Compensatory Expression of MRP3 in the Livers of MRP2-Deficient EHBRs Is Promoted by DHA Intake

We have hypothesized a suppressive mechanism against dietary docosahexaenoic acid (22:6n-3; DHA)-induced tissue lipid peroxidation, in which the degradation products, including their conjugates, are excreted into the urine by xenobiotic or organic anion transporters. In this study, we employed parent-strain Sprague-Dawley rats (SDRs), together with their mutant strain, Eisai hyperbilirubinuria rats (EHBRs). EHBRs are deficient in multidrug resistance-associated protein (MRP) 2, and show defective urinary excretion of numerous xenobiotics and organic anions. Both strains of rats were fed a diet containing DHA at 8.4% of total energy for 31 d. In the livers of the DHA-fed rats, the level of free malondialdehyde (MDA) + 4-hydroxy-2-alkenals (HAE) fell, and conversely glutathione S-transferase (GST) activity increased in MRP2-deficient EHBRs as compared to the SDRs, suggesting that the glutathione (GSH)-conjugation reaction for the aldehydes generated on DHA intake was accelerated in the MRP2-deficient EHBRs. Since the gene expression of liver MRP3 in the MRP2-deficient EHBRs was amplified to compensate for DHA intake, it is thought that the transport of MRP3 substrates into the bloodstream, rather than MRP2-mediated excretion of its substrates into the bile, was promoted. Indeed, excretion of mercapturic acid (acetylcysteine conjugates derived metabolically from the conjugate of each aldehyde with GSH) into the urine increased significantly in MRP2-deficient EHBRs fed DHA.     

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

Free radical mediated oxidative stress and toxic side effects in brain induced by the anti cancer drug adriamycin: insight into chemobrain

Adriamycin (ADR) is a chemotherapeutic agent useful in treating various cancers. ADR is a quinone-containing anthracycline chemotherapeutic and is known to produce reactive oxygen species (ROS) in heart. Application of this drug can have serious side effects in various tissues, including brain, apart from the known cardiotoxic side effects, which limit the successful use of this drug in treatment of cancer. Neurons treated with ADR demonstrate significant protein oxidation and lipid peroxidation. Patients under treatment with this drug often complain of forgetfulness, lack of concentration, dizziness (collectively called somnolence or sometimes called chemobrain). In this study, we tested the hypothesis that ADR induces oxidative stress in brain. Accordingly, we examined the in vivo levels of brain protein oxidation and lipid peroxidation induced by i.p. injection of ADR. We also measured levels of the multidrug resistance-associated protein (MRP1) in brain isolated from ADR- or saline-injected mice. MRP1 mediates ATP-dependent export of cytotoxic organic anions, glutathione S-conjugates and sulphates. The current results demonstrated a significant increase in levels of protein oxidation and lipid peroxidation and increased expression of MRP1 in brain isolated from mice, 72 h post i.p injection of ADR. These results are discussed with reference to potential use of this redox cycling chemotheraputic agent in the treatement of cancer and its chemobrain side effect in brain.     

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E₂ (PGE₂) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE₂ can bind to four receptor subtypes, EP₁–EP₄, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE₂, dexamethasone, and agonists for the EP₁ and EP₂ receptor subtypes. An EP₁ antagonist, SC-19220, inhibited the induction of enzyme activity by PGE₂ or 17-phenyltrinor-PGE₂, an EP₁ agonist. Sulprostone, an EP₃ agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.     

Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats

We have shown that aluminum (Al) induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2. This work aims to investigate whether these harmful effects are mediated by the oxidative stress caused by the metal. For this purpose, the capability of the antioxidant agent, vitamin E, to counteract these alterations was studied in male Wistar rats. Aluminum hydroxide (or saline in controls) was administered ip (27 mg/kg body weight, three times a week, for 90 d). Vitamin E (600 mg/kg body weight) was coadministered, sc. Al increased lipid peroxidation (+50%) and decreased hepatic glutation levels (-43%) and the activity of glutation peroxidase (-50%) and catalase (-88%). Vitamin E counteracted these effects total or partially. Both plasma and hepatic Al levels reached at the end of the treatment were significantly reduced by vitamin E (-40% and -44%, respectively;p< 0.05). Al increased 4 times the hepatic apoptotic index, and this effect was fully counteracted by vitamin E. Bile flow was decreased in Al-treated rats (-37%) and restored to normality by vitamin E. The antioxidant normalized the hepatic handling of the Mrp2 substrates, rose bengal, and dinitrophenyl-S-glutathione, which was causally associated with restoration of Mrp2 expression. Our data indicate that oxidative stress has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage, and the impairment in liver transport function induced by Al and that vitamin E counteracts these harmful effects not only by preventing free-radical formation but also by favoring Al disposal.     

B4GALT family mediates the multidrug resistance of human leukemia cells by regulating the hedgehog pathway and the expression of p-glycoprotein and multidrug resistance-associated protein 1

β-1, 4-Galactosyltransferase gene (B4GALT) family consists of seven members, which encode corresponding enzymes known as type II membrane-bound glycoproteins. These enzymes catalyze the biosynthesis of different glycoconjugates and saccharide structures, and have been recognized to be involved in various diseases. In this study, we sought to determine the expressional profiles of B4GALT family in four pairs of parental and chemoresistant human leukemia cell lines and in bone marrow mononuclear cells (BMMC) of leukemia patients with multidrug resistance (MDR). The results revealed that B4GALT1 and B4GALT5 were highly expressed in four MDR cells and patients, altered levels of B4GALT1 and B4GALT5 were responsible for changed drug-resistant phenotype of HL60 and HL60/adriamycin-resistant cells. Further data showed that manipulation of these two gene expression led to increased or decreased activity of hedgehog (Hh) signaling and proportionally mutative expression of p-glycoprotein (P-gp) and MDR-associated protein 1 (MRP1) that are both known to be related to MDR. Thus, we propose that B4GALT1 and B4GALT5, two members of B4GALT gene family, are involved in the development of MDR of human leukemia cells, probably by regulating the activity of Hh signaling and the expression of P-gp and MRP1.     

Modulation of TRPV2 by endogenous and exogenous ligands: A computational study

Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.