Chilling of Arabidopsis thaliana (L.) Heynh. callus tissue to 4 degrees C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H2O2 was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 degrees C. In callus held at 23 degrees C, levels of reduced glutathione remained static whereas they rose in callus held at 4 degrees C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 degrees C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 degrees C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 degrees C. Exposure of callus to abscisic acid at 23 degrees C also led to increased activities of ascorbate peroxidase and glutathione reductase.
The effects of oxidant stress were studied in immortalised hamster (BHK-21) and rat (208F) cell lines before and after transformation to the malignant state with polyoma virus, or activated H-ras, respectively. Whilst intracellular superoxide production was detectable in both transformed and immortalised cells the rate was somewhat higher in the transformed cells which have lower levels of superoxide dismutase. Because growth of transformed cells was particularly depressed in the presence of MTT, a tetrazolium compound reduced by superoxide, the possible role of active oxygen species in the promotion of cell growth was examined. Low levels of hydrogen peroxide were stimulatory towards both immortalised and transformed cells. In the case of H-ras transformed rat cells, paraquat was also stimulatory provided serum was present in the growth medium. In the absence of serum, paraquat was notably inhibitory but inhibition could be alleviated by addition of low concentrations of alpha-tocopherol (10(-8)M) to the serum-depleted medium. Although depletion of serum from the growth medium also leads to lower cell proliferation, subsequent experiments showed that alpha-tocopherol addition to serum-free medium was sufficient to restimulate growth. In the case of transformed cells, yields of cells were even greater than that encountered in the presence of 10% serum. Thus whilst certain active oxygen species (e.g. hydrogen peroxide) may have a role in promoting the growth of transformed and immortalised cells the necessity for antioxidant protection is important.
Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H2O2, such as the superoxide dismutase mimic, copper II-(3,5-diisopropylsalicylate)2, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 microM exogenous H2O2 to the culture medium. Under these conditions, however, intracellular levels of H2O2 are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H2O2 and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H2O2, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to "trigger" their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H2O2.
Antioxidants such as mannitol, butylated hydroxytoluene and alpha-tocopherol enhance the growth of polyoma virus transformed and non-transformed BHK-21 cells. In the case of mannitol this is observed even in the absence of added calf serum. In part these effects may operate to protect cellular growth control mechanisms. On the other hand oxidants such as H2O2 and t-butyl hydroperoxide can inhibit growth and overall cellular protein synthesis, through mechanisms that are likely to involve radicals. In the case of H2O2 the inhibitory effects can nevertheless be reduced by 'prestressing' the cells with mild heat or with H2O2 itself. Paradoxically very low concentrations (10(-8) M) of H2O2 or t-butyl hydroperoxide can actually stimulate cell growth, even in the absence of serum. These stimulatory effects however do not appear to involve radicals as they are enhanced by inclusion of mannitol or DMSO in the medium.
Addition of H2O2 at 100 microM, or 1 mM, to the culture medium of BHK-21 fibroblasts results in increased intracellular levels of H2O2. Whilst exposure of BHK-21 cells to lower levels of H2O2 (1 microM) actually stimulates proliferation, these higher oxidant concentrations not only depress proliferation rates but also lead to an increase in the appearance of apoptotic-like cells in the cultures. Other agents such as inhibitors of glutathione peroxidase and catalase, or mimics of superoxide dismutase, which also bring about elevated cellular levels of H2O2 in BHK-21 cells, similarly lead to decreased proliferation and an apparent increase in cells with apoptopic features. Thus intracellular conditions which are considered more prooxidant than normal, appear to favour apoptosis over proliferation in BHK-21 fibroblasts. Additionally these abnormal cellular conditions also appear to favour excessive DNA replication, in remaining non-apoptotic cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.