Membrane vesicles from trophoblast cells as models for placental exchange studies

Micro- and nanoplastics (MNPs) are ubiquitous in the environment and have recently been found in human lungs, blood and placenta. However, data on the possible effects of MNPs on human health is extremely scarce. The potential toxicity of MNPs during pregnancy, a period of increased susceptibility to environmental insults, is of particular concern. The placenta provides a unique interface between maternal and fetal circulation which is essential for in utero survival and healthy pregnancy. Placental toxicokinetics and toxicity of MNPs are still largely unexplored and the limited studies performed up to now focus mainly on polystyrene particles. Practical and ethical considerations limit research options in humans, and extrapolation from animal studies is challenging due to marked differences between species. Nevertheless, diverse in vitro and ex vivo human placental models exist e.g., plasma membrane vesicles, mono-culture and co-culture of placental cells, placenta-on-a-chip, villous tissue explants, and placental perfusion that can be used to advance this research area. The objective of this concise review is to recapitulate different human placental models, summarize the current understanding of placental uptake, transport and toxicity of MNPs and define knowledge gaps. Moreover, we provide perspectives for future research urgently needed to assess the potential hazards and risks of MNP exposure to maternal and fetal health.

Pregnant women are daily exposed to a wide selection of foreign substances. Sources are as different as lifestyle factors (smoking, daily care products, alcohol consumption, etc.), maternal medication or occupational/environmental exposures. The placenta provides the link between mother and foetus, and though its main task is to act as a barrier and transport nutrients and oxygen to the foetus, many foreign compounds are transported across the placenta to some degree and may therefore influence the unborn child. Foetal exposures to environmental and medicinal products may have impact on the growth of the foetus (e.g. cigarette smoke) and development of the foetal organs (e.g. methylmercury and thalidomide). The scope of this review is to give insight to the placental anatomy, development and function. Furthermore, the compounds physical properties and the transfer mechanism across the placental barrier are evaluated. In order to determine the actual foetal risk from exposure to a chemical many studies regarding the topic are necessary, including means of transportation, toxicological targets and effects. For this purpose several in vivo and in vitro models including the placental perfusion system are models of choice.

Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta.

The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture.

Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.

The human placenta is a complex specialized structure that mediates the interchange of molecules, ions, and gases between maternal and foetal circulation. We have investigated the distribution and expression of caveolae/caveolin in the placenta. Using immunochemical, immunocytochemical, and ultrastructural methods, we show that the placenta expresses caveolin-1 and caveolin-2, which are marker proteins for caveolae. These proteins and caveolae were expressed at high levels in endothelium of placental capillaries and in endothelial and smooth muscle cells of larger vessels. In addition, fibroblasts in areas of the placenta with high connective tissue content also expressed caveolin. However, we were unable to detect these proteins or caveolae-like structures in the syncytiotrophoblast layer or in cytotrophoblasts. These results have important implications for further understanding placental biology and for the role of caveolae in cell regulation in this organ.

Placental transfer of maternal calcium (Ca²⁺) is a crucial process for fetal development although the biochemical mechanisms responsible for this transfer are largely unknown. We have investigated the characteristics of Ca²⁺uptake by the human placental trophoblast cell line BeWo. The kinetics studies revealed an active extracellular Ca²⁺uptake by BeWo cells, which was rapid in the first 2 min (initial velocity (V_i) of 4.17±0.25 nmol/mg/min), and showed a subsequent plateau. Uptake experiments performed at V_iwith increasing concentrations of Ca²⁺resulted in a typical saturation curve (K_mof 0.54±0.07 m $\text{m}$ and V_maxof 7.07±0.28 nmol/mg protein/min). Lowering the pH of the incubation medium from 7.4 to 5.5 led to Ca²⁺uptake inhibition of 40–50 per cent. The presence of voltage-sensitive ($\text{l}$ -type) Ca²⁺channels in BeWo cells was demonstrated by Western blot. Therefore, the implication of such channels in basal Ca²⁺uptake of BeWo cells was investigated. Cell depolarization with extracellular high potassium concentration (40 m $\text{m}$), and hyperpolarization with extracellular high chloride concentration (60 m $\text{m}$) or with valinomycin (10 μ$\text{m}$) did not influence the basal Ca²⁺uptake of BeWo cells. The L-type Ca²⁺channel modulators (Bay K 8644 and Nitrendipine) had no effect on the Ca²⁺uptake. An antagonist of receptor-mediated, store-operated and voltage-gated Ca²⁺channels (SKF-96365) also did not modulate the Ca²⁺uptake of BeWo cells. Therefore, our results indicate that the basal Ca²⁺uptake of BeWo cells is inhibited by lowering pH of the incubation medium, is voltage independent, and is not influenced by $\text{l}$ -type Ca²⁺channel and capacitative Ca²⁺conductance modulators.

Sulfohydrolase activities of microsomal membranes from human term placenta and of a homogenous sterylsulfatase preparation isolated from these membranes were studied using a number of steryl sulfates and nonsteroidal aryl sulfates as substrates. The results of these experiments clearly indicate that in vitro both classes of substrates can be hydrolyzed by the sterylsulfatase. The enzyme catalyzed (in the order of decreasing specific activity) the hydrolysis of sulfuric acid esters of non-steroidal phenols, of a phenolic steroid, and of neutral 3β, 21-, and (though at a very low rate) 17β-hydroxysteroids. Triiodothyronine sulfate and indoxyl sulfate likewise were cleaved by the sulfatase. However, among all substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited a considerably high affinity towards the sterylsulfatase.

The products of the hydrolytic reaction only slightly inhibited the sulfatase activity. K_I values derived for free steroids were ten- to hundred-fold higher than K_M values obtained for the respective sulfoconjugates. Inorganic sulfate was a rather weak inhibitor of the enzyme's activity. Its inhibitory potency, however, increased significantly in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sterylsulfatase activity.

Treatment of the sulfatase with amino acid side-chain modifying reagents directed against arginine, cysteine, cystine, serine, or tyrosine did not result in significant alteration of its activity. The enzyme, however, was severely inactivated by diethylpyrocarbonate which is known to react preferentially with histidyl groups on proteins. In the presence of substrate, this inactivation was significantly reduced. Histidine thus appears to be essential for the sulfatase activity. Taken together, our results reveal a considerable similarity between the reaction mechanism of human placental sterylsulfatase and the ones reported for other sulfatases.

Salicylate, taurocholate, and 4,4′-substituted stilbene-2,2′-disulfonates, well known inhibitors of protein-mediated transport of anions across biological membranes, likewise interfered with the sulfatase activity. The dose dependency pattern of these interferences resembled the one reported for inhibition of anion transport by the same substances. Since the sterylsulfatase in vivo is strongly associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of the anionic steryl sulfates.