Subchronic effects of cyanobacterial cells on the transcription of antioxidant enzyme genes in tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis><Emphasis Type="Italic">niloticus</Emphasis>)

The increasing occurrence of toxic cyanobacterial blooms in eutrophic water bodies is nowadays of worldwide concern due to their ability to produce toxins such as microcystins (MCs). These cyanobacterial toxins have been shown to affect aquatic organisms such as fish, resulting in oxidative stress. Among the antioxidant enzymes, glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST) play an important role in the detoxification of MCs. In the present work tilapia (Oreochromis niloticus) were orally exposed to cyanobacterial cells containing MCs and non-containing MCs for 21 days. The activity and relative mRNA expression by real-time PCR of both enzymes and the GST protein abundance by Western blot analysis were evaluated in liver and kidney. Also the induction of lipid peroxidation (LPO) was assayed. MCs containing cyanobacterial cells induced an increase of LPO products in both organs, and MCs containing and MCs non-containing cyanobacterial cells altered the activity, gene expression and protein abundance of the enzymes, indicating the importance of GPx and sGST in MCs detoxification. Moreover, liver, the main organ involved in biodegradation and biotransformation, experienced an adaptative response to the toxic insult. These results show for the first time that the subchronic exposure to cyanobacterial cells causes changes in antioxidant and detoxification enzymes and that GPx and GST gene expression are good markers of these alterations in tilapia.     

Distribution of microcystins in various organs (heart, liver, intestine, gonad, brain, kidney and lung) of Wistar rat via intravenous injection

The distribution of microcystins (MCs) in various tissues of Wistar rats was studied under laboratory conditions. Rats were injected intravenously (i.v.) with extracted MCs at a dose of 80 μg MC-LR_equivalent/kg body weight. MCs concentrations in various tissues were detected at 1, 2, 4, 6, 12 and 24 h post-injection using liquid chromatography-mass spectrometry (LC-MS). The highest concentration of MCs was found in kidney (0.034-0.295 μg/g dry weight), followed by lung (0.007-0.067 μg/g dry weight), stomach (0.010-0.058 μg/g dry weight) and liver (0.003-0.052 μg/g dry weight). The maximum MCs content in the whole body of rat, 2.9% of the injected dose, was observed at 2 h post-injection. MCs concentration was higher in kidney than in liver during the experiment, and two peaks of MCs concentration (at 2 and 24 h, respectively) were observed in kidney, indicating that MCs can be excreted directly via kidney of rat. Though heart, intestine, spleen, brain, gonad and stomach contained less than 0.2% of injected MCs during the whole experiment stage, the presence of MCs in these tissues represents potential damage to them.     

GlutathioneS-Transferase-Catalyzed Conjugation of Bioactivated Aflatoxin B1in Rabbit Lung and Liver

Aflatoxin B₁(AFB₁) requires bioactivation to AFB₁-8,9-epoxide for carcinogenicity, and glutathioneS-transferase (GST)-catalyzed conjugation of activated AFB₁with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [³H]AFB₁, rabbit liver microsomes, an NADPH-generating system, 1 mMGSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB₁-8,9-epoxide. [³H]AFB₁–GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [³H]AFB₁–GSH formation rates were significantly lower for lung than for liver (0.3 ± 0.1 and 1.7 ± 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [³H]AFB₁–GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB₁-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB₁exo- andendo-epoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB₁exo- andendo-epoxide–GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only theexostereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB₁exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB₁in lung may be an important factor in the susceptibility of the lung to AFB₁toxicity.     

Rabbit Aorta GlutathioneS-Transferases and Their Role in Bioactivation of Trinitroglycerin

The pharmacological action of glyceryl trinitrate (GTN), a widely used drug for the treatment of angina pectoris, is thought to be mediated through release of nitric oxide (NO) during its biotransformation. Since glutathioneS-transferases (GST) can utilize GTN as substrate and GST inhibitors can attenuate GTN-induced relaxation of rabbit aortain vitroit has been suggested that these enzymes are involved in the bioactivation of GTN in rabbit aorta. Because GSTs are multifunctional enzymes and a multitude of GST isozymes with varying substrate preferences are present in mammalian tissues, the role of specific GST isozymes in bioactivation of GTN in rabbit aorta needs to be established. Therefore, during the present studies we have purified and characterized GST isozymes from rabbit aorta and evaluated their possible roles in the biotransformation of GTN. The results of these studies showed that rabbit aorta contained three GST isozymes having pI values of 9.4, 7.7, and 5.4. Structural, immunological, and kinetic studies showed that GST 9.4, GST 7.7, and GST 5.4 belonged to the α-, π-, and μ-classes, respectively. The relative abundance of these enzymes in rabbit aorta was α > π > μ. The α- and μ-class GST isozymes had similar activities toward GTN (0.71 U/mg and 0.86 U/mg, respectively) while the π-class GST showed much lower activity toward GTN. The catalytic efficiencyk_cat/K_mof the μ- and α-class GSTs toward GTN were similar but these activities were differentially inhibited by ethacrynic acid, its GSH conjugate, bromosulfophthalein (BSP), and hematin. These results suggest that in rabbit aorta GSTs may be involved in bioactivation of GTN, and because of their higher abundance the α-class GSTs may be more important for the pharmacological effects of GTN than the μ-class GSTs. The results on kinetics of inhibition by various inhibitors suggest that hematin may be an effective inhibitor to delineate the role of specific GST isozymes in the bioactivation of GTN.     

Cytochrome p450 and glutathione s-transferase mRNA expression in human fetal liver hematopoietic stem cells.

During fetal development, the liver serves as the primary hematopoietic organ in which hematopoietic stem cells (HSC) capable of initiating long-term hematopoiesis comprise a large proportion of the hepatic cell population. Although HSC are potential targets for transplacental chemicals, little is known regarding their xenobiotic biotransformation ability. We quantitated the steady-state mRNA expression of six cytochrome P450 (P450) and 11 glutathione S-transferase (GST) isoforms in CD34(+)-selected HSC isolated from second trimester human fetal liver donors, genotyped donors for polymorphic hGSTM1 and hGSTT1 status, and analyzed gene expression in HSC relative to total liver from donors of similar gestational ages. Several P450 isoforms, including CYP1A1, CYP2E1, CYP3A4, and CYP3A5, were expressed at low levels in HSC (relative mRNA expression CYP3A5 > CYP1A1 > CYP2E1 > CYP3A4). CYP1A2 and CYP3A7 were not detected in HSC. The CYP3A4/5 mRNA expression in HSC was accompanied by detectable CYP3A protein and low midazolam oxidation activity. Several GST isoforms, including hGSTM1, hGSTM2, hGSTM4, and hGSTP1, were significantly higher in HSC as compared with total fetal liver. With the exception of hGSTA4, alpha class GST were not detected in HSC. GST expression in HSC was accompanied by substantial GST catalytic activity toward 1-chloro-2,4-dinitrobenzene. In summary, our data indicate that fetal liver CD34(+)-derived HSC constitutively express several P450 isoforms at low levels relative to total hepatic cell populations but have a higher capacity for GST conjugation reactions through mu and pi class isoforms. The functional ramifications of these observations are discussed relative to the sensitivity of human fetal HSC to transplacental chemical injury.     

Glutathione transferases from Anguilla anguilla liver: identification, cloning and functional characterization

Glutathione transferases (GSTs) constitute a class of detoxifying enzymes involved in Phase II metabolism. Using GSH-affinity chromatografy followed by HPLC analysis, two GST isoforms were isolated from the Anguilla anguilla liver cytosol. The major GST belongs to the piscine-specific rho class and accounted for about 59% of total GST affinity eluted fraction, while the remaining 41% was represented by a Pi class GST. Both isoforms were cloned, heterologously expressed in Escherichia coli and their enzyme activities were characterized with respect to a broad spectrum of well-known GST substrates. Our data indicate that only a fraction of prototypical GST substrates are conjugated by these enzymes and that Pi class GST has higher specific activity than rho class GST against 1-chloro-2,4-dinitrobenzene (CDNB), ethracrynic acid, 4-nitroquinoline-1-oxide and p-nitrophenyl acetate while trans-2-nonenal is detoxified more efficiently by rho class GST. Analysis of the kinetics parameters of the conjugation against CDNB indicated that the utilization ratio K(cat)/K(m) is slightly higher for rho class GST with respect to pi class GSTs. Finally, to determine the potential for environmental inhibition of the GST isoforms, we examined the effect of the widely used herbicide atrazine as an inhibitor of catalytic activity. The inhibition studies revealed that atrazine was an effective inhibitor of GST-CDNB catalytic activities of both isoforms at micromolar concentrations, suggesting the sensitivity of these isoforms to pesticide inhibition at environmentally relevant concentrations.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Sex and species differences in glutathione S-transferase activities

Glutathione S-transferases (GSTs) are one of the important enzymes in terms of not only drug metabolism but also physiological functions. The marked sex difference in GST activity has been found in rat and mouse liver cytosol, and such differences in rat liver are suggested to be primarily due to the differences in the subunit composition of GSTs in both sexes. In addition, GST activities of rat liver cytosol are known to be largely influenced by treatment with inducers such as phenobarbital and 3-methylcholanthrene and various hormones. GSTs are widely distributed in mammalian species, and multiplicity of GST has been demonstrated so far. The present review also describes multiple forms of GST from the viewpoint of enzymology and immunology.     

In vitro and in vivo assessment of hepatic and extrahepatic metabolism of diazepam in the rat

Since diazepam is metabolized by many organs in the rat, the microsomal fractions of the liver, kidney, and lung from male Wistar rats were assayed for NADPH-dependent metabolism of diazepam and enzymatic parameters. The predicted extraction ratios were obtained from this in vitro experimental system. The organ clearances of the liver, kidney, and lung were then calculated for the determination of the relative contribution of each eliminating organ to the total body clearance (CLtot) of diazepam in the rat. The liver was the most effective eliminating organ, followed by the kidney and the lung, in that order. The hepatic extraction ratio of diazepam was determined in vivo after portal and femoral vein administrations of diazepam. The validity of the in vitro experimental system for the liver was demonstrated by a good agreement between the calculated hepatic extraction ratio of diazepam from in vitro enzymatic parameters (0.616) and that derived in vivo (0.648). However, the sum of organ clearances of the liver, kidney, and lung did not account for CLtot of diazepam in the rat, suggesting the possible contribution of the metabolism in the other organs or tissues, or an underestimation of the pulmonary and renal metabolism.     

Role of alpha class glutathione S-transferases as antioxidant enzymes in rodent tissues

Glutathione S-transferases (GST) are multifunctional proteins. alpha class GSTs are known to catalyze glutathione peroxidase reactions, in addition to their major activity, i.e., conjugation of electrophiles to glutathione. In the present work, the contribution of rat and mouse alpha class GSTs to glutathione-dependent reduction of phospholipid hydroperoxides has been studied., Results of these studies indicate that the alpha class GST fraction, which consists of three isoforms, has glutathione peroxidase activity toward phospholipid hydroperoxides residing in biological membranes, without the need of prior phospholipase C action. Immunotitration studies using antibodies specific to the alpha class GSTs, GSTA1-1, GSTA2-2, and GSTA3-3, indicate that these GST isozymes account for approximately half of the glutathione peroxidase activity toward phospholipid hydroperoxides present in the 28,000g supernatant fractions of rat and mouse liver extracts. GSTs contribute proportionally lesser fraction of this activity in other tissues in which alpha class GSTs are less prevalent. In mice, the contribution of alpha class GSTs to the overall glutathione peroxidase activity is indistinguishable in wild-type mice and knockout mice lacking the major selenoenzyme, glutathione peroxidase 1, an enzyme that does not act on intact phospholipid hydroperoxides. These results are consistent with our previous studies on human alpha class GSTs (Yang, et al. J. Biol. Chem. 276, 19220-19230, 2001) and demonstrate that alpha class GSTs are of physiological importance, not only in the conjugative detoxification of electrophiles, but are also an essential component of cellular antioxidant defense mechanisms.