Original ContributionA novel role for vitamin B12: Cobalamins are intracellular antioxidants in vitro
Section snippets
Experimental procedures
Sk-Hep-1 (ECACC 91091816) cells were maintained in MegaCell MEME (Sigma; M-4067) with 3% serum and 200 mM l-glutamine at 37 °C in 5% CO2. The Sk-Hep-1 cell line was chosen as it is highly sensitive to oxidative stress. Cells were plated into 96-well microtiter plates and cultured for 24 h. Medium was replaced with 100 μl fresh medium containing various concentrations of dl-homocysteine or H2O2 as oxidants for up to 24 h. Cells were preincubated with cobalamin, thiol (NAC or GSH), or cobalamin
Results
Preliminary experiments established that a Hcy concentration of 30 μM achieved > 90% cell death in Sk-Hep-1 cells. Dose-dependent protection from cell death was observed when Sk-Hep-1 cells were pretreated with increasing concentrations of either GSCbl or NACCbl (2–64 μM) for 2 h before exposure to Hcy (30 μM) (Fig. 1). Cell survival (> 90%) from Hcy toxicity was achieved with GSCbl (30 μM) and NACCbl (30 μM) (Fig. 1). Cobalamin and thiol (NAC and GSH) concentrations were optimized in terms of
Discussion
In this study, preincubation of Sk-Hep-1 cells with GSH or NAC only partially protected against Hcy- or H2O2-induced damage. Their respective thiolatocobalamin derivatives (GSCbl and NACCbl) demonstrated significantly greater protective capabilities than the thiol alone or the thiol combined with standard cobalamin derivatives.
GSH and NAC, unlike the cobalamins, are well-characterized antioxidants. GSH is a thiol-containing tripeptide and a major antioxidant defense molecule. Reduced GSH levels
Acknowledgments
GSCbl and NACCbl are currently the subjects of U.S. Patent applications by N.E.B., C.S.B., and J.H.H.W. (U.S. Application 20080113900, “Pharmaceutical compositions and therapeutic applications for the use of a synthetic vitamin B12 derivative, glutathionylcobalamin,” and U.S. Application 20080076733, “Pharmaceutical compositions and therapeutic applications for the use of a novel vitamin B12 derivative, N-acetyl-l-cysteinylcobalamin”) and by A.M. (U.S. Application 20040157783, “Method for
References (31)
- et al.
Cobalamin-mediated regulation of transcobalamin receptor levels in rat organs
Arch. Biochem. Biophys.
(2007) - et al.
Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases
Free Radic. Biol. Med.
(2008) - et al.
Redox modifications of protein-thiols: emerging roles in cell signaling
Biochem. Pharmacol.
(2006) - et al.
Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes
Biochem. Biophys. Res. Commun.
(1990) - et al.
Measuring the secretion of heat shock proteins from cells
Methods
(2007) - et al.
B12-responsive internal ribosome entry site (IRES) element in human methionine synthase
J. Biol. Chem.
(2005) - et al.
Folic acid protects motor neurons against the increased homocysteine, inflammation and apoptosis in SOD1G93A transgenic mice
Neuropharmacology
(2008) - et al.
The inhibition of corrinoid-catalyzed oxidation of mercaptoethanol by methyl iodide: mechanistic implications
J. Inorg. Biochem.
(1993) A scarlet pimpernel for the resolution of inflammation? The role of supra-therapeutic doses of cobalamin, in the treatment of systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic or traumatic shock
Med. Hypotheses
(2006)Disorders of cobalamin (vitamin B12) metabolism: emerging concepts in pathophysiology, diagnosis and treatment
Blood Rev.
(2007)