Estimate of the digestibility, assimilability and intestinal permeability of butyltins occurring in wine

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Abstract

An estimate of the digestibility and assimilability of butyltins occurring in contaminated wines (Port, red and white) was obtained by means of in vitro studies of gastrointestinal digestion. The influence of the wine matrix on the intestinal permeability was explored by studying the accumulation of butyltins in Caco-2 monolayers either when these species are dissolved in buffer only or in the dialysates of digested wines. Some important information about the fate of the butyltin compounds ingested from contaminated wines could be achieved. Only a very small fraction of the ingested DBT and TBT, the two most toxic forms, appear to be able to reach the epithelium as judged by the small dialyzable fraction found (<2%). This is probably independent from the food/drink matrix introducing these contaminants, since the influence of the involved enzymes appear to be dominant, especially for DBT and TBT. Additionally, the intestinal permeability of the three butyltins was also very low, the wine matrix possibly having a hindrance effect in a few cases.

Introduction

The anthropogenic butyltin compounds, monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT), are industrially produced for their application mainly as biocides (TBT) and polymer stabilisers (MBT and DBT) (Hoch, 2001). The high toxicity of these compounds for biota is well documented, especially in the aquatic environment. Concerning the exposure of human species to butyltins and its effects, the available information is minimal. The occurrence of butyltin compounds in human blood and liver has been reported (Kannan et al., 1999, Takahashi et al., 1999) and constitutes direct evidence of human exposure. The two main routes of human exposure are the direct ingestion of contaminated food and drink and the indirect exposure from household items. For example, seafood collected from coastal areas and lakes contain varying amounts of butyltin compounds, while organotins leach from PVC and related materials, resulting in contamination of foodstuff and beverages (Hoch, 2001). Butyltin compounds, mainly DBT with concentrations up to 138 μg/L, were found in wines of the Canadian market (Forsyth et al., 1992, Forsyth et al., 1994). Apparently, this occurrence was associated with the use of PVC for wine transportation. At present there is not an official tolerable daily intake (TDI) value for DBT, although 5 μg of Sn/kg of body weight has been advanced by Summer et al. (1996) as a proposed TDI. An adult weighing 60 kg who drinks 1 L of wine containing 138 μg/L as Sn of DBT would imply an intake of 2.3 μg/kg of body weight. This intake value poses high concern because, considering exposure to other possible sources (dietary or household items related), the proposed TDI may be exceeded. More recently a survey of butyltin compounds in Portuguese wines has found measurable DBT (0.05–0.15 μg/L as Sn) in 14% of the analysed samples (Azenha and Vasconcelos, 2002). A further study in a Portuguese winery showed that high-density polyethylene containers used in the transfer of wine in an early stage of the vinification process could have been sources of these contaminants (Azenha and Vasconcelos, 2002). Agglomerated cork stoppers appear to be another possible source of contamination (Jiang et al., 2004). The Portuguese wines contaminated with 0.15 μg/L as Sn of DBT would imply, for the same 60 kg weight adult and consumption an intake of only 0.0025 μg/kg of body weight, which is 2000 times lower than the referred proposed TDI. However, history shows that TDI values tend to be lowered over the years, which means that lower values than that proposed by Summer et al. may be expected to be established in the future by national and international organizations. Therefore, it may be advisable and safer to produce wine (and other drinks and food) with as low butyltin and other organotin concentrations as possible. Advance on the knowledge about the fate of these contaminants after their intake constitutes also an important issue.

The butyltin compounds ingested with contaminated wine, or other beverage/foodstuff, face the gastrointestinal tract, and specially the intestinal epithelium, as the main barrier for reaching the bloodstream. The lack of objective data on the intestinal permeability of these compounds led the authors to use the Caco-2 in vitro intestinal cell line model for a study of the intestinal permeation of the butyltins (Azenha et al., 2004). The study was conducted with synthetic butyltin solutions in an appropriate buffer, the results suggesting a low in vivo permeability of the butyltins. The authors found that the reported permeability pattern correlated well with the general in vivo toxicity pattern (trialkyltin > dialkyltin  monoalkyltin). Additionally, the transport of MBT and DBT was found to be dependent on the paracellular route status. The results of this work about butyltin accumulation and transport by the epithelial Caco-2 cell line incubated in an appropriate buffer, prompted the authors to move on to more complex matrices such as beverage gastrointestinal digests, where matrix–butyltin and matrix–cell line interactions may dictate substantially different permeation scenarios.

The aims of the research presented in this paper are: (i) to estimate the digestibility and assimilability of butyltins occurring in contaminated wines (Port, red and white) by performing in vitro studies of gastrointestinal digestion; (ii) to check for matrix influences on the intestinal permeability by comparing the accumulation of butyltins in Caco-2 monolayers observed either when these species are dissolved in buffer only or in the dialysates of digested wines.

Section snippets

Reagents and materials

Solvents and common laboratory reagents were of analytical grade. Deionised water with conductivity below 0.1 μS/cm was used throughout the work. The butyltin compounds and tetramethylammonium hydroxide 25% w/v solution (TMAH) were obtained from Sigma–Aldrich. The enzymes were supplied by Sigma: pancreatin from porcine pancreas grade VI; bile salts approximately 50% sodium cholate and approximately 50% sodium deoxycholate; pepsin from porcine stomach mucosa (pepsin A, EC 3.4.23.1); α-amylase

Simulation of gastrointestinal digestion

The procedure of simulation of wine’s gastrointestinal digestion was carried out as before (Azenha and Vasconcelos, 2000), by an adaptation of the procedure described by Crews et al. (1985). Briefly, 50 mL wine (fortified with 10 μg/L (as Sn) MBT, DBT and TBT or non-fortified) were mixed with 25 mL of gastric juice (10 g/L pepsin, NaCl 0.15 M, HCl 0.02 M) and left with stirring for 3 h. After 3 h, 20 mL of gastric juice were collected from the reactor and filtered through 0.45 μm pore size filters. Then

Solubility and dialysability of butyltins in wine digests

Before subjecting the doped wines (10 μg/L as Sn) to the simulation of gastrointestinal digestion, certain warranties were assured. There was no butyltin contamination coming from the materials and reagents used for the simulation, as proven by blank trials performed with water in the place of wine. The possible adsorption of butyltins to the dialysis membrane was checked and found to be negligible.

The solubility of the butyltin compounds after the gastric and intestinal stages of the wine

Solubility and dialysability of butyltins in wine digests

Prior to the discussion of the results a foreword about the employed in vitro model is necessary. There are well established in vitro methods for estimating the element solubility (a required, although not sufficient, condition for nutrient absorption) in animals with a simple stomach (Boisen and Eggum, 1991) which comprise a two-phase simulation of the gastrointestinal physiology, the stomach and intestinal phases. However, the soluble fraction found for an element at the end of the simulated

Conclusion

The present work provides some important indications about the possible fate of the butyltin compounds when ingested from contaminated wines. Only a very small fraction of the ingested DBT and TBT, the two most toxic forms, appear to be able to reach the epithelium. Binding to enzymes appear to play a major role in the poor assimilability found for the butyltins. Additionally, the intestinal permeability of the three butyltins is also very low, the wine matrix possibly having a hindrance effect

Acknowledgement

The authors thank the National Institute of Environmental Studies, Japan, for providing the NIES 11 CRM.

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