Bilirubin-induced cell death during continuous and intermittent phototherapy and in the dark.

AIM: To compare continuous and intermittent light exposure in the presence of bilirubin with respect to cellular damage. Furthermore, it was of interest to characterize the nature of cellular toxicity of bilirubin in the dark. METHOD: A murine lymphoma cell line, L5178Y-R (LY-R), was exposed to solutions of bilirubin (160 microM) supplemented with human serum albumin (200 microM) and irradiated with phototherapy light (Philips 20W/52) at a constant total dose of approximately 500 kJ/m2. The irradiation was given either as intermittent or continuous treatment with light of variable irradiance. The three lower irradiance levels were clinically relevant. Cells treated with bilirubin were also kept in the dark for various periods of time. Cell toxicity was determined by measuring apoptosis and necrosis. Apoptosis was measured by terminal deoxynucleotide transferase and propidium iodide staining assay, while trypan blue assay was used for detection of necrosis. RESULTS: There was no difference (n = 6, p > 0.05) between continuous and intermittent irradiation in the induction of early and late apoptotic cell death. Necrosis was more pronounced after intermittent treatment. Bilirubin dark toxicity was observed and classified as both apoptotic and necrotic. CONCLUSION: Continuous and intermittent light exposure caused the same degree of apoptotic cell death, while the cells underwent more necrotic death after intermittent exposure. Bilirubin was cytotoxic in the dark by both cell death mechanisms.

Effects of bilirubin and phototherapy on osmotic fragility and haematoporphyrin-induced photohaemolysis of normal erythrocytes and spherocytes.

AIM: To study the effects of phototherapy on erythrocyte haemolysis in vitro and to determine possible differences in sensitivity to phototherapy between normal erythrocytes and spherocytes. METHODS: Erythrocytes from four normal healthy donors and two donors with hereditary spherocytosis were treated with bilirubin (160 microM) in the presence of human serum albumin in the molecular ratio bilirubin/albumin 0.8. Treated cells were maintained either in the dark or in blue light (450 nm, 8 mW/cm2, 30 min). The experimental light dose was comparable to 2 h of clinical phototherapy. The osmotic fragility of the treated cells was measured by scoring haemolysis in hypo-osmolar solutions (0.10-0.90% NaCl). The sensitivity to photohaemolysis of cells pre-treated with bilirubin (BR) and/or phototherapy was tested by exposing the cell suspensions to haematoporphyrin and UVA radiation. The delayed (18 h) photohaemolysis was measured by spectrophotometry. RESULTS: Osmotic fragility, expressed as percentage haemolysis, of normal erythrocytes was more than doubled in the presence of BR combined with phototherapy (n = 6, p < 0.05). In contrast, osmotic fragility of spherocytes was unaffected by either treatment (n = 8, p < 0.05). Increased photohaemolysis was seen in spherocytes treated with BR (n = 13, p < 0.05), phototherapy (n = 13, p < 0.05) and a combination of the two agents (n = 13, p < 0.05) compared with spherocytes without BR in the dark (n = 6). CONCLUSION: Bilirubin may make the plasma membrane of normal erythrocytes more fragile. Newborns with hereditary spherocytosis may be sensitive to phototherapy.

Formation of photoproducts and cytotoxicity of bilirubin irradiated with turquoise and blue phototherapy light.

AIM: To compare a new turquoise ("green") fluorescent phototherapy lamp (490 nm) with a conventional blue phototherapy lamp (450 nm) with respect to cytotoxicity and photochemical effects of bilirubin. METHODS: Mouse lymphoma cells (L5178Y-R) in the presence of bilirubin solutions were exposed to phototherapy light. Occurrence of necrosis and apoptosis, reduction of mitotic index and inhibited cell growth was assayed by appropriate methods. The presence of bilirubin and its photoisomers was measured by high-pressure liquid chromatography analysis and absorption spectroscopy. RESULTS: At constant and equal light irradiances, the cytotoxic effects in the presence of bilirubin bound to human serum albumin showed that the green lamp caused significantly less necrosis (n = 4, p < 0.05) and less inhibition of cell multiplication (n = 3, p < 0.05) than the blue lamp. A slightly lower apoptotic fraction, although not statistically significant, was observed in cells exposed to the blue lamp. Photo-oxidation of bilirubin was more prominent with blue light irradiation. The photoequilibria between geometric isomers of bilirubin were different for the two lamps; more geometric photoisomers were formed by blue irradiation (n = 6, p < 0.05). The amounts of the most water-soluble isomers (presumably mainly lumirubin) were rather similar for the two lamps. CONCLUSION: The two lamps were similar in the formation of therapeutically relevant photoproducts, but the blue lamp showed potential in forming more photo-oxidation products and in causing more severe cellular damage in the presence of bilirubin.

In vitro cell death induced by irradiation and chemicals relevant for dental applications; dose-response and potentiation effects.

Resin-based dental materials polymerized using blue light are frequently used in dental practice and may come in contact with the oral mucosa. Remnants from oral hygiene product ingredients, such as sodium lauryl sulfate (SLS), add to the chemical exposure of the mucosa. The aim of the present in vitro study was to elucidate the cytotoxic effects in terms of apoptosis and necrosis after exposures to combinations of an adhesive (0.5% and 0.6%), SLS (concentration range 0.0025%-0.0075%), and irradiation from a dental curing lamp (radiant exposure of 8 J cm(-2)). The test system chosen was rat submandibular salivary gland acinar cells, and the cytotoxic effects were measured by fluorescence microscopy and flow cytometry methods. Cytotoxicity was observed as a result of irradiation. The most pronounced cytotoxic effects were seen in cells exposed to a combination of adhesive and SLS compared with those exposed to either agent alone. Necrosis was the dominating form of cell death for all exposures, except for the highest concentration of SLS. Apoptosis was dose-dependent on SLS in the rat submandibular acinar cells. Cytotoxic considerations of dental materials should include contributions from irradiation and other chemicals that might be present in the oral cavity.