Publication: Dapagliflozinin karaciğer üzerindeki etkisinin in vitro araştırılması
Abstract
Amaç: Tip 2 diabetes mellitus (T2DM) dünya genelinde en yaygın görülen diyabet türü olup, metabolik disfonksiyonla ilişkili yağlı karaciğer hastalığı (MAFLD) riskini artırmaktadır. T2DM tedavisinde dünya çapında onaylanan dapagliflozinin (DAPA) karaciğere etkileri üzerine literatürde çeşitli ve tutarsız bulgular mevcuttur. Bu yüksek lisans tez çalışmasının amacı, dapagliflozininin olası hepatotoksisitesinin in vitro değerlendirilmesi, ilaca bağlı karaciğer hasarı riski açısından taşıdığı potansiyelin aydınlatılmasıdır. Gereç ve Yöntem: İnsan hepatosellüler karsinom hücre hattı (HepG2) üzerinde uygulanacak dozların belirlenmesi için MTT testinden, reaktif oksijen türlerinin (ROS) seviyelerinin ölçülmesi için DCF-DA testinden, biyokimyasal parametreler olan aspartat aminotransferaz (AST), alanin aminotransferaz (ALT), bilirubin ve üre ölçümü için ELİSA kitlerinden, apoptotik/ nekrotik hücre ölümü analizi için flow sitometrik Anneksin V/ PI testinden faydalanıldı. Bulgular: HepG2 hücre hattında yapılan MTT testi sonucunda, DAPA’nın hiçbir son konsantrasyonunda ve sürede IC50 değeri hesaplanamadı. Bu yüzden, tüm testler DAPA’nın yaklaşık Cmax (0.4 μM) değerinin 1 ve 10 katı olan 0.4 ve 4 μM final konsantrasyonlarında gerçekleştirildi. DCF-DA testi sonucunda; 4 saatlik maruziyette DAPA’nın tüm konsantrasyonlarında kontrole kıyasla ROS düzeylerinde istatistiksel anlamlı olmayan azalma gözlendi (p>0.05). Hücre süpernatantında AST seviyesinde 0.4 μM DAPA final konsantrasyonunda istatistiksel anlamlı olmayan artış (p>0.05), yine hücre süpernatantında ALT seviyesinde her iki konsantrasyonda kontrole kıyasla istatistiksel anlamlı olmayan artış gözlendi (p>0.05). Hücre ekstraktında her iki konsantrasyonda AST seviyesinde istatistiksel anlamlı olmayan artış (p>0.05), ALT seviyesinde yalnızca 0.4 μM DAPA konsantrasyonunda kontrole kıyasla istatistiksel anlamlı olmayan artış gözlendi (p>0.05). Bilirubin seviyesinde 0.4 μM DAPA konsantrasyonunda, üre seviyesinde her iki konsantrasyonda kontrole kıyasla istatistiksel anlamlı olmayan artış gözlendi (p>0.05). DAPA her iki konsantrasyonunda erken apoptotik hücre popülasyonununda istatistiksel anlamlı olmayan artış gözlendi (p>0.05). Sonuç: Dapagliflozinin tek sefer 24 saat uygulamasında karaciğer hasarı ile ilgili incelenen ölçümlerdeki istatistiksel anlamlı olmayan artış, olası hepatotoksisite riski açısından dikkatle değerlendirilmelidir.
Objective: Type 2 diabetes mellitus (T2DM) is the most common type of diabetes worldwide and increases the risk of metabolically associated fatty liver disease (MAFLD). Dapagliflozin, a globally approved treatment option for T2DM, has shown variable and inconsistent findings in the literature regarding its hepatic effects. The aim of this master's thesis was to evaluate the potential hepatotoxicity of dapagliflozin in vitro and to investigate its potential risk of drug- induced liver injury. Materials and Methods: To determine the dosing for HepG2 human hepatocellular carcinoma cells, the MTT assay was employed. The DCF-DA assay was used to measure reactive oxygen species (ROS) levels, ELISA kits were employed to measure biochemical markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, and urea. Apoptotic and necrotic cell death was evaluated by flow cytometric analysis using the Annexin V/ PI assay. Results: The MTT assay revealed that no IC50 value could be calculated for any concentration or exposure duration of DAPA in HepG2 cells. Therefore, all tests were conducted at final concentrations of 0.4 μM and 4 μM, corresponding to approximately 1× and 10× the estimated human plasma Cmax value (0.4 μM). In the DCF-DA assay, ROS levels showed a non- significant decrease at both DAPA concentrations after 4-hour exposure compared to control (p>0.05). In the cell supernatant, AST levels exhibited non-significant increases at 0.4 μM, while ALT levels evels exhibited non-significant increases at both concentrations compared to the control (p>0.05). In the cell extract, AST levels insignificantly increased at both concentrations, whereas ALT levels increased only at 0.4 μM (p>0.05). Bilirubin levels showed a non-significant elevation at 0.4 μM, and urea levels showed non-significant increases at both concentrations compared to the control group (p>0.05). A non-significant increase in the early apoptotic cell population was also observed at both concentrations of DAPA (p>0.05). Conclusion: Although statistically non-significant, the observed increases in hepatotoxicity- related parameters following a single 24-hour exposure to dapagliflozin warrant careful evaluation of its potential liver toxicity.
Objective: Type 2 diabetes mellitus (T2DM) is the most common type of diabetes worldwide and increases the risk of metabolically associated fatty liver disease (MAFLD). Dapagliflozin, a globally approved treatment option for T2DM, has shown variable and inconsistent findings in the literature regarding its hepatic effects. The aim of this master's thesis was to evaluate the potential hepatotoxicity of dapagliflozin in vitro and to investigate its potential risk of drug- induced liver injury. Materials and Methods: To determine the dosing for HepG2 human hepatocellular carcinoma cells, the MTT assay was employed. The DCF-DA assay was used to measure reactive oxygen species (ROS) levels, ELISA kits were employed to measure biochemical markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, and urea. Apoptotic and necrotic cell death was evaluated by flow cytometric analysis using the Annexin V/ PI assay. Results: The MTT assay revealed that no IC50 value could be calculated for any concentration or exposure duration of DAPA in HepG2 cells. Therefore, all tests were conducted at final concentrations of 0.4 μM and 4 μM, corresponding to approximately 1× and 10× the estimated human plasma Cmax value (0.4 μM). In the DCF-DA assay, ROS levels showed a non- significant decrease at both DAPA concentrations after 4-hour exposure compared to control (p>0.05). In the cell supernatant, AST levels exhibited non-significant increases at 0.4 μM, while ALT levels evels exhibited non-significant increases at both concentrations compared to the control (p>0.05). In the cell extract, AST levels insignificantly increased at both concentrations, whereas ALT levels increased only at 0.4 μM (p>0.05). Bilirubin levels showed a non-significant elevation at 0.4 μM, and urea levels showed non-significant increases at both concentrations compared to the control group (p>0.05). A non-significant increase in the early apoptotic cell population was also observed at both concentrations of DAPA (p>0.05). Conclusion: Although statistically non-significant, the observed increases in hepatotoxicity- related parameters following a single 24-hour exposure to dapagliflozin warrant careful evaluation of its potential liver toxicity.