Ethanol withdrawal hastens the aging of cytochrome c oxidase

Abstract

We investigated whether abrupt ethanol withdrawal (EW) age-specifically inhibits a key mitochondrial enzyme, cytochrome c oxidase (COX), and whether estrogen mitigates this problem. We also tested whether this possible effect of EW involves a substrate (cytochrome c) deficiency that is associated with proapoptotic Bcl2-associated X protein (BAX) and mitochondrial membrane swelling. Ovariectomized young, middle age, and older rats, with or without 17β-estradiol (E2) implantation, underwent repeated EW. Cerebelli were collected to measure COX activity and the mitochondrial membrane swelling using spectrophotometry and the mitochondrial levels of cytochrome c and BAX using an immunoblot method. The loss of COX activity and the mitochondrial membrane swelling occurred only in older rats under control diet conditions but occurred earlier, starting in the young rats under EW conditions. E2 treatment mitigated these EW effects. EW increased mitochondrial BAX particularly in middle age rats but did not alter cytochrome c. Collectively EW hastens but E2 delays the age-associated loss of COX activity. This EW effect is independent of cytochrome c but may involve the mitochondrial overload of BAX and membrane vulnerability.

Figure 1.. Diet schedules.

All experimental groups underwent repeated withdrawals except for an ethanol exposure group. Some female rats were ovariectomized and recovered for two weeks from ovariectomy before a liquid diet began. All withdrawal groups received a 25-day control dextrin or ethanol (6.5% w/v) diet and 5-day abrupt withdrawal. This cycle was repeated three times. Rats were then sacrificed two weeks after the last dose of ethanol for brain collection. Rats in the ethanol exposure group continuously received an ethanol-diet and were gradually withdrawn from the diet for 7 days with step-down concentrations of ethanol at the end of the diet regimen. They were then sacrificed next morning.

Figure 2.. Effects of age on COX activity.

Ovariectomized rats were implanted with oil or E2 pellets, received a 25-day control dextrin diet followed by a 5-day chow pellet diet. This cycle was repeated three times. Two weeks after the last dextrin diet, COX activity was measured in the cerebellar whole-cell lysates. Older rats had a lower activity of COX than young rats (*p < 0.01). The activity of middle-age rats did not differ from that of young or older rats. E2 per se did not alter COX activity. *p < 0.01 vs. young dextrin rats. ^†p < 0.01 vs. young dextrin + E2 rats. Data (%) are presented relative to a control-dextrin diet value in young rats. Depicted are mean ± SEM for 7–10 rats/group.

Figure 3.. Effects of age-EW combination on COX activity.

Ovariectomized rats were implanted with oil or E2 pellets, received a 25-day control dextrin or ethanol (6.5% v/v) diet followed by 5-day abrupt withdrawal. This cycle was repeated three times. The ethanol exposure group continuously received an ethanol-diet without withdrawal. COX activity was measured in the cerebellum obtained two weeks after or at the end of the last dose of ethanol for the condition of EW or ethanol exposure, respectively. EW suppressed COX activity in young rats, further so in middle-age rats, and thereafter, did not significantly alter the activity in older rats. This effect of EW was prevented by E2. *p < 0.01 vs. young EW rats. ^†p < 0.01 vs. young EW + E2 rats. ^‡p < 0.01 vs. control diet rats (100%) at each age. Some statistical symbols are omitted for the clarity of a figure. Depicted are mean ± SEM for 7–10 rats/group.

Figure 4.. Effects of endogenous E2 on COX activity.

This experiment used gonadally intact young male and female rats and middle-age female rats and ovariectomized young or middle-age female rats. They received a 25-day control dextrin or ethanol (6.5% v/v) diet followed by 5-day abrupt withdrawal and repeated this cycle three times. Ovary-intact female rats were on estrus on the first day of EW when the most severe EW stress occurred. Two weeks after the last ethanol-diet, COX activity was measured in the cerebellar whole-cell lysates. COX activity during EW was lower in young male rats vs. young female rats, in ovariectomized rats vs. ovary-intact rats, and in middle-age female rats vs. young female rats. All data (%) in this figure are from ethanol-withdrawn groups and presented relative to a control-diet value at 100 %. Depicted are mean ± SEM for 5–10 rats/group. The number indicates a p value between two groups indicated with a horizontal line.

Figure 5.. Effects of EW on the expression of mitochondrial cytochrome c.

Ovariectomized rats received a 25-day control dextrin or ethanol (6.5% v/v) diet followed by a 5-day abrupt withdrawal. This cycle was repeated three times. Two weeks after the last ethanol-diet, the protein level of cytochrome c was measured using an immunoblot method in the mitochondrial fraction of cerebellum. The bar graph indicates no significant difference between diet and age groups. β-actin was used as a loading control. A clear image was selected from 5–6 rats/group.

Figure 6.. Effects of EW on the expression of mitochondrial BAX.

Ovariectomized rats received an identical diet regimen as described in Figure 3 legend. Two weeks after or at the end of the last dose of ethanol, the protein level of BAX was measured using an immune blot method in the mitochondrial fraction of cerebellum. EW provoked an increase in the expression of BAX in middle-age rats among diet or age groups (*p < 0.01) (Figure 6A). *p < 0.01 vs. all other groups. Figure 6B illustrates that E2 does not alter the mitochondria level of BAX in middle-age rats. ^†p < 0.01 vs. dextrin. β-actin was used as a loading control. A clear image was selected from 3 rats/group.

Figure 6.. Effects of EW on the expression of mitochondrial BAX.

Ovariectomized rats received an identical diet regimen as described in Figure 3 legend. Two weeks after or at the end of the last dose of ethanol, the protein level of BAX was measured using an immune blot method in the mitochondrial fraction of cerebellum. EW provoked an increase in the expression of BAX in middle-age rats among diet or age groups (*p < 0.01) (Figure 6A). *p < 0.01 vs. all other groups. Figure 6B illustrates that E2 does not alter the mitochondria level of BAX in middle-age rats. ^†p < 0.01 vs. dextrin. β-actin was used as a loading control. A clear image was selected from 3 rats/group.

Figure 7.. Effects of age-EW combination on mitochondrial membrane swelling.

Ovariectomized rats received an identical diet regimen as described in Figure 3 legend. Two weeks after or at the end of the last dose of ethanol, cerebelli were collected to measure mitochondrial membrane swelling by recording an absorbance decline at 540 nm (Figure 7, upper panel). In the lower panel of Figure 7, the smaller area-under-curve (AUC) indicates the more rapid swelling of mitochondrial membranes. The magnitude of EW-induced mitochondrial membrane swelling relative to a dextrin condition is illustrated as a grey area in the upper panel and a vertical bar (%) next to a dextrin in the lower panel. Compared to a control-diet, EW provoked more severe mitochondrial membrane swelling in middle-age (27%) (^†p < 0.01) than young (17%) or older (15%) age groups in a manner mitigated by E2 treatment. *p < 0.01 vs. a dextrin diet, ethanol exposure, or EW + E2 at each age. ^‡p < 0.01 vs. young dextrin group. ^††p < 0.01 vs. young or middle-age dextrin group. Depicted are mean ± SEM for 5–10 rats/group.

Figure 8.. Effects of EW on mitochondrial respiration.

HT22 cells (600 cells/well) were plated in 24-well microplates, exposed to ethanol (100 mM) for 3 days, and withdrawn for 4 hours. The cells were treated with E2 (10 μM) during the 4 hour-EW period. The microplate was then placed on the O₂ sensor cartridge and inserted to XF respirometry immediately or 4 hours after the 3-day ethanol exposure for an ethanol exposure condition or EW condition, respectively (Figure 8A). COX inhibitor (NaN3, 1 mM) was applied to cells approximately 21 minutes after XF respirometry began to read basal O₂ consumption rate (Figure 8B). Compared to a control condition, EW suppressed mitochondrial respiration more severely than ethanol (p^† < 0.01, Figure 8A) in a manner mitigated by E2 (^‡p < 0.01) treatment (Figure 8B). COX inhibitor suppressed the basal level of mitochondrial respiration (p * < 0.001 vs. the basal level of Control) and blunted E2’s protection (p^† < 0.01 vs. the basal level of EW+E2). N=4 wells/each condition.