Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1.

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.

Inhibition of Na+/K(+)-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis.

To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener, 86Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti-CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of 86Rb+, however, the uptake of 86Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of 86Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K(+)-ATPase inhibitor), demonstrating Na+/K(+)-ATPase dependent K+ uptake. Ouabain induced significant 86Rb+ efflux in untreated cells, as well as it seemed to compete with 86Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K(+)-ATPase in the CD95-mediated 86Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na+/K(+)-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.

Dithiocarbamate toxicity toward thymocytes involves their copper-catalyzed conversion to thiuram disulfides, which oxidize glutathione in a redox cycle without the release of reactive oxygen species.

We have reported previously that diethyldithio-carbamate (DDC) and pyrrolidine dithiocarbamate (PDTC) induce apoptosis in rat thymocytes. Apoptosis was shown to be dependent upon the transport of external Cu ions into the cells and was accompanied by the oxidation of intracellular glutathione, indicating the inducement of pro-oxidative conditions (C. S. I. Nobel, M. Kimland, B. Lind, S. Orrenius, and A. F. G. Slater, J. Biol. Chem. 270, 26202-26208, 1995). In the present investigation we have examined the chemical reactions underlying these effects. Evidence is presented to suggest that dithiocarbamates undergo oxidation by CuII ions, resulting in formation of the corresponding thiuram disulfides, which are then reduced by glutathione, thereby generating the parent dithiocarbamate and oxidized glutathione (glutathione disulfide). Although DDC and PDTC were found to partially stabilize CuI ions, limited redox cycling of the metal ion was evident. Redox cycling did not, however, result in the release of reactive oxygen species, which are believed to be scavenged in situ by the dithiocarbamate. DDC and PDTC were, in fact, shown to prevent copper-dependent hydroxyl radical formation and DNA fragmentation in model reaction systems. The thiuram disulfide disulfiram (DSF) was found to induce glutathione oxidation, DNA fragmentation, and cell killing more potently than its parent dithiocarbamate, DDC. Of particular importance was the finding that, compared with DDC, the actions of DSF were less prone to inhibition by the removal of external copper ions with a chelating agent. This observation is consistent with our proposed mechanism of dithiocarbamate toxicity, which involves their copper-catalyzed conversion to cytotoxic thiuram disulfides.

Mechanism of dithiocarbamate inhibition of apoptosis: thiol oxidation by dithiocarbamate disulfides directly inhibits processing of the caspase-3 proenzyme.

Dithiocarbamates (DCs) have been reported to be potent inhibitors of apoptosis in several different model systems, which suggests a target common to the apoptotic machinery. Without further investigation, this has been assumed to reflect an antioxidant activity of the DCs. However, we have recently shown that DCs exert prooxidant effects on T cells [Nobel et al. (1995) J. Biol. Chem. 270, 26202-26208], which are dependent on their transfer of external copper into the cells and can be inhibited by the inclusion of high-affinity external copper chelators in the medium. Investigating antiapoptotic actions of DCs, we found that inclusion of a membrane-impermeable copper chelator severely compromised the inhibitory activity of reduced DCs. Since copper can promote DC oxidation to the respective DC disulfides, the inhibitory effect on lymphocyte apoptosis might be mediated by the DC disulfides. In agreement with this we observed that DC disulfides were more potent inhibitors of T cell apoptosis than their reduced counterparts. Inhibition of apoptosis by DC disulfides correlated with the inhibition of caspase-3 proenzyme processing and activation. Similar results were obtained in a cell-free model system of caspase-3 activation. Significantly, dithiothreitol reduction of the DC disulfide abolished its inhibition of in vitro proenzyme processing, thereby demonstrating thiol-disulfide exchange between the DC disulfide and a free thiol group on an activator(s) of caspase-3. Since T cell apoptosis involves the generation of mature caspase-3 and requires caspase-3-like activity, we propose that (1) DC disulfides are the active agents behind DC inhibition of apoptosis and (2) their site of action is the proteolytic activation of this enzyme. These findings also reveal the potential for other thiol-oxidizing toxicants to inhibit apoptosis by preventing the proteolytic activation of caspases.

Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors.

n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.

Disulfiram is a potent inhibitor of proteases of the caspase family.

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.

Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody.

Although human JURKAT T lymphocytes induced to undergo apoptosis with anti-Fas/APO-1 antibody were observed to rapidly lose reduced glutathione (GSH), increased concentrations of oxidized products were not detectable. Unexpectedly, the reduced tripeptide was instead quantitatively recovered in the incubation medium of the cells. As GSH loss was blocked by bromosulfophthalein and dibromosulfophthalein, known inhibitors of hepatocyte GSH transport, a specific export rather than nonspecific leakiness through plasma membranes is proposed to be responsible. Apoptosis was delayed when GSH-diethylesters were used to elevate intracellular GSH, although the high capacity of the activated efflux system quickly negated the benefit of this treatment. Stimulation of GSH efflux provides a novel mechanism whereby Fas/APO-1 ligation can deplete GSH. We speculate that it enhances the oxidative tonus of a responding cell without requiring an increase in the production of reactive oxygen species.

The role of intracellular oxidants in apoptosis.

Apoptotic cell death is a complex process whose biochemistry is poorly understood. Direct exposure of various cell types of oxidants such as hydrogen peroxide or lipid hydroperoxides can directly induce apoptosis, while in many experimental models pretreatment of cells with antioxidants has been shown to protect against this form of cell death. Recent experimental evidence suggests that multiple forms of thymocyte apoptosis can be inhibited by free radical spin traps, spin probes and thiol reductants, and that this inhibition correlates with a lower oxidative burden within the treated cells. Possible sites of production of these oxidants include mitochondrial electron transport and phospholipase A2-activated arachidonic acid metabolism, while intracellular targets may include redox sensitive transcription factors and inhibitory proteins that must be tagged for proteolysis before apoptosis can commence.

Induction of interleukin-1 beta mRNA and enkephalin mRNA in the rat adrenal gland by lipopolysaccharides studied by in situ hybridization histochemistry.

Interleukin-1 is a macrophage-derived cytokine, which can also be synthesized in other cell types. It has been shown to exert several activities in the nervous and endocrine system, including a potent activating effect on the hypothalamo-pituitary-adrenal axis. High levels of interleukin-1 have previously been found in the adrenal gland. The effect of bacterial lipopolysaccharides (2.0 mg/kg) on interleukin-1 beta mRNA was studied in the rat adrenal gland by in situ hybridization histochemistry using a synthetic oligonucleotide probe. A transient induction was observed, with the strongest hybridization signal seen after 1.5 h and subsequent decrease to near basal levels at 3 h. Similarly, the effect of lipopolysaccharides on preproenkephalin A mRNA expression in the adrenal gland was analyzed. Preproenkephalin A is a precursor for methionine-enkephalin, a neuropeptide known to be produced in the chromaffin cells, and also known to affect immunological functions. A low level of preproenkephalin A mRNA was seen in the adrenal medulla in animals injected with saline and 0.5 h after lipopolysaccharide administration. A small, but distinct increase in hybridization signal appeared at 1.5 h and a marked increased was observed at 3 h after administration of lipopolysaccharides. In addition to the different kinetics of expression after LPS administration, the two mRNA species showed a somewhat different morphological distribution in that IL-1 beta mRNA could be seen in both adrenal medulla and cortex, whereas preproenkephalin A expression was confined to the adrenal medulla.

Nitrone spin traps and a nitroxide antioxidant inhibit a common pathway of thymocyte apoptosis.

Oxidative stress has recently been suggested to be a mediator of apoptotic cell death [Buttke and Sandstrom (1994) Immunology Today 15, 7-10], although evidence that this phenomenon is a widespread component of apoptosis is lacking. When rat thymocytes were exposed to the glucocorticoid methylprednisolone (MPS), a progressive increase in intracellular peroxides and a decrease in glutathione (GSH) were observed to accompany the onset of apoptosis. Using Percoll density gradients to isolate subpopulations of thymocytes at different stages of apoptosis, the increase in peroxide content was found to be restricted to apoptotic cells, while a significant depletion of GSH and reduced protein thiol was detected in both pre-apoptotic and fully apoptotic cells. To investigate the biological significance of these redox changes, the free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TMPO), and the related nitroxide-radical antioxidant 2,2,6,6-tetramethyl-1-piperidinyl-1-oxyl (TEMPO) were tested as inhibitors of thymocyte apoptosis. The cell shrinkage and DNA fragmentation induced by four different initiators of apoptosis were reduced by each compound. TEMPO inhibition of both etoposide- and MPS-induced thymocyte DNA fragmentation was also found to correlate with an increase in intracellular GSH, providing support for the proposal that its antioxidant properties were responsible for the observed protective activity. We conclude that some form of intracellular oxidation (here measured indirectly by changes in intracellular GSH and peroxide levels) is required during thymocyte apoptosis even when this process is initiated by an agent that does not exert a direct oxidant action.