Aging - oxidative damage
The oxidative damage theory of aging states that the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives, is the primary cause of aging. EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), are excellent tools to test this theory. Melov and colleagues (Melov et al., 2000) previously reported that treatment with EUK-8 or EUK-134 significantly extended lifespan in C. elegans in a dose-independent manner. On the other hand, no extension of lifespan, but a dose-dependent reduction in fecundity and lifespan was found in both David Gems’ lab (Keaney and Gems, 2003) and our own upon administration of these mimetics. No extension of lifespan was found in other animal models such as the housefly (Bayne & Sohal, 2002). We are currently collaborating with the David Gems laboratory to further examine the effect of EUK-8 and EUK-134 both in vitro and in vivo in C. elegans.
Firstly, we checked SOD activity in vitro
and found comparable results to previous publications. Moreover, we
confirmed that the SOD activity of both compounds was inhibited by EDTA
et al., 1993).
Our next step was to test the in vivo SOD activity of EUK-8 and EUK-134. This was done using our high-throughput SOD assay (Lenaerts et al., 2002), with or without EDTA in the assay mixture in order to discriminate between endogenous SOD (not inhibited by EDTA) and SOD activity by EUK only (inhibited by EDTA). In addition, Michelle Keaney (Gems lab) tested the in vivo effect of EUK-8 by evaluating the protective effect of EUK-8 in C. elegans upon administration of the superoxide generator paraquat. Finally, using subcellular fractionation techniques, we have tested whether the SOD mimetics act at the most important cellular superoxide production site, i.e. the mitochondria (Keany et al., 2004).
There are still many issues to be
in the EUK story. Sensitive assays to measure in vivo superoxide and hydrogen
peroxide production could yield lots of information on this matter. It
would also be very interesting
to thoroughly examine the effect of EUK on oxidative damage in the
aging animal. Currently, we are working on a sensitive protein
carbonylation asay. We are also eager to find out if it is
possible to separate the toxic and curative effect of EUK-8. This
would help us to conclude whether the dose-dependent toxic effect is
due to toxic components in the EUK-preparation or to an
'over-scavenging' of superoxides. Finally,
the in vivo effect of EUK-8
on mev-1 mutants, which are believed
to live short because of superoxide overproduction, remains to be