Effects of transient overexpression or knockdown of cytochrome P450 reductase on reactive oxygen species generation and hypoxia reoxygenation injury in liver cells.

1. Literature data suggest that the electron-donating enzyme, cytochrome P450 reductase (CPR), might act as a source of reactive oxygen species (ROS). However, the role of CPR in pathophysiological conditions associated with oxidative stress is unknown. The aim of the present study was to study the role of CPR in the generation of ROS and cellular injury under basal conditions, and after simulated in vitro ischaemia-reperfusion (IR). 2. Plasmid DNA or siRNA approaches were used to transiently overexpress or knockdown the human CPR gene in rat liver epithelial (WB-F344) or human hepatoblastoma (HepG2) cells, respectively. The generation of ROS and/or cellular injury was then studied under the basal conditions and after simulated IR (4 h of ischaemia plus 30 min of reoxygenation). 3. Under the basal conditions, transient overexpression of CPR protein in WB-F344 cells caused a 90% increase in the CPR activity, which was associated with a 100% increase in the ROS production. In contrast, after simulated IR, a 2.5-fold higher CPR activity did not significantly affect the magnitude of ROS generation or cell death. Similarly, although the knockdown of CPR protein resulted in a significant reduction (∼30%) in the CPR activity, the ROS production was not substantially altered after simulated IR in HepG2 cells. 4. Our data suggest that CPR plays a major role in the ROS generation by liver cells under the basal conditions. However, the role of CPR in the ROS generation during simulated in vitro IR injury in these cells is minimal, if any.

Protective effect of a phytocompound on oxidative stress and DNA fragmentation against paracetamol-induced liver damage.

The hepatoprotective potential DTS (1.5 g/kg bw, Denshici-to-Chiusei, Kyotsu Jigyo, Tokyo, Japan) was evaluated against either toxic (1.5 g/kg bw) and sub-toxic (150 mg/kg bw) dosage of paracetamol-induced liver injury in Sprague-Dawley rat. Paracetamol intoxication caused a reduction of serum total protein and increase levels of serum alkaline phosphatase (ALP), aspartate aminotranferase (AST) and serum alanine aminotranferase (ALT) at higher extent in the toxic group. This phenomenon was paralleled by an impaired liver redox status (reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) and increased MDA in both paracetamol-administered groups. Moreover, a marked reduction of ATPase and thiols together with DNA fragmentation occurred in liver tissue. Animals pretreated with DTS showed a marked mitigation of the severity of liver enzyme and of the impaired redox status of the liver. Moreover, DTS partly prevented the DNA fragmentation and the decline of liver tissue ATPase and protein thiol assay as compared with both groups treated with paracetamol alone. Although more detailed studies are awaited to ascertain the detailed mode of action of DTS, it wouls seem to be related to the prevention of formation of the reactive oxygen groups thereby preventing the damage on the hepatocytes and possibly modulating the genes responsible for synthesis of liver antioxidant enzymes thus providing marked DNA protection.

Alcohol dehydrogenase: a potential new marker for diagnosis of intestinal ischemia using rat as a model.

AIM: Intestinal ischemia (Ii) is an abdominal emergency due to blockade of the superior mesenteric artery resulting in 60-100% mortality if diagnosed late. Changes in several biochemical parameters such as D (-)-lactate, Creatinine kinase isoenzymes and lactate dehydrogenase suggested for early diagnosis, lack specificity and sensitivity. Therefore a biochemical parameter with greater sensitivity needs to be identified. METHODS: Wistar male rats were randomly assigned into two groups; control sham operated (n = 24) and ischemic test (n = 24) group. Superior mesenteric arterial occlusion was performed in the ischemic test group for 1 h. Alcohol dehydrogenase (ADH) was estimated in blood from portal vein, right ventricle of heart, dorsal aorta (DA) and inferior vena cava (IVC). The Serum glutamic acid pyruvate transaminase (SGPT) was also estimated in blood from portal vein and right ventricle of heart. RESULTS: A significant increase (P<0.001) in the levels of ADH in both portal blood as well as heart blood of the test group (232.72+/-99.45 EU and 250.85+/-95.14 EU, respectively) as compared to the control group (46.39+/-21.69 EU and 65.38+/-30.55 EU, respectively) were observed. Similarly, increased levels of ADH were observed in blood samples withdrawn from DA and IVC in test animals (319.52+/-80.14 EU and 363.90+/-120.68 EU, respectively) as compared to the control group (67.68+/-63.22 EU and 72.50+/-58.45 EU, respectively). However, in test animals there was significant increase in SGPT in portal blood (P = 0.054) without much increase in heart blood. CONCLUSION: Significant increase in the levels of ADH in portal and heart blood within 1 h of SMA occlusion without increase in SGPT in heart blood, suggests that the origin of ADH is from ischemic intestine and not from liver. Similarly, raised ADH levels were found in DA and IVC as well. IVC blood does represent peripheral blood sample. A raised level of ADH in test animals confirms it to be a potential marker in the early diagnosis of Ii.

2-methoxyestradiol binding of GPR30 down-regulates angiotensin AT(1) receptor.

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [(3)H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Tannic acid down-regulates the angiotensin type 1 receptor through a MAPK-dependent mechanism.

In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 µg/ml (7.34-58.78 µm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression.