non-enzymatic), causing tissue injury and, in many cases, apoptosis. Among xenobiotic agents, the pesticide dichlorodiphenylethylene (DDE) is the most persistent metabolite of the insecticide dichlorodiphenyltrichloroethane (DDT) and causes hepatoxicity, nephrotoxicity and hormonal disorders [8,9]. Moreover, it produces mitochondrial dysfunction [10] and oxidative stress in different organisms, such as marine species [11], terrestrial vertebrates [12] and cell culture [13]. Today, DDT utilization against the principal disease vectors is restricted to equatorial countries, where malaria is still endemic [14]. Nevertheless, residues of DDT and DDE are still observed in soils of many occidental countries, and in mother’s milk [15], in maternal blood serum [16] and in grapes [17]. DDT was listed by the Convention on Persistent Organic Pollutants in the “Dirty Dozen” substances in 2001. However, apart from the tropical countries where DDT is still currently used, several other countries are considering the possibility to reintroduce it [18]. In literature it was reported that DDE toxicity is due mostly to ROS production [19]. In the hepatocytes, the first line of defense from free radicals is represented by the superoxide dismutase (SODs) that catalyze the dismutation of superoxide in H2O2 and oxygen. Three isoforms of SODs have been identified, each expression of a different gene and with distinct subcellular localizations. Cu/ZnSOD (SOD1) is a cytosolic enzyme, MnSOD (SOD2) has a mitochondrial localization, and EC-SOD (SOD3) is localized in the extracellular matrix, being secreted from cells [20]. SOD1 is constitutively expressed, but can be induced by redox-active metals, superoxide, and xenobiotics. SOD2 is the most inducible form, raising its levels up to10-fold in presence of drugs and cytokines. Defects in SOD2 expression cause oxidative damage in liver, while the overexpression generally plays a protective role [21]. SOD3 does not have a significant role in superoxide detoxification in hepatocytes [22]. Another class of intracellular antioxidant enzyme are known as glutathione peroxidase (GPx). GPx are tetrameric enzymes containing a seleno-cysteine in their active site [23]. These enzymes occur in different isoforms (eight in humans), all able to degrade hydroperoxides, alkyl peroxide, and fatty acid hydroperoxides to lipid alcohols and oxygen. In particular, GPx1 seems to be the major isoform that converts hydrogen peroxide to water and oxygen and catalyzes the reduction of peroxide radicals to alcohols and oxygen; mainly cytosolic, a small fraction is also present within the mitochondrial matrix [24]. GPx1 exerts its action via oxidation of reduced GSH into its disulfide form [25]. It is known that the mitochondrial respiratory chain is the major site of intracellular ROS generation and, at the same time, an important target for the ROS damaging effects. The superoxide anion produced in the matrix side of inner mitochondrial membrane has been proposed to activate the uncoupling proteins (UCPs) that in turn might reduce the generation of further superoxide anions [26]. UCPs are a family of mitochondrial proteins formed by six trans-membrane segments into the phospholipid matrix of the inner mitochondrial membrane [27], present in animal and plant in five isoforms, from UCP1 to UCP5 [28]. UCPs isoforms are expressed in different tissues and may have different functions. The isoform1 is present in brown adipose tissue (BAT), isoform 2 is expressed almost ubiquitously, isoform 3 in BAT and in skeletal muscle, isoforms 4 and 5 are present predominantly in the central nervous system [29]. Moreover, in mitochondria, UCPs functional structure is constituted by a dimer stabilized with a disulfide bridge between the cysteines present in the hydrophilic C-terminal segment [30]. These proteins are mitochondrial anion carriers [31] whose function was initially associated to uncoupling respiration from ATP synthesis performed by UCP1, the first isoform to be discovered by Nicholls and coworkers (1978) [32], that determines releasing of heat from the oxidation of substrates in brown adipocytes. Besides the adaptive thermogenesis [33], UCPs may regulate a lot of biological processes, such as the ATP synthesis and all the mechanisms directly or indirectly linked to ATP utilization, for example the inhibition of insulin secretion by UCP2 from the pancreatic beta cells [34]. The ubiquitously UCP2 is described as mitochondrial scavenger of ROS produced by mitochondria [35, 36]. The antioxidant effect of UCP2 has been reported by in vitro and in vivo studies using UCP2 overexpression, genetic ablation and pharmacological inhibition [37, 38, 39]. Moreover, different research works suggest that UCP2 could be involved in lipid metabolism: it could stimulate fatty acids oxidation and/or prevent the oxidative damage due to high lipid levels [40]. In many tissues it has been found a modulation of UCP2 expression, with both a basal and stimulated synthesis of the protein [41]. In liver, under physiological conditions, UCP2 is essentially localized in the immunocompetent cells [42], while in conditions of oxidative stress with mitochondrial ROS accumulation, the protein is up-regulated and expressed in hepatocytes [43], suggesting that UCP2 plays an important role as a negative regulator of mitochondrial ROS production [35]. To clarify if DDE toxicity on liver is due predominantly to the oxidative stress caused by this pesticide, we administered male Wistar rats with a daily dose of DDE comparable to human daily absorption or blood concentration [44, 45]. We also compared the effect of DDE to the effect of HFD treatment on hepatic oxidative stress onset. Finally, we analyzed the effect of the simultaneous exposure to both HFD and DDE on the same markers of oxidative stress. To this end, we determined the activation of the antioxidant enzymatic systems (SOD1-SOD2) and GPx1 response and we tested the hypothesis of mitochondrial uncoupling involvement to prevent ROS production in terms of UCP2 gene expression and protein synthesis in rat livers. Our findings showed, together with the activation of the antioxidant enzymatic systems, a difference in UCP2 modulation according to the treatments used, with the highest induction of UCP2 in the hepatocytes of DDE-treated animals. From these data we assumed that UCP2 plays a protective role to limit cell damage and liver injury mitigating mitochondrial ROS production with an increasing functional impact at increasing levels of oxidative stress.

This work was carried out to study the cellular response, if any, to the toxic actions of the pesticide DDE in rat liver following a treatment of four weeks, in which DDE was administered alone or in combination with hyperlipidic diet to compare the effects of single exposure (xenobiotic or HFD) and simultaneous exposure to both environmental stimuli. Our findings revealed the presence of a different degree of oxidative stress for the different experimental groups used, together with the modulation of gene expression and protein synthesis of UCP2, the uncoupling protein that seemed to be involved in the regulation of oxidative damage as adaptive cellular response, so supporting the endogenous antioxidant system of hepatocytes. Moreover, our research confirmed that UCP2 in the hepatocytes under oxidative stress conditions was mainly induced by DDE.