Influence of the long-term exposure to tartrazine and chlorophyll on the fibrogenic signalling pathway in liver and kidney of rats: the expression patterns of collagen 1-α, TGFβ-1, fibronectin, and caspase-3 genes

  • Yasmina M. Abd-ElhakimEmail author
  • Gihan G. Moustafa
  • Mohamed M. Hashem
  • Haytham A. Ali
  • Khaled Abo-EL-Sooud
  • Abeer E. El-Metwally
Research Article


Colouring agents are highly present in diverse products in the human environment. We aimed to elucidate the fibrogenic cascade triggered by the food dyes tartrazine and chlorophyll. Rats were orally given distilled water, tenfold of the acceptable daily intake of tartrazine, or chlorophyll for 90 consecutive days. Tartrazine-treated rats displayed a significant rise (p < 0.05) in the mRNA levels and immunohistochemical localization of the renal and hepatic fibrotic markers collagen 1-α, TGFβ-1, and fibronectin and the apoptotic marker caspase-3. Moreover, a significant increment (p < 0.05) in the levels of AST, ALP, creatinine, and urea was evident in both experimental groups but more significant differences were noticed in the tartrazine group. Furthermore, we found a marked increment in the MDA level and significant declines (p < 0.05) in the levels of the SOD, CAT, and GSH enzymes in the kidney and liver from tartrazine-treated rats. The histological investigation reinforced the aforementioned data, revealing hepatocytes with fibrous connective tissue proliferation, apoptotic hepatocytes and periportal fibrosis with tubular necrosis, and shrunken glomeruli and interstitial fibrous tissue proliferation. We concluded that, even at the exposure to high concentrations for long durations, chlorophyll exhibited a lower propensity to induce fibrosis, apoptosis, and histopathological perturbations than tartrazine.


Tartrazine Chlorophyll Collagen 1-α TGFβ-1 Fibronectin Caspase-3 


Funding information

This work was financially supported by Cairo University in a project entitled “Assessment of Residual Patterns and Genotoxicity of Some Food Additives using LMRS (laser molecular Raman spectroscopy)”.

Compliance with ethical standards

The experimental procedures were done in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals in scientific investigations (NRC 2011) and were accepted by the Ethics of Animal Use in Research Committee of Cairo University, Egypt.

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Abd-Elhakim YM, Hashem MM, Anwar A, el-Metwally AE, Abo-el-Sooud K, Moustafa GG, Mouneir SM, Ali HA (2018a) Effects of the food additives sodium acid pyrophosphate, sodium acetate, and citric acid on hemato-immunological pathological biomarkers in rats: relation to PPAR-α, PPAR-γ and tnfα signaling pathway. Environ Toxicol Pharmacol 62:98–106. CrossRefGoogle Scholar
  2. Abd-Elhakim YM, Hashem MM, El-Metwally AE, Anwar A, Abo-El-Sooud K, Moustafa GG, Ali HA (2018b) Comparative haemato-immunotoxic impacts of long-term exposure to tartrazine and chlorophyll in rats. Int Immunopharmacol 63:145–154. CrossRefGoogle Scholar
  3. Abo-EL-Sooud K, Hashem MM, Badr YA, Eleiwa MME, Gab-Allaha AQ, Abd-Elhakim YM, Bahy-EL-Dien A (2018) Assessment of hepato-renal damage and genotoxicity induced by long-term exposure to five permitted food additives in rats. Environ Sci Pollut Res 25:26341–26350.
  4. Aboel-Zahab H, El-Khyat Z, Sidhom G, Awadallah R, Abdel-Al W, Mahdy K (1997) Physiological effects of some synthetic food colouring additives on rats. Boll Chim Farm 136:615–627Google Scholar
  5. Amin K, Hameid IIHA, Elsttar AA (2010a) Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food Chem Toxicol 48:2994–2999CrossRefGoogle Scholar
  6. Amin KA, Abdel Hameid H, Abd Elsttar AH (2010b) Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food Chem Toxicol 48:2994–2999. CrossRefGoogle Scholar
  7. Barham D, Trinder P (1972) Enzymatic determination of uric acid. Analyst 97:142–145CrossRefGoogle Scholar
  8. Beutler E (1969) Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. J Clin Invest 48:1957–1966CrossRefGoogle Scholar
  9. Bhatt D, Vyas K, Singh S, John PJ, Soni I (2018) Tartrazine induced neurobiochemical alterations in rat brain sub-regions. Food Chem Toxicol 113:322–327. CrossRefGoogle Scholar
  10. Chung K-T, Stevens SE, Cerniglia CE (1992) The reduction of azo dyes by the intestinal microflora. Crit Rev Microbiol 18:175–190CrossRefGoogle Scholar
  11. Coulombe J, Favreau L (1963) A new simple semimicro method for colorimetric determination of urea. Clin Chem 9:102–108Google Scholar
  12. Dhandayuthapani S, Marimuthu P, Hörmann V, Kumi-Diaka J, Rathinavelu A (2013) Induction of apoptosis in HeLa cells via caspase activation by resveratrol and genistein. J Med Food 16:139–146CrossRefGoogle Scholar
  13. El-Wahab HMFA, Moram GSE-D (2013) Toxic effects of some synthetic food colorants and/or flavor additives on male rats. Toxicol Ind Health 29:224–232CrossRefGoogle Scholar
  14. Escobar J, Rubio M, Lissi E (1996) SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic Biol Med 20:285–290CrossRefGoogle Scholar
  15. Farrington C, Novak D, Liu C, Haafiz AB (2010) Immunohistochemical localization of transforming growth factor β-1 and its relationship with collagen expression in advanced liver fibrosis due to biliary atresia. Clin Exp Gastroenterol 3:185Google Scholar
  16. FDA (2002) Listing of color additives exempt from certification; sodium copper chlorophyllin. Fed Regist 67(97)Google Scholar
  17. Hashem MM, Abd-Elhakim YM, Abo-EL-Sooud K, Eleiwa MME (2019) Embryotoxic and teratogenic effects of tartrazine in rats. Toxicol Res 35:75–81CrossRefGoogle Scholar
  18. Inoue K, Yoshida M, Takahashi M, Shibutani M, Takagi H, Hirose M, Nishikawa A (2009) Induction of kidney and liver cancers by the natural food additive madder color in a two-year rat carcinogenicity study. Food Chem Toxicol 47:184–191CrossRefGoogle Scholar
  19. Issa N, El-Sherif N (2015) Histological and immunohistochemical study on the toxic effects of Anthracene on the lung and liver of adult male albino rats and the possible protective role of Ocimum gratissimum extract. J Cell Biol Histol 1:103Google Scholar
  20. JECFA (1996) Joint FAO/WHO Expert Committee on Food Additives [JECFA] 1996. Summary of evaluations performed by the joint FAO/WHO expert committee on food additives (JECFA) 1956–1995 (first through 44th meetings). International Life Sciences Institute (ILSI) Press, WHO, Washington, DC, p T-3Google Scholar
  21. Kojima S, Nara K, Rifkin DB (1993) Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells. J Cell Biol 121:439–448CrossRefGoogle Scholar
  22. Lausen K (1972) Creatinine assay in the presence of protein with LKB 8600 Reaction Rate Analyser. Clin Chim Acta 38:475–476CrossRefGoogle Scholar
  23. Mak KM, Chen LL, Lee TF (2013) Codistribution of collagen type IV and laminin in liver fibrosis of elderly cadavers: immunohistochemical marker of perisinusoidal basement membrane formation. Anat Rec 296:953–964CrossRefGoogle Scholar
  24. Mehedi N, Ainad-Tabet S, Mokrane N, Addou S, Zaoui C, Kheroua O, Saidi D (2009) Reproductive toxicology of tartrazine (FD and C Yellow No. 5) in Swiss albino mice. Am J Pharmacol Toxicol 4:130–135CrossRefGoogle Scholar
  25. Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175Google Scholar
  26. Mohammadzadeh-Aghdash H, Sohrabi Y, Mohammadi A, Shanehbandi D, Dehghan P, Dolatabadi JEN (2018) Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives. Food Chem 257:211–215CrossRefGoogle Scholar
  27. Moutaouakkil A, Zeroual Y, Dzayri FZ, Talbi M, Lee K, Blaghen M (2003) Purification and partial characterization of azoreductase from Enterobacter agglomerans. Arch Biochem Biophys 413:139–146CrossRefGoogle Scholar
  28. Mpountoukas P, Pantazaki A, Kostareli E, Christodoulou P, Kareli D, Poliliou S, Mourelatos C, Lambropoulou V, Lialiaris T (2010) Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine. Food Chem Toxicol 48:2934–2944CrossRefGoogle Scholar
  29. Mustafa HN, El Awdan SA, Hegazy GA, Jaleel GAA (2015) Prophylactic role of coenzyme Q10 and Cynara scolymus L on doxorubicin-induced toxicity in rats: biochemical and immunohistochemical study. Indian J Pharmacol 47:649CrossRefGoogle Scholar
  30. Nair V, Turner GA (1984) The thiobarbituric acid test for lipid peroxidation: structure of the adduct with malondialdehyde. Lipids 19:804–805CrossRefGoogle Scholar
  31. NRC (2011) National Research Council Committee for the Update of the Guide for the, Care Use of Laboratory, Animals. The National Academies Collection: reports funded by National Institutes of Health. In: th (ed) Guide for the Care and Use of Laboratory Animals. National Academies Press (US) National Academy of Sciences., Washington (DC).
  32. Ozaki A, Kitano M, Furusawa N, Yamaguchi H, Kuroda K, Endo G (2002) Genotoxicity of gardenia yellow and its components. Food Chem Toxicol 40:1603–1610CrossRefGoogle Scholar
  33. Paget GE, Barnes GM (1964) Evaluation of drug activities, vol 1. Academic Press, LondonGoogle Scholar
  34. Poul M, Jarry G, Elhkim MO, Poul J-M (2009) Lack of genotoxic effect of food dyes amaranth, sunset yellow and tartrazine and their metabolites in the gut micronucleus assay in mice. Food Chem Toxicol 47:443–448CrossRefGoogle Scholar
  35. Rafati A, Nourzei N, Karbalay-Doust S, Noorafshan A (2017) Using vitamin E to prevent the impairment in behavioral test, cell loss and dendrite changes in medial prefrontal cortex induced by tartrazine in rats. Acta Histochem 119:172–180. CrossRefGoogle Scholar
  36. Raposa B et al (2016) Food additives: sodium benzoate, potassium sorbate, azorubine, and tartrazine modify the expression of NFκB, GADD45α, and MAPK8 genes. Physiol Int(Acta Physiologica Hungarica) 103:334–343Google Scholar
  37. Reitman S, Frankel S (1957) A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28:56–63CrossRefGoogle Scholar
  38. Saxena B, Sharma S (2015) Food color induced hepatotoxicity in Swiss albino rats, Rattus norvegicus. Toxicol Int 22:152CrossRefGoogle Scholar
  39. Sherif IO, Al-Gayyar MM (2013) Antioxidant, anti-inflammatory and hepatoprotective effects of silymarin on hepatic dysfunction induced by sodium nitrite. Eur Cytokine Netw 24:114–121Google Scholar
  40. Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47:389–394CrossRefGoogle Scholar
  41. Suvarna KS, Layton C, Bancroft JD (2012) Bancroft’s theory and practice of histological techniques E-book. Elsevier Health SciencesGoogle Scholar
  42. Tietz N et al (1983) A reference method for measurement of alkaline phosphatase activity in human serum. Clin Chem 29:751–761Google Scholar
  43. Toyoda T, Cho Y-M, Mizuta Y, Akagi J-i, Nishikawa A, Ogawa K (2014) A 13-week subchronic toxicity study of sodium iron chlorophyllin in F344 rats. J Toxicol Sci 39:109–119CrossRefGoogle Scholar
  44. Van der Oost R, Beyer J, Vermeulen NP (2003) Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol 13:57–149CrossRefGoogle Scholar
  45. Yilmaz S, Atessahin A, Sahna E, Karahan I, Ozer S (2006) Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology 218:164–171CrossRefGoogle Scholar
  46. Zhang Z-F, Fan S-H, Zheng Y-L, Lu J, Wu D-M, Shan Q, Hu B (2009) Purple sweet potato color attenuates oxidative stress and inflammatory response induced by d-galactose in mouse liver. Food Chem Toxicol 47:496–501CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Forensic Medicine and Toxicology, Faculty of Veterinary MedicineZagazig UniversityZagazigEgypt
  2. 2.Department of Pharmacology, Faculty of Veterinary MedicineCairo UniversityGizaEgypt
  3. 3.Department of Biochemistry, Faculty of Veterinary MedicineZagazig UniversityZagazigEgypt
  4. 4.Department of Biochemistry, Faculty of ScienceJeddah UniversityJeddahSaudi Arabia
  5. 5.Pathology DepartmentAnimal Reproduction Research InstituteGizaEgypt

Personalised recommendations