Vol 59, No 4 (2021)
Original paper
Published online: 2021-12-09

open access

Page views 6533
Article views/downloads 596
Get Citation

Connect on Social Media

Connect on Social Media

Effect of e-cigarette flavoring agents on the neural retina of chick embryo: histological and gene expression study

Malak Alshareef1, Aziza Alrafiah2, Sara Abed1, Fatemah Basingab1, Aisha Alrofaidi1
Pubmed: 34897642
Folia Histochem Cytobiol 2021;59(4):245-258.


Background: An electronic cigarette (e-cigarette) is initially marketed as an assistant product to quit smoking or limit its use. However, recent studies suggest the opposite, describing it as a product that lacks adequate quality and user safety. The present study aimed to investigate the effect of e-cigarette flavoring agent (cinnamon flavor) on the neural retina development of chick embryos and apoptosis induction after the early and late apoptosis stages by quantitative detection of gene expression CASP-3 at both embryonic days E9 and E17.

Methods: Fertilized chicken eggs were divided into two groups: control and treatment, and each group included two embryonic days; E9 and E17. For each treatment stage, two dosages of the treatment were applied, 2% and 5%. The neural retinas were dissected from the sclera and retinal pigment epithelium for subsequent RNA extraction and histological examination.

Results: This study indicated that aerosol of the subtle cinnamon flavor e-liquid causes downregulated expression of CASP3 in neural retina development. In addition, the hematoxylin and eosin (H&E)-stained sections showed multiple structural changes in the retinal layers and evidence of apoptotic cell death.

Conclusion: Cell death was visible and abundant in E9, and E17 concludes that flavor vapor condensate treatment caused neuronal cell death. CASP-3 was downregulated, which indicates that cell death occurred independently of CASP-3.

Article available in PDF format

View PDF Download PDF file


  1. Nguyen T, Electronic Cigarettes: Neurological Effects on Murine Offspring and the Response of Neuronal Cells. 2020.
  2. Dutra LM, Grana R, Glantz SA. Philip Morris research on precursors to the modern e-cigarette since 1990. Tob Control. 2017; 26(e2): e97–e9e105.
  3. Leigh NJ, Lawton RI, Hershberger PA, et al. Flavourings significantly affect inhalation toxicity of aerosol generated from electronic nicotine delivery systems (ENDS). Tob Control. 2016; 25(Suppl 2): ii81–ii87.
  4. Barrington-Trimis JL, Samet JM, McConnell R. Flavorings in electronic cigarettes: an unrecognized respiratory health hazard? JAMA. 2014; 312(23): 2493–2494.
  5. Kaur G, Muthumalage T, Rahman I. Mechanisms of toxicity and biomarkers of flavoring and flavor enhancing chemicals in emerging tobacco and non-tobacco products. Toxicol Lett. 2018; 288: 143–155.
  6. Krüsemann EJZ, Boesveldt S, de Graaf K, et al. An E-Liquid Flavor Wheel: A Shared Vocabulary Based on Systematically Reviewing E-Liquid Flavor Classifications in Literature. Nicotine Tob Res. 2019; 21(10): 1310–1319.
  7. Clapp PW, Pawlak EA, Lackey JT, et al. Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function. Am J Physiol Lung Cell Mol Physiol. 2017; 313(2): L278–L292.
  8. Merecz-Sadowska A, Sitarek P, Zielinska-Blizniewska H, et al. A Summary of In Vitro and In Vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment. Int J Mol Sci. 2020; 21(2).
  9. Theron AJ, Feldman C, Richards GA, et al. Electronic cigarettes: where to from here? J Thorac Dis. 2019; 11(12): 5572–5585.
  10. Bitzer ZT, Goel R, Reilly SM, et al. Effect of flavoring chemicals on free radical formation in electronic cigarette aerosols. Free Radic Biol Med. 2018; 120: 72–79.
  11. Sussan TE, Gajghate S, Thimmulappa RK, et al. Hasan, Exposure to electronic cigarettes impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model, PLoS One. 2015; 10: e0116861.
  12. Scheffler S, Dieken H, Krischenowski O, et al. Evaluation of E-cigarette liquid vapor and mainstream cigarette smoke after direct exposure of primary human bronchial epithelial cells. Int J Environ Res Public Health. 2015; 12(4): 3915–3925.
  13. D’amelio M, Cavallucci V, Cecconi F. Neuronal caspase-3 signaling: not only cell death, Cell Death Differ. 2010; 17(7): 1104–1114.
  14. Allen JG, Flanigan SS, LeBlanc M, et al. Flavoring Chemicals in E-Cigarettes: Diacetyl, 2,3-Pentanedione, and Acetoin in a Sample of 51 Products, Including Fruit-, Candy-, and Cocktail-Flavored E-Cigarettes. Environ Health Perspect. 2016; 124(6): 733–739.
  15. Faden A, Yakovlev A. Caspase-Dependent Apoptotic Pathways in CNS Injury. Mol Neurobiol. 2001; 24(1-3): 131–144.
  16. Carnevali S, Petruzzelli S, Longoni B, et al. Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol. 2003; 284(6): L955–L963.
  17. Goel R, Durand E, Trushin N, et al. Highly reactive free radicals in electronic cigarette aerosols. Chem Res Toxicol. 2015; 28(9): 1675–1677.
  18. Trachsel-Moncho L, Benlloch-Navarro S, Fernández-Carbonell Á, et al. Oxidative stress and autophagy-related changes during retinal degeneration and development. Cell Death & Disease. 2018; 9(8).
  19. Wisely CE, Sayed JA, Tamez H, et al. The chick eye in vision research: An excellent model for the study of ocular disease. Prog Retin Eye Res. 2017; 61: 72–97.
  20. Vecino E, Hernández M, García M. Cell death in the developing vertebrate retina. Int J Dev Biol. 2004; 48(8-9): 965–974.
  21. Olmedo P, Navas-Acien A, Hess C, et al. A direct method for e-cigarette aerosol sample collection. Environ Res. 2016; 149: 151–156.
  22. J.D. Bancroft, M. Gamble, Theory and practice of histological techniques, 7th ed, Elsevier health sciences, USA: Churchill Livingston. ; 2008.
  23. Khanagar SB, Siddeeqh S, Khinda V, et al. Impact of electronic cigarette smoking on the Saudi population through the analysis of literature: A systematic review. J Oral Maxillofac Pathol. 2019; 23(3): 473.
  24. Behar RZ, Luo W, Lin SC, et al. Distribution, quantification and toxicity of cinnamaldehyde in electronic cigarette refill fluids and aerosols. Tob Control. 2016; 25(Suppl 2): ii94–ii9ii102.
  25. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27(2-3): 126–139.
  26. Tommasi S, Bates S, Behar R, et al. Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro. Lung Cancer. 2017; 112: 41–46.
  27. Doonan F, Groeger G, Cotter TG. Preventing retinal apoptosis--is there a common therapeutic theme? Exp Cell Res. 2012; 318(11): 1278–1284.
  28. Méndez-Armenta M, Nava-Ruíz C, Juárez-Rebollar D, et al. Oxidative stress associated with neuronal apoptosis in experimental models of epilepsy. Oxid Med Cell Longev. 2014; 2014: 293689.
  29. Y.-C. Chuang, Mitochondrial dysfunction and oxidative stress in seizure-induced neuronal cell death, Acta Neurol Taiwan. 2010; 19(1): 3–15.
  30. Lee WH, Ong SG, Zhou Y, et al. Modeling Cardiovascular Risks of E-Cigarettes With Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells. J Am Coll Cardiol. 2019; 73(21): 2722–2737.
  31. Behar RZ, Davis B, Wang Y, et al. Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids. Toxicol In Vitro. 2014; 28(2): 198–208.
  32. C.A. Lerner, I.K. Sundar, H. Yao, J. Gerloff, D.J. Ossip, S. McIntosh, R. Robinson, I. Rahman, Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung, PLoS One. 2015; 10: e0116732.
  33. Cadet J, Wagner JR. DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation. Cold Spring Harb Perspect Biol. 2013; 5(2).
  34. Mangal D, Vudathala D, Park JH, et al. Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress. Chem Res Toxicol. 2009; 22(5): 788–797.
  35. Muthumalage T, Prinz M, Ansah KO, et al. Inflammatory and Oxidative Responses Induced by Exposure to Commonly Used e-Cigarette Flavoring Chemicals and Flavored e-Liquids without Nicotine. Front Physiol. 2017; 8: 1130.
  36. Clapp PW, Lavrich KS, van Heusden CA, et al. Cinnamaldehyde in flavored e-cigarette liquids temporarily suppresses bronchial epithelial cell ciliary motility by dysregulation of mitochondrial function. Am J Physiol Lung Cell Mol Physiol. 2019; 316(3): L470–L486.
  37. Chen S-D, Yang D-I, Lin T-K, et al. Chuang, Roles of oxidative stress, apoptosis, PGC-1α and mitochondrial biogenesis in cerebral ischemia, Int. J Mol Sci. 2011; 12(10): 7199–7215.
  38. Anderson C, Majeste A, Hanus J, et al. E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells. Toxicol Sci. 2016; 154(2): 332–340.
  39. Zhao J, Zhang Y, Sisler JD, et al. Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches. J Hazard Mater. 2018; 344: 549–557.