Gsta 4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity

Abstract

The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4-4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)₃], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)₃ treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.

Keywords: Glutathione Transferase; Lipid Peroxidation; Oxidants; Protein Adducts; Stress Kinases.

Figure 1

(a): Expression of GSTA4–4 in MEF cells prepared from WT and Gsta4 null mice analyzed by Western blot; (b)-(d): Viability of wild-type and Gsta4 null MEF cells analyzed with the CCK-8 kit after treatment with different concentrations of 4-HNE(Ac)₃ (0 – 25 µM) , paraquat (0 – 250 µM) and H₂O₂ (0 – 700 µM).

Figure 2

Analysis of 4-HNE-protein adducts in control and treated MEF cells by ELISA: Cells (2 × 10⁶) were incubated with 20 µM 4-HNE(Ac)₃ for 2 h and subjected to ELISA analysis using antibodies against 4-HNE protein adducts as described in Methods section. Data presented are Mean ± SD of two separate experiments done in triplicate (n = 6).

Figure 3

4-HNE or H₂O₂ induced activation of caspases in MEF cells; (a) Activation of caspase-3 in MEF cells treated with H₂O₂ (100 µM) and 4-HNE(Ac)₃ (20 µM) measured by ELISA; (b) Western blot analyses of 4-HNE induced activation of caspase3. Cells were treated with 4-HNE(Ac)₃ (20 µM) for 0 h - 5 hr, harvested, washed, and lysed in RIPA buffer. 25 µg of protein from control and treated samples were resolved by SDS-PAGE and immunobloted on nitrocellulose membrane. Immunoblots were probed with caspase3 antibodies; (c) and (d). 4-HNE induced activation of cas-pase-8 and 9 in MEF cells: WT and null MEF cells (5000 cells) were treated with 4-HNE(Ac)₃ (20 µM) for 2 h. Control and 4-HNE treated cells were analyzed for the activation of caspase-8 and 9 by ELISA as described in the Methods section. Data presented are Mean ± SD of two separate experiments done in quadruplicates (n = 8).

Figure 4

DNA damage determined by alkaline comet assay. MEFs from wild-type and mGsta4 null mice were treated for 4 hr with vehicle or with 4-HNE(Ac)_3. 60 cells from each treatment were scored for DNA damage [25] using Comet-Score (http://autocomet.com). Box: 25 – 75 percentile; whiskers: 10 – 90 percentile; horizontal line: median, circle: mean. Untreated WT and mGsta4null cells do not differ (p > 0.4), but cells treated with 20 µM 4-HNE(Ac)₃ are significantly different (p= 0.005, Wilcoxon rank-sum test).

Figure 5

Activation of MAP kinases in MEF cells after treatment with kinase inhibitors and 4-HNE(Ac)₃. Cells (2 × 10⁶) were treated separately with the SP600125 (JNK inhibitor), UO126 (ERK inhibitor) and SB202190 (p38 inhibitor) and exposed to 4-HNE(Ac)₃ (20 µM) for 2 hr. Cells treated only with 4-HNE(Ac)₃ and vehicle alone were used as controls. Extracts of control and treated cells were subjected to Western blot analyses and probed separately with antibodies against pERK [anti-pp44/42(Thr202/Tyr204)], p38 [anti-phospho-Thr180/Tyr204] and pJNK [anti-pJNK(Thr183/ Tyr185)]. Antibodies against β actin were used to ascertain equal loading of proteins. Appropriate lanes in the Western blot have been marked.

Figure 6

Effect of inhibitors of p38 (a); ERK (b) and JNK (c) on viability of MEF cells:WT and null cells (2 × 10⁴) were seeded in a 96 well plate and treated with fixed concentration of inhibitors for 1 h then treated with 10 µM of 4-HNE(Ac)₃ for 24 h. Control cells received equal volume of DMSO. After completion of treatment cell viability was assayed by MTT as described in the Methods section. Data shown are Mean ± SD of two experiments with eight replicate wells.

Figure 7

(a) Verification of GST isozyme uptake in Gsta4 null MEF cells after delivery with BioTrek reagent: mGsta4–4, hGSTA1–1, and DmGSTD1–1 were delivered into Gsta4 null MEF cells using BioTrek (BT) reagent as described in Methods section. Delivery of each isozyme in Gsta4 null MEF was confirmed by Western blot analyses of GST tran-sfected and control cell extracts (BT reagent treated and untreated). (b) Effect on stress kinase activation by exogenous introduction of GST protein into Gsta4 null MEF cells: Lane 1: untreated wild-type cells. Lane 2: wild-type cells treated with protein delivery reagent only. Lane 3: wild-type cells treated with protein delivery reagent and 20 µM 4-HNE(Ac)₃. Lanes 4–10:mGsta4 null cells. Lane 4: untreated. Lane 5: treated with 20 µM 4-HNE(Ac)₃. Lane 6: treated with delivery reagent only. Lane 7: treated with delivery reagent and 20 µM 4-HNE(Ac)₃. Lanes 8–10: treated with 20 µM 4-HNE(Ac)₃ with delivery reagent plus 100 µg/dish of mGSTA4–4 (lane 8), hGSTA1–1 (lane 9), or DmGSTD1–1 (lane 10), respectively. The core comparison: delivery of no protein (lane 7) or the three GSTs into KO cells (lane 8–10) exposed to 4-HNE(Ac)₃.