Intestinal oxidative state can alter nutrient and drug bioavailability

Abstract

Organic cations (OCs) are substances of endogenous (e.g. dopamine, choline) or exogenous (e.g. drugs like cimetidine) origin that are positively charged at physiological pH. Since many of these compounds can not pass the cell membrane freely, their transport in our out of cells must be mediated by specific transport systems. Transport by organic cation transporters (OCTs) can be regulated rapidly by altering their trafficking and/or affinities in response to a stimuli. However, for example, a specific disease could lead to modifications in the expression of OCTs. Chronic exposure to oxidative stress has been suggested to alter regulation and functional activity of proteins through several pathways. According to results from a previous work, oxidation-reduction pathways were thought to be involved in intestinal organic cation uptake modulation. The present work was performed in order to evaluate the influence of oxidative stressors, especially glutathione, on the intestinal organic cation absorption. For this purpose, the effect of compounds with different redox potential (glutathione, an endogenous antioxidant, and procyanidins, diet antioxidants) was assessed on MPP+ (1-methyl-4-phenylpyridinium iodide) uptake in an enterocyte cell line (Caco-2). Caco-2 cells were subcultured with two different media conditions (physiological: 5 mM glucose, referred as control cells; and high-glucose: 25 mM glucose, referred as HG cells). In HG cells, the uptake was significantly lower than in control cells. Redox changing interventions affected MPP+ uptake, both in control and in high-glucose Caco-2 cells. Cellular glutathione levels could have an important impact on membrane transporters activity. The results indicate that modifications in the cellular oxidative state modulate MPP+ uptake by Caco-2 cells. Such modifications may reflect in changes of nutrient and drug bioavailability.

Figure 1

(A) In the y-axis it is represented ³H-Mpp⁺ apical uptake (as pmol/mg protein/5 min) of Caco-2, Caco-2 cells maintained with 25 mM glucose (HG Caco-2) and Caco-2 cells depleted of reduced glutathione (GSH depleted Caco-2). Confluent Caco-2 monolayers were preincubated at 37°C for 60 min with Hanks buffer and then incubated with 200 nM ³H-MPP⁺ for 5 min. (B) Effect of reduced glutathione (GSH) (500 µM, 1 mM and 5 mM) on ³H-MPP⁺ apical uptake (represented as % of the control treatment) by Caco-2 cells and Caco-2 cells maintained with 25 mM glucose (HG Caco-2). Confluent monolayers were preincubated at 37°C for 3 and 60 min (PI = 3 min and PI = 60 min, respectively) and then incubated with 200 nM ³H-MPP⁺ for 5 min in the presence of H₂O (control) or the tested compounds. each value represents the mean ± SEM (n = 6). *p < 0.05.

Figure 2

Effect of reduced (GSH) and oxidized (GSSG) glutathione (1 mM) on ³H-MPP⁺ apical uptake (represented as % of the control treatment) by Caco-2 cells. Confluent Caco-2 monolayers were preincubated at 37°C for 3 and 60 min and then incubated with 200 nM ³H-MPP⁺ for 5 min in the presence of H₂O (control) or the tested compounds. each value represents the mean ± SEM (n = 6). *p < 0.05.

Figure 3

Effect of oxidized procyanidin fractions (FI-FV) on ³H-MPP⁺ apical uptake (respresented as %, considering control treatment as 100%) by Caco-2 cells and Caco-2 cells maintained with 25 mM glucose (HG Caco-2). Confluent Caco-2 monolayers were preincubated for 60 min with oxidized procyanidins and then incubated with 200 nM ³H-MPP⁺ for 5 min in the presence of etOh (control) or the tested compounds. each value represents the mean ± SEM (n = 3–27). *p < 0.05.

Figure 4

Schematic representation of the investigated pathways involved in OCT activity modulation, through changing oxidative cellular status. (A) Control cells. Caco-2 cells were grown in Minimum essential Medium containing 5 mM (physiological) glucose. In these cells the uptake of the organic cations was more pronounced compared to HG cells (B). (B) High Glucose cells (HG). Caco-2 cells were grown in Minimum Essential Medium containing 25 mM (high) glucose. In these cells, uptake was significantly reduced by oxidative stress. It is possible that oxidative stress, increased in these cells, modifies OCTs activity, either directly or indirectly through glutathione pathways. Similarly, glutathione, directly or through indirect pathways, modifies oxidative status of OCTs, and consequently their activity.

Procyanidins were fractionated using low pressure chromatography and recovered during a period of time. Average molecular weights (in Dalton) of procyanidins in grape seeds fractions was determined by Liquid Secondary Ion Mass Spectrometry.