Guesmi Fatma， Amit K Tyagi， Bellamine Houda， and Landoulsi Ahmed

1. Laboratory of Biochemistry and Molecular Biology， Faculty of Sciences of Bizerte， University of Carthage 1054，Tunisia； 2. Department of Experimental Therapeutics， The University of Texas M. D. Anderson Cancer Center，1515 Holcombe Boulevard， Houston， TX 77030， USA； 3. Service of Anatomo-Pathology of Menzel Bourguiba，Bizerte 1054， Tunisia

Original Article

Antioxidant Machinery Related to Decreased MDA Generation by Thymus Algeriensis Essential Oil-induced Liver and Kidney Regeneration

Guesmi Fatma1，2，#， Amit K Tyagi2， Bellamine Houda3， and Landoulsi Ahmed1

1. Laboratory of Biochemistry and Molecular Biology， Faculty of Sciences of Bizerte， University of Carthage 1054，Tunisia； 2. Department of Experimental Therapeutics， The University of Texas M. D. Anderson Cancer Center，1515 Holcombe Boulevard， Houston， TX 77030， USA； 3. Service of Anatomo-Pathology of Menzel Bourguiba，Bizerte 1054， Tunisia

Objective This study was conducted to determine the histopathological and biochemical effects of Thymus algeriensis essential oil （TEO） on hydrogen peroxide （H2O2）-induced oxidative stress in liver and kidney tissues of rats.

Methods Rats were treated in six groups and were exposed for 2 weeks to low （LD； 100 μmol/L） and high doses （HD； 1 mmol/L） of H2O2in the presence or absence of TEO （180 mg/kg）. Liver and kidney atrophy was measured by using biochemical and histopathological assays.

Results Our study demonstrated that H2O2induced liver and kidney atrophy， as evidenced by the significant elevation of serum aminotransferase， urea， and creatinine levels compared with those in the control rats. Urea levels were estimated by evaluating the activity of serum urease that hydrolyzes urea into CO2and ammonia. However， TEO treatment significantly alleviated oxidative stress in the H2O2-induced liver and kidney toxicity model by reducing the levels of malondialdehyde concomitantly with marked elevations in superoxide dismutase， catalase， glutathione peroxidase， and glutathione S-transferase， as well as decrease in glutathione activity.

Conclusion Our data demonstrated that TEO protected against H2O2toxicity by decreasing oxidant levels and DNA damage， as well as increasing antioxidant levels， indicating that TEO has a spectrum of antioxidant and DNA-protective properties.

Liver and kidney atrophy； Hydrogen peroxide； Thymus algeriensis； Antioxidants； Toxicity

INTRODUCTION

H ydrogen peroxide （H2O2） is an important cause of oxidative injury because it can easily transform into a hydroxyl radical，which is one of the most destructive free radicals. Moreover， the half-life of H2O2is comparatively longer than that of other reactive oxygen species（ROS）［1］. H2O2uses aquaporins to cross cell membranes rapidly［2］and therefore has the ability to diffuse in and out through the cell membrane. In a previous study， it was found that H2O2， rather than the superoxide anion， is the most toxic species that induces alterations in cellular functioning［3］.

The liver is very susceptible to toxicity produced by reactive metabolites because it is a major site of xenobiotic metabolism. Other than the liver， the kidney， which is susceptible to toxicity， isalso a highly specialized organ that maintains the internal environment of the body through selective excretion of unwanted substances or retention of various substances according to the needs of the body［4］. ROS have been implicated in the pathogenesis of several harmful diseases， including multiple sclerosis and liver cirrhosis. It is also a universal risk factor for the development of liver cancer （human hepatocellular carcinoma）［5］. Because hepatocyte inflammation is the major cause of liver disorders， one important therapeutic strategy may include the use of anti-inflammatory and hepatoprotective interventions， either alone or in combination［6］. Furthermore， the use of safe natural compounds as anti-inflammatory agents could be a better alternative for the detoxification of the liver and kidney.

Numerous studies revealed that plant remedies are useful in treating liver diseases［7］. As many plants and herbs have effective natural antioxidants， such as carbolic acid， isoprene derivatives， carotene，terpene， and polyphenol，［8］extracts or essential oils from these plants could serve as effective hepatoprotective and nephroprotective agents. Collective evidence supports that H2O2-induced cell injury can be prevented by antioxidants such as the essential oil of Thymus algeriensis （TEO）［9］. As described in our previous reports， TEO has the potential to prevent acute gastric injury and testis toxicity in rats by correcting the cellular imbalance between oxidants and antioxidants［10-11］.

In this study， we determined whether TEO has the potential to protect the liver and kidney from H2O2-induced injuries. We present extensive data showing the biochemical and pathological alterations induced by H2O2， and the preventive effects of TEO against H2O2-induced tissue damage in rats.

MATERIALS AND METHODS

Chemicals

All reagents， including Ellman’s reagent， reduced glutathione （GSH）， 5，5’-dithiobis-（2-nitrobenzoic acid）， bovine serum albumin， H2O2， thiobarbituric acid （TBA）， 2，4-dinitrochlorobenzene （CDNB）， and Tris-HCl buffer were purchased from Sigma （St. Louis，MO， USA）， Fluka Chemie （Buchs， Switzerland）， and Merck （Nottingham， UK）.

Plant Material

The aerial portions of wild Thymus algeriensis plants were collected during the flowering stage in March 2013 from Jebel Orbata in Gafsa， which is located on the middle west of Tunisia at a latitude and longitude of 34.39641 and 9.12914， respectively. Plant specimens （Voucher #1188） were identified and deposited in the herbarium of medicinal plants at the Agronomic National Institute of Tunis， Tunisia. The essential oil of TEO was extracted with a Clevenger apparatus［12］and dried over Na2SO4. Purified TEO was stored in sealed dark vials at 4 °C.

Experimental Animals

Thirty-six male Sprague Dawley rats （6-8 weeks old） weighing between 180 and 200 g were obtained from the animal laboratory of the Pasteur Institute of Tunis. The Ethical Committee for animal experiments of the Faculty of Sciences of Bizerte，University of Carthage， Tunisia， approved all animal protocols （Ethic# LNSP/Pro 152012） governing the experiments. Before the experiment， rats were acclimatized for 7 days to human contact to minimize their physiological responses to handling for subsequent protocols［12］. Rats were randomized into six groups comprising six animals each for treatments as follows： （1） control （C）， （2） low dose（100 μmol/L） H2O2（LD H2O2）， （3） high dose （1 mmol/L）H2O2（HD H2O2）， （4） TEO （180 mg/kg per day dissolved in normal saline）， （5） TEO and LD H2O2（180 mg/kg per day and 100 μmol/L， respectively）， and （6）TEO and HD H2O2（180 mg/kg per day and 1 mmol/L，respectively）. TEO was administered 1 h prior to H2O2treatment in animals receiving both agents. Rats were then housed in a pathogen-free environment （clean polypropylene cages） with a 12 h light/dark cycle， relative humidity of 55%±10%， and a temperature of 20-25 °C. The animals were permitted access to standard pellets and water ad libitum. The dosage of TEO used had been optimized in our previous study［10］. All treatments were administered orally for 15 d.

Body weights were measured daily for 15 d. At the end of this experiment， rats were anesthetized and blood was collected from tail vein to measure the enzymatic activities. Subsequently， rats were euthanized by using cervical dislocation. The livers and kidneys of treated and untreated animals were carefully dissected， and portions were fixed in 10% buffered formaldehyde for routine histological evaluations after staining with hematoxylin and eosin （H&E）. Unfixed portions of the livers and kidneys were weighed and subjected to extraction by using a mechanical rotary homogenizer in0.1 mol/L phosphate buffer at a pH of 7.4. The supernatant obtained after centrifugation at 8000 × g for 15 min was used as a source of enzymes for biochemical analyses.

Assessment of Hepatic and Renal Functional Marker Enzymes

The collected blood was also used for the estimation of serum marker enzymes. Serum urease activity was determined in the presence of urea by assessing CO2and ammonia production. The addition of phenol-hypochloride led to formation of the indophenol-blue complex with an absorbance at 600 nm［13］. Serum creatinine levels were estimated by using a reaction with picric acid in alkaline buffer to form a yellow-orange complex， whose color intensity is proportional to the creatinine concentration in the sample［14］. Alanine aminotransferase and aspartate aminotransferase activities were determined by using pyruvate and oxaloacetate as substrates， wherein NADH is converted into NAD+proportional to the activities of those enzymes according to the method described by Wilkinson［15］.

Assessment of Enzymatic Antioxidants and Lipid Profile

The supernatant was collected for the estimation of the protein concentration by using the Lowry method［16］， using crystalline bovine serum albumin as a standard. The level of malondialdehyde（MDA） in liver tissue homogenates was estimated by measuring TBA-reactive substances by using the TBA method and expressed as nmoles of MDA/mg protein［17］.

Antioxidant levels were evaluated by measuring the activities of superoxide dismutase （SOD）［18］，catalase （CAT）［19］， glutathione peroxidase （GPx）［20］，and glutathione transferase （GST）［21］， and the levels of reduced glutathione （GSH）［22］. The specific activities of the antioxidant enzymes （SOD， CAT， GST，and GPx） and antioxidant levels （GSH） were expressed in U/mg protein.

Histopathological Examination

For histopathological observations， a portion of the liver and kidney from each animal was preserved in 10% neutral buffered formalin and processed for paraffin embedding by using the standard methods described by Drury and Wallington［23］. Tissue sections were prepared and stained with H&E. Statistical Analysis

Statistical analysis was performed by using Statistica software version 5.1 and Microsoft Excel software. The significance of the differences between various groups was analyzed by using one-way analysis of variance followed by Duncan’s multiple range tests. The level of significance was set at P＜0.05.

RESULTS

Body， Liver， and Kidney Weights

In all experimental groups， rats remained active and vigorous throughout the treatment duration. The body and relative organ weights of untreated，low- and high-dose H2O2-exposed， and TEO-pretreated animals were examined. The results showed that treatment with both high and low doses of H2O2induced a significant （P＜0.05） body weight loss in rats compared to that in controls after 2 weeks （Figure 1）. At dosages of both 100 μmol/L and 1 mmol/L， H2O2induced dyspnea and paralysis. However， body and organ weights were found to be enhanced on TEO administration. Significant reductions in relative liver and kidney weights were also observed in H2O2-treated animals. However，administration of TEO prevented H2O2-induced liver，kidney， and weight loss （Figure 1）.

Effect of H2O2on Serum Hepatic and Kidney Markers

The effects of H2O2treatment as well as the preventive effects of TEO on serum biochemical markers are shown in Table 1. A significant increase（P＜0.05） in functional marker enzyme levels （AST and ALT） was observed in LD-H2O2- and HD-H2O2-treated rats compared to that in the untreated rats. However， prophylactic treatment with TEO before H2O2administration significantly attenuated these effects.

The levels of renal products， urea and creatinine，significantly increased in LD-H2O2- and HD-H2O2-treated rats compared to those in the control group. TEO significantly （P＜0.05） reduced renal serum product levels in H2O2-treated animals compared to that in the control group （Table 1）. This indicates the possible nephroprotective efficacy of the thyme species against H2O2toxicity.

Hepatic Lipid Profile and Antioxidant Enzyme Activities

Lipid peroxidation （oxidative degradation of lipids） is a widely accepted measure for assessing oxidative stress. The effects of H2O2treatment and the preventive effects of TEO on lipid peroxidation and antioxidant activity in liver tissue are shown in Figure 2. H2O2-mediated oxidative stress and antioxidant ability in rats was partially or completely inhibited by pretreatment with TEO （Figure 2A-G）. The administration of H2O2led to a dose-dependent increase in MDA activity relative to that in control（Figure 2B）. In addition， H2O2treatment at both 100 μmol/L and 1 mmol/L doses significantly reduced the activities of GST， GPx， GSH， CAT， and SOD （Figure 2C-G） in a dose-dependent manner compared to those in the control group. While pretreatment with TEO partially recovered CAT activity （Figure 2F） and GSH levels （Figure 2E）， it fully recovered SOD （Figure 2G） and GPx （Figure 2D） activities to those of the control group （P＜0.05）. TEO induced a significant increase in GPx and GSH activities （Figure 2D， 2E） in liver tissues.

Renal Lipid Profile and Antioxidant Enzyme Activities

The effects of H2O2treatment and the preventive effects of TEO on lipid peroxidation and antioxidant activity in the kidney are shown in Figure 3. H2O2-mediated oxidative stress and antioxidant ability in kidney tissues were partially or completely inhibited by TEO pretreatment （Figure 3A-G）. Administration of H2O2increased MDA activity in a dose-dependent manner relative to that in the control （Figure 3B）. However， MDA induction in the kidney was not as high as that in the liver. H2O2treatment significantly （P＜0.05） reduced the activities of GST， GPx， GSH， CAT， and SOD （Figure 3C-G） in a dose-dependent manner compared with those in the control group. Similar to the trend found in liver tissues， TEO pretreatment partially recovered CAT activity （Figure 3F） and GSH levels （Figure 3E）， while fully recovering SOD （Figure 3G） and GPx （Figure 3D）activities to those of the control group （P＜0.05）. TEO significantly induced the activity of GPx and levels of GSH （Figure 3D， 3G） in kidney tissues.

Correlation among Total Protein， Antioxidant Enzymes， and Daily Body Weight Gain

Linear correlation coefficients were established to explore the trend of association among total protein， antioxidant enzymes， daily body weight gain，and hepatic MDA levels （Table 2）. As presented in Table 2， AST （r=0.95） and ALT （r=0.86） levels showed a significant （P＜0.05） positive correlation with MDA levels. A similar result was obtained betweenurea （r=0.3）， creatinine （r=0.34）， and MDA. These results indicated the parallel evolution of the above-mentioned parameters with MDA levels. Therefore， to evaluate the contribution of total protein， antioxidant enzymes， and daily body weight gain to renal MDA levels （Table 3） and its antioxidant capacity， linear correlation coefficients were determined. A negative （P＜0.05） correlation was found between protein concentration （r=-0.89；r=-0.24）， CAT （r=-0.88； r=-0.23）， SOD （r=-0.95； r=-0.24），GPx （r=-0.94； r=-0.24）， GST （r=-0.89； r=-0.16）， GSH（r=-0.47； r=-0.47）， and MDA in kidney and liver tissues， suggesting that antioxidant enzymes might be responsible， among other factors， for antioxidant defense. There was no significant correlation between daily weight gain and MDA content in the liver， but a positive correlation （r=0.056） was found in the kidney.

Table 1. Effect of TEO on Biochemical Parameters

Histological Changes in the Liver and Kidney

Table 2. Linear Correlation Coefficients （r） and P Value of Total Protein， Antioxidant Status， and Daily Body Weight Gain versus the Hepatic MDA Level

Table 3. Linear Correlation Coefficients （r） and P Value of Total Protein， Antioxidant Status， and Daily Body Weight Gain versus the Kidney MDA Level

Histological appearance plays an important role in the study of the hepatoprotective and renoprotective potential of TEO in rats. Representative photomicrographs of the control，H2O2-treated， and TEO-treated liver and kidney tissues are presented in Figures 4 and 5， respectively. Sections were stained with H&E to examine cell structure. The necropsy of the euthanized rats showed hemorrhage in the liver. Histological features of the livers of untreated rats appeared normal （Figure 4A）. In contrast， in the H2O2-treated groups， higher doses of H2O2administered frequently（＜3 weeks） caused hepatotoxicity to a larger extent than did lower doses spaced more than a week apart. The liver showed loss of normal hepatocyte architecture， inflammatory cell infiltration，cytoplasmic vacuolization of liver cells， and disorganization of the hepatic parenchyma， the severity of which increased with increase in the dose. Furthermore， the oral administration of TEO revealed no histopathological changes to the liver and kidney compared with those in the untreated controls. The glomerulus and tubules appeared normal in the kidney tissue of the control rats （Figure 5）. Low doses of H2O2induced mild vascular and inflammatory changes with signs of vascular congestion， tubular necrosis， and glomerular atrophy，which are a sign of toxicity. Proteinaceous casts in the lumen of renal tubules， decreased cell numbers（glomerulosclerosis）， and disruption of the normal renal architecture were observed in the high-dose H2O2-treated group. Notably， histopathological analysis showed that TEO markedly alleviated injuries in the glomeruli and proximal tubules.

DISCUSSION

Essentially， oxidative stress is an imbalance between the production of ROS and reactive nitrogen species （RNS） in the body， and has been considered an important etiological factor involved in various chronic diseases， such as atherosclerosis，arthritis， neurodegenerative diseases， diabetes mellitus， and cancer［24］. Oxidative damage also plays a critical role in different pathophysiological conditions including liver and kidney diseases. To prevent oxidative stress and harmful diseases，consumption of antioxidant-rich phytochemicals is recommended. Besides several antioxidant medications， the effects of safe and inexpensive natural compounds need to be evaluated. One of these natural phytochemicals is TEO， which has been reported to act as an effective antioxidant in reversing the cellular imbalance between ROS and RNS， thus preventing acute gastric injury in rats［10］. Therefore， in this study， we determined whether TEO has the potential to inhibit H2O2-induced hepatotoxicity and renal toxicity in vivo.

H2O2is a weak oxidizing agent that can directly inactivate some enzymes， usually by the oxidation of essential thiol （-SH） groups. It can cross cell membranes rapidly and inside the cell， H2O2probably reacts with Fe2+or other redox metals such as Cu2+to form hydroxyl radicals， which may be the origin of many of its toxic effects［25］. H2O2can induceOH-accumulation in liver and kidney cells by breaking down endogenous antioxidant defense mechanisms， and TEO effectively reduces H2O2-induced ROS production. The detrimental effects of H2O2on the liver and kidney in rats are strongly associated with biochemical changes including impairment of function， metabolic disorders， oxidative stress， and histological alterations. In the present study， higher serum aspartate aminotransferase and alanine aminotransferase activities， which generally serve as indicators of hepatocyte mitochondrial damage，were found in response to H2O2treatment. Thus，H2O2-induced oxidative stress increased enzymatic activity and subsequently altered the permeability of the liver cell membrane， resulting in the leakage of these enzymes into the circulation［26］. It was also reported that the release of transaminases （AST and ALT） and lactate dehydrogenase from the cell cytosol leads to cellular necrosis［27］， and these are therefore considered biomarkers for liver dysfunction. Of the two enzymes， ALT is considered the most liver-specific enzyme in rats because it is present in higher quantities in hepatocyte cytosol. Therefore，its presence in the serum is considered a marker of hepatocellular necrosis［28-29］. AST has also been found in cytosolic and mitochondrial forms， and is produced in the liver， as well as the kidney， pancreas，and erythrocytes of rats［30］. As we have observed，TEO has significant potential to attenuate H2O2-induced serum AST and ALT level elevations，and to function as an antioxidant in liver and kidney cells.

Oxidative stress affects many cellular functions through various mechanisms， including alteration of gene expression through transcriptional factor activation and alteration of protein expression levels，which are responsible for most cellular functional processes［31］. In the present study， we have observed that H2O2induced protein degradation in a dose-dependent manner， which was recovered by TEO treatment in liver and renal cells （Figure 2A and 3A）. H2O2treatment increased lipid peroxidation through elevated hepatic and renal MDA levels，decreased hepatic and renal enzymatic levels （SOD，GST， GPx， and CAT）， and decreased GSH antioxidant levels. MDA is a physiological ketoaldehyde produced by the peroxidative decomposition of unsaturated lipids as a by-product of arachidonate metabolism［32］. Direct treatment with hydrogen peroxide elevated serum levels of renal dysfunction markers such as urea， creatinine， and uric acid （Table 1）. H2O2-induced the elevation of MDA levels， which was prevented by TEO pretreatment. MDA levels reflect the extent of cell damage due to oxidative stress［33］. It was also previously observed that the production of excess MDA due to tissue injury can combine with free amino acids and proteins， possibly altering their biological properties［31］.

Maintenance of intracellular redox homeostasis is dependent on a complex web of antioxidant molecules［34］， and a pivotal component is the tripeptide glutathione （g-L-glutamyl-L-cysteinylglycine）， whose free thiol group in the cysteine residue is extremely reactive［35］. Moreover， the liver and kidney contain remarkably high levels of glutathione than those in other organs. Similarly， CAT is a tetramer of four polypeptide chain antioxidants found in nearly all living organisms exposed to oxygen. It is derived from the epididymis and seminal vesicle and detoxifies both intracellular and extracellular H2O2by reducing H2O2to H2O and O2， thus eliminating the potential ROS toxicity［36］. Oxidative stress conditions can be indicated by decreases in antioxidant enzyme levels （i.e. SOD， CAT， GPx， GST， and GSH）， and this may be responsible for triggering cell apoptosis in rats. These findings suggest that a decrease in GSH may be used to monitor the severity and progress of hepatorenal toxicity. However， total GSH concentrations are high in these organs in healthy rats［37-38］. GSH peroxidase metabolizes H2O2in both cytosolic and mitochondrial compartments. In this context， GPx converts H2O2into water before it can produce ROS［39］. As we evaluated in our study，pre-administration of TEO prevented hepatorenal injuries and regulated serum hepatorenal biomarkers.

As per the histopathology of liver and kidney，oral administration of H2O2to rats for 2 weeks resulted in liver and kidney injury （Figure 4）. However， the extent of injury in TEO pre-treated rats was significantly lower than that in the H2O2-treated groups. Liver and kidney injury was associated with elevated serum alanine aminotransferase and alkaline phosphatase levels， as well as pathological lesions in the liver［40］. It was also reported that oxidative stress associated with the formation of ROS plays an important role in the pathogenesis of hepatic injury［33］and renal failure［41］， which can affect all cells within the liver by inducing chronic inflammation， ischemia， apoptosis， and necrosis［42］. The histopathological results revealed that oral exposure to H2O2at different doses produced a remarkable dose-dependent damaging effect to both the liver and the kidney of rats， supporting our biochemical observations.

As the injurious effects of oxidative stress are counteracted by natural antioxidant defense mechanisms to protect the biological system against ROS， the protective effect of TEO against H2O2-induced oxidative stress could be either direct by inhibiting lipid peroxidation and scavenging free radicals or indirect through the enhancement of the activities of SOD and CAT， which act as intracellular free radical scavengers.

CONCLUSIONS

We demonstrated that TEO is a potent antioxidant that was reported to ameliorate the effect of H2O2-induced hepatorenal toxicity through inhibition of lipid peroxidation， restoration of antioxidant enzyme activity， and alteration of serum biochemical parameters. This study helps to clarify the physiological changes caused by many known chemotherapeutic agents， pesticides， and drugs， the number of which has increased notably in the past few decades.

CONFLICT OF INTEREST

The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

AUTHORS' CONTRIBUTIONS

GF conceived and designed the study； GF and BH performed the animal experiments and histological assessment； GF and AKT drafted the manuscript； GF performed the statistical analysis； and AKT revised the manuscript. All authors read and approved the final manuscript.

Supplementary materials

The online version of this article contains supplementary materials （Table S1 and Figure S1）.

Abbreviations

ALT： Alanine aminotransferase

AST： Aspartate aminotransferase

CAT： Catalase

CDNB： 2，4-Dinitrochlorobenzene

DTNB： 5，5’-dithiobis-（2-nitrobenzoic acid）

GSH： Reduced glutathione

GPx： Glutathione peroxidase

GSH： Glutathione

GSSG： Glutathione disulfide

GST： Glutathione S-transferase

HD： High dose

LD： Low dose

LPO： Lipid peroxidation

MDA： Malondialdehyde

ROS： Reactive oxygen species

RNS： Reactive nitrogen species

SOD： Superoxide dismutase

TBA： Thiobarbituric acid

U： Unit

Received： June 26， 2016；

Accepted： September 2， 2016

1. Clarkson PM， Thompson HS. Antioxidants： what role do they play in physical activity and health. Am J Clin Nutr， 2000； 72，637S-46S.

2. Henzler T， Steudle E. Transport and metabolic degradation of hydrogen peroxide in chara corallina： model calculations and measurements with the pressure probe suggest transport of H2O2across water channels. J Exp Bot， 2000； 51， 2053-66.

3. Gamaley IA， Klyubin IV. Roles of reactive oxygen species：signaling and regulation of cellular functions. Int Rev Cytol，1999； 188， 203-55.

4. Liu CM， Ma JQ， Sun YZ. Quercetin protects the rat kidney against oxidative stress-mediated DNA damage and apoptosis induced by lead. Environ Toxicol Pharmacol， 2010； 30， 264-71.

5. Major GN， Collier JD. Repair of DNA lesion O6-methylguanine in hepatocellular carcinogenesis. J Hepatobiliary pancreat Surg，1998； 5， 355-66.

6. Catherine B， Lorenzo G， Oliver K， et al. Decoding cell death signals in liver inflammation. J Hepatol， 2013； 59， 583-94.

7. Thyagarajan SP， Jayaram S， Gopalakrishnan V， et al. Herbal medicine for liver diseases in India. J Gastroenterol Hepatol，2002； 39， 293-304.

8. Duthie GG. Parsley， polyphenols and nutritional antioxidants. Br J Nutr， 1999； 81， 425-626.

9. Guesmi F， Ben Farhat M， Mejri M， et al. In-vitro assessment of antioxidant and antimicrobial activities of methanol extracts and essential oil of Thymus hirtus sp. algeriensis. Lipids Health Dis， 2014； 13，114.

10.Guesmi F， Ben Ali M， Barkaoui T， et al. Effects of Thymus hirtus sp. algeriensis Boiss. et Reut. （Lamiaceae） essential oil on healing gastric ulcers according to sex. Lipids Health Dis， 2014；13， 138.

11.Guesmi F， Beghalem H， Tyagi AK， et al. Prevention of H2O2Induced Oxidative Damages of Rat Testis by Thymus algeriensis. Biomed Environ Sci， 2016； 29， 275-85.

12.Ma XM， Lightman SL. The arginine vasopressin and corticotrophin releasing hormone gene transcription responses to varied frequencies of repeated stress in rats. J Physiol， 1998；510， 605-14.

13.Fawcett JK， Scoth JE. A rapid and precise method for determination of urea. J Clin Pathol 1960； 13， 156-9.

14.Andrade BF， Braga CP， Dos Santos KC， et al. Effect of Inhaling Cymbopogon martinii Essential Oil and Geraniol on Serum Biochemistry Parameters and Oxidative Stress in Rats. Biochem Res Int， 2014； 2014， 493.

15.Wilkinson JH. Introducción al diagnóstico enzimático. Ediciones Toray， Barcelona， 1972； 310.

16.Lowry OH， Rosebrough NJ， Farr AL， et al. Protein measurement with the Folin phenol reagent. J Biol Chem， 1951； 93， 265-275.

17.Ohkawa H， Ohishi N， Yagi K. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem， 1979； 95，351-8.

18.Marklund SL. Pyrogallol autooxidation. In Handbook of Methods for Oxygen Radical Research. Edited by Greenwald RA. Boca Raton， Florida： CRC Press； 1985； 243-7.

19.Takahara S， Hamilton HB， Neel JV， et al. Hypocatalasemia： a new genetic carrier state. J Clin Invest， 1960； 39， 610-9.

20.Hafeman DG， Sunde RA， Hoekstra WG. Effect of dietary selenium and erythrocyte and liver glutathione peroxidise in the rat. J Nutr， 1973； 104， 580.

21.Habig WH， Pabst MJ， Jakoby WB. Glutathione s-transferase：the first enzymatic step in mercapturic acid formation. J Biol Chem， 1974； 249， 7130-9.

22.Sedlak J， Lindsay RH. Estimation of total， protein-bound， and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem， 1968； 25， 192.

23.Drury RAB， Wallington EA. Carleton’s Histological Technique，5thedn， Oxford university Press， Oxford， 1980； 250-1.

24.Hogg N. Free radicals in disease. Semin Reprod Endocrinol，1998； 16， 241-8.

25.Zar JH. Biostatistical Analysis， Prentice-Hall， Inc.USA， 1984； 620.

26.Shohda A， Maraghy EL， Sherine M， et al. Hepatoprotective potential of crocin and curcumin against iron overload-induced biochemical alterations in rat. AJBR， 2009； 3， 215-21.

27.Gaskill CL， Miller LM， Mattoon JS， et al. Liver histopathology and liver serum alanine aminotransferase and alkaline phosphatase activities in epileptic dogs receiving Phenobarbital. Vet Pathol， 2005； 42， 147-60.

28.Boyd JW. The mechanisms relating to increases in plasma enzymes and isozymes in diseases of animals. Vet Clin Pathol，1983； 12， 9-24.

29.Clampitt RB， Hart RJ. The tissue activities of some diagnostic enzymes in ten mammalian species. J Comp Pathol， 1978； 88，607-21.

30.Tennant B. Hepatic function. In： Clinical Biochemistry of Domestic Animals （5th ed.）， edited by Kaneko JJ， Harvey JW，Bruss ML. Toronto： Academic， 1997； 327-52.

31.Dalle-Donne I， Rossi R， Colombo R， et al. Biomarkers of oxidative damage in human disease. Clin Chem， 2006； 52 ，601-23.

32.Stocker R， Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev， 2004； 84， 1381-478.

33.Jiang J， Yu S， Jiang Z， et al. N-Acetyl-Serotonin Protects HepG2 Cells from Oxidative Stress Injury Induced by Hydrogen Peroxide. Oxid Med Cell Longev， 2014； 15.

34.Toren F. Signal transduction by reactive oxygen species. J Cell Biol， 2011； 194， 17-25.

35.Meister A， Anderson ME. Glutathione. Ann Rev Biochem， 1983；52， 71160.

36.Aitken J. Mechanisms of prevention of lipid peroxidation in human spermatozoa. In： Human acrosome reaction （Eds：Fenichel， P. and Parinaud， J.）， 1995； 339-53.

37.Husain K， Scott BR， Reddy SK， et al. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol， 2001； 25， 89-97.

38.Liang Q， Sheng Y， Jiang P， et al. The gender-dependent difference of liver GSH antioxidant system in mice and its influence on isoline-induced liver injury. Toxicology， 2011； 280，61-9.

39.El-Desoky GE， Bashandy SA， Alhazza IM， et al. Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina platensis in Rats. PLoS One， 2013； 8，e59177.

40.Sharma V， Singh P， Pandey AK， et al. Induction of oxidative stress， DNA damage and apoptosis in mouse liver after sub-acute exposure to zinc oxide nanoparticles. Mutat Res，2012； 745， 84-91.

41.Adedara IA， Abolaji AO， Odion BE， et al. Impairment of Hepatic and Renal Functions by 2，5-Hexanedione Is Accompanied by Oxidative Stress in Rats. J Toxicol， 2014； 239-40.

42.Lieber CS. Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver diseases. Adv Pharmacol， 1997；38， 601-28.

Environ Sci， 2016； 29（9）： 639-649

10.3967/bes2016.086 ISSN： 0895-3988 www.besjournal.com （full text） CN： 11-2816/Q Copyright ?2016 by China CDC

#Correspondence should be addressed to Guesmi Fatma， Tel： 216-99542528， E-mail： guesmif10@gmail.com Biographical note of the first author： Guesmi Fatma， PhD， majoring in medicinal plants and molecular biology.