Enzymatic catalysis of the reversible sulfitolysis of glutathione disulfide and the biological reduction of thiosulfate esters

Rat liver supernatants were shown to contain an enzymatic activity catalyzing in both forward and reverse directions the reversible sulfitolysis of glutathione disulfide. The enzymatic sulfitolysis has maximal activity at pH 7. S-Sulfoglutathione, which is a product of the sulfitolysis, was isolated by passage through an ion-exchange column. Three different assays were applied to determine S-sulfoglutathione, viz., methods based on the ninhydrin reaction, the formation of a thiazoline derivative in strong acid, and the use of radioactively labeled glutathione. The reversal of the sulfitolysis, i.e., the reaction of S-sulfoglutathione with glutathione, was studied directly by determination of sulfite with radioactive N-ethylmaleimide, or indirectly by coupling to the NADPH- and glutathione reductase-linked reduction of glutathione disulfide.Chromatographic analysis of rat liver supernatants demonstrated that all fractions catalyzing the reversible sulfitolysis did also catalyze the previously studied thiol-disulfide interchange of glutathione and the mixed disulfide of cysteine and glutathione.The reduction of thiosulfate esters, such as S-sulfocysteine and trimethylammonium-ethylthiosulfate, with glutathione was also catalyzed by the enzyme active in the sulfitolysis, which indicates an important biosynthetic role of the enzyme in microorganisms synthesizing cysteine via S-sulfocysteine. The enzyme is also capable of participating in the formation of the naturally occurring S-sulfoglutathione.     

Identification of plasma proteins containing sulfite-reactive disulfide bonds

Plasma protein S-sulfonate compounds (RS-SO-3) have previously been shown to form, presumably by sulfitolysis of disulfide bonds, as a result of exposure to sulfite. In the investigations reported here, we identify two proteins in rabbit plasma, namely albumin and plasma fibronectin, which contain reactive sites for S-sulfonate formation. Separation and identification of these proteins following in vitro and in vivo exposure to sulfite was accomplished primarily by column chromatographic and electrophoretic techniques. In addition, the structure of presumed S-sulfonate groups was confirmed by the identification of cysteinyl-S-sulfonate residues in protein hydrolysates generated by enzymatic digestion. The molar ratio of RS-SO-3 in both albumin and plasma fibronectin was less than one. Data from our experiments suggest that the mixed disulfide site of non- mercaptalbumin is the reactive site for S-sulfonate formation. The site(s) of formation within the plasma fibronectin molecule was not investigated. The possible physiological significance of disulfide sulfitolysis of albumin and plasma fibronectin is discussed.     

Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

Multiple Forms of Inorganic Pyrophosphatase from the Leaf of Amarantus blitum L.

The existence of a carrier-bound pathway for inorganic sulfate assimilation has been proposed in Chlorella and Escherichia coli. The possibility that the sulfonyl group of active sulfate is transferred to a specific organic acceptor to form thiosulfate ester was examined with Salmonella typhimurium LT-2. Some 11% of the radioactive products from [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate were transferred to high molecular weight compounds, and the remainder of the product is identified as free inorganic sulfite. Apparent thiosulfonate reductase activity was detected in the reaction mixtures containing S-sulfoglutathione and NADPH as conceivable substrates, but not with partially purified sulfite reductase. The former activity was attributable to the nonenzymatic reaction, sulfitolysis. Through these in vitro experiments the existence of the carrier-bound pathway was disproved.     

Reaction of selenium with immunoglobulin molecules.

Radioactive selenite reacts with purified human and goat immunoglobulins at acidic and neutral pH. The antigenic properties of the immunoglobulins are retained during the selenium labelling as shown by immunoelectrophoresis and autoradiography. Pepsin digests of 75Se-labelled IgG possess 75Se both in the (Fab')2 fraction and in the low molecular weight peptides derived from the Fc domains. Alpha-1-acid glycoprotein, ribonuclease, and lysozyme are also labelled by this procedure. Enhancement of 75Se incorporation by urea, guanidinium chloride, mercaptoethanol, sodium sulfite and carrier selenite is interpreted as an effect of destabilization of IgG disulfide bonds. Up to 1.4 g atoms Se per mol IgG have been incorporated. We assume that selenite is cleaving disulfides by a process akin to sulfitolysis. The lability of the isolated 75Se-labelled IgG to high concentrations of mercaptans and sulfite is consistent with this idea. These 75Se-labelled proteins may be useful in structure studies and radioimmunoassay.     

Sulfate-reducing pathway in Escherichia coli involving bound intermediates.

Although a sulfate-reducing pathway in Escherichia coli involving free sulfite and sulfide has been suggested, it is shown that, as in Chlorella, a pathway involving bound intermediates is also present. E. coli extracts contained a sulfotransferase that transferred the sulfonyl group from a nucleosidephosphosulfate to an acceptor to form an organic thiosulfate. This enzyme was specific for adenosine 3'-phosphate 5'-phosphosulfate, did not utilize adenine 5'-phosphosulfate, and transferred to a carrier molecule that was identical with thioredoxin in molecular weight and amino acid composition. In the absence of thioredoxin, only very low levels of the transfer of the sulfo group to thiols was observed. As in Chlorella, thiosulfonate reductase activity that reduced glutathione-S-SO3- to bound sulfide could be detected. In E. coli, this enzyme used reduced nicotinamide adenine dinucleotide phosphate and Mg2+, but did not require the addition of ferredoxin or ferredoxin nicotinamide adenine dinucleotide phosphate reductase. Although in Chlorella the thiosulfonate reductase appears to be a different enzyme from the sulfite reductase, the E. coli thiosulfonate reductase and sulfite reductase may be activities of the same enzyme.     

Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1

A keratinolytic enzyme produced by Bacillus subtilis KS-1 isolated from poultry waste was purified and characterized using ultrfiltration, DEAE-Sephadex, and Sephadex G-100 chromatographies. The specific activity of the purified protease was 538.2 units/mg. The enzyme was shown to have a relative molecular mass of 25.4 kDa. The enzyme was made completely inactive by PMSF, which indicates a serine-protease. Dithiothreitol enhanced keratinolytic activity by 1.6 times at a concentration of 5.0 mM. These results suggest that the cleavage of the disulfide bonds with reducing agents can occur directly or by excretion of sulfite, which causes the sulfitolysis of the disulfide bonds. The first 10 amino acids of the N-terminal sequence are Ala-Gin-Pro-Val-Glu-Trp-Gly-Ile-Ser-Gln. The enzyme hydrolyzed casein and feather, but hydrolyzed casein more effectively than it did feather.     

Cystine catabolism in mycelia of Microsporum gypseum,a dermatophytic fungus

The fate of ³⁵S label was studied during cystine degradation by mycelia of the dermatophytic fungus Microsporum gypseum. Excess free cystine in the medium was readily taken up and its sulfur moiety excreted as inorganic sulfate and sulfite. At intervals after 3–60 min of incubation with ³⁵S cystine the products of cystine catabolism were extracted from the mycelia by boiling water and separated by thin layer chromatography and electrophoresis. A total of 10 sulfur-containing compounds were identified, and their relative radioactivity was assessed. After 3 min the mycelia contained, in addition to cystine, labeled cysteine and particularly cysteine sulfinic acid which was accompanied by a smaller amount of cysteic acid. Later on, oxidized and reduced glutathione, inorganic sulfate and taurine appeared consecutively. In all extracts, small amounts of labeled S-sulfocysteine were found, not, however, sulfite.The results suggest that the intermediates of cysteine degradation in the fungal mycelia are cysteine, cysteine sulfinate, unstable sulfinylpyruvate, sulfite and sulfate, i.e., that the catabolic pattern is similar to that of higher organisms.The formation and the role of S-sulfocysteine, cysteic acid, and of taurine is not yet completely understood, although certainly autoxidative processes are involved in the formation of the latter two compounds, and sulfitolysis in that of the former compound.     

A conserved mechanism for sulfonucleotide reduction

Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor.     

New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase

Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon Km PAPS (22.5 microM). Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.     

Gpx1 is a stationary phase-specific thioredoxin peroxidase in fission yeast

The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1⁺). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H₂O₂, it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.     

Production of recombinant oxytocin through sulfitolysis of inteincontaining fusion protein

An artificial gene consisting of seven copies of an oxytocinoyl-lysine encoding sequence arranged in a tandem was synthesized and inserted downstream of the SspDnaB intein gene in a pTWIN1 plasmid. The corresponding fusion protein Dnab-7oxy contained 16 cysteine residues and formed inclusion bodies when expressed in E. coli. The standard protocol involving solubilization of the fusion protein and its autocatalytic cleavage on a chitin resin was not effective because of a very low yield of the cleavage reaction. Attempts to perform a refolding of the intein part of the fusion protein in solution were also unsuccessful because of a high level of protein aggregation. Sulfitolysis of cysteine residues is known to increase a solubility of proteins and peptides. Therefore we suggested a one-step approach that combines solubilization of inclusion bodies and sulfitolysis of a hybrid protein. The fusion protein was completely reduced and solubilized in 8M urea at pH 9.0 in the presence of sodium sulfite and sodium tetrathionate. The sulfitized protein was loaded onto a chitin column, an efficient cleavage was induced by a pH shift from 9.0 to 6.5, and seven successively connected oxytocinoyl- lysine units were released. The heptamer was subjected to trypsinolysis yielding sulfitized monomers of oxytocinoyllysine. Oxytocinoyl-lysine was refolded as described previously and treated by carboxypeptidase B to form the oxytocinic acid. The target oxytocin amide was then synthesized via methyl ester intermediate. Using this approach 6 mg of recombinant oxytocin can be obtained from 1 g of biomass.     

Intron position as an evolutionary marker of thioredoxins and thioredoxin domains

In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequencedArabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence ofChlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.     

Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite - a compound in the aqueous fraction of wine - could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu(2+)) initiated LDL oxidation. As transition metal ions can catalyse the auto-oxidation of sulfite, we studied the influence of sulfite on Cu(2+) initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu(2+). The LDL-oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu(2+)/SO(3)(2-) was not inhibited by SOD but by catalase. We propose that formation of Cu(+), sulfite radicals (SO(3)*(-)) and hydroxyl radicals (OH(*)) is a mechanism by which sulfite could act as a pro-atherogenic agent in presence of transition metal ions.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x

In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.     

Biophysical and biochemical characterization of the thioredoxin system from Colwellia psychrerythraea

The thioredoxin system is a ubiquitous oxidoreductase system consisting of the enzyme thioredoxin reductase, the protein thioredoxin, and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, were characterized using biophysical and biochemical techniques. Analysis of the complete genome sequence of the C. psychrerythraea thioredoxin system suggested the presence of a putative thioredoxin reductase and at least three thioredoxin. In this study, these identified putative thioredoxin system components were cloned, overexpressed, purified, and characterized. Our studies have indicated that the thioredoxin system proteins from E. coli were more stable than those from C. psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from E. coli had a higher optimal temperature than that from C. psychrerythraea.     

Functional and physicochemical characterization of the thioredoxin system in Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of -267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mm- 1 cm-1, there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/trypanothione reductase system.     

Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.     

Regulation of thioredoxin mRNA in the rat uterus by gonadal steroids

Estradiol has been shown to increase the level of thioredoxin mRNA in the uterus of the ovariectomized (ovx) rat. In this study the influence of progesterone, androgens, the anti-estrogen ICI 182780 and the anti-androgen Flutamid on thioredoxin expression, has been studied in the rat uterus. Thioredoxin mRNA concentrations were determined by solution hybridization. Ovx rats treated with progesterone alone showed no effect on thioredoxin expression. Combined treatment of ICI 182780 and estradiol attenuated the estradiol-induced increase in thioredoxin mRNA. When ovx rats were treated with a testosterone depot, the amount of thioredoxin mRNA was increased five-fold after 48 h and remained at that level during the rest of the 168 h monitored. A similar increase in thioredoxin mRNA could be seen after 5-dihydrotestosterone treatment, indicating a true androgenic effect. In addition, the anti-androgen Flutamid attenuated the thioredoxin mRNA increase seen after 5-dihydrotestosterone treatment alone.

It is concluded that thioredoxin mRNA is regulated by growth promoting gonadal steroids in the rat uterus. The attenuation of the estrogen and androgen-induced increases of the thioredoxin mRNA with ICI 182780 and Flutamid, indicate that the effect is mediated via the estrogen receptor and androgen receptor respectively. None of these hormones affected the hepatic thioredoxin mRNA level in the same animals.