Research Article Influence of Different Doses of Levofloxacin on...

Research ArticleInfluence of Different Doses of Levofloxacin onAntioxidant Defense Systems and Markers of Renal andHepatic Dysfunctions in Rats

Ebenezer Tunde Olayinka Ayokanmi Ore and Olaniyi Solomon Ola

Biochemistry Unit Department of Chemical Sciences Ajayi Crowther University PMB 1066 Oyo Oyo State 211213 Nigeria

Correspondence should be addressed to Ebenezer Tunde Olayinka etolayinkaacuedung

Received 22 September 2014 Revised 9 December 2014 Accepted 9 December 2014

Academic Editor Arezoo Campbell

Copyright copy 2015 Ebenezer Tunde Olayinka et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Levofloxacin (LFX) is a broad spectrum fluoroquinolone antibiotic used in the treatment of infections such as pneumonia chronicbronchitis and sinusitis The present study assessed the likely toxic effect of LFX on hepatic and renal tissues in rats Twentymale Wistar rats were randomly divided into four treatment groups A control B 5mgkg bw LFX (half therapeutic dose) C10mgkg bw LFX (therapeutic dose) and D 20mgkg bw LFX (double therapeutic dose) After seven days of administration resultindicated significant (119875 lt 005) increase in plasma ALT AST and ALP activities in the treated groups compared to control Alsothere was a significant increase in plasma creatinine urea and total bilirubin in the treated groups relative to control Plasmatotal cholesterol HDL-cholesterol LDL-cholesterol and triglycerides also increased significantly in the treated groups relative tocontrol Also hepaticMDA level increased significantly in all the treated groups However hepatic SOD catalase andGST activitieswere significantly reduced in the LFX-treated animals Moreover GSH and ascorbic acid levels were significantly decreased in theLFX-treated groups relative to control In conclusion three doses of levofloxacin depleted antioxidant defense system and inducedoxidative stress and hepatic and renal dysfunctions in rats

1 Introduction

Levofloxacin (LFX) belongs to fluoroquinolone antimicrobialagents that have broad spectrum bactericidal effect againstboth Gram-positive and Gram-negative bacteria [1 2] It isa chosen drug with better efficacy in the treatment of typhoidfever especially when compared with other members of thesame family such as ciprofloxacin [3] Its usage has beenextended to treatment of varieties of infections in adults suchas sinus infections bronchitis pneumonia and genitourinaryinfections including both complicated and uncomplicatedurinary tract infections [4 5]

LFX is a chiral fluorinated carboxyquinolone (Figure 1)with fluorine at position 9 a pure (minus)-S-enantiomer of theracemic drug substance ofloxacin [6] It has lowest phototoxicpotential [7 8] The reduced risk of photosensitivity is due tothe fluorine at position 9 rather than position 8 of quinolonenucleus that is related to the risk of phototoxicity as itis found in lomefloxacin and sparfloxacin [9] LFX being

a fluoroquinolone primarily targets bacterial topoisomeraseIV and DNA gyrase in Gram-positive and Gram-negativebacteria respectively as its mechanism of action [10] LFXundergoes limited metabolism and is primarily excretedunchanged [11 12] Two metabolites desmethyl-levofloxacinN-oxide have been identified in humans and they contributeminimal pharmacological activities [13]

Generally fluoroquinolone has been shown to be associ-ated with certain adverse effects like Juvenile joint toxicitygastrointestinal discomfort adverse central nervous systemeffect and cutaneous reaction such as phototoxicity [14 15]However LFX had been reported to have low potential tocause adverse hepatobiliary reaction [16] Despite its clinicalacceptability and usage there are little reports on its effecton antioxidant defense system Several in vivo and in vitrostudies have related phototoxicity of fluoroquinolone to thegeneration of reactive oxygen species (ROS) such as superox-ide anion hydrogen peroxide and hydroxyl radical [17 18]

Hindawi Publishing CorporationAdvances in ToxicologyVolume 2015 Article ID 385023 7 pageshttpdxdoiorg1011552015385023

2 Advances in Toxicology

HN

N N

F

O

O O

OH

Figure 1 Structure of levofloxacin

These ROS caused severe damage tomacromolecules tissuesand organs through the process of lipid peroxidation (LPO)protein modification and DNA damage [19 20] Oxidativestress results when these ROS overwhelm the antioxidant sys-tem [21] Oxidative stress has been linked with pathogenesisof some disease conditions such as cardiovascular disordersaging cancer and neurodegenerative disease [22 23]

Cell protects itself from effect of ROS by the actionof nonenzymatic antioxidants (Vitamins A E and C andreduced glutathione GSH) and enzymatic antioxidant suchas superoxide dismutase catalase and glutathione peroxidase(GPX) [24] In the present study we investigate the toxicity oflevofloxacin using the status of antioxidant defense systemmarkers of kidney and renal damage and some biochemicalindices in rats

2 Materials and Methods

21 Chemicals and Reagents Levofloxacin (Devox) is a prod-uct of Waves-Biotech Pvt Ltd New Delhi India Glu-tathione (GSH) 1-chloro-24-dinitrobenzene (CDNB) 551015840-dithio-bis-2-nitrobenzoic acid (DTNB) thiobarbituric acid(TBA) epinephrine and hydrogen peroxide were purchasedfrom Sigma chemical company (London UK) Alkalinephosphatase (ALP) alanine amino transferase (ALT) aspar-tate aminotransferase (AST) gamma glutamyl transferase(GGT) urea creatinine bilirubin total cholesterol HDL-cholesterol LDL-cholesterol and triglycerides assay kits wereproducts of Randox Laboratories Ltd (Antrim UK) Allother chemicals and reagents were of analytical grade and ofhighest purity

22 Animals and Treatments Male rats (Wistar strain)weighing between 180 and 220 g were used in this studyThe rats were bred and housed in the animal house of theDepartment of Chemical Sciences Ajayi Crowther Univer-sity Oyo Nigeria They were kept in wire meshed cagesat room temperature and under controlled light cycle (12hr light dark) They were fed with commercial rat chow(Ladokun feeds Ibadan Nigeria) and water ad libitum Theexperimental animals were handled and used in accordancewith the international guide for the care and use of laboratoryanimals (National Research Council) [25]

23 Experimental Design Twenty male albino rats (Wistarstrain) were randomly divided into four groups of 5 rats eachGroup 1 animals were used as control and received distilledwaterGroup 2 animalswere treatedwith LFX-1 (equivalent to5mgkg body weight of levofloxacin) Group 3 animals weretreated with LFX-2 (equivalent to 10mgkg body weight oflevofloxacin) while Group 4 animals were treated with LFX-3 (equivalent to 20mgkg body weight of levofloxacin) 1mLof each of the prepared LFX solutionswas administered orallyto the animals in the different groups using an oral cannulaonce daily for a period of seven days (the normal durationof therapy in humans) The animals were sacrificed 24 hoursafter the last treatment

24 Collection of Blood Samples for Plasma PreparationBlood was collected from the retroorbital plexus of theanimals into heparinized tubes and the rats were sacrificedby cervical dislocation Plasma was prepared by centrifugingblood samples for tenminutes at 3000 rpm in a cencombenchcentrifugeThe clear supernatant was used for the estimationof urea creatinine bilirubin lipid profiles and enzymes

25 Preparation of Cytosolic Fractions The liver excised fromrat blotted of blood stains and rinsed in 115 KCl washomogenized in 4 volumes of ice-cold 001M potassiumphosphate buffer (pH74)Thehomogenateswere centrifugedat 12500 g in an Eppendorf (UK) refrigerated centrifugefor 15min at 4∘C and the supernatants termed as thepostmitochondrial fractions (PMF) were used for enzymeassays

26 Renal and Liver Functions Test Plasma creatinine ureaand bilirubin determinationwas done usingRandox diagnos-tic kitsMethods for creatinine assays are based on colorimet-ric alkaline picratemethods [26]with creatinine-picrate com-plex measured at 492 nm The urea determination methodwas based on the Fearon reaction [27] with the Diazinechromogen formed being absorbed strongly at 540 nm Thedimethyl sulphoxide method by Tietz et al [27] was usedfor bilirubin determination The dimethyl sulphoxide formsa coloured compound with maximum absorption at 550 nm

27 Determination of Plasma AST ALT ALP and GGTActivities Plasma AST ALT ALP and GGT activities weredetermined using Randox diagnostic kits Determination ofAST and ALT activities was based on the principle describedby Tietz et al [27] AST was measured by monitoring theconcentration of oxaloacetate hydrazone formed with 24-dinitrophenylhydrazine at 546 nm and ALT was measuredby monitoring the concentration of pyruvate hydrazoneformed with 24-dinitrophenylhydrazine at 546 nm ALP wasdetermined in accordance with the principles of Tietz [28]The p-nitrophenol formed by the hydrolysis of p-Nitrophenylphosphate confers yellowish colour on the reaction mixtureand its intensity can bemonitored at 405 nm to give ameasureof enzyme activity GGT activity was measured based on amodification of the method described by Hoslashrder et al [29]using Abbott diagnostic kit

Advances in Toxicology 3

Table 1 Effect of levofloxacin treatments on plasma creatinine urea and bilirubin levels in rats

Treatment Creatinine (mgdL) Urea (mgdL) Bilirubin (mgdL)Control 072 plusmn 002 36 plusmn 082 024 plusmn 002LFX-1 080 plusmn 001 (19)lowast 47 plusmn 05 (31)lowast 048 plusmn 003 (100)lowast

LFX-2 088 plusmn 003 (22)lowast 52 plusmn 129 (44)lowast 056 plusmn 003 (133)lowast


The values are means plusmn SD for five rats in each group lowastSignificantly different from the control 119875 lt 005 Values in parenthesis represent percentage () of theincrease

Table 2 Effects of levofloxacin treatments on plasma alkaline phosphatase (ALP) alanine aminotransferase (ALT) and aspartateaminotransferase (AST) activities in rats

Treatment ALP (UL) ALT (UL) AST (UL)Control 187 plusmn 244 33 plusmn 330 60 plusmn 309LFX-1 2208 plusmn 256 (18)lowast 48 plusmn 492 (45)lowast 82 plusmn 125 (37)lowast




28 Determination of Plasma Lipid Profiles The plasma totalcholesterol HDL-cholesterol LDL-cholesterol and triglyc-erides were determined using Randox diagnostic kits andthe determination was based on CHOD-PAD enzymaticcolorimetric method of Trinder [30]

29 Assay of Nonenzymatic Antioxidants and Lipid Peroxi-dation Hepatic vitamin C was determined according to themethod of Erel et al [31] using dinitro phenyl hydrazine(DNPH) while hepatic glutathione was determined accord-ing to the method of Jollow et al [32] The chromophoricproduct resulting from the reaction of Ellmanrsquos reagentwith the reduced glutathione 2-nitro-5-thiobenzoic acidpossesses a molar absorption at 412 nm which was read ina spectrophotometer Reduced GSH is proportional to theabsorbance at 412 nmThe extent of lipid peroxidation (LPO)was estimated by the method of Varshney and Kale [33]the method involved the reaction between malondialdehyde(MDA) and thiobarbituric acid to yield a stable pink chro-mophore with maximum absorption at 532 nm

210 Determination of Antioxidant Enzymes The procedureof Misra and Fridovich [34] as described by Magwere et al[35] was used for the determination of hepatic superoxidedismutase (SOD) activity by measuring the inhibition ofautooxidation of epinephrine at pH 102 and 30∘C SODactivity was expressed in Umg protein Hepatic catalaseactivity was determined according to the method of Sinha[36] by measuring the reduction of dichromate in aceticacid to chromic acetate at 570 nm Catalase activity wasexpressed as 120583molH

2O2consumedminmg protein Hepatic

glutathione S-transferase (GST) activity was determined bythe method described by Habig et al [37] using 1-chloro-24-dinitrobenzene (CDNB) as substrate GST activity wasexpressed in 120583molminmg protein

211 ProteinDetermination Protein content of plasma and allfractions was estimated by the method of Lowry et al [38]using bovine serum albumin as standard

212 Histopathological Studies The method of Baker andSilverton [39] was employed for the processing of liver forhistopathological studies

213 Statistical Analysis Results were expressed as mean of5 replicates plusmn SD Data obtained were subjected to one-way Analysis of Variance (ANOVA) and complementedwith Duncanrsquos multiple range test using Stat Pac StatisticalSoftware Statement of statistical significance was based on119875 lt 005

3 Results

31 Effect of Levofloxacin Treatment on Plasma CreatinineUrea and Bilirubin in Rats Table 1 shows the effect of LFXtreatment on plasma creatinine urea and bilirubin Plasmacreatinine urea and bilirubin were significantly increased(119875 lt 005) in the treated groups by 19 22 and 26 3144 and 53 100 133 and 175 respectively relative tothe control

32 Effect of Levofloxacin Treatment on Plasma ALP ALT andAST Activities in Rats The effect of LFX treatment on plasmaALP ALT and AST is presented in Table 2 Administrationof different doses of LFX significantly increased the plasmaactivities of ALP ALT and AST by 18 37 and 4845 58 and 67 37 43 and 57 respectively whencompared to the control

33 Effect of LevofloxacinTreatments on Lipid Profiles Table 3represents the effect of LFX treatment on lipid profilesPlasma total cholesterol HDL cholesterol LDL cholesterol


Table 3 Effects of levofloxacin treatments on plasma lipid profile in rats

Treatment Total cholesterol (mgdL) HDL-cholesterol (mgdL) LDL-cholesterol (mgdL) Triglyceride (mgdL)Control 68 plusmn 330 38 plusmn 434 45 plusmn 05 135 plusmn 035LFX-1 878 plusmn 329 (29)lowast 51 plusmn 216 (34)lowast 66 plusmn 08 (23)lowast 20 plusmn 10 (54)lowast

LFX-2 102 plusmn 221 (50)lowast 55 plusmn 330 (45)lowast 78 plusmn 06 (56)lowast 235 plusmn 078 (77)lowast



Table 4 Effects of levofloxacin treatments on hepatic superoxide dismutase (SOD) and catalase in rats

Treatment Superoxide dismutase (Unitsmg protein) Catalase (120583molH2O2 consumedminmg protein)Control 752 plusmn 010 063 plusmn 001LFX-1 58 plusmn 023 (23)lowast 058 plusmn 001 (8)lowast

LFX-2 35 plusmn 023 (52)lowast 049 plusmn 003 (14)lowast

LFX-3 27 plusmn 038 (66)lowast 033 plusmn 002 (25 )lowast


and triglyceride levels were significantly increased by 2950 and 66 34 45 and 53 23 56 and 83 5477 and 115 in all treated groups respectively relative tothe control

34 Effect of Levofloxacin Treatment on Enzymatic Antioxi-dants Effect of LFX treatment on hepatic SOD and catalaseis shown in Table 4 Hepatic SOD and catalase activities weresignificantly reduced in all the treated groups by 23 52and 66 8 14 and 25 respectively when comparedto control Similarly hepatic GST activity was significantlyreduced (Figure 2) in all the treated groups by 33 50 and57 respectively relative to control

35 Influence of Levofloxacin Treatment on Hepatic Nonen-zymatic Antioxidants and Lipid Peroxidation Figure 3 showsthe hepatic vitamin C level following treatment with differentdoses of LFXThe vitamin C level was significantly decreasedby 38 53 and 70 in the treated groups when comparedwith the control Similarly hepatic GSH level was decreasedsignificantly (Figure 4) in all the treated groups by 19 38and 60 respectively relative to control However hepaticlevel of malondialdehyde (MDA) (Figure 5) was increasedsignificantly in the treated groups by 15 24 and 34respectively

4 Discussion

The fluoroquinolone antibiotics have been reported to gen-erate reactive oxygen species which may result in oxidativestress and cellular damage to the liver and kidney [40ndash42]In this study levofloxacin used at three different doses (half-therapeutic therapeutic and double-therapeutic) inducedmarked renal and hepatic damage and oxidative stress anddepleted the hepatic antioxidant reserves

Plasma ALT and AST are reliable marker enzymes ofliver function and integrity [43] When body tissue or an

0

5

10

15

20

25

30

Control LFX-1 LFX-2 LFX-3

lowast

lowast

lowast

GST

(120583m

olm

inm

g pr

otei

n)

Figure 2 Influence of levofloxacin on hepatic glutathione-S-transferase (GST) activity in rats The values are the means plusmn SDfor five rats in each group lowastSignificantly different from the control119875 lt 005

0

2

4

6

8

10

12

14


Vita

min

C (120583

gm

L)

lowast

lowast

lowast

Figure 3 Influence of levofloxacin treatment on hepatic vitamin Cconcentration in rats The values are the means plusmn SD (range) forfive rats in each group lowastSignificantly different from the control119875 lt 005


0123456789

10


GSH

(120583g

g liv

er) lowast

lowast

lowast

Figure 4 Influence of levofloxacin treatment on hepatic GSHconcentration in rats The values are the means plusmn SD (range) forfive rats in each group lowastSignificantly different from the control119875 lt 005

0

50

100

150

200

250

300


lowast

lowast

lowast

MD

A (n

mol

mg

prot

ein)

Figure 5 Influence of levofloxacin treatments on hepatic lipidperoxidation (MDA) level in rats The values are the means plusmn SD(range) for five rats in each group lowastSignificantly different from thecontrol 119875 lt 005

organ such as the heart or liver is diseased or damagedadditional AST and ALT are released into the bloodstreamIncrease in plasma ALT and AST has been reported inconditions involving necrosis of hepatocytes [44]myocardialcells erythrocyte and skeletal muscle cells [45] Alkalineand total acid phosphatases are produced by several partsof the body especially the liver Plasma ALP elevation hasbeen attributed to increased osteoblastic activity such as inhyperparathyroidism osteomalacia and neoplasm and alsoin hepatobiliary diseases [45] These enzymes AST ALPand ALT are diagnostic enzymes and their release abovenormal physiological levels indicates a disease conditionincluding various bone disorders and jaundice (ALP) viralhepatitis (ALT) and myocardial infarction (AST) [46] Theelevation in the activity of these enzymes by LFX may be asa result of their release in response to tissue damage duringroutine normal destruction of erythrocytes leukocytes andother cells like liver cells [47] Both urea and creatinineare metabolic waste products that are freely filtered by theglomeruli of the kidneys [48] Serumplasma concentrationsare commonly used to screen for renal or cardiovasculardiseases [49] More so plasma urea has been reported to

increase in acute and chronic intrinsic renal disease and alsowhen there is decreased effective circulating blood volumewith decreased renal perfusion [50] Elevation of the plasmalevels of creatinine and urea by LFX is an indication ofabnormal renal function [51] while elevated level of biliru-bin has been associated with hepatocellular damage intra-and extrahepatic biliary tract obstruction intravascular andextracellular haemolysis and neonatal jaundice [52]

Cells have a number ofmechanisms to protect themselvesfrom the toxic effects of ROS These include free radicalscavengers and chain reaction terminators enzymes likeglutathione (GSH) vitamins C and E SOD CAT and GSHperoxidase [53] Inhibition of these protective mechanismsor the reduction in their activities would result in enhancedsensitivity of the cells to free radical-induced cellular dam-age due to accumulation of superoxide ions and hydrogenperoxide SOD removes superoxide ion (O

2

minus) by convertingit to hydrogen peroxide (H

2O2) which could be rapidly

converted to water and oxygen by CAT [54] SOD has ahigh catalytic effect and it is present in high concentrationsin all tissues protecting cells against O

2

minus under normalconditions [55] A decrease in the activity of SOD observedin this study might increase the cellrsquos susceptibility to attackby O2

minus Catalase catalyzes the breakdown of H2O2generated

by the actions of SOD to water and O2[56] Therefore the

reduction in the activities of CAT and SOD by the drugmay render the liver more susceptible to H

2O2and hydroxyl

radical-induced oxidative stress Ascorbic acid is a watersoluble compound which scavenges biologically relevant freeradicals by reacting with oxygen radicals to generate semi-aldehyde ascorbate radical (a less reactive radical that canbe scavenged by ascorbic acid) Thus decrease in ascorbatersquosactivity occasioned by LFX administration makes the cellmore susceptible to free radicals The level of reduced glu-tathione (GSH) is a measure of the cellular redox status [57]Hence alteration in glutathione concentration may affect theoverall redox status of the cell GST catalyzes the conjugationof reduced glutathione by conjugating these compounds(often electrophilic and somewhat lipophilic in nature) withreduced glutathione (GSH) to facilitate dissolution in theaqueous cellular and extracellularmedia and from there outof the body Severe oxidative stress might result in decrease inGST with concomitant depletion of glutathione [58] Thusdecrease in GST observed from the result correlates withthe depletion of GSH in the liver confirming drug toxicityOther researchers have reported the induction of oxidativestress and depletion of antioxidant status by fluoroquinolones[59 60]

The increase in HDL-cholesterol LDL-cholesterol totalcholesterol and triglycerides indicates a disorder in themetabolism of lipoproteins and lipid It has been suggestedthat cholesterol is a general indicator of the level of lipidin circulation [61] and the more the lipid the greater theamount of lipid peroxidation activity and the greater theamount of lipid peroxidation products such asMDA Increasein the level of lipid peroxidation has been described as abiomarker of tissue damage [62] The increased hepatic lipidperoxidation as evidenced by the increased production of


MDA in this study indicates the involvement of free radical-induced oxidative cell injury in mediating the toxicity offluoroquinolone This observation is in agreement with thereports of several authors [42 60]

5 Conclusion

Overall our results from this study reveal that levofloxacin atdifferent doses induced renal and liver damage and oxidativestress and altered both enzymatic and nonenzymatic antiox-idant defense system in rats

Conflict of Interests

The authors declare that there is no conflict of interests

References

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[2] S Shenoy S Chakravarty A Nayak P Z Candita and TShanbhag ldquoAnxiogenic effect of moxifloxacin in wistar ratsrdquoThe International Journal of Applied Biology and PharmaceuticalTechnology vol 3 no 4 pp 158ndash162 2011

[3] R H H Nelwan K C Lie S Hadisaputro et al ldquoA single-blind randomized multicenter comparative study of efficacyand safety of levofloxacin vs ciprofloxacin in the treatment ofuncomplicated typhoid feverrdquo inProceedings of theASTMH55thAnnual Meeting abstract 2517 Atlanta Ga USA November2006

[4] H D Langtry and H M Lamb ldquoLevofloxacin Its use ininfections of the respiratory tract skin soft tissues and urinarytractrdquo Drugs vol 56 no 3 pp 487ndash515 1998

[5] K F Croom and K L Goa ldquoLevofloxacin a review of its use inthe treatment of bacterial infections in theUnited StatesrdquoDrugsvol 63 no 24 pp 2769ndash2802 2003

[6] T Foster ldquoPlasmid determined resistance to antimicrobialdrugs and toxic metal ions in bacteria Levofloxacin SideEffectsrdquo Biochemistry vol 97564 pp 35ndash65 2009

[7] N Hayashi Y Nakata and A Yazaki ldquoNew findings on thestructurephototoxicity relati onship a nd photos tabili ty offluoroquinolones with various substituents at position 1rdquo Anti-microbial Agents and Chemotherapy vol 48 no 3 pp 799ndash8032004

[8] B A Lipsky andC A Baker ldquoFluoroquinolone toxicity profilesa review focusing on newer agentsrdquo Clinical Infectious Diseasesvol 28 no 2 pp 352ndash364 1999

[9] R Stahlmann andH Lode ldquoToxicity of quinolonesrdquoDrugs vol58 no 2 pp 37ndash42 1999

[10] D C Hooper ldquoMode of action of fluoroquinolonesrdquoDrugs vol58 supplement 2 pp 6ndash10 1999

[11] J Child D Mortiboy J M Andrews A T Chow and R WiseldquoOpen-label crossover study to determine pharmacokineticsand penetration of two dose regimens of levofloxacin intoinflammatory fluidrdquo Antimicrobial Agents and Chemotherapyvol 39 no 12 pp 2749ndash2751 1995

[12] D N Fish and A T Chow ldquoThe clinical pharmacokinetics oflevofloxacinrdquo Clinical Pharmacokinetics vol 32 no 2 pp 101ndash119 1997

[13] A Hemeryck R N V SMamidi M Bottacini DMacphersonM Kao and M F Kelley ldquoPharmacokinetics metabolismexcretion and plasma protein binding of 14C-levofloxacin after asingle oral administration in the Rhesus monkeyrdquo Xenobioticavol 36 no 7 pp 597ndash613 2006

[14] J S Wolfson and D C Hooper ldquoThe fluoroquinolones struc-tures mechanisms of action and resistance and spectra ofactivity in vitrordquo Antimicrobial Agents and Chemotherapy vol28 no 4 pp 581ndash586 1985

[15] M L Grayson A Kucers S M Crowe and J F HoyTheUse ofAntibiotics A Clinical Review of Antibacterial Antifungal andAntiviral Drugs The Bath Press 1999

[16] I Harding and I Simpson ldquoLevofloxacin low potential forhepatobiliary adverse reactionsrdquoClinical Bacterial Infection vol7 supplement 1 p 164 2001 Abstract P851

[17] L J Martınez R H Sik and C F Chignell ldquoFluoroquinoloneantimicrobials singlet oxygen superoxide and PhototoxicityrdquoPhotochemistry and Photobiology vol 67 no 4 pp 399ndash4031998

[18] M Goswami S H Mangoli and N Jawali ldquoInvolvement ofreactive oxygen species in the action of ciprofloxacin againstEscherichia colirdquo Antimicrobial Agents and Chemotherapy vol50 no 3 pp 949ndash954 2006

[19] B Halliwell and JM C Gutteridge ldquoFree radical other reactivespecies and diseaserdquo in Free Radicals in Biology and Medicinepp 617ndash783 Clarendon Press 1999

[20] S M Zaidi and N Banu ldquoAntioxidant potential of vitamins AE and C in modulating oxidative stress in rat brainrdquo ClinicaChimica Acta vol 340 no 1-2 pp 229ndash233 2004

[21] BHalliwell ldquoEstablishing the significance and optimal intake ofdietary antioxidants the biomarker conceptrdquoNutritionReviewsvol 57 no 4 pp 104ndash113 1999

[22] A Y Sun and Y M Chen ldquoOxidative stress and neurodegener-ative disordersrdquo Journal of Biomedical Science vol 5 no 6 pp401ndash414 1998

[23] P M Abuja and R Albertini ldquoMethods for monitoringoxidative stress lipid peroxidation and oxidation resistance oflipoproteinsrdquo Clinica Chimica Acta vol 306 no 1-2 pp 1ndash172001

[24] S Gupta A Agarwal J Banerjee and J G Alvarez ldquoThe roleof oxidative stress in spontaneous abortion and recurrent preg-nancy loss a systematic reviewrdquo Obstetrical and GynecologicalSurvey vol 62 no 5 pp 335ndash347 2007

[25] National Research Council Guide for the Care and Use ofLaboratory Animals The National Academies Press Nationalresearch Washington DC USA 8th edition 2011

[26] B Jaffe ldquoWhat made the radical breakrdquo The New EnglandJournal of Medicine vol 286 pp 156ndash157 1972

[27] N W Tietz E L Pruden and O Siggaard-Andersen ldquoLiverfunctionrdquo in Tietz Textbook of Clinical Chemistry A C Burtisand E R Ashwood Eds pp 1354ndash1374WB Saunders LondonUK 1994

[28] N W Tietz Clinical Guide to Laboratory Tests WB SaundersPhiladelphia Pa USA 3rd edition 1995

[29] M Hoslashrder E Magid E Pitkanen et al ldquoRecommendedmethod for the determination of creatine kinase in bloodmodified by the inclusion of EDTAThe committee on enzymesof the Scandinavian Society for Clinical Chemistry and ClinicalPhysiology (SCE)rdquo Scandinavian Journal of Clinical and Labo-ratory Investigation vol 39 no 1 pp 1ndash5 1979


[30] P Trinder ldquoCHOD-PAD enzymatic colorimetric method ofcholesterol determinationrdquoAnnals of Clinical Biochemistry vol6 pp 24ndash27 1969

[31] O Erel A Kocyigit S Avci N Aktepe and V Bulut ldquoOxidativestress and antioxidative status of plasma and erythrocytes inpatients with vivax malariardquo Clinical Biochemistry vol 30 no8 pp 631ndash639 1997

[32] D J Jollow J R Mitchell N Zampaglione and J R GilletteldquoBromobenzene induced liver necrosis protective role of glu-tathione and evidence for 34-bromobenzene oxide as thehepatotoxic metaboliterdquo Pharmacology vol 11 no 3 pp 151ndash169 1974

[33] R Varshney and R K Kale ldquoEffects of calmodulin antagonistson radiation-induced lipid peroxidation in microsomesrdquo Inter-national Journal of Radiation Biology vol 58 no 5 pp 733ndash7431990

[34] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquo Journal of Biological Chemistry vol 247 no 10 pp3170ndash3175 1972

[35] T Magwere Y S Naik and J A Hasler ldquoEffects of chloroquinetreatment on antioxidant enzymes in rat liver and kidneyrdquo FreeRadical Biology and Medicine vol 22 no 1-2 pp 321ndash327 1996

[36] A K Sinha ldquoColorimetric assay of catalaserdquo Analytical Bio-chemistry vol 47 no 2 pp 389ndash394 1972

[37] W H Habig M J Pabst and W B Jakoby ldquoGlutathione trans-ferases the first enzymatic step in mercapturic acid formationrdquoJournal of Biological Chemistry vol 249 no 22 pp 7130ndash71391974

[38] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[39] F J Baker and R E Silverton Introduction to Medical Labora-tory Technology Butter Worth London UK 6th edition 1985

[40] V R Dharnidharka K Nadeau C L Cannon H W Harrisand S Rosen ldquoCiprofloxacin overdose acute renal failure withprominent apoptotic changesrdquo American Journal of KidneyDiseases vol 31 no 4 pp 710ndash712 1998

[41] F Pouzaud M Dutot C Martin M Debray J M Warnetand P Rat ldquoAge-dependent effects on redox status oxida-tive stress mitochondrial activity and toxicity induced byfluoroquinolones on primary cultures of rabbit tendon cellsrdquoComparative Biochemistry andPhysiology Part CToxicology andPharmacology vol 143 no 2 pp 232ndash241 2006

[42] O K Afolabi and E B Oyewo ldquoEffects of ciprofloxacin andlevofloxacin administration on some oxidative stress markersin the ratrdquo International Journal of Biological Veterinary Agri-cultural and Food Engineering vol 8 no 1 pp 80ndash84 2014

[43] S R Naik and V S Panda ldquoAntioxidant and hepatoprotectiveeffects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodentsrdquo Liver International vol 27 no3 pp 393ndash399 2007

[44] D S Pratt and M M Kaplan ldquoEvaluation of abnormal liver-enzyme results in asymptomatic patientsrdquo The New EnglandJournal of Medicine vol 342 no 17 pp 1266ndash1271 2000

[45] S K Singh U Dimri M Kataria and P Kumari ldquoAmeliorativeactivity ofWithania somnifera root extract on paraquat-inducedoxidative stress in micerdquo Journal of Pharmacology and Toxicol-ogy vol 6 no 4 pp 433ndash439 2011

[46] J B Whitfield ldquoGamma glutamyl transferaserdquo Critical Reviewsin Clinical Laboratory Sciences vol 38 no 4 pp 263ndash355 2001

[47] I Macafarlane A Bomford and R A Sherwood Liver Diseaseand Laboratory Medicine ACB Ventures London UK 2000

[48] D Johnson ldquoCKD screening and management overviewrdquo inHandbook of Chronic Kidney DiseaseManagement D John Edchapter 4 Lippincott Williams ampWilkins 2011

[49] M A Ferguson and S S Waikar ldquoEstablished and emergingmarkers of kidney functionrdquo Clinical Chemistry vol 58 no 4pp 680ndash689 2012

[50] L A Stevens J Coresh T Greene and A S Levey ldquoAssessingkidney functionmdashmeasured and estimated glomerular filtra-tion raterdquo The New England Journal of Medicine vol 354 no23 pp 2473ndash2483 2006

[51] R Mouton and K Holder ldquoLaboratory tests of renal functionrdquoAnaesthesia and Intensive Care Medicine vol 7 no 7 pp 240ndash243 2006

[52] Y Nagao H Watanabe and Y Syun-ichiro ldquoPercentile analysisof plasma total bilirubinmdashhow different will the rate of pho-totherapy for jaundice of neonates be by different standardsrdquoOpen Journal of Pediatrics vol 2 pp 133ndash137 2012

[53] P H Proctor and J E McGinness ldquoThe function of melaninrdquoArchives of dermatology vol 122 no 5 pp 507ndash508 1986

[54] B Halliwell J M C Gutteridge and C E Cross ldquoFree radicalsantioxidants and human disease where are we nowrdquo TheJournal of Laboratory and Clinical Medicine vol 119 no 6 pp598ndash620 1992

[55] M Morikawa Y Hirata and T Imanaka ldquoA study on thestructure-function relationship of lipopeptide biosurfactantsrdquoBiochimica et Biophysica Acta vol 1488 no 3 pp 211ndash218 2000

[56] J Cejkova S Stıpek J Crkovska T Ardan and A MidelfartldquoReactive oxygen species (ROS)-generating oxidases in thenormal rabbit cornea and their involvement in the cornealdamage evoked byUVB raysrdquoHistology andHistopathology vol16 no 2 pp 523ndash533 2001

[57] B Chance H Sies andA Boveris ldquoHydroperoxidemetabolismin mammalian organsrdquo Physiological Reviews vol 59 no 3 pp527ndash605 1979

[58] Y Aniya and A Naito ldquoOxidative stress-induced activationof microsomal glutathione S-transferase in isolated rat liverrdquoBiochemical Pharmacology vol 45 no 1 pp 37ndash42 1993

[59] S Altinordulu and G Eraslan ldquoEffects of some quinoloneantibiotics on malondialdehyde levels and catalase activity inchicksrdquo Food and Chemical Toxicology vol 47 no 11 pp 2821ndash2823 2009

[60] V Talla and P R Veerareddy ldquoOxidative stress induced byfluoroquinolones on treatment for complicated urinary tractinfections in Indian patientsrdquo Journal of Young Pharmacists vol3 no 4 pp 304ndash309 2011

[61] D I Thurnham R Singkamani R Kaewichit and K Wong-worapat ldquoInfluence of malaria infection on peroxyl-radicaltrapping capacity in plasma from rural and urban Thai adultsrdquoBritish Journal of Nutrition vol 64 no 1 pp 257ndash271 1990

[62] J M C Gutteridge ldquoLipid peroxidation and antioxidants asbiomarkers of tissue damagerdquo Clinical Chemistry vol 41 no 12pp 1819ndash1828 1995

Submit your manuscripts athttpwwwhindawicom

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MEDIATORSINFLAMMATION

of


HN

N N

F

O

O O

OH

Figure 1 Structure of levofloxacin

These ROS caused severe damage tomacromolecules tissuesand organs through the process of lipid peroxidation (LPO)protein modification and DNA damage [19 20] Oxidativestress results when these ROS overwhelm the antioxidant sys-tem [21] Oxidative stress has been linked with pathogenesisof some disease conditions such as cardiovascular disordersaging cancer and neurodegenerative disease [22 23]

Cell protects itself from effect of ROS by the actionof nonenzymatic antioxidants (Vitamins A E and C andreduced glutathione GSH) and enzymatic antioxidant suchas superoxide dismutase catalase and glutathione peroxidase(GPX) [24] In the present study we investigate the toxicity oflevofloxacin using the status of antioxidant defense systemmarkers of kidney and renal damage and some biochemicalindices in rats

2 Materials and Methods

21 Chemicals and Reagents Levofloxacin (Devox) is a prod-uct of Waves-Biotech Pvt Ltd New Delhi India Glu-tathione (GSH) 1-chloro-24-dinitrobenzene (CDNB) 551015840-dithio-bis-2-nitrobenzoic acid (DTNB) thiobarbituric acid(TBA) epinephrine and hydrogen peroxide were purchasedfrom Sigma chemical company (London UK) Alkalinephosphatase (ALP) alanine amino transferase (ALT) aspar-tate aminotransferase (AST) gamma glutamyl transferase(GGT) urea creatinine bilirubin total cholesterol HDL-cholesterol LDL-cholesterol and triglycerides assay kits wereproducts of Randox Laboratories Ltd (Antrim UK) Allother chemicals and reagents were of analytical grade and ofhighest purity

22 Animals and Treatments Male rats (Wistar strain)weighing between 180 and 220 g were used in this studyThe rats were bred and housed in the animal house of theDepartment of Chemical Sciences Ajayi Crowther Univer-sity Oyo Nigeria They were kept in wire meshed cagesat room temperature and under controlled light cycle (12hr light dark) They were fed with commercial rat chow(Ladokun feeds Ibadan Nigeria) and water ad libitum Theexperimental animals were handled and used in accordancewith the international guide for the care and use of laboratoryanimals (National Research Council) [25]

23 Experimental Design Twenty male albino rats (Wistarstrain) were randomly divided into four groups of 5 rats eachGroup 1 animals were used as control and received distilledwaterGroup 2 animalswere treatedwith LFX-1 (equivalent to5mgkg body weight of levofloxacin) Group 3 animals weretreated with LFX-2 (equivalent to 10mgkg body weight oflevofloxacin) while Group 4 animals were treated with LFX-3 (equivalent to 20mgkg body weight of levofloxacin) 1mLof each of the prepared LFX solutionswas administered orallyto the animals in the different groups using an oral cannulaonce daily for a period of seven days (the normal durationof therapy in humans) The animals were sacrificed 24 hoursafter the last treatment

24 Collection of Blood Samples for Plasma PreparationBlood was collected from the retroorbital plexus of theanimals into heparinized tubes and the rats were sacrificedby cervical dislocation Plasma was prepared by centrifugingblood samples for tenminutes at 3000 rpm in a cencombenchcentrifugeThe clear supernatant was used for the estimationof urea creatinine bilirubin lipid profiles and enzymes

25 Preparation of Cytosolic Fractions The liver excised fromrat blotted of blood stains and rinsed in 115 KCl washomogenized in 4 volumes of ice-cold 001M potassiumphosphate buffer (pH74)Thehomogenateswere centrifugedat 12500 g in an Eppendorf (UK) refrigerated centrifugefor 15min at 4∘C and the supernatants termed as thepostmitochondrial fractions (PMF) were used for enzymeassays

26 Renal and Liver Functions Test Plasma creatinine ureaand bilirubin determinationwas done usingRandox diagnos-tic kitsMethods for creatinine assays are based on colorimet-ric alkaline picratemethods [26]with creatinine-picrate com-plex measured at 492 nm The urea determination methodwas based on the Fearon reaction [27] with the Diazinechromogen formed being absorbed strongly at 540 nm Thedimethyl sulphoxide method by Tietz et al [27] was usedfor bilirubin determination The dimethyl sulphoxide formsa coloured compound with maximum absorption at 550 nm

27 Determination of Plasma AST ALT ALP and GGTActivities Plasma AST ALT ALP and GGT activities weredetermined using Randox diagnostic kits Determination ofAST and ALT activities was based on the principle describedby Tietz et al [27] AST was measured by monitoring theconcentration of oxaloacetate hydrazone formed with 24-dinitrophenylhydrazine at 546 nm and ALT was measuredby monitoring the concentration of pyruvate hydrazoneformed with 24-dinitrophenylhydrazine at 546 nm ALP wasdetermined in accordance with the principles of Tietz [28]The p-nitrophenol formed by the hydrolysis of p-Nitrophenylphosphate confers yellowish colour on the reaction mixtureand its intensity can bemonitored at 405 nm to give ameasureof enzyme activity GGT activity was measured based on amodification of the method described by Hoslashrder et al [29]using Abbott diagnostic kit




















3 Results


















4 Discussion



0

5

10

15

20

25

30


lowast

lowast

lowast

GST

(120583m

olm

inm

g pr

otei

n)


0

2

4

6

8

10

12

14


Vita

min

C (120583

gm

L)

lowast

lowast

lowast



0123456789

10


GSH

(120583g

g liv

er) lowast

lowast

lowast


0

50

100

150

200

250

300


lowast

lowast

lowast

MD

A (n

mol

mg

prot

ein)





2




2





2O2and hydroxyl





5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















3 Results


















4 Discussion



0

5

10

15

20

25

30


lowast

lowast

lowast

GST

(120583m

olm

inm

g pr

otei

n)


0

2

4

6

8

10

12

14


Vita

min

C (120583

gm

L)

lowast

lowast

lowast



0123456789

10


GSH

(120583g

g liv

er) lowast

lowast

lowast


0

50

100

150

200

250

300


lowast

lowast

lowast

MD

A (n

mol

mg

prot

ein)





2




2





2O2and hydroxyl





5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















4 Discussion



0

5

10

15

20

25

30


lowast

lowast

lowast

GST

(120583m

olm

inm

g pr

otei

n)


0

2

4

6

8

10

12

14


Vita

min

C (120583

gm

L)

lowast

lowast

lowast



0123456789

10


GSH

(120583g

g liv

er) lowast

lowast

lowast


0

50

100

150

200

250

300


lowast

lowast

lowast

MD

A (n

mol

mg

prot

ein)





2




2





2O2and hydroxyl





5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0123456789

10


GSH

(120583g

g liv

er) lowast

lowast

lowast


0

50

100

150

200

250

300


lowast

lowast

lowast

MD

A (n

mol

mg

prot

ein)





2




2





2O2and hydroxyl





5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Influence of Different Doses of Levofloxacin on...

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