Antioxidant role of melatonin against nicotine’s teratogenic effects on embryonic bone development

Document Type : Original Article


1 Department of Therapy and Rehabilitation, Kozakli Vocational School, Nevsehir Haci Bektas Veli University, Nevsehir, Turkey

2 Department of Anatomy, Faculty of Medicine, Afyonkarahisar Health Sciences University, Afyonkarahisar, Turkey

3 Department of Anatomy, Faculty of Medicine, Erciyes University, Kayseri, Turkey

4 Department of Anatomy, Faculty of Medicine, Bozok University, Yozgat, Turkey

5 Department of Therapy and Rehabilitation, Sungurlu Vocational School, Hitit University, Corum, Turkey


Objective(s): This study investigated the possible effects of low (3 mg/kg) and high (6 mg/kg) doses of nicotine on the skeletal development of rat fetuses by the double staining method and the protective role of melatonin (10 mg/kg) against these effects.
Materials and Methods: Eighteen adult female Wistar-Albino rats were divided into six groups (n=3, each) as control, low-dose nicotine, high-dose nicotine, low-dose nicotine+melatonin, high-dose nicotine + melatonin and melatonin. While nicotine was given to the experimental groups on gestation days 1–20, nicotine and melatonin were administered together to the treatment groups. The fetuses were delivered by cesarean section on the 20th day of pregnancy. The skeletal systems of the fetuses were stained using the double staining method. The forelimbs and hindlimbs of the fetuses were firstly investigated under a stereomicroscope, and then their photos were taken. The total bone length, the length of the ossified part and the ossification rate were calculated using the ImageJ program.
Results: The degree of ossification in the bones of the feet and the hands was determined. When the total bone length and the length of the ossified part were evaluated, they were significantly decreased in the nicotine groups (P<0.05), but were close to each other in the treatment and the control groups (P<0.05).
Conclusion: It has been found that the use of nicotine during pregnancy delays skeletal ossification and that melatonin, a powerful antioxidant, eliminates the teratogenic effects of nicotine.


Main Subjects

1. Abd El-Aziz GS, El-Fark MM, Saleh HA. The prenatal toxic effect of methylmercury on the development of the appendicular skeleton of rat fetuses and the protective role of vitamin E. Anat Rec 2012; 295:939-949.
2. Junqueira LC, Carneiro J. Temel Histoloji. Aytekin Y and Solakoğlu S (eds). İstanbul: Nobel Medical Bookstores, 2006, pp. 158.
3. Soysal H, Unur E, Düzler A, Karaca Ö, Ekinci N. Effects of intraperitoneal administration of the phenytoin on the skeletal system of rat fetus. Seizure 2011; 20:187-193.
4. Yilmaz S, Ozden B, Bas B, Altun G, Altunkaynak BZ. Could calcified triglyceride bone cement be an alternative graft material in maxillary sinus augmentation?. J Craniofac Surg 2017; 28:97-103.
5. Khaksary Mahabady M, Najafzadeh Varzi H, Ranjbar R, Mehrzadi S. Melatonin and vitamin E protects against sodium arsenite-induced skeletal malformations in rats. AEJTS 2011; 3:184-189.
6. Khaksary Mahabady M, Najafzadeh Varzi H, Zareyan Jahromi S. L-Carnitine protect against cyclophosphamide induced skeletal and neural tube malformations in rat fetuses. Acta Med Iran 2015; 53:703-710.
7. Uçar I, Ertekin T, Nisari M, Ceylan D, Al Ö, Ülger H. The potential teratogenic effects of interferon beta-1a and interferon beta-1b on in vitro embryonic development. Folia Morphol 2016; 75:257-263.
8. Benowitz NL, Hukkanen J, Jacob P. Nicotine chemistry, metabolism, kinetics and biomarkers. Handbi Exp Pharmacol 2009; 192:29-60.
9. Altun I, Yuksel KZ. An experimental study on the effects of smoking in the perinatal period and during lactation on the intervertebral disks of newborns. World Neurosurg 2017; 99:1-5.
10. Mitchell JA, Hammer RE. Effects of nicotine on oviducal blood flow and embryo development in the rat. J Reprod Fertil 1985; 74:71-76.
11. Mızrak S, Turan V, Caglayan O, Ercan G. The effect of long term pre/postnatal low/high dose nicotine exposure on tissue oxidant/antioxidant status and dna damage in rats. Drug Res 2015; 65:432-436.
12. Carmines EL, Gaworski CL, Faqi AS, Rajendran N. In utero exposure to 1R4F reference cigarette smoke: evaluation of developmental toxicity. Toxicol Sci 2003; 75:134-147.
13. Rocha MIPNM. Nicotine effects on bone metabolism: in vitro studies with human osteoclasts and co-cultures of osteoclasts and osteoblasts in an hydroxyapatite surface. Master Thesis, Faculdade de Engenharia da Universidade do Porto, Portugal, 2011, pp. 27-29.
14. Fang Y, Svoboda KK. Nicotine inhibits human gingival fibroblast migration via modulation of Rac signalling pathways. J Clin Periodontol 2005; 32:1200–1207.
15. Abate M, Vanni D, Pantalone A, Salini V. Cigarette smoking and musculoskeletal disorders. Muscles Ligaments Tendons J 2013; 3:63-69.
16. Behnke M, Smith VC. Prenatal substance abuse: short- and long-term effects on the exposed fetus. Pediatrics 2013; 131:1009-1024.
17. Xiao D, Wang L, Huang X, Li Y, Dasgupta C, Zhang L. Protective effect of antenatal antioxidant on nicotine-induced heart ischemia-sensitive phenotype in rat offspring.  PLoS One 2016; 11:e0150557.
18. Dubocovich ML, Delagrange P, Krause DN, Sugden D, Cardinali DP, Olcese J. International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, classification, and pharmacology of G protein-coupled melatonin receptors. Pharmacol Rev 2010; 62:343-380.
19. Dollins AB, Zhdanova IV, Wurtman RJ, Lynch HJ, Deng MH. Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature and performance. Proc Natl Acad Sci U S A 1994; 91:1824-1828.
20. Tan DX, Chen LD, Poeggeler B, LC Manchester,  RJ Reiter. Melatonin: a potent endogenous hydroxyl radical scavenger. Endocrine Journal 1993; 1:57-60.
21. Nesil T, Kanit L, Ugur M, Pogun S. Nicotine withdrawal in selectively bred high and low nicotine preferring rat lines. Pharmacol Biochem Behav 2015; 131:91-97.
22. El-Sokkary GH, Cuzzocrea S, Reiter RJ. Effect of chronic nicotine administration on the rat lung and liver: beneficial role of melatonin. Toxicology 2007; 239:60-67.
23. Akpak YK, Çekmez Y, Erdoğan ÇA, Karaca N, Batmaz G, Gülşen S, et al. An animal model of effects of nicotine exposure on endometrial receptivity and embryo implantation in pregnancy. J Matern Fetal Neonatal Med 2016; 20:1-6.
24. Tizabi Y, Russell LT, Nespor SM, Perry DC, Grunberg NE. Prenatal nicotine exposure: effects on locomotor activity and central [125I] alpha-BT binding in rats. Pharmacol Biochem Behav 2000; 66:495-500.
25. Kurtoglu S, Gunes T, Koklu E, Bastug O, Canoz O, Kula M, et al. Influence of maternal nicotine exposure on neonatal rat bone: protective effect of pentoxifylline. Exp Biol Med 2007; 232:398-405.
26. Gunes T, Akin MA, Canoz O, Coban D, Ozcan B, Kose M, et al. Aortic intima-media thickness in nicotine-exposed rat pups during gestation and lactation period. Eur J Pediatr 2011; 170:1257-62.
27. Gunes T, Koklu E, Gunes I, Narin F, Koklu S. Influence of maternal nicotine exposure on neonatal rat oxidant-antioxidant system and effect of ascorbic acid supplementation. Hum Exp Toxicol 2008; 27:781-786.
28. Baykan A, Narin N, Narin F, Akgün H, Yavaşcan S, Saraymen R. The protective effect of melatonin on nicotine-induced myocardial injury in newborn rats whose mothers received nicotine. Anadolu Kardiyol Derg 2008; 8:243-248.
29. Koklu E, Gunes T, Gunes I, Canoz O, Kurtoglu S, Duygulu F, et al. Influence of maternal nicotine exposure on neonatal rat bone: protective effect of ascorbic acid. Am J Perinatol 2006; 23:387-395.
30. Chen T, Yan YE, Liu S, Liu HX, Yan HY, Hou LF, et al. Increased fetal thymocytes apoptosis contributes to prenatal nicotine exposure-induced Th1/Th2 imbalance in male offspring mice. Sci Rep 2016; 6:39013.
31. Wang H, Gondré-Lewis MC. Prenatal nicotine and maternal deprivation stress deregulate the development of CA1, CA3 and dentate gyrus neurons in hippocampus of infant rats. PLoS One 2013; 8:e65517.
32. Braun JM, Daniels JL, Poole C, Olshan AF, Hornung R, Bernert JT, et al. A prospective cohort study of biomarkers of prenatal tobacco smoke exposure: the correlation between serum and meconium and their association with infant birth weight. Environ Health 2010; 9:53
33. Sunay BF. İkili iskelet boyamaları. Uludağ Üniversitesi Tıp Fakültesi Dergisi 2005; 31:119-126.
34. Sunay FB, Ersoy S, Kahveci Z. İkili iskelet boyamasında mikrodalga ışınının kullanılması. Uludağ University Medical Journal 2009; 35:113-118.
35. Khaksary Mahabady M, Gholami MR, Najafzadeh Varzi H, Zendedel A, Doostizadeh M. Protective effect of quercetin on skeletal and neural tube teratogenicity induced by cyclophosphamide in rat fetuses. Vet Res Forum 2016; 7:133-138.
36. Öztürk M. Sıçan ön ekstremitesine ait kemiklerin gelişimi esnasında ortaya çıkan kemik ve kıkırdak alanların ikili boyama yöntemi ile tespiti ve stereolojik yöntemle oranlarının hesaplanması. PhD thesis, University of Erciyes, Turkey, 2014.
37. Xiao D, Huang X, Yang S, Zhang L. Antenatal nicotine induces heightened oxidative stress and vascular dysfunction in rat offspring. Br J Pharmacol 2011; 164:1400-1409.
38. Xiao D, Huang X, Li Y, Dasgupta C, Wang L, Zhang L. Antenatal antioxidant prevents nicotine-mediated hypertensive response in rat adult offspring. Biol Reprod 2015; 93:66.
39. Akkoyun HT, Karadeniz A. Investigation of the protective effect of ellagic acid for preventing kidney injury in rats exposed to nicotine during the fetal period. Biotech Histochem 2016; 91:108-115.
40. Voiculescu SE, Zygouropoulos N, Zahiu CD, Zagrean AM. Role of melatonin in embryo fetal development. J Med Life 2014; 7:488–492.
41. Australian Government Therapeutic Goods Administration. Australian public assessment report for melatonin. AusPAR Circadin Melatonin Commercial Eyes Pty Ltd PM 2008; 2125:1-57.
42. Sener G, Kapucu C, Paskaloglu K, Ayanoglu-Dülger G, Arbak S, Ersoy Y, et al. Melatonin reverses urinary system and aorta damage in the rat due to chronic nicotine administration. J Pharm Pharmacol 2004; 56:359-366.
43. Baykan H, Ozyazgan I, Selçuk CT, Altiparmak M, Ozköse M, Ozyurt K. Effect of sildenafil citrate in nicotine-induced ischemia: An experimental study using a rat model. Can J Plast Surg 2013; 21:217-220.