Probenecid, an organic anion transporter 1 and 3 inhibitor, increases plasma and brain exposure of N-acetylcysteine.

1. N-acetylcysteine (NAC) is being investigated as an antioxidant for several conditions including traumatic brain injury, but the mechanism by which it crosses membrane barriers is unknown. We have attempted to understand how the transporter inhibitor, probenecid, affects NAC pharmacokinetics and to evaluate the interaction of NAC with transporters. 2. Juvenile Sprague-Dawley rats were administered NAC alone or in combination with probenecid intraperitoneally. Plasma and brain samples were collected serially and NAC concentrations were measured. Transporter studies were conducted with human embryonic kidney-293 cells that overexpress organic anion transporter (OAT)1 or OAT3 and with human multi-drug resistance-associated protein (MRP)1 or MRP4 membrane vesicles. 3. NAC area under the curve was increased in plasma (1.65-fold) and brain (2.41-fold) by probenecid. The apparent plasma clearance was decreased by 65%. Time- and concentration-dependent NAC uptake that was inhibitable by probenecid was observed with OAT1 and OAT3. No uptake of NAC was observed with MRP1 or MRP4. 4. Our results indicate for the first time that NAC is substrate for OAT1 and OAT3 and that probenecid increases NAC plasma and brain exposure in vivo. These data provide insight regarding how NAC crosses biological barriers and suggest a promising therapeutic strategy to increase NAC exposure.

Lipid peroxidation is not the primary mechanism of bilirubin-induced neurologic dysfunction in jaundiced Gunn rat pups.

BACKGROUND: Hazardous levels of bilirubin produce oxidative stress in vitro and may play a role in the genesis of bilirubin-induced neurologic dysfunction (BIND). We hypothesized that the antioxidants taurourosdeoxycholic acid (TUDCA), 12S-hydroxy-1,12-pyrazolinominocycline (PMIN), and minocycline (MNC) inhibit oxidative stress and block BIND in hyperbilirubinemic j/j Gunn rat pups that were given sulfadimethoxine to induce bilirubin encephalopathy. METHODS: At peak postnatal hyperbilirubinemia, j/j Gunn rat pups were dosed with sulfadimethoxine to induce bilirubin encephalopathy. Pups were given TUDCA, PMIN, MNC, or vehicle pretreatment (15 min before sulfadimethoxine). After 24 h, BIND was scored by using a rating scale of neurobehavior and cerebellar tissue 4-hydroxynonenal and protein carbonyl dinitrophenyl content were determined. Nonjaundiced heterozygous N/j pups served as controls. RESULTS: Administration of sulfadimethoxine induced BIND and lipid peroxidation but not protein oxidation in hyperbilirubinemic j/j pups. TUDCA, PMIN, and MNC each reduced lipid peroxidation to basal levels observed in nonjaundiced N/j controls, but only MNC prevented BIND. CONCLUSION: These findings show that lipid peroxidation inhibition alone is not sufficient to prevent BIND. We speculate that the neuroprotective efficacy of MNC against BIND involves action(s) independent of, or in addition to, its antioxidant effects.

Unconjugated bilirubin efflux by bovine brain microvascular endothelial cells in vitro.

OBJECTIVES: The passage of unconjugated bilirubin (UCB) across the blood-brain barrier into the central nervous system is a crucial first step in the development of kernicterus. The objective of the current study was to characterize the passage of UCB across primary bovine brain microvascular endothelial cell (BBMVEC) monolayers in vitro. DESIGN: Experimental study. SETTING: Research institute. SUBJECTS: BBMVECs. INTERVENTIONS: Tritiated UCB (H-UCB) transport at 60, 80, 100, 200, 300, and 400 nM concentrations was tested in both the apical to basolateral (A--> B) and basolateral to apical (B-->A) directions in BBMVEC monolayers in vitro with or without preincubation with pharmacologic active transport inhibitors cyclosporine A, indomethacin, or MK571. MEASUREMENTS AND MAIN RESULTS: The rate of H-UCB transport in the B-->A direction was 6.2- to 7.3-fold higher than in the A-->B direction, suggesting active efflux of UCB. Cyclosporine A (5 microM), a model inhibitor of P-glycoprotein, enhanced A-->B while decreasing B-->A UCB transport, resulting in an overall decrease in BBMVEC UCB efflux of between 46% and 54%. Indomethacin (10 microM) and MK-571 (50 microM), respectively a substrate and potent inhibitor of multidrug resistance-associated protein-1, had no effect. CONCLUSIONS: We conclude that 1) UCB is transported by BBMVEC monolayers in vitro in a net B-->A direction (i.e., active efflux); and 2) cyclosporine A partially inhibits such transport. We speculate that the blood-brain barrier limits the passage and central nervous system retention of UCB by active transport and that this may be accounted in part by P-glycoprotein.

Calculated in vivo free bilirubin levels in the central nervous system of Gunn rat pups.

In vitro studies suggest a free bilirubin (B(F)) concentration in the range of 71-770 nmol/L can induce neurotoxicity. In vivo data regarding central nervous system (CNS) B(F) levels have not been determined. We calculated in vivo CNS B(F) levels in Gunn rat pups (15-19 d old; heterozygous nonjaundiced Gunn rats (J/j) and homozygous jaundiced Gunn rats (j/j); saline or sulfadimethoxine treated) based on 1) total brain bilirubin (TBB) content, 2) brain albumin level, 3) CNS bilirubin binding capacity attributable to brain albumin determined using an ultrafiltration technique, and 4) published Gunn rat albumin-bilirubin binding constants (k). Gunn rat brain bilirubin binding capacity was approximately 22 x 10(-3) micromol/g, of which two thirds was accounted for by brain albumin. Using a Gunn rat pup in vivo, k of 9.2 L/micromol, calculated CNS B(F) levels ranged from 72 to 112 nmol/L [95% confidence interval (CI)] in saline and from 59 to 156 nmol/L (95% CI) in sulfadimethoxine-treated J/j pups. These animals demonstrated no neurobehavioral abnormalities and normal cerebellar weight. Calculated CNS B(F) levels were severalfold higher (p < 0.001) in saline (95% CI: 556-1110 nmol/L) and sulfadimethoxine-treated (95% CI: 3461-8985 nmol/L) j/j pups; the former evidenced reduced cerebellar weight; the latter both reduced cerebellar weight and acute neurobehavioral abnormalities. We conclude that calculated CNS B(F) values in j/j pups are substantially higher than those in J/j animals. Given the absence of CNS abnormalities in J/j pups, the presence of such in j/j animals, and the CNS B(F) levels in these groups, we speculate that the CNS B(F) neurotoxicity threshold in vivo is subsumed within the range (71-770 nmol/L) reported in vitro.

Sex-specific regional brain bilirubin content in hyperbilirubinemic Gunn rat pups.

BACKGROUND: The hyperbilirubinemic j/j Gunn rat is frequently used to study the effects of neonatal hyperbilirubinemia on the developing central nervous system (CNS). Despite evidence that the cerebellar region and males are predisposed to bilirubin-induced brain injury in this animal model, there are limited regional and no sex-specific brain bilirubin content data. OBJECTIVE/METHODS: To characterize and contrast the regional (cortex, brainstem, cerebellum) and sex-specific CNS bilirubin contents of hyperbilirubinemic j/j Gunn rat pups and their age-matched (15-19 days) nonjaundiced J/j counterparts. Pups were injected 24 h prior to sacrifice with sulfadimethoxine (200 mg/kg i.p.) to enhance the CNS bilirubin content. RESULTS: The CNS bilirubin contents in each region and total serum bilirubin levels were significantly greater in jaundiced j/j pups versus nonjaundiced J/j pups. Within the sulfadimethoxine-treated male j/j cohort, the mean brain bilirubin content was highest in the cerebellum (18.9 +/- 7.8 microg/g), intermediate in the brainstem (10.7 +/- 8.0 microg/g), and lowest in the cortex (4.7 +/- 3.0 microg/g) (F = 11.31, p < 0.001 by ANOVA), and the cerebellar bilirubin level was significantly higher than in the littermate-matched sulfadimthoxine-treated j/j female pups (p < 0.02). The serum albumin levels were not different between j/j male and j/j female pups. CONCLUSIONS: We conclude that the brain bilirubin content of hyperbilirubinemic j/j Gunn rat pups is greater than in nonjaundiced J/j pups and varies as a function of CNS region and sex. We speculate that the higher cerebellar bilirubin content may preferentially predispose male j/j Gunn rat pups to bilirubin-induced neurotoxicity.

IUGR alters postnatal rat skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1 gene expression in a fiber specific manner.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of insulin resistance in humans and rats. Aberrant skeletal muscle lipid metabolism contributes to the pathogenesis of insulin resistance. Peroxisome proliferator-activated receptor-gamma co-activator-1 (PGC-1) is a transcriptional co-activator that affects gene expression of key lipid metabolizing enzymes such as carnitine palmitoyl-transferase I (mCPTI). Because gene expression of lipid metabolizing enzymes is altered in IUGR postnatal skeletal muscle, and we hypothesized that PGC-1 expression would be similarly affected. To prove this hypothesis, bilateral uterine artery ligation and sham surgery were used to produce IUGR and control rats respectively. Western Blotting demonstrated that PGC-1 hind limb skeletal muscle protein levels were increased in perinatal and postnatal IUGR rats. Conventional RT-PCR demonstrated that PGC-1 mRNA levels were similarly increased in perinatal hind limb skeletal muscle and juvenile extensor digitorum longus (EDL), but were decreased in juvenile soleus. Because a gender specific trend was noted in PGC-1 mRNA levels, real time RT-PCR was used for further differentiation. Real time RT-PCR revealed that changes in postnatal skeletal muscle PGC-1 expression were more marked in male IUGR rats versus female IUGR rats. Down stream targets of PGC-1 followed a similar pattern of expression. We conclude that PGC-1 expression is altered in rat IUGR skeletal muscle and speculate that it contributes to the pathogenesis of insulin resistance in the IUGR rat.

Understanding neonatal hyperbilirubinaemia in the era of genomics.

The genomics revolution offers novel approaches to scientific investigation. Application of genomics technologies including microarray gene chips will provide a more complete picture of biological phenomena and help define the genetic contribution to disease by monitoring changes in expression across thousands of genes in physiological and clinical contexts. We briefly summarize identified genetic components that contribute to the genesis of neonatal hyperbilirubinaemia with a focus on inborn errors of hepatic bilirubin conjugation and discuss the potential use of microarray gene expression profiling technology to enhance our understanding of the pathogenesis of hyperbilirubinaemic neuronal cell injury. Expanded study using the tools of genomics will shed insights into the genetics of newborn jaundice and the pathogenesis of hyperbilirubinaemic encephalopathy.

P-glycoprotein expression in mouse brain increases with maturation.

The mdr1a isoform of P-glycoprotein (Pgp) is an integral plasma membrane efflux pump expressed in adult brain capillary endothelial cells and astrocytes of the blood-brain barrier. We determined the developmental pattern of Pgp expression in brain tissue at embryonic day 16 (E16), day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adults (Ad). The relative expression of Pgp mRNA and protein was indexed as a percent (mean +/- SEM) of D0 levels. Pgp mRNA levels increased significantly (p < 0.01) with maturation (E16: 75 +/- 8%; D21: 303 +/- 37%, and Ad: 1,160 +/- 120%). Similarly, Pgp protein expression was observed at E16 and increased significantly (p < 0.01) during development (E16: 52 +/- 8%; D7: 187 +/- 23%; D21: 440 +/- 48%, and Ad: 441 +/- 56%). This developmental pattern of enhanced blood-brain barrier Pgp expression with maturation was confirmed by immunohistochemistry. We conclude that (i) Pgp expression in mouse brain is limited during late embryogenesis and the newborn period; (ii) Pgp expression increases markedly with postnatal maturation, and (iii) by D21 brain Pgp protein expression approximates adult levels.