Placental Transfer Mechanism Of T-2 Toxin And Its Major Metabolite HT-2 Toxin In BeWo Cells

T-2 toxin is a type A-trichothecene group mycotoxin synthesized by a variety of Fusarium fungi. These soil fungi are common in temperate and warm countries, and are regular contaminants of cereal crops worldwide. T-2 toxin can cause mammals serious maternal toxicity and fetotoxicity when dosed at different pregnancy day. Fetotoxicity of T-2 toxin is secondary to maternal toxicity. Fetotoxicity is not secondary to maternal toxincity but a direct toxicity to fetuses. Placental barriers is the only channel for nutrients exchanging between maternal blood and fetal bloode, but it is also the pathway of the virus, toxin, drug, hormone, etc. to fetus. Placental transfer of mycotoxins could present a potential risk for direct effects on fetus. However, placental transfer has not been verified in any species by quantitative determination of foetal concentrations of T-2 toxin itself or its metabolites. It is therefore relevant to study the ability of T-2 toxin and HT-2 toxin to pass into the foetus of mammals.In this study, we aimed to investigate the possible carrier-mediated transport of T-2 toxin and HT-2 toxin on Be Wo cell monolayers, which represents a placental barrier by transport studies. To elucidate the role of ABC transporters (P-gp, MRP1, MRP2, BCRP) in the trans-trophoblast transport, uptake of T-2 toxin and HT-2 toxin by means of uptake and transcellular transport studies. In order to correctly evaluate the toxicity upon the health and vability of the developing fetus.In this study, based on the cell growth characteristics we established the stable and reliable BeWo cell model in our lab. The model was judged through three quality control standards to confirm the reliability and authenticity of the results. It indicated that the Be Wo cells grown in Transwell at a density of 2.24 X 105 cells/mL for 6-7 days can form an intact cell monolayer, the TEER measured was higher than required and the papp of fluorescein showed a density barrier.In transport study, to calculate the carries-mediate transport, we conducted the bidirection transcellular transport experiment. The result showed a asymmetric profile of transfer for T-2 toxin, AP-BL transfer being higher than the BL-AP one. Concentration dependence of T-2 toxin saturation was not achieved due to its toxicity in Be Wo cells. The strong asymmetry in AP-BL and BL-AP transport of T-2 toxin suggested the involvement of facilated or active transport. The absence of effect of verapamil, MK571, Ko143 and ATP production inhibitors (NaN3+2-DOG) suggested no involvement of ABC transporters. Otherwise, the metabolite HT-2 toxin showed a completely different way. The transport rate of HT-2 toxin was similar in the AP to BL and BL to AP directions, with no significant difference, and the transport in the two directions was stictly proportional to the concentration over the entire range. Neither verapamil, an inhibitor of P-gp, nor MK571, an inhibitor of MRPs, did affect the transport of HT-2 toxin in both AP-BL or BL-AP directions. These data are compatible with either a transcellular or a paracellular passive transport mechanism, as well as with a combination of both.In uptake study, we conducted uptake experiments to calculate the temperature,concentration,pH-depend uptake of T-2 toxin and HT-2 toxin, in order to explain the uptake mechanism for mycotoxin transport from maternal to fetal. Kinetic analysis reveled that T-2 toxin but not HT-2 toxin uptake by BeWo cells was predominantly saturable, suggest that the T-2 toxin uptake into Be Wo cells is carrier-mediated. DIDS, PAH and probencid are well-known inhibitors of OATs, they had significant inhibitory effect, indicating that OAT family members are involved in T-2 toxin uptake. Otherwise, TEA and cimetidine are inhibitors of OCTs, they also inhibit T-2 toxin uptake significantly indicated the OCT involvment. These results suggest that more than two carriers are involved in T-2 toxin uptake in Be Wo cells. But these inhibitors showed little effects on HT-2 toxin uptake, indicating the passive diffusion transport mechanism. In conclusion, our reslut indicated that T-2 toxin and HT-2 txoin could easily transport across the BeWo cell monolayer which was an in vitro model reprsented placenta barries. We proved first that ABC efflux transporters (P-gp, MRP1, MRP2, BCRP) played a minor role in the trans-trophoblast transport of T-2 toxin and HT-2 toxin, the transport rate of HT-2 toxin was similar in the AP to BL and BL to AP directions, while T-2 toxin was higher in the AP to BL direction, the involvement of facilated or active transport mechanism for T-2 toxin and a paracellular passive transport mechanism for HT-2 toxin, and OATs may play an important role for toxin passing from maternal to fetal which would lead to serious toxicities. Finally the study may be benefit for drawing up possible protection measures to reduce the risk of adverse effects of T-2 toxin.