Journal of Hepatology
Volume 34, Issue 3 , Pages 467-470, March 2001

New concepts in bilirubin neurotoxicity and the need for studies at clinically relevant bilirubin concentrations

  • J.Donald Ostrow

      Affiliations

    • Research Service, GI/Hepatology Division, VA Puget Sound Health Care System-Seattle Division, and University of Washington, Seattle, WA 98108-1597, USA
    • ,
  • Claudio Tiribelli

      Affiliations

    • Centro Studi Fegato, Department BBCM, University of Trieste, Via Giorgeri 1, 34127 Trieste, Italy
    • Corresponding Author InformationCorresponding author. Tel.: +39-040-399-4927; fax: +39-040-399-4924

Received 31 October 2000; accepted 6 November 2000.

See Article, pages 402–408

Article Outline

 

The fetus cannot detoxify unconjugated bilirubin (UCB) by hepatic conjugation and is protected from accumulation of UCB by its passage across the placenta, for conjugation and secretion by the maternal liver [1], [2]. At birth, this protection is suddenly lost and the newborn must cope with an acute ≥2× increase in production of UCB [3], [4], [5] using its own immature mechanisms for hepatic uptake, conjugation and biliary secretion of bilirubin [6]. As a result, virtually all newborn infants have temporary, mild to moderate ‘physiologic’ jaundice, which is believed to be beneficial, since UCB is a powerful antioxidant [7]. In some infants, however, especially those who are premature and/or have associated hemolytic disorders, concentrations of UCB may rise to levels that cause bilirubin encephalopathy, sometimes with deposition of bilirubin in selected central nervous system (CNS) centers (kernicterus) [1], [2]. These neurotoxicities may cause severe, lifelong motor, auditory and mental impairment, but subtler, permanent impairment is increasingly appreciated in a larger proportion of less severely jaundiced newborns. [2].

Although phototherapy and metalloporphyrins are available to prevent and treat severe neonatal jaundice [8], kernicterus still occurs and is increasing in incidence due to early discharge of newborns from hospitals [2], [9]. Prediction is uncertain as to which neonates are at risk and should receive such treatment, due largely to imprecise concepts regarding the ionization, binding, transport, and mechanisms of neurotoxicity of UCB [10]. These uncertainties are largely traceable to the performance of most studies with impure UCB and/or at unrealistically high UCB concentrations, exceeding both its very low aqueous solubility of 70 nM [11] and the maximum capacity of the high-affinity binding site of plasma albumin to bind one molecule of UCB [12]. High UCB concentrations are used because: (a) it is difficult to measure the very low unbound concentrations of UCB found even in icteric plasma [13], and (b) it is generally believed that toxicity is cumulative and determined by UCB concentration×exposure time, so that high UCB concentrations are required to observe effects during the brief study periods necessitated by the instability of UCB [13], [14]. Unfortunately, in such supersaturated systems, the excess unbound UCB diacid forms microprecipitates, an altered physical state that confounds interpretation of such studies [10] and renders the findings irrelevant to UCB levels that seldom exceed 1 mol per mol albumin in neonates with bilirubin encephalopathy [6], [15]. Alternatively, studies have been performed at high pH values, at which UCB solubility is greater [11], but at which the pigment is less stable [13] and the proportions of the three ionization species of UCB differ from those at physiological pH values [10].

These factors likely contribute to the over 100-fold variation in published values for the binding affinity of human serum albumin (HSA) for UCB [16] and the confusion regarding the mechanisms by which accumulated UCB causes neurotoxicity. For example, oxidative phosphorylation was markedly impaired and uncoupled in brain mitochondria exposed to micromolar concentrations of unbound UCB but not when the UCB/albumin ratios and unbound UCB concentrations were realistic [17], [18], [19]. The landmark studies from Brites’ laboratory of UCB toxicity to cultured rat neurones and astrocytes in this issue of the Journal of Hepatology [20] and in astrocytes [21], notably utilized purified UCB and studied concentrations well below saturation of the HSA. These are among the few in vitro studies to demonstrate that very modestly elevated concentrations of purified, unbound UCB can be toxic to cells from the CNS. Previous studies in a murine neuroblastoma cell line (NBR10A) [22] had demonstrated impaired uptake of 3H-thymidine after exposure for 22 h, but not for 2 h, to UCB/HSA ratios as low as 0.8 in the presence of 100 or 200 μM HSA; cell viability, assessed by the MTT test, was impaired only at UCB/HSA molar ratios of 1.1–1.5. Such different responses of different functional activities of the cell after exposure to UCB highlight the need to carefully select the indicators of toxicity for study. The comparable cell line from rats (N115) was less sensitive by either assay, indicating the importance of species differences and caution in extrapolation to humans of data obtained in rodents.

The Lisbon group showed that uptake of the neurotransmitter, glutamate, by cultured rat astrocytes was inhibited significantly, in a dose-dependent fashion, by UCB added at UCB/HSA ratios of 0.8 or above [21]. More importantly, there was a linear decrease in glutamate uptake as UCB/HSA ratios increased from 0.2–1.0, indicating that UCB was inhibitory at ratios well below saturation. The inhibition was absent at pH 7.0, but evident at pH 7.4 or higher, suggesting that the monoanionic species of UCB was responsible for the inhibition. Such impaired reuptake of glutamate by astrocytes would promote accumulation of glutamate in the synapses of adjacent neurones, favoring excitotoxic damage to the neurones. In support of this concept, Gunn rats given unilateral intrastriatal injections of N-methyl-d-aspartate (NMDA), an excitotonic analogue of glutamate, developed atrophy of the striatum and hippocampus, accompanied by ventricular dilatation on the affected side [23]. This injury was strikingly greater in jaundiced (jj) than in non-jaundiced (Jj) Gunn rats, was exaggerated by displacement of UCB from albumin by treatment with a sulfa drug, and was ameliorated by concurrent treatment with an NMDA-type glutamate channel antagonist. These early effects contrast with the 75% reduction glutamate concentrations in the hypoplastic cerebelli of 8-month-old jj Gunn rats, as compared with non-jaundiced Jj littermates [24].

In the Journal of Hepatology paper [20], apoptosis, evaluated by both Hoechst nuclear staining and the terminal dUTP nick-end-labeling (TUNEL) assay, was induced in a dose- and time-dependent fashion in primary cultures of rat astrocytes and neurones by exposure to UCB. Neurones were more sensitive, developing apoptosis when exposed for only 4 h to UCB/HSA ratios as low as 1.0, although not at a ratio of 0.5. Unfortunately, unlike the earlier paper [21], this was the only low molar ratio tested, so that possible effects throughout the range below saturation were not evaluated. It is also possible that, had higher, more physiologic albumin concentrations been used at similar low UCB/HSA ratios, the higher but still-relevant concentrations of unbound UCB would have caused apoptosis. The need for such studies is reinforced by the demonstrated decreases in the affinity of HSA for UCB as HSA concentrations increase [25], [26]. These in vitro effects of UCB are concordant with the appearance of what are now known to be classical histological and ultrastructural changes of neuronal apoptosis and astrocyte swelling in the cerebellum of jj Gunn rat pups as early as 3 days after birth, before peak plasma UCB levels are attained in the 3rd week of life.

If UCB is toxic to the CNS cells, how is the cellular content of the pigment regulated? Penetration of drugs and toxic compounds into the cerebrospinal fluid (CSF) and brain parenchyma is limited by the choroid plexus and the blood-brain barrier (BBB), respectively. In the BBB the tight junctions of the capillary endothelial cells are the barrier [27]. In the choroid plexus (CP), where the endothelial cells lack tight junctions and are fenestrated [28], the associated CP epithelial cells provide the barrier. Nonetheless, the non-ionized forms of relatively non-polar compounds diffuse freely across these barriers [29]; this includes UCB diacid [30], [31], [32]. Therefore, the low concentrations of UCB found in the brain, even of kernicteric Gunn rats [31], [33], might be due to the presence of transporters in the barriers and parenchymal cells that export UCB from the brain or CSF back into the blood. Alterations in any of these factors can increase the deposition of UCB into the CNS, raising its intracellular concentration and consequently its neurotoxicity.

Although UCB rapidly diffuses across membranes [32], uptake of UCB follows saturative kinetics in freshly isolated liver cells [34] and in basolateral liver plasma membrane vesicles [25], although the putative carrier(s) is still to be fully elucidated. It is uncertain whether this carrier-mediated basolateral uptake of UCB by the hepatocyte involves a member of the organic anion transport polypeptide family [35], [36]. This issue may be relevant to passage of UCB into the CNS, since oatp-1 protein is expressed and functional in the apical pole of the choroid plexus [37], [38], [39] and oatp-3 is expressed in rat brain [40].

An involvement of the MRP proteins in the ATP-dependent UCB cellular excretion has been suggested in the yeast, Saccharomyces cerevisiae [41]. In the brain, MRP1/mrp1 is highly expressed in CP epithelial cells of rat, mouse and human [42], in astrocytes from rats, humans, and other primates [42], [43]; and in the parenchymal cells of rats [42], [44]. In the knockout mrp1(−/−) mouse, the loss of mrp1 protein from the CP is associated with a 10× increase in the penetration of etoposide from blood into CSF in vivo [45]. MRP/mrp proteins are also detectable in the endothelium of brain microvessels of rats [44], humans and lower primates [46], but the expression is weak, probably due to downregulation when these cells are in contact with astrocytes, as shown in co-cultures [44]. Mrp1 is localized facing the blood and thus oriented to pump its substrates (including UCB?) into the blood, and limit their net passage from blood into the CSF and parenchymal cells respectively. Another cytoprotective ABC transporter, MDR1 (P-glycoprotein, Pgp) [46], [47], [48], is highly expressed at the apical pole of cerebral capillary endothelial cells (facing the blood) [46], [47], [48], and in CP epithelial cells (facing the CSF) [42], [45].

By exporting various substrates from these cells, the MRP and MDR transporters might play a major role in preventing accumulation of toxic levels of UCB in CNS neurones and astrocytes [42], [49]. Although the transport of UCB in yeast by analogues of MRP, but not of MDR [41], favors MRP1/mrp1 for this role in mammals, the possible involvement of MDR1/mdr1 in protecting the CNS against influx of UCB cannot be excluded. This hypothesis needs to be experimentally tested.

In conclusion, the new in vitro studies from Lisbon and studies of the function of CNS cells and organelles in untreated, jaundiced (jj) Gunn rats provide convincing evidence for a meaningful toxic effect of UCB at realistic concentrations in vivo. The evolving concepts of the mechanisms of neonatal jaundice, the penetration of UCB into the CNS and the neurotoxicity of UCB, may offer new prospects for prevention and treatment of bilirubin encephalopathy and for more accurate identification of newborns at risk who need such treatment. Led by the excellent examples of the studies of UCB neurotoxicity by the Lisbon group [20], [21] it is hoped that future studies will be performed with purified UCB at concentrations that are clinically relevant. Likewise, in vivo studies will be most meaningful if performed in untreated, jaundiced (jj) Gunn rats at various degrees of neurological impairment, with non-jaundiced (Jj) littermates as controls.

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Supported by grants from Italian Ministry of Health (ICS060.1/RF98.67), and Fondo Studi Fegato, Trieste, Italy and intramural research grant from the University of Trieste, Italy (60%-99).

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PII: S0168-8278(00)00051-9

doi:10.1016/S0168-8278(00)00051-9

Journal of Hepatology
Volume 34, Issue 3 , Pages 467-470, March 2001

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