Hepatic Glutathione S-transferase Research Articles

Effect of naturally occurring phenolic acids on the expression of glutathione S-transferase isozymes in the rat

Naturally occurring plant phenols, protocatechuic and tannic acids, have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Our previous studies, have shown that these compounds modulate the activity of phases 1 and 2 enzymes in rodents. The aim of the present study was to investigate whether these compounds affect protein levels of rat hepatic and renal glutathione S-transferase (GST) isozymes. Male Wistar rats were treated intraperitoneally with protocatechuic or tannic acid at 50 mg/kg body weight five times during 14 days. 3-Methylcholanthrene (MC) was administered at 20 mg/kg body weight on day 13 (the last treatment with phenolic compounds) and on day 14. Tissues were obtained from rats terminated 24 h after the last treatment. Western blot analysis with specific antibodies showed significant differences in the effect of the phenolic compounds in the liver and kidney. In the liver, protocatechuic acid significantly increased the constitutive GSTμ, while tannic acid reduced the GSTα protein level by 60%. Both plant phenols decreased all classes of constitutive GST isozymes in the kidney including GSTπ, and also the MC-induced GSTα and/or π protein levels. These results, as well as our previous reports, suggest that protocatechuic and tannic acids interfere with the pathways related to xenobiotic toxicities and carcinogenesis. This effect may be important for chemoprotective activity of these plant phenols.

Influence of cooking duration of cabbage and presence of colonic microbiota on the excretion ofN-acetylcysteine conjugates of allyl isothiocyanate and bioactivity of phase 2 enzymes in F344 rats

Isothiocyanates have been implicated in the cancer-protective effects of brassica vegetables. When cabbage is consumed, sinigrin is hydrolysed by plant or microbial myrosinase partly to allyl isothiocyanate (AITC), which is mainly excreted as N-acetylcysteine conjugates (NAC) of AITC in urine. The effect of cooking cabbage on the excretion of NAC of AITC, and glutathione-S-transferase (GST) and uridine 5'-diphospho-glucuronosyl transferase (UGT) activity in rat liver and colon was investigated. Germ-free (GF) and human faecal microbiota-associated (HFM) rats were fed a control diet containing 20 % raw, lightly cooked, or fully cooked cabbage for 14 d. When plant myrosinase was present, excretion of NAC of AITC/24 h was increased by 1.4 and 2.5 times by the additional presence of microbial myrosinase after consumption of raw and lightly cooked cabbage respectively. When plant myrosinase was absent, as after consumption of fully cooked cabbage, excretion of the AITC conjugate was almost zero in GF and HFM rats. None of the cabbage diets modified hepatic GST activity. When microbiota was absent, colonic GST was 1.3-fold higher after fully cooked cabbage, and hepatic UGT was increased by 1.4-1.8-fold after all cabbage diets, compared with the control feed. There were no differences in GST or UGT following cabbage consumption when microbiota was present. It is possible that other constituents of cabbage, rather than metabolites of glucosinolates per se, may be responsible for changes in phase 2 enzyme activity. The main effect of cooking cabbage and altering colonic microbiota was on excretion of NAC of AITC.

Protective effects of puerarin on carbon tetrachloride-induced hepatotoxicity

Puerarin, the main isoflavone glycoside found in the root of Pueraria lobata, has been used for various medicinal purposes in traditional Chinese medicine for thousands of years. The purpose of this study was to investigate the protective effects of puerarin against hepatotoxicity induced by carbon tetrachloride (CCl4) and the mechanism of its hepatoprotective effect. In mice, pretreatment with puerarin prior to the administration of CCl4 significantly prevented the increased serum enzymatic activity of alanine aspartate aminotransferase and hepatic malondialdehyde formation in a dose-dependent manner. In addition, pretreatment with puerarin significantly prevented both the depletion of reduced glutathione (GSH) content and the decrease in glutathione S-transferase (GST) activity in the liver of CCl4-intoxicated mice. Hepatic GSH levels and GST activity were increased by treatment with puerarin alone. CCl4-induced hepatotoxicity was also prevented, as indicated by liver histopathology. The effects of puerarin on cytochrome P450 (CYP) 2E1, the major isozyme involved in CCl4 bioactivation, were also investigated. Treatment of the mice with puerarin resulted in a significant decrease in the CYP2E1-dependent aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the CYP2E1 protein levels were also lowered. Puerarin exhibited anti-oxidant effects on FeCl2-ascorbate induced lipid peroxidation in mouse liver homogenates, and on superoxide radical scavenging activity. These results suggest that the protective effects of puerarin against the CCl4-induced hepatotoxicity possibly involve mechanisms related to its ability to block CYP-mediated CCl4 bioactivation, induction of GST activity and free radical scavenging effects.

Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P < .05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P < .05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2-) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.

Induction of Hepatic CYP2E1 by a Subtoxic Dose of Acetaminophen in Rats: Increase in Dichloromethane Metabolism and Carboxyhemoglobin Elevation

Dichloromethane (DCM) is metabolically converted to carbon monoxide mostly by CYP2E1 in liver, resulting in elevation of blood carboxyhemoglobin (COHb) levels. We investigated the effects of a subtoxic dose of acetaminophen (APAP) on the metabolic elimination of DCM and COHb elevation in adult female rats. APAP, at 500 mg/kg i.p., was not hepatotoxic as measured by a lack of change in serum aspartate aminotransferase, alanine aminotransferase, and sorbitol dehydrogenase activities. In rats pretreated with APAP at this dose, the COHb elevation resulting from administration of DCM (3 mmol/kg i.p.) was enhanced significantly. Also blood DCM levels were reduced, and its disappearance from blood appeared to be increased. Hepatic CYP2E1-mediated activities measured with chlorzoxazone, p-nitrophenol, and p-nitroanisole as substrates were all induced markedly in microsomes of rats treated with APAP. Aminopyrine N-demethylase activity was also increased slightly, but significantly. Western blot analysis showed that APAP treatment induced the expression of CYP2E1 and CYP3A proteins. Neither hepatic glutathione contents nor glutathione S-transferase activity was changed by the dose of APAP used. The results indicate that, contrary to the well known hepatotoxic effects of this drug at large doses, a subtoxic dose of APAP may induce CYP2E1, and to a lesser degree, CYP3A expression. This is the first report that APAP can increase cytochrome P450 (P450)-mediated hepatic metabolism and the resulting toxicity of a xenobiotic in the whole animal. The pharmacological/toxicological significance of induction of P450s by a subtoxic dose of APAP is discussed.

Antioxidant and chemoprotective properties of Momordica charantia L. (bitter melon) fruit extract

Momordica charantia, commonly known as bitter melon, is used as a vegetable in number of countries. Extracts of M. charantia plant, fruit pulp, and seed have been reported to have a wide medicinal use in the traditional medical systems, most often as hypoglycemic and anti-diabetic agents. We have studied the effect of M. charantia, collected from Kazdaglari (Mount Ida) in Balikesir, fruit extract on glutathione S-transferases (GSTs), cytochrome P450s (CYPs), and antioxidant enzymes in rats. Male Wistar rats, aged 12 weeks and weighing 200-250 g, were given 200 mg M. charantia fruit extract per kg body weight, i.p., for four consecutive days. At the end of the experimental period, the animals were sacrificed, and liver, kidney, and lung were isolated. Our results have indicated significant increase in especially hepatic antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. The strongest increase (about 9-fold) was observed in GPx activities while about 2 to 5-fold increases were observed in SOD and CAT. M. charantia fruit extract also exhibited hepatoprotective effects in CCl4-intoxicated rats. In addition, about 50% increase was also noted with hepatic cytosolic GSTs. On the other hand, treatments of rats with M. charantia significantly reduced both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities in rat liver microsomes, which are known to be catalyzed by CYP1A isoforms These results suggest that the M. charantia fruit extract possesses the anti-oxidant effects besides having protective activities in rats.

A diet of red ginseng‐chungkukjang fermented with Bacillus licheniformis improves streptozotocin‐induced physiological symptoms and oxidative stress

The protective effects of red ginseng-chungkukjang fermented with Bacillus licheniformis were observed in streptozotocin (STZ)-induced diabetic rats by measuring levels of blood glucose, serum lipid profiles, and hepatic enzyme activities. Eight groups of male Sprague-Dawley rats weighing 195±5 g (10 animals/group) were fed for 3 weeks with the following: nondiabetic control (N); STZ-treated diabetic (D); 0.16% chungkukjang-treated (C); 0.4% red ginseng-treated (R); 2 % the combined red ginseng and chungkukjang-treated (RC); diabetic C (D-C); diabetic R (D-R); diabetic RC (D-RC). Concentrations of the dietary supplements in the diet were based on a kg diet. Body weight, feed efficiency ratio, and levels of blood glucose and serum lipids in the D group were significantly improved by the treatment of RC. Dietary treatments in diabetes led to the increases of HDL-cholesterol levels in the D-C, D-R, and D-RC groups relative to the D group. The RC diet greatly improved levels of hepatic triglycerides and lipid peroxides in the D group similar to those of the N group. The significantly elevated activity of xanthine oxidase in diabetes was also greatly decreased by the RC diet. The treatment of RC showed the improved hepatic glutathione S-transferase activities in the diabetic animals. The present study indicates that the combined diet of red ginseng and chungkukjang, neither red ginseng nor chungkukjang alone, could ameliorate STZ-induced diabetic symptoms such as aggravated blood glucose levels, serum lipid profiles, and even the conditions of oxidative stress.

Characterization of hepatic glutathione S-transferases in coho salmon ( Oncorhynchus kisutch)

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon ( Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24–26 kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS–MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Heterogeneity of Damage Between Segments of Rat Liver After Inflow-Outflow Obstruction

Background Total vascular exclusion (TVE) causes warm liver ischemia. The complete explanation of the events during inflow and outflow obstruction of the liver during selective TVE has not yet been studied. The aim of this study was to investigate the liver injury caused by inflow-outflow obstruction in the rat liver. Materials and methods Forty Wistar-Albino rats were divided into four groups. Liver inflow occlusion (groups A and C) or inflow-outflow occlusion (groups B and D) was applied for 30 minutes. Samples were collected at the end of the ischemia period. We examined oxidative injury in the liver tissue and liver histopathology. Results Oxidative stress and histopathologic alterations were more prominent with TVE application. Significant alterations were shown in hepatic superoxide dismutase, glutathione, and glutathione S-transferase levels. Central segments of the rat liver were affected significantly from inflow occlusion, whereas dome segments were significantly damaged from inflow-outflow occlusion. Conclusions Inflow-outflow occlusion of the liver caused more tissue damage compared with inflow occlusion. The pattern of distribution of the damage due to TVE seemed different from other well-known ischemia-reperfusion injuries.

Evaluation of Remediation of Coal Mining Wastewater by Chitosan Microspheres Using Biomarkers

Acidic mine waters have a marked influence on the surrounding environment and pose a serious threat through long-term environmental degradation. Therefore, it is important to improve and monitor water quality with the aim of decreasing the hazard presented by this effluent emission. The aim of this work was to evaluate the remediation of mining wastewater effluents by chitosan microspheres using biomarkers of exposure and effect. DNA damage (Comet assay) and several biomarkers of oxidative stress, such as lipoperoxidation levels (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activities, and contents of reduced glutathione (GSH), were measured in blood and liver of tilapia (Oreochromis niloticus) exposed for 7, 15, and 30 days to dechlorinated tap water, 10% coal mining wastewater (CMW), and coal mining wastewater treated with chitosan microspheres (RCM). The results indicate that hepatic TBARS levels were significantly higher in fish exposed to CMW after 7, 15, and 30 days (100%, 86%, and 63%, respectively), and after remediation there was no significant difference in relation to the control group. Hepatic GSH concentrations were lower than control values for CMW after 7 and 15 days of exposure (34% decrease at both times), and this concentration was normalized by treatment with chitosan. SOD showed increased activity in liver after 15 and 30 days of exposure, 30% and 36%, respectively, and in fish exposed to RCM there was no change in this activity compared with the control group. Increased CAT activity in liver was observed during all experimental periods in fish exposed to CMW (46%, 50%, and 56% at 7, 15, and 30 days, respectively) compared with the control or treated-water groups. The highest increase in hepatic GST activity (106%) was observed only in fish exposed to CMW for 30 days. There was an increase in DNA damage in liver (50% at 7 and 15 days) and blood (79%, 77%, and 48% at 7, 15, and 30 days, respectively) after exposure to CMW. In contrast, the fish exposed to wastewater treated with chitosan microspheres exhibited DNA fragmentation indexes similar to the control group. The results obtained indicate the use of oxidative stress biomarkers as useful tools for the toxicity evaluation of coal mining effluents and also suggest that chitosan microspheres may be used as an alternative approach for remediation of coal mining wastewaters.

Ameliorative effect of melatonin against gamma-irradiation-induced oxidative stress and tissue injury

While radiation hazards, due to free radical generation, present an enormous challenge for biological and medical safety, melatonin is a potent scavenger of a variety of free radicals. The aim of this study was to investigate the radioprotective effect of melatonin against oxidative stress and tissue injury induced by gamma radiation. Rats were subjected to two doses of 2 and 4 Gy from cesium-137 source. Four days prior to irradiation, animals received melatonin daily (10 mg/kg body weight i.p.). In the irradiated animals, the oxidative stress markers malondialdehyde (MDA) and protein carbonyl were significantly increased in the liver, while a marked decrease in hepatic contents of DNA, RNA, and glutathione (GSH) as well as activity of glutathione- S-transferase (GST) was demonstrated. In addition, catalase (CAT) activity was increased in the liver 5 days after irradiation. The levels of total lipids, cholesterol, triglyceride (TG), low-density lipoprotein (LDL), urea, and creatinine, as well as activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT), were significantly increased in sera of the irradiated rats. This is coupled with decreased serum levels of high-density lipoprotein (HDL), total protein and albumin, and total globulins by irradiation. The administration of melatonin alone daily for 4 days caused significant decreases in MDA and protein carbonyl content and produced significant elevations of GSH content and GST activity in the liver. Moreover, significant decreases in total lipids, cholesterol, and TG without change in LDL or HDL levels in serum were demonstrated. Treatment with melatonin for 4 days before acute irradiation significantly abolished radiation-induced elevations in MDA and protein carbonyl levels in the liver and significantly maintained hepatic GSH content, GST, and CAT activities close to the control values. Preirradiation treatment with melatonin showed significantly higher hepatic DNA and RNA contents than irradiated rats. The levels of total lipids, cholesterol, TG, HDL, LDL, total proteins, albumin, total globulins, creatinine, and urea, as well as the activities of AST, ALT, and GGT in serum were significantly ameliorated when melatonin was injected before irradiation. In conclusion, the increase in oxidative stress markers and the concomitant change in antioxidant levels indicate the role of oxidative stress in radiation-induced tissue damage. Moreover, melatonin shows a radioprotective impact against ionizing-radiation-induced oxidative stress and organ injury.

Protective effects of fruit extracts of Hippophae rhamnoides L. against arsenic toxicity in Swiss albino mice

Seabuckthorn (Hippophae rhamnoides L.) berry has a long history of applications as a food and medicinal ingredient in eastern countries. The present study was carried out to investigate the effect of different fruit extracts of H. rhamnoides on altered biochemical parameters indicative of haematological alterations, tissue oxidative stress, and arsenic concentration in arsenic-exposed mice (2.5 mg/kg body weight, intraperitoneally). Two aqueous extracts (at room temperature and under reflux condition) and an ethanolic extract of H. rhamnoides at a dose of 500 mg/kg body weight were co-administered daily during arsenic exposure in mice for 3 weeks. Exposure to arsenic led to a significant inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) activity, suggesting disturbed haem synthesis pathway. Arsenic also caused significant depletion of reduced hepatic glutathione (GSH) level, glutathione S-transferase (GST) and glutathione peroxidase and catalase activities, while it increased the level of thiobarbituric acid reactive substance (TBARS), suggesting liver oxidative stress. Most of the altered biochemical variables responded favorably to the co-supplementation of H. rhamnoides, particularly the aqueous fruit extract, extracted at room temperature (HF-WRT). However, arsenic concentration in blood and tissues remained unchanged, suggesting the lack of chelating property of fruit extract of H. rhamnoides. The present study, thus, led us to conclude that the fruit extract of H. rhamnoides has a significant protective role against arsenic-induced oxidative injury. However, it lacks the ability to remove arsenic from the binding sites, suggesting that the herbal extract could be co-administered with a chelating agent of known efficacy during treatment of arsenic to achieve the optimum effect of chelation treatment.

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50-60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (approximately 40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to trans-plancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.

Antioxidative function and biodistribution of [Gd@C 82(OH) 22] n nanoparticles in tumor-bearing mice

Oxidative stress is considered to be one of the important mechanisms involved in carcinogenesis. In our previous study, gadolinium endohedral metallofullerenol ([Gd@C 82(OH) 22] n nanoparticles) have shown high inhibitory activity on hepatoma cell (H22) growth in mice. To explore the antioxidative functions of nanoparticles, we investigated the biodistribution of [Gd@C 82(OH) 22] n nanoparticles, the changes of blood coagulation profiles, the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice as well as the possible relationships between nanoparticles treatment and ROS production in this paper. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and catalase (CAT) as well as the levels of reduced glutathione (GSH), protein-bound thiols and malondialdehyde (MDA) were compared between the tumor-bearing mice and normal mice. Transplanted tumors were grown in mice by subcutaneous injection of murine hepatoma cells in the mice. The comparison of the above parameters between nanoparticles and cyclophosphamide (CTX) therapy were also investigated. [Gd@C 82(OH) 22] n administration can efficiently restore the damaged liver and kidney of the tumor-bearing mice. All the activities of enzymes and other parameters related to oxidative stress were reduced after [Gd@C 82(OH) 22] n treatment and tended closely to the normal levels. The results suggest that [Gd@C 82(OH) 22] n nanoparticle treatment could regulate ROS production in vivo.

Naturally occurring coumarins inhibit 7,12-dimethylbenz[ a ]anthracene DNA adduct formation in mouse mammary gland

Naturally occurring coumarins (NOCs) are anti-carcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases (GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g. imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively. Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3 weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week dose-response study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8- benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.

Evaluation of Liver Damage After Application of TVE in the Rat Model

Introduction The aim of this study was to investigate the effects of total vascular exclusion (TVE) on the liver during the early period of reperfusion. Materials and methods Forty Wistar-Albino rats were divided into four groups. Portal pedicle clamping (groups 1 and 2) or TVE (groups 3 and 4) were applied for 10 minutes. Samples were collected at the time of clamp release (groups 1 and 3) and at 30 minutes of reperfusion (groups 2 and 4). We examined oxidative injury to and histopathology of the liver. Results Oxidative stress was more prominent with TVE application. Significant alterations were shown in hepatic superoxide dismutase, catalase, glutathione, and glutathione S-transferase levels. The levels of malondialdehyde and myeloperoxidase were not altered significantly. Conclusion Inflow-outflow occlusion of the liver causes more oxidative stress compared with inflow occlusion.

Seasonal Variations of Selected Biomarkers in Sand Gobies Pomatoschistus minutus from the Guadalquivir Estuary, Southwest Spain

Sand gobies, Pomatoschistus minutus, were collected monthly from September 2002 to August 2003 at a station situated 8 km upstream from the mouth of the Guadalquivir estuary (southwest Spain). Physical parameters of the water and selected biomarkers of organic pollution were recorded in the fish to discuss its potential as a sentinel species in estuaries. The biomarkers selected were the activities of catalase (CAT), 7-ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST) in the liver and acetylcholinesterase (AChE) and lipid peroxidation (LP) in the head. The results showed an increase in total protein synthesis in late spring and early summer coinciding with the reproductive period as well as the release of fresh water from a dam situated 110 km upstream. During the same period, a significant depletion of hepatic GST and head AChE but higher LP levels in this tissue suggest exposure to pesticides such as those applied to crops established along the course of the river and reaching the estuary mostly when the freshwater discharges occur. Changes in CAT and EROD activities fluctuated randomly and were not noted as seasonally dependent. Biomarker fluctuations in sand goby are discussed as normal seasonal variations, but other variables-such as potential local pollution inputs-cannot be disregarded.

Hepatic glutathione S-transferase activity in flounder collected from contaminated and reference sites along the Polish coast

The aim of the present study was to examine the activity of hepatic glutathione S-transferase (GST) in flounder, Platichthys flesus, as a potential biomarker of exposure to xenobiotics present in the environment. The fish were collected along known pollution gradients and from areas regarded relatively free of anthropogenic input. GST was measured spectrophotometrically in each sampled specimen of flounder. The databases from the studies performed by two research institutes were compiled, and generalized linear models were used to analyze the dependence of the enzyme activity on the time and area of sampling and on selected biological parameters of the fish. Fish sex, sampling area, year, month, water temperature, and somatic indices (gonado-somatic index, hepato-somatic index) were significant in the model of GST activity. The three annual datasets showed that the biomarker activities exhibited in the Gulf of Gdansk were significantly different from those measured in the areas off the coast.

Rubber additives induce oxidative stress in rainbow trout

Several studies have demonstrated the toxicity of rubber leachate, mainly from rubber tires, to aquatic organisms. In the present study rainbow trout ( Oncorhynchus mykiss) were exposed to water provided to aquaria through a rubber hose. Increased hepatic ethoxyresorufin- O-deethylase (EROD) activity, and glutathione reductase (GR) activity were observed in the exposed fish. Two common rubber additives, 2-mercaptobenzothiazole (MBT) and diphenylamine (DPA) and structurally related compounds, were identified by chemical analyses of water samples as were hydroxylated polycyclic aromatic hydrocarbons. Metabolites of these compounds were also detected in the bile of exposed fish, as were some of the parent compounds. In a following experiment , we injected rainbow trout with DPA or MBT. Both compounds affected total glutathione (tGSH) concentration in liver and MBT caused an increase in hepatic GR and glutathione S-transferase (GST) activity as well. In DPA injected fish, hydroxylated DPA was the main metabolite in the bile. Our results indicate that rubber chemicals may leach into the water surroundings where they can be taken up and metabolised by fish. Some of these chemicals can lead to up-regulation of antioxidant defences as demonstrated with DPA and MBT injections.

Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid

5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.