Cherukury Madhu

Induction of hepatic metallothionein by paraquat

Paraquat, a frequently used contact herbicide, produces oxidative stress by undergoing redox cycling and generating reactive oxygen species. Paraquat is also effective at increasing hepatic levels of metallothionein (MT). The mechanism(s) by which agents that induce oxidative stress produce increases in MT concentrations is not yet known. Therefore, the goal of the current study was to characterize the elevation in hepatic MT produced by paraquat administration to mice and to examine potential mechanism(s) of this increase. A dose-response study for increases in MT showed that administration of 0.1 to 0.5 mmol/kg of paraquat, sc, increased hepatic MT with a maximal increase of 36-fold. Subsequent studies were carried out with paraquat at a dose (0.3 mmol/kg, sc) that caused oxidative stress, as shown by a 35-fold increase in the biliary excretion of oxidized glutathione. There were coordinate elevations of both hepatic MT-I and MT-II mRNA of approximately 5-fold with peaks at both 6 and 24 hr after paraquat. The time course for the elevation in hepatic MT protein following paraquat treatment showed that MT levels had a maximal increase of 18-fold obtained at 36 hr. Paraquat appears to be an indirect MT inducer, in that there were no elevations in MT when cultured mouse hepatocytes were exposed to paraquat. No rise in liver Zn was observed prior to the increase in hepatic MT, thus, a Zn redistribution to the liver did not cause the increase in hepatic MT following paraquat administration. Adrenalectomy did not abolish the increase in MT produced by paraquat, suggesting that adrenal gland products are not required for the increase in MT produced by paraquat. In conclusion, the chemical mediator responsible for the increase in hepatic MT after paraquat was not determined, but the elevation in MT concentration appears to be due to increased transcription.

Effect of pregnenolone-16α-carbonitrile and dexamethasone on acetaminophen-induced hepatotoxicity in mice☆

Recently, we demonstrated that a microsomal enzyme inducer with a steroidal structure, pregnenolone-16 alpha-carbonitrile (PCN), markedly decreased the hepatotoxicity of acetaminophen (AA) in hamsters. Therefore, it was of interest to determine if PCN, as well as another steroid microsomal enzyme inducer, dexamethasone (DEX), would decrease the toxicity of AA in mice, another species sensitive to AA hepatotoxicity. Mice were pretreated with PCN or DEX (100 and 75 mg/kg, ip, for 4 days, respectively) and were given AA (300-500 mg/kg, ip). Twenty-four hours after AA administration, liver injury was assessed by measuring serum activities of sorbitol dehydrogenase and alanine aminotransferase and by histopathological examination. Neither PCN nor DEX protected markedly against AA hepatotoxicity in mice; PCN tended to decrease AA-induced hepatotoxicity, whereas DEX was found to enhance AA-induced hepatotoxicity and it produced some hepatotoxicity itself. DEX decreased the glutathione concentration (36%) in liver and increased the biliary excretion of AA-GSH, which reflects the activation of AA, whereas PCN produced neither effect. Thus, whereas PCN has been shown to markedly decrease the hepatotoxicity of AA in hamsters, apparently by decreasing the isoform of P450 responsible for activating AA to N-acetyl-p-benzoquinoneimine, this does not occur in mice after induction with either PCN or DEX. In contrast, DEX enhances AA hepatotoxicity apparently by decreasing liver GSH levels and increasing the activation of AA to a cytotoxic metabolite.

Protective effect of pregnenolone-16α-carbonitrile on acetaminophen-induced hepatotoxicity in hamsters☆

Overdosage of acetaminophen (AA) is known to produce acute liver toxicity in both humans and laboratory animals. Hamsters are especially sensitive to the hepatotoxic effect of AA. In the present study, hamsters pretreated with pregnenolone-16 alpha-carbonitrile (PCN; 75 mg/kg, ip, daily for 4 days) were given a single dose of AA (350-1200 mg/kg, ip) and liver function was determined 24 hr later. Serum activities of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as well as histopathology were used as indices of hepatotoxicity. PCN pretreatment decreased AA-induced mortality. PCN dramatically decreased ALT (93-97%) and SDH (63-98%) activities relative to control values from hamsters treated with AA alone, and remarkably decreased hepatic centrilobular necrosis produced by AA. To investigate the mechanism of this protective effect, the biliary and urinary excretion of AA metabolites were measured for 1 hr after administration of AA (150 mg/kg, iv) in bile-duct-cannulated hamsters. PCN pretreatment resulted in increased urinary and biliary excretion of AA-glucuronide and decreased biliary excretion of AA-glutathione. Microsomes from PCN-pretreated hamsters produced less benzoquinoneimine intermediate than controls, as determined by the formation of AA-glutathione. In addition, hepatic UDP-glucuronic acid and UDP-glucuronosyltransferase were significantly increased in PCN-pretreated hamsters. In conclusion, PCN pretreatment protected against AA-induced hepatotoxicity. The mechanism of this protection appears to be due to decreased formation of the reactive metabolite by the cytochrome P450 pathway, and an increased detoxication by enhanced glucuronidation of AA.

Simple method for analysis of diquat in biological fluids and tissues by high-performance liquid chromatography☆

A simple HPLC method has been described to quantify diquat in biological fluids and tissues. This method permits separation and quantification of diquat from blood, bile, urine, liver and kidney. It does not require special pretreatment of the samples prior to analysis, nor a specially prepared analytical column. Various concentrations of diquat were added (10-300 nmol/ml or g) to fluids or tissues. Analysis of blank samples revealed no substances that interfere with diquat elution. Excellent recovery (95-105%) was obtained. Diquat (120 mumol/kg, i.v.) was injected to rats and quantified in bile, blood and liver. Concentration of diquat was higher in blood and bile than liver. Therefore, this method is applicable for quantification of diquat in toxicological samples, and may be used to determine structurally similar compounds such as paraquat.

Bromobenzene-glutathione excretion into bile reflects toxic activation of bromobenzene in rats

This investigation was designed to determine whether biliary excretion of bromobenzene(BB)-glutathione(GSH) conjugate can be used as an index of in vivo activation of BB. In order to test this hypothesis, the effect of chemicals known to alter the toxicity and biotransformation of BB (i.e., cytochrome P-450 inducers and inhibitors) on the biliary excretion of BB-GSH was studied in rats. BB-GSH was the major BB metabolite in bile. A linear relationship was observed between the dosage of BB administered and BB-GSH excreted into bile, up to a dosage of 250 mumol/kg of BB. Of the inducers tested, phenobarbital, which is known to increase the toxicity of BB, dramatically increased (700%) the rate of biliary excretion of BB-GSH over that in control animals. In contrast, 3-methylcholanthrene, which is known to decrease the hepatotoxicity of BB, decreased the biliary excretion of BB-GSH (56%). Inhibitors of P-450, such as SKF 525-A and piperonyl butoxide which are known to decrease the activation and hepatotoxicity of BB, also decreased the biliary excretion of BB-GSH. These findings are in agreement with the hypothesis that the biliary excretion of BB-GSH reflects the formation of the reactive BB metabolite in liver and the rate of biliary excretion can be used to determine factors that are important in determining the toxicity of BB.

Peroxide Scavenging by Cu(II) Sulfate and Cu(II) (3,5-Diisopropylsalicylate)2

The production of reactive oxygen species (ROS) in biological systems provides key mediators of physiological and pathological processes. Arguably the most important of this class are the partially reduced oxygen species, which include the superoxide anion radical (O2 ÷), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH). These species have been proposed as mediators of both inflammation1–3 and in various stages in carcinogenesis3–5. One line of evidence supporting a role for ROS in these processes drives from the ability of exogenous scavengers for ROS to inhibit the process.

Inducibility of glutathione S-transferases in hamsters

The effects of 3-methylcholanthrene (3-MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenobarbital, trans-stilbene oxide (TSO), pregnenolone-16 alpha-carbonitrile (PCN), dexamethasone, ethanol, isoniazid and butylated hydroxyanisole (BHA) on hepatic glutathione S-transferase (GST) activities toward six substrates were determined in hamsters. TCDD and 3-MC, which are comparatively poor inducers of GSTs in rats, were most effective in enhancing GST activities in hamster liver. In contrast, TSO, BHA and phenobarbital, which are very effective inducers of hepatic GSTs in rats and mice, were ineffective or poor inducers of GSTs in hamster liver. While dexamethasone increased some GST activities, treatments with PCN, ethanol and isoniazid were without effect. The findings indicate that not only the control activity but also the inducibility of hepatic GSTs are different in hamsters from those in other species.