Ss

Aa

b

h

����

a

ARR2AA

KCILSSS

1

tuaeY

tflr3c

(

0h

Toxicology Letters 217 (2013) 197– 204

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epage: www.elsev ier .com/ locate / tox le t

unset yellow FCF, a permitted food dye, alters functional responses ofplenocytes at non-cytotoxic dose

shish Yadava,b, Arvind Kumarb, Anurag Tripathia,∗, Mukul Dasa,∗

Food Toxicology Division, CSIR-Indian Institute of Toxicology Research, M.G. Marg. Post Box No. 80, Lucknow 226001, IndiaMolecular Immunology Lab, School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India

i g h l i g h t s

Sunset yellow FCF (SY), a permitted food dye, induces immune suppression.SY alters the relative expression of surface receptors in T and B cells.SY causes lymphocyte hypo-responsiveness on stimulation with Con A or LPS.SY shows suppressive effect on the Con A or LPS stimulated release of cytokines.

r t i c l e i n f o

rticle history:eceived 27 September 2012eceived in revised form8 November 2012ccepted 20 December 2012vailable online 1 January 2013

eywords:

a b s t r a c t

Sunset yellow FCF (SY), a permitted food color, is extensively used in various food preparations andquite often exceeds the permissible levels (100–200 mg/kg). Several toxicity studies on SY are reported,however immunomodulatory properties have not been explored yet. To investigate the immunotoxicproperties of SY, splenocytes were isolated, cultured and subjected to mitogen stimulated prolifera-tion assay (lipopolysaccharide, LPS or concanavalin A, Con A), mixed lymphocyte reaction (MLR) assay,immunophenotypic analysis of cell surface receptor expression and assay for cytokines release in theculture supernatants were performed in the presence of SY. Since SY did not exhibit any cytotoxicity

ytokinesmmunotoxicityymphoproliferationplenocytesunset Yellow FCFurface receptors

up to 250 �g/ml, this dose was used for further studies. It was observed that SY (250 �g/ml) signifi-cantly (p < 0.05) suppressed the mitogen induced proliferation of splenocytes and MLR response. Further,immunophenotypic analysis revealed that SY alters the relative expression of CD3e/CD4/CD8 in T cellsand CD19 in B-cells. Consistent with the suppression of T-cell and B-cell responses and altered surfacereceptor expression, SY also lowered the expression of IL2, IL4, IL6, IL-17, IFN-� and TNF-� cytokines.These results suggest that non-cytotoxic dose of SY may have immunomodulatory effects.

. Introduction

Studies have shown the association of some azo dyes such asartrazine, amaranth, with allergic responses including contacttricaria, angioneurotic edema, asthma, contact anaphylaxis

nd immunosuppression (Thune and Granholt, 1975; Mikkelsent al., 1978; Shari et al., 1995; Koutsogeorgopoulou et al., 1998;adav et al., 2012). Some azo dyes have been found to induce

Abbreviations: APC-Cy7, allophycocyanin-cyanine 7; CD, clusters of differen-iation; Con A, concanvalin A; DMEM, Dulbecco’s modified Eagles medium; FITC,uorescein isothiocyanate; IFN, interferon; IL, interleukin; LPS, lipopolysaccha-ide; MFI, mean fluorescence intensity; MLR, mixed lymphocyte reaction; MTT,-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PerCP, peridininhlorophyll protein; PI, propidium iodide; TNF, tumor necrosis factor.∗ Corresponding authors. Tel.: +91 522 2963826; fax: +91 522 2628227.

E-mail addresses: anurag1706@gmail.com, anurag1706@rediffmail.comA. Tripathi), mditrc@rediffmail.com (M. Das).

378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2012.12.016

© 2012 Elsevier Ireland Ltd. All rights reserved.

bronchioconstriction in perennial asthmatics, food intolerance,hypersensitivity and behavioral hyperactivity in children (Weberet al., 1979; Ibero et al., 1982; McCann et al., 2007).

Sunset Yellow FCF (SY) (Disodium 6-hydroxy-5-[(4-sulfophenyl)azo]-2-napthalenesulfononate) is an azo dyepermitted for food usage in several countries including India(FSSR, 2011). It is extensively used in food preparations suchas confectionary products, ice candy, sweets, savory samples,frozen desserts and beverages, cosmetics, medicines, and dietarysupplements, etc. (Tripathi et al., 2007, 2010; Dixit et al., 2008,2011; Rao and Sudershan, 2008; Husain et al., 2006; FSA, 2006).The maximum permissible level of SY in food commodities is100–200 ppm, however, various studies have reported that thislimit is frequently exceeded manifolds (Tripathi et al., 2007; Dixit

et al., 2008; Husain et al., 2006). Also, SY has been observed to bepresent in those food commodities like tomato sauce where its useas a colorant is prohibited under regulatory guidelines (Dixit et al.,2008).

1 y Lette

gNe1t1ddc(ahr

eaPaiIHfeta

F(s

98 A. Yadav et al. / Toxicolog

Earlier acute, chronic and long term studies in mice did not sug-est any significant adverse effect of SY (Gaunt et al., 1967, 1974).o mutagenic or carcinogenic potential of SY has been observedither in vitro or in vivo studies (Price et al., 1978; Yoshimoto et al.,984). However, reports suggest that dietary exposure of SY altershe reproductive and neurobehavioral parameters in mice (Tanaka,996). In a recent study it has been reported that although SYid not affect the body weight and spleen weight but significantlyecreased the thymus weight along with alteration in monocyteounts and suppressed delayed type hypersensitivity responseHashem et al., 2010). However, immunomodulatory effects of SYt cellular level have not been explored yet. Further, US FDA (2000)as also expressed concerns regarding the need for immunotoxicityisk assessment of chemicals added to food articles.

The immune response is largely governed by the type of for-ign assault, portal of antigen encounter with immune componentsnd the levels of different cytokines at the site of action (Seder andaul, 1994). Effectors of immune system (activated lymphocytesnd phagocytes) play a major role in immune reactions by talk-ng to each other through proinflammatory cytokines, such as IL-2,L-4, IL-6, IFN-�, TNF-� and IL-17 (Feldman and Brennan, 2001).enceforth, functional assays of lymphocyte and analysis of dif-

erent cytokine levels provides a better platform to understand theffect of xenobiotics on the immune system. The present study aimso determine the alteration in the functional responsiveness of thedaptive and innate immune system on SY exposure to splenocytes.

ig. 1. Cytotoxicity of sunset yellow FCF in splenocytes. Splenocytes from Balb/c were

A) Propidium Iodide staining followed by flow cytometric analysis and (B) MTT assay (7plenocytes in 72 h. Data represent mean ± SE of three experiments as a percent of untrea

rs 217 (2013) 197– 204

2. Materials and methods

2.1. Chemicals and reagents

Sunset Yellow FCF was purchased from Sarabhai Chemicals, Mumbai. Dulbecco’smodified Eagles medium (DMEM) was purchased from Invitrogen Co. (Carlsbad, CA).3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), concanvalinA (Con A), lipopolysaccharide (LPS), mitomycin C, N-acetyl cysteine, non-essentialamino acids, propidium iodide (PI) and other reagents were purchased fromSigma Chemical Co (St. Louis, MO). Antibodies (Anti-CD3e-APC-Cy7, Anti-CD4-FITC,Anti-CD8-PerCP, Anti-CD19-Alexafluor700) for immunophenotyping and cytomet-ric bead array kit for TH1/TH2/TH17 cytokines were purchased from BD Biosciences(San Diego, CA). Tritiated thymidine was purchased from Amersham Lifescience(Uppsala, Sweden) and scintillation cocktail-W was a product of Sisco ResearchLaboratories Pvt. Ltd. (Mumbai, India).

2.2. Animals

Female Balb/c mice and Swiss mice (Inbred strains, 8–10 weeks old, 18–20grams) were procured from the animal breeding colony of Indian Institute ofToxicology Research (Lucknow, India). Before preceding the experiments, animalswere acclimatized under standard laboratory condition for one week. Animalswere housed in polycarbonate cage maintained in a controlled atmosphere of 12 hdark/light cycle, 22 ± 2 ◦C temperature and 50–60% humidity as per rules laid downby Animal Welfare Committee of CSIR-IITR. Animals were fed a normal diet andwater ad libitum before sacrifice.

2.3. Splenocytes culture

Animals were sacrificed according to CSIR-IITR guidelines for the care and useof laboratory animals. Spleens were dissected out, washed with incomplete DMEM

incubated with Sunset Yellow FCF for 72 h and cytotoxicity was determined with2 h). Toxicity of non-cytotoxic dose of SY in naïve and ConA (C) or LPS (D) inducedted cells (percent control) (*p < 0.05, significant with respect to control).

y Letters 217 (2013) 197– 204 199

awtpi1pa

2

c(sPws

2

a(uTTfi�

2

aRcaSp

2

fiTsroPitp

2

fbao

2

aPs

3

3

it1ss

Fig. 2. Effect of sunset yellow FCF on (A) Con A and (B) LPS induced splenocytesproliferative response. Splenocytes were cultured with or without Con A (5 �g/ml)

A. Yadav et al. / Toxicolog

nd minced into a cell suspension in incomplete DMEM. Cell suspension was treatedith erythrocytes lysis buffer (0.15 M NH4Cl, 1 mM NaHCO3, 0.1 mM EDTA, pH 7.4)

o remove erythrocytes. The cells were subsequently washed two times with incom-lete medium and centrifuged (300 × g) for 5 min at 4 ◦C. Cells were resuspended

n supplemented DMEM (DMEM with 10% fetal bovine serum, 2 mM l-glutamine,00 U/ml of penicillin, 100 �g/ml of streptomycin, 25 mM HEPES, 1 mM sodiumyruvate, 25 mM dextrose and 50 �M 2-mercaptoethanol). The cells were culturedt a concentration of 2 × 106 cells/ml and incubated overnight for acclimatization.

.4. Cytotoxicity assays

MTT assay and PI staining was used to determine the cytotoxicity of SY in spleno-ytes for 72 h. Cells were cultured and treated with different concentrations of SY0–1000 �g/ml). MTT assay was carried out according to Mosmann (1983). For PItaining, cells were washed twice to get rid of SY, suspended in PI solution (1 �g/mlI in PBS) and studied by flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA)ithin 1 h. Further, non-cytotoxic dose of SY was derived from ConA or LPS induced

plenocytes experiments using PI staining assay for 72 h.

.5. Lymphoproliferation assays

T and B cells in splenocytes were induced with Con A and LPS to prolifer-te, respectively. Cultured splenocytes were treated with Con A (5 �g/ml) or LPS10 �g/ml) in presence of SY and incubated at 37 ◦C for 72 h. Tritiated thymidineptake assay was used to determine the relative fold proliferation in splenocytes.ritiated thymidine (2 �Ci/ml) was added to the cultures 18 h prior to the end point.he cells were then harvested with nunc cell harvester in glass fiber filters. Theselters were transferred to scintillation cocktail-W and �-counts were recorded on-counter (Hewlett-Packard, Palo Alto, CA).

.6. Mixed lymphocyte reaction

For mixed lymphocyte reaction (MLR), splenocyte culture media was addition-lly supplemented with 1% non-essential amino acids and 10 mM N-acetyl cysteine.esponder cells (Balb/c splenocytes without mitomycin C treatment) were co-ultured with stimulator cells (mitomycin C treated Swiss albino splenocytes) in

ratio of 1:4 (0.5 × 105: 2 × 105 cells per 200 �l). The co-culture was treated withY and incubated at 37 ◦C in humidified CO2 incubator for 5 days. The relative foldroliferation of cells was determined by tritiated thymidine uptake assay.

.7. Immunophenotyping

Splenocytes were labeled with surface marker specific antibodies for the identi-cation of B cells and T cells in control and SY treated groups after 72 h of treatment.he cells were suspended in staining buffer (2% FBS, 1% sodium azide in PBS) andtained with Alexafluor 700 rat anti-mouse CD19, FITC rat anti-mouse CD4, PerCPat anti-mouse CD8 and APC-Cy7 hamster anti-mouse CD3e antibodies for 25 minn ice. The stained cells were washed twice with wash buffer (0.01% Sodium azide inBS) and finally suspended in phosphate buffered saline. The samples were kept once and analyzed within 1 h by flow cytometer. Live cells were analyzed for the rela-ive distribution and expression of CD19+ and CD3e+ population in dot plot. CD3e+opulation was further resolved into CD4+ and CD8+ sub-populations.

.8. Cytokines analysis

Culture supernatants from Con A or LPS stimulated splenocytes were collectedor the estimation of IL2, IL4, IL6, IL17, IFN-� and TNF-� cytokines with cytometricead array kit (BD Biosciences, San Jose, CA). Samples were prepared for cytokinesnalysis as directed by the kit manufacturer and analyzed on the same day. Levelsf cytokines present in the samples were evaluated with CBA FCAP array software.

.9. Data analysis

Data are expressed as mean ± SE. The results were analyzed using $t$-test andnalysis of variance (one-way ANOVA). Each statistical analysis was performed usingrism version 5, Graph Pad Software Inc. A value of p < 0.05 was considered astatistically significant.

. Results

.1. Cytotoxicity assays

Cytotoxicity assays for SY showed a dose dependent responsen splenocytes. The highest non-cytotoxic dose of SY after 72 h of

reatment was observed to be 250 �g/ml, but higher doses (500 and000 �g/ml) were significantly toxic at 72 h, as depicted by both PItaining (Fig. 1A) and MTT assay (Fig. 1B). Therefore, 250 �g/ml wascreened out as the highest non-cytotoxic dose to study the effects

or LPS (10 �g/ml) in presence of sunset yellow FCF for 72 h and proliferation wasassessed using [3H] thymidine uptake assay. Data represent mean ± SE of 5 sets(*p < 0.05, significant with respect to control).

of SY on functional responses of T-cells and B-cells. Furthermore, PIstaining assay revealed that this non-cytotoxic dose of SY does notcauses any significant toxicity in ConA or LPS induced splenocytes(Fig. 1C and D).

3.2. Lymphoproliferation assays

SY (250 �g/ml) showed decrease in proliferative response of ConA or LPS stimulated splenocytes. SY (250 �g/ml) exposure signifi-cantly down regulates the functional activity of T cells (52%) withrespect to Con A treated splenocytes (Fig. 2A), and B cells (48%)when compared to LPS stimulated splenocytes (Fig. 2B).

3.3. Mixed lymphocyte reaction

The MLR was also suppressed by SY (250 �g/ml). SY significantlyinhibited the MLR (29%) of Balb/c splenocytes against allogenicantigens (Swiss mice splenocytes) (Fig. 3).

3.4. Immunophenotyping

SY disturbs the relative distribution of T and B cells. It alters therelative percentage of CD3e, CD4 and CD19 positive populationsin splenocytes. SY treatment showed significantly enhanced levelof CD3e+ (16%) and CD4+ (13%) T cells (Fig. 4C and G) and sup-

pressed levels of CD19+ B cells (11%) in splenocytes (Fig. 5C). SYalso, enhances the expression of CD3e surface receptor (11%) andsuppresses the expression of CD4 (15%) and CD8 (26%) in T cells(Fig. 4D, H and J).

200 A. Yadav et al. / Toxicology Lette

Fig. 3. Effect of sunset yellow FCF on mixed lymphocyte response. The co-cultureof stimulator cells from splenocytes of Swiss albino mice and responder cells fromsplenocytes of Balb/c mice were treated with Sunset Yellow FCF as depicted undermaterial and methods and [3H] thymidine uptake assay was analyzed. Data repre-s

Fftw

ent mean ± SE of 5 sets (*p < 0.05, significant with respect to control).

ig. 4. Relative percentage of CD3e+, CD4+ and CD8+ population and expression of CD3or 72 h. T-cells (CD3e+) were gated and resolved into CD4+ and CD8+ sub-populations. Hhree experiments (A, B, E and F) and corresponding graph representing the relative perceith respect to control).

rs 217 (2013) 197– 204

3.5. Cytokine analysis

Treatment of SY to Con A stimulated splenocytes showeddecreased levels of cytokines IL-2 (35%), IL-4 (31%), IL-6 (99%), IFN-�(85%), TNF-� (41%) and IL-17 (77%) (Fig. 6A–F). Similarly, SY abro-gated the LPS stimulated secretion of IL-6 (34%), IFN-� (50%) andTNF-� (20%) cytokines (Fig. 7A–C).

4. Discussion

In vivo animal studies are most appropriate for immunotoxicityassessment, but in vitro screening of compounds for risk assessmentturns out to be a better option prior to animal studies. The huntfor alternative animal models have led to the emergence of novelin vitro approaches, which offer an upper edge over in vivo studies,apart from minimizing time and efforts. European Centre for thevalidation of alternative methods (ECVAM) is also promoting the

methods that can help in the minimal use of laboratory animals(Gennari et al., 2005).

SY has been under study for various systemic and generaltoxicity evaluations in different animal models. Besides general

e, CD4 and CD8 on T cells following exposure of sunset yellow FCF to splenocytesistogram from a typical experiment is shown while values represent mean ± SE ofntage (C, G and I) and mean fluorescence intensity (D, H and J) (*p < 0.05, significant

A. Yadav et al. / Toxicology Letters 217 (2013) 197– 204 201

Fig. 5. Relative percentage of CD19+ population and expression of CD19 on B cells following exposure of sunset yellow FCF to splenocytes for 72 h. Histogram from at ts (A afl

tls2itislasfawpsprtcCuccCgt

ypical experiment is shown while values represent mean ± SE of three experimenuorescence intensity (D) (*p < 0.05, significant with respect to control).

oxicity of SY (Gaunt et al., 1967, 1974; Tanaka, 1996), some pre-iminary reports have shown alteration in monocyte counts anduppressed delayed type hypersensitivity response (Hashem et al.,010). However, detailed immunotoxic studies on SY are still lack-

ng. Therefore, the present investigation was undertaken to studyhe effect of SY on functional responses of normal and mitogennduced splenocytes, relative distribution of T and B cells, expres-ion levels of surface markers, lymphoproliferative response, mixedymphocyte response and cytokine expression levels. To enumer-te the immunotoxic potential of SY, cultured splenocytes wereubjected to various concentration of SY for 72 h. It was concludedrom PI and MTT cell viability assays that the SY does not showny significant cytotoxicity up to 250 �g/ml and hence this doseas selected for further immunotoxic experiments. The lympho-roliferation is an essential parameter to indicate the immuneuppression (Descotes, 2006). It was found that SY (250 �g/ml) sup-ressed the proliferation of Con A and LPS induced T and B cells,espectively. Further, immunophenotyping studies indicate that SYreatment affect the relative distribution of T and B cells in spleno-yte population, and simultaneously alters the expression of CD3e,D4 and CD8 cell surface receptors in T cells with respect to theirntreated controls. CD3e is a component of T cell receptor (TCR)omplex, which is involved in the initiation of signals in receptor

omplex (Pitcher and van Oers, 2003; Alarcon et al., 2003) whileD4/CD8 molecules act as a co-receptor and modulate the signalenerated by TCR (Barber et al., 1989). Recent studies have shownhat CD4/CD8 molecules play an important role in the TCR signaling

nd B) and corresponding graph representing the relative percentage (C) and mean

and in absence of the CD4/CD8 co-stimulation, activation and pro-liferation of T cells is hampered (Sykulev, 2010; Janeway, 1992).In the present study it was found that SY enhances the relativepercentage and expression of CD3e surface marker in CD3e+ popu-lation but simultaneously decreases the expression of CD4 and CD8co-receptor on CD4+ and CD8+ T cells respectively, indicating theabsence of a co-stimulus in primarily activated cells which mightbe the cause for suppressed T cell proliferation in Con A inducedsplenocytes. MLR, an indicator of allogenic responses of T cellsalso suggests that SY suppresses the activity of T cells against allo-genic splenocytes. CD19 co-receptor expressed in B cells interactswith B cell receptor and induces B cell activation and differen-tiation (Tedder et al., 1997). Splenocytes treated with SY showsdeclined percentage of CD19+ B cells concurrently SY induces thesuppression in lymphoproliferative response of LPS stimulatedsplenocytes.

To understand the mechanism underlying the SY inducedimmunosuppression to investigate the effect of SY on cytokine pro-duction by ConA or LPS activated immune cell types. Cytokines arethe major regulators of the immune system and directly participatein the cross talk of the cells involved in immune responses. IL-2,is a potent T cell growth factor which regulates various immunefunctions including the differentiation of TH1, TH2 and TH17 cells

(Kim et al., 2006; Cote-Sierra et al., 2004; Liao et al., 2011); IL-4 andIL-6, participate in the proliferation and differentiation of T and Bcells (Kishimoto, 2010; Le Gros et al., 1990; Haynes et al., 2004);IFN-�, is a T cell growth promoting factor and also potentiates the

202 A. Yadav et al. / Toxicology Letters 217 (2013) 197– 204

Fig. 6. Effect of sunset yellow FCF on cytokine responses in Con A stimulated Balb/c splenocytes in vitro. Splenocytes were stimulated with or without Con A (5 �g/ml) inp nd ana( , signifi

rcsmVpSai

(cibi

resence of sunset yellow FCF. The culture supernatants were collected after 48 h aC), IFN-� (D), TNF-� (E) and IL-17 (F). Data represent mean ± SE of 5 sets (*p < 0.05

esponses to mitogens (Smeltz et al., 2002); TNF-�, enhances the Tell proliferation in response to mitogens and do not require IL-2timulus (Heller and Kronke, 1994); and IL-17 is known to play aajor role in the innate immunity (Reynolds et al., 2010; Van deeerdonk et al., 2009; Liu et al., 2009). Consistent with the sup-ression of T-cell and B-cell responses, our results indicated thatY generally suppress the expression of IL2, IL4, IL6, IL-17, IFN-�nd TNF-� cytokines involved in stimulation of immune responsesn mitogen induced splenocytes.

The immunomodulatory effects of SY at the non-cytotoxic dose250 �g/ml) observed in the present investigation finds signifi-

ance in relation to the studies of McCann et al. (2007), whichndicates several food additives including SY cause hyperactiveehavior in 3-year-old and 8/9-year-old children. The present study

s of importance as several countries including Philippines (FAIRS,

lyzed by cytometric bead array kit with a flow cytometer for IL-2 (A), IL-4 (B), IL-6cant with respect to Con A treatment).

2009), permit SY in food items at the concentration of 300 �g/ml,thereby making the children susceptible to immunomodulation.Our recent studies showed that the intake of SY exceeded the ADIin Indian children by 88% (Dixit et al., 2011) which is far morecompared to European or American population, where the intakesaturates the ADI by <0.2–12% (Pentilla et al., 1988; Hunziker andZimmerli, 1984; Quattrucci and Saletti, 1983; IFT, 1986) therebyindicating the risk of immunomodulation in children and hencethere could be increased incidences of impaired resistance againstmicrobial pathogens (Klein et al., 2001; Sia and Paya, 1998) andvirus related malignancies (Vial and Descotes, 2003). Further, our

recent studies on Orange II, a non-permitted azo dye encountered inseveral food commodities, also showed immunomodulatory effectsat a 5 fold lower dose than SY (Yadav et al., 2012), implying thateither the parent molecule, azo dye, or their metabolite(s) are

A. Yadav et al. / Toxicology Lette

Fig. 7. Effect of sunset yellow FCF on cytokine responses in LPS stimulated Balb/csplenocytes in vitro. Splenocytes were stimulated with or without LPS (10 �g/ml) inpresence of sunset yellow FCF. The culture supernatants were collected after 48 ha�r

re

swtes

nd analyzed by cytometric bead array kit with a flow cytometer for IL-6 (A), IFN- (B) and TNF-� (C). Data represent mean ± SE of 5 sets (*p < 0.05, significant withespect to LPS treatment).

esponsible for immunomodulatory responses and needs furtherlucidation.

Conclusively, the present investigation suggests that SY expo-ure suppresses the functional properties of T-cells and B-cells

hich are further corroborated by simultaneous decline in

he cytokines production and altered relative percentage andxpression of receptors (CD3e, CD4, CD8 and CD19) in theplenocytes.

rs 217 (2013) 197– 204 203

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Funding

This work was supported by funds from CSIR-Supra InstitutionalProject-08.

Acknowledgements

We are grateful to the Director of our Institute for his keen inter-est in the study. AY is thankful to Council of Scientific and IndustrialResearch (CSIR)/University Grant Commission (UGC), New Delhifor the award of Senior Research Fellowship. This Manuscript isCSIR-IITR communication #3091.

References

Alarcon, B., Gil, D., Delgado, P., Schamel, W.W.A., 2003. Initiation of TCR signaling:regulation within CD3 dimers. Immunological Reviews 191, 38–46.

Barber, E.K., Dasgupta, J.D., Schlossman, S.F., Trevillyan, J.M., Rudd, C.E., 1989. TheCD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) thatphosphorylates the CD3 complex. Proceedings of the National Academy of Sci-ences of the United States of America 86 (9), 3277–3281.

Cote-Sierra, J., Foucras, G., Guo, L., Chiodetti, L., Young, H.A., Hu-Li, J., Zhu, J., Paul,W.E., 2004. Interleukin 2 plays a central role in Th2 differentiation. Proceedingsof the National Academy of Sciences of the United States of America 101,3880–3885.

Descotes, J., 2006. Methods of evaluating immunotoxicity. Expert Opinion on DrugMetabolism & Toxicology 2 (2), 249–259.

Dixit, S., Mishra, K.K., Khanna, S.K., Das, M., 2008. Benzoate and synthetic color riskassessment for fast food sauces served at street food joints of Lucknow, India.American Journal of Food Technology 3, 183–191.

Dixit, S., Khanna, S.K., Das, M., 2011. Usage pattern of synthetic food colours in differ-ent states of India and exposure assessment through commodities preferentiallyconsumed by children. Food Additives and Contaminants 28, 996–1005.

FAIRS, 2009. Food and Agricultural Import Regulations and Standards CountryReports. Food and Agricultural Import Regulations and Standards – Narra-tive, Philippines, Available from: http://gain.fas.usda.gov/Recent%20GAIN%20Publications/Food%20and%20Agricultural%20Import%20Regulations%20and%20Standards%20-%20Narrative Manila Philippines 7-17-2009.pdf

Feldman, M., Brennan, F.M., 2001. Cytokines and disease. In: Oppenheim, J.J., Feld-man, M., Durum, S.K. (Eds.), Cytokine References. Academic Press Inc., New York,pp. 35–51.

FSA, 2006. Food Standards Agency. Study on Illegal Dyes on ImportedFoods, Available from: http://www.foodstandards.gov.uk/multimedia/pdfs/illegaldyereport.pdf [cited 10.12.08] [Internet].

FSSR, 2011. Substances added to food. In: Handbook of Food Adulteration & SafetyLaws. Eastern Book Company, Lucknow, India, p. 348.

Gaunt, I.F., Farmer, M., Grasso, P., Gangolli, S.D., 1967. Acute (rat and mouse) andshort term (rat) toxicity studies on sunset yellow FCF. Food and Cosmetics Tox-icology 5, 747–754.

Gaunt, I.F., Mason, P.L., Grasso, P., Kiss, I.S., 1974. Long term toxicity of sunset yellowFCF in mice. Food and Cosmetics Toxicology 12, 1–10.

Gennari, A., Ban, M., Braun, A., Casati, S., Corsini, E., Dastych, J., Descotes, J., Hartung,T., Hooghe-Peters, R., House, R., Pallardy, M., Pieters, R., Reid, L., Tryphonas, H.,Tschirhart, E., Tuschl, H., Vandebriel, R., Gribaldo, L., 2005. The use of in vitrosystem for evaluating immunotoxicity: the report and recommendations of anECVAM workshop. Journal of Immunotoxicology 2, 61–83.

Hashem, M.M., Atta, A.H., Arbid, M.S., Nada, S.A., Asaad, G.F., 2010. Immunologicalstudies on Amaranth, Sunset Yellow and Curcumin as food colouring agent inalbino rats. Food and Chemical Toxicology 48, 1581–1586.

Haynes, L., Eaton, S.M., Burns, E.M., Rincon, M., Swain, S.L., 2004. Inflammatorycytokines overcome age-related defects in CD4 T cell responses in vivo. Journalof Immunology 172, 5194–5199.

Heller, R.A., Kronke, M., 1994. Tumor necrosis factor receptor-mediated signalingpathways. Journal of Cell Biology 126, 5–9.

Hunziker, H.R., Zimmerli, B., 1984. Intake estimation of food additives: red syntheticfood colors. Mittelungen aus dem Gebiete der Lebensmittelunterschung undHygiene 75, 77–92.

Husain, A., Sawaya, W., Al-omair, A., Al-zenki, S., Al-amiri, H., Ahmed, N., Al-sinan,M., 2006. Estimates of dietary exposure of children to artificial food colours inKuwait. Food Additives and Contaminants 23, 245–251.

Ibero, M., Eseverri, J.L., Barroso, C., Botey, J., 1982. Dyes, preservatives and salicy-lates in the induction of food intolerance and/or hypersensitivity in children.Allergologia et Immunopathologia 10, 263–268.

Institute of Food Technologists (IFT), 1986. Food Colors a Scientific Status Summary.Expert Panel on Food Safety and Nutrition, IFT, Chicago, IL.

2 y Lette

J

K

K

K

K

L

L

L

M

M

M

P

P

P

Q

04 A. Yadav et al. / Toxicolog

aneway Jr., C.A., 1992. The T cell receptor as a multicomponent signalling machine:CD4/CD8 coreceptors and CD45 in T cell activation. Annual Review of Immunol-ogy 10, 645–674.

im, H.P., Imbert, J., Leonard, W.J., 2006. Both integrated and differential regulationof components of the IL-2/IL-2 receptor system. Cytokine and Growth FactorReviews 17, 349–366.

ishimoto, T., 2010. IL-6: from its discovery to clinical applications. InternationalImmunology 22, 347–352.

lein, N.C., Go, C.H., Cunha, B.A., 2001. Infections associated with steroid use. Infec-tious Disease Clinics of North America 15, 423–432.

outsogeorgopoulou, L., Maravelias, C., Methenitou, G., Koutselinis, A., 1998.Immunological aspects of the common food colorants, amaranth and tartrazine.Veterinary and Human Toxicology 40, 1–4.

e Gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., Paul, W.E., 1990. Generation ofinterleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are requiredfor in vitro generation of IL-4-producing cells. Journal of Experimental Medicine172, 921–929.

iao, W., Lin, J., Wang, L., Li, P., Leonard, W.J., 2011. Cytokine receptor modulationby interleukin-2 broadly regulates T helper cell lineage differentiation. NatureImmunology 12 (June (6)), 551–559.

iu, Z., Yuan, X., Luo, Y., He, Y., Jiang, Y., Chen, Z.K., Sun, E., 2009. Evaluating the effectsof immunosuppressants on human immunity using cytokine profiles of wholeblood. Cytokine 45, 141–147.

cCann, D., Barrett, A., Cooper, A., Crumpler, D., Dalen, L., Grimshaw, K., Kitchin,E., Lok, K., Porteous, L., Prince, E., Sonuga-Barke, E., Warner, J.O., Stevenson, J.,2007. Food additives and hyperactive behaviour in 3-year-old and 8/9-year-oldchildren in the community: a randomised, double-blinded, placebo-controlledtrial. Lancet 370, 1560–1567.

ikkelsen, H., Larsen, J.C., Tarding, F., 1978. Hypersensitivity reactions to foodcolours with special reference to the natural colour annatto extract (buttercolour). Archives of Toxicology. Supplement 1, 141–143.

osmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. Journal of ImmunologicalMethods 65, 55–63.

entilla, P.-L., Salminen, S., Niemi, E., 1988. Estimates on the intake of food addi-tives in Finland. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung 186,11–15.

itcher, L.A., van Oers, N.S.C., 2003. T-cell receptor signal transmission: who givesan ITAM? Trends in Immunology 24, 554–560.

rice, P.J., Suk, W.A., Freeman, A.E., Lane, W.T., Peters, R.L., Verron, M.L.,Huebner, R.J., 1978. In vitro and in vivo indications of the carcinogeni-

city and toxicity of food dyes. International Journal of Cancer 21, 361–367.

uattrucci, E., Saletti, M.C., 1983. Determinazione analitica ed ingestion potenzialedi coloranti artificiali negli alimenti. Rivista della Societa Italiana di ScienzaDellAlimentazione 12, 28–36.

rs 217 (2013) 197– 204

Rao, P., Sudershan, R.V., 2008. Risk assessment of synthetic colors: a case studyin Hyderabad, India. International Journal of Food Safety, Nutrition and PublicHealth 1, 68–86.

Reynolds, J.M., Angkasekwinai, P., Dong, C., 2010. IL-17 family member cytokines:regulation and function in innate immunity. Cytokine and Growth FactorReviews 21 (6), 413–423.

Seder, R.A., Paul, W.E., 1994. Acquisition of lymphokine-producing phenotype byCD4+ T cells. Annual Review of Immunology 12, 635–673.

Shari, L.S., Joseph, F., Fowler Jr., 1995. Contact anaphylaxis: a review. AmericanJournal of Contact Dermatitis 6, 133–142.

Sia, I.G., Paya, C.V., 1998. Infectious complications following renal transplantation.Surgical Clinics of North America 78, 95–112.

Smeltz, R.B., Chen, J., Ehrhardt, R., Shevach, E.M., 2002. Role of IFN-� in Th1 dif-ferentiation: IFN-� regulates IL-18R� expression by preventing the negativeeffects of IL-4 and by inducing/maintaining IL-12 receptor 2 expression. Journalof Immunology 168, 6165–6172.

Sykulev, Y., 2010. T cell receptor signaling kinetics takes the stage. Science Signalling3, pe50.

Tanaka, T., 1996. Reproductive and neurobehavioral effects of sunset yellow FCFadministered to mice in the diet. Toxicology and Industrial Health 12 (1), 69–79.

Tedder, T.F., Inaoki, M., Sato, S., 1997. The CD19–CD21 complex regulates sig-nal transduction thresholds governing humoral immunity and autoimmunity.Immunity 6, 107–118.

Thune, P., Granholt, A., 1975. Provocation tests with antiphlogistica and food addi-tives in recurrent urticaria. Dermatologica 151, 360–367.

Tripathi, M., Dixit, S., Khanna, S.K., Das, M., 2010. Intake pattern of synthetic coloursby different age and socio-economic consumer groups of Lucknow, India. Inter-national Journal of Food Safety, Nutrition and Public Health 3, 19–31.

Tripathi, M., Khanna, S.K., Das, M., 2007. Surveillance on use of synthetic colours ineatables vis a vis Prevention of Food Adulteration Act of India. Food Control 18,211–219.

Van de Veerdonk, F.L., Gresnigt, M.S., Kullberg, B.J., van der Meer, J.W., Joosten, L.A.,Netea, M.G., 2009. Th17 responses and host defense against microorganisms: anoverview. BMB Reports 42 (12), 776–787.

Vial, T., Descotes, J., 2003. Immunosuppressive drugs and cancer. Toxicology 185,229–240.

Weber, R.W., Hoffman, M., Raine Jr., D.A., Nelson, H.S., 1979. Incidence of bron-choconstriction due to aspirin, azo dyes, non-azo dyes, and preservatives in apopulation of perennial asthmatics. Journal of Allergy and Clinical Immunology64, 32–37.

Yadav, A., Kumar, A., Dwivedi, P.D., Tripathi, A., Das, M., 2012. In vitro studies on

immunotoxic potential of Orange II in splenocytes. Toxicology Letters 208 (3),239–245.

Yoshimoto, M., Yamaguchi, M., Hatano, S., Watanabe, T., 1984. Configurationalchanges in Rat liver nuclear chromatin caused by azo dyes. Food and ChemicalToxicology 22 (5), 337–344.