Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury.
4-Hydroxynonenal (4-HNE) is a mutagenic alpha,beta-unsaturated aldehyde produced during oxidative injury that is conjugated by several glutathione S-transferase (GST) isoforms. The alpha class human GSTA4-4 enzyme (hGSTA4-4) has a particularly high catalytic efficiency toward 4-HNE conjugation. However, hGST4-4 expression is low in most human cells and there are other aldehyde metabolizing enzymes that detoxify 4-HNE. In the current study, we determined the effect of over-expression of hGSTA4 mRNA on the sensitivity of HepG2 cells to 4-HNE injury. HepG2 cells transfected with an hGSTA4 vector construct exhibited high steady-state hGSTA4 mRNA, high GST-4-HNE catalytic activities, but lower basal glutathione (GSH) concentrations relative to insert-free vector (control) cells. Exposure to 4-HNE elicited an increase in GSH concentrations in the control and hGSTA4 cells, although the dose-response of GSH induction differed among the two cell types. Specifically, hGSTA4 cells had significantly higher GSH concentrations when exposed to 5-15 microM 4-HNE, but not at 20 microM 4-HNE, suggesting extensive GSH utilization at high concentrations of 4-HNE. The hGSTA4 cells exhibited a significant growth advantage relative to control cells in the absence of 4-HNE, and a trend towards increased growth at low dose exposures to 4-HNE. However, the hGSTA4 cells did not exhibit a growth advantage relative to control cells at higher 4-HNE exposures associated with increased GSH utilization. As expected, the hGSTA4 cells showed resistance to 4-HNE stimulated lipid peroxidation at all 4-HNE doses. In summary, our data indicates that over-expression of hGSTA4 at levels conferring high GST-4-HNE conjugating activity confers a partial growth advantage to HepG2 cells and protects against 4-HNE oxidative injury. However, the loss of proliferative capacity of hGSTA4 cells challenged with levels of 4-HNE associated with severe oxidative stress indicates a role of other aldehyde metabolizing enzymes, and/or GSH-electrophile transporter proteins, in providing full cellular protection against 4-HNE toxicity.
Gallagher
Evan P
EP
School of Public Health and Community Medicine, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105-6099, USA. evang3@u.washington.edu
Huisden
Christiaan M
CM
Gardner
James L
JL
D000447:Aldehydes / Q000633:toxicity*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
D000447:Aldehydes; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; C027576:4-hydroxy-2-nonenal