Galactosamine prevents ethinylestradiol-induced cholestasis.

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

A functional role for histidyl residues of the UDP-glucuronic acid carrier in rat liver endoplasmic reticulum membranes.

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.