Glutathione modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor toxicity.

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha causes much of the hepatocellular injury and cell death that follows toxin-induced liver damage. The mechanism by which toxic liver injury sensitizes hepatocytes to TNF-alpha cytotoxicity is unknown. The aim of this study was to determine the role of the antioxidant glutathione in this process. METHODS: A rat hepatocyte cell line and primary hepatocytes sensitized to TNF-alpha toxicity by the addition of actinomycin D were examined for changes in glutathione levels and for the effects of glutathione depletion or supplementation on cell death. The in vivo effects of glutathione depletion were determined in mice treated with galactosamine plus lipopolysaccharide. RESULTS: Treatment of hepatocytes with actinomycin D and TNF-alpha induced apoptotic cell death without affecting cellular glutathione levels or production of the reactive oxygen intermediate H2O2. Glutathione depletion induced by diethyl maleic acid significantly increased TNF-alpha-induced cell death even when this agent was administered 2 hours after TNF-alpha treatment. Hepatocyte cell death was not affected by glutathione supplementation. In mice treated with galactosamine plus lipopolysaccharide, glutathione depletion increased mortality from liver injury from 32% to 72%. CONCLUSIONS: TNF-alpha-induced cytotoxicity in hepatocytes occurs in the absence of glutathione depletion. However, a preexisting reduction in glutathione levels can significantly increase cell death from TNF-alpha.

Salinity change and cell volume: the response of tissues from the estuarine mussel Geukensia demissa.

The response of cell volume to changes in external salinity was assessed in four tissues (gill, mantle, hemolymph cells and ventricle) of the estuarine mussel Geukensia demissa by using one or more of the following three indicators of cell volume response: changes in cell dimensions, cell water space and cell solute content. All three techniques indicated that short-term volume regulation was generally absent from gill tissue. Lateral cell height in gills, measured using differential interference contrast (DIC) microscopy, increased by approximately 20% after an abrupt exposure to reduced salinity (60% artificial sea water, ASW). There was significant variability in the observance of a regulatory volume decrease (RVD) subsequent to the initial swelling; cells remained swollen for 1 h after low-salinity exposure in two-thirds of the trials, while there was a return of cell volume towards control values in the remaining one-third of the trials. Lateral cell height increased linearly when salinity was gradually decreased from 100 to 60% ASW over 135 min. Cell height then returned to control values when the salinity was abruptly returned to 100% ASW, indicating that an RVD was not elicited by a slow change in salinity of the type normally encountered by estuarine mussels. Cumulative cell water space in gills increased by 47% after exposure to 60% ASW and the cells remained swollen for at least 4 h, returning to control values when gills were returned to 100% ASW. Consistent with the overall lack of an RVD, there was only a small decrease (approximately 5%) in cumulative osmolyte content (primarily taurine, betaine and K+) after 4 h in 60% ASW. Decreases in both cell water space and osmolyte content after 3 weeks of acclimation to 60% ASW indicated a long-term RVD of approximately 60%. Individual cells in the mantle epithelium also generally lacked an RVD in response to lowered salinity. Both abrupt and gradual decreases in salinity caused an increase in mantle cell height to a maximum of 25-30%, and cell height returned to the control height when salinity was abruptly returned to 100% ASW. Corresponding with the lack of an RVD in individual mantle cells, there was no change in solute content of the mantle tissue after 4 h of exposure to low salinity. The response of the volume of spherical hemolymph cells to 1 h of abrupt exposure to low salinity, calculated from measured cell diameters, likewise indicated that an RVD is generally lacking in these hemolymph cells. In the ventricle, however, there was a significant decrease in amino acid and betaine content after 4 h of exposure to low salinity, suggesting tissue-specific variability in the cellular response to salinity change. The consistent lack of a short-term RVD in many tissues may serve to avoid large energetic expenditures associated with repeated volume regulation in the face of the frequent, short-term changes in salinity encountered by estuarine mussels.

Response of cell volume in Mytilus gill to acute salinity change.

The response of gill cell volume in Mytilus californianus and Mytilus trossolus (=edulis) to acute changes in salinity was assessed using three independent indicators: optical measurement of lateral cell height, measurement of intracellular water content using radiolabeled tracers and measurement of the contents of the major osmolytes of the gills. Optical measurements indicated significant variation in the response of individual lateral cells of M. californianus to acute low-salinity shock. Lateral cell height increased by approximately 20% shortly after abrupt exposure to 60% artificial sea water (ASW). Following this initial swelling, we estimate that a substantial regulatory volume decrease (RVD) was present in 25% of the trials. More commonly, however, an RVD was either absent or minimal: cell height remained elevated for at least 1 h, then returned to the control height when gills were re-exposed to 100% ASW. Changes in the combined water space of all cells in the gill, measured as the difference between total water space and extracellular space ([14C]polyethylene glycol space), indicated that cell volume regulation in the gill as an organ was also absent or minimal. Cell water space was 2.16 ml g-1 dry mass in isolated gills of M. californianus acclimated to 100% sea water in the laboratory and increased to 2.83 ml g-1 dry mass after a 6 min exposure to 60% ASW. Cell water space was still 2.81 ml g-1 dry mass after 1 h in 60% ASW and returned to 2.06 ml g-1 dry mass upon re-exposure to 100% ASW. Consistent with these observations, the gill contents of the principal cytoplasmic osmolytes (taurine, betaine and K+) were unchanged (approximately 450, 250 and 230 mu mol g-1 dry mass, respectively) following exposure of gills from 100% ASW-acclimated mussels to 60% ASW. A decrease in cell water space to 2.66 ml g-1 dry mass after 4 weeks of acclimation to 60% ASW corresponded with a 37% decrease in betaine content; taurine and K+ contents were unchanged. The changes in water space and solute content of gills from freshly collected M. californianus and M. trossolus were also consistent with the absence of volume regulation; cell water space remained elevated for at least 1 h after low-salinity exposure, and solute contents were unchanged after this period. We calculated the potential energetic cost of cell volume regulation for mussels exposed to 12 h of sinusoidal fluctuations between 100% and 50% sea water; solute uptake for full volume regulation in all tissues would cost a minimum of approximately 30% of the standard metabolic rate during the period of salinity increase. The routine absence of substantial cell volume regulation in Mytilus gill may reflect the potentially high energetic cost of volume regulation in the face of the large and frequent salinity fluctuations that are regularly encountered by estuarine bivalves.

Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills.

We found that the basolateral surface of the gill epithelium of the marine mussel Mytilus californianus possesses a carrier-mediated process capable of concentrating taurine within epithelial cells. We used retrograde perfusion of gill sections to demonstrate the kinetics, specificity and ion-dependence of taurine transport. [3H]taurine was concentrated relative to a space marker ([14C]mannitol); this accumulation was blocked by the inclusion of 10 mmol l-1 unlabeled taurine in the perfusate. The drop in [3H]taurine uptake at increasing concentrations of unlabeled taurine was fitted to Michaelis-Menten kinetics and indicated a basolateral process with a taurine concentration at which transport is half-maximal (Kt) of 35.3 mumol l-1 and a maximal flux (Jmax) of 0.35 mumol g-1 wet mass h-1. Taurine accumulation on the apical surface had a higher affinity (Kt = 9.5 mumol l-1) and a higher maximum rate of transport (Jmax = 1.23 mumol g-1 h-1). Basolateral transport was inhibited by inclusion in the perfusate of 1 mmol l-1 of another beta-amino acid (beta-alanine), but not by inclusion of alpha-alanine, glutamic acid or betaine. The dependence of basolateral taurine transport on Na+ (when replaced with N-methyl-D-glucamine) was sigmoidal with an apparent Hill coefficient of 2.3, indicating that more than one Na+ is necessary for the transport of each taurine molecule. Complete substitution of Cl- in bathing media reduced taurine accumulation by 90% and 70% on the apical and basolateral surfaces, respectively. Taurine accumulation on both surfaces was reduced by only 20% when Cl- was reduced from 496 to 73 mmol l-1, suggesting that taurine uptake is not significantly influenced by the changes in Cl- concentration accompanying the salinity fluctuations normally encountered by mussels. We estimate that the various Na+ and Cl- gradients naturally encountered by epithelial cells are capable of providing ample energy to maintain a high intracellular concentration of taurine. We suggest that the ability of epithelial cells to accumulate taurine across the basolateral surface from the hemolymph plays a significant role in the intracellular regulation of this important osmolyte and may effect osmolality-dependent changes in the intracellular concentration of taurine.

HLA-DR alpha gene regulation in immortalized human thyroid cancer cells.

The induction of HLA class II antigens on human thyroid epithelial cells has been shown to be an intimate part of the pathology of autoimmune thyroid disease and may also be relevant to the natural history of thyroid neoplasia since thyroid cancer cells may show spontaneous HLA class II antigen expression. We have, therefore, analyzed the regulation of HLA-DR-alpha-specific mRNA transcripts, as a model for HLA class II antigen induction, in three established human thyroid cancer cell lines (papillary thyroid cell line NPA, and follicular thyroid cell lines RO-82-W1 and MRO-87-1). Each of the lines expressed 1.4 kb HLA-DR-alpha-specific mRNA transcripts, either constitutively or after cytokine induction, but showed markedly different regulatory characteristics. For example, the induction of HLA-DR alpha mRNA in response to recombinant human interferon-gamma (IFN-gamma) was inversely proportional to the degree of constitutive DR alpha mRNA expression; when there was constitutive absence there was a greater degree of induction. Similarly, the half-lives of the HLA-DR alpha mRNA transcripts varied from 80 to 420 min with the longest degradation time occurring in those cells lacking constitutive expression of HLA-DR alpha mRNA. These data indicated that the degradation rate was not a determinant of their constitutive expression. We also sequenced the 5' promoter region of the HLA-DR alpha gene (nucleotides -88 to -277 with respect to the translation start site) in each of the three cell lines. Sequence analyses revealed identity to the previously published normal human genomic sequence. Taken together, therefore, these data indicate that transactivating factors, rather than changes in mRNA degradation or promoter abnormalities, are the likely causes of variation in constitutive and cytokine-induced HLA-DR alpha gene expression in human thyroid cancer cells.

Strain-specific determination of the degree of thyroid cell MHC class II antigen expression: evaluation of established Wistar and Fisher rat thyroid cell lines.

The major histocompatibility complex (MHC) genes, primarily of the MHC class II region, are linked to increased susceptibility to autoimmune thyroid disease in animals and humans. The quantity of MHC class II antigens that are expressed on the appropriate cell surface, as well as their allotype, are of vital importance to immune function. We have, therefore, compared MHC class II (RT1.D) gene activation in a newly available Wistar rat thyroid (WRT) cell line (a thyroiditis-susceptible strain) with a cloned cell (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) (a thyroiditis-resistant strain) to determine differences in their degree of MHC class II gene activation and antigen expression. There was no detectable constitutive MHC class II antigen or RT1.D alpha-chain messenger RNA expression in either WRT or 1B-6 cells. Cultures of both cells expressed MHC class II gene induction in response to recombinant rat gamma-interferon (gamma IF). After 72 h of exposure to 25 U/ml gamma IF, over 80% of WRT cells expressed class II antigen, detected by flow cytometric assessment, as compared to only 15% of 1B-6 cells. This earlier and greater expression of MHC class II antigen was reflected in the RT1.D alpha-chain mRNA responses with peak levels in WRT cells after only 24 h of exposure to gamma IF, a period in which 1B-6 cells showed only minor increases in mRNA. To examine whether these differences in class II MHC expression were thyroid-cell specific, primary pulmonary fibroblast lines from Wistar and Fisher rats were developed and a similar, although less marked, variation in susceptibility to gamma IF was observed. Hence, those rats strains that are resistant to autoimmune thyroid disease exhibit a fundamental difference in their MHC class II gene responsiveness to cytokines, which may contribute to their disease susceptibility profiles.

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.