Anti-aging effects of deuterium depletion on Mn-induced toxicity in a C. elegans model
Highlights
► Manganese exposure causes life span reduction in C. elegans. ► Manganese causes reduction in DAF-16 and SOD-3 levels. ► D-depletion treatment reverses the Mn-induced reduction in life-span caused by and restores DAF-16 levels to control values.
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.
Section snippets
DDW production
DDW was produced from ordinary water containing the natural amount of D (150 ppm, equivalent to16.8 mmol/L), using fractional distillation to decrease the D concentration to 120–90 ppm. The production of DDW is based on the differences between the physical and chemical characteristics of normal water (H2O) and heavy water (D2O). When producing DDW, advantage is taken of the fact that as a consequence of the different volatility, at the boiling point of normal water, the steam in equilibrium with
DDW effects on life span
Fig. 1 shows that 48-h treatment with DDW alone did not affect life-span in C. elegans at either of the two tested concentrations.
DDW protects from Mn-induced aging
When worms were pre-exposed to Mn, there was a statistically significant reduction in their life-span (Fig. 2, p < 0.05). In parallel experiments, at the end of DDW treatment the proteins were extracted for further analysis. Mn exposure caused a decrease in DAF-16 levels, which was also reflected in decreased levels of a downstream gene product, SOD (Fig. 3A and B).
Discussion
Though the concentration of D in all living organisms is greater than 10 mmol/L, its biological role has yet to be defined. Recent studies have shown that depletion of naturally occurring D can result in tumor regression in mice, dogs, cats and humans (Kovács et al., 2011, Krempels et al., 2008, Somlyai et al., 1998a, Somlyai et al., 1998b). The present study demonstrates that subnormal D concentrations reversed the Mn-induced reduction in the nematode's life-span and modified the DAF-16
Conclusions
The present investigation shows that deuterium depletion in water reverses the intracellular effects of Mn exposure in C. elegans. We show that Mn caused reduction in DAF-16 and SOD-3 levels, which was associated with reduced life-span. Notably, treatment of Mn exposed worms with DDW (90 ppm) restored life-span, DAF-16 and SOD-3 levels to control levels. In conclusion, our study strongly suggest that low D concentrations can restore the Mn-induced reduced life-span in C. elegans, reiforcing the
Conflict of interest
There is no conflict of interest in this study and in the preparation of this manuscript.
Acknowledgments
NIH ES R01 10563 and 07331 (MA).
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