Dorothy C. Dziedzic

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 by cultured rabbit lens epithelial cells

The relative roles of the glutathione redox cycle and catalase in the detoxification of H2O2 were investigated in cultured rabbit lens epithelial cells. Exposure of cells to H2O2 was carried out following inhibition of either of the two antioxidant systems. Two different procedures were used to expose the cells to extracellular H2O2, one in which a low, steady state level of 0.025 mM H2O2 was maintained in the culture medium with the use of glucose oxidase and the other in which H2O2 was added to the medium as a single pulse at levels ranging from 0.03 to 0.5 mM. When lens cells were treated with a low, steady state level of H2O2, the glutathione redox cycle was the primary means of defense against oxidative damage. Cells with fully active catalase but with inhibited glutathione reductase were not able to resist the cytotoxic effects of a 0.025 mM level of extracellular H2O2. Under these conditions the cells were nearly completely depleted of reduced glutathione within 15 min. The cellular damage observed after 1.5 hr of culture included loss of cell-to-cell contact, rounding up of the cells and formation of numerous blebs. In contrast, cells with completely inhibited catalase but with an unimpaired glutathione redox cycle suffered few damaging effects from a 3-hr exposure to 0.025 mM H2O2. When lens cells were pulsed with a single challenge of 0.5 mM H2O2, both the glutathione redox cycle and catalase were found to be essential for survival of the cells. While control cells were able to withstand the pulse of H2O2, cells with impaired activities of either the glutathione redox cycle or catalase were killed. Control cells treated with 0.5 mM H2O2 may have been protected from damage by the fact that the cellular level of GSH never dropped below 35% of normal. The cause of cell death following inhibition of catalase appeared to be related to an inability of the cells to remove peroxide from the culture medium, at a rapid rate, following the H2O2-pulse. Although cells with impaired glutathione reductase activity removed H2O2 from the medium at a rate comparable to that of control cells (due to uninhibited catalase activity), they did not survive the challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Propyl gallate is a superoxide dismutase mimic and protects cultured lens epithelial cells from H2O2 insult

n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain. We investigated whether nPG protected cultured lens epithelial cells from H(2)O(2)-induced damage. Cells were treated with H(2)O(2) or with nPG and then H(2)O(2). H(2)O(2) inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD(+), and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H(2)O(2)-induced growth inhibition, membrane blebbing, drop in NAD(+) and single-strand breaks in DNA. The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to microM) was mixed with a large excess of O(2)(-)*, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O(2)(-)* in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O(2)(-)* was ca. 10(6)M(-1)s(-1). nPG had a very low specific binding constant for Fe(2+) as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.

Detoxification of H2O2 by cultured rabbit lens epithelial cells: Participation of the glutathione redox cycle

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

Abstract A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.

Influence of the activity of glutathione reductase on the response of cultured lens epithelial cells from young and old rabbits to hydrogen peroxide.

Our previous studies on cultured rabbit lens epithelial cells from 4-day-old rabbits showed that the glutathione redox cycle plays an important role in detoxifying H2O2, a potentially damaging oxidant present in the aqueous humor. Here we report the effect of donor age and cell density on the ability of cultured rabbit lens epithelial cells to detoxify H2O2. Lens epithelial cells (8 x 10(5] from a 4-day-old and an 8-year-old rabbit were cultured for 3 hr in minimal essential medium (MEM) or in MEM containing 0.01-0.1 mM H2O2 maintained with glucose oxidase. We determined the effect of H2O2 on the level of reduced glutathione (GSH), hexose monophosphate shunt activity, cell growth, and morphology. For growth studies, cells were exposed to the desired concentration of H2O2 for 3 hr and then cultured in MEM plus 10% rabbit serum for 7 days and counted. Young and old untreated cells contained high levels (30-40 nmol/8 x 10(5) cells) of GSH. Cells from 4-day-old rabbits tolerated 0.03 mM H2O2 with no effect on GSH and a minimal decrease in subsequent cell growth. However, in the older cells, GSH and growth were substantially diminished following treatment with 0.03 mM H2O2. Cells plated out at high density (8 x 10(5] were more tolerant of 0.03 mM H2O2 than cells plated out at low density (5 x 10(4]. Maximum shunt activity in the younger cells exposed to H2O2 was twice that of the older cells and occurred at a higher level of H2O2 (0.04 compared with 0.03 mM). Enzyme activities in untreated young and old cells were comparable for hexokinase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase. However, glutathione reductase activity was 50% lower in the cells from the 8-year-old rabbit. The toxicity of H2O2 to cultured lens epithelial cells was directly related to donor age and inversely related to cell density. The damage in the older lens epithelial cells at 0.03 mM H2O2 was apparently due, in part, to a diminished response of the glutathione redox cycle to oxidative challenge.

Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture

Human lens cells from 5-91-year old individuals were cultured in 8 different basal media containing fetal bovine, adult bovine, rabbit or human serum or human plasma or in a serum-containing medium supplemented with insulin, epidermal growth factor, fibroblast growth factor plus other hormones or trace elements. Cultures were initiated from explants of the capsule and epithelium or following enzymatic dissociation of cells from the capsule. Under all conditions the epithelial cells had a limited doubling potential. As a function of time in culture, cells enlarged, displayed numerous filaments and exhibited apparent in vitro senescence. Lens epithelia from 4-6 day old rabbits cultured under identical conditions mimicked the behavior of human lens cells. Lens epithelia from newborn rabbits may be a suitable model for investigating the basis of apparent in vitro senescence in this cell type and could help in defining the conditions required for the long-term growth of human lens cells. The limited growth of human lens epithelia suggests that these cells require tissue-specific nutrients or hormonal supplements not present in standard tissue culture media.

Establishment and characterization of a lens epithelial cell line from an eight year old rabbit

Although information is available on the in vitro properties of lens epithelia of young adult animals from several species, few, if any reports document the conditions required for the initiation and long-term culture of lens epithelium from animals beyond their medium life-span. We report here on the conditions required for the culture of lens cells from an 8 year old rabbit. New Zealand White rabbits have a median life-span of approximately 7 years. Primary culture was initiated in MEM supplemented with 10% rabbit serum. Cells reached confluency within 25 days, responded to serum in a dose dependent manner and had an average doubling time of 23 h during the logarithmic growth phase. Cells increased in number in a dose dependent manner when insulin, insulin growth factor, epidermal growth factor (EGF), or fibroblast growth factor (FGF) was added to the culture medium. Thus, lens epithelia from this very old rabbit retained the ability to respond to highly purified growth factors. Cells exposed to a medium supplemented with insulin, EGF and FGF showed a five-fold increase in number at day 7 of culture, a value exceeding that brought about by the individual growth factors. An examination of chromosomal preparations indicated that the cells were aneuploid. Whether the aneuploidy was acquired in vitro or is a normal adjunct of aging in the lens in vivo is unknown. Proteins extracted from this line contained polypeptides that migrated to the position of and had apparent molecular weights of lens proteins.