Nathan J Cherrington

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.

PMID: 18474683;PMCID: PMC3693743;Abstract:

Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2^-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR^-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.

Treatment with the microsomal enzyme inducer trans-stilbene oxide (TSO) can decrease biliary excretion of acetaminophen-glucuronide (AA-GLUC) and increase efflux of AA-GLUC into blood. The hepatic canalicular multidrug resistance protein (Mrp) 2 and sinusoidal protein Mrp3 transport AA-GLUC conjugates into bile and blood, respectively. Thus, TSO-induced alterations in the vectorial excretion of AA-GLUC may occur via increased hepatic Mrp3 levels. The goal of this study was to determine whether TSO, diallyl sulfide (DAS), and oltipraz (OLT) treatments can up-regulate Mrp3 protein expression, and whether treatment with DAS and OLT can correspondingly increase hepatovascular efflux of AA metabolites. Rats were administered phenobarbital, TSO, DAS, OLT, or vehicle for 4 days. Interestingly, all of the chemicals increased the plasma concentration and urinary excretion of AA-GLUC and decreased its biliary excretion. In control animals, approximately 77% and 23% of AA-GLUC was excreted into bile or urine, respectively, whereas with inducer-pretreated animals, 32% of AA-GLUC was excreted into bile and >68% was excreted into urine. Correspondingly, all of the compounds increased hepatic Mrp3 mRNA levels by 13- to 37-fold and protein levels by 2- to 6-fold, respectively. In conclusion, these studies correlate increased Mrp3 protein levels in liver with increased hepatovascular excretion of AA-GLUC and suggest that induction of Mrp3 affects the route of drug excretion.

Nonalcoholic fatty liver disease (NAFLD) is represented by a spectrum of liver pathologies ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Liver damage sustained in the progressive stages of NAFLD may alter the ability of the liver to properly metabolize and eliminate xenobiotics. The purpose of the current study was to determine whether NAFLD alters the disposition of the environmental toxicant arsenic. C57BL/6 mice were fed either a high-fat or a methionine-choline-deficient diet to model simple steatosis and NASH, respectively. At the conclusion of the dietary regimen, all mice were given a single oral dose of either sodium arsenate or arsenic trioxide. Mice with NASH excreted significantly higher levels of total arsenic in urine (24 h) compared with controls. Total arsenic in the liver and kidneys of NASH mice was not altered; however, NASH liver retained significantly higher levels of the monomethyl arsenic metabolite, whereas dimethyl arsenic was retained significantly less in the kidneys of NASH mice. NASH mice had significantly higher levels of the more toxic trivalent form in their urine, whereas the pentavalent form was preferentially retained in the liver of NASH mice. Moreover, hepatic protein expression of the arsenic biotransformation enzyme arsenic (3+ oxidation state) methyltransferase was not altered in NASH animals, whereas protein expression of the membrane transporter multidrug resistance-associated protein 1 was increased, implicating cellular transport rather than biotransformation as a possible mechanism. These results suggest that NASH alters the disposition of arsenical species, which may have significant implications on the overall toxicity associated with arsenic in NASH.

PMID: 23639800;PMCID: PMC3684844;Abstract:

The blood-testis barrier (BTB) prevents the entry of many xe-nobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiologic nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR, 6-S- [(4-nitrophenyl)methyl]-6-thioinosine), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir disoproxil fumarate, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.