Worm Breeder's Gazette 17(2): 42 (April 1, 2002)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The superoxide dismutase mimetic EUK-8 shortens lifespan

Michelle Keaney, David Gems

Department of Biology, University College London, WC1E 6BT, UK

If the free radical theory of aging is correct, augmentation of antioxidant defenses should retard ageing and increase lifespan. Yet over the years, studies of administration of antioxidants to model organisms have not generally fulfilled this prediction. By contrast, a recent report demonstrated that synthetic catalytic antioxidants increase lifespan in C. elegans, on average by 44% (1). The antioxidants used were the salen manganese compounds EUK-8 and EUK-134 which have superoxide dismutase (SOD) activity in vitro and in vivo. The effect was seen over a 0.05 - 10 mM range, and was not dose dependent. These findings provide robust support for the view that free radical damage is a primary cause of ageing.

Hoping to use EUK-8 as an experimental tool to investigate mechanisms of ageing, we tested its effect on lifespan in C. elegans. EUK-8 was obtained from the same source as the Melov et al. study (Eukarion Inc., Bedford, MA, USA), and also from the Dept. of Pharmacy and Biological Science, University of Brighton, UK (R.G. Faragher and F. Fucassi). We examined the effect on lifespan of 0.05mM, 0.5mM and 5mM EUK-8, using methods as described (1). Worms were maintained in monoxenic liquid culture, either singly in 50-70ml of S medium containing an E. coli concentration of 5 x 108/ml - 1 x 109cells/ml, in 96 well microtitre plates; or in groups of approximately 20 in shallow plate culture. Medium was replenished at 2-3 day intervals. We examined effects on N2 hermaphrodites (2 trials, 661 animals) and males (2 trials, 132 animals), and nulliparous fog-2(q71) females (3 trials, 389 animals). In the case of the N2 hermaphrodite studies, the E. coli OP50 stock used was newly obtained from the Caenorhabditis Genetics Center.

In all trials we observed a dose-dependent reduction in longevity. For example, in a representative trial using N2 hermaphrodites, mean lifespans (20oC) were as follows: Control, 20.0±0.6 days; 0.05mM EUK-8, 20.6±0.5 days; 0.5mM, 14.7±0.4 days; and 5.0mM, 3.8±0.1 days (average sample size per test, 84 animals; range 72-100). We also measured the effect of EUK-8 on fertility in self-fertilizing hermaphrodites. As previously seen (1), no major reduction in fertility resulted from treatment with 0.05mM or 0.5mM EUK-8 (progeny numbers: control, 159±36 [S.D.], N=54 broods counted; 0.05mM, 151±38, N=56; 0.5mM, 143±33, N=61; when animals are raised from hatching liquid culture, brood sizes are reduced relative to those of animals maintained on agar plates). However, in 5mM EUK-8 brood size fell to 14±7 (N=11) (figures are means from four separate trials).

Thus, in our hands, EUK-8 did not increase lifespan, but rather, proved to be mildly toxic. It remains unclear whether the failure of EUK-8 to increase lifespan is because a) increased SOD activity does not retard ageing in C. elegans; b) EUK-8 does not have SOD activity in C. elegans; or c) EUK-8 is not taken up by worms (however, the dose-dependent reduction of lifespan seen suggests that EUK-8, or a derivative of it, is entering the worms). One possibility is that EUK-8 is inactivated by E. coli. With this in mind, we tested the effect of EUK-8 on axenically cultured N2 hermaphrodites (2). However, under these conditions a similar dose-dependent shortening of lifespan was seen. Mean lifespans for a representative trail are: Control, 33.8±0.8 days; 0.05mM EUK-8, 33.5±0.7 days; 0.5 mM, 24.8±1.1 days; and 5.0mM, 5.5±0.3 days (2 trials, 288 animals; note that culture in axenic medium increased lifespan, as previously observed). Thus, the toxicity of EUK-8 is not the result of an unusual interaction between EUK-8 and E. coli.

The reason for the differences between our results and those previously reported remain unclear. Our findings will only be fully interpretable when it is known whether administration of EUK-8 to C. elegans results in increased intracellular SOD levels, and protection against superoxide.

1. Melov, S., et al. Science 289, 1567 (2000). 2. Vanfleteren, J.R., et al. J. Gerontol. 53, B393 (1998).