The modulation of transcriptional expression and inhibition of multidrug resistance associated protein 4 (MRP4) by analgesics and their primary metabolites

During the course of a toxic challenge, changes in gene expression can manifest such as induction of metabolizing enzymes as a compensatory detoxification response. We currently report that a single 400 mg/kg acetaminophen (APAP)dose to C57BL/6J mice led to an increase in multidrug resistance-associated (Mrp) 4 (Abcc4) mRNA 12 h after administration. Alanine aminotransferase, as a marker of liver injury, was also elevated indicating hepatotoxicity had occurred. Therefore, induction of Mrp4 mRNA was likely attributable to APAP-induced liver injury. Mrp4 has been shown to be upregulated during oxidative stress, and it is well-established that APAP overdose causes oxidative stress due to depletion of glutathione. Given the importance of Mrp4 upregulation as an adaptive response during cholestatic and oxidative liver injury, we next investigated the extent by which human MRP4 can be inhibited by the analgesics, APAP, diclofenac (DCF), and their metabolites. Using an in vitro assay with inside out human MRP4 vesicles, we determined that APAP-cysteine inhibited MRP4 mediated transport of leukotriene C4 with an apparent IC50 of 125 μM. APAP-glutathione also attenuated MRP4 activity though it achieved only 28% inhibition at 300 μM. Diclofenac acyl glucuronide (DCF-AG) inhibited MRP4 transport by 34% at 300 μM. The MRP4 in vitro inhibition occurs at APAPcysteine and DCF-AG concentrations seen in vivo after toxic doses of APAP or DCF in mice, hence the findings are important given the role that Mrp4 serves as a compensatory response during oxidative stress following toxic challenge.Fil: Scialis, Renato J.. University of Connecticut; Estados UnidosFil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Manautou, José E.. University of Connecticut; Estados Unido

Differential \u3cem\u3eFmo3\u3c/em\u3e gene expression in various liver injury models involving hepatic oxidative stress in mice

Flavin-containing monooxygenase-3 (FMO3) catalyzes metabolic reactions similar to cytochrome P450 monooxygenase, however, most metabolites of FMO3 are considered non-toxic. Recent findings in our laboratory demonstrated Fmo3 gene induction following toxic acetaminophen (APAP) treatment in mice. The goal of this study was to evaluate Fmo3 gene expression in other diverse mouse models of hepatic oxidative stress and injury. Fmo3 gene regulation by Nrf2 was also investigated using Nrf2 knockout (Nrf2 KO) mice. In our studies, male C57BL/6J mice were treated with toxic doses of hepatotoxicants or underwent bile duct ligation (BDL, 10 days). Hepatotoxicants included APAP (400 mg/kg, 24–72 h), alpha-naphthyl isothiocyanate (ANIT; 50 mg/kg, 2–48 h), carbon tetrachloride (CCl4; 10 or 30 μL/kg, 24 and 48 h) and allyl alcohol (AlOH; 30 or 60 mg/kg, 6 and 24 h). Because oxidative stress activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), additional studies investigated Fmo3 gene regulation by Nrf2 using Nrf2 knockout (Nrf2 KO) mice. At appropriate time-points, blood and liver samples were collected for assessment of plasma alanine aminotransferase (ALT) activity, plasma and hepatic bile acid levels, as well as liver Fmo3 mRNA and protein expression. Fmo3 mRNA expression increased significantly by 43-fold at 12 h after ANIT treatment, and this increase translates to a 4-fold change in protein levels. BDL also increased Fmo3 mRNA expression by 1899-fold, but with no change in protein levels. Treatment of mice with CCl4 decreased liver Fmo3 gene expression, while no change in expression was detected with AlOH treatment. Nrf2 KO mice are more susceptible to APAP (400 mg/kg, 72 h) treatment compared to their wild-type (WT) counterparts, which is evidenced by greater plasma ALT activity. The Fmo3 mRNA and protein expression increased in Nrf2 KO mice after APAP treatment. Collectively, not all hepatotoxicants that produce oxidative stress alter Fmo3 gene expression. Along with APAP, toxic ANIT treatment in mice markedly increased Fmo3 gene expression. While BDL increased the Fmo3 mRNA expression, the protein level did not change. The discrepancy with Fmo3 induction in cholestatic models, ANIT and BDL, is not entirely clear. Results from Nrf2 KO mice with APAP suggest that the transcriptional regulation of Fmo3 during liver injury may not involve Nrf2

Constitutive activation of nuclear factor‐E2‐related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte‐specific deletion of Kelch‐like ECH‐associated protein 1

Chemicals that activate nuclear factor‐E2‐related factor 2 (Nrf2) often increase multidrug‐resistance‐associated protein (Mrp) expression in liver. Hepatocyte‐specific deletion of Kelch‐like ECH‐associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte‐specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild‐type and hepatocyte‐specific Keap1‐null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate–cysteine ligase catalytic subunit expression were increased in livers of Keap1‐null mice. Organic anion‐transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild‐type mice. By contrast, Mrp 1–5 mRNA and protein levels were increased in Keap1‐null mouse livers, with Mrp4 expression being more than 15‐fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions

Epidermal growth factor receptor (EGFR) activation induces the expression of multidrug resistance associated protein 4 (MRP4/ABCC4) in a pancreatic cancer human cell line

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent type of pancreatic cancer and has one of the worse prognosis. The poor overall survival is associated with the overexpression of epidermal growth factor receptor (EGFR), a known member of the ErbB family of receptor tyrosine kinases and multidrug resistance associated protein 4 (MRP4/ABCC4). Our previous results show that high levels MRP4 are associated with increase in tumor cell proliferation, metastatic invasion and up-regulation of EGFR protein levels in PDACs cell lines. The aim of our study was to evaluate the regulation of MRP4 by EGFR activation in a pancreatic cancer cell model. To accomplish our objective, we treated the pancreatic cancer cell line BxPC-3 with EGF (0.1ng/ul). EGFR activation was confirmed by ERK phosphorylation at 5, 10, 20, 30, and 40 min after EGF treatment. MRP4 protein expression was evaluated by western blot using whole cell extracts following incubation with EGF for 0, 24 and 48 h, using histone as loading control. MRP4 expression levels were also assessed 48 h after treatment with EGF alone or in combination with the EGFR inhibitor CL 387-785 (1µM). Our results confirm that EGFR is quickly activated upon incubation with EGF, as evidenced by a 4-fold increase in the pERK/total ERK ratio detected (P<0.001) at 5 min and normalized at 40 min. Maximal induction of MRP4 expression (86%, P<0.001) was observed in cells treated with EGF for 48 h. Furthermore, EGF-mediated MRP4 induction was abolished by co-treatment with CL387-785 (P<0.05), while its expression does not change by treatment with this EGFR inhibitor alone. These data demonstrate that EGFR activation produces increments in MRP4 protein levels in a PDAC cell line. In summary, it is possible that MRP4 and EGFR, both PDAC poor prognosis markers, are co-regulated by a positive feed-back which ultimately enhances their effect upon each other.Fil: Lago, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Barosso, Ismael Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Sahores, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Imperiale, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Manautou, José E.. University of Connecticut; Estados UnidosFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaReunión Anual de la Sociedad Argentina de FisiologíaRosarioArgentinaSociedad Argentina de Fisiologí

Hepatobiliary excretion of acetaminophen glutathione conjugate and its derivatives in transport-deficient (TR) hyperbilirubinemic rats. Drug Metab Dispos 31:798–804.

ABSTRACT: The involvement of the canalicular multidrug resistance protein 2 (Mrp2) in the hepatobiliary excretion of acetaminophen (APAP)-glutathione (GSH) conjugate and its derivatives was investigated using transport-deficient (TR Acetaminophen (APAP 1 ) is biotransformed through phase I and phase II reactions into negatively charged, hydrophilic metabolites that are eliminated in urine and bile. Cytochrome P450 converts APAP to the highly reactive electrophile N-acetyl-p-benzoquinoneimine Measuring the amount of APAP metabolites in urine and/or bile is considered a useful in vivo method for examining the effects of other chemicals on APAP metabolism, because changes in urinary and/or biliary excretion of APAP conjugates correlate well with changes in APAP biotransformation produced by xenobiotics We recently showed that the biliary concentration of APAP-GSH, APAP-NAC, and APAP-GLU, but not that of APAP itself, APAP-SUL or APAP-CG/CYS, was significantly decreased by coadministration of the nonmetabolizable organic anion indocyanine green (ICG) in male CD-1 mice A primary active transporter on the canalicular domain of hepatocytes, known as multidrug resistance protein 2 (Mrp2), mediates the hepatobiliary transport of a wide range of organic anions, including GSH-S-conjugates (e.g., leukotriene C4 and 2,4-dinitrophenyl-S-GSH), oxidized GSH (GSSG), glucuronide conjugates (e.g., glucu

From hepatoprotection models to new therapeutic modalities for treating liver diseases: a personal perspective [version 2; referees: 2 approved]

A variety of rodent models of hepatoprotection have been developed in which tolerance to acetaminophen-induced hepatotoxicity occurs. Autoprotection/heteroprotection is a phenomenon where prior exposure to a mildly toxic dose of toxicant confers protection against a subsequently administered higher dose of the same toxicant (as in the case of autoprotection) or to a different toxicant (referred to as heteroprotection). Multiple mechanisms regulate this adaptive response, including hepatocellular proliferation, proteostasis, enhanced expression of cytoprotective genes, and altered tissue immune response. In this review, we will discuss recent findings that highlight the complexity of these adaptive mechanisms and we also outline the usefulness of these findings to devise therapeutic and/or diagnostic tools for acetaminophen-induced liver damage in patients

Ontogenic mRNA expression of RNA modification writers, erasers, and readers in mouse liver.

RNA modifications are recently emerged epigenetic modifications. These diverse RNA modifications have been shown to regulate multiple biological processes, including development. RNA modifications are dynamically controlled by the "writers, erasers, and readers", where RNA modifying proteins are able to add, remove, and recognize specific chemical modification groups on RNAs. However, little is known about the ontogenic expression of these RNA modifying proteins in various organs, such as liver. In the present study, the hepatic mRNA expression of selected RNA modifying proteins involve in m6A, m1A, m5C, hm5C, m7G, and Ψ modifications was analyzed using the RNA-seq technique. Liver samples were collected from male C57BL/6 mice at several ages from prenatal through neonatal, infant, child to young adult. Results showed that most of the RNA modifying proteins were highly expressed in prenatal mouse liver with a dramatic drop at birth. After birth, most of the RNA modifying proteins showed a downregulation trend during liver maturation. Moreover, the RNA modifying proteins that belong to the same enzyme family were expressed at different abundances at the same ages in mouse liver. In conclusion, this study unveils that the mRNA expression of RNA modifying proteins follows specific ontogenic expression patterns in mice liver during maturation. These data indicated that the changes in expression of RNA modifying proteins might have a potential role to regulate gene expression in liver through alteration of RNA modification status

Acquired Resistance to Acetaminophen Hepatotoxicity is Associated with Induction of Multidrug Resistance-Associated Protein 4 (Mrp4) in Proliferating Hepatocytes

Treatment with hepatotoxicants such as acetaminophen (APAP) causes resistance to a second, higher dose of the same toxicant (autoprotection). APAP induces hepatic mRNA and protein levels of the multidrug resistance-associated proteins (Mrp) transporters in mice and humans. Basolateral efflux transporters Mrp3 and Mrp4 are the most significantly induced. We hypothesized that upregulation of Mrp3 and Mrp4 is one mechanism by which hepatocytes become resistant to a subsequent higher dose of APAP by limiting accumulation of xeno-, endobiotics, and byproducts of hepatocellular injury. The purpose of this study was to evaluate Mrp3 and Mrp4 expression in proliferating hepatocytes in a mouse model of APAP autoprotection. Plasma and livers were collected from male C57BL/6J mice treated with APAP 400 mg/kg for determination of hepatotoxicity and protein expression. Maximal Mrp3 and Mrp4 induction occurred 48 h after APAP. Mrp4 upregulation occurred selectively in proliferating hepatocytes. Additional groups of APAP-pretreated mice were challenged 48 h later with a second, higher dose of APAP. APAP-pretreated mice had reduced hepatotoxicity after APAP challenge compared to those pretreated with vehicle. A more rapid recovery of glutathione (GSH) in APAP-pretreated mice corresponded with increases in GSH synthetic enzymes. Interestingly, mice pretreated and challenged with APAP had dramatic increases in Mrp4 expression as well as enhanced hepatocyte proliferation. Inhibition of hepatocyte replication with colchicine not only restored sensitivity of APAP-pretreated mice to injury, but also blocked Mrp4 induction. Mrp4 overexpression may be one phenotypic property of proliferating hepatocytes that protects against subsequent hepatotoxicant exposure by mechanisms that are presently unknown

Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice

Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400 mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, NAD(P)H:quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24 h after APAP treatment. Nrf2 nuclear content increased by 58% at 12 h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention.Fil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Rudraiah, Swetha. University Of Connecticut; Estados UnidosFil: Bataille, Amy M.. University Of Connecticut; Estados UnidosFil: Vigo, María B.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas (i); Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Goedken, Michael J.. Rutgers University. Office of Translational Science; Estados UnidosFil: Manautou, José E.. University Of Connecticut; Estados Unido