Tetrazolium-Based Cytotoxicity Tests May Not Always Reflect Accurate Results

Öz

Background: Cell viability and/or cytotoxicity analysis is one of the most important tools used for biological evaluation in vitro studies. The selection of the right cytotoxicity tests is critical to form the basis for in vivo and preclinical studies, specifically for cancer research. In the present study, we aimed to investigate the cytotoxic effects of bromelain, a widely-used phytochemical product in the medical field, and idarubicin, an anthracycline antibiotic used in the treatment of cancer, in normal lymphocytes and a promyelocytic leukemia cell line (HL-60) with MTT, WST-1, and luminescent ATP assays and to compare the results of these tests..
Materials and Methods: We obtained peripheral blood lymphocytes from healthy, young, non-smoker male volunteers and obtained the HL-60 cell line from the American Type Culture Collection (ATCC). Bromelain and idarubicin were added in increasing concentrations to both cell lines. Cells were incubated at 37°C in a carbon dioxide incubator for 24 h. After incubation, cytotoxicity levels were determined by MTT, WST-1, and ATP assays, and morphological evaluations were performed by fluorescent staining.
Results: The MTT and WST-1 assays demonstrated that cell viability/formazan formation increased with bromelain concentration; however, the luminescent ATP assay demonstrated that cell viability decreased with increasing concentrations of bromelain. Whereas fluorescent staining methods confirmed the ATP assay results, the MTT and WST-1 assays contradicted the ATP assay results. The cytotoxic effects of idarubicin were similar in the two cell lines according to the three different measurement methods and were positively correlated with the results of the fluorescent staining methods.
Conclusion: The detection of cell viability and cytotoxicity by bromelain with the MTT and WST-1 assays in lymphocytes and HL-60 cells is limited. To obtain accurate and reliable results from cytotoxicity studies, a measurement method should be carefully selected by considering that the phytochemicals to be tested could interfere with the results, and the results should be verified by other methods.

Anahtar Kelimeler

• Bromelain,idarubicin,cytotoxicity tests,interference,tetrazolium salts

Tetrazolium Bazlı Sitotoksisite Testleri Her Zaman Doğru Sonuçları Yansıtmayabilir

Öz

Amaç: Hücre canlılığı ve/veya sitotoksisite analizleri in vitro çalışmalarda biyolojik değerlendirme için kullanılan en önemli göstergelerdendir. Özellikle kanser araştırmalarında, in vivo ve preklinik çalışmalara kaynaklık etmesi bakımından sitotoksisite testlerinin seçimi kritik öneme sahiptir. Bu çalışmada medikal kullanımı yaygın fitokimyasal bir ürün olan bromelain ve kanser tedavisinde kullanılan antrasiklin antibiyotik bir ilaç olan idarubisin’in MTT, WST-1 ve Luminesan ATP yöntemleriyle sitotoksik etkilerinin, normal lenfosit hücre kültürü ve HL-60 promyelositik lösemi hücre hattında araştırılması ve testler arasındaki ilişkinin incelenmesi amaçlandı.
Materyal ve Metod: Periferal kan lenfositleri sigara içmeyen, sağlıklı, gönüllü, genç erkek bireyden, HL-60 promyelosik lösemi hücre hattı ise ATCC firmasından sağlandı. Bromelain ve idarubisinin artan konsantrasyonları her iki hücre hattına eklendi. Hücreler, 24 saat 37 °C’de karbondioksit inkübatöründe inkübe edildi. İnkübasyon sonrası MTT, WST-1 ve ATP analizleriyle sitotoksisite düzeyleri araştırılırken, floresan boyama yöntemleriyle de morfolojik incelemeler yapıldı.
Bulgular: MTT ve WST-1 analizlerinde, bromelain konsantrasyon artışına paralel olarak hücre canlılığının/formazan oluşumunun arttığı, ATP testinde ise konsantrasyon artışıyla hücre canlılığının azaldığı bulundu. Floresan boyama yöntemleri ile ATP analiz sonuçları doğrulanırken, MTT ve WST-1 negatif ilişkiliydi. İdarubisin’in sitotoksik etkisinin her iki hücre hattında 3 ölçüm yöntemi ile benzer olduğu, floresan boyama yöntemleri ile de pozitif ilişkili olduğu bulundu
Sonuç: Bromelain’in lenfosit ve HL-60 hücre dizileri üzerinde MTT ve WST-1 analiz yöntemleri ile hücre canlılığı/sitotoksisite tespitinde bazı ölçüm sınırlamalarına neden olduğu bulunmuştur. Sitotoksisite çalışmalarında doğru ve güvenilir sonuçlar elde etmek için analiz edilecek fitokimyasalların interferenslere sebep olabileceği göz önüne alınarak yöntem seçimi dikkatlice yapılmalı ve başka metodlarla sonuçlar doğrulanmalıdır.

Anahtar Kelimeler

• Bromelain,idarubisin,sitotoksisite testleri,interferans,tetrazolyum

Kaynakça

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