Formik asidin insan lenfositleri üzerindeki in vitro genotoksik ve sitotoksik etkisi

Öz

Amaç: Formik asit, bitkiler, arılar, toprak, araçlar, gübre yanması ve fotokimyasal reaksiyonlar gibi çeşitli kaynaklarca üretilen ve çevrede yaygın olarak bulunan kimyasal bir bileşimdir. Mevcut çalışma, insan lenfositlerinde sitokinez-blok mikronükleus tayini ve kromozomal aberasyon analizi gibi sitogenetik testler kullanarak formik asidin sitotoksik ve genotoksik etkilerinin in vitro analizine odaklanmıştır. Yöntem: Bu çalışma 18-22 yaş arası 10’u erkek, 6’sı kadın sağlıklı, sigara kullanmayan yetişkinlerinden kan örnekleri kullanılarak gerçekleştirilmiştir. Lenfosit kültürüne kromozomal aberasyon CA testi için formik asidin farklı konsantrasyonları 0,07, 0,1, 0,2, 0,3, 0,4, 0,5, 0,8 mM eklendi. Pozitif kontrol olarak mitomycin-C 0,3 µg/ml kullanıldı. Sitokinez-blok mikronükleus tayini CBMN için, insan periferik kan lenfositleri 20, 40, 60, 80 mM derişimde formik asitle 48 saat işleme tabi tutuldu. Lenfosit kültürüne, pozitif kontrol olarak mitomycin-C 0,50 µg/ml eklendi. Bu araştırma, sitokinezblok mikronükleus tayinini CBMN ve kromozomal aberasyon CA analizini kullanarak in vitro insan periferal lenfositlerinde formik asidin sitotoksik ve genotoksik etkilerini değerlendirmek için yapılmıştır. Bulgular: Pozitif kontrol olarak kullanılan mitomisin C MMC, 0,5 µg/ml ile negatif kontrol karşılaştırıldığında formik asitin tüm dozları mikronükleus frekansını belirgin bir düzeyde arttırdığı gözlenmiştir. Pozitif kontrol olarak kullanılan mitomisin C MMC, 0,3 µg/ml ’nın negatif kontolle karşılaştırıldığında formik asidin 24 saatte tüm uygulama derişimlerinde, kromozomal aberasyonların sıklığını belirgin bir şekilde arttırdığı gözlenmiştir. Mikronükleus sıklığı ve kromozomal aberasyonlar, doza bağımlı olarak artmıştır. Sonuçlar formik asit konsantrasyonu, mikronükleus frekansı r=0,92 , nekrotik hücrelerin sayısı r=0,95 ve apoptotik hücreler r=0,91 arasında anlamlı korelasyonların var olduğunu göstermektedir. Sonuç: Verilerimiz, in vitro olarak formik asit konsantrasyonu ve mikronükleus frekansı, apoptotik hücreler ile nekrotik hücreler gibi kromozomal aberasyonlar arasında anlamlı bir korelasyon olduğu kanıtını sağlamıştır

Anahtar Kelimeler

• Formik asit, insan lenfosit kültürü, kromozomal aberasyon, mikronükleus, sitotoksisite

Genotoxic and cytotoxic effects of formic acid on human lymphocytes in vitro

Öz

Objective: Formic acid is an ubiquitous chemical constituent in the environment, being produced by sources as diverse as vegetation, ants, soil, vehicles, biomass burning, and photochemical reactions. The present work is focused on in vitro analysis of cytotoxic and genotoxic effects of formic acid, using cytogenetic tests such as the cytokinesis-block micronucleus assay CBMN and chromosomal aberration analysis, in human lymphocytes. Method: This study was carried out using blood samples from healthy, non-smoking adults aged 18–22 years, of whom 10 were male and 6 were famale. Different concentrations 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 0.8 mM of formic acid was added to the lymphocyte culture test for chromosomal aberration CA analysis. Mitomycin-C 0.3 mg/ml was used as the positive control. Human peripheral blood lymphocyte cells were treated with 20, 40, 60, 80 mM concentrations of formic acid for 48 h. for the CBMN test. Mitomycin-C 0.5 mg/ml was added to the Lymphocyte culture as a positive control. The present research was carried out to assess the cytotoxic and genotoxic effects of formic acid on human peripheral lymphocytes in vitro using the cytokinesis-block micronucleus assay CBMN , as well as chromosomal aberration CA analysis. Results: A significant increase was observed for induction of micronucleus frequency in all treatments of formic acid concentrations for 48 h. comparing with the negative control and mitomycin C MMC, 0.5 µg/ ml which was used as positive control. When compared with negative control and with mitomycin C MMC, 0.3 µg/ml which is used as positive control, it is observed that formic acid is rising the frequency of chromosomal aberration significantly at all appliance concantrations in 24 h. The frequency of micronuclei and chromosomal aberrations increased in a dose dependent manner. The results showed that there were significant correlations between formic acid concentration and micronuclei frequency r= 0.92 , numbers of necrotic cells r= 0.95 , and apoptotic cells r= 0.91 . Conclusion: Our data provided evidence that there is a significant correlation between the concentration of formic acid and the following chromosomal aberrations: frequency of micronuclei, apoptotic cells, and necrotic cells in vitro.

Anahtar Kelimeler

• Formic acid, human lymphocyte culture, chromosomal aberration, micronucleus, cytotoxicity

Kaynakça

1. 1. Barceloux DG, Bond GR, Krenzelok EP, Cooper H, Vale JA. American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning. Clin Toxicol, 2002; 40: 415-46.
2. 2. Boeniger MF. Formate in urine as a biological indicator of formaldehyde exposure-a Review. Am Ind Hyg Assoc J, 1987; 48: 900-8.
3. 3. Morita T, Takeda K, Okumura K. Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells. Mutation Research/Genetic Toxicology, 1990; 240 (3): 195-202.
4. 4. Tephly TR. The toxicity of methanol. Life Sci, 1991; 48 (11): 1031-41.
5. 5. Lazutka JR, Mierauskiene J, Slapsyte G, Dedonyte V. Genotoxicity of dill (Anethum graveolens L.), peppermint (Mentha x piperita L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster. Food Chem Toxicol, 2001; 39: 485-92.
6. 6. Roncada T, Vicentini VEP, Mantovani MS. Possible modulating actions of plant extracts on the chromosome breaking activity of MMC and Ara-C in human lymphocytes in vitro. Toxicol in Vitro, 2004; 18: 617-22.
7. 7. Yuzbasioglu D, Celik M, Yilmaz S, Unal F, Aksoy H. Clastogenicity of the fungicide a fugan in cultured human lymphocytes. Mutat Res-Gen Tox En, 2006; 604 (1-2): 53-9.
8. 8. Demsia G, Vlastos D, Goumenou M, Matthopoulos DP. Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow. Mutat Res-Gen Tox En, 2007; 634: 32-9.

9. 9. Fenech M, Crot J, Turner J, Brown S. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis, 1999; 14: 605-12.
10. 10. Eastmond DA, Tucker JD. Identification of aneuploidy-inducing agents using cytokinesisblocked human-lymphocytes and an antikinetochore antibody. Environ Mol Mutagen, 1989; 13 (1): 34-43.
11. 11. Carrano AV, Natarajan AT. Considerations for population monitoring using cytogenetic techniques. Mutation Research/Genetic Toxicology, 1988; 204: 379-406.
12. 12. Hagmar L, Brogger A, Hansteen IL, Heim S, Hogstedt B, Knudsen L, et al. Cancer risk in human predicted by increased levels of chromosomal aberrations in lymphocytes: Nordic study group on the health risk of chromosome damage. Cancer Res, 1994; 54 (11): 2919-22.
13. 13. Savage JRK. Update on target theory as administered to chromosomal aberrations. Environ Mol Mutagen, 1993; 22: 198-207.
14. 14. Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, et al. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat Res-Rev Mutat, 2000; 463: 111-72.
15. 15. Boffetta P, Van der Hel O, Norppa H, Fabianova E, Fucic A, Gundy S, et al. Chromosomal aberrations and cancer risk: Results of a cohort study from central europe. Am J Epidemiol, 2007; 165: 36-43.
16. 16. Norppa H, Bonassi S, Hansteen IL, Hagmar L, Stromberg U, Rossner P, et al. Chromosomal aberrations and SCEs as biomarkers of cancer risk. Mutat Res-Fund Mol M, 2006; 600(1-2): 37-45.
17. 17. Fenech M. Biomarkers of genetic damage for cancer epidemiology. Toxicology, 2002; 181-182: 411-6.
18. 18. Heddle JA, Cimino MC, Hayashi M, Romagna F, Shelby MD, Tucker JD, et al. Micronuclei as an index of cytogenetic damage: Past, present, and future. Environ Mol Mutagen, 1991; 18 (4): 277-91.
19. 19. Bonassi S, Znaor A, Ceppi M, Lando C, Chang WP, Holland N, et al. An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis, 2007; 28 (3): 625-31.
20. 20. Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutation Research/Environmental Mutagenesis and Related Subjects, 1985; 147: 29-36.
21. 21. Surralles J, Xamena N, Creus A, Marcos R. The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair. Mutation Research/Genetic Toxicology, 1995; 342: 43-59.
22. 22. Mahboob S, Al-Balwai HFA, Al-Misned F, Ahmad Z. Investigation on the genotoxicity of mercuric chloride to freshwater Clarias gariepinus. Pak Vet J, 2014; 34 (1): 100-3.
23. 23. Fries I, Rosenkranz P. Number of reproductive cycles of Varroa jacobsoni in honey bee colonies. Exp Appl Acarol, 1996; 20: 103-12.
24. 24. Al-Tarazi YH, Alshawabkeh K. Effect of dietary formic and propionic acids on Salmonella pullorum shedding and mortality in layer chicks after experimental infection. J Vet Med Infect Dis Vet Public Health, 2003; 50: 112-7.
25. 25. Wang HF, Xu LH, Sun HF, Xue B, Liu YF, Peng S, et al. High binding of formic acid to biomacromolecules in mice. Nucl Instrum Meth B, 2004; 223: 745-9.
26. 26. Celikler S, Yildiz G, Vatan O, Bilaloglu R. In vitro antigenotoxicity of Ulva rigida C. Agardh (Chlorophyceae) extract against induction of chromosome aberration, sister chromatid exchange and micronuclei by mutagenic agent MMC. Biomed Environ Sci, 2008; 21: 492-8.
27. 27. Ogura H, Takeuchi T, Morimoto K. A comparison of the 8-hydroxydeoxyguanosine, chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes. Mut ResRev Gen Toxicol, 1996; 340: 175-82.
28. 28. Panneerselvam N, Sinha S, Shanmugam G. Genotoxicity of the herbicide fluchloralin on human lymphocytes in vitro: chromosomal aberration and micronucleus tests. Mutation Research/Genetic Toxicology, 1995; 344: 69-72.
29. 29. Anwar WA, Khalil MM, Wild CP. Micronuclei, chromosomal-aberrations and aflatoxin-albumin adducts in experimental-animals after exposure to aflatoxin B-1. Mut Res/Gen Toxicol, 1994; 322: 61-7.
30. 30. Buyukleyla M, Rencuzogullari E. The effects of thymol on sister chromatid exchange, chromosome aberration and micronucleus in human lymphocytes. Ecotox Environ Safe, 2009; 72: 943-7.