Anti-aging effects of deuterium depletion on Mn-induced toxicity in a C. elegans model

Abstract

Work with sub-natural levels of deuterium (D) in animals has demonstrated an anti-cancer effect of low D-concentration in water. Our objective was to investigate whether deuterium-depleted water (DDW) can overturn reverse manganese (Mn)-induced reduction in life span, using the Caenorhabditis elegans (C. elegans) as a model system. DDW per se had no effect on worm's life span 48 h after treatment; however, it reversed the Mn-induced decrease in C. elegans life span. Mn reduced DAF-16 levels, a transcription factor strongly associated with life-span regulation. Low D-concentration (90 ppm) restored the Mn-induced changes in DAF-16 to levels indistinguishable from controls, suggesting DDW can regulate the DAF-16 pathway. We further show that insulin-like receptor DAF-2 levels were unaltered by Mn exposure, tAKT levels increased, whilst superoxide dismutase (SOD-3) levels were decreased by Mn. DDW (90 ppm) restored the levels of tAKT and superoxide dismutase (SOD) to control values without changing DAF-2 levels. Treatment of Mn exposed worms with DDW (90 ppm) restored life-span, DAF-16 and SOD-3 levels to control levels, strongly suggesting that low D concentrations can protect against Mn toxic effects.

Introduction

Water is an essential requirement for life. It is the largest constituent of living organisms; the human body consists of 60–70% water dependent upon age. Intra- and extracellular water plays numerous physiological roles, providing an appropriate medium for chemical reactions in cells (Mitchell et al., 1945, Wang et al., 1999).

Hydrogen has a naturally occurring stable isotope, deuterium (D) with a mass number of 2, and it is present in surface water in the form of HDO [semiheavy water; water with light hydrogen (protium, 1H) and deuterium (D or 2H) in the mix] or D₂O at a concentration of 16.8 mmol/L or 150 ppm (Katz and Crespi, 1971, Rundel et al., 1988). The two isotopes have the largest mass ratio among stable isotopes of the same element, resulting in significantly different chemical and physical behavior (Jancsó, 2003, Katz and Crespi, 1971, Rundel et al., 1988). Although the effect of D₂O – due to its isotopic effect – at an elevated concentration in biological systems has been investigated (Czajka et al., 1961, Katz et al., 1962), the significance of naturally occurring D-concentration has yet to be addressed. It was first reported in 1993 that reduced D concentration in water affects living organisms (Somlyai et al., 1993). In vitro studies noted that D-depletion triggers apoptosis, exerts influence on proto-oncogenes and tumor suppression genes and weakens the expression of genes induced by exposures to carcinogens (Cong et al., 2010, Somlyai et al., 1993, Somlyai et al., 1998a, Somlyai et al., 1998b). A study in four patients with brain metastases secondary to lung cancer found that DDW prolonged survival time compared with the average life expectancy in these cases. Survival times of 9.7, 26.6, 33.4, and 54.6 months are unique in the annals of brain metastases secondary to lung tumors (Krempels et al., 2008). In a randomized, double-blind phase 2 study and in a prospective study in patients with prostate cancer consuming DDW parallel to the conventional therapy, prolonged survival was noted (Kovács et al., 2011). Based on these observations, DDW has been posited to offer anticancer activity as an adjunct to conventional therapy.

A link between aging and D is well established. D₂O concentrations exceeding the natural level resulted in numerous adverse effects: (a) increased viral mutation rates (Konrad, 1960); (b) deuteration of synthetic estrogen hormones weakened its estrogenic properties (Thompson, 1963); (c) deuterated enzymes exhibited conformational changes, affecting their active sites (Van Hook, 1971); (d) the skin became enriched in deuterium along a temporal aging axis (Griffiths, 1973); (e) reduced the life-span of mice (Czajka and Finkel, 1960). Notably, the effect of lower than natural D concentrations on longevity has yet to be investigated.

Caenorhabditis elegans (C. elegans) offers a unique animal model for aging studies (Baumeister et al., 2006, Lee et al., 2009). This small soil nematode has a short life-cycle, which is significant for assays where the analysis of the whole life-span is necessary (Johnson and Wood, 1982). The progeny is large, and therefore, high throughput assays are possible (Helmcke et al., 2010, Leung et al., 2008). Furthermore, the nematode can be genetically manipulated to provide strains with loss-of-function mutations or complete gene knockout (Brenner, 1974).

DAF-16 is the orthologue of the mammalian FOXO (forkhead O), a transcription factor responsible for activation of genes that codify antioxidant enzymes, which neutralize reactive oxygen species (ROS) involved in the aging process. Notably, daf-16 knockout reduces longevity (Murphy, 2006, Zhang et al., 2009) and, consequently, this pathway has been implicated in the regulation of stress resistance and life-span (Baumeister et al., 2006, Lee et al., 2003). DAF-16 belongs to the DAF-2 insulin-like cascade, which is negatively regulated by phosphorylation. The signaling pathway, via the PI3K receptor DAF2 (insulin-like receptor homologue), regulates levels of 3′-phosphorylated phosphatidylinositol lipids, directing the related kinases AKT1,2 (PKB homologues) to phosphorylate the FOXO homologue, DAF-16 (Hertweck et al., 2004, Murphy, 2006, Paradis and Ruvkun, 1998). This signaling is likely mediated by activation of age1-associated protein (AAP1), the homologue of mammalian heterodimer p85–p110 (Burgering and Kops, 2002). AKT1,2 phosphorylation, in turn, phosphorylates nuclear DAF-16 and translocates it into the cytosol. Conversely, inhibition of this phosphorylation cascade causes the migration of DAF-16 to the nucleus, which binds to DAF-16 binding-element (a core sequence TTGTTTAC of the DNA), thus increasing transcriptional activation of genes with context-dependent effects on cellular stress, consequently reducing oxidative stress and slowing-down the aging process (Murphy, 2006).

Mn has been shown to reduce the life-span in C. elegans (Benedetto et al., 2010) and this model has proven an excellent tool for mechanistically dissecting out aging processes (Baumeister et al., 2006, Hertweck et al., 2004, Lee et al., 2009). Accordingly, the present study was designed to test the hypothesis that DDW has the ability to increase the life span of Mn-exposed worms and that the DAF-16 pathway is modulated by low D concentrations, thus supporting a role for this transcription factor as a key target for pharmacological interventions aimed at prolonging life-span.