Phenobarbital increases rat hepatic prostaglandin F2 alpha, glutathione S-transferase activity and oxidative stress

Eight-week-old female F344/N rats were fed 3.0 or 6.0% of calories (kcal%) as linoleate with or without 0.05% phenobarbital (PB) for 35 days. PB treatment increased glutathione S-transferase (GST) activity by 80% and prostaglandin (PG) F2 alpha levels 4-fold (p less than 0.05). PB decreased hepatic alpha-tocopherol significantly. Hepatic linoleate was decreased by PB in rats fed 6 kcal% but not 3 kcal% linoleate. Increased dietary linoleate had no significant effect on hepatic PGF2 alpha or alpha-tocopherol levels or GST activity. This study suggests that PB hepatotoxicity and tumor-promoting ability may be mediated, at least in part, by PGF2 alpha. PB's effect on PGF2 alpha could be a result of both GST-mediated prostaglandin synthesis and oxidative stress. The removal of significant amounts of hepatic alpha-tocopherol during oxidative stress induced by PB might diminish endogenous inhibition of hepatic PG synthesis by a-tocopherol.     

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).     

The effects of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.     

Chain-shortening of prostaglandin F2 alpha by rat liver peroxisomes

Liver peroxisomes were isolated from di(2-ethylhexyl)phthalate treated rats by isopycnic sucrose gradient centrifugation of a light mitochondrial fraction. Incubation of prostaglandin F2 alpha with purified peroxisomes resulted in conversion into a more polar product(s). In contrast, incubation with mitochondrial fractions and microsomal fractions under the same conditions did not result in any detectable conversion. The polar material obtained from a preparative incubation was purified by high performance liquid chromatography and characterized by radio-gas chromatography and gas chromatography-mass spectrometry. The structure of the polar compound was shown to be 5,7,11-trihydroxy-tetranorprost-9-enoic acid (tetranor-prostaglandin F1 alpha). Prostaglandin F2 alpha was thus chain-shortened by four carbon atoms.     

The role of selenium-dependent and selenium-independent glutathione peroxidases in the formation of prostaglandin F2 alpha

In recent years, growing evidence suggests that glutathione peroxidases (GSH-Pxs), both selenium-dependent GSH-Px (Se-GSH-Px) and selenium-independent GSH-Px (non-Se-GSH-Px) play an important role in the biosynthesis of prostaglandins and leukotrienes and in the regulation of key enzymes associated with the arachidonic acid cascade. The precise nature of their involvement in eicosanoid metabolism, however, is not yet completely understood. In the study reported here, we have systematically determined the catalytic efficiencies of Se-GSH-Px and non-Se-GSH-Px toward prostaglandin (PG) G2 (PGG2) and PGH2. Se-GSH-Px exhibited high catalytic activity for the reduction of PGG2 as indicated by Km and Vmax values of 12 microM and 78 mumol/min/mg, respectively, whereas PGH2 was found to be a poor substrate, an indication that Se-GSH-Px reduces the hydroperoxide moiety but not the endoperoxide moiety of PGG2. The kinetic constants of Se-GSH-Px toward PGG2 were comparable to those determined for such classical substrates as H2O2 and cumene hydroperoxide. In contrast to Se-GSH-Px, non-Se-GSH-Px associated with cationic isozyme II of glutathione S-transferases (GSTs) from sheep lung cytosol was very active in the conversion of PGH2 to PGF2 alpha with a Vmax of 960 nmol/min/mg and a Km of 77 microM. This study shows that PGF2 alpha formation by non-Se-GSH-Px occurred in a GSH-dependent reduction of either PGG2 or PGH2. When PGG2 was used as the substrate for non-Se-GSH-Px, a novel intermediate compound appeared and was later identified by several methods of structural analysis as 15-hydroperoxy PGF2 alpha. Thus, the reductive cleavage of the endoperoxide occurs faster than the 15-hydroperoxide reduction allowing 15-hydroperoxy PGF2 alpha to accumulate briefly. A study of GSTs from several different tissues and species indicated that the transformation of PG endoperoxides to PGF2 alpha is catalyzed specifically by GST isozymes, which contain Ya size subunits. This specificity of GST isozymes in PG biosynthesis, coupled with their tissue-specific expression, may be a mechanism by which the body modulates the type of PGs produced in these tissues. Also, these results suggest a possible interaction of Se-GSH-Px and non-Se-GSH-Px in the biosynthesis of PGF2 alpha.     

Identification of heterogeneous and microheterogeneous subunits of glutathione S-transferase in rat liver cytosol

Subunits of multiple molecular forms of dimeric glutathione S-transferase in rat liver cytosol were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-electrophoresis) followed by staining with Coomassie blue dye. The five subunits, Ya, Yb, Yb', Yc, and Yp (Mr's 26,500, 27,500, 27,500, 28,500, and 26,000, respectively) of seven molecular forms, A2, AC, C2, B2, BL, L2, and GST-P, were identified by comparison of molecular weights and pI values with those of purified molecular forms and by immunoadsorption of the molecular forms in the cytosol as well as those synthesized in vitro using antibodies against the seven forms. Yp is the subunit of placental glutathione S-transferase, GST-P (YpYp), which is markedly increased in carcinogen-treated rat livers [A. Kitahara et al. (1984) Cancer Res. 44, 2698-2703; K. Satoh et al. (1985) Proc. Natl. Acad. Sci. USA 82, 3964-3968]. Microheterogeneity was detectable within Yb, Yb', and Yp subunits, the different forms, termed Yb1, Yb2, Yb'1, Yb'2, and Yp1, Yp2, being similar in size but differing by approx. 0.3 pI unit within each subunit. These microheterogeneous forms were also detectable in the polypeptides translated in vitro in a rabbit reticulocyte lysate translation system from liver poly(A)-containing RNAs, suggesting that they are translatable from distinct mRNAs.     

The development of glutathione S-transferase subunits in rat liver. Sensitive detection of the major subunit forms of rat glutathione S-transferase by using an e.l.i.s.a. method.

The development of the subunits of glutathione S-transferase in rat liver shows that there is a co-ordinated development of the Ya, Yb1, Yb2 and Yc subunits but that the Yf and Yk subunits show unique patterns of development. The Yk subunit is the only form that is expressed at relatively high levels during the foetal period as well as during the adult period. In contrast with all other forms, the Yf subunit in the rat declines rapidly during the last few days before parturition and is virtually undetectable in hepatocytes of adult animals. The expression of the Yf subunit in foetal liver presents a 'patchy' appearance that is similar to that induced by the administration of lead acetate and may reflect cell-cycle-associated regulation of expression.     

Down-regulation of prostaglandin F2 alpha receptors in rat liver during chronic endotoxemia.

Prostaglandin (PG) F2 alpha binding parameters were measured in purified plasma membrane preparations isolated from livers of chronically endotoxin-(ET) treated rats and corresponding controls. Two classes of binding sites were detected in both groups: high affinity, low capacity, with a KD of 44.4 +/- 8.8 nM for saline- and 28.6 +/- 11.3 nM for ET-treated rats (n = 5 for both, p greater than 0.05) and low affinity, high capacity with a KD of 1.12 +/- 0.49 microM for saline- and 1.24 +/- 0.43 microM for ET-treated rats (p greater than 0.05). Bmax values for high affinity sites were 1.01 +/- 0.18 fmol.mg-1 protein for saline- and 1.02 +/- 0.54 (same units) for ET-treated rats (p greater than 0.05). There was a significant difference (p less than 0.01) between the Bmax values for low affinity sites in saline- (675 +/- 332 fmol.mg-1 protein) and ET-treated rats (12 +/- 1, same units). This decrease in the amount of PGF2 alpha low affinity high capacity binding sites may underlie the depression of the PGF2 alpha stimulatory effect on hepatic gluconeogenesis induced by non-lethal, chronic ET treatment of rats, recently described by us (9).     

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.     

CCAAT enhancer-binding protein alpha suppresses the rat placental glutathione S-transferase gene in normal liver

The rat placental glutathione S-transferase (GST-P), an isozyme of glutathione S-transferase, is not expressed in normal liver but is highly induced at an early stage of chemical hepatocarcinogenesis and in hepatomas. Recently, we reported that the NF-E2 p45-related factor 2 (Nrf2)/MafK heterodimer binds to GST-P enhancer 1 (GPE1), a strong enhancer of the GST-P gene, and activates this gene in preneoplastic lesions and hepatomas. In addition to the positive regulation during hepatocarcinogenesis, negative regulatory mechanisms might work to repress GST-P in normal liver, but this remains to be clarified. In this work, we identify the CCAAT enhancer-binding protein alpha (C/EBPalpha) as a negative regulator that binds to GPE1 and suppresses GST-P expression in normal liver. C/EBPalpha binds to part of the GPE1 sequence, and the binding of Nrf2/MafK and C/EBPalpha to GPE1 is mutually exclusive. In a transient-transfection analysis, C/EBPalpha activated GPE1 in F9 embryonal carcinoma cells but strongly inhibited GPE1 activity in hepatoma cells. The expression of C/EBPalpha was specifically suppressed in GST-P-positive preneoplastic foci in the livers of carcinogentreated rats. A chromatin immunoprecipitation analysis showed that C/EBPalpha bound to GPE1 in the normal liver in vivo but did not bind in preneoplastic hepatocytes. Introduction of the C/EBPalpha gene fused with the estrogen receptor ligand-binding domain into hepatoma cells, and subsequent activation by beta-estradiol led to the suppression of endogenous GST-P expression. These results indicate that C/EBPalpha is a negative regulator of GST-P gene expression in normal liver.     

Characterization of the basic glutathione S-transferase B1 and B2 subunits from human liver.

The basic glutathione S-transferases in human liver are composed of at least two immunochemically distinct polypeptides, designated B1 and B2. These subunits exist as homodimers, but can hybridize to form the B1B2 heterodimer [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Although these basic glutathione S-transferases possess similar catalytic properties, the B2 subunit exhibits significantly greater selenium-independent glutathione peroxidase activity than subunit B1. The use of the ligands haematin, tributyltin acetate and Bromosulphophthalein as inhibitors of 1-chloro-2,4-dinitrobenzene-GSH-conjugating activity clearly discriminate between the B1 and B2 subunits and should help facilitate their identification. Peptide mapping experiments showed that B1 and B2 are structurally distinct, but related, subunits; subunit B1 yielded 43 tryptic peptides, seven of which were unique, whereas subunit B2 yielded 40 tryptic peptides, four of which were unique.     

Cloning and sequence analysis of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits.

We describe the construction and characterization of a cDNA plasmid for one of the rat liver glutathione S-transferase subunits. Poly(A)-RNA isolated from rat livers was enriched for glutathione S-transferase mRNA activity and used as templates to synthesize double stranded cDNA. The double stranded cDNAs were annealed to pBR322 through terminal deoxynucleotidyl transferase generated GC-tails followed by transformation into E. coli. Several candidate clones were selected by colony hybridization using polynucleotide kinase labeled liver and testis poly(A)-RNA probes. These candidate clones were further characterized by hybrid-selected translation of mRNA followed by immunoprecipitation and SDS gel electrophoresis. The positive clone, pGTR112 was mapped with restriction endonuclease analysis and sequenced by the chemical method of Maxam and Gilbert. The largest upen reading frame contains 142 amino acids very rich in Arg and Lys residues. The C-terminal residue phenylalanine of this open reading frame is consistent with what was reported for one of the ligandin subunits by Bhargava et al., (J. Biol. Chem. 253, 4116-4119, 1978). Among the 352 nucleotides covered by both pGTR112 and pGST94 described by Kalinyak and Taylor (J. Biol. Chem. 257, 523-530, 1982), there are only 9 nucleotide differences resulting in four changes of amino acid sequences.     

The induction of labour by the intravenous infusion of prostaglandin F2alpha

Labour was induced by the intravenous infusion of prostaglandin F2alpha in 106 patients at 36-44 weeks of pregnancy. The induction was successful in 80% of the women. The total dose needed ranged from 0.1 to 14.2 mg of PGF2alpha. The uterine activity and fetal heart rate were recorded by cardiotocography. The contraction pattern and induction-delivery time were the same as reported for induction with oxytocin. In one case uterine hyperactivity occurred after rupture of the membranes. No serious adverse effects were seen, but in a few cases local irritation was noted at the site of infusion. The condition of the infants was generally good. It might be concluded that PGF2alpha seems valuable for the induction of labour, but due to the risk for over-stimulation careful supervision is needed.     

Subcellular distribution of N-ethylmaleimide-stimulatable glutathione S-transferase activity in rat liver. Evidence of localization of glutathione S-transferase in peroxisomal membrane

Subcellular distribution of glutathione S-transferase activity was investigated as stimulated form by N-ethylmaleimide in rat liver. The stimulated glutathione S-transferase activity was localized in mitochondrial and lysosomal fractions besides microsomes. Among N-ethylmaleimide-treated submitochondrial fractions, glutathione S-transferase activity was stimulated only in outer mitochondrial membrane fraction. In lysosomal fraction, it was suggested that glutathione S-transferase activity in peroxisomes, which is immunochemically related to microsomal transferase, was also stimulated, but not in lysosomes.     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.