International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491

Vol 3, Suppl 3, 2011

ResearchArticle

A90DAYORALTOXICITYSTUDYOFTARTRAZINE,ASYNTHETICFOODDYE,INWISTARRATS

IMANEHIMRI1*,SAIDBELLAHCEN2,FAIZASOUNA1,FATIMABELMEKKI2,MOHAMMEDAZIZ2,MOHAMED
BNOUHAM2,JOUHARZOHEIR3,ZOLIKHABERKIA4,HASSANEMEKHFI2,ENNOUAMANESAALAOUI1
:UniversitMohamedIer,FacultdesSciences,LaboratoiredeBiochimie,2:UniversitMohamedIer,FacultDesSciences,Laboratoire
DePhysiologieEtEthnopharmacologie,3:LaboratoiredAnalyseZoheir,Oujda,4:CabinetdAnatomiePathologique,Oujda

Received:28Feb2011,RevisedandAccepted:30March2011
ABSTRACT
ThefollowingstudyisaboutTartrazine(E102)whichisknown asanazodyeusedinfoodproducts,drugsandcosmetics.Asa partofthesafety
assessmentofTartrazine,a13weeksubchronicoraltoxicitystudywasperformedonWistarratsofbothsexes.Theanimalsweredividedinto5
groupsof6animalseach,3ofeachsex,andfedadietcontaining5,7.5,or10mg/kgb.w,ofTartrazineand3.75mg/kgb.w,ofSulfanilicacid.
Therewerenotreatmentrelatedadverseeffectswithregardtobodyweight,foodandwaterconsumption.Theirbloodsampleswereanalyzedfor
hematological measurements, Glucose, Creatinine, Blood urea nitrogen, Cholesterol total, Triglecerid, alanine aminotransferase, aspartate
aminotransferase.TheStomach,Jejunum,Liver,Kidneystissueswerealsoprocessedforhistologicalexamination.
ThepresentstudyshowsthatTartrazineandSulfanilicacidinducedamorphologicalchangefromthediscoidshapetoanechinocyticforminrat
RBCs.Relativeweightsoftheliverweresignificantlyincreasedingrouptreatedwith10mg/kgb.w,ofTartrazine.Ourdatashowedasignificant
increaseinGLU,CREA,CHOL,TG,AST,andtotalProteininserumofratstreatedwithTartrazineandSulfanilicacidcomparedtocontrolratsand
thesesignificantchangesweremoreapparentinhighdosesthanlowones.ThehistopathologicalchangesofLiverandKidneywereinaccordance
withthebiochemicalfindings.
Keywords:Tartrazine,Subchronictoxicity,Hepatotoxicity,Nephrotoxicity,Wistarrats.

INTRODUCTION
Food additives are products added to the basic foodstuffs with an
aimofimprovingitsaspect,savour,taste,colour,texture,foodvalue
andconservation.
Food dyes are added with a principal aim to give a colour to a
foodstuff, or to restore its natural colour. From the organoleptic
pointofview,thevisualaspectisanimportantfactorforthechoice
of the products by the consumer. Thus, the synthetic food dyes
occupyanimportantplaceintheclassoftheessentialadditivesfor
food industry in the conquest of markets. Among the food dyes
whicharewidelyusedisTartrazine.Itisanorangecoloured, water
solublepowderused worldwideasfoodadditivestocolourseveral
foods,drugsandcosmetics.Ithasthefollowingchemicalstructure
illustratedinFig.1.

The study of the carcinogenetic and mutagenetic effects of

Tartrazine were established by some authors which gives variable
results616.
WhentheTartrazinereachestheintestine,itcanundergometabolic
reduction by intestinal microflora 17, and the reductive cleavage
productsarerapidlyabsorbed18.
Following reductive cleavage of the azo linkage by intestinal
bacteria, Sulfanilic acid and aminopyrazolone are produced. The
pyrazolone fragment is further degraded by intestinal bacteria to
yieldasecondmoleculeofSulfanilicacid19.
The studies described here were performed to investigate the oral
subchronictoxicityofTartrazineinWistarrats.
MATERIALSANDMETHODS

Chemicals
Tartrazine(CAS1934210,Purity86.7%),Sulfanilicacid(CAS 122
573, Purity) were purchased from Alfa Aesar (Germany), Sigma
Aldrich(Japan)respectively.
Animalsandhousing
MaleandfemaleWistarratsweighingbetween170and200gwere
housed in a controlled room with a 12 h lightdark cycle and
temperatureof222C(animalhouseofthedepartmentofbiology,
facultyofsciences,Oujda,Morocco).Theywerekeptintransparent
polypropylene cages with free access to water and dry rat pellets
feeds(SocietSONABETAIL,Oujda,Morocco).
Experimentaldesign
Fig.1:ChemicalstructureofTartrazine(trisodiumsaltof3
carboxy5hydroxy1(psulphophenyl)4(psulphophenylazo)
pyrazole

Moreover,thisfoodcolorantisusedincookinginmanydeveloping
countries as a substitute for saffron 1. The Acceptable Daily Intake
(ADI)forhumansis07.5mgkg1bodyweight2.
Tartrazine has been implicated as the food additive which is most
often responsible for allergic reactions in specific human
populations3,4,5.

Theanimalsweredividedinto5groupsof6animalseach,3ofeach
sex.Allanimalsweretreatedbydailyoralgavagefor90dayswitha
volumeof10ml/kg.TartrazineandSulfanilicacidweredissolvedin
distilled water. Tartrazine was administered at 5, 7.5, or 10 mg/kg
b.w. Sulfanilic acid was administered at 3.75 mg/kg b.w. For the
controlgroupitwasadministeredwithdistilledwater.
Theanimalswereobserveddailyforgeneralconditions.Theywere
weighed once every ten days during the administration period, on
thefirstandthelastdaysoftheperiod,andonthedayofnecropsy.
Dailyfoodconsumptionwasmeasuredonceaweek.

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Attheendofweek13,allratsweredeprivedoffood,butnotwater,
overnightandthenbloodsampleswerecollectedviatheabdominal
aorta for hematology and serum biochemistry. Animals were then
killedbyexsanguinationfromtheabdominalaorta.

Organ weights were obtained for the heart, lung, liver, spleen, and
stomach. The left and right kidneys were weighed separately.
Relative organ weights were calculated based on body weights
measuredonthedayofsacrifice.

Hematology

Aportionofeachorganwasfixedin10%neutralbufferedformalin
for histopathology, and was further processed using standard
method.Themicrosectionsof5mthicknesseswerestainedwith
hematoxylineosin and the prepared slides were examined under
lightmicroscopeforhistopathological.

Hematological measurements and calculations were performed by

using Coulter ACT 5diff Hematology Analyzer (Beckman Coulter Inc.,
Fullerton,CA,USA).Hematologicalevaluationsincludedredbloodcell
count (RBC), hemoglobin concentration (HGB), hematocrit (HCT),
mean corpuscular volume (MCV), mean corpuscular haemoglobin
(MCH),meancorpuscularhemoglobinconcentration(MCHC),redcell
volumedistribution(RDW),bloodplateletcount(PLT),meanplatelet
volume (MPV), and white blood cell count (WBC), differential
leukocytepercentageandreticulocyteratioweremeasured.

Statisticalanalysis
Data are presented in tables or figures as the mean SEM. The
statistical significance of the differences between control and
experimental groups was evaluated by Students ttest using
GraphPadInstat3.0626

For morphological examination differential counts of leukocytes

were made by light microscopical observation of smear specimens
stainedwitharoutineMayGlnwaldGiemsaprotocol.

RESULTS
Clinical observations, Body weight, Organ weights and intake
food

Clinicalbiochemistry

Comparedtothewatercontrolgroup,treatmentwithTartrazineand
Sulfanilicaciddidnotaffectmortality,clinicalsigns,intakefoodand
body weights. Body weight curves for Wistar rats during the
treatment period are shown in Fig.2. Data for the intact food and
OrganweightareshowninTable1.

ClinicalchemistrydeterminationswereperformedbyusingILab300
(Instrumentation Laboratory Corporate Headquarters, Barcelona,
Spain). Parameters included Aspartate aminotransferase (AST),
Alanine aminotransferase (ALT), blood urea nitrogen (BUN),
CREatinine (CRE), GLUcose (GLU), Total CHOlesterol (TCHO),
Triglyceride(TG),TotalProtein(TP).

Withregardtoorganweights,statisticallysignificantdecreaseofthe
absoluterightkidneyweightandincreasesoftherelativeweightof
the liver were observed in group treated with 10 mg/kg b.w of
Tartrazine. Moreover, absolute lung and stomach weights were
significantly increased in group treated with 3.75 mg/kg b.w, of
Sulfanilicacid.

Activity of serum ALT and AST were determined by the method of

Henryandal.(1960) 20.Totalproteinwasdeterminedaccordingto
the method of Gornall and al. (1949) 21. Urea was determined in
serum by the method of Tiffany and al. (1972) 22. Creatinine in
serum was estimated by the method of Fabiny and al. (1971) 23.
Totalcholesterolwasestimatedinserumbyenzymaticcolorimetric
method according the method of Allain and al. (1974) 24.
Triglycerides in serum were estimated by enzymatic colorimetric
methodofTrinder(1969a) 25a.Glucoseconcentrationinserumwas
estimated by enzymatic colorimetric method according to Trinder
(1969b)25b.

Hematologicalexamination
Hematology results after the treatment period did not revealed
significant changes in group control and group treated with 10
mg/kg b.w of Tartrazine. But we revealed a significantly higher
meanplateletvolume,neutrophileandbasophile;andasignificantly
lower blood platelet count in group treated with 7.5 mg/kg b.w of
Tartrazine. In group treated with 3.75 mg/kg b.w of Sulfanilic acid
changes included a significantly lower blood platelet count,
neutrophile, lymphocyte, monocyte, basophile, and eosinophile.
HematologydataareshowninTable2.

Clinicalpathologyandhistopathology
At the end of the treatment, animals were exsanguinated by
transectingtheabdominalaorta.Thebodysurface,theintrathracic,
intraabdominalorgansandtissueswereobservedmacroscopically.

Body weight curves of wistar rats fed different concentration of

Tartrazine diets for 90 days

350,00
300,00
250,00

Group control
5 mg/ (kg BW) of
Tartrazine
7.5 mg/ (kg BW) of
Tartrazine
10 mg/ (kg BW) of
Tartrazine
3.5 mg/ (kg BW) of
Sulfanilic acid

200,00
150,00
100,00
50,00

,0
0
90

,0
0
80

,0
0
70

,0
0

,0
0

,0
0

,0
0
60

50

40

30

,0
0
20

10

,0
0

Fig.2:BodyweightsofWistarratstreatedorallywithdifferentconcentrationofTartrazineandSulfanilicacidfor90days.

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Table1:IntactfoodandorganweightforWistarratssacrificedonday90ofsubchronicfeedingofTartrazineandSulfanilicacid

Groupcontrol

Intactfood(g)
Absolue(g)
Liver
Rightkidney
Leftkidney
Heart
Lung
Stomach
Spleen
Relative(g/100gBW)
Liver
Rightkidney
Leftkidney
Heart
Lung
Stomach
Spleen

9.190.39

7.120.62
0.750.04
0.760.05
0.810.03
1.570.06
2.250.12
0.550.03

2.860.10
0.3040.008
0.3110.009
0.330.018
0.650.04
0.9250.05
0.220.012

5mg/(kgBW)of
Tartrazine
10.180.60

7.070.37
0.710.04
0.690.05
0.860.05
1.660.08
2.140.14
0.520.12

3.030.07
0.3080.006
0.290.01
0.370.01
0.720.05
0.920.03
0.220.04

7.5mg/(kgBW)of
Tartrazine
10.200.58

10mg/(kgBW)of
Tartrazine
9.810.51

8.710.71
0.770.07
0.760.06
0.850.06
1.840.1
2.350.13
0.570.04

8.171.27
0.740.08*
0.770.09
0.790.06
1.740.11
2.170.22
0.520.05

3.230.15
0.280.02
0.280.01
0.310.01
0.7010.06
0.880.04
0.210.01

2.970.15*
0.2700.007
0.2800.008
0.300.01
0.670.05
0.820.05
0.1980.007

3.75mg/(kgBW)of
Sulfanilicacid
10.020.33

9.431.13
0.840.04
0.820.04
0.830.03
1.830.09*
2.520.12*
0.570.04

3.230.16
0.290.009
0.2910.009
0.290.01
0.640.03
0.890.02
0.2010.009

Note:valuesrepresentthemeanSEM(n=6);*p<0.05.Significantlydifferentfromcontrols

Table2:HematologicaldataofWistarratsFedwithTartrazineandSulfanilicacidfor90days
Parameters

Groupcontrol

WBC(10^3/l)
RBC(10^6/l)
HGB(g/dl)
HCT(%)
MCV(fL)
MCH(pg)
MCHC(g/dl)
RDW(%)
PLT(10^3/l)
MPV(fL)
NE(%)
LY(%)
MO(%)
EO(%)
BA(%)

4.531.18
7.390.26
13.130.33
38.931.19
52.500.42
17.810.26
33.760.26
12.000.38
725.0011.56
6.450.08
19.431.86
72.751.27
3.480.63
3.861.18
0.460.03

5mg/(kgBW)of
Tartrazine
4.580.43
6.990.24
12.460.51
36.231.59
52.330.71
17.410.37
33.410.28
12.100.79
627.5043.90
6.860.23
32.64.55
59.703.97
3.981.66
3.281.21
0.310.03

7.5mg/(kgBW)of
Tartrazine
6.261.54
7.730.22
13.710.38
40.401.18
52.160.16
17.750.14
33.980.22
12.150.23
609.8345.58*
6.800.03**
25.352.17*
65.783.009
6.082.10
1.880.55
0.900.16*

10mg/(kgBW)of
Tartrazine
6.801.25
7.560.19
13.510.24
40.360.88
53.330.42
17.910.22
33.500.21
11.130.35
727.1616.75
6.380.01
22.402.19
68.981.97
4.050.69
4.201.41
0.360.03

3.75mg/(kgBW)of
Sulfanilicacid
9.211.25*
7.750.23
13.650.18
40.710.90
52.500.56
17.660.32
33.560.30
11.980.37
654.1623.33*
6.630.07
18.500.74*
70.050.43*
5.581.07*
5.360.76*
0.500.03*

Note:valuesrepresentthemeanSEM(n=6);*p<0.05.significantlydifferentfromcontrols.**p<0.01.verysignificantlydifferentfromcontrols.

The morphological changes of rat RBCs were made by light

microscopical observation. All the treated groups showed
morphologicalchangesintheformofechinocytes.Thepercentageof

echinocytes significantly increased among the treated groups in a

doseresponsemanner(***p<0.001)(Fig3,Fig4).

120
100
***

80

***

***

***

%GR
***

***

***

***

%EC

60
40
20
0
Group control 5 mg/ (kg BW)
of Tartrazine

7.5 mg/ (kg

BW) of
Tartrazine

10 mg/ (kg
BW) of
Tartrazine

3.75 mg/ (kg

BW) of
Sulfanilic acid

Fig.3:Thepercentageofechinocytessignificantlyincreasedamongthetreatedgroupsinadoseresponsemanner(P<0.001)
Note:valuesrepresentthemeanSEM(n=12);***p<0.001.extremelysignificantlydifferentfromcontrols
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Fig.4:EffectsofTartrazineonthemorphologyofratRBC.(A):untreatedRBCandRBCtreatedwith(B):5mg/(kgBW),(C):7.5mg/(kg
BW),(D):10mg/(kgBW)and(E):treatedwith3.75mg/(kgBW)ofSulfanilicacid.Theechinocytescanbeobservedin(B),(C)and(D)

Biochemicalexamination

acid(Fig.7,Fig.8).Therewasnosignificantdifferenceinthelevelof
BUN and ALT among the different groups. The level of AST was
significantly in group treated with 10 mg/kg b.w of Tartrazine and
group treated with 3.75 mg/kg b.w of Sulfanilic acid. The total
Protein was significantly increased in group treated with 7.5, 10
mg/kgb.wofTartrazine.

The levels of GLU, CREA, were significantly increased in all groups

compared to group control (Fig.5, Fig.6). The levels of CHOL, TG
weresignificantlyincreasedingrouptreatedwith7.5,10mg/kgb.w
of Tartrazine and group treated with 3.75 mg/kg b.w of Sulfanilic
Glucose
3
2,5

***

**
**

***
Glucose

1,5
1
0,5
0
Group control

Group 1

Group 2

Group 3

Group A.S

Fig.5:EffectsofTartrazineonplasmaGlucose.(Groupcontrol)untreatedandtreatedwith(Group1)5mg/(kgBW),(Group2)7.5mg/
(kgBW),(Group3)10mg/(kgBW),andgrouptreatedwithSulfanilicacid
Note:valuesrepresentthemeanSEM(n=6)**p<0.01.verysignificantlydifferentfromcontrols;***p<0.001.extremelysignificantlydifferentfrom
controls
7

Creatinine

**

6,5
6

***

***

5,5
Creatinine

5
4,5
4
3,5
3
Group control

Group 1

Group 2

Group 3

Group A.S

Fig.6:EffectsofTartrazineonplasmaCreatinine.(Groupcontrol)untreatedandtreatedwith(Group1)5mg/(kgBW),(Group2)7.5mg/
(kgBW),(Group3)10mg/(kgBW),andgrouptreatedwithSulfanilicacid
Note:valuesrepresentthemeanSEM(n=6);*p<0.05.significantlydifferentfromcontrols;**p<0.01.verysignificantlydifferentfromcontrols;
***p<0.001.extremelysignificantlydifferentfromcontrols
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Cholesterol
0,7
0,65

Group 2

Group 3

0,6
0,55
0,5
0,45
0,4
0,35
0,3
Group control

Group 1

Group A.S

Fig.7:EffectsofTartrazineonplasmaCholesterol.(Groupcontrol)untreatedandtreatedwith(Group1)5mg/(kgBW),(Group2)7.5
mg/(kgBW),(Group3)10mg/(kgBW),andgrouptreatedwithSulfanilicacid
Note:valuesrepresentthemeanSEM(n=6);*p<0.05.significantlydifferentfromcontrols

Triglecerid

0,7

*
*

0,6

**

0,5
0,4
0,3
0,2
0,1
0
Group control

Group 1

Group 2

Group 3

Group A.S

Fig.8:EffectsofTartrazineonplasmaTriglecerid.(Groupcontrol)untreatedandtreatedwith(Group1)5mg/(kgBW),(Group2)7.5
mg/(kgBW),(Group3)10mg/(kgBW),andgrouptreatedwithSulfanilicacid
Note:valuesrepresentthemeanSEM(n=6);*p<0.05.significantlydifferentfromcontrols;**p<0.01.verysignificantlydifferentfromcontrols.

Table3:EffectsofTartrazineonplasmaUrea,TotalProtein,AST,ALT.(Groupcontrol)untreatedandtreatedwith(Group1)5mg/(kg
BW),(Group2)7.5mg/(kgBW),and(Group3)10mg/(kgBW)
Parameters

Groupcontrol

5mg/(kgBW)of
Tartrazine

7.5mg/(kgBW)of
Tartrazine

10mg/(kgBW)of
Tartrazine

3.75mg/(kgBW)of
Sulfanilicacid

Urea(g/l)
TotalProtein
(g/l)
AST(U/L)
ALT(U/L)

0.3860.024
63.52.56

0.3950.018
58.830.47

0.360.02
71.52.41*

0.400.039
69.830.70*

0.390.03
57.660.91

1205.64
54.165.33

131.337.50
67.834.27

1197.68
583.66

165.1617.82*
622.84

159.1612.755*
60.661.97

Note:valuesrepresentthemeanSEM(n=6);p<0.05.Significantlydifferentfromcontrols.

Histopathologicalstudies
The results of histological examination are shown in Table 4. In
control group no structural changes were identified by
histopathologyintheliver,kidneys,stomach,orjejunum,suggesting
thattheseanimalswerehealthyandtheconditionsunderwhichthe
experimentwasconductedwereproper.
All the tissue sections obtained from the stomach of experimental
Wistarratsfedwith5,7.5,10mg/(kgBW)ofTartrazine,and with
3.75mg/kgb.wofSulfanilicacidwerenotdifferentfromthecontrol
animalstissues.Allthesectionswereessentiallynormalwithoutany
inflammatorylesion(Fig.9).

The histopathological examination showed lymphoid infiltrates in

the Jejunum of experimental Wistar rats fed with 7.5, 10 mg/ (kg
BW)ofTartrazine(Fig.10).
Thehistopathologicalstudiesshowed brownpigmentdepositionin
theKpffercellsandfattydegenerationoftheliveringroupstreated
with7.5,10mg/(kgBW)ofTartrazine(Fig.11).
They also revealed tubular dilatation with thickened basement
membrane in group treated with 5 mg/ (kg BW) of Tartrazine,
tubulardegeneration,anddilatationoftheglomerularcapillariesin
grouptreatedwith7.5mg/(kgBW)ofTartrazine,andintercapillary
sclerosis, atrophy of glomerulus in group treated with 10 mg/ (kg
BW)ofTartrazine(Fig.12).

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Table4:HistologicaldataforthemainorgansofWistarratstreatedorallywithdifferentconcentrationofTartrazineandSulfanilicacid
for90days

Kidneys

Liver

Stomach

Jejunum

Dose
No.ofanimalsexamined
Tubularregeneration
Eosinophilicbody
Interstitialnephritis
Lymphoidinfiltrates
Glomerularchanges
Tubulardilatation
Centrilobularnecrosis
Fattydegeneration
Inflammation
Vascularchanges
Pigmentation
Regeneration,glandular
Epithelium,focal
Inflammation,erosions
Ulcerationofthegastric
Atrophy
Inflammationandulceration
Hypertrophyandhyperplasia
Lymphoidinfiltrates

Tartrazine
(mg/kgb.w)
0
6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6

5
6
0/6
0/6
0/6
0/6
1/6
3/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6

7.5
6
1/6
0/6
0/6
0/6
5/6*
6/6**
0/6
5/6*
0/6
1/6
5/6*
0/6
0/6
0/6
0/6
0/6
0/6
0/6
5/6*

10
6
1/6
0/6
0/6
0/6
6/6**
6/6**
0/6
5/6*
0/6
2/6
5/6*
0/6
0/6
0/6
0/6
0/6
0/6
0/6
5/6*

Sulfanilicacid
(mg/kgb.w)
3.75
6
0/6
0/6
0/6
0/6
1/6
1/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6

Note:valuesrepresentthemeanSEM(n=6);*.Significantlydifferentfromcontrolsatp<0.05byFisherexacttest;**.Verysignificantlydifferent
fromcontrolsp<0.01byFisherexacttest

Fig.9:Photomicrographiesofparaffinembeddedratstomach.Allsectionswerestainedwithhematoxylinandeosin
(A): Section of control rats showing normal histological appearance of the stomach. (B): Section of rat stomach treated with 5 mg/ (kg BW) of
Tartrazineshowingnormalarchitecture.(C):Sectionofratstomachtreatedwith7.5mg/(kgBW)ofTartrazineshowingnormalarchitecture.(D):
Sectionofratstomachtreatedwith10mg/(kgBW)ofTartrazineshowingnormalarchitecture.(E):Sectionofratstomachtreatedwith3.75mg/
(kgBW)ofSulfanilicacidshowingnormalarchitecture.(Scalebar=40m).

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Fig.10:Photomicrographiesofparaffinembeddedratjejunum. Allsectionswerestainedwithhematoxylinandeosin.
(A):Jejunumsectionofcontrolratsshowingnormalhistologicalappearanceofthejejunum.(B):Sectionofratjejunumtreatedwith5mg/(kgBW)
ofTartrazineshowinglymphoidinfiltration.(C):Sectionofratjejunumtreatedwith7.5mg/(kgBW)ofTartrazineshowinglymphoidinfiltration.
(D):Sectionofratjejunumtreatedwith10mg/(kgBW)ofTartrazineshowinglymphoidinfiltration.(E):Sectionofratjejunumtreatedwith3.75
mg/(kgBW)ofSulfanilicacidshowinglymphoidinfiltration.(Scalebar=40m).

Fig.11:Photomicrographiesofparaffinembeddedratliver.Allsectionswerestainedwithhematoxylinandeosin
(A): Liver section of control rats showing normal histological appearance of the liver. (B): Section of rat liver treated with 5 mg/ (kg BW) of
Tartrazine revealed fatty degeneration (arrow). (C): Section of rat liver treated with 7.5 mg/ (kg BW) of Tartrazine revealed fatty degeneration
(blackarrow)and brownpigmentdepositioninKpffer cells(white arrow). (D):Section ofrat liver treated with 10 mg/ (kgBW) ofTartrazine
revealedfattydegeneration(blackarrow)andbrownpigmentdepositioninKpffercells(whitearrow).(E):Sectionofratlivertreatedwith3.75
mg/(kgBW)ofSulfanilicacidshowingnearnormalarchitecture.(Scalebar=40m).

165

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Fig.12:Photomicrographiesofparaffinembeddedratkidney.Allsectionswerestainedwithhematoxylinandeosin
(A):kidneysectionofcontrolratsshowingnormalhistologicalappearanceofthekidney.(B):Sectionofratkidneytreatedwith5mg/(kgBW)of
Tartrazinerevealedtubulardilatationwiththickenedbasementmembrane(whitearrow).(C):Sectionofratkidneytreatedwith7.5mg/(kgBW)of
Tartrazine showed revealed tubular degeneration, (white arrow) and dilatation of the glomerular capillaries (black arrow). (D): Section of rat
kidneytreatedwith10mg/(kgBW)ofTartrazinereveletedintercapillarysclerosis(whitearrow)andatrophyofglomerulus(blackarrow).(E):
Sectionofratkidneytreatedwith3.75mg/(kgBW)ofSulfanilicacidshowingnearnormalarchitecture.(Scalebar=40m).

DISCUSSION
Duringthe administrationperiod,nodeathsoccurredinanimals of
anygroup.Therewerenoabnormalsignsinallgroups.
Thedifferencesinmeanbodyweight,organweightsandintakefood
between control and treated groups (5, 7.5, 10 (mg/kg b.w) of
Tartrazine and 3.75 (mg/kg b.w) of Sulfanilic acid) were non
significant. These results are in accordance with the data from
BorzellecaandHallagan(1988b) 27whosuggestedthatthedecrease
of body weight was due to decreased caloric intake due to the
Tartrazine component of the diet, and that there were a few
differences between control and treated groups in the
haematological,clinicalchemistryandurineanalysisparametersbut
noneofthedifferencesappearedtobetreatmentrelated.
Regarding the hematological finding in group treated with 3.75
mg/kgbwofSulfanilicacid,theobservedincreaseofWBCsuggestsa
physiologicalinflammatoryresponseofitsabsorption.
ThepresentstudyshowsthatTartrazineandSulfanilicacidinduced
a morphological change from the discoid shape to an echinocytic
forminratRBCs.ThepercentageofRBCechinocytesformstreated
by Tartrazine and Sulfanilic acid were shown in a doseresponse
manner. Some studies had shown that RBCs respond to various
treatments by various agents by altering their morphological
features(Przybylskaandal.,1998;Taibandal.,2009) 2829.
Our work revealed that rats which consumed 7.5, 10 mg/kg b.w of
Tartrazine showed a significant increase in serum total protein
concentrationwhencomparedtocontrolrats.
TheseresultsareinaccordancewithMekkawyandal.(1998) 30who
foundasignificantincreaseinserumtotalprotein,alsothesearein
agreementwithAboelZahaband al. (1997) 31 who foundthe same
effect on serum total protein with rats whose diets were
supplemented with chocolate colours A and B. In addition, Sharma
and al.(2006) 32 found thattotal proteinwas significantly elevated
whenTomatoRedwasconsumedbyswissalbinomice.

Sharma and al. (2005) 33 observed a significant increase in serum

total protein and globulin in rats whose diets were supplemented
withchocolatecoloursAandB.
K.A. Amin and al. (2010) 34 demonstrated that high dose of
Tartrazine caused a significant increase in serum total protein and
serum albumin concentration when rats consumed high dose of
Tartrazine (500 mg/kg bw) or low dose of Tartrazine (15 mg/kg
bw).
Our study demonstrated that the daily intake for 90 day of
Tartrazine exhibited a significant increase in serum Creatinine
concentrationwhencomparedwithcontrolratsinadoseresponse
manner.ThisisinagreementwithHelalandal.(2000) 35whofound
a significant elevation in serum Creatinine and Urea in rats which
consumed a synthetic or natural food colorants after 30 days of
treatment. Moreover , the present results are in accordance with
data reported by Ashour and Abdelaziz (2009) 36 who observed a
significantelevationinserumCreatinineandurealevelofratsdosed
withorganicazodye(fastgreen)orallyfor35days.Ourstudyisalso
inaccordancewithdatareportedbyK.A.Aminandal.(2010) 34who
observed a significant elevation in serum Creatinine and urea level
whenratsconsumedhighdoseofTartrazine(500mg/kgbw)orlow
doseofTartrazine(15mg/kgbw).
Despitethefactthatourresultsareinagreementwiththepreceding
studies, they are also in a contrary because the serum Urea
concentration showed no significant increase. The mechanism for
thesechangeareunclearfromthepresentresults,butthesechanges
may be due to the low concentration of Tartrazine given to Wistar
rat.
Also, these results are at the same time correlated with those
reported by AboelZahab and al. (1997) 31 who observed no
significantincreasesinserumUreaconcentration,andarealsoina
contrary because the serum Creatinine concentration showed no
significant increase in rats whose diets were supplemented with
chocolate colours A and B that Tartrazine and Carmoisine were

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IntJPharmPharmSci,Vol3,Suppl3,2011,159169
among ofthem(sunset yellow,Tartrazine,Carmoisine and brilliant
blue)invaryingconcentrations.
TheincreaseinserumCholesterolandTrigleceridlevelsobtainedin
this study are in accordance with results recorded by AboelZahab
and al. (1997) 31 who observed significant increases in serum total
lipids, cholesterol and triglycerides in rats whose diets were
supplemented with chocolate colours A and B that Tartrazine and
Carmoisine were among of them (sunset yellow, Tartrazine,
Carmoisineandbrilliantblue)invaryingconcentrations.Ourresults
areinacontrarywithSharmaetal.(2006) 32whoreportedthattwo
dosesofTomatoRed(blendofCarmoisineandponceau4R)showed
a significant decrease in serum total cholesterol and triglycerides
when swiss albino mice consumed these colorants for 21 days as
short term or 42 days as long term. Also, these results are in
opposite with those reported by Ashour and Abdelaziz (2009) 36
whoobtainedasignificantreductioninserumtotalcholesteroland
triglycerides level when food colour azo dye (fast green) was
consumedorallytomalealbinoratsfor35days.
In the present study, the daily intake for 90 day of Tartrazine
exhibited a significant increase in serum Glucose concentration
when compared with control rats in a doseresponse manner. Our
results are in a contrary with AboelZahab and al. (1997) 31 who
observednosignificantincreasesinserumGlucoseconcentrationin
ratswhosedietsweresupplementedwithchocolatecoloursAandB
thatTartrazineandCarmoisinewereamongofthem(sunsetyellow,
Tartrazine,Carmoisineandbrilliantblue)invaryingconcentrations.
OurresultarealsoinacontrarywithK.A.Aminandal.(2010)34who
demonstrated that high dose of Tartrazine caused no significant
increase serum Glucose concentration when rats consumed high
dose of Tartrazine (500 mg/kg bw) or low dose of Tartrazine (15
mg/kgbw).
The present study revealed that rats which consumed high dose of
Tartrazine(10mg/kgbw)exhibitedasignificantincreaseinserum
ASTwhencomparedtocontrolrats,whilethesamedoseshowedno
significantincreaseinserumALTwhencomparedtocontrolrats.
TheseresultareinaccordancewithdatareportedbyK.A.Aminand
al. (2010) 34 who revealed that rats which consumed high dose of
Tartrazine(500mg/kgbw)exhibitedasignificantincreaseinserum
ALT, AST and alkaline phosphatase activities when compared to
controlrats,whilelowdoseofTartrazine(15mg/kgbw)showeda
significant increase in serum alkaline phosphatase activity when
comparedtocontrolrats.
Thepresentstudyrevealedthatalltissuesectionsobtainedfromthe
stomach of experimental Wistar rats fed with Tartrazine, and
Sulfanilic acid were not different from the control animals tissues.
TheseresultsareinaccordancewithdatareportedbyK.A.Aminand
al. (2010) 34 who revealed that no histopathological effects were
recorded in the stomach tissue. Our results are also in a contrary
withMoutinho,ILDandal.(2007)37whoobtainedanincreaseinthe
number of lymphocytes and eosinophils of the gastric antrum
mucosa when 7.5 mg.kg1.day1 of Tartrazine was offered in
drinkingwateradlibitumtoWistarratstenmonthsfromweaningto
theageoftwelvemonths.
These studies showed lymphoid infiltrates in the Jejunum of
experimentalWistarratsfedwith7.5,10mg/(kgBW)ofTartrazine.
TheseresultsareinaccordancewithdatareportedbySasakiandal.
(2002) 38whostudiedthegenotoxicityof39chemicalscurrentlyin
use as food additives. They treated groups of four male ddY mice
onceorallywitheachadditiveatuptohalfitsLD50orthelimitdose
(2000 mg/kg bw) and performed Comet assays on glandular
stomach,colon,liver,kidney,urinarybladder,lung,brain,andbone
marrow, 3 and 24 hours after treatment. Tartrazine induced dose
relatedDNAdamageintheglandularstomach,colon,and/orurinary
bladder. All 7 food dyes tested induced DNA damage in the
gastrointestinal organs at low doses (10 or 100 mg/kg). Among
them, Amaranth, Allura Red, New Coccine, and Tartrazine induced
DNA damage in the colon. Tartrazine also induced DNA damage in
thestomachatdosesof10and2000mg/kgbwwithoutadoseeffect
relationship.

The brown pigment deposition in the Kpffer cells and fatty

degenerationoftheliveringroupstreatedwith7.5,10mg/(kgBW)
of Tartrazine obtained in this study are in accordance with results
recorded by AboelZahab and al. (1997) 31 who observed brown
pigmentdepositionintheportaltractsandKupffercellsoftheliver
as well as in the interstitial tissue and renal tubular cells of the
kidney. Congested blood vessels and areas of haemorrhage in both
liver and renal sections were revealed in rats receiving mixture B.
OurresultsarealsoinaccordancewithdatareportedbyV.Rusand
al. (2009) 39 who described changes in the liver when guinea pigs
received Tartrazine in drinkingwater in concentrations of 1,2and
3%for3weeks.Fortheconcentrationof1%thechangesconsistof
slight congestion, in both intralobular and extralobular vessels and
discrete perivascular edema. In the external third of lobules they
observedsomeapoptotichepatocytes.Fortheconcentrationof2%,
the liver vascular phenomena are more pronounced, capillary
congestion is present in many lobules, determining a slight
compression atrophy of hepatocyte cords. Hepatocytes in various
stagesofapoptosiswereobservedinsmallnumberswithintheliver
lobe, the number of hepatocytes in apoptosis being greater at the
lobe periphery. For 3% concentration, liver lesions are more
advancedthaninotherconcentrations.
The groups treated with 10 mg/ (kg BW) of Tartrazine revealed a
marked increase in the average liver weight in the experimental
groups and severe histological changes in the liver; But we did not
revealed congested blood vessels and areas of haemorrhage in the
liver.
In the early phase of fatty degeneration, vacuoles appear in the
cytoplasm around the nucleus, because their lipid content is
dissolved in the course of embedding. The vacuoles appear empty.
As the damage to the cells progresses, the hepatocytes become
swollen and a single large vacuole will occupy their entire
cytoplasm, pushing aside the nucleus and making the hepatocyte
signetring shaped. The degenerated hepatocytes form wide
trabeculae which compress and narrow the lumen of the sinusoids
40.
The present histopathological findings revealed tubular dilatation,
tubular degeneration, dilatation of the glomerular capillaries,
intercapillary sclerosis, and atrophy of glomerulus. These changes
areinconsistencywiththereportingofV.Rusandal.(2009) 39who
described changes in the kidney when guinea pigs received
Tartrazineindrinkingwaterinconcentrationsof1,2and3%for3
weeks. The changes in the kidney are somewhat comparable to
those of liver, meaning that there is congestion of different
intensities and perivascular edema. In addition to these, abnormal
glomerular filtration and glomerular or tubular stasis were
observed. In animals treated with low dose (1%), vascular
congestion is relatively moderate and for the group treated with
Tartrazinepresentalsoalteredglomerularfiltrationprocesses,with
accumulationofproteinmaterialinthecapsularspace.Inthegroups
treated with 2% concentration, the changes are comparable to a
certain point, only to have higher intensity. Glomerular filtration
disorders are present in animals treated with carmoisine, but are
more pronounced at this dose for 2% Tartrazine, where stasis
occurs with atrophy of renal corpuscles. Furthermore, area
nephrociteapoptosisappears.Concentrationof3%ledtothekidney
level the most pronounced changes. Renal corpuscles atrophy is
presentinbothgroups,forthegrouptreatedwithTartrazinehaving
inadditionseveralcorpusclesandtubularnecrosis.
Theglomerulusistheprimarysiteofactionofseveralchemicalsand
it may be injured by any toxic, metabolic and immunologic
mechanism 4142.Thetoxicirritantsubstancesbroughttothekidney
by circulatory blood cause degenerative changes in the kidney
tissues 43. According to Varely, H., (1987) 44, the blood Urea can be
increased in all forms of kidney diseases. Also plasma Creatinine
increasesinrenaldiseasesgaveprognosticsignificancethanthoseof
othernitrogenoussubstances.
Tartrazine is transformed into aromatic amine sulfanilic acid after
beingmetabolizedbythegastrointestinalmicroflora 37.Theformed
aromatic amines can generate reactive oxygen species as part of
their metabolism by interaction of these amino groups with nitrite
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or nitrate containing foods or in the stomach. The reactive oxygen
species could be produced in the metabolism of nitrosamines and
increaseoxidativestress45.
Reactive oxygen species play an essential role in pathological
changes in the liver 46. Increased generation of Reactive oxygen
speciesorfreeradicalsisabletocauseautooxidationofthehepatic
cells,resultinginmarkedhepaticlesions 47.
CONCLUSION
In conclusion, the present 13week subchronic study indicates that
Tartrazinenotonlycauseschangesinhepaticandrenalparameters
but also their effect become more risky at higher doses because it
caninduceoxidativestressbyformationoffreeradicals.
Therefore, it is necessary that people should be aware about the
hazardouseffectsofconsumingTartrazine.
ABBREVIATIONS
Acceptable Daily Intake (ADI); Alanine aminotransferase (ALT);
Aspartate aminotransferase (AST); Basophile (BA); Blood platelet
count (PLT); Blood urea nitrogen (BUN); Body weight (b.w);
Creatinine (CRE); Eosinophile ( EO); Glucose (GLU); Hematocrit
(HCT); Hemoglobin concentration (HGB); Lymphocyte (LY); Mean
corpuscular haemoglobin (MCH); Mean corpuscular hemoglobin
concentration (MCHC); Mean corpuscular volume (MCV); Mean
platelet volume (MPV); Monocyte (MO); Neutrophile (NE); Red
blood cell count (RBC); Red cell volume distribution (RDW); Total
cholesterol (TCHO); Total protein (TP); Triglycerid (TG); White
bloodcellcount(WBC).
ACKNOWLEDGEMENT
This research is financially sponsored by the CUD Commission
Universitaire pour le Developpement, Laboratoire dAnalyse
Zoheir and Cabinet dAnatomie Pathologique . We thank El
MostaphaBedraouiandKarimRamdaouiforhelpinginanimalcare.
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