Kenichiro Ogura

Characterization of Organic Anion Transporting Polypeptide 1b2-null Mice: Essential Role in Hepatic Uptake/Toxicity of Phalloidin and Microcystin-LR

The liver-specific importer organic anion transporting polypeptide 1b2 (Oatp1b2, Slco1b2, also known as Oatp4 and Lst-1) and its human orthologs OATP1B1/1B3 transport a large variety of chemicals. Oatp1b2-null mice were engineered by homologous recombination and their phenotype was characterized. Oatp1b2 protein was absent in livers of Oatp1b2-null mice. Oatp1b2-null mice develop normally and breed well. However, adult Oatp1b2-null mice had moderate conjugated hyperbilirubinemia. Compared with wild-types, Oatp1b2-null mice had similar hepatic messenger RNA expression of most transporters examined except a higher Oatp1a4 but lower organic anion transporter 2. Intra-arterial injection of the mushroom toxin phalloidin (an Oatp1b2-specific substrate identified in vitro) caused cholestasis in wild-type mice but not in Oatp1b2-null mice. Hepatic uptake of fluorescence-labeled phalloidin was absent in Oatp1b2-null mice. Three hours after administration of microcystin-LR (a blue-green algae toxin), the binding of microcystin-LR to hepatic protein phosphatase 1/2a was much lower in Oatp1b2-null mice compared with wild-type mice. In contrast, Oatp1b2-null mice were transiently protected from decrease in bile flow induced by estradiol-17beta-D-glucuronide, a common substrate for Oatps. Oatp1b2-null mice were completely resistant to the hepatotoxicity induced by phalloidin and microcystin-LR, but were similarly sensitive to alpha-amanitin-induced hepatotoxicity compared with wild-type mice. In conclusion, Oatp1b2-null mice display altered basic physiology and markedly decreased hepatic uptake/toxicity of phalloidin and microcystin-LR. Oatp1b2-null mice are useful in elucidating the role of Oatp1b2 and its human orthologs OATP1B1/1B3 in hepatic uptake and systemic disposition of toxic chemicals and therapeutic drugs.

Reverse geometrical selectivity in glucuronidation and sulfation of cis- and trans-4-hydroxytamoxifens by human liver UDP-glucuronosyltransferases and sulfotransferases.

The phenolic active metabolites, cis-4-hydroxytamoxifen (cis-HO-TAM) and trans-4-hydroxytamoxifen (trans-HO-TAM), of the anti-breast-cancer drug, trans-tamoxifen (TAM), were geometrically selectively glucuronidated in the manner of cis>>trans by microsomes and sulfated in the manner of trans>>cis by cytosol from the liver of 10 human subjects (7 females and 3 males). There was a large individual difference in the microsomal glucuronidation of cis-HO-TAM, which correlated well with glucuronidation of 4-hydroxybiphenyl by human liver microsomes. However, there was only a slight correlation between the glucuronidation of cis-HO-TAM and trans-HO-TAM or 4-nitrophenol (NP). A small individual difference was observed for the human liver cytosolic sulfation of trans-HO-TAM, which correlated well with the sulfation of NP. Recombinant human UDP-glucuronosyltransferase (UGT)2B15 catalyzed the cis-selective glucuronidation of geometrical isomers of HO-TAM. UGTs1A1, 1A4, 1A9 and 2B7 had weak activity toward HO-TAMs with a much smaller cis-selectivity than did UGT2B15. UGTs1A3 and 1A6 had no detectable activity toward these substrates. Among the four known major sulfotransferases (SULTs) occurring in the human liver, SULT1A1 was strongly suggested to play the most important role in the hepatic cytosolic trans-selective sulfation of HO-TAM isomers. A good correlation was observed between the hepatic cytosolic sulfation of trans-HO-TAM and NP, a standard substrate for SULT1A1. SULT1E1 had slight activity toward the HO-TAMs. SULTs1A3 and 2A1 had no detectable activity toward HO-TAMs.

Suicidal inactivation of human dihydropyrimidine dehydrogenase by (E)-5-(2-bromovinyl)uracil derived from the antiviral, sorivudine.

An enzymatic study was performed to clarify the mechanism of 18 acute deaths in patients who had received the new oral antiviral drug, sorivudine (SRV), during anticancer chemotherapy with 5-fluorouracil (5-FU) prodrugs. Human dihydropyrimidine dehydrogenase (hDPD), playing a key role in the liver as the rate-limiting enzyme in catabolism of 5-FU, was expressed in E. coli, purified and incubated in the presence of NADPH with SRV or (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of SRV produced by human gut flora. hDPD was rapidly and irreversibly inactivated by BVU, but not by SRV. Radioactivity of [14C]BVU was incorporated into hDPD in the presence of NADPH in a manner reciprocal to the enzyme inactivation. In the absence of NADPH, hDPD was not inactivated by BVU, nor radiolabeled with [14C]BVU. Thus, as we demonstrated previously with studies using the rat, the acute deaths were strongly suggested to be attributable to markedly elevated tissue 5-FU levels which were responsible for irreversible inhibition of hDPD by covalent binding of a reduced form of BVU as a suicide inactivator.

Inhibitory effects of various beverages on ritodrine sulfation by recombinant human sulfotransferase isoforms SULT1A1 and SULT1A3

PurposeRitodrine is known to undergo extensive presystemic sulfation in the intestinal mucosa, and its bioavailability is as low as 30%. Accordingly, inhibition of intestinal sulfation may lead to an increase in the bioavailability of ritodrine. In this study, we aimed to investigate the activities of ritodrine sulfation by SULT1A1, which is expressed predominantly in the liver, and SULT1A3, which is expressed predominantly in the intestine, as well as the inhibitory effects of beverages on their activities.MethodsWe investigated ritodrine sulfation by using recombinant human sulfotransferase (SULT) 1A1 and SULT1A3 in an in vitro study. Next, we investigated the inhibitory effects of grapefruit juice, orange juice, green tea, and black tea on ritodrine sulfation.ResultsSulfation of ritodrine by SULT1A3 was much higher than that by SULT1A1, suggesting that the bioavailability of ritodrine may be limited by intestinal SULT1A3. The ritodrine sulfation activities of SULT1A1 and SULT1A3 were significantly inhibited by all beverages examined at a concentration of 10%. Green tea and black tea exhibited potent inhibition; even at a concentration of 5%, they both inhibited SULT1A1 by 100% and SULT1A3 by ≥95%.ConclusionOur results suggest that concomitant ingestion of beverages such as green tea and black tea may increase the bioavailability of orally administered ritodrine, and perhaps other β2-agonists, and lead to an increase in the clinical effects or adverse reactions.