The Role of Oxidative Stress and Antioxidants in Liver Diseases

Free radicals are atoms or molecules that have unpaired electrons, usually unstable and highly reactive [ 1 ]. In biology system, oxygen based radicals and nitrogen based radicals are two types of free radicals. Oxygen free radicals, such as superoxide, hydroxyl radicals, and peroxyl radicals, with the addition of non-radicals, such as hydrogen peroxide, hypochlorous acid and ozone, are known as reactive oxygen species (ROS), which are generated during the metabolism process of oxygen. Reactive nitrogen species (RNS), including nitrogen based radicals and non-radicals, such as nitrogen dioxide, nitric oxide radicals and peroxynitrite, are derived from nitric oxide and superoxide via inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, respectively [ 2 , 3 ]. Due to their special chemical characteristics, ROS/RNS can initiate lipid peroxidation, cause DNA strand breaks, and indiscriminately oxidize virtually all molecules in biological membranes and tissues, resulting in injury. However, since the body is able to remove ROS/RNS to a certain degree, these reactive species are not necessarily a threat to the body under physiological conditions [ 3 , 4 ]. As a matter of fact, ROS are required at certain level in the body to perform its important physiological functions. The generation of ROS is a natural part of aerobic life, which is responsible for the manifestation of cellular functions including signal transduction pathways, defense against invading microorganisms and gene expression to the promotion of growth or death [ 1 ]. Oxidative/nitrosative stress represents the bodies’ imbalance in the production and the elimination of reactive oxygen and nitrogen species as well as decreased production of antioxidants. In terms of oxidative stress, in specific physiological conditions, it is actually useful. For example, it could strengthen biological defense mechanisms during appropriate physical exercise and ischemia, and induce apoptosis to prepare the birth canal for delivery [ 2 , 3 ]. However, this is confined to particular situations, and in most other cases, large levels of ROS and oxidative stress will induce cell death through necrotic and/or apoptotic mechanisms, leading to cellular and tissue injury. Liver is a major organ attacked by ROS [ 5 ]. Parenchymal cells are primary cells subjected to oxidative stress induced injury in the liver. The mitochondrion, microsomes and peroxisomes in parenchymal cells can produce ROS, regulating on PPARα, which is mainly related to the liver fatty acid oxidation gene expression. Moreover, Kupffer cells, hepatic stellate cells and endothelial cells are potentially more exposed or sensitive to oxidative stress-related molecules. A variety of cytokines like TNF-α can be produced in Kupffer cells induced by oxidative stress, which might increase inflammation and apoptosis. With regard to hepatic stellate cells, the proliferation and collagen synthesis of hepatic stellate cells is triggered by lipid peroxidation caused by oxidative stress [ 6 , 7 , 8 ]. In mammals, a sophisticated antioxidant system has been developed to maintain the redox homeostasis in the liver ( Figure 1 ). When the ROS is excessive, the homeostasis will be disturbed, resulting in oxidative stress, which plays a critical role in liver diseases and other chronic and degenerative disorders [ 9 ]. The oxidative stress not only triggers hepatic damage by inducing irretrievable alteration of lipids, proteins and DNA contents and more importantly, modulating pathways that control normal biological functions. Since these pathways regulate genes transcription, protein expression, cell apoptosis, and hepatic stellate cell activation; oxidative stress is regarded as one of the pathological mechanisms that results in initiation and progression of various liver diseases, such as chronic viral hepatitis, alcoholic liver diseases and non-alcoholic steatohepatitis [ 10 , 11 ]. It has also been suggested that there are complicated cross-talks among pathological factors, inflammation, free radicals and immune responses [ 11 , 12 ]. The general mechanism scheme of oxidative stress induced by various factors on liver disease is concluded in Figure 2 . Moreover, systemic oxidative stress arising during liver disease can also cause damage to extra-hepatic organs, such as brain impairment and kidney failure [ 13 ]. It was suggested systemic oxidative stress might be a significant “first hit”, acting synergistically with ammonia to induce brain edema in chronic liver failure [ 14 ]. With regard to kidney failure, systemic oxidative stress is considered to play a critical role in the pathophysiology of several kidney diseases [ 15 , 16 ]. Figure 1 The redox homeostasis in the liver. Figure 2 The general mechanism scheme of oxidative stress induced by various factors on liver disease. Both enzymatic and non-enzymatic antioxidant system are essential for cellular response in o

The liver is the most frequently targeted organ in terms of drug toxicity. The production of radical species, specifically ROS and RNS, has been proposed as an early event of drugs hepatotoxicity and as an indicator of hepatotoxic potential [ 48 ]. It has been discovered that a lot of drugs could induce oxidative stress including increase of cellular oxidants and lipid peroxidation, depletion of antioxidants in the liver, such as anti-inflammation drugs, anti-analgesic drugs, anti-cancer drugs and antidepressants. For example, sulfasalazine, a drug to treat inflammatory bowel diseases, has been found to induce hepatic oxidative damage [ 49 ]. Oral sulfasalazine administration could reduce SOD but increase CAT activity significantly. It is also suggested that oxidative damage is involved in hepatotoxicity of sulfasalazine treatment. As for zoledronic acid, it is a nitrogen-bearing bisphosphonate, and used to treat the cancer-associated hypercalcemia. It has been shown that zoledronic acid significantly elevated MDA and nitric oxide levels, whereas reduced GSH levels, which indicated that zoledronic acid could induce oxidative stress and decrease antioxidant level in liver [ 18 ]. Furthermore, liver antioxidant capacity in hepatic injury induced by paracetamol, an extensively used analgesic compound in mice was evaluated [ 50 ]. It was shown that paracetamol induced a remarkable increase of MDA and nitrite as well as nitrate in the liver, with potent decrease of total SOD and Cu/Zn-SOD activity. Samarghandian et al . [ 51 ] studied effect of long-term treatment of morphine on enzymes, oxidative stress indices and antioxidant status in male rat liver. The results showed that the levels of ALT, AST and lactate dehydrogenase (LDH) in serum as well as MDA in liver were significantly elicited, while the activities of SOD, glutathione-s-transfrase and CAT were remarkably reduced by morphine. Oxidative stress generated by anticancer drugs including doxorubicin, paclitaxel and docetaxel in the liver of rats have been indicated. It was found that all three drugs increased thiobarbituric acid-reactive substances (TBARS), and the administration of docetaxel significantly decreased the activity of SOD. Furthermore, combined administration of two drugs generated greater changes in oxidative stress related molecules than single agents [ 52 ]. Nimesulide, nonsteroidal anti-inflammatory drug, could increase the activities of ALT, AST, ALP and the content of bilirubin in the serum. The activities of SOD and CAT and GSH-Px in the liver were decreased by nimesulide in mice [ 53 ]. Chronic administration of fluoxetine (15 mg/kg/day) or clozapine (20 mg/kg/day) was measured in rats exposed to chronic social isolation and controls. The increased serum ALT activity, MDA, decreased GSH levels and compromised SOD expression suggests a link between drugs and hepatic oxidative stress [ 54 ]. Anti-tuberculosis agent isoniazid (INH) resulted in both oxidative and nitrosative stress, but the correlation of hepatotoxicity severity with RNS rather than ROS suggested that ONOO − generation and mitochondrial dysfunction are responsible mechanisms for hepatotoxicity of INH in vivo [ 55 , 56 ]. Although hepatotoxicity induced by various drugs in humans has been demonstrated in a great number of clinical trials, report concerning the role of oxidative stress in patients with drug induced liver disease is limited by far. For example, mitochondrial dysfunction and DNA damage are found to be critical events in the underling mechanism of paracetamol induced hepatotoxicity in patients, which is supposed to partly attribute to oxidative stress, but, accurate and direct evidence to show the status and role oxidative stress in patients is lacking [ 57 ]. As a matter of fact, currently, in addition to animal model study, the investigation of hepatotoxicity induced by drugs is mainly based on the results of retrospective study, whereas there are few clinical studies with large numbers of patients. Moreover, models using human cells have been attempted to mimic pathogenesis of drug induced hepatotoxicity in humans [ 55 ]. Overall, clinical data and appropriate experimental model, which could closely resemble the human pathophysiology, is critical for future study of antioxidant treatment for hepato-toxicity caused by drugs.

Other factors such as radiation and temperature may also induce hepatic oxidative stress. The oxidative stress induced through exposure of mobile phone-like radiation has been investigated in the liver of guinea pigs [ 61 ]. The results showed that after radiation exposure, the levels of MDA and total nitric oxide were significantly increased and the activities of SOD, myeloperoxidase and GSH-Px were reduced in the liver of guinea pigs. Additionally, the severity of oxidative damage was increased along with the duration of radiation exposure. The results suggested that mobile phone-like radiofrequency radiation could induce oxidative damage in liver, implying the adverse effect of mobile phone use. Moreover, study observed that cold stress could lead to decrease in CAT, SOD and GSH-Px activities in rat liver when the rats were kept at 10 °C for a week, which indicated that cold stress may cause hepatic damage which is associated with oxidative stress [ 62 ]. Benzoyl peroxide is a substance with strong oxidizing capacity, and broadly used as flour bleaching agent. The hepatic antioxidant status and ATPases were affected by benzoyl peroxide in mice [ 63 ]. Following benzoyl peroxide exposure, SOD activity was reduced significantly, whereas the content of MDA was increased in liver tissue. The activities of Ca 2+ -ATPase and Mg 2+ -ATPase in liver were also significantly decreased by benzoyl peroxide. In another study, the effect of ZnO 2 nanoparticles, a common cosmetic component, on cellular oxidative stress in mouse liver was investigated [ 64 ]. After exposure to ZnO 2 nanoparticles, viability of hepatic cells was decreased in concentration-dependent manner, and decrease in antioxidant enzyme levels as well as increase in DNA adduct. Studies have suggested that maternal high-fat diet feeding could raise the incidence of metabolism-related diseases in offspring, including chronic liver disease. Zhang et al . [ 65 ] found that maternal high-fat diet increased the level of plasma triglyceride and hepatic TBARS significantly. The size of lipid droplets in the liver of rat offspring was also increased. Expression of antioxidant defense genes, such as GSH-Px-1, Cu/Zn-SOD, and paraoxonase enzymes, were significantly lowered in the liver. Up-regulation of the inhibitor of cyclooxygenase-2 and cyclin dependent kinase 4a, and down-regulation of cyclin D1 and phosphorylation of retinoblastoma protein were found in the offspring. These results suggested that maternal high-fat diet might reduce the capacity of antioxidant defense and speed up cellular senescence in hepatic tissue of older offspring. In another study, the effect of high dietary salt on hepatic antioxidant defensing enzyme of fructose-fed rats was investigated [ 66 ]. Feeding fructose-fed rats with high-salt diet could trigger hyperinsulinemia and insulin resistance resulting in membrane perturbation. This potentially enhanced hepatic lipid peroxidation in the presence of steatosis, and led to decrease in antioxidant defenses, as observed by reduction of GSH, SOD and CAT activities. These results indicated that consumption of salt-rich diet by insulin-resistant subjects could lead to sodium reabsorption, which may aggravate hepatic lipid peroxidation related to damage antioxidant defenses. In addition to those liver injury induced by exogenous substances, hepatic oxidative stress has been revealed in other liver diseases and functional disorders. For instance, Messarah et al. [ 67 ] has found that thyroid dysfunction would increase lipid peroxidation and oxidative stress status in rat liver. In another study, oxidative stress and antioxidant status in patients with autoimmune cholestatic liver diseases (AC) or autoimmune hepatitis (AIH) were investigated [ 68 ]. Several markers of oxidative injury and antioxidant components in whole blood, serum, and urine of 49 patients with AC and 36 patients with AIH as well as healthy subjects were assessed. The results showed that both AC and AIH patients had increased levels in oxidation products of lipid and protein while significant decreased of whole blood GSH level. Protein carbonyl and isoprostane levels were increased and GSH level was gradually decreased with disease severity level (mild to severe fibrosis and cirrhosis) in both AC and AIH patients. In addition, AIH patients had higher levels of aldehydes and GSH-Px activity and lower protein carbonyl levels compared to AC patients. In patients with nonalcoholic fatty liver disease (NAFLD), the oxidative stress and antioxidant status were changed as well [ 69 ]. It was shown that level of TBARS in NAFLD patients was significantly higher than subjects with viral hepatitis or healthy controls. Moreover, the ferric reducing ability of plasma in patients with NAFLD was significantly higher than healthy controls, and diseased control group of patients. These results implied that lipid peroxidation and oxidative stress were significantly increased in patients with

NAFLD is characterized by abnormal fatty acids deposition in the liver cells of patients without excessive alcohol intake, viral infection or other hepatoxins, including a broad spectrum of histological irregularities [ 98 ]. Notably, obesity is considered to be the main risk factor for the development of NAFLD and the main driver of rapid rise of NAFLD prevalence [ 99 ]. The oxidative stress of endoplasmic reticulum induced by free fatty acid in the liver might contribute to the hepatic injury, progressive fibrosis and even cirrhosis [ 100 ]. In Table 2 , certain antioxidants or plants were attempted to reduce liver injury induced by high fat diet in experimental animals, which indicated that most of them showed both antioxidant and hepato-protective effects. Furthermore, in a clinical trial that aims to systematically evaluate the effect of antioxidant supplements, it was found that AST levels, but not of ALT levels were reduced significantly in patients with NAFLD by antioxidant intervention. It should be pointed out that, however, data obtained is so far insufficient to figure out whether dietary supplements is beneficial or useless for patients with NAFLD [ 98 ]. To address this issue, large-scaled of prospective randomized clinical studies on this topic is quite necessary. It has also been indicated that insulin resistance, oxidative stress, and the inflammatory cascade play a vital role in the pathogenesis of NAFLD by animal study. Data from clinic trial indicated that insulin resistance is a high risk factor of NAFLD. Recent studies have shown that insulin resistance is present in surrounding tissue and live of almost all NAFLD patients [ 44 ]. The severity of insulin resistance is correlated with the progression of disease. However, the role of oxidative stress and inflammation in the pathogenesis of NAFLD cascade need to be further studied in human. In the setting of obesity, increased fatty acids and other related metabolites enhance oxidative phosphorylation and ATP generation, leads to increase ROS/RNS production and oxidative stress. Multiple stress-sensitive kinase signaling cascades, such as JNK and IKKβ, are activated by the increased oxidative stress. Once activated, these kinases are able to phosphorylate multiple targets, including the insulin receptor and the family of IRS proteins [ 101 ]. Insulin action is impaired by the abnormal serine/threonine phosphorylation in insulin receptor and IRS proteins such as IRS-1 and IRS-2, resulting in insulin resistance. In hyperglycemia caused by insulin resistance, intensive redox reactions occur during the process of protein glycation, generating a great deal of ROS [ 102 ]. Additionally, hyperglycemia and high insulin levels stimulate fatty acids synthesis and result in increasing lipid droplets storage within hepatocytes. The excessive intracellular levels of lipid can induce hepatocytes dysfunction or death. The increased ROS also act on large molecules such as poly-unsaturated fatty acids to initiate lipid per-oxidation, which further change the fluidity and permeability of the cell membrane. The inflammatory infiltration induced by lipid per-oxidation may also result in liver inflammation and necrosis, and even fibrosis. In mitochondrion, lipid peroxidation reduces the activity of mitochondrial respiratory chain, and thereby produces more ROS and increase oxidative stress. The prolonged oxidative stress may favor insulin resistance circularly, acting like a vicious circle. Then, the persistent exposure of oxidative stress and hyperglycemia contribute to NAFLD [ 103 , 104 ] . In addition to obesity, other risk factors such as drugs, re-feeding syndrome and other disorders are considered. For example, streptozotocin-induced diabetic rats constitutes as the model of oxidative stress. It was indicated that supplementation of alpha-tocopherol increased alpha-tocopherol in liver, but not in plasma [ 105 ]. Diet supplementation of acai, a promising source of natural antioxidants, could increase mRNA levels of gamma-glutamylcysteinesynthetase and GSH-Px in liver tissue, and decrease ROS produced by neutrophils. In addition, supplementation with acai could decrease thiobarbituric acid-reactive substances levels, and increase reduced GSH content in the liver. Moreover, the effect of dietary supplementation of vitamins C and E on oxidative stress and antioxidant redox systems was studied in streptozotocin-induced aged diabetic rats [ 106 ]. GSH-Px activity and the concentration of vitamin E in liver were lower, whereas lipid peroxidation levels in liver, and contents of ALT and AST in plasma were higher in the diabetic group than in the control group and were mostly restored by vitamins C and E treatment. Furthermore, the combined treatment with vitamin C, vitamin E, and Se showed a curative effect against the liver injury in streptozotocin-induced diabetic rats [ 107 ]. The effects of some antioxidants/plants on liver of streptozotocin-induced diabeti

Clinical trials are extremely vital and indispensable for the development of anti-oxidative therapy. We looked up the related information of current anti-oxidative therapy in clinic at http://www.ClinicalTrials.gov website. Vitamins, especially vitamin E, are the most frequently studied antioxidant as dietary supplement in clinical trials for liver disease, primarily in phase 2/3. Some other nutritional antioxidants such as zinc and Coenzyme Q10 were studied in phase 2. Compounds including silymarin, metadoxine, N -acetylcystein, propofol, and mitoquinone mesylate, which partially act as antioxidant, have been used as drugs or supplement for liver disease. Some of them, such as silymarin, metadoxine and N -acetylcysteine, are studied for NAFLD or NASH or ALD in phase 4. For example, the application of antioxidants supplement consisted of siliphos, selenium, methionine, and alpha lipoic acid has been approved in patients with fatty liver and non-alcoholic steatohepatitis in Mexico. Plants and foods such as ginger, green tea extract, and chocolate have been adopted as food supplement for their anti-oxidative properties for liver disease. Furthermore, quercetin and resveratrol, two well-known bioactive compounds isolated from plants, have been studied as food supplement as antioxidants for liver disease in phase 3. Despite certain promising results have obtained in clinical trials, anti-oxidative therapy still has a long way to go. As a matter of fact, many antioxidants are highly effective for prevention or treatment in animal models, but in humans it does not appear to be effective for the treatment of established disease. For example, anti-TNF, which shows desirable treatment effects in animal model, appears not to be effective in patients with acute alcoholic hepatitis. Therefore, translational research is highly important for the application of antioxidant therapy in clinic. In the future, natural plants and bio-active compounds isolated from plants as well as endogenous antioxidants such as melatonin, which have shown strong anti-oxidative ability and hepato-protection effects, should be studied by clinic trials with large patient samples and longer duration time.