Investigation of the Effects of Pre-Incubation Formaldehyde Exposure on DNA Methyltransferase (DNMT) mRNA Expression in Muscle and Liver Tissues of Chicken Embryos

Öz

The DNA methyltransferase (DNMT) family is a group of enzymes that play an important role in epigenetic gene control and regulate the DNA methylation process. While DNMT3A and DNMT3B are responsible for establishing new methylation patterns, DNMT1 maintains existing ones. These enzymes play an active role during embryonic development, and their expression levels are markedly reduced in differentiated cells. This study aimed to determine the effects of pre-incubation formaldehyde (FA) treatment on the expression levels of DNA methyltransferase (DNMT) genes in liver and muscle tissues of chicken embryos. Fertilized chicken eggs were disinfected with FA, and liver and muscle tissues were collected from embryos on day 18 of incubation. DNMT1, DNMT3A, and DNMT3B gene expression levels were assessed using the RT-qPCR method. The results indicated that FA treatment significantly increased DNMT1 gene expression in liver tissue (p<0.05) and induced a non-significant upward trend in DNMT3A and DNMT3B gene expression. Conversely, DNMT3A gene expression significantly decreased in muscle tissue (p<0.05), while non-significant downward trends were observed for DNMT1 and DNMT3B genes. These findings suggest that formaldehyde treatment differentially modulates DNMT gene expression in chicken embryos in a tissue-specific and gene-specific manner, potentially influencing epigenetic mechanisms.

Anahtar Kelimeler

• Gene expression
• DNMT genes
• Fumigation
• Chick embryo

Tavuk Embriyolarında Kuluçka Öncesi Formaldehit Uygulamasının Kas ve Karaciğer Dokularında DNA Metiltransferaz (DNMT) mRNA Ekspresyonu Üzerine Etkilerinin Araştırılması

Öz

DNA metiltransferaz (DNMT) ailesi, epigenetik gen kontrolünde önemli rol oynayan, DNA metilasyon sürecini düzenleyen enzim grubudur. DNMT3A ve DNMT3B yeni metilasyon desenleri oluştururken, DNMT1 var olan desenleri korumaktan sorumludur. Bu enzimler embriyonal gelişimde aktif rol oynar ve farklılaşma sonrası hücrelerde ekspresyon seviyeleri büyük ölçüde azalır. Bu çalışma, tavuk embriyolarına kuluçka öncesi uygulanan formaldehitin (FA), karaciğer ve kas dokularında DNMT genlerinin ekspresyon düzeyleri üzerindeki etkilerini belirlemek amacıyla gerçekleştirilmiştir. Araştırmada, döllü tavuk yumurtaları FA ile dezenfekte edilmiş ve kuluçkanın 18. gününde embriyolardan karaciğer ve kas dokuları alınarak DNMT1, DNMT3A ve DNMT3B genlerinin ekspresyon seviyeleri qRT-PCR yöntemiyle araştırılmıştır. Sonuçlar, FA uygulamasının karaciğer dokusunda DNMT1 gen ekspresyonunu anlamlı düzeyde artırdığını (p<0.05), DNMT3A ve DNMT3B genlerinde ise anlamlı olmayan bir artış eğilimi oluşturduğunu göstermiştir. Aksine, kas dokusunda DNMT3A geninin ekspresyonu anlamlı düzeyde azalırken (p<0.05), DNMT1 ve DNMT3B genlerinde anlamlı olmayan azalma eğilimleri gözlenmiştir. Elde edilen bulgular, formaldehit uygulamasının tavuk embriyolarında DNMT genlerinin ekspresyonunu dokuya özgü ve gen bazında farklı şekillerde etkileyerek, epigenetik mekanizmaları modüle edebileceğini ortaya koymuştur.

Anahtar Kelimeler

• Gen ekspresyonu
• DNMT genleri
• Fumigasyon
• Civciv embriyo

Kaynakça

1. Avila, L. P., Sweeney, K. M., Schaeffer, C., Holcombe, N., Selby, C., Montiel, E., Wilson, J. L., 2023. Broiler breeder feed treatment with a formaldehyde-based sanitizer and its consequences on reproduction, feed and egg contamination, and offspring livability. Journal of Applied Poultry Research. 32(2): 100330.
2. Bernardini, L., Barbosa, E., Charão, M. F., Goethel, G., Muller, D., Bau, C., Steffens, N. A., Santos Stein, C., Moresco, R. N., Garcia, S. C., Souza Vencato, M., Brucker, N., 2021. Oxidative damage, inflammation, genotoxic effect, and global DNA methylation caused by inhalation of formaldehyde and the purpose of melatonin. Toxicology Research. 9(6): 778–789.
3. Bönsch, D., Lenz, B., Fiszer, R., Frieling, H., Kornhuber, J., Bleich, S., 2006. Lowered DNA methyltransferase (DNMT-3b) mRNA expression is associated with genomic DNA hypermethylation in patients with chronic alcoholism. Journal of Neural Transmission. 113(9): 1299–1304.
4. Bourc’his, D., Xu, G.-L., Lin, C.-S., Bollman, B., Bestor, T. H., 2001. Dnmt3L and the establishment of maternal genomic imprints. Science. 294(5551): 2536–2539.
5. Branco, J. R. O., Dallago, B. S. L., Bernal, F. E. M., 2021. Efficiency of ultraviolet light for disinfection of fertile broiler eggs. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 73(5): 1137–1146.
6. Cadirci, S., 2009. Disinfection of hatching eggs by formaldehyde fumigation - A review. Archiv Fur Geflugelkunde. 73(2): 116–123.
7. Cheng, X., 1995. Structure and function of DNA methyltransferases. Annual Review of Biophysics and Biomolecular Structure. 24(1): 293–318.
8. Coulibaly, F., Onbaşılar, E. E., Bakır, B., Sarıçam İnce, S., 2024. The effects of using UV light instead of formaldehyde in disinfection of hatching eggs on shell microbial load, embryo development, hatchability, and chick characteristics. International Journal of Environmental Health Research. 34(8): 2852–2862.

9. Hamburger, V., Hamilton, H. L., 1992. A series of normal stages in the development of the chick embryo. Developmental Dynamics. 195(4): 231–272.
10. Iyer, L. M., Abhiman, S., Aravind, L., 2011. Natural history of eukaryotic DNA methylation systems. Progress in Molecular Biology and Translational Science. 101: 25–104.
11. Jackson, P. G. G., Cockcroft, P. D., 2002. Clinical Examination of Farm Animals. Oxford, UK: Wiley-Blackwell. (Book format)
12. Johnson, P., 2018. Evaluation of the effects of formaldehyde on growth parameters of broiler chicks. Master Thesis. University of Arkansas. (Thesis format)
13. Ju, X., Wang, Z., Cai, D., Bello, S. F., Nie, Q., 2023. DNA methylation in poultry: a review. Journal of Animal Science and Biotechnology. 14(1): 138.
14. Kang, D. S., Kim, H. S., Jung, J.-H., Lee, C. M., Ahn, Y.-S., Seo, Y. R., 2021. Formaldehyde exposure and leukemia risk: a comprehensive review and network-based toxicogenomic approach. Genes and Environment. 43(1): 13.
15. Li, E., Beard, C., Jaenisch, R., 1993. Role for DNA methylation in genomic imprinting. Nature. 366(6453): 362–365.
16. Li, S., Zhu, Y., Zhi, L., Han, X., Shen, J., Liu, Y., Yao, J., Yang, X., 2016. DNA methylation variation trends during the embryonic development of chicken. PLOS ONE. 11(7): e0159230.
17. Liu, Q., Yang, L., Gong, C., Tao, G., Huang, H., Liu, J., Zhuang, Z., 2011. Effects of long-term low-dose formaldehyde exposure on global genomic hypomethylation in 16HBE cells. Toxicology Letters. 205(3): 235–240.
18. Lyko, F., 2018. The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. Nature Reviews Genetics. 19(2): 81–92.
19. Magras, I. N., 1996. Formaldehyde vapour effects in chicken embryo. Anatomia Histologia Embryologia. 25(3): 197–200.
20. Okano, M., Bell, D. W., Haber, D. A., Li, E., 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 99(3): 247–257.
21. Oliveira, G. D. S., dos Santos, V. M., Nascimento, S. T., 2021. Essential oils as sanitisers for hatching eggs. World’s Poultry Science Journal. 77(3): 605–617.
22. Özdemir, M., Sajid, G. A., Büyükkılıç Beyzi, S., Kızılaslan, M., Arzık, Y., Yalçın, S., White, S. N., Cinar, M. U., 2025. RNA-Seq of chicken embryo liver reveals transcriptional pathways influenced by egg formaldehyde treatment. Genes. 16(5): 1–10.
23. Pham, V. N., Bruemmer, K. J., Toh, J. D., Ge, E. J., Tenney, L., Ward, C. C., Chang, C. J., 2023. Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism. Science. 382(6670): eabp9201.
24. Ricke, S. C., Richardson, K., Dittoe, D. K., 2019. Formaldehydes in feed and their potential interaction with the poultry gastrointestinal tract microbial community. Frontiers in Veterinary Science. 6: 1–10.
25. Salthammer, T., Mentese, S., Marutzky, R., 2010. Formaldehyde in the indoor environment. Chemical Reviews. 110(4): 2536–2572.
26. Tajima, S., Tsuda, H., Wakabayashi, N., Asano, A., Mizuno, S., Nishimori, K., 1995. Isolation and expression of a chicken DNA methyltransferase cDNA1. The Journal of Biochemistry. 117(5): 1050–1057.
27. Taşdemir, A. N., Onbaşılar, E. E., Yalçın, S., Boyalı, B., Aygören, H., Tülek, E., Sarıçam, S., Akan, M., 2021. Effects of oregano juice on eggshell microbial load, layer embryo development, hatching results, and growth at the first 2 weeks after hatch. Tropical Animal Health and Production. 53(3): 1–9.
28. Tong, Z., Han, C., Qiang, M., Wang, W., Lv, J., Zhang, S., He, R., 2015. Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease. Neurobiology of Aging. 36(1): 100–110.
29. Yokomine, T., Hata, K., Tsudzuki, M., Sasaki, H., 2006. Evolution of the vertebrate DNMT3 gene family: a possible link between existence of DNMT3L and genomic imprinting. Cytogenetic and Genome Research. 113(1–4): 75–80.
30. Zeweil, H. S., Rizk, R., Bekhet, G. M., Ahmed, M. R., 2015. Comparing the effectiveness of egg disinfectants against bacteria and mitotic indices of developing chick embryos. The Journal of Basic and Applied Zoology. 70: 1–15.
31. Zhang, J., Gao, Y.-Y., Huang, Y.-Q., Fan, Q., Lu, X.-T., Wang, C.-K., 2018. Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers. Italian Journal of Animal Science. 17(2): 540–546.