Glutathione S-transferase mRNA Levels Research Articles

Assessing the impact of weathered polystyrene collected from the marine environment on oxidative stress responses in Zophobas morio larvae: A preliminary study

Foamed polystyrene (PS) is a prevalent material in consumer products and thus represents one of the largest constituents of marine litter. PS may interact with and be ingested by various organisms, including insects, which are starting to be used in biodegradation of plastic wastes. This study examines the physiological performances and the potential oxidative stress related to dietary environmental PS exposure of Z. morio larvae. Experimental groups were fed different diets: bran and oatmeal (control), weathered PS collected from the marine environment, and virgin PS. Over a 30-day feeding period, larvae growth, survival, and PS consumption were measured, together with antioxidant enzymatic activities and gene expression profiles. Results showed that PS ingestion supports larval growth similarly to bran, but the two PS-fed groups consumed a different amount of plastic, suggesting that weathered PS might affect the patterns of PS consumption by larvae. We denoted that PS consumption was associated with a decline in total antioxidant capacity, which showed the highest decrease in the environmental PS group. Marked increases in oxidative stress biomarkers such as superoxide dismutase (SOD) activity/mRNA levels and malondialdehyde content were also observed in the environmental PS-fed group, indicating an elevated oxidative stress response, probably due to pollutants adsorbed/absorbed on PS. Differently to SOD, catalase at both mRNA and enzyme activity levels was found to be significantly reduced by PS digestion, regardless of PS type. The mRNA levels of glutathione s-transferase (GST) were significantly higher in the environmental PS-fed larvae compared to the virgin PS-fed group. In addition, Principal Component Analysis clearly distinguished between larvae fed different PS types, highlighting the enhanced oxidative stress caused by the ingestion of PS collected from marine environments. These results support the potential of insects in plastic biodegradation processes but also demonstrate the health hazards of using environmental PS as principal dietary component in Z. morio larvae, thus questioning their use in downstream productions.

Lipid changes and molecular mechanism inducing cuproptosis in Cryptocaryon irritans after copper–zinc alloy exposure

Cryptocaryons irritans is a ciliate parasite responsible for cryptocaryoniasis, leading to considerable economic losses in aquaculture. It is typically managed using a copper–zinc alloy (CZA), effectively diminishing C. irritans infection rates while ensuring the safety of aquatic organisms. Nevertheless, the precise mechanism underlying cuproptosis induced C. irritans mortality following exposure to CZA remains enigmatic. Therefore, this study delves into assessing the efficacy of CZA, investigate cuproptosis as a potential mechanism of CZA action against C. irritans, and determine the alterations in antioxidant enzymes, peroxidation, and lipid metabolism. The mRNA expression of dihydrolipoamide S-acetyltransferase was upregulated after 40 and 70 min, while aconitase 1 was implicated in cuproptosis following 70 min of CZA exposure. Furthermore, the relative mRNA levels of glutathione reductase experienced a significant increase after 40 and 70 min of CZA exposure. In contrast, the relative mRNA levels of glutathione S-transferase and phospholipid-hydroperoxide glutathione peroxidase were significantly decreased after 70 min, suggesting a disruption in antioxidant defense and an imbalance in copper ions. Lipidomics results also unveiled an elevation in glycerophospholipids metabolism and the involvement of the lipoic acid pathway, predominantly contributing to cuproptosis. In summary, exposure to CZA induces cuproptosis in C. irritans, impacts glutathione-related enzymes, and alters glycerophospholipids, consequently triggering lipid oxidation.

Glutathione metabolism in Cryptocaryon irritans involved in defense against oxidative stress induced by zinc ions

BackgroundCryptocaryon irritans is a fatal parasite for marine teleosts and causes severe economic loss for aquaculture. Galvanized materials have shown efficacy in controlling this parasite infestation through the release of zinc ions to induce oxidative stress.MethodsIn this study, the resistance mechanism in C. irritans against oxidative stress induced by zinc ions was investigated. Untargeted metabolomics analysis was used to determine metabolic regulation in C. irritans in response to zinc ion treatment by the immersion of protomonts in ZnSO4 solution at a sublethal dose (20 μmol). Eight differential metabolites were selected to assess the efficacy of defense against zinc ion stimulation in protomonts of C. irritans. Furthermore, the mRNA relative levels of glutathione metabolism-associated enzymes were measured in protomonts following treatment with ZnSO4 solution at sublethal dose.ResultsThe results showed that zinc ion exposure disrupted amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in C. irritans. Four antioxidants, namely ascorbate, S-hexyl-glutathione, syringic acid, and ubiquinone-1, were significantly increased in the Zn group (P < 0.01), while the glutathione metabolism pathway was enhanced. The encystment rate of C. irritans was significantly higher in the ascorbate and methionine treatment (P < 0.05) groups. Additionally, at 24 h post-zinc ion exposure, the relative mRNA level of glutathione reductase (GR) was increased significantly (P < 0.01). On the contrary, the relative mRNA levels of glutathione S-transferase (GT) and phospholipid-hydroperoxide glutathione peroxidase (GPx) were significantly decreased (P < 0.05), thus indicating that the generation of reduced glutathione was enhanced.ConclusionsThese results revealed that glutathione metabolism in C. irritans contributes to oxidative stress resistance from zinc ions, and could be a potential drug target for controlling C. irritans infection.Graphical

Dietary supplemental microalgal astaxanthin modulates molecular profiles of stress, inflammation, and lipid metabolism in broiler chickens and laying hens under high ambient temperatures

This research was to determine effects of supplemental dietary microalgal astaxanthin (AST) on hepatic gene expression and protein production of redox enzymes, heat shock proteins (HSPs), cytokines, and lipid metabolism in broilers (BR) and laying hens (LH) under high ambient temperatures. A total of 240 (day old) Cornish male BR and 50 (19 wk old) White Leghorn Shavers LH were allotted in 5 dietary treatments with 6 and 10 cages/treatment (8 BR or 1 LH/cage), respectively. The birds were fed corn-soybean meal basal diets supplemented with microalgal (Haematococcus pluvialis) AST at 0, 10, 20, 40, and 80 mg/kg diet for 6 wk. Supplemental AST to the BR diet linearly decreased (P < 0.10, R2 = 0.18–0.36) hepatic mRNA levels of several redox status-controlling genes, heat shock protein 70 (HSP70), heat shock transcription factor 1 (HSTF1), c-Jun N-terminal kinase 1 (JNK1), tumor necrosis factor-α, and sterol regulatory element-binding protein 1 (SREBP1). The supplementation linearly elevated (P = 0.04, R2 = 0.20) diacylglycerol acyltransferase 2 (DGAT2) mRNA level and produced quadratic changes (P < 0.10, R2 = 0.15–0.47) in mRNA levels of glutathione S-transferase (GST), serine/threonine kinase (AKT1), P38 mitogen-activated protein kinase (P38MAKP), lipid metabolism–controlling genes, and the protein production of HSP90 and P38MAPK in the liver. Supplementing AST to the LH diets linearly decreased (P < 0.10, R2 = 0.18–0.56) mRNA levels of GST, HSF1, JNK1, and interleukin 10; lipogenesis genes; and JNK1 protein production. However, supplemental dietary AST produced quadratic changes (P < 0.10, R2 = 0.26–0.72) in mRNA levels of most antioxidant-, stress-responsive, and lipid metabolism–related genes in the liver of LH. In conclusion, supplemental dietary AST affected the hepatic gene expression and protein production related to redox status, heat stress and inflammation, and lipid metabolism in both BR and LH. The impacts varied with the chicken type and demonstrated linear and quadratic regressions with the inclusion levels of AST.

Impairment of gill structural integrity by manganese deficiency or excess related to induction of oxidative damage, apoptosis and dysfunction of the physical barrier as regulated by NF-κB, caspase and Nrf2 signaling in fish

This study is for the first time to explore the possible effects of dietary manganese (Mn) on structural integrity and the related signaling in the gills of fish. Grass carp (Ctenopharyngodon idella) were fed with six diets containing graded levels of Mn [3.65–27.86 mg Mn/kg diet] for 8 weeks. The results firstly demonstrated that Mn deficiency aggravated inflammation indicated by up-regulation of pro-inflammatory cytokines (tumour necrosis factor α, interleukin 8, and interleukin 1β mRNA levels) and down-regulation of anti-inflammatory cytokines (interleukin 10, transforming growth factor-β1) mRNA levels, which might be partially related to the up-regulation of nuclear factor kappa B (NF-κB p65) and down-regulation of nuclear inhibitor factor κBα (iκBα) mRNA levels in the gills of fish. Meanwhile, Mn deficiency caused DNA fragmentation, which might be partially associated with the up-regulation of the apoptosis signaling (caspase-3, caspase-8 and caspase-9) in the gills of fish. Furthermore, Mn deficiency-caused apoptosis might be partly related to the increases of oxidative damage that indicated by increases of lipid peroxidation and protein oxidation, and decreases of antioxidant enzyme activities [included Mn superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST)]. However, Mn deficiency only down-regulated MnSOD and GST mRNA levels, which might be partially related to the up-regulation of NF-E2-related factor-2 (Nrf2) inhibitor (Keap1), and only down-regulated the gene expression of claudin-b and claudin-15 to disrupt the TJ in the gills of fish. Excessive Mn led to negative effects on partial parameters studied in the gills of fish. The optimal levels of Mn based on protecting against ROS, MDA and PC in the gills of grass carp were 17.04, 16.86 and 21.20 mg/kg diet, respectively. Collectively, Mn deficiency or excess could cause inflammation, apoptosis, antioxidant system disruption and change tight junction protein (claudin-b and claudin-15) transcription abundances, which might be partially related to the NF-κB p65, caspase-(3,8,9) and Nrf2 signaling, in the gills of fish.

Co- and Post-Treatment with Lysine Protects Primary Fish Enterocytes against Cu-Induced Oxidative Damage.

The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1–19.6 g kg-1 diet) for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0–300 mg/L in media) prior to (pre-treatment), along with (co-treatment) or following (post-treatment) with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD), glutathioneperoxidase (GPx), glutathione-S-transferase (GST) and reductase (GR)), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) OD values, along with alkaline phosphatase (ALP) and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b) followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT) and GST activities (P<0.01 and P<0.001). GR activity and gene expression, and glutathione (GSH) content followed an opposite trend to GST activity (P<0.05). Thus, post-treatment of lysine elevated protein and DNA repair abilities and ameliorated the cellular redox state of enterocytes. The overall results suggest that lysine plays a significant role in the protection of fish intestine in vivo and in vitro through the induction of key antioxidant protection.

Silver nanoparticles affects the expression of biomarker genes mRNA in rainbow trout (Oncorhynchus mykiss)

Due to their strong antiseptic properties, silver nanoparticles (AgNPs) are widely used in consumer products, and therefore there is a high risk of their release to the aquatic environment. In the present study, we investigated chronic effect of waterborne AgNPs on the mRNA expression of four important biomarker genes including metallothionein (MT), glutathione S-transferase (GST), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in the gill tissue of rainbow trout. Twenty-one-day exposure to AgNPs significantly increased MT and GST mRNA levels in the gills in a dose-dependent manner. Although mRNA levels of SOD significantly increased in the gills of fish exposed to AgNPs, but its increase was not dose-dependent. In contrast, exposure to AgNPs significantly decreased GPX mRNA levels in the gills. The results suggest that exposure to AgNPs can significantly change the expression of biomarker genes mRNA in rainbow trout.

Hepatitis C virus structural proteins can exacerbate or ameliorate acetaminophen-induced liver injury in mice.

Chronic hepatitis C virus (HCV) infection predisposes patients to develop liver failure after acetaminophen (APAP) overdose. Mechanisms involved in this were explored using transgenic mice expressing the HCV structural proteins core, E1 and E2. Treatment of C57BL/6J mice with 200 mg/kg body weight APAP resulted in significant liver injury at 6 h as indicated by elevated ALT levels, focal centrilobular necrosis and nuclear DNA fragmentation. HCV transgenic mice showed a variable response, with approximately half the animals showing exacerbation of all parameters of liver injury, while the other half was protected. HCV transgenic mice with higher liver injury had lower liver glutathione levels, elevated mitochondrial oxidative stress and enhanced release of apoptosis-inducing factor (AIF) from the mitochondria. This was accompanied by induction of a higher ER stress response and induction of autophagy. Transgenic animals showing protection against liver injury had a robust recovery of liver glutathione content at 6 h when compared to wild-type animals, accompanied by reduction in mitochondrial oxidative stress and AIF release. This was accompanied by an elevation in glutathione S-transferase mRNA levels and activity, which suggests that an efficient clearance of the reactive intermediate may contribute to the protection against APAP hepatotoxicity in these mice. These results demonstrate that while HCV infection could exacerbate APAP-induced liver injury due to induction and amplification of mitochondrial oxidant stress, it could also protect against injury by activation of APAP scavenging mechanisms.

Dietary aloe vera gel powder and extract inhibit azoxymethane- induced colorectal aberrant crypt foci in mice fed a high- fat diet.

Aloe vera gel exhibits protective effects against insulin resistance as well as lipid-lowering and anti-diabetic effects. The anti-diabetic compounds in this gel were identified as Aloe-sterols. Aloe vera gel extract (AVGE) containing Aloe-sterols has recently been produced using a new procedure. We previously reported that AVGE reduced large-sized intestinal polyps in Apc-deficient Min mice fed a high fat diet (HFD), suggesting that Aloe vera gel may protect against colorectal cancer. In the present study, we examined the effects of Aloe vera gel powder (AVGP) and AVGE on azoxymethane-induced colorectal preneoplastic aberrant crypt foci (ACF) in mice fed a HFD. Male C57BL/6J mice were given a normal diet (ND), HFD, HFD containing 0.5% carboxymethyl cellulose solution, which was used as a solvent for AVGE (HFDC), HFD containing 3% or 1% AVGP, and HFDC containing 0.0125% (H-) or 0.00375% (L-) AVGE. The number of ACF was significantly lower in mice given 3% AVGP and H-AVGE than in those given HFD or HFDC alone. Moreover, 3% AVGP, H-AVGE and L-AVGE significantly decreased the mean Ki-67 labeling index, assessed as a measure of cell proliferation in the colonic mucosa. In addition, hepatic phase II enzyme glutathione S-transferase mRNA levels were higher in the H-AVGE group than in the HFDC group. These results suggest that both AVGP and AVGE may have chemopreventive effects on colorectal carcinogenesis under the HFD condition. Furthermore, the concentration of Aloe-sterols was similar between 3% AVGP and H-AVGE, suggesting that Aloe-sterols were the main active ingredients in this experiment.

Lipopolysaccharide-induced chorioamnionitis causes acute inflammatory changes in the ovine central nervous system.

To better understand the inflammatory response in the central nervous system (CNS) after lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal sheep were exposed to intra-amniotic LPS 2 or 14 days before preterm delivery at 125 days of gestation. mRNA levels of cytokines, TLRs and anti-oxidants were determined in different CNS regions. Interleukin 1β levels increased in hippocampus, cortex and cerebellum 2 days after LPS exposure, while Interleukin 8 levels increased in the periventricular white matter as well. Levels returned back to control levels after 14 days. Tumor necrosis factor-α levels increased in hippocampus and cortex after 2 days. Toll like receptor 4 levels was upregulated in all grey matter regions 2 and 14 days after exposure. Glutathione s-transferase mRNA levels were lower after 2 and 14 days in all grey matter regions. Intra-amniotic LPS exposure causes acute and region-specific changes in inflammatory markers in the fetal brain, with grey matter being more affected than white matter. Intra-amniotic LPS exposure causes acute and region-specific changes in cytokines, TLR and anti-oxidants levels, with grey matter being more affected than white matter.

Emamectin benzoate treatment alters the expression and activity of CYP1A, FMO and GST in different tissues of rainbow trout (Oncorhynchus mykiss)

Abstract Emamectin benzoate (EMB) is one of the drugs used for the treatment and control of infections by Caligus rogercresseyi on salmonids. EMB is orally delivered to the salmon in food, which could be affecting the activity of biotransformation markers in these fish. The aim of this study was to determine the effect of EMB treatment on the mRNA and protein expression levels and enzymatic activity of cytochrome P450 1A (CYP1A), flavin-monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues of rainbow trout (Oncorhynchus mykiss). Rainbow trout were collected between 10 and 25 days after completion of medication with EMB by a standard seven-day treatment in different salmon farms from southern Chile. The CYP1A, FMO and GST mRNA, protein levels and activity in the liver, muscle, gill, kidney and intestine tissues from rainbow trout were measured. Most mRNA and protein levels were significantly affected by treatment with EMB. The highest mRNA transcriptional up-regulations were observed with CYP1A in the liver and FMO in the liver, muscle and kidney and were very marked with regard to the increases of CYP1A and FMO in the gill and GST in the gill and intestine. These effects only correlated slightly with FMO protein levels in the liver muscle and gill. The highest up-regulations for protein levels were observed for GST in the kidney. While, the down-regulation measured in CYP1A was similar between protein levels in the liver, gill and intestine, and also in CYP1A activity levels in all tissues examined. On the other hand, GST mRNA levels were essentially without great changes, except slight increases in the gill and intestine, while GST protein and activity levels increased in all tissues measured. These results show that treatment with EMB induces changes of the expression of detoxification enzymes in most trout tissues, and this effect was consistent with increases of activity for FMO in the intestine and was comparable to the increases of activity for GST observed in all trout tissues analyzed. By contrast, EMB decreased the CYP1A activity levels in all tissues examined. These results show that treatment with EMB in trout alters the expression and activity of CYP1A, FMO and GST, which could be affecting detoxification processes and immuno-modulatory mechanisms. Moreover, the data obtained will allow insight into the mechanisms involved in the biotransformation of EMB in salmonids and its putative relation with drug resistance observed in EMB treatments.

Transgenic Expression of the Human MRP2 Transporter Reduces Cisplatin Accumulation and Nephrotoxicity in Mrp2-Null Mice

The chemotherapeutic drug cisplatin is actively transported into proximal tubules, leading to acute renal injury. Previous studies suggest that the multidrug resistance-associated protein 2 (Mrp2) transporter may efflux cisplatin conjugates from cells. We sought to determine whether the absence of Mrp2 alters the accumulation and toxicity of platinum in the kidneys of mice and whether transgenic expression of the human MRP2 gene could protect against cisplatin injury in vivo. Plasma, kidneys, and livers from vehicle- and cisplatin-treated wild-type and Mrp2-null mice were collected for quantification of platinum and toxicity. By 24 hours, twofold higher concentrations of platinum were detected in the kidneys and livers of Mrp2-null mice compared with wild types. Enhanced platinum concentrations in Mrp2-null mice were observed in DNA and cytosolic fractions of the kidneys. Four days after cisplatin treatment, more extensive proximal tubule injury was observed in Mrp2-null mice compared with wild-type mice. Kidneys from naive Mrp2-null mice had elevated glutathione S-transferase mRNA levels, which could increase the formation of cisplatin-glutathione conjugates that may be metabolized to toxic thiol intermediates. Transgenic expression of the human MRP2 gene in Mrp2-null mice reduced the accumulation and nephrotoxicity of cisplatin to levels observed in wild-type mice. These data suggest that deficiency in Mrp2 lowers platinum excretion and increases susceptibility to kidney injury, which can be rescued by the human MRP2 ortholog.

Tissue-Specific Antioxidant Responses in Pale Chub (Zacco platypus) Exposed to Copper and Benzo[a]pyrene

In this study, antioxidant responses including lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), were evaluated in the liver, gill and muscle tissues of pale chub (Zacco platypus) exposed to copper (Cu) and benzo[a]pyrene (BaP). Cu exposure induced significant antioxidant responses in Z. platypus, particularly in the liver, whereas BaP exposure had a negligible effect. Following Cu exposure, both SOD and CAT activity increased in a concentration-dependent manner, showing significant correlations with malondialdehyde (MDA) levels as a measure of LPO (r=0.646 and 0.663, respectively). SOD, CAT and GST mRNA levels were also enhanced following Cu exposure, except at 20μgL(-1), although significant correlations with antioxidant enzyme activities were not found. The results of this study suggest that combined information on SOD and CAT activities together with LPO levels in the liver could be a useful indicator for assessing oxidative stress in freshwater fish.

Tumor promoting and co-carcinogenic effects in medium-term rat hepatocarcinogenesis are not modified by co-administration of 12 pesticides in mixture at acceptable daily intake

The purpose of this investigation was to evaluate the possible influence of a mixture of pesticides on medium-term carcinogenesis using improved hepatocarcinogenesis protocols. We performed a 12 commercially available pesticides combination with alachlor, atrazine, carbofuran, chlorpyrifos, diazinon, dicofol, endosulfan, iprodione, mancozeb, maneb, procymidone and rotenone. The mixture was given at 1-fold and 10-fold the acceptable daily intake (ADI) level in a set of Solt–Farber-derived protocols involving diethylnitrosamine, 2-acetylaminofluorene treatments and a partial hepatectomy. Co-carcinogenic effect and promoting activity were evaluated using γ-glutamyl transpeptidase (GGT) positive altered hepatocyte foci, as well, protein and mRNA levels of glutathione S-transferase P (GSTP) in liver extracts as molecular biomarkers of carcinogenic effects. The pesticide treatments when compared to vehicle treatments always produced the same number of hepatocyte lesions and an equal GSTP expression on liver extracts independently of carcinogenic-protocol utilized. On this base, we concluded that the pesticide mixture evaluated in this report does not have tumor promoting activity or co-carcinogenic effect in the rat medium-term liver carcinogenesis. Altogether these data contribute to the confidence that the ADI represents a safe intake level to mixture of pesticides at dietary exposure.

Decreased expression of glutathione S-transferase M1 in HPV16-transfected human cervical keratinocytes in culture.

Glutathione S-transferase (GST) M1 is a member of the GST mu family of cytosolic enzymes that have been hypothesized to catalyze the conjugation of glutathione to a large number of hydrophobic substances, including carcinogens such as polynuclear aromatic hydrocarbons present in tobacco smoke, leading to their excretion. Epidemiologic and experimental evidence suggests that the risk of cervical cancer is related to both human papillomavirus (HPV) infection and cigarette smoking. We compared the enzymatic activities and mRNA levels of GSTs in GSTM1-positive human cervical keratinocytes (HCKs) that had been transfected with HPV16 with those in the parental cells. The GSTM1 activity toward the substrate trans-stilbene oxide was 5- to 7-fold lower than in the parental cells. The relative mRNA level in HCK transfected with HPV16 E6/E7, as quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) with normalization against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, was 6% that of the parental cells. It was 16 and 82%, respectively, in cells that were transfected with HPV16 E6 alone or HPV16 E7 alone. When quantified by competitive RT-PCR using an exogenous nuclease-resistant synthetic cyclophilin RNA transcript as control, the mRNA level in HCK transfected with HPV16 E6 was approximately 10-fold lower that that in the parental cells. It was approximately 5- to 7-fold lower in the HPV16 E7 or HPV16 E6/E7 cells. Our results suggest that viral infections, through the modulation of cellular xenobiotic-metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.

Lipopolysaccharide inhibition of rat hepatic microsomal epoxide hydrolase and glutathione S-transferase gene expression irrespective of nuclear factor-κB activation

Lipopolysaccharide (LPS) is an endotoxin involved in septic shock syndrome and potentiates toxicant-induced liver injury. The effects of LPS on the constitutive and inducible expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) genes were studied in rats. Northern blot analysis showed that treatment of rats with LPS caused suppression in mEH and GST gene expression. The mEH mRNA level was decreased in a time-dependent manner following a single dose of LPS (1 mg/kg, i.v.), resulting in levels of 52%, 22%, 17%, and 94% of those in untreated animals at 2, 6, 12, and 24 hr, respectively. The levels of rGSTA2 and rGSTA3 mRNA were suppressed in response to an LPS injection to the similar extents as observed in mEH mRNA, whereas rGSTM1 and rGSTM2 mRNA levels were less affected. LPS inhibited mEH gene expression at the doses of 1 μg or greater. Whereas treatment of rats with allyl disulfide (ADS), oltipraz (OZ) or pyrazine (PZ) at the dose of 50 mg/kg caused increases in the mEH mRNA level at 12 hr, a concomitant LPS injection (1 mg/kg) resulted in 80%–95% suppression of the inducible gene expression. The inducible rGSTA2, rGSTA3, rGSTM1, and rGSTM2 mRNA levels were also 50%–90% decreased at 12 hr after LPS treatment, with the relative change in rGSTA being greater than that in rGSTM. Three consecutive daily treatments with LPS (10 μg/kg/day) resulted in significant decreases of the constitutive and PZ (50 mg/kg/day, i.p. for 3 days)-inducible mEH and GST mRNA levels, which were consistent with those in the protein levels. Gel shift retardation analysis showed that LPS substantially activated the hepatic nuclear p65/p50 nuclear factor-κB (NF-κB) complex with the maximal effect observed at 1 hr at the doses of 1 μg/kg or greater. LPS-induced activation of nuclear NF-κB (1 μg/kg, i.v.) failed to be inhibited by concomitant treatment with the mEH and GST inducers, including ADS (300 mg/kg, p.o.), OZ (300 mg/kg, p.o.), and PZ (300 mg/kg, i.p.), indicating that NF-κB activation was not required for suppression of the gene expression by LPS. In contrast, GdCl 3, an inhibitor of mEH and GST expression, inhibited LPS-induced activation of the p65/p50 NF-κB. These gel shift analyses provided evidence that LPS-induced activation of the NF-κB was not responsible for alterations in the gene expression. In summary, the results of this research demonstrate that LPS effectively inhibits constitutive and inducible mEH and GST expression with decreases in their mRNA levels, and that LPS suppression in the expression of the detoxifying enzymes is not mediated with its activation of NF-κB.

Enhancement of radiation-inducible hepatic glutathione-S-transferases Ya, Yb1, Yb2, Yc1, and Yc2 gene expression by oltipraz: possible role in radioprotection.

Previous studies have shown that radiation in combination with oltipraz enhances hepatic microsomal epoxide hydrolase expression. The effects of gamma-ray radiation exposure in combination with oltipraz on the expression of hepatic glutathione-S-transferase (GST) subunits Ya, Yb1, Yb2, Yc1, and Yc2 were examined in the rat. Northern RNA blot analyses revealed that GST mRNA levels were altered in response to daily 3- or 0.5-Gy doses of radiation. The hepatic GST mRNA levels were transiently decreased at 3 and 8 hr after a single 3-Gy dose of radiation. The GST Ya, Yb1, Yb2, Yc1, and Yc2 mRNA levels were increased by 2-4-fold at 15 and 24 hr after irradiation with 3 Gy, followed by return to the levels of untreated rats at 48 hr after treatment. The treatment of animals with oltipraz alone resulted in dose-related increases in the GST Ya, Yb1, Yc1, and Yc2 mRNA levels, whereas Yb2 mRNA levels were, minimally increased. Although a single dose of oltipraz (30 mg/kg orally) caused a minimal 2-fold elevation in the hepatic GST Ya mRNA level, exposure of animals to both oltipraz and 3-Gy radiation resulted in a 4-fold relative increase in GST Ya mRNA level, indicating that the Ya mRNA expression was additively enhanced by the combination treatment. The Yb1/2 and Yc1/2 mRNA expressions were also enhanced by oltipraz in combination with radiation. Multiple exposure of rats to daily 0.5-Gy radiation caused time-related increases in GST gene expression. The greatest enhancement in GST expression was observed at 24 hr after a single 0.5-Gy dose of radiation in conjunction with oltipraz (e.g., a 9-fold relative increase in GST Ya), whereas the relative additive increases in GST mRNA were less pronounced at day 3 or 5 after treatment. These increases in the GST mRNA levels were consistent with those in the immunochemically detectable GST protein levels. Histopathological examinations revealed that exposure of rats to radiation (0.5 Gy/day for 3-5 days) caused mild-to-moderate hepatocyte degeneration with sinusoidal congestion, whereas oltipraz (30 mg/kg/day for 3 days) was effective in blocking the radiation-induced liver injury. The enhanced expression of these GST isoforms by oltipraz may be associated in part with its hepatoprotective effect against the injury caused by ionizing radiation.

Ozone-Induced Expression of Stress-Related Genes in Arabidopsis thaliana.

Ozone is a major gaseous pollutant that is known to have detrimental effects on plant growth and metabolism. We have investigated the effects of ozone on Arabidopsis thaliana growth and the pattern of expression of several stress-related genes. A. thaliana plants treated with either 150 or 300 parts per billion (ppb) ozone daily for 6 h exhibited reduced growth and leaf curling. Fresh and dry weights of ozone-treated plants were reduced 30 to 48% compared to ambient air controls. RNA blot analyses demonstrated that mRNA levels for glutathione S-transferase (GST), phenylalanine ammonia-lyase (PAL), a neutral peroxidase, and a cytosolic Cu/Zn superoxide dismutase (SOD) were higher in plants treated with 300 ppb ozone than in ambient air-treated controls. The mRNA levels of lipoxygenase and a catalase were not affected by ozone treatment. Of the transcripts examined, GST mRNA levels increased the most, showing a 26-fold induction 3 h after the initiation of ozone treatment. PAL mRNA was also rapidly induced, reaching 3-fold higher levels than controls within 3 h of ozone treatment. The neutral peroxidase and SOD mRNA levels rose more slowly, with both reaching maximum levels corresponding to 5-fold and 3-fold induction, respectively, approximately 12 h after ozone treatment. These studies indicate that ozone-induced expression of stress-related genes in A. thaliana provides an excellent model system for investigating the molecular and genetic basis of ozone-induced responses in plants.

Effects of altered calcium homeostasis on the expression of glutathione S- transferase isozymes in primary cultured rat hepatocytes

The effects of altered Ca 2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca 2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class α and μ specific antibodies showed an ∼1.6-and 1.5-fold increase in the class α, Ya and Yc subunits, respectively, whereas no significant increase (∼1.2-fold) in class μ GST expression was observed following A23187 treatment. Northern blot analysis revealed an ∼5-fold increase in GST class α and an ∼7-fold increase in class μ GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Inununoblot analysis showed a significant increase in the expression of GST class α, Ya and Yc subunits, ∼1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class μ GSTs. Northern blot analysis showed ∼3- and 2-fold increases, respectively, in class α and μ GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca 2+ homeostasis produced by either Ca 2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.

Transcriptional- and post-transcriptional-dependent regulation of glutathione S-transferase expression in rat hepatocytes as a function of culture conditions

Transcriptional activity of the glutathione S-transferase (GST) α (subunits 1 and 2), μ (subunits 3 and 4) and π (subunit 7) gene families has been analyzed using the nuclear ‘run-on’ technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co-culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co-cultured for 4 days maintain approximately 30–70% of the α gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co-culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the μ gene family is maintained at 40–160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4-fold) and dimethylsulphoxide (approximately 2-fold) in conventional culture and/or in co-culture. In contrast to freshly isolated hepatocytes, GST π gene transcriptional activity is observed in conventional and co-cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST α, μ and π genes in conventional and co-cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.