Mitochondria are a major source of free radicals in cells, and longevity studies indicate that women have a longer life span than men. Similar findings have also been reported for animal models, including rats and mice.

In the present study, we have investigated mitochondrial oxidative stress in male and female Wistar rats aged between 4-6 months to evaluate the molecular mechanisms enabling females to live longer than males. Animals were sacrificed by decapitation using approved animal handling procedures. As described previously (Sastre et al. 1996), mitochondria were isolated from liver homogenates by differential centrifugation (3 X 1000 g for 10 min and 3 X 10000 g for 10 min). The rate of peroxide production was measured fluorometrically, reduced glutathione (GSH) determined using the glutathione transferease method (Brigelius et al. 1983), and mRNA levels for glutathione peroxidase and Mn-superoxide dismutase (Mn-SOD) relative to 26 S rRNA by RT-PCR.

Our results show that the rate of peroxide production was significantly higher in liver mitochondria from male (n = 13) compared to female (n = 14) rat (0.097 ± 0.028 vs. 0.070 ± 0.02 nmol H₂O₂ min^-1 (mg protein)^-1, P < 0.01, n = 5 rats, Student’s unpaired t test). In ovariectomized rats (n = 6), peroxide levels were similar to those of males (0.112 ± 0.045 nmol H₂O₂ min^-1 (mg protein)^-1, n = 6). Oestrogen replacement (1 µg kg^-1 day^-1 oestradiol administered subcutaneously for 4 weeks) prevented the effect of ovariectomy (0.044 ± 0.017 nmol H₂O₂ min^-1 (mg protein)^-1, n = 7). In addition, hepatic mitochondria from female rats had higher GSH levels than those from males (9.76 ± 1.8 vs.6.41 nmol GSH/mg protein, n = 4, P < 0.01) or from ovariectomized controls (6.34 ± 1.27 nmol GSH (mg protein)^-1, n = 4). Oestrogen replacement restored GSH levels to values in non-ovariectomized rats (10.29 ± 1.66 nmol GSH (mg protein)^-1, n = 6).

Oxidative damage to hepatic mitochondrial DNA was also higher in males compared to female rats, as assayed by determining levels of 8{nbh}oxodeoxyguanosine. Moreover, mRNA expression and activities of glutathione peroxidase and Mn-SOD were also elevated in mitochondria from female compared to male rats (n = 5, P < 0.01). In addition, 16S rRNA expression, which is known to decrease significantly with aging, was found to be higher in mitochondria from female compared to age-matched male rats (n = 5, P < 0.01).

In conclusion, the difference between genders in average life span could be explained, at least in part, by the different rates of oxidant species generated in mitochondria and by differences in mitochondrial antioxidant activity.