In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin.

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.

Bilirubin/biliverdin-Cu(II) induced DNA breakage; reaction mechanism and biological significance.

Bilirubin and its metabolic precursor biliverdin are heme degradation products but have been proposed as physiological antioxidants. Reports from another laboratory as well as from ours have shown bilirubin to form a complex with the transition metal ion-Cu(II). Such a complex was shown by us to cause oxidative DNA damage. Further, biliverdin was also shown to be capable of causing similar DNA damage. In the present studies we have aimed to elucidate the mechanism of DNA breakage reaction by these bile pigments. Absorption and fluorescence studies indicate binding of bile pigments to DNA and copper ions. Cu(II) is reduced by these compounds to Cu(I) which is an essential intermediate in the DNA breakage reaction. Redox recycling of Cu(II) leads to generation of reactive oxygen species. Strand scission by the bile pigments-Cu(II) system is found to be biologically significant as assayed by bacteriophage inactivation. Our results, therefore are suggestive of one of the mechanisms through which endogenous DNA damage may occur.

Antiviral activity of a bile pigment, biliverdin, against human herpesvirus 6 (HHV-6) in vitro.

Biliverdin (BV), a bile pigment, was examined for its antiviral activity against human herpesvirus-6 (HHV-6) in vitro. BV (10 micrograms/ml) markedly inhibited HHV-6 replication in MT-4 cells when the cells were treated during a virus adsorption period. Its antiviral effect was weakened when cells were treated after adsorption. Treatment of cells with BV (40 micrograms/ml) 3 hr after virus infection had no inhibitory effect on virus replication. Virus replication was also significantly inhibited by treatment of MT-4 cells with BV (10 micrograms/ml) before infection, while the virions were not inactivated by BV (20 micrograms/ml). Bilirubin and urobilin, metabolic derivatives of BV, showed slight inhibitory effects on virus replication in the cells. On the other hand, BV had no potent inhibitory activity in the replication of herpes simplex virus-1 or human cytomegalovirus. These observations suggest that BV could interact with MT-4 cells to inhibit an early stage of HHV-6 infection in a virus-specific manner.

[Modification of the cellular cytoskeleton and hepatic tumor promotion]. / Altérations du cytosquelette cellulaire et promotion tumorale hépatique.

Non transformed epithelial hepatic cells (established cell line and adult rat hepatocytes) treated by liver tumor promoters, phenobarbital and biliverdin, for 24 and 48 h showed a fragmentation and loss of F-actin and a depolymerisation of microtubules. This pattern closely resembles that of transformed cells which were not susceptible to the action of promoters. In liver preneoplastic nodules obtained from rats submitted to an initiation-promotion process, actin almost completely disappeared with the concomitant appearance of a characteristic enzymatic pattern rich in GGT and GST-P. Therefore, cytoskeleton of hepatic cells is a target for tumor promoters and could play a role in promotion mechanism.