Individual, Culture‐Specific Alterations in the Human Endothelial Glutathione System: Relationships to Oxidant Toxicity

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Abstract
In this comunication we report on the effects of serial passage of human umbilical vein endothelial cells on the levels of GSH and activities of selenium‐dependent GSH peroxidase (SeGSHpx) and GSSG reductase (GSSGred) in confluent monolayers of human umbilical vein endothelial cells derived from 4 individual donors. The mean levels of GSH in confluent monolayers of individual human umbilical vein endothelial cells batches fell from an average of 35 nmol/mg protein to 16 nmol/mg protein from primary culture to the third passage, with little interindividual variation noted. Simultaneously, the mean activities of SeGSHpx and GSSGred remained unaltered during serial culture, again with little interindividual variation noted. However, serial passage of the cells from different individuals did not affect the sensitivity of the cells to sublethal injury induced by H2O2 (10 or 100 μM), as judged by the stimulated release of (3H)‐deoxyglucose. When these GSH‐dependent parameters were assayed during the coarse of the growth of an individual passage of cells, GSH levels declined following trypsinisation, but the activities of SeGSHpx and GSSGred remained essentially unaltered, when the data were standardised to cell number. On the other hand, standardisation of these data to cell protein revealed decreases in all three parameters following trypsinisation, indicating the introduction of artefacts into the data and the unsuitability of this standardisation procedure. For the first four hours following plating, all of the GSH‐dependent parameters remained essentially unaltered, but began increasing thereafter. Thus, GSH levels climbed to a peak prior to the onset of cell division and the activity of GSSGred rose to a peak by 24 hr, whilst the activity of SeGSHpx rose steadily during the first 72 hr of culture. When these low density cell cultures were tested for their sublethal response to H2O2 (3, 10 and 100 μM), a dose‐dependent increase in the release of (3H)‐deoxyglucose was noted, which remained constant during the first 72 hr of culture. Due to the relative lack of interindividual variations in the GSH parameters tested and in the sensitivity to H2O2, these data suggest that confluent cultures of pooled cells of any passage up to the third may be used for model studies of the sublethal effects of H2O2 in these human cells. The data also suggest that the sensitivity of confluent human umbilical vein endothelial cells derived from different individuals to the sublethal effects of H2O2 is not predictable from their individual GSH‐dependent potential to detoxicate the peroxide. Additionally, fluctuations in GSH‐dependent potential to detoxicate H2O2 may be related to the onset of growth in sub‐confluent cultures of human umbilical vein endothelial cells, but appear, once again, not to be related to the sensitivity of the cells to sublethal effects of H2O2.