Evaluation of bilirubin inducing apoptotic and necrotic cell death in astrocyte cell culture

SUMMARY As early as 1875 it was established that there is an association between high serum bilirubin levels and clinical encephalopathy characterized by yellowish staining of certain central nervous system sites on pathological examination. However, even today the mechanism underlying the bilirubin neurotoxicity is not fully understood. Bilirubin cytotoxicity has been demonstrated in different cell types, including astrocytes and neurons. Astrocytes form the barrier function of the brain capillaries. These cells comprise about one-third of the total cellular mass in the human brain cortex, astrocytes also play a vital role in the transport of hormones and micronutrients into the brain. Therefore, astrocytes may be the first line cells that come in contact with bilirubin when the blood-brain barrier fails. Their role in the development of bilirubin encephalopathy has not been fully investigated. Astrocyte cell cultures may contribute to other models in the demonstration of bilirubin neurotoxicity. In this study performed on astrocyte cell cultures we demonstrated that serum from patients with hyperbilirubinemia caused more cytotoxicity then bilirubin added directly to the culture media, even when the level of bilirubin in the serum was much less then the directly added bilirubin. Also, inactivated serum samples caused less cytotoxicity then not inactivated samples. Results of the current study show that both serum from patients with indirect hyperbilirubinemia and bilirubin itself causes cytotoxicity in astrocyte cell cultures. This study for the first time shows that serum from patients with indirect hyperbilirubinemia and bilirubin itself have cytotoxic effects on rat astrocytes. The study also tried to identify whether serum from patients with indirect hyperbilirubinemia and bilirubin directly caused apoptotic cell death in astrocyte cell cultures. The results show that both serum from patients with indirect hyperbilirubinemia and bilirubin lead to dose and time dependent apoptosis in astrocytes. This thesis is the first study showing that serum from patients with indirect hyperbilirubinemia and bilirubin itself induce cell death via apoptosis. Apostain immunofluorescent staining showed that both patient serum and bilirubin lead to dose dependent cell death by apoptosis in astrocyte cell cultures. Similar to the effect of LDH, bilirubin caused most of its cytotoxic effect within 48 hours.In conclusion, serum from patients with indirect hyperbilirubinemia and bilirubin directly induce dose and time dependent apoptotic cell death in newborn rat astrocyte cell cultures. Keywords: indirect bilirubin, astrocyte cell culture, apoptosis, cytotoxicity