Alternatively Spliced Methionine Synthase in SH‐SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

Abstract

The folate and cobalamin (Cbl-) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S-adenosylmethionine (SAM). OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

Vitamin B12 protects against DNA damage induced by hydrochlorothiazide

“…The results of our study revealed that vitamin B12 has significantly reduced HCT-induced SCEs, indicating the ability of vitamin B12 to reduce DNA damage induced by HCT. This finding is consistent with the results of other studies that have shown that vitamin B12 plays a significant role in protecting and maintaining the stability of the human genome against mutagenicity induction by some genotoxic drugs such as ribavirin (Joksic et al, 2006), pioglitazone (Alzoubi et al, 2012), Paclitaxel (Alzoubi et al, 2014), thimerosal (Waly et al, 2016), and in hemodialysis patients (Stopper et al, 2008). The evidence in our study illustrate that vitamin B12 is important determinant of DNA damage.…”

“…Various studies have shown that vitamin B12 plays a significant role in protecting and maintaining the stability of the human genome. As an example, the protective effects of B12 were tested against the DNA damage induced by ribavirin (Joksic et al, 2006), pioglitazone (Alzoubi et al, 2012), Paclitaxel (Alzoubi et al, 2014), thimerosal (Waly et al, 2016), and in hemodialysis patients (Stopper et al, 2008). All of these studies and many more have given positive promising results that support the growing role of vitamin B12 in preventing DNA damage.…”

<p>Andrographolide attenuates bupivacaine-induced cytotoxicity in SH-SY5Y cells through preserving Akt/mTOR activity</p>

“…Oxidative status is an important cell characteristic included in the response to cellular environment transformation. Generation of ROS and GSH mediates apoptosis in neuroblastoma SH-SY5Y cells 19,20. As shown in Figure 3, A and B, Bup significantly increased the level of ROS and decreased the level of total GSH in SH-SY5Y cells compared with the control group.…”

Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System

“…Depletion of GSH has been shown to occur after exposure to Hg compounds in vitro and in vivo using experimental animals (47,204). In primary cultures of neurons and astrocytes, GSH was decreased by 30-50% following a 30-min treatment with 5 lM MeHg (98).…”