review article some in vitro/in vivo chemically-induced...

Review ArticleSome In VitroIn Vivo Chemically-Induced ExperimentalModels of Liver Oxidative Stress in Rats

Rumyana Simeonova Magdalena Kondeva-Burdina Vessela Vitcheva and Mitka Mitcheva

Laboratory of Drug Metabolism and Drug Toxicity Department of Pharmacology Pharmacotherapy and ToxicologyFaculty of Pharmacy Medical University 2 Dunav Street 1000 Sofia Bulgaria

Correspondence should be addressed to Rumyana Simeonova rsimeonovayahoocom

Received 27 April 2013 Accepted 24 October 2013 Published 16 January 2014

Academic Editor Afaf K El-Ansary

Copyright copy 2014 Rumyana Simeonova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Oxidative stress is critically involved in a variety of diseases Reactive oxygen species (ROS) are highly toxic molecules that aregenerated during the bodyrsquos metabolic reactions and can react with and damage some cellular molecules such as lipids proteins orDNA Liver is an important target of the oxidative stress because of its exposure to various prooxidant toxic compounds as well as ofits metabolic function and ability to transform some xenobiotics to reactive toxic metabolites (as ROS) To investigate the processesof liver injuries and especially liver oxidative damages there are many experimental models some of which we discuss further

1 Introduction

Oxidative stress is an imbalance between the production andscavenging of reactive oxygen and nitrogen species (ROSand RNS) and free radicals that can induce lipid peroxida-tion DNA fragmentation and protein oxidation [1] Thesedamages result in the loss of membrane integrity structuraland functional changes in proteins and gene mutations [2]Normally the affected cells are trying to neutralise reac-tive molecules by deploying their antioxidative defense thatinclude reduced glutathione (GSH) alpha-tocopherol ascor-bic acid antioxidant enzymes catalase (CAT) superoxidedismutase (SOD) glutathione peroxidase (GPx) glutathionereductase (GR) and glutathione-S-transferase (GST)

Oxidative stress is critically involved in a variety of dis-eases ROS are highly dangerousmolecules that are generatedduring the bodyrsquos metabolic reactions and can react with anddamage some cellular molecules such as lipids proteins orDNA

Liver plays a pivotal role in the regulation of variousphysiological processes in the body such as carbohydratemetabolism and storage fat metabolism bile acid synthesisand so forth besides being themost important organ involvedin the detoxification of various drugs as well as xenobiotics inour body [3]

It is highly susceptible to damage by xenobiotics owingto its continuous exposure to these toxicants via the portalblood circulation [4] Various chemicals like carbon tetra-chloride (CCl

4) tert-butyl hydroperoxide (t-BHP) alcohol

paracetamol galactosamine (GalN) and others can causepotential damage to the liver cells leading to progressivedysfunctionMost of the hepatotoxic chemicals cause damageto the hepatocytes by inducing lipid peroxidation [5 6]Thusthe disorders associated with liver are numerous and varied

One of the most important liver toxicity mechanismsmight be a consequence of cell damage by ROS and RNSKupffer cells release reactive oxygen species (ROS) cytokinesand chemokines which induce neutrophil extravasationand activation Also the liver expresses many cytochromeP450 isoforms including ethanol-induced CYP2E1 CYP2E1generates ROS activates many toxicologically importantsubstrates and may be the central pathway by which somesubstances cause oxidative stress (ethanol carbon tetrachlo-ride etc) [7]

In this review we summarize some commonly usedtoxic models employed in the study of hepatotoxicity andhepatoprotection A number of models of hepatic disorderssupport the notion that ROS have a causal role in liverinjuries Experimental liver injuries are induced by specific

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 706302 6 pageshttpdxdoiorg1011552014706302

2 BioMed Research International

toxic compounds because the formation of ROS is stimulatedby a number of xenobiotics

2 Carbon Tetrachloride (CCl4)

Carbon tetrachloride (CCl4) is the most widely used model

to develop oxidative stress and liver toxicity in rats Hepaticinjury through carbon tetrachloride induced lipid peroxi-dation is well known and has been extensively used in theexperimental models to understand the cellular mechanismsbehind oxidative damage and further to evaluate the thera-peutic potential of drugs and dietary antioxidants [8]

CCl4is activated by cytochrome CYP2E1 CYP2B1 or

CYP2B2 and possibly CYP3A to form the trichloromethylradical CCl3lowast [9]This radical can bind to cellular molecules(nucleic acid protein lipid) impairing crucial cellular pro-cesses such as lipid metabolism with the potential outcomeof fatty degeneration (steatosis) [10] This radical can alsoreact with oxygen to form the trichloromethylperoxy radicalCCl3OOlowast a highly reactive species CCl

3OOlowast initiates the

chain reaction of lipid peroxidation which attacks anddestroys polyunsaturated fatty acids [9] Among the degra-dation products of fatty acids are reactive aldehydes mal-ondialdehyde (MDA) and 4-hydroxynonenal which bindeasily to functional groups of proteins and inhibit importantenzyme activities Disturbed cellular processes aremost likelydue to increased levels of these thiobarbituric acid reactivespecies (TBARS) [11] lactate dehydrogenase (LDH) leakageas a result ofmembrane breakdown and concomitant increasein membrane permeability [12] loss of cell protection wit-nessed byGSHdepletion and as a result of all these changesmdashcell death

In our laboratory we use some in vitro and in vivohepatotoxicity models based on CCl

4-induced liver damage

in Wistar rats and in spontaneously hypertensive rats (SHR)In vitro experiments are carried out in primary isolated rathepatocytes [13] or liver microsomes [14] Cell incubationwith CCl

4(86 120583mol Lminus1) leads to a significant decrease in

cell viability increased LDH leakage decreased levels ofcellular GSH and elevation in MDA quantity Enzyme-induced LPO is started with 20mM CCl

4in the presence of

1mM NADPH [14] For in vivo experiments Wistar rats arechallenged with a single dose (2mLkg) of 20 of CCl

4in

olive oil [15] These in vitroin vivo CCl4-induced liver injury

models are useful for investigations on hepatoprotective andantioxidant properties of some plant-derived biologicallyactive compounds [13ndash17]

We found that ROS produced byCCl4 decrease the activ-

ities not only of antioxidant enzymes such as catalase (CAT)superoxide dismutase (SOD) glutathione peroxidase (GPx)glutathione reductase (GR) and glutathione-S-transferase(GST) [18] but also the activities of some drug metabolizingenzymes such as CYP2E1 and CYP3A involved in theirproduction [15]

3 Tert-Butyl Hydroperoxide (t-BHP)

The cellular system of energy supply localized in mitochon-dria is another target of many hepatotoxic substances causing

oxidative stress and is one of themost importantmechanismsthrough which hepatotoxic factors induced apoptotic andnecrotic processes [19]

Tert-butyl hydroperoxide caused necrosis through induc-ing mitochondrial reactive oxygen formation [20] As aprooxidant t-BHP was widely used and many effects oncell metabolism have been described for example changesin calcium homeostasis [21] increase of lipid peroxidationor decrease of mitochondrial membrane potential [22 23]Two mechanisms for t-BHP action were proposed depletionof cellular stores of GSH and oxidation of functionallyimportant SH groups on mitochondrial enzymes [24] andorchanges of mitochondrial membrane integrity induced byperoxidation of membrane lipids [22 23] The metabolismof t-BHP to free radicals undergoes through several stepsIn microsomal suspension in the absence of NADPH ithas been shown to undergo one-electron oxidation to aperoxyl radical (1) whereas in the presence of NADPH it hasbeen shown to undergo one-electron reduction to an alkoxylradical (2) In isolated mitochondria and intact cells thet-BHP has been shown to undergo 120573-scission to the methylradical (3) All these radicals cause lipid peroxidation process[25 26]

(CH3)3COOH 997888rarr (CH

3)3COO∙ + eminus +H+ (1)

(CH3)3COOH + eminus 997888rarr (CH

3)3CO∙ +OHminus (2)

(CH3)3CO∙ 997888rarr (CH

3)2CO + ∙CH

3(3)

Experiments on isolated hepatocytes are thus a useful modelsystem for evaluation of the toxic effect of various prooxidantswhich act directly on mitochondrial enzymes In our exper-iments using freshly isolated rat hepatocytes we found thatt-BHP (75 120583mol Lminus1) decreases cell viability [27 28] It causesleakage of lactate dehydrogenase (LDH) and formation ofmalondialdehyde in hepatocytes Furthermore t-BHP causesthe depletion of cellular GSH levelsThese data correlate withthe results obtained by many authors [23ndash25]

Enhanced formation of ROS has been suggested to playa role in some liver disease processes including alcohol-induced liver injury [29ndash31] paracetamol-induced liver fail-ure [32 33] andmany othersMany other drugs as isoniazideamiodarone and valproic acid as well as widely used andabused substances as nicotine and cocaine damage livercells by producing toxic ROS Because of their widespreadconsumption they are also used as experimental models ofliver injuries

4 Ethanol

Acute and chronic ethanol treatments increase the produc-tion of ROS lower cellular antioxidant levels and enhanceoxidative stress in many tissues especially the liver Ethanol-induced oxidative stress plays amajor role in themechanismsby which ethanol produces liver injury [34]

The liver expresses many cytochrome P450 isoformsincluding ethanol-induced CYP2E1 CYP2E1 generates ROSactivates many toxicologically important substrates and may

BioMed Research International 3

be the central pathway by which ethanol causes oxidativestress [7]

CYP2E1 metabolizes and activates many toxicologicallyimportant substrates including ethanol carbon tetrachlo-ride acetaminophen and N-nitrosodimethylamine to moretoxic products [35 36] Induction of CYP2E1 by ethanolis a central pathway by which ethanol generates oxidativestress In our intragastric model of ethanol feeding (3 gkg14 days) of spontaneously hypertensive rats (SHR) a promi-nent induction of CYP2E1 occurs along with significantalcohol liver injury [37] Lipid peroxidation also occursand ethanol-induced liver pathology correlates with CYP2E1levels and elevated lipid peroxidation [38] Chronic ethanolconsumption is associatedwith reduced liver GSH and alpha-tocopherol level and with reduced superoxide dismutase(SOD) catalase (CAT) and glutathione peroxidase (GPx)activity [39] Our results concerning normotensive rats(Wistar-Kyoto) are in accordance with these data whereasalcohol intake in SHR increases significantly SOD and CATactivities by approximately 50 [37] We suggested that thedifferences in antioxidant status and the effect of ethanolbetween the strains might be due to the oxidative stressstate in the hypertensive pathological model Additionallywe found that multiple ethanol treatment resulted in lesspronounced effect on the assessed parameters (MDA GSHnNOS) in the female SHR compared tomale SHR [38]Theseresults might be due to a protective effect of estrogens onthe oxidative stress and to a stimulation of the antioxidantdefense systems in liver

5 Paracetamol

Paracetamol (PCM) is primarilymetabolized by sulfation andglucuronidation but with an increasing dose rate these path-ways become saturated and a greater proportion of the drugis available for oxidation by the microsomal cytochrome P-450 system [40] N-Acetyl-P-benzoquinone Imine (NAPQI)is the product of this pathway which is thought to beresponsible for the subsequent hepatic damage [41] N-acetyl-P-benzoquinone imine (NAPQI) is a highly reactiveelectrophile and is detoxified in liver by either reductionto the parent compound acetaminophen or conjugation atthe metaposition with glutathione in which both reactionsconsume GSH [42]

Glutathione (GSH) plays an important role in protect-ing cells from electrophilic compounds and free radicalssuch as reactive oxygen species generated during cellu-lar metabolism Reduced glutathione can act as a reduc-tant reducing hydrogen peroxide and lipid hydroperoxidesdirectly to H

2O a reaction catalyzed by GSH-Px [43] Deple-

tion of intracellular GSH under conditions of continuousintracellular oxidative stress leads to oxidation and damageof lipids proteins and DNA by the reactive oxygen species[44 45]

The importance of glutathione in PCM toxicity is furtheremphasized by the large body of evidence which indicatesthat interventions which increase GSH content can dra-matically reduce PCM and NAPQI-induced hepatic injury[46 47] Our previous studies have shown that GSH content

in animal livers decreases after PCM overdose (2 gkg ipsingle dose) [15] and have proved that biologically activecompounds derived from plants are useful for treatment ofPCM-induced liver disorders because of a stimulation ofGSH synthesis

Oxidative stress is also considered to be involved in theinduction of hepatotoxicity by PCM Oxidation of PCM byCYPs may generate ROS Hydrogen peroxide and superoxideare produced during metabolic activation of PCM in themixed function oxidase system [47]

6 Nicotine

During smoking nicotine is rapidly absorbed into the cir-culatory system where more than 80 is metabolized in theliver [48] Liver is an important organ and is responsible forbiotransformation of drugs and other toxins to remove themfrom the body Nicotine from heavy smoking increases therisk of developing some dangerous liver disorders by one ofthe main mechanisms being the oxidative stress Increasedproduction of free radicals or decreased function of thedefense system play an important role in nicotine toxicity[49] Also maternal nicotine exposure induces oxidativestress and causes histopathological changes in the lung andliver of lactating offspring [50] Nicotine induces oxida-tive stress both in vivo and in vitro that causes a peroxi-dantantioxidant imbalance in blood cells blood plasma andother tissues [51] Some authors [50 52] reported that nico-tine induces oxidative stress and depleted antioxidant defensemechanisms through reduction of glutathione peroxidase inliver lung and kidney Oxidative stress generates free radicalsthat attack the membrane lipids resulting in the formationof malondialdehyde (MDA) which causes peroxidative tis-sue damage [53] Animalrsquos studies have shown significantlyhigher liver and serum levels of MDA conjugated dieneshydroperoxides and free fatty acids in rats intoxicated bynicotine [54 55]

Nicotine is not recognized as a common experimentalmodel for liver injuries but because of its well-establishedprooxidant mechanisms of hepatotoxicity and widespreadconsumption is used from many authors [51 53 55 56]for investigations of antioxidant and protective properties ofnatural compounds

In our previous experiments [57 58] enhanced level oftissue lipid peroxides in nicotine treated rats (1mgkg ip65mgkg po) has been shown to be accompanied by a sig-nificant decrease in the levels of GSH glutathione peroxidase(GPx) superoxide dismutase (SOD) and catalase (CAT) andincreased glutathione reductase (GR) activity in Wistar ratliver

7 D-Galactosamine (GAL)

D-Galactosamine is a well-known experimental hepatotoxinusually used to produce acute toxicity in rat liver Galac-tosamine metabolism depletes the uridine pool of hepato-cytes thus inducing transcriptional arrest and causing anincrease in sensitization to cytokines such as TNF-120572 and an


increase in oxidative stress and GSH depletion which lead tomitochondrial dysfunction and cell death [59] Both oxida-tive and nitrosative stress play a key role in the pathogenesisof GAL-induced hepatic injury [60]

Usually rats are injected (ip) with GAL (400mgkg bw)as a single dose [61]

8 Cocaine

Cocaine is a psychoactive drug that has been recognizedas one of the most significant examples of drug abuse Itsmisuse can induce severe toxic effects including neurotox-icity cardiotoxicity and hepatotoxicity One of the mainmechanisms discussed for cocaine-induced liver injury ispromotion of lipid peroxidation by ROS which are pro-duced during cocaine bioactivation to norcocaine throughN-demethylation by cytochrome P 450 and flavin adeninedinucleotide containing monooxygenases [62]

A large body of evidence in both human and experimentalmodels suggests that impairment of the antioxidant defensesystem by cocaine and its metabolites plays a role in thepathogenesis of cocaine hepatotoxicity [62ndash64] In particularglutathione seems to play an important protective role againstcocaine-induced hepatic injury For example the acuteadministration of cocaine to rats [65] andmultiple treatmentsof mice [63] have been shown to deplete the cellular reducedglutathione to decrease the activity of superoxide dismutase(SOD) catalase (CAT) and glutathione peroxidase (GPx)and to increase glutathione reductase (GR) activity TheGSH depletion induced by cocaine administration observedin these and other studies [62 66] might be explained byincreased utilization of GSH for detoxification of ROS andlipid peroxidation products The critical role of ROS andoxidative stress in the pathogenesis of cocaine-induced liverdamage was demonstrated by the observed amelioratingeffects of bioactive compounds with an antioxidant activityadministered several days before cocaine treatment [65 67]The bioactive compounds were found to decrease cocainetoxicity both by increasing GSH levels and antioxidantenzyme activities

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] B V Reddy J S Sundari E Balamurugan and V P MenonldquoPrevention of nicotine and streptozotocin treatment inducedcirculatory oxidative stress by bis-17-(2-hydroxyphenyl)-hepta-16-diene-35-dione in diabetic ratsrdquo Molecular and CellularBiochemistry vol 331 no 1-2 pp 127ndash133 2009

[3] S K Sharma S M Arogya D H Bhaskarmurthy A Agarwaland C C Velusami ldquoHepatoprotective activity of the Phyllan-

thus species on tert-butyl hydroperoxide (t-BH)-induced cyto-toxicity inHepG2 cellsrdquo PharmacognosyMagazine vol 7 no 27pp 229ndash233 2011

[4] V M Pineiro-Carrero and E O Pineiro ldquoLiverrdquo Pediatrics vol113 no 4 pp 1097ndash1106 2004

[5] A Subramoniam and P Pushpangadan ldquoDevelopment of phy-tomedicines for liver diseasesrdquo Indian Journal of Pharmacologyvol 31 no 3 pp 166ndash175 1999

[6] M Joyeux A Rolland J Fleurentin FMortier and P DorfmanldquoTert-butyl hydroperoxide-induced injury in isolated rat hep-atocytes a model for studying anti-hepatotoxic crude drugsrdquoPlanta Medica vol 56 no 2 pp 171ndash174 1990

[7] H Jaeschke G J Gores A I Cederbaum J A Hinson DPessayre and J J Lemasters ldquoMechanisms of hepatotoxicityrdquoToxicological Sciences vol 65 no 2 pp 166ndash176 2002

[8] S Basu ldquoCarbon tetrachloride-induced lipid peroxidation eic-osanoid formation and their regulation by antioxidant nutri-entsrdquo Toxicology vol 189 no 1-2 pp 113ndash127 2003

[9] L W D Weber M Boll and A Stampfl ldquoHepatotoxicity andmechanism of action of haloalkanes carbon tetrachloride as atoxicological modelrdquo Critical Reviews in Toxicology vol 33 no2 pp 105ndash136 2003

[10] Y Masuda ldquoLearning toxicology from carbon tetrachloride-induced hepatotoxicityrdquo Yakugaku Zasshi vol 126 no 10 pp885ndash889 2006

[11] M Bhadauria S K Nirala and S Shukla ldquoPropolis protectsCYP 2E1 enzymatic activity and oxidative stress induced bycarbon tetrachloriderdquoMolecular and Cellular Biochemistry vol302 no 1-2 pp 215ndash224 2007

[12] S Sahreen M R Khan and R A Khan ldquoHepatoprotectiveeffects of methanol extract of Carissa opaca leaves on CCl

4-

induced damage in ratrdquo BMC Complementary and AlternativeMedicine vol 11 article 48 2011

[13] M Kondeva M Mitcheva and S Nikolov ldquoEffect of theDiosgenin in fresh isolated rat hepatocytes treated with carbontetrachloriderdquo European Journal of Drug Metabolism and Phar-macokinetics vol 28 no 1 pp 1ndash3 2003

[14] R L Simeonova V B Vitcheva M S Kondeva-Burdina I NKrasteva S D Nikolov and M K Mitcheva ldquoEffect of purifiedsaponin mixture from Astragalus corniculatus on enzyme- andnon-enzyme-induced lipid peroxidation in liver microsomesfrom spontaneously hypertensive rats and normotensive ratsrdquoPhytomedicine vol 17 no 5 pp 346ndash349 2010

[15] V Vitcheva R Simeonova I Krasteva S Nikolov and MMitcheva ldquoProtective effects of a purified saponin mixturefromAstragalus corniculatus Bieb in vivohepatotoxicity mod-elsrdquo Phytotherapy Research vol 27 no 5 pp 731ndash736 2013

[16] S A Ali M Z Rizk N A Ibrahim M S Abdallah H M Sha-rara and M M Moustafa ldquoProtective role of Juniperus phoe-nicea and Cupressus sempervirens against CCl

4rdquo World Journal

Gastrointestinal Pharmacology andTherapeutics vol 1 no 6 pp123ndash131 2010

[17] F Sun E Hamagawa C Tsutsui Y Ono Y Ogiri and SKojo ldquoEvaluation of oxidative stress during apoptosis andnecrosis caused by carbon tetrachloride in rat liverrdquo Biochimicaet Biophysica Acta vol 1535 no 2 pp 186ndash191 2001

[18] R Simeonova I Krasteva M Kondeva-Burdina and N Ben-bassat ldquoEffects of extract from Astragalus Glycyphylloides onCarbon tetrachloride-induced hepatotoxicity in Wistar ratsrdquoInternational Journal of Pharma and Bio Sciences vol 4 no 3pp 179ndash186 2013


[19] G Kroemer B Dallaporta and M Resche-Rigon ldquoThe mito-chondrial deathlife regulator in apoptosis and necrosisrdquoAnnual Review of Physiology vol 60 pp 619ndash642 1998

[20] Z Drahota P Krivakova Z Cervinkova et al ldquoTert-butyl hyd-roperoxide selectively inhibits mitochondrial respiratory-chainenzymes in isolated rat hepatocytesrdquo Physiology Research vol54 pp 67ndash72 2005

[21] P Nicotera D McConkey S-A Svensson G Bellomo and SOrrenius ldquoCorrelation between cytosolic Ca2+ concentrationand cytotoxicity in hepatocytes exposed to oxidative stressrdquoToxicology vol 52 no 1-2 pp 55ndash63 1988

[22] R Rubin and J L Farber ldquoMechanisms of the killing of culturedhepatocytes by hydrogen peroxiderdquo Archives of Biochemistryand Biophysics vol 228 no 2 pp 450ndash459 1984

[23] E Kmonıckova Z Drahota L Kamenıkova Z Cervinkova KMasek and H Farghali ldquoModulatory effect of cyclosporin Aon tert-butyl hydroperoxide-induced oxidative damage in hep-atocytesrdquo Immunopharmacology Immunotoxicology vol 23 pp43ndash54 2001

[24] N Masaki M E Kyle A Serroni and J L Farber ldquoMitochon-drial damage as a mechanism of cell injury in the killing ofcultured hepatocytes by tert-butyl hydroperoxiderdquo Archives ofBiochemistry and Biophysics vol 270 no 2 pp 672ndash680 1989

[25] K Ollinger and U T Brunk ldquoCellular injury induced by oxi-dative stress is mediated through lysosomal damagerdquo Free Rad-ical Biology and Medicine vol 19 no 5 pp 565ndash574 1995

[26] V OrsquoDonnell and M J Burkitt ldquoMitochondrial metabolismof a hydroperoxide to free radicals in human endothelialcells an electron spin resonance spin-trapping investigationrdquoBiochemical Journal vol 304 no 3 pp 707ndash713 1994

[27] M Mitcheva M Kondeva-Burdina V Vitcheva I Krastevaand S Nikolov ldquoEffect of purified saponin mixture from Astr-agalus corniculatus on toxicity models in isolated rat hepato-cytesrdquo Pharmaceutical Biology vol 46 no 12 pp 866ndash8702008

[28] M Mitcheva M Kondeva-Burdina I Krasteva and S NikolovldquoProtective effect of purified saponin mixture from Astragaluscorniculatus on toxicity models in vitrordquo in Medical Manage-ment of Chemical and Biological Casualties S Tonev K Kanevand C Dishovsky Eds pp 239ndash251 Publishing house IRITASofia Bulgaria 2009

[29] M Adachi and H Ishii ldquoRole of mitochondria in alcoholic liverinjuryrdquoFree Radical Biology andMedicine vol 32 no 6 pp 487ndash491 2002

[30] G E Arteel ldquoOxidants and antioxidants in alcohol-inducedliver diseaserdquo Gastroenterology vol 124 no 3 pp 778ndash7902003

[31] A Dey and A I Cederbaum ldquoAlcohol and oxidative liverinjuryrdquo Hepatology vol 43 no 2 pp S63ndashS74 2006

[32] H Jaeschke andM L Bajt ldquoIntracellular signalingmechanismsof acetaminophen-induced liver cell deathrdquo Toxicological Sci-ences vol 89 no 1 pp 31ndash41 2006

[33] H Jaeschke T R Knight and M L Bajt ldquoThe role of oxi-dant stress and reactive nitrogen species in acetaminophen hep-atotoxicityrdquoToxicology Letters vol 144 no 3 pp 279ndash288 2003

[34] A I Cederbaum Y Lu and D Wu ldquoRole of oxidative stress inalcohol-induced liver injuryrdquo Archives of Toxicology vol 83 no6 pp 519ndash548 2009

[35] F P Guengerich D-H Kim and M Iwasaki ldquoRole of humancytochrome P-450 IIE1 in the oxidation of many low molecularweight cancer suspectsrdquoChemical Research in Toxicology vol 4no 2 pp 168ndash179 1991

[36] D R Koop ldquoOxidative and reductive metabolism by cytoch-rome P450 2E1rdquo The FASEB Journal vol 6 no 2 pp 724ndash7301992

[37] R Simeonova V Vitcheva andMMitcheva ldquoEffect of multipletreatments with alcohol on some liver antioxidant biochemicalparameters in spontaneously hypertensive rats (SHRs) versusnormotensive rats (NTRs)rdquo Toxicology Letters vol 189 2009

[38] R Simeonova V Vitcheva and M Mitcheva ldquoEffect of ethanolon some hepatic and brain parameters in male and femalespontaneously hypertensive rats (SHRs)rdquo Toxicology Lettersvol 196 2010

[39] T Radosavljevic D Mladenovic and D Vucevic ldquoThe role ofoxidative stress in alcoholic liver injuryrdquoMedicinski Pregled vol62 no 11-12 pp 547ndash553 2009

[40] P J Amar and E R Schiff ldquoAcetaminophen safety and hepato-toxicity where do we go from hererdquo Expert Opinion on DrugSafety vol 6 no 4 pp 341ndash355 2007

[41] L M Aleksunes S N Campion M J Goedken and J E Man-autou ldquoAcquired resistance to acetaminophen hepatotoxicityis associated with induction of multidrug resistance-associatedprotein 4 (Mrp4) in proliferating hepatocytesrdquo ToxicologicalSciences vol 104 no 2 pp 261ndash273 2008

[42] S U Ruepp R P Tonge J ShawNWallis and F Pognan ldquoGen-omics and proteomics analysis of acetaminophen toxicity inmouse liverrdquo Toxicological Sciences vol 65 no 1 pp 135ndash1502002

[43] M Anoush M A Eghbal F Fathiazad H Hamzeiy and NS Kouzehkonani ldquoThe protective effects of garlic extract agai-nst acetaminophen-induced oxidative stress and Glutathionedepletionrdquo Pakistan Journal of Biological Sciences vol 12 no 10pp 765ndash771 2009

[44] N Kaplowitz ldquoMechanisms of liver cell injuryrdquo Journal of Hep-atology vol 32 no 1 pp 39ndash47 2000

[45] J Nordberg and E S J Arner ldquoReactive oxygen species anti-oxidants and themammalian thioredoxin systemrdquo Free RadicalBiology and Medicine vol 31 no 11 pp 1287ndash1312 2001

[46] J R Mitchell D J Jollow and W Z Potter ldquoAcetaminopheninduced hepatic necrosis IV Protective role of glutathionerdquoJournal of Pharmacology and Experimental Therapeutics vol187 no 1 pp 211ndash217 1973

[47] T Amimoto T Matsura S-Y Koyama T Nakanishi K Yam-ada and G Kajiyama ldquoAcetaminophen-induced hepatic injuryin mice the role of lipid peroxidation and effects of pretreat-ment with coenzyme Q10 and 120572- tocopherolrdquo Free RadicalBiology and Medicine vol 19 no 2 pp 169ndash176 1995

[48] A-R El-Zayadi ldquoHeavy smoking and liverrdquo World Journal ofGastroenterology vol 12 no 38 pp 6098ndash6101 2006

[49] K Chattopadhyay and B D Chattopadyay ldquoEffect of Nicotineon lipid profile peroxidation amp antioxidant enzymes in femalerats with restricted dietary proteinrdquo Indian Journal of MedicalResearch vol 127 no 6 pp 571ndash576 2008

[50] B H Ozukutat K U Ozkan C F Ibrahim E Guldur M SKilinc and F Inan ldquoEffects of maternal nicotine exposure dur-ing on breast-fed rat pupsrdquo Biology in Neonats vol 88 no 2 pp113ndash117 2005

[51] H SuleymanKGumustekin S Taysi et al ldquoBeneficial effects ofHippophae rhamnoides L on nicotine induced oxidative stressin rat blood compared with vitamin Erdquo Biological and Pharma-ceutical Bulletin vol 25 no 9 pp 1133ndash1136 2002

[52] G H El-Sokkary S Cuzzocrea and R J Reiter ldquoEffect of chr-onic nicotine administration on the rat lung and liver beneficialrole of melatoninrdquo Toxicology vol 239 no 1-2 pp 60ndash67 2007


[53] K N Srinivasan and K V Pugalendi ldquoEffect of excessive intakeof thermally oxidized sesame oil on lipids lipid peroxidationand antioxidantsrsquo status in ratsrdquo Indian Journal of ExperimentalBiology vol 38 no 8 pp 777ndash780 2000

[54] L Ashakumary and P L Vijayammal ldquoAdditive effect of alcoholand nicotine on lipid peroxidation and antioxidant defencemechanism in ratsrdquo Journal of Applied Toxicology vol 16 pp305ndash308 1996

[55] J Zhang S Jiang and R RWatson ldquoAntioxidant supplementa-tion prevents oxidation and inflammatory responses induced bysidestream cigarette smoke in old micerdquo Environmental HealthPerspectives vol 109 no 10 pp 1007ndash1009 2001

[56] AMGawishAM IssaN S Bassily and SMManaa ldquoRole ofgreen tea on nicotine toxicity on liver and lung of mice hist-ological and morphometrical studiesrdquo African Journal of Biot-echnology vol 11 no 8 pp 2013ndash2025 2012

[57] R Simeonova V Vitcheva G Gorneva and M Mitcheva ldquoEff-ects of myosmine on antioxidative defence in rat liverrdquoArhiv zaHigijenu Rada i Toksikologiju vol 63 no 1 pp 7ndash14 2012

[58] M Micheva M Kondeva-Burdina and V Vicheva ldquoStudy onhepatotoxicity of cytisine (Tabex) compared with nicotine infreshly isolated rat hepatocytesrdquo Pharmacia vol 56 no 1ndash4 pp27ndash32 2009

[59] N Alva D Cruz S Sanchez J Ma Valentin and T C Berm-udez ldquoNitric oxide as a mediator of fructose 1 6-bisphosphateprotection in galactosamine-induced hepatotoxicity in ratsrdquoNitric Oxide vol 28 pp 17ndash23 2013

[60] J Das J Ghosh A Roy and P C Sil ldquoMangiferin exerts hepato-protective activity against D-galactosamine induced acute tox-icity and oxidativenitrosative stress via Nrf2-NF120581B pathwaysrdquoToxicology and Applied Pharmacology vol 260 no 1 pp 35ndash472012

[61] G Pushpavalli C Veeramani andK V Pugalendi ldquoEffect of Pi-per betle on plasma antioxidant status and lipid profile againstD-galactosamine-induced hepatitis in ratsrdquo Redox Report vol14 no 1 pp 7ndash12 2009

[62] R Labib R Turkall and M S Abdel-Rahman ldquoOral cocaineproduces dose-related hepatotoxicity in male micerdquo ToxicologyLetters vol 125 no 1ndash3 pp 29ndash37 2001

[63] R Labib R Turkall andM SAbdel-Rahman ldquoInhibition of co-caine oxidative metabolism attenuates endotoxin potentiationof cocaine mediated hepatotoxicityrdquo Toxicology vol 179 no 1-2 pp 9ndash19 2002

[64] P Kovacic ldquoRole of oxidative metabolites of cocaine in toxicityand addiction oxidative stress and electron transferrdquo MedicalHypotheses vol 64 no 2 pp 350ndash356 2005

[65] V Vitcheva R Simeonova I Krasteva M Yotova S Nikolovand M Mitcheva ldquoHepatoprotective effects of saponarin iso-lated from Gypsophila trichotoma wend on cocaine-inducedoxidative stress in ratsrdquo Redox Report vol 16 no 2 pp 56ndash612011

[66] T Visalli R Turkall and M S Abdel-Rahman ldquoCocaine hepa-totoxicity and its potentiation by lipopolysaccharide treatmentand gender effectsrdquo International Journal of Toxicology vol 23no 3 pp 163ndash170 2004

[67] Q-Y Cai H-B Chen S-Q Cai et al ldquoEffect of roots of Ficushirta on cocaine-induced hepatotoxicity and active compone-ntsrdquo Zhongguo Zhongyao Zazhi vol 32 no 12 pp 1190ndash11932007

Submit your manuscripts athttpwwwhindawicom

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Volume 2014

ToxinsJournal of

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MEDIATORSINFLAMMATION

of


toxic compounds because the formation of ROS is stimulatedby a number of xenobiotics

2 Carbon Tetrachloride (CCl4)

Carbon tetrachloride (CCl4) is the most widely used model

to develop oxidative stress and liver toxicity in rats Hepaticinjury through carbon tetrachloride induced lipid peroxi-dation is well known and has been extensively used in theexperimental models to understand the cellular mechanismsbehind oxidative damage and further to evaluate the thera-peutic potential of drugs and dietary antioxidants [8]

CCl4is activated by cytochrome CYP2E1 CYP2B1 or

CYP2B2 and possibly CYP3A to form the trichloromethylradical CCl3lowast [9]This radical can bind to cellular molecules(nucleic acid protein lipid) impairing crucial cellular pro-cesses such as lipid metabolism with the potential outcomeof fatty degeneration (steatosis) [10] This radical can alsoreact with oxygen to form the trichloromethylperoxy radicalCCl3OOlowast a highly reactive species CCl

3OOlowast initiates the

chain reaction of lipid peroxidation which attacks anddestroys polyunsaturated fatty acids [9] Among the degra-dation products of fatty acids are reactive aldehydes mal-ondialdehyde (MDA) and 4-hydroxynonenal which bindeasily to functional groups of proteins and inhibit importantenzyme activities Disturbed cellular processes aremost likelydue to increased levels of these thiobarbituric acid reactivespecies (TBARS) [11] lactate dehydrogenase (LDH) leakageas a result ofmembrane breakdown and concomitant increasein membrane permeability [12] loss of cell protection wit-nessed byGSHdepletion and as a result of all these changesmdashcell death

In our laboratory we use some in vitro and in vivohepatotoxicity models based on CCl

4-induced liver damage

in Wistar rats and in spontaneously hypertensive rats (SHR)In vitro experiments are carried out in primary isolated rathepatocytes [13] or liver microsomes [14] Cell incubationwith CCl

4(86 120583mol Lminus1) leads to a significant decrease in

cell viability increased LDH leakage decreased levels ofcellular GSH and elevation in MDA quantity Enzyme-induced LPO is started with 20mM CCl

4in the presence of

1mM NADPH [14] For in vivo experiments Wistar rats arechallenged with a single dose (2mLkg) of 20 of CCl

4in

olive oil [15] These in vitroin vivo CCl4-induced liver injury

models are useful for investigations on hepatoprotective andantioxidant properties of some plant-derived biologicallyactive compounds [13ndash17]

We found that ROS produced byCCl4 decrease the activ-

ities not only of antioxidant enzymes such as catalase (CAT)superoxide dismutase (SOD) glutathione peroxidase (GPx)glutathione reductase (GR) and glutathione-S-transferase(GST) [18] but also the activities of some drug metabolizingenzymes such as CYP2E1 and CYP3A involved in theirproduction [15]

3 Tert-Butyl Hydroperoxide (t-BHP)

The cellular system of energy supply localized in mitochon-dria is another target of many hepatotoxic substances causing

oxidative stress and is one of themost importantmechanismsthrough which hepatotoxic factors induced apoptotic andnecrotic processes [19]

Tert-butyl hydroperoxide caused necrosis through induc-ing mitochondrial reactive oxygen formation [20] As aprooxidant t-BHP was widely used and many effects oncell metabolism have been described for example changesin calcium homeostasis [21] increase of lipid peroxidationor decrease of mitochondrial membrane potential [22 23]Two mechanisms for t-BHP action were proposed depletionof cellular stores of GSH and oxidation of functionallyimportant SH groups on mitochondrial enzymes [24] andorchanges of mitochondrial membrane integrity induced byperoxidation of membrane lipids [22 23] The metabolismof t-BHP to free radicals undergoes through several stepsIn microsomal suspension in the absence of NADPH ithas been shown to undergo one-electron oxidation to aperoxyl radical (1) whereas in the presence of NADPH it hasbeen shown to undergo one-electron reduction to an alkoxylradical (2) In isolated mitochondria and intact cells thet-BHP has been shown to undergo 120573-scission to the methylradical (3) All these radicals cause lipid peroxidation process[25 26]

(CH3)3COOH 997888rarr (CH

3)3COO∙ + eminus +H+ (1)

(CH3)3COOH + eminus 997888rarr (CH

3)3CO∙ +OHminus (2)

(CH3)3CO∙ 997888rarr (CH

3)2CO + ∙CH

3(3)

Experiments on isolated hepatocytes are thus a useful modelsystem for evaluation of the toxic effect of various prooxidantswhich act directly on mitochondrial enzymes In our exper-iments using freshly isolated rat hepatocytes we found thatt-BHP (75 120583mol Lminus1) decreases cell viability [27 28] It causesleakage of lactate dehydrogenase (LDH) and formation ofmalondialdehyde in hepatocytes Furthermore t-BHP causesthe depletion of cellular GSH levelsThese data correlate withthe results obtained by many authors [23ndash25]

Enhanced formation of ROS has been suggested to playa role in some liver disease processes including alcohol-induced liver injury [29ndash31] paracetamol-induced liver fail-ure [32 33] andmany othersMany other drugs as isoniazideamiodarone and valproic acid as well as widely used andabused substances as nicotine and cocaine damage livercells by producing toxic ROS Because of their widespreadconsumption they are also used as experimental models ofliver injuries

4 Ethanol

Acute and chronic ethanol treatments increase the produc-tion of ROS lower cellular antioxidant levels and enhanceoxidative stress in many tissues especially the liver Ethanol-induced oxidative stress plays amajor role in themechanismsby which ethanol produces liver injury [34]

The liver expresses many cytochrome P450 isoformsincluding ethanol-induced CYP2E1 CYP2E1 generates ROSactivates many toxicologically important substrates and may




5 Paracetamol








6 Nicotine









8 Cocaine





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AddictionJournal of






Autoimmune Diseases




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review article some in vitro/in vivo chemically-induced...

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