Simultaneous measurement of gene expression for hepatotoxicity in thioacetamide-administered rats by DNA microarrays

DNA microarray technology was developed as a tool for simultaneously measuring a number of gene expression changes, and has been applied for investigations of toxicity assessments of chemicals. In this study, we used a typical hepatotoxicant, thioacetamide (TA), to find correlations between the extent of hepatotoxicity and certain gene expression patterns or specific gene expression profiles. TA was intraperitoneally administered at high (400 mg/kg), medium (150 mg/kg) or low (50 mg/kg) dose (four rats per group) and then the serum and liver were collected at the indicated time (6, 12, 24, 36 and 48 h). Serum biochemical markers were measured and hepatic mRNA expression profiles were analyzed by a DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased by TA-administration in a dose-dependent manner and reached the maximum at 24h. Hierarchical clustering analysis of all dosage groups revealed in 2 major clusters, distinguished by an early (6 and 12h) and a late (24, 36 and 48 h) phase. The early and late phase clusters were sorted in time- and dose-dependent manners. The major gene expression profile obtained by quality-threshold (QT) clustering analysis showed the same maximal toxic time as that estimated by the serum biochemical markers. The individual expression profiles of the candidate genes selected in our previous studies and the simultaneous gene expression patterns measured by five typical hepatotoxicants including TA also reflected the hepatotoxicity of TA. These findings suggest that the potential toxic effects appearing as gene expression changes are independent of the dosage of TA. This study suggested that the major gene expression profile estimated by QT clustering would be a sensitive marker of hepatotoxicity.     

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.     

溴化1-己基-3-甲基咪唑对斜生栅藻谷胱甘肽及其代谢酶的影响

研究了浓度为0、1、5、10、15、20 mg/L的新兴离子液体溴化1-己基-3-甲基咪唑([C6mim]Br)在24h、48h、72h和96h对斜生栅藻还原型谷胱甘肽（GSH）及其代谢酶-谷胱甘肽过氧化物酶（GPX）、谷胱甘肽转硫酶（GST）和谷胱甘肽还原酶（GR）的影响。结果表明：GSH含量在24h、48h和72h时,在最低处理浓度下不变,其他处理浓度下随胁迫浓度增加而降低,96h时则与对照无差异或较小;GPX和GST的活性在72h之前明显升高（最高浓度组的GST活性有波动）,96h时均降低至对照水平;GR活性在24h时,[C6mim]Br=1 mg/L时升高,之后降低,在48h增高至对照水平,72h时,[C6mim]Br≥10 mg/L的处理组高于对照水平,96h时,除最低处理组外,均降至对照水平以下。GR是GSH系统中的限速酶,GST则是该系统中活性和灵敏性最高的酶,可作为[C6mim]Br胁迫时的敏感的生物标志物。1 mg/L的[C6mim]Br可引起藻细胞的氧化胁迫,具有环境毒性。     

Triiodothyronine downregulates the periportal expression of alpha class glutathione S-transferase in rat liver

Most drug-metabolizing phase I and phase II enzymes, including the glutathione S-transferases (GST), exhibit a zonated expression in the liver, with lower expression in the upstream, periportal region. To elucidate the involvement of pituitary-dependent hormones in this zonation, the effect of hypophysectomy and 3,3',5-triiodo-L-thyronine (T3) on the distribution of GST was studied in rats. Hypophysectomy increased total GST activity both in the periportal and perivenous liver region. Subsequent T3 treatment counteracted this effect in the perivenous zone. However, analysis for either mu class M1/M2-specific (1,2-dichloro-4-nitrobenzene) or alpha class A1/A2-specific (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) GST activity revealed that T3 treatment did not significantly affect the perivenous activity of these GST classes. In contrast, T3 was found to significantly counteract the increase of alpha class GST activity caused by hypophysectomy in the periportal zone. To establish whether this effect was T3-specific, hepatocytes were isolated from either the periportal and perivenous zone by digitonin/collagenase perfusion and cultured either as pyruvate-supplemented monolayer or as co-culture with rat liver epithelial cells. Only in the latter it was found that T3 suppressed the A1/A2-specific GST activity and alpha class proteins predominantly in periportal cells. The data demonstrate that T3 is an important factor responsible for the low expression of alpha GST in the periportal region. T3 may be involved in the periportal downregulation of other phase I and II enzymes as well.     

Expression of glyoxalase, glutathione peroxidase and glutathione S-transferase isoenzymes in different bovine tissues

(1) The tissue-specific expression of various glutathione-dependent enzymes, including glutathione S-transferase (GST), glutathione peroxidase and glyoxalase I, has been studied in bovine adrenals, brain, heart, kidney, liver, lung and spleen. Of the organs studied, liver was found to possess the greatest GST and glyoxalase I activity, and spleen the greatest glutathione peroxidase activity. The adrenals contained large amounts of these glutathione-dependent enzymes, but significant differences were observed between the cortex and medulla. (2) GST and glyoxalase I activity were isolated by S-hexylglutathione affinity chromatography. Glyoxalase I was found in all the organs examined, but GST exhibited marked tissue-specific expression. (3) The alpha, mu and pi classes of GST (i.e., those that comprise respectively Ya/Yc, Yb/Yn and Yf subunits) were all identified in bovine tissues. However, the Ya and Yc subunits of the alpha class GST were not co-ordinately regulated nor were the Yb and Yn subunits of the mu class GST. (4) Bovine Ya subunits (25.5-25.7 kDa) were detected in the adrenal, liver and kidney, but not in brain, heart, lung or spleen. The Yc subunit (26.4 kDa) was expressed in all those organs which expressed the Ya subunit, but was also found in lung. The mu class Yb (27.0 kDa) and Yn (26.1 kDa) subunits were present in all organs; however, brain, lung and spleen contained significantly more Yn than Yb type subunits. The pi class Yf subunit (24.8 kDa) was detected in large amounts in the adrenals, brain, heart, lung and spleen, but not in kidney or liver. (5) Gradient affinity elution of S-hexylglutathione-Sepharose showed that the bovine proteins that bind to this matrix elute in the order Ya/Yc, Yf, Yb/Yn and glyoxalase I. (6) In conclusion, the present investigation has shown that bovine GST are much more complex than previously supposed; Asaoka (J. Biochem. 95 (1984) 685-696) reported the purification of mu class GST but neither alpha nor pi class GST were isolated.     

Perfluorodecanoic acid decreases the enzyme activity and the amount of glutathione S-transferases proteins and mRNAs in vivo

The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H(2)O(2)), sodium azide (NaN(3)), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H(2)O(2), BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN(3) in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames' test, as well as that of the cytosolic enzyme GST. A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Characterization of a cytochrome P450 gene (CYP4G) and modulation under different exposures to xenobiotics (tributyltin, nonylphenol, bisphenol A) in Chironomus riparius aquatic larvae

Cytochrome P450 family members participate in xenobiotic transformation as a detoxification mechanism. We have characterized a CYP gene, assigned to the 4G family, in Chironomus riparius, a reference organism in aquatic toxicology. Due to the potential interest of CYP genes and P450 proteins for monitoring pollution effects at the molecular level, the alterations in the pattern of expression of this gene, induced by different xenobiotics, were analyzed. Different compounds, such as the biocide tributyltin (TBTO) and two other well-known endocrine disruptors, nonylphenol (NP) and bisphenol A (BPA), were tested at different concentrations and acute exposures. Upregulation of the CrCYP4G gene was found after exposures to TBTO (1 ng/L 24h-0.1 ng/L 96 h) and, as measured by RT-PCR mRNA quantification, its level was up to twofold that of controls. However, in contrast, NP (1, 10, 100 μg/L, 24h) and BPA (0.5mg/L 24h-3mg/L 96 h) downregulated the gene (by around a half of the control level) suggesting that this gene responds specifically to particular chemicals in the environment. Glutathione-S-transferase (GST) enzymatic activity was also evaluated for each condition. A fairly good correlation was found with CYP4G gene behavior, as it was activated by TBTO (96 h), but inhibited by NP and BPA (24h). Only the higher concentration of BPA tested activated GST, whereas it inhibited CYP4G activity. The results show that different xenobiotics can induce distinct responses in the detoxification pathway, suggesting multiple xenobiotic transduction mechanisms. This work confirms that specific P450 codifying genes, as well as GST enzyme activities, could be suitable biomarkers for ecotoxicological studies.     

Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare)

The effect of the herbicide safener mefenpyr-diethyl on glutathione S -transferase (GST, EC 2.5.1.18) activities of dark-grown barley ( Hordeum vulgare cv. Alexis) was examined. Mefenpyr-diethyl treatment increased the GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) and the herbicide fenoxaprop as substrates. Glutathione (GSH) peroxidase activity was markedly increased. GST subunits were analysed by high performance liquid chromatography (HPLC). The quantities of two major subunits were increased by the safener treatment, while the quantities of two other major subunits remained constant. A cDNA encoding the most abundant inducible GST (HvGST6) was cloned and expressed in E. coli . The purified enzyme exhibited a low activity with herbicides as substrates. By contrast, it exhibited a strong GSH peroxidase activity.     

Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30-month-old rats following a necrogenic dose of thioacetamide

The influence of aging on the mechanisms of liver injury and regeneration was studied in a model of hepatotoxicity induced in 2-, 6-, 12-, 18- and 30-month-old rats by a sublethal dose of thioacetamide (500 mg/kg body weight), a soft nucleophilic and hepatotoxic compound metabolized by the hepatic microsomal FAD monooxygenase system. Samples-blood and hepatocytes-were obtained at 0, 12, 24, 48, 72 and 96 h following thioacetamide intoxication. Parameters of liver injury in serum (NADPH-isocitrate dehydrogenase (ICDH) activity) indicate that the severity of injury was significantly higher in the adult groups (6 and 12 months old) when compared either with the youngest (2 months old) or oldest (18 and 30 months old) groups. Parameters related to biotransformation, such as microsomal FAD monooxygenase, followed mainly the same pattern of age-dependent changes as those observed for injury. The profile of glutathione-S-transferase activity showed an initial induction parallel to liver injury and opposite to the levels of reduced glutathione and protein -SH groups. Enzyme activities and gene expression of the systems involved in the cell endogenous antioxidant defense, such as Mn- and Cu,Zn-superoxide dismutases (SOD), catalase and glutathione peroxidase (GPX) showed significant age-dependent changes that can be summarized as follows: an increase in all enzyme activities and gene expression and a decreased ability to restore the initial activities following 96 h of thioacetamide. We conclude, first, that the gene expression and activity of the enzymes involved in the intracellular antioxidant defense system increased with aging, which can be considered a consequence of the enhanced oxidative state of the cell (decreased in GSH level); and second, that the lower and delayed response in the aged groups significantly influenced the restoration towards normal of GSH and the antioxidant enzyme activities.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

Boron ameliorates fulminant hepatic failure by counteracting the changes associated with the oxidative stress

Boron has well-defined biological effects and may be of therapeutic benefit. In the current paper, the effect of boron in the form of borax was tested in experimental animal model of fulminant hepatic failure (FHF). The syndrome was induced in female Wistar rats by three consecutive daily intraperitoneal injections of thioacetamide (400 mg/kg). In the treatment groups, rats received borax (4.0 mg/kg) orally for three consecutive days followed by thioacetamide. The group administered with thioacetamide plus vehicle, and the borax alone treated rats served as controls. In all groups, rats were terminated 4 h after administering the last dose of thioacetamide, and the tissue/serum was used to measure hepatic levels of thiobarbituric acid reactive substances, reduced glutathione, and various enzymes associated with oxidative stress including peroxide metabolizing enzymes and xanthine oxidase. In thioacetamide treated group, many fold increase in the activity level of serum marker enzymes suggesting FHF was observed that could be brought down significantly in rats receiving boron. Modulation and a correlation in the activity level of oxidant generating enzyme and lipid peroxidation as well as hepatic glutathione level was also observed in rats receiving thioacetamide. In the group receiving boron followed by thioacetamide, these changes could be minimized moderately. The activity level of the peroxide metabolizing enzymes and the tripeptide glutathione, which decreased following thioacetamide treatment were moderately elevated in the group receiving boron followed by thioacetamide. The data clearly shows that borax partly normalizes the liver and offsets the deleterious effects observed in FHF by modulating the oxidative stress parameters.     

Role of selenium on antioxidant capacity in methomyl-treated mice

Methomyl carbamate is a pesticide widely used in the control of insects. The present work aims at studying the effect of selenium on the antioxidant system of methomyl-treated mice. Swiss albino mice were intraperitoneally administered a single dose of methomyl (7 mg/Kg body weight). Mice of another group were injected with sodium selenite (5 pmole/Kg b.wt.) 7 days before methomyl intoxication. After 24 hours, methomyl exposure resulted in significant increase in lactic dehydrogenase activity (LDH). The antioxidant capacity of hepatic cells in terms of the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) content was diminished. It appears that methomyl exerts its toxic effect via peroxidative damage to hepatic, renal and splenic cell membranes. Also, methomyl induced DNA damage in these organs as detected by alkaline filter elution technique. The distribution of methomyl in different organs of mice was detected by HPLC. Selenium administration prior to methomyl injection produced pronounced protective action against methomyl effects. It is observed that selenium enhances the endogenous antioxidant capacity of the cells by increasing the activities of SOD, CAT, GR and GST as well as increasing GSH content. The activity of LDH was decreased in blood and the damage of DNA was suppressed comparable to controls. In conclusion, the adverse effects of methomyl in mice could be ameliorated by selenium.     

Effect of thioacetamide,growth hormone or partial hepatectomy on spermidine acetylase activity of rat liver cytosol

Rat liver cytosol extracts catalyzed the formation of monoacetylspermidine when incubated with acetyl-CoA and spermidine.This activity was enhanced 15-fold by administration of thioacetamide (150 mg/kg). The peak of activity occurred 18–24 h after treatment with the drug and then declined reaching control levels by 76 h. Previous studies have shown that ornithine decarboxylase activity was also greatly increased over this time period. Putrescine content in the liver was increased 80–90-fold at 18–24 h and then declined. Spermidine levels were decreased significantly over the period 12–24 h after thioacetamide treatment and then increased substantially at later times. These results are consistent with the hypothesis that, at early times after administration of thioacetamide, the increase in putrescine content is brought about both by decarboxylation of ornithine and by degradation of monoacetylspermidine.Spermidine acetylase activity was also measured in liver extracts prepared after two other physiological stimuli known to enhance ornithine decarboxylase activity were used. Both growth hormone treatment and partial hepatectomy produced an early 2–3-fold increase in the cytosolic spermidine acetylase activity.     

Effects of shark hepatic stimulator substance on the function and antioxidant capacity of liver mitochondria in an animal model of acute liver injury

This study was carried out to investigate whether shark hepatic stimulator substance （HSS） can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model of thioacetamide （TAA）-induced liver injury. The acute liver injury was induced by two intraperitoneal injections of TAA （400 mg/kg） in a 24 h interval. In the TAA plus shark HSS group, rats were treated with shark HSS （80 mg/kg） 1 h prior to each TAA injection. In this group, serum liver enzyme activities were significantly lower than those in the TAA group. The mitochondrial respiratory control ratio was improved, and the mitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group. The mitochondrial antioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in the TAA group. These results suggest that the protective effect of shark HSS against TAA-induced acute liver injury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defenses and decreased oxygen stress.     

The effect of ovarian hormones on antioxidant enzyme activities in the brain of male rats

The brain is widely responsive to gonadal hormones. The functional significance of ovarian hormones in the brain is evident from biochemical studies indicating that estradiol or progesterone treatment of testectomized rats produces changes of antioxidant enzyme activities. The effect of estradiol benzoate (EB) and progesterone (P) in the control of antioxidant (AO) enzyme activities was studied in the brain of adult male Wistar rats. The activities of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and glutathione reductase (GR) were measured in appropriate subcellular fractions, prepared from brains of animals belonging to various experimental groups. These groups were designed with the intention to follow changes in enzyme activities 2 h or 24 h after systemic administration of 5 microg EB or 2 mg P to testectomized (TX) animals. The obtained results show that both EB and P increase CAT activity, whereas EB decreases GSH-Px, GST and GR activities. These findings clearly show the modulatory role of EB and P in the control of enzymes responsible for the protection of rat nerve cells against oxidative damage caused by free oxygen radicals.