Selective Serotonin Reuptake Inhibitor Use During Pregnancy and Major Malformations: The Importance of Serotonin for Embryonic Development and the Effect of Serotonin Inhibition on the Occurrence of Malformations.

Bioelectric signaling is transduced by neurotransmitter pathways in many cell types. One of the key mediators of bioelectric control mechanisms is serotonin, and its transporter SERT, which is targeted by a broad class of blocker drugs (selective serotonin reuptake inhibitors [SSRIs]). Studies showing an increased risk of multiple malformations associated with gestational use of SSRI have been accumulating but debate remains on whether SSRI as a class has the potential to generate these malformations. This review highlights the importance of serotonin for embryonic development; the effect of serotonin inhibition during early pregnancy on the occurrence of multiple diverse malformations that have been shown to occur in human pregnancies; that the risks outweigh the benefits of SSRI use during gestation in populations of mild to moderately depressed pregnant women, which encompass the majority of pregnant depressed women; and that the malformations seen in human pregnancies constitute a pattern of malformations consistent with the known mechanisms of action of SSRIs. We present at least three mechanisms by which SSRI can affect development. These studies highlight the relevance of basic bioelectric and neurotransmitter mechanism for biomedicine.

Establishing the Embryonic Axes: Prime Time for Teratogenic Insults.

A long standing axiom in the field of teratology states that the teratogenic period, when most birth defects are produced, occurs during the third to eighth weeks of development post-fertilization. Any insults prior to this time are thought to result in a slowing of embryonic growth from which the conceptus recovers or death of the embryo followed by spontaneous abortion. However, new insights into embryonic development during the first two weeks, including formation of the anterior-posterior, dorsal-ventral, and left-right axes, suggests that signaling pathways regulating these processes are prime targets for genetic and toxic insults. Establishment of the left-right (laterality) axis is particularly sensitive to disruption at very early stages of development and these perturbations result in a wide variety of congenital malformations, especially heart defects. Thus, the time for teratogenic insults resulting in birth defects should be reset to include the first two weeks of development.

Is VACTERL a laterality defect?

To date the etiology of the association called VACTERL remains a mystery. Interestingly, clues as to the origin of this collection of defects may reside in an old hypothesis concerning the midline as a developmental field as postulated by Dr. John Opitz. This theory suggested that the midline was not a separate entity, but could be influenced by other developmental signals. With new information concerning the origin of the left-right axis (laterality) and the importance of communications between this axis and the cranio-caudal (anterior-posterior) axis for normal development, it has become clear that coordination of the molecular signals responsible for specification of these domains is essential for normal development. In fact, if the signals regulating laterality are disrupted, then midline and other defects can occur as has been observed in cases of heterotaxy, presumably because of a disruption in this coordinated signaling effort. Thus, the origins of the defects commonly observed in the VACTERL association may be due to altered signaling responsible for establishing the left-right axis.

Deciphering the genetic basis of microcystin tolerance.

BACKGROUND: Cyanobacteria constitute a serious threat to freshwater ecosystems by producing toxic secondary metabolites, e.g. microcystins. These microcystins have been shown to harm livestock, pets and humans and to affect ecosystem service and functioning. Cyanobacterial blooms are increasing worldwide in intensity and frequency due to eutrophication and global warming. However, Daphnia, the main grazer of planktonic algae and cyanobacteria, has been shown to be able to suppress bloom-forming cyanobacteria and to adapt to cyanobacteria that produce microcystins. Since Daphnia's genome was published only recently, it is now possible to elucidate the underlying molecular mechanisms of microcystin tolerance of Daphnia. RESULTS: Daphnia magna was fed with either a cyanobacterial strain that produces microcystins or its genetically engineered microcystin knock-out mutant. Thus, it was possible to distinguish between effects due to the ingestion of cyanobacteria and effects caused specifically by microcystins. By using RNAseq the differentially expressed genes between the different treatments were analyzed and affected KOG-categories were calculated. Here we show that the expression of transporter genes in Daphnia was regulated as a specific response to microcystins. Subsequent qPCR and dietary supplementation with pure microcystin confirmed that the regulation of transporter gene expression was correlated with the tolerance of several Daphnia clones. CONCLUSIONS: Here, we were able to identify new candidate genes that specifically respond to microcystins by separating cyanobacterial effects from microcystin effects. The involvement of these candidate genes in tolerance to microcystins was validated by correlating the difference in transporter gene expression with clonal tolerance. Thus, the prevention of microcystin uptake most probably constitutes a key mechanism in the development of tolerance and adaptation of Daphnia. With the availability of clear candidate genes, future investigations examining the process of local adaptation of Daphnia populations to microcystins are now possible.

Physiological interaction of Daphnia and Microcystis with regard to cyanobacterial secondary metabolites.

Cyanobacterial blooms in freshwater ecosystems are a matter of high concern with respect to human health and ecosystem services. Investigations on the role of cyanobacterial secondary metabolites have largely been confined to microcystins, although cyanobacteria produce a huge variety of toxic or inhibitory secondary metabolites. Mass occurrences of toxic cyanobacteria strongly impact freshwater zooplankton communities; especially the unselective filter feeder Daphnia. Daphnids have been shown to successfully suppress bloom formation. However, the opposite situation, i.e. the suppression of Daphnia populations by cyanobacteria can be observed as well. To understand these contradictory findings the elucidation of the underlying physiological mechanisms that help daphnids to cope with cyanotoxins is crucial. We fed Daphnia magna with the cyanobacterium Microcystis aeruginosa PCC7806 for 24h and used high-resolution LCMS analytics to analyze the Microcystis cells, the Daphnia tissue and the surrounding medium in order to investigate the fate of seven investigated cyanobacterial compounds (cyanopeptolins A-C, microcyclamide 7806A and aerucyclamides B-D). For none of these bioactive compounds evidence for biotransformation or biodegradation by Daphnia were found. Instead feeding and subsequent release experiments point at the importance of transport mechanisms in Daphnia with regard to the cyanopeptolins A and C and microcyclamide 7806A. In addition we found hints for new inducible defense mechanism in Microcystis against predation by Daphnia. These putative defense mechanisms include the elevated production of toxic compounds other than microcystins, as could be demonstrated here for aerucyclamide B and D, cyanopoeptolin B and microcyclamide 7806A. Moreover, our data demonstrate the elevated active export of at least one cyanobacterial compound (microcyclamide 7806A) into the surrounding medium as a response to grazer presence, which might constitute an entirely new not yet described cyanobacterial defense mechanism.

Dietary exposure of Daphnia to microcystins: no in vivo relevance of biotransformation.

Anthropogenic nutrient input into lakes has contributed to the increased frequency of toxic cyanobacterial blooms. Daphnia populations have been shown to be locally adapted to toxic cyanobacteria and are able to suppress bloom formation; little is known about the physiology behind this phenomenon. Microcystin-LR (MCLR) is the most widespread cyanobacterial toxin, and, based on in vitro experiments, it is assumed that the enzyme glutathione-S-transferase (GST) might act as the first step of detoxification in Daphnia by conjugating MCLR with glutathione. In the present study Daphnia magna was fed a diet of 100% Microcystis aeruginosa PCC7806, a cyanobacterial strain that contains MCLR in high amounts (4.8-5.6 fg cell(-1)), in order to test for a possible conjugation of MCLR with GST in Daphnia in vivo. We used high-resolution LCMS to analyze incubation water, cyanobacterial cells and Daphnia tissue for the presence of MCLR conjugation products as well as unconjugated MCLR. Newly formed conjugation products were detected neither in Daphnia tissue nor in the incubation water. Moreover, the presence of Daphnia led to a decrease in unconjugated MCLR in the cyanobacterial cell fraction due to grazing, in comparison to a control without daphnids, which was well reflected by a similar increase of MCLR in the respective incubation water. As a consequence, the MCLR content did not change due to Daphnia presence within the entire experimental setup. In summary, MCLR ingestion by Daphnia led neither to the formation of conjugation products, nor to a decrease of unconjugated MCLR. GST-mediated conjugation thus seems to be of minor relevance for microcystin (MC) tolerance in Daphnia in vivo. This finding is supported by the fact that GST activity in Daphnia feeding on the MC-containing wildtype or a MC-free mutant of M. aeruginosa PCC7806 revealed an identical increase of specific activity in comparison to a cyanobacteria-free diet. Therefore, the frequently observed induction of GST activity upon exposure to toxic cyanobacteria is not a specific MC effect but a general cyanobacterial effect. This suggests that GST in Daphnia is involved in an oxidative stress response rather than in the specific detoxification of MCs. Furthermore, our results indicate the presence of an efficient transport mechanism which efficiently removes unconjugated MCLR from the Daphnia tissue. Further studies are needed to elucidate the nature of this transport mechanism.

Effect of nutrient limitation of cyanobacteria on protease inhibitor production and fitness of Daphnia magna.

Herbivore-plant interactions have been well studied in both terrestrial and aquatic ecosystems as they are crucial for the trophic transfer of energy and matter. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is to a large extent represented by Daphnia and cyanobacteria. The occurrence of cyanobacterial blooms in lakes and ponds has, at least partly, been attributed to cyanotoxins, which negatively affect the major grazer of planktonic cyanobacteria, i.e. Daphnia. Among these cyanotoxins are the widespread protease inhibitors. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the gut of Daphnia, i.e. trypsins and chymotrypsins, and to reduce Daphnia growth. In this study we grew cultures of the cyanobacterium Microcystis sp. strain BM25 on nutrient-replete, N-depleted or P-depleted medium. We identified three different micropeptins to be the cause for the inhibitory activity of BM25 against chymotrypsins. The micropeptin content depended on nutrient availability: whereas N limitation led to a lower concentration of micropeptins per biomass, P limitation resulted in a higher production of these chymotrypsin inhibitors. The altered micropeptin content of BM25 was accompanied by changed effects on the fitness of Daphnia magna: a higher content of micropeptins led to lower IC50 values for D. magna gut proteases and vice versa. Following expectations, the lower micropeptin content in the N-depleted BM25 caused higher somatic growth of D. magna. Therefore, protease inhibitors can be regarded as a nutrient-dependent defence against grazers. Interestingly, although the P limitation of the cyanobacterium led to a higher micropeptin content, high growth of D. magna was observed when they were fed with P-depleted BM25. This might be due to reduced digestibility of P-depleted cells with putatively thick mucilaginous sheaths. These findings indicate that both the grazer and the cyanobacterium benefit from P reduction in terms of digestibility and growth inhibition, which is an interesting starting point for further studies.

Examining the evidence for vascular pathogenesis of selected birth defects.

Vascular mechanisms have been proposed to be involved in the pathogenesis of a number of defects, including transverse-limb defects, intestinal atresias, gastroschisis, hydranencephaly, porencephaly, oromandibular-limb hypogenesis sequence, and oculoauriculovertebral spectrum (OAVS). Here, we examine the available clinical, epidemiologic, and experimental evidence for four defects (transverse-limb defects, intestinal atresias, gastroschisis, and OAVS) for which vascular pathogenesis has been hypothesized. Based on our review, transverse-limb defects appear to sometimes be due to vascular events related to placental vascular abnormalities, hypoperfusion, abnormal development of blood vessels, intrauterine compression, hemoglobinopathies, or exposure to vasoactive agents, although epidemiological studies have not consistently demonstrated the latter association. However, transverse-limb defects can also be due to abnormal developmental events, such as aberrant molecular signaling in the apical ectodermal ridge. Some intestinal atresias may have a vascular origin, with the hypothesis supported by experiments in canines. However, evidence is accumulating that a more common mechanism might be related to improper molecular signaling related to gut specification early in development. In contrast, evidence to support vascular pathogenesis for gastroschisis and OAVS is less compelling. Instead, these defects probably arise from interference with basic developmental events [e.g., body wall closure (gastroschisis) and neural crest cell development (OAVS)]. These conclusions are important for counseling parents of children with these defects and for guiding the design of future epidemiological studies and experiments to further characterize the causes of these defects.

The embryologic origin of ventral body wall defects.

Ventral body wall defects include ectopia cordis, bladder exstrophy, and the abdominal wall malformations gastroschisis and omphalocele. The etiology of ectopia cordis, gastroschisis, and bladder exstrophy is not known, but they may be linked to abnormalities in the lateral body wall folds responsible for closing the thoracic, abdominal, and pelvic portions of the ventral body wall. These folds form in the fourth week (postfertilization) of development as a combination of the parietal layer of lateral plate mesoderm and overlying ectoderm and must move ventrally to meet in the midline. There are differential rates of cell proliferation in the folds and asymmetries in their movement that may be involved in teratogenic effects of toxic factors. Also, the fusion process between the folds is complex, involving cell-to-cell adhesion, cell migration, and cell reorganization and all of these phenomena may be targets for disruption, leading to malformations. In this regard, closure of the ventral body wall is likened to neural tube closure and involves similar processes. It also encompasses a similar time frame during development, such that most neural tube and ventral body wall defects have their origins during the fourth week of development. Omphalocele is a separate entity whose etiology is known. This defect is attributed to a failure of gut loops to return to the body cavity after their normal physiological herniation into the umbilical cord from the 6th to 10th week of development. Thus, the origin of this defect is completely different from that of the ventral body wall malformations.

Development of gastroschisis: review of hypotheses, a novel hypothesis, and implications for research.

Gastroschisis, a ventral body wall defect, is a continuing challenge and concern to researchers, clinicians, and epidemiologists seeking to identify its cause(s) and pathogenesis. Concern has been renewed in recent years because, unlike most other birth defects, rates of gastroschisis are reportedly increasing in many developed and developing countries. No tenable explanation or specific causes have been identified for this trend. Rates of gastroschisis are particularly high among pregnancies of very young women. Such an intriguing association, not observed to this degree with other birth defects, may afford clues to the defect's cause. Understanding the causes of gastroschisis may provide insight to the defect's origin. In pursuing such causal studies, it would be helpful to understand the embryogenesis of gastroschisis. To date, four main embryologic hypotheses have been proposed: (1) Failure of mesoderm to form in the body wall; (2) Rupture of the amnion around the umbilical ring with subsequent herniation of bowel; (3) Abnormal involution of the right umbilical vein leading to weakening of the body wall and gut herniation; and (4) Disruption of the right vitelline (yolk sac) artery with subsequent body wall damage and gut herniation. Although based on embryological phenomena, these hypotheses do not provide an adequate explanation for how gastroschisis would occur. Therefore, we propose an alternative hypothesis, based on well described embryonic events. Specifically, we propose that abnormal folding of the body wall results in a ventral body wall defect through which the gut herniates, leading to the clinical presentation of gastroschisis. This hypothesis potentially explains the origin of gastroschisis as well as that of other developmental defects of the ventral wall.

Fumonisins disrupt sphingolipid metabolism, folate transport, and neural tube development in embryo culture and in vivo: a potential risk factor for human neural tube defects among populations consuming fumonisin-contaminated maize.

Fumonisins are a family of toxic and carcinogenic mycotoxins produced by Fusarium verticillioides (formerly Fusarium moniliforme), a common fungal contaminant of maize. Fumonisins inhibit ceramide synthase, causing accumulation of bioactive intermediates of sphingolipid metabolism (sphinganine and other sphingoid bases and derivatives) as well as depletion of complex sphingolipids, which interferes with the function of some membrane proteins, including the folate-binding protein (human folate receptor alpha). Fumonisin causes neural tube and craniofacial defects in mouse embryos in culture. Many of these effects are prevented by supplemental folic acid. Recent studies in LMBc mice found that fumonisin exposure in utero increases the frequency of developmental defects and administration of folate or a complex sphingolipid is preventive. High incidences of neural tube defects (NTD) occur in some regions of the world where substantial consumption of fumonisins has been documented or plausibly suggested (Guatemala, South Africa, and China); furthermore, a recent study of NTD in border counties of Texas found a significant association between NTD and consumption of tortillas during the first trimester. Hence, we propose that fumonisins are potential risk factors for NTD, craniofacial anomalies, and other birth defects arising from neural crest cells because of their apparent interference with folate utilization.

Perturbations in choline metabolism cause neural tube defects in mouse embryos in vitro.

A role for choline during early stages of mammalian embryogenesis has not been established, although recent studies show that inhibitors of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), produce neural tube defects in mouse embryos grown in vitro. To determine potential mechanisms responsible for these abnormalities, choline metabolism in the presence or absence of these inhibitors was evaluated in cultured, neurulating mouse embryos by using chromatographic techniques. Results showed that 90%-95% of 14C-choline was incorporated into phosphocholine and phosphatidylcholine (PtdCho), which was metabolized to sphingomyelin. Choline was oxidized to betaine, and betaine homocysteine methyltransferase was expressed. Acetylcholine was synthesized in yolk sacs, but 70 kDa choline acetyltransferase was undetectable by immunoblot. DMAE reduced embryonic choline uptake and inhibited phosphocholine, PtdCho, phosphatidylethanolamine (PtdEtn), and sphingomyelin synthesis. ET-18-OCH3 also inhibited PtdCho synthesis. In embryos and yolk sacs incubated with 3H-ethanolamine, 95% of recovered label was PtdEtn, but PtdEtn was not converted to PtdCho, which suggested that phosphatidylethanolamine methyltransferase (PeMT) activity was absent. In ET-18-OCH3 treated yolk sacs, PtdEtn was increased, but PtdCho was still not generated through PeMT. Results suggest that endogenous PtdCho synthesis is important during neurulation and that perturbed choline metabolism contributes to neural tube defects produced by DMAE and ET-18-OCH3.