Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione

BACKGROUND: Increased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation. METHODS: Cells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies. RESULTS: At low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels. CONCLUSION: These results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions

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 cell 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-arabino-furanosyl-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

MODIFICATIONS OF CELLULAR THIOLS DURING GROWTH AND SQUAMOUS DIFFERENTIATION OF CULTURED HUMAN BRONCHIAL EPITHELIAL-CELLS

Thiol modifications during growth and differentiation of cultured normal human bronchial epithelial cells was studied by analysis of their content and redox state of low-molecular-weight thiols and protein thiols. Subculture of the cells with trypsin decreased the cellular content of the major low-molecular-weight thiol, i.e., reduced glutathione, although the glutathione content had returned to levels comparable to those before subculture already after 4 h in conjunction with cell attachment. During subsequent culture, increases in the cellular contents of glutathione, total cysteine equivalents, and total protein thiols occurred. These modifications in the amounts and redox balance of thiols were transient and preceded the major growth phase. Exposure of cells at clonal density to either diethylmaleate, a thiol-depleting agent, or buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the proliferative ability of the cells as demonstrated by a markedly decreased colony forming efficiency. Moreover, in mass cultures exposed to buthionine sulfoximine, a marked depletion of the glutathione content was again accompanied by inhibition of growth. Exposure of the cells to agents known to induce growth arrest and terminal squamous differentiation, i.e., fetal bovine serum, Ca2+, or transforming growth factor-beta 1, resulted in increased levels of reduced glutathione. No consistent alteration in the contents of the other thiols was noted. Overall, the results demonstrate consistent variations in the amounts and redox state of cellular thiols, particularly reduced glutathione, supporting a role of thiols in regulation of growth and squamous differentiation of human bronchial epithelial cells. (C) 1994 Academic Press, Inc

Thiol modification and cell signalling in chemical toxicity.

Oxidant interaction with cell signalling may either mimic growth factor stimulation and stimulate cell proliferation or inhibit growth signals and activate programmed cell death in the same cell systems

PATHOBIOLOGICAL EFFECTS OF ACETALDEHYDE IN CULTURED HUMAN EPITHELIAL-CELLS AND FIBROBLASTS

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a Ih exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed