Nuclear receptor and nuclear receptor target gene messenger ribonucleic acid levels at different sites of the gastrointestinal tract and in liver of healthy dogs

Nuclear receptors (NR) are ligand-activated transcription factors that regulate different metabolic pathways by influencing the expression of target genes. The current study examined mRNA abundance of NR and NR target genes at different sites of the gastrointestinal tract (GIT) and the liver of healthy dogs (Beagles; n = 11). Samples of GIT and liver were collected postmortem and homogenized, total RNA was extracted and reverse transcribed, and gene expression was quantified by real-time reverse-transcription PCR relative to the mean of 3 housekeeping genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and ubi-quitin). Differences were observed (P < or = 0.05) in the mRNA abundance among stomach (St), duodenum (Du), jejunum (Je), ileum (Il), and colon (Col) for NR [pregnane X receptor (Du, Je > Il, Col > St), peroxisome proliferator-associated receptor gamma (St, Du, Col > Je, Il), constitutive androstane receptor (Je, Du > Il, Col), and retinoid x receptor alpha (Du > Il)] and NR target genes [glutathione-S-transferase A3-3 (Du > Je > St, Il; St > Col), phenol-sulfating phenol sulfotransferase 1A1 (Du, Je > Il, St; Col > St), cytochrome P450 3A12 (Du, Je > St, Il, Col), multiple drug resistance gene 1 (Du, Je, Il, Col > St), multiple drug resistance-associated protein 2 (Je, Du > Il > St, Col), multiple drug resistance-associated protein 3 (Col > St > Il; Du > Je, Il; St > Il), NR corepressor 2 (St > Il, Col), and cytochrome P450 reductase (St, Du, Je > Il, Col)], but not for peroxisome proliferator-associated receptor alpha. Differences (P > 0.05) in mRNA abundance in the liver relative to the GIT were also observed. In conclusion, the presence of numerous differences in expression of NR and NR target genes in different parts of the GIT and in liver of healthy dogs may be associated with location-specific functions and regulation of GIT regions.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

Hypoxic preconditioning reverses protection after neonatal hypoxia-ischemia in glutathione peroxidase transgenic murine brain

The effect of hypoxic preconditioning (PC) on hypoxic-ischemic (HI) injury was explored in glutathione peroxidase (GPx)-overexpressing mice (human GPx-transgenic [hGPx-tg]) mice. Six-day-old hGPx-tg mice and wild-type (Wt) littermates were pre-conditioned with hypoxia for 30 min and subjected to the Vannucci procedure of HI 24 h after the PC stimulus. Histopathological injury was determined 5 d later (P12). Additional animals were killed 2 h or 24 h after HI and ipsilateral cerebral cortices assayed for GPx activity, glutathione (GSH), and hydrogen peroxide (H2O2). In line with previous studies, hypoxic PC reduced injury in the Wt brain. Preconditioned Wt brain had increased GPx activity, but reduced GSH, relative to naive 24 h after HI. Hypoxic PC did not reduce injury to hGPx-tg brain and even reversed the protection previously reported in the hGPx-tg. GPx activity and GSH in hGPx-tg cortices did not change. Without PC, hGPx-tg cortex had less H2O2 accumulation than Wt at both 2 h and 24 h. With PC, H2O2 remained low in hGPx-tg compared with Wt at 2 h, but at 24 h, there was no longer a difference between hGPx-tg and Wt cortices. Accumulation of H2O2 may be a mediator of injury, but may also induce protective mechanisms.

Oxidative stress in lungs of mice infected with influenza A virus

As oxidative stress has been implicated in the pathogenesis of certain viral diseases we determined antioxidant and prooxidant parameters in lungs and bronchoalveolar lavage fluid (BALF) of mice infected with a lethal dose of influenza A/PR8/34 virus. Viral infection was characterized by massive infiltration of leukocytes, mainly polymorphonuclear leukocytes, into the alveolar space. The total number of BALF cells increased up to 8-fold (day 3 post-infection) and these cells appeared activated as judged by their increased rates of superoxide anion radical (O2-.) generation upon stimulation. Maximal rates of radical generation by BALF cells during the early stages of infection were 15- or 70-fold higher than those of cells from control animals when expressed per cell or total BALF cells, respectively. At the terminal stages of infection the total capacity of BALF cells to release O2-. declined to approximately 35-fold the control values. Infection also resulted in increased in vivo formation of hydrogen peroxide (H2O2) within the lungs at a time that coincided with the maximal capacity of BALF cells to release O2-.. Whereas pulmonary activities of glutathione peroxidase and reductase remained unaltered, levels of ascorbate in the cell-free BALF decreased significantly during the early stages of the infection and then returned to normal levels and above, late in infection. The oxidation state of the dehydroascorbic acid/ascorbate couple increased concomitantly with the decrease in ascorbate concentrations early in infection and remained elevated throughout the infection. As assessed by the prevention of peroxyl radical-induced loss of phycoerythrin fluorescence, the total antioxidant capacity present in lung tissue homogenate from terminally ill animals was not diminished when compared to that prepared from lungs of control mice. We conclude that although early stages of influenza infection are associated with the presence of oxidative stress in the lung tissue and alveolar fluid lining the epithelial cells, this stress does not appear to overwhelm local antioxidant defenses. The results therefore do not support a direct causative role of oxidative tissue damage in the pathogenesis of influenza virus infection.

Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

Reductase inhibitory activity of some flavones: Topological descriptors in modeling the activity

In healthy people, glucose is metabolized through Embden-Meyerhoff pathway. In cases of diabetes mellitus, with the increased levels of glucose in insulin-insensitive tissues the Aldose Reductase (AR) in polyol pathway facilitates the conversion of glucose to sorbitol. In this cascade of events the accumulated sorbitol is attributed to be responsible for cataract, neuropathy and retinopathy in diabetic cases.1,2 Thus, the inhibition of AR in polyol pathway may prevent and lead to the cure of the complications arising out of the diabetes mellitus. In this background, Matsuda and coworkers3 studied the AR inhibitory activity of large number of flavones and related compounds from traditional antidiabetic remedies. Here, many of these compounds shared 2-Aryl-benzpyran-4-one as scaffold for different chemical groups surrounding this moiety. This offers scope to investigate the AR inhibitory activity of these compounds in relation to the functional group environment surrounding this core

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

Studies on the interaction of NADPH cytochrome P-450 reductase and cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the K$\sb{\rm m}$ of the reductase for cytochrome P-450b or cytochrome P-450c. Modification of carboxyl groups on the reductase with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, and taurine, respectively. The apparent K$\sb{\rm m}$ for cytochrome P-450c or cytochrome P-450b was increased 1.3 to 5.2 fold. There were varied effects on the V$\sb{\rm max}$. There was no significant change in the conformation of the reductase upon chemical modification. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450. Cytochrome P-450 protected 0.8 moles of carboxyl residues on the reductase from being modified with EDC. These protected amino acids on the reductase are presumably involved in binding to cytochrome P-450. The specific peptide containing these amino acids has been identified. ^

The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

Cytochrome P-450 reductase FAD binding domain: Cloning, enzymatic activity and flavin binding characterization

The Mixed Function Oxidase System metabolizes a wide range of biochemicals including drugs, pesticides and steroids. Cytochrome P450 reductase is a key enzymatic component of this system, supplying reducing equivalents from NADPH to cytochrome P450. The electrons are shuttled through reductase via two flavin moieties: FAD and FMN. Although the exact mechanism of flavins action is not known, the enzymatic features of reductase greatly depleted of either FMN of FAD have been characterized. Additionally, flavin location within reductase has been proposed by homology and chemical modification studies. This study seeks to extend the flavin depletion analysis in a more controlled system by eliminating the proposed FMN binding domain with recombinant DNA techniques and biochemical analysis. Two P450 reductase cDNA clones containing only the FMN and NADPH binding domain were isolated, expressed and the protein products purified and analysed. This study confirms the proposed FAD binding site, role of FAD in electron shuttling pathway and provides new methods to study the FAD binding domain. ^

Characterization of NARJ: A molecular chaperone involved in assembly of nitrate reductase

The major goal of this work was to define the role of accessory protein, NARJ, in assembly of nitrate reductase which is a membrane-bound multisubunit enzyme that can catalyze the reduction of nitrate to nitrite under anaerobic growth in E. coli. Nitrate reductase is encoded by the nar GHJI operon under control of the narG promoter. The purified nitrate reductase is composed of three subunits: $\alpha,\ \beta,$ and $\gamma.$ The NARJ protein which is encoded by the third gene (narJ) is not found to be associated with any of the purified preparations of the enzyme, but is required for active nitrate reductase. In this study the product of the narJ gene was identified. NARJ appeared to be produced at a reduced level, compared to the other proteins encoded by the nar operon. Since NARJ could not be overexpressed to a level for an efficient purification, NARJ was expressed and purified as a recombinant protein with polyhistidine tag. The recombinant protein NARJ-6His could functionally replace native NARJ. Purified NARJ-6His is a dimeric protein which contains no identifiable cofactors or unique secondary structure. NARJ was localized in the cytoplasm, and was not associated with nitrate reductase in the membrane. In vivo NARJ activated the $\alpha\beta$ complex and stabilized the $\alpha$ subunit against protease degradation. In the absence of the membrane-bound $\gamma$ subunit, NARJ formed an intermediate complex with $\alpha\beta$ in the cytosol. Based on these studies, NARJ fits the formal definition of a molecular chaperone. It appears to be required only for the biogenesis of nitrate reductase and, therefore, is defined as a private chaperone specifically involved in the assembly of nitrate reductase system. ^

The role of BCL-2 in regulation of apoptosis susceptibility by modulation of glutathione

The role of oxidative stress and apoptosis has recently been recognized as an important determinant in the development of a variety of diseases known to man. The oncogene BCL-2 is known to regulate sensitivity to induction of apoptosis and appears to function in an antioxidant pathway by regulating glutathione. We have investigated various steps in the oxidative stress cascade to determine possible sites of action for BCL-2. The fluorescent probes H2DCFDA, dihydroethidium and cis-parinaric acid were used to quantitate generation of peroxides, superoxide and lipid peroxidation, respectively. While each of these agents was able to detect substantial increases in oxidative stress following exposure of cells to ionizing radiation, there was no significant difference between cells expressing high or low levels of BCL-2. Investigation of mitochondrial dysfunction during apoptosis revealed a possible site of bcl-2 intervention, but, analysis of kinetic events occurring during apoptosis suggested that the observed effect is not in the direct apoptotic effector pathway. When glutathione was studied, localization to the nucleus was observed in cells overexpressing BCL-2 that did not occur in cells lacking BCL-2. Additionally, nuclear accumulation of glutathione was sufficient to block granzyme b-mediated nuclear DNA fragmentation, poly (ADP-ribose) polymerase cleavage and caspase activity suggesting that nuclear accumulation of glutathione via a bcl-2 dependent process is functionally relevant to suppression of apoptosis. Thus, a model system emerges where BCL-2 is able to regulate a cell's ability to prevent apoptosis by modifying the cell's antioxidant systems at the organelle level to compensate for oxidative stresses placed upon it. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^