Thiol status and cytopathological effects of acrolein in normal and xeroderma pigmentosum skin fibroblasts.

Thiol redox status was determined in normal human skin fibroblasts and a DNA repair-deficient xeroderma pigmentosum (XP) fibroblast cell line (XP12BE, group A), and cytotoxic and genotoxic effects of the thiol-reactive aldehyde acrolein were studied in these cell types. Normal cells contained higher amounts of the reduced glutathione and cysteine respectively, and higher amounts of these thiols as protein-bound disulfides than the XP cells. However, in both cell types total glutathione was present in 6- to 7-fold higher amounts than total cysteine, and total protein thiols corresponded to approximately 30% of total thiols. A 1 h exposure to acrolein caused a quantitatively similar depletion of reduced glutathione and free protein thiols in both cell types, without causing changes in the thiol redox state. However, acrolein caused higher toxicity measured as trypan blue exclusion, and also a higher extent of DNA single-strand breaks in the XP cells than in the normal cells. Exposure to acrolein, followed by incubation in fresh medium resulted in continued formation of DNA single-strand breaks in the normal cells, whereas no such accumulation occurred in the XP cells. In the normal cells, the DNA single-strand breaks accumulated to a similar extent as in the presence of 1-beta-D-arabinofuranosyl-cytosine and hydroxyurea, i.e. two agents which together efficiently inhibit DNA repair synthesis. The results indicate quantitative and qualitative differences in the thiol redox state between normal and XP cells, and that these differences may contribute to the higher cytotoxicity and genotoxicity of acrolein in XP cells. Moreover, the results indicate that acrolein is a potent inhibitor of DNA excision repair.

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts.

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.

Reactivity of fecapentaene-12 toward thiols, DNA, and these constituents in human fibroblasts.

Micromolar concentrations of fecapentaene-12, a mutagen found in human feces, decrease survival measured as colony-forming efficiency and membrane integrity of cultured human fibroblasts. Fecapentaene-12 also decreases the content of cellular free low-molecular-weight thiols including glutathione. Fecapentaene-12 reacts directly with glutathione by causing both decreased levels of free thiol and some concomitant formation of oxidized glutathione, indicating that thiol depletion is a result of both alkylation and oxidative reactions. Exposure of cells to 2 or 5 microM fecapentaene-12 causes significant amounts of DNA-interstrand cross-links and DNA-single strand breaks, respectively, whereas exposure to a higher concentration of fecapentaene-12, i.e., 10 microM, also causes significant DNA-protein cross-links. Results from the reaction of fecapentaene-12 with isolated plasmid DNA parallel the cellular pattern of DNA damage; primarily interstrand cross-links and strand breaks occur also in plasmid DNA. Taken together, these studies show that fecapentaene-12 is a potent cytotoxic and genotoxic agent which can react with cellular thiols and cause several types of DNA damage.