Protective effect of ginger (Zingiber officinale) against PCB-induced acute hepatotoxicity in male rats

After absorption by the organism, polychlorinated biphenyls (PCBs) cross cellular membranes and pass into blood vessels and the lymphatic system. It is generally in the liver, adipose tissues, brain and skin that we find the strongest concentrations of PCBs. Herbal medicine remains as a discipline intended to treat and to prevent certain functional disorders and/or pathologies caused by oxidative stress, which can be induced by pesticides, medicines or pollutants. The objective of this study is to verify the toxic and oxidative effects of PCBs and to investigate the protective effect of ginger (Zingiber officinale) in the liver of male rats of the “Wistar” strain. These rats are divided into 6 groups: a control group (T), two groups treated with PCB at two different concentrations (P1 and P2), a group treated with ginger extract (G), a group pretreated with ginger extract and then injected with the first concentration of PCBs (P1G), and a group pretreated with ginger and then injected with the second concentration of PCBs (P2G). The results showed that the administration of PCBs led to an increase in the relative weight of the liver, and a significant increase in all of the hepatic biomarker levels (glucose, cholesterol, triglycerides, AST, ALT, and LDH) in the serum. Furthermore, an increase in the rate of lipid peroxidation and a decrease in the antioxidant enzyme activities (catalase, superoxide dismutase and glutathione peroxidase) were observed under the influence of PCBs in the liver. The histological test showed that the PCBs induced hepatocyte vacuolization, prominent and peripheralized nuclei, hepatocellular hypertrophy and turgor of the vein in the centriacinar regions. Pretreatment with ginger extract restored all of the biochemical and oxidative parameters to the normal values and reduced the injuries caused by the PCBs. In conclusion, in our experimental conditions, ginger effectively protects the liver against the hepatotoxic effects induced by PCBs.


Introduction
Polychlorinated biphenyls (PCBs) are a group of POPs that include chemicals such as aldrin, heptachlor, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, as dened by the Stockholm Convention. 1 PCBs are a class of synthetic organic chemicals containing 209 congeners with one to ten chlorine atoms attached to the biphenyl ring, and were previously used in a variety of commercial applications. 2 They are ubiquitous pollutants chemically characterized by their lipophilicity, high persistence, chemical stability and low biodegradability. 3 They have a renowned toxicity; they are bioaccumulative contaminants and are found in certain fatty tissues in humans, including human milk. [4][5][6][7] In the body, polychlorinated biphenyls cross cellular membranes and pass into the blood vessels and lymphatic system; then, they are stored in the liver, adipose tissues, brain and skin. 8,9 PCBs can undergo anaerobic reductive dechlorination and lead to the formation of less chlorinated congeners. 10 The highly chlorinated PCBs accumulate more in the body than the lower chlorinated ones, but they are considered less toxic. 11,12 The biotransformation of PCBs could also occur via enzyme-mediated oxidation, which leads to the formation of hydroxylated polychlorinated biphenyls (OH-PCBs). [11][12][13] OH-PCBs are considered to be more toxic than their parent PCBs and could lead to the disruption of thyroid hormone metabolism. 14 Some research work has shown that PCBs as endocrine disruptors and enzyme inducers can perturb metabolism. 15 Exposure to PCBs has harmful effects on the nervous system, 16 reproduction because of the hormonal disruption, alteration in thyroid function, and the development of the immune system, and PCBs also have carcinogenic effects. 17,18 Most of the past studies also reported that PCBs induce oxidative stress by the high generation of free radicals such as O 2 c À and H 2 O 2 . 19 These ROS are thought to contribute to lipid peroxidation, DNA damage and protein degradation. 20 Indeed, in physiological conditions, there is a perfect balance between the production of reactive oxygen species (ROS) and antioxidant defense systems. Thus, oxidative stress will be dened when there is a serious imbalance between pro-oxidants and antioxidants in favor of the latter. 21 The liver, being the primary site for xenobiotic detoxication, is the principal target organ for toxic effects induced by environmental pollutants, including PCBs. 22 It has been shown that polychlorinated biphenyls disrupt the function of the liver. Commercial mixtures of PCBs and their congeners have been discovered to be hepatically carcinogenic by their induction of mono-oxygenase cytochrome P450-dependent pathways in the liver. 23 This is due to the activity of tumor initiation of the lower chlorinated congeners and tumor promoting activity of highly chlorinated PCB congeners. 24 Furthermore, it was found that PCB 3 is responsible for the induction of gene mutation, which is a typical characteristic of tumor initiation, in the liver and lungs of rats by increasing ROS levels. 25 For several years, the wealth of medicinal plants was the remedy and solution to health problems and increasing attention has been paid to the protective effects of these natural antioxidants on drug-induced toxicities.
Ginger, Zingiber officinale, has been used for 6000 years, and it is the panacea of Asian medicine. It has been used to treat transport and pregnancy nausea, has been used as an antioxidant, and has antimicrobial and antifungal properties, in addition to its culinary uses. 26,27 Several studies have shown that gingerol, the active ingredient of ginger, has anti-inammatory and analgesic activities. 28 Besides, "in vitro" it has been shown that Zingiber officinale has an antioxidant action and can protect against free radicals in animals, 29 hence its anticancer activity. 30 In addition, it was shown that ginger acts on the liver to reduce cholesterol biosynthesis, stimulate its conversion into bile acids and increase its fecal excretion. 31 Therefore, we assume that ginger can prevent against the hepatotoxic effects of PCBs.
In this study, we investigated whether pretreatment with an aqueous extract of ginger for 6 weeks could prevent PCBinduced hepatotoxicity in male Wistar rats. The serum levels of glucose, cholesterol, triglycerides, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and activities of antioxidant enzymes (catalase, SOD, GPx, TBARS) were measured in the liver.

Preparation of the aqueous extract
Commercialized powder of Zingiber officinale (ZOE) was purchased from a local market, homogenized in boiling distilled water and soaked for about 24 h. Then the mixture was ltered, and the ltrates were stored in a refrigerator for subsequent analysis.

PCB sample
In this study, we used Aroclor 1260 provided from an electricity company's dead stock.

Phytochemical studies of Zingiber officinale extract
Determination of total phenolic content. The total phenolic content of ZOE (1 mg ml À1 ) was determined using the Folin-Ciocalteu method. 32 Briey, 250 ml of the extract was mixed with 0.125 ml of Folin-Ciocalteu reagent (diluted 10 times with distilled water) and 1 ml of 7.5% saturated sodium carbonate (w/v). Aer 2 h of incubation at 45 C, the absorbance was measured at 765 nm by a Shimadzu 1240 model spectrophotometer. The amount of total phenolics is expressed as gallic acid equivalents (GAE, mg gallic acid per g of ZOE) through a calibration curve ranging from 0-100 mg ml À1 (R 2 ¼ 0.9927) and all tests were carried out in triplicate.
Determination of total avonoid content. The total avonoid content in ZOE (1 mg ml À1 ) was determined using a method described by Djeridane et al. 33 A volume of 500 ml of ZOE was mixed with 150 ml of NaNO 2 and 150 ml of AlCl 3 $6H 2 O methanolic solution (2%). Then, aer 15 min of incubation at room temperature, the absorbance of the mixture was measured at 430 nm. The amount of total avonoid content is expressed as rutin equivalents (mg RE per g of ZOE) through the calibration curve ranging from 0-400 mg ml À1 (R 2 ¼ 0.9644) and all tests were carried out in triplicate.
Determination of total condensed tannins (proanthocyanidin). The total condensed tannins of ZOE were determined by the vanillin-H 2 SO 4 method. 34 3 ml of vanillin methanolic (4%) was added to 400 ml of ZOE (1 mg ml À1 ) and 1.5 ml of concentrated sulfuric acid. The mixture was then incubated for 15 min at room temperature and the absorbance was measured at 430 nm. The amount of proanthocyanidin is expressed as catechin equivalents (mg CE per g ZOE). The calibration curve ranged from 0-350 mg ml À1 (R 2 ¼ 0.9978).

In vitro antioxidant activity of ZOE
DPPH radical scavenging activity. The method reported by Grzegorczyk et al. 35 was used to estimate the free radical scavenging activity of ZOE with the DPPH radical assay. 1 ml of various concentrations of ZOE (0-400 mg ml À1 ) was mixed with 1 ml of a methanolic solution of DPPH (0.1 mM) and incubated for 30 min at 37 C. A second range of concentrations was prepared with 1 ml of methanol to serve as a control solution. Ascorbic acid was used as a reference in the same concentration range as the test extract. Then, the absorbance of each sample was measured at 517 nm. All the analyses were done in triplicate. The ZOE antioxidant activity was calculated as follows: where A DPPH is the absorbance of the DPPH solution without sample extract, A sample is the absorbance of the sample extract mixed with DPPH solution and A control is the absorbance of the sample extract tested without DPPH. The IC 50 value is the concentration of ZOE capable of scavenging 50% of the DPPH radicals.
Ferric reducing antioxidant power. The capacity of the ZOE, in different concentrations (0-500 mg ml À1 ), to reduce the ferric ion (Fe 3+ ) present in the K 3 [Fe(CN) 6 ] complex to a ferrous ion (Fe 2+ ) was evaluated by the method described by Chu et al. 36 Briey, 2.5 ml of potassium phosphate buffer (0.1 M, pH 6) and 2.5 ml of potassium ferricyanide (1% w/v) were mixed with 1 ml of the extract (0-500 mg ml À1 ). The reaction mixture was incubated for 20 min at 50 C in a water bath. Subsequently, 2.5 ml of trichloroacetic acid solution (10%, w/v) was added, and the mixture was centrifuged at 3000 rpm for 10 min. Then, 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl 3 and nally incubated at 20 C for 30 min. The absorbance of the samples was measured at 700 nm. Ascorbic acid was used as a standard for comparison and the tests were carried out in triplicate.

Acute toxicity test
The ZOE was investigated in toxicity studies. A total of 30 rats were divided randomly into 5 groups supplemented orally with gradually increasing concentrations (100 mg to 1000 mg per rat). The animals were directly observed for toxic symptoms, aer the rst 4 h of dosing. Aer 24 h, the surviving animals were maintained under daily observation for two weeks.
Live subject statement. The experimental protocol was approved by the Faculty of Ethics Committee in our institution with ethics approval number 1204. The animals were maintained in accordance with the International Guidelines for the Care and Use of Living Animals in Scientic Investigations (Council of European Communities 1986).

Animal treatments
Three-month-old Wistar male rats, about 111 g in body weight, fed on 15% protein food (SNA, Sfax, Tunisia), were kept in a breeding farm, at 22 C, with a stable hygrometry, under a constant photoperiod.
These rats were divided into 6 batches each containing 6 rats: Group T, which served as the control group. Group G, which was treated by drinking 200 mg per kg b.w. of the aqueous ginger extract 37 throughout the duration of the treatment.
Two groups P 1 and P 2 , which were treated with PCBs at two different concentrations (P 1 ¼ 470 mg per kg of b.w. and P 2 ¼ 980 mg per kg of b.w.) by using intra-gastric intubation, 38 for 7 and 5 days, respectively.
Two groups P 1 G and P 2 G, which were pretreated with the aqueous extract of ginger in drinking water for 6 weeks and then administered the PCBs at concentrations P 1 and P 2 for 7 and 5 days, respectively.
The animals were weighed daily and aer 49 days of treatment they were sacriced by cervical disruption. The liver was quickly removed and weighed, a portion was stored at À80 C until analysis and a portion was xed in formalin immediately for histopathological examination. Blood was centrifuged and serum aliquots were stored at À80 C.

Preparation of the liver extracts
About 1 g of liver was cut into small pieces and homogenized in 2 ml of ice-cold Tris buffer (TBS, pH 7,4) using a crusher (homogenizing Ultra-Turax), and then centrifuged at 9000 rpm for 15 min at 4 C. The supernatants (S1) were collected and stored at À80 C until use.
Estimation of lipid peroxidation. According to Yagi, 39 the level of lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS). For the assay, 125 ml of supernatant (S1) was mixed with 175 ml of 20% trichloroacetic acid containing 1% butylhydroxy-toluene (BHT) and centrifuged (1000 Â g, 10 min, 4 C). Then, 200 ml of the supernatant (S2) was mixed with 40 ml of HCl (0.6 M) and 160 ml of thiobarbituric acid (0.72 mM) and the mixture was heated at 80 C for 10 min.
The absorbance was measured at 530 nm. The amount of TBARS was calculated using an extinction coefficient of 156 mM À1 cm À1 and expressed in nmol mg À1 of protein.
Catalase activity. Catalase activity was measured according to Aebi. 40 The reaction mixture (1 ml) contained 100 mM phosphate buffer (pH 7), 100 mM H 2 O 2 and 20 ml (about 1-1.5 mg of protein) of liver homogenate. H 2 O 2 decomposition was followed by measuring the decrease in absorbance at 240 nm for 1 min. The enzyme activity was calculated using an extinction coefficient of 0.043 mM À1 cm À1 and expressed in international units (IU), i.e., in mmol H 2 O 2 destroyed per min per mg protein, at 25 C.
Superoxide-dismutase activity. The total (Cu-Zn and Mn) superoxide-dismutase (SOD) activity was determined by measuring its ability to inhibit the photo-reduction of nitroblue tetrazolium (NBT). 41 One unit of SOD represents the amount inhibiting the photo-reduction of NBT by 50%. The activity was expressed as units per mg protein, at 25 C.
Glutathione peroxidase activity (GPx). GPx activity was assayed according to the method of Flohe & Gunzler. 42 The activity was expressed as mmol of GSH oxidized per min per g of protein, at 25 C.
Protein content. Protein content in tissue extracts was determined according to Lowry's method 43 using bovine serum albumin as the standard.

Histopathological examination
Formalin-xed livers were processed routinely, embedded in paraffin, sectioned at 3-4 mm and stained with hematoxylin and eosin (H&E). An expert in histopathological evaluation 44 examined the slides.

Statistical analysis
Two independent experiments were performed. Data were expressed as means AE standard deviation (SD). Statistical signicance was assessed using Student's t-test. p # 0.05 was considered signicant.

Acute toxicity test
The tested animals were administered with different doses of ginger extract (100-1000 mg kg À1 ). The data of the acute toxicity test showed no toxicity or lethality observed up to 1000 mg kg À1 . In this study, a dose of 200 mg kg À1 BW was chosen to investigate the antioxidant and hepatoprotective activities of the aqueous extract of Z. officinale in experimental animals.

In vitro antioxidant capacity
DPPH radical scavenging activity. The antiradical activity of the Zingiber officinale extract, in vitro, against the DPPH radical is shown on Fig. 1. Indeed, as the concentration of the extract increases, the anti-DPPH activity increases also until reaching a maximum concentration of 0.4 mg mL À1 . The antioxidant capacity was determined from IC 50 , which corresponds to the concentration necessary to reduce 50% of the DPPH radicals. The IC 50 of the Z. officinale extract is mathematically calculated and valued as 79.70 AE 0.27 mg mL À1 , which is signicantly lower than that of ascorbic acid used as a positive control (19.41 AE 2.71 mg mL À1 ) ( Table 2).
Ferric reducing antioxidant power. As shown in Fig. 2, ginger has the capacity to reduce Fe 3+ to Fe 2+ , at different concentration ranges. The reducing power of ZOE and its concentration are dose-dependent. It was found to be 0.129 AE 0.008 absorbance units at 500 mg mL À1 with the effective concentration EC 50 being 19.4 AE 1.23 mg mL À1 . This activity appeared significantly (p < 0.05) lower compared with that of the positive control (ascorbic acid), which was 0.52 AE 0.003 absorbance units at 500 mg mL À1 with an EC 50 of 0.47 AE 0.002 mg mL À1 .

Liver weight
Aer sacrice of the rats, the livers were weighed and the relative weight was estimated. The results showed an important increase in liver relative weight in all PCB-treated groups: P 1 , P 2 , P 1 G and P 2 G (+77%, 68%, 87% and 54%, respectively). There was no signicant improvement noticed even with ginger pretreatment (Fig. 3).

Serum markers of cell damage
Lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are released into the blood when certain organs or tissues, particularly the liver and heart, are injured. As shown in Fig. 4, PCB treatment induced a signicant increase in the serum levels of LDH, AST and ALT (+33%, 42% and 64%, respectively) in (P 1 ) and (P 2 ) rats, as compared to controls (T). These effects were signicantly decreased in the PCB-treated rats drinking ginger extract (P 1 G and P 2 G groups) (À26%, 20% and 19%, respectively) compared to the PCB-treated groups (Fig. 4).
The serum levels of glucose (+24%), triglycerides (+180%) and total cholesterol (+98%) were also found to be signicantly increased in (P 1 ) and (P 2 ) rats, as compared to controls. However, these levels were signicantly decreased in the rats pretreated with ginger extract (P 1 G and P 2 G) ( Table 3).

Estimation of lipid peroxidation levels (TBARS) in liver extract
In this study, we showed that the administration of PCBs (at concentrations P 1 and P 2 ) induces an important increase in the TBARS rates (+117% and 140%, respectively) in the liver as compared to control rats (T). However, in rats pretreated with singer extract (P 1 G and P 2 G), the rates of TBARS decreased and the results are comparable with those obtained for the control rats (T) (Fig. 5).

Changes of antioxidant enzyme activities in liver extracts
PCB treatment induced a signicant increase (+142%) in SOD activity in the liver of (P 1 ) and (P 2 ) rats and about a À53% decrease in CAT and GPx activities (Fig. 5). However, the ginger extract pretreatment (P 1 G and P 2 G groups) showed important changes in the activities of these enzymes (À45% decrease in SOD activity, +88.5% and 63.5% increase, respectively, in CAT and GPx activities).

Liver histopathological changes
Histological examination showed that the PCBs (for both concentrations of 470 mg kg À1 and 940 mg kg À1 ) lead to an increase in the size of the hepatic lobules due to hepatocyte hypertrophy characterized by large areas of cytoplasmic pallor (hydropic degeneration), mild vacuolation of hepatocytes (arrows, c and d, Fig. 6), prominent and peripheralized nuclei, and hypertrophy and turgor in the central vein (CL, c and d, Fig. 6). These changes were moderately improved in gingerpretreated rats (e and f). In the control (a) and ginger groups (b), there was no evidence of hepatic abnormality; we observed mild cytoplasmic clearing without vacuolization and with centrally located nuclei consistent with glycogen.

Discussion
In this study, we intended to determine the toxic effect of polychlorinated biphenyls (PCBs) on hepatic function, and to verify the protective effects of ginger extract against the toxicity induced by PCBs.     3 Liver relative weight of control rats (T), rats consuming ginger (G), rats treated for 7 and 5 days (P 1 and P 2 , respectively) and rats pretreated with ginger for 6 weeks (P 1 G and P 2 G). Values correspond to the mean of 6 measurements AE SD. Student test: **(p # 0.01) indicates significant differences between (P 1 and P 2 ) and control rats (T).
In the animal study, we found that the oral administration of PCBs to male rats from the "Wistar" strain induced liver hypertrophy. In accordance with our study, Lai et al. 45 explained this liver hypertrophy by the accumulation of lipids due to disruption of hepatic lipid intake and metabolism.
The PCB treatment induced a highly signicant increase in the serum levels of glucose, cholesterol and triglycerides, which is associated with an increase in the hepatic biomarkers AST, ALT and LDH. This could be explained by the strong accumulation of PCBs in the liver and the severe alteration of hepatocytes. Indeed, the transaminases (AST and ALT), which are involved in protein renewal and the synthesis of new peptides, strongly affect the metabolism due to their inhibition by PCBs. Consistent with our results, Pereira & Rao 46 found that the administration of PCBs (Clophen A60) at 2.8 mg per kg of b.w. per day signicantly increased the level of glucose, LDH, cholesterol and triglycerides. In addition, our results are harmonized with other studies reporting negative effects of PCBs in the liver, such as that by Wang et al. 47 AST, being a primarily mitochondrial enzyme, allows us to deduce that, at the cellular level, there was an increase in respiratory burst and mitochondrial involvement in the hepatocytes of rats treated with PCB. Pereira & Rao 46 explained the increase of hepatic biomarkers with the increase of lipid peroxidation that could affect mitochondrial function and the leakage of mitochondrial enzymes due to the injury of the mitochondrial membranes.
Aer 49 days of treatment, we observed in rats pretreated with ginger aqueous extract (P 1 G and P 2 G) that the toxicity of PCB was greatly reduced. The aqueous extract of ginger decreased signicantly the AST, ALT, cholesterol, triglyceride, glucose and LDH levels. Several studies clearly reinforce our results, showing the hepatoprotective effect of ginger against liver toxicity induced by ethanol, carbon tetrachloride, bromobenzene and acetaminophen, accompanied by a signicant decrease of AST and ALT. [48][49][50][51] It is the same for the hypocholesterolemic effect of ginger, which is probably due to the inhibition of cellular synthesis of cholesterol. The possible mechanism of the plant to reduce serum triglycerides is due to Fig. 4 Activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in control rats (T), rats consuming ginger (G), rats treated for 7 and 5 days (P 1 and P 2 , respectively) and rats pretreated with ginger for 6 weeks (P 1 G and P 2 G). Values correspond to the mean of 6 measurements AE SD. Student test: **(p # 0.01) indicates significant differences between (P 1 and P 2 ) and control rats (T). ++ (p # 0.01) indicates significant difference between (P 1 G and P 2 G) and (P 1 and P 2 ) rats. Table 3 Serum levels of glucose, triglycerides and cholesterol in control rats (T), rats consuming ginger (G), rats treated for 7 and 5 days (P 1 and P 2 , respectively) and rats pretreated with ginger for 6 weeks (P 1 G and P 2 G) a Controls (T) G P 1 P 2 P 1 G P 2 G a Student test: **(p # 0.01) indicates signicant differences between (P 1 and P 2 ) and control rats (T). ++ (p # 0.01) indicates signicant difference between (P 1 G and P 2 G) and (P 1 and P 2 ) rats. Values correspond to the mean of 6 measurements AE SD.
the increase in the expression and activity of the lipoprotein lipase enzyme in the vessels. This enzyme increases the breakdown of triglycerides in the blood vessels and reduces the blood levels of triglycerides. 52 Ginger also inhibits hepatic fatty acid and triglyceride synthesis by lowering key enzyme activity. 53 In the research of Heeba & Abd-Elghany, 54 it was also shown that ginger stimulates the conversion of cholesterol into bile acids, and increasing the excretion of cholesterol and phospholipids in the stools aer taking ginger can also be considered as a potential mechanism for the effects of ginger in reducing serum cholesterol levels. 55,56 In the study of Gao et al., 57 it was shown that ginger improves insulin sensitivity in the body, Fig. 5 Levels of TBARS and the activities of superoxide-dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in the liver of control rats (T), rats consuming ginger (G), rats treated for 7 and 5 days (P 1 and P 2 , respectively) and rats pretreated with ginger for 6 weeks (P 1 G and P 2 G). Values correspond to the mean of 6 measurements AE SD. Student test: **(p # 0.01) indicates significant differences between (P 1 and P 2 ) and control rats (T). ++ (p # 0.01) indicates significant difference between (P 1 G and P 2 G) and (P 1 and P 2 ) rats. which may explain the mechanism of the decrease in blood glucose levels in rats pretreated with ginger.
Our experimental study showed that the induction of oxidative stress by PCBs has been demonstrated by the highly signicant increase of TBARS in liver tissues. We found that the levels of lipid peroxidation are responsible for the formation of lipid hydroperoxides in membranes, leading to lesions in the membrane structure and inactivation of enzyme membrane binding. 58 Research by Twaroski et al. 59 showed that the toxic manifestations induced by PCBs may be associated with the high production of ROS and the initiation and self-propagating reaction of lipid peroxidation. The oxygen radicals react with poly-unsaturated fatty acid residues in the phospholipids resulting in the production of excessive amounts of products, which can damage proteins and DNA. In fact, PCBs may interact with hydrogen peroxide to form hydroxyl radicals, which are the most active form of ROS in biological systems.
It seems quite clear that the presence of ginger reduced the TBARS in pretreated rats (P 1 G and P 2 G). This urged us to deduce that ginger plays a protective role against oxidative stress induced by PCBs. The research of Rajkumar & Rao 60 showed an important inhibition of lipid peroxidation by dehydrozingerone, which is a synthetic analogue of zingerone. It is important to note that dietary ginger concomitant to 1% w/w during the administration of malathion (20 ppm) for 4 weeks signicantly attenuated lipid peroxidation in the liver. 61 The high lipid peroxidation may also be due to decreased activities of catalase and superoxide dismutase, which are scavenger enzymes of free radicals. In our study, we found that PCBs increased the activity of superoxide dismutase and decreased the catalase and glutathione peroxidase activities. Several studies have associated changes in the activity of superoxide dismutase with the reduced synthesis, high degradation or inactivation of this enzyme. 16,20,62 Other studies have investigated the increase in superoxide dismutase activity and they explained these results by the increase of the concentration of superoxide anions (O 2 c À ), which causes an increase in the concentration of H 2 O 2 due to the inhibition of catalase and glutathione peroxidase, as they are the responsible enzymes for scavenging H 2 O 2 . In the work of Venkataraman et al., 16 the decrease in glutathione peroxidase activity in rats treated with PCBs is correlated to the reduction of the substrate, meaning reduced glutathione (GSH) level and high peroxide level. In other research studies, the decrease observed in our results is evaluated by decreased synthesis and/or inactivation of the enzyme. 20 However, the administration of ginger as a pretreatment (200 mg per kg of b.w.) for 6 weeks restored the activities of antioxidant enzymes. This could be explained by the presence of several antioxidant compounds in ginger, such as gingerols, shogaol derivatives, ketones, phenolics, avonoids and volatile oils. 58 [6]-Gingerol as the major constituent of ginger has been shown to exert an inhibitory effect on xanthine oxidase, the enzyme responsible for the generation of reactive oxygen species. 63 This antioxidant activity of ginger extract is explained by the results found in our phytochemical study, which revealed signicant levels of polyphenols and a lesser amount of avonoids and tannins, which is correlated with the ndings of Gabr et al. 64 These antioxidant substances are well known to protect the body against free radicals. The antioxidant activity of Z. officinale aqueous extract was evaluated in vitro using the DPPH and FRAP tests. Our results showed important reducing properties due to the presence of compounds that reduce the ferricyanide complex of Fe 3+ to the ferrous (Fe 2+ ) form by donating a proton. In addition, the ginger aqueous extract exhibits a signicant scavenging activity based on the reduction of the stable and free radical DPPH (purple color) thanks to the hydrogen atoms existing on the antioxidant contents of the ZOE. Our observations are consistent with those demonstrated by Gabr et al. 64 These antioxidant potentials of the ginger extract might be due to its richness in polyphenols and avonoids. These bioactive compounds are known by their redox properties and might play an important role in chelating transition metals and scavenging free radicals. 65 The studies of Shanmugam et al. 58 showed that the activities of antioxidant enzymes SOD and GPx were improved in the liver tissue of animals pretreated with an ethanolic extract of ginger at a concentration of 100 mg per kg b.w. compared to animals exposed to bromobenzene. The same research team reported that the extract of ginger decreases the activity of cytochrome P450 by reducing the metabolism of bromobenzene into reactive metabolites.
Histological examination showed that the PCBs (at both concentrations of 470 mg kg À1 and 940 mg kg À1 ) induced vacuolization of hepatocytes, prominent and peripheralized nuclei and hypertrophy and turgor of the central vein. The results of this study are consistent with the works of Lai et al. 45 and Wang et al., 47 which showed severe changes in hepatocytes with an increase in the size of the lobules and capsular irregularity. However, the aqueous extract of ginger slightly attenuated these cytological manifestations. This has been demonstrated in the studies of Heeba & Abd-Elghany, 54 showing that the administration of ginger plays an important role in reducing liver damage and preserves the integrity of the hepatocyte membranes.
In conclusion, it seems that ginger is able to protect the liver against oxidative stress and biochemical manifestations induced by polychlorinated biphenyls. The high protection capacity of ginger could be due to its content of antioxidant phytochemicals, which neutralize the high production of free radicals generated by the PCBs.

Data availability
The data used to support the ndings of this study are available from the corresponding author upon request.

Conflicts of interest
The authors declare that there are no conicts of interest. Macromolecular Biochemistry and Genetics, Faculty of Sciences of Gafsa and Laboratory of Environmental Pathophysiology, Development of Bioactive Molecules and Mathematical Modeling, Faculty of Science of Sfax, with collaboration with the Laboratory of Biochemistry at the Regional Hospital of Gafsa, Tunisia.