Transport Measurements Across Caco-2 Monolayers of Different Organic and Inorganic Selenium: Influence of Sulfur Compounds

Abstract

The transport and uptake of the most common Se compounds, selenate (SeO42-), selenite (SeO3(2-)), selenomethionine, and selenocystine, were investigated using confluent monolayers of Caco-2 cells, a human carcinoma cell line. Comparative measurements were performed in the absorptive (apical to basolateral side) and exsorptive (basolateral to apical side) directions. Apparent permeability coefficients (Papp), calculated from transport experiments in the absorptive direction, showed increasing values in the following rank order: about 1 x 10(6) cm/s < mannitol < SeO3(2-) < or = selenocystine < selenomethionine < SeO4(2-) < or = about 16 x 10(4) cm/s. The ratios of the Papp measured in the absorptive versus exsorptive directions indicated that only the organic forms presented a net polarized transport (Papp ratio >> 1), suggesting the presence of a transcellular pathway. No significant excretion was observed. The transport of selenomethionine was inhibited by its sulfur analog, methionine, suggesting a common transport mechanism. In contrast, an inhibition of the transport of selenocystine by cysteine was not observed. From the two substrates tested, sulfate and thiosulfate, only thiosulfate inhibited the transport of SeO4(2-) . This effect was also observed for SeO32- (i.e., was unspecific), which questioned the assertion of a common transport for sulfate and SeO4(2-) and may confirm the paracellular pathway of SeO42- suggested by the Papp ratio of about 1. The addition of glutathione (GSH) in large excess had no consequence on the passage of SeO3(2-) but strongly increased the uptake (about fourfold). The liquid chromatography - mass spectrometry (LC-MS) data showed that, in the ionic condition of incubation medium, GSH promptly reduced SeO3(2-) (< or = 2 min) in its elemental form Se0, which cannot ascribe to selenodiglutathione a direct role in the effect of GSH.

Basal Bioenergetic Abnormalities in Skeletal Muscle from Ryanodine Receptor Malignant Hyperthermia-susceptible R163C Knock-in Mice

“…Considering that the R163C RyR1 mutation in skeletal muscle resulted in a significantly increased cytosolic Ca 2ϩ (about 2-fold of the normal resting concentration, this study and that by Yang et al (30), an activation of several calcium-dependent pathways was expected leading to the activation of a slow-twitch fiber program to increase the resistance to fatigue, the oxidative capacity, and the calcium buffering capacity of mitochondria. The values for total calcium contents of WT mitochondria evaluated in this study were within the range of those previously reported for rodent skeletal muscle using ICP-MS or other techniques (82)(83)(84)(85)(86). The significantly higher concentrations of mitochondrial calcium content in R163C reported here may stem from the chronically elevated (2-4-fold) resting intracellular Ca 2ϩ concentrations in intact MHS skeletal muscle from various species (76 -78).…”

“…Given that exposure of mitochondria to a sustained higher calcium concentration may lead to higher calcium uptake and net calcium accumulation, dissipation of the electrochemical gradient, decreased ATP production, and/or organelle swelling or bursting (79 -81), the calcium concentrations in mitochondrial and PM fractions were evaluated in WT and R163C skeletal muscle by ICP-MS. The values for total calcium contents of WT were within those reported for rodent skeletal muscle using ICP-MS or other techniques (690 -1500 nmol/g muscle wet weight) (82)(83)(84)(85)(86). Skeletal muscle mitochondria from R163C mice had 5.7 times more Ca 2ϩ than WT, and the Ca 2ϩ concentration in the cytosolic fraction was 1.8-fold higher than WT (Table 4).…”

Estimating Intestinal Absorption of Inorganic and Organic Selenium Compounds by in Vitro Flux and Biotransformation Studies in Caco-2 Cells and ICP-MS Detection

“…The Se amounts were quantified by atomic absorption spectrometry. Only the selenoamino acids, SeMet and (SeCys) 2 , showed a net absorptive flux across the Caco-2 cell monolayer, suggesting that a transporter-mediated transcellular pathway was involved, while a paracellular pathway for selenate was suggested [15]. The Caco-2 cell model was in another study used to assess the production of the selenoprotein, glutathione peroxidase (GPx), from Semethylselenocysteine (MeSeCys), SeMet or selenite applied in concentrations between 15.6 and 125 nM Se, in time intervals between 8 and 72 h. GPx activity was determined by an enzyme assay while total Se was determined by AAS [16].…”

“…Thus, absorptive flux describes the transport from the intestinal lumen to the serosal side, while exsorptive flux describes transport in the opposite direction. A thorough investigation on selenium permeability across filter grown Caco-2 cell monolayers was performed by Leblondel et al [15] who compared the permeabilities of the four seleno compounds (10 μM) selenite, selenate, SeMet, and selenocysteine (SeCys) 2 in the absorptive as well as exsorptive directions. The Se amounts were quantified by atomic absorption spectrometry.…”

“…Excess selenium is excreted either as the volatile dimethylselenide (DMeSe) (exhaled) or as selenosugars (urine) as such but probably as GSH conjugates and/or other reaction products. In previous experiments, it has been demonstrated that the addition of GSH in large excess of selenite strongly increased the intracellular uptake but did not affect the overall apparent permeability (P app ) of selenite across the Caco-2 cell monolayer [15]. Permeability studies performed in cell models may therefore mimic in vivo conditions better if reducible compounds, like selenite (+4) and MeSeA (+2), are applied together with GSH.…”