Letters to the Editor pubs.acs.org/crt
More on the in Vitro Characterization of the Intestinal Absorption of Methylmercury was high across the artificial lipid membranes (52 ± 2.3%), thereby confirming the observations of Vázquez et al.3 pointing to an important participation of passive transcellular diffusion. However, the presence of rising concentrations of L-Cys in the medium reduced the transport of CH3Hg across the lipid layer (25 μM, 27 ± 0.6%; 200 μM, 0.23 ± 0.03%; 500 μM, 0.10 ± 0.01%). This shows that CH3Hg-Cys complexes are being formed; these complexes are polar and give rise to a notorious decrease in passive transcellular transport. The new assay has also involved the addition of concentrations of Zn2+, Cu2+, and Mg2+ similar to that of CH3Hg (50 μM) in the presence of L-Cys (200 and 500 μM). The results show the presence of these cations to favor CH3Hg transport across the lipid membranes since the recorded fluxes were similar to those recorded in the absence of L-Cys (53 ± 1.6%). This indicates that L-Cys, in the presence of these cations for which it exhibits comparatively greater affinity, does not bind to CH3Hg even if there is an excess of L-Cys in the medium. Consequently, in such cases, CH3Hg is mainly transported through passive diffusion. These results demonstrate that both routes coexist and that the predominance of one or the other mechanism depends on different factors, including the concentration of other cations within the intestinal lumen.
W
e thank Dr. Guzzi et al. for their observation. Because of its octanol−water partition coefficient, methylmercury (CH3Hg) can be considered lipophilic,1 in the same way as verapamil.2 The study by Vázquez et al.3 in artificial membranes (PAMPA) has shown the transport of both compounds to be high and of the same order. This means that CH3Hg can cross the epithelial cell membrane through passive diffusion in the lipid bilayer, as has been previously described for verapamil.2 However, this does not mean that the above-mentioned mechanism is the only form of transport across the membrane. In fact, Vázquez et al.3 reports that passive diffusion can coexist with other types of transcellular transport: “Although the transcellular passive route appears to be an important form of transport of CH3Hg, the kinetic parameters (Figure 2A) obtained in the present study also evidence the participation of saturable transcellular transport and therefore the possible mediation of a carrier in the net transport of CH3Hg from apical to basolateral compartment.” Furthermore, the possibility of transcellular transport through binding to thiol groups has also been described by Vázquez et al.,3 as also mentioned by Guzzi et al. in their letter to the Editor: “It has been postulated that CH3Hg is transported through the membrane by means of neutral amino acid transporters, as a result of its structural similarity to methionine when CH3Hg is bound to L-Cys or other thiol groups.” All these transport mechanisms and their magnitudes influence the toxicity of the mercurial species. It is difficult to establish the proportions of CH3Hg in salt form or bound to thiol groups within the intestinal lumen. These proportions are dependent upon factors such as diet. It must be emphasized that the affinity of divalent mercury (Hg2+) for cysteine (L-Cys) is less than that of other cations such as Zn2+, Cu2+, Mg2+,Ca2+, Be2+, Li+, or Na+.4,5 Although no studies have been published on the affinity of CH3Hg compared with that of other cations, it can be expected to be similar to that observed for Hg2+ or even lower, due to the presence of the methyl group. Most of these cations are found in foods at concentrations higher than those of mercury. In this respect, in seafood products (the main source of CH 3 Hg), the concentrations of Zn2+ range between 2.7 and 106 mg/kg,6−9 those of Cu2+ between 0.096 and 6.48 mg/kg,10,11,6,12,8,9 and those of Mg2+ between 56 and 225 mg/kg,13 while the maximum concentrations of CH3Hg rarely exceed 1 mg/ kg.14,15,13,9 Therefore, depending on the type of diet, and in the presence of these cations, the salt form of CH3Hg may possibly exceed CH3Hg bound to thiol groups within the intestinal lumen. Taking into account the comments made by Guzzi et al., an additional study was made to elucidate the influence of cysteine in the passive diffusion of CH3Hg. The assay was carried out in artificial lipid membranes created following the protocol developed by Vázquez et al.3 The assessment of CH3HgCl (10.8 mg/L; 50 μM) transport was made in the absence and presence of rising concentrations of L-Cys (25, 200, and 500 μM). As expected, CH3HgCl transport in the absence of L-Cys © 2014 American Chemical Society
M. Vázquez D. Vélez V. Devesa*
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Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avenida Agustín Escardino 7, 46980 Paterna, Valencia, Spain
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 963 900 022. Fax: +34 963 636 301. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Halbach, S. (1985) The octanol/water distribution of mercury compounds. Arch. Toxicol. 57, 139−141. (2) Velicky, M., Bradley, D. F., Tam, K. Y., and Dryfe, R. A. (2010) In situ artificial membrane permeation assay under hydrodynamic control: permeability-pH profiles of warfarin and verapamil. Pharm. Res. 27, 1644−1658. (3) Vazquez, M., Velez, D., and Devesa, V. (2014) In vitro characterization of the intestinal absorption of methylmercury using a Caco-2 cell model. Chem. Res. Toxicol. 27, 254−264. (4) Belcastro, M., Marino, T., Russo, N., and Toscano, M. (2005) Interaction of cysteine with Cu2+ and group IIb (Zn2+, Cd2+, Hg2+) metal cations: a theoretical study. J. Mass Spectrom. 40, 300−306.
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Chemical Research in Toxicology
Letters to the Editor
(5) Shankar, R., Kolandaivel, P., and Senthilkumar, L. (2011) Interaction studies of cysteine with Li+, Na+, K+, Be2+, Mg2+, and Ca2+ metal cation complexes. J. Phys. Org. Chem. 24, 553−567. (6) Yi, Y., Wang, Z., Zhang, K., Yu, G., and Duan, X. (2008) Sediment pollution and its effect on fish through food chain in the Yangtze River. Int. J. Sediment Res. 23, 338−347. (7) Wang, Y., Chen, P., Cui, R., Si, W., Zhang, Y., and Ji, W. (2010) Heavy metal concentrations in water, sediment, and tissues of two fish species (Triplohysa pappenheimi, Gobio hwanghensis) from the Lanzhou section of the Yellow River, China. Environ. Monit. Assess. 165, 97−102. (8) Medeiros, R. J., dos Santos, L. M. G., Freire, A. S., Santelli, R. E., Braga, A. M. C. B., Krauss, T. M., and Jacob, S. D. C. (2012) Determination of inorganic trace elements in edible marine fish from Rio de Janeiro State, Brazil. Food Control 23, 535−541. (9) Wei, Y., Zhang, J., Zhang, D., Tu, T., and Luo, L. (2014) Metal concentrations in various fish organs of different fish species from Poyang Lake, China. Ecotoxicol. Environ. Saf. 104C, 182−188. (10) Uluozlu, O. D., Tuzen, M., Mendil, D., and Soylak, M. (2007) Trace metal content in nine species of fish from the Black and Aegean Seas, Turkey. Food Chem. 104, 835−840. (11) Türkmen, M., Türkmen, A., Tepe, Y., Ates, A., and Gökkus, K. (2008) Determination of metal contaminations in sea foods from Marmara, Aegean and Mediterranean seas: Twelve fish species. Food Chem. 108, 794−800. (12) Bilandžic, N., Dokic, M., and Sedak, M. (2011) Metal content determination in four fish species from the Adriatic Sea. Food Chem. 124, 1005−1010. (13) Perrault, J. R., Buchweitz, J. P., and Lehner, A. F. (2014) Essential, trace and toxic element concentrations in the liver of the world’s largest bony fish, the ocean sunfish (Mola mola). Mar. Pollut. Bull. 79, 348−353. (14) Fabris, G., Turoczy, N. J., and Stagnitti, F. (2006) Trace metal concentrations in edible tissue of snapper, flathead, lobster, and abalone from coastal waters of Victoria, Australia. Ecotoxicol. Environ. Saf. 63, 286−292. (15) Calatayud, M., Devesa, V., Virseda, J. R., Barbera, R., Montoro, R., and Velez, D. (2012) Mercury and selenium in fish and shellfish: occurrence, bioaccessibility and uptake by Caco-2 cells. Food Chem. Toxicol. 50, 2696−2702.
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