Embryotoxicity and teratogenicity study with alpha-cyclodextrin in rabbits.

In a standard embryotoxicity/teratogenicity study, alpha-cyclodextrin (alpha-CD) was administered to groups of sixteen, artificially inseminated New Zealand White rabbits at dietary concentrations of 0, 5, 10, or 20%. An additional group received a diet containing 20% lactose. Treatment started on day 0 of gestation and ended on day 29 when the animals were killed. Except for the occurrence of transient diarrhoea in one rabbit of the 20% alpha-CD group for a few days, the treatment was well tolerated. A reduced food intake in the 20% alpha-CD group during the first week of treatment resulted in a reduced weight gain from day 0 to 12 of the study. However, the difference to the controls was not significant and at termination of the study body weights were similar in all groups. Even at the highest dose level, which corresponds to an intake of 5.9-7.5 g/kg bw/day, no signs of maternal toxicity were observed. Maternal reproductive performance was not affected by the treatment. Uterine weight, placental weight, fetal weight, number of fetuses, sex ratio, number of implanation sites, resorptions, and corpora lutea did not differ among the groups. Visceral and skeletal examinations of the fetuses did not reveal any malformations, anomalies or variations that could be attributed to treatment. It was concluded that dietary alpha-CD is generally well tolerated by pregnant rabbits, has no adverse effect on maternal reproductive performance and is not embryotoxic, fetotoxic, or teratogenic at dietary concentrations of up to 20%, the highest dose level tested.

Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development.

Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. The formation of spermatozoa cisplatin-DNA adducts showed dose and time-dependent increases both in vitro, and in vivo up to 168 h (7 days) after dosing. Treatment of rats with 10 mg cisplatin/kg resulted in spermatozoa Pt-GG adduct levels of approximately 1.0 fmol/microg DNA. When cisplatin-treated male rats were bred to untreated females 6-24 h after cisplatin administration, no adverse developmental effects or decreases in body weight were seen in the offspring although there was a trend towards increased early embryo mortality.

Embryotoxicity and teratogenicity study with gamma-cyclodextrin in rabbits.

In a standard embryotoxicity/teratogenicity study, gamma-cyclodextrin (gamma-CD) was administered to groups of 16, artificially inseminated New Zealand White rabbits at dietary concentrations of 0, 5, 10, or 20%. A comparison group received a diet containing 20% lactose. Treatment started on day 0 of gestation and ended on day 29 when the animals were killed. Except for the occurrence of transient diarrhea in 2 and 3 rabbits of the 10 and 20% gamma-CD groups, respectively, in the first few days, the treatment was well tolerated. A reduced food intake in the 20% gamma-CD group during the first week of treatment resulted in a reduced weight gain during this period. However, after week 1 there were no differences in weight gains between the groups, and at termination of the study body weights were similar in all groups. Even at the highest dose level, which corresponds to an intake of 5-7 g/kg body wt/day, no signs of maternal toxicity were observed. Reproductive performance was not affected by the treatment. Uterine weight, placental weight, fetal weight, number of fetuses, sex ratio, number of implantation sites, resorptions, and corpora lutea did not differ among the groups. Visceral and skeletal examinations of the fetuses did not reveal any malformations, anomalies, or variations that could be attributed to treatment. It was concluded that dietary gamma-CD is well tolerated by pregnant rabbits, has no adverse effect on reproductive performance, and is not embryotoxic, fetotoxic, or teratogenic at dietary concentrations of up to 20%.

Embryotoxicity/teratogenicity study with isomaltulose (Palatinose) in rats.

The embryotoxicity/teratogenicity of the natural sweetener isomaltulose (Palatinose) was studied in Wistar rats. Groups of 24 mated females were fed diets containing isomaltulose at levels of 0, 2.5, 5 and 10% from day 0 to day 21 of pregnancy. The dams were killed on day 21 of pregnancy. No maternal toxicity occurred and no effects on reproductive performance, nor on embryonic or foetal development, including visceral and skeletal examination, were seen in any of the groups fed isomaltulose. The dietary level of 10% isomaltulose was equivalent to about 7 g/kg body weight/day.

Embryotoxicity and teratogenicity study with erythritol in rats.

The embryotoxicity/teratogenicity of erythritol, a low-calorie polyol sugar substitute, was examined in Wistar Crl:(WI) WU BR rats. Erythritol was fed at dietary concentrations of 0, 2.5, 5, and 10% to groups of 32 female rats from Day 0 to 21 of gestation. The treatment was generally well tolerated and no mortality occurred in any group. Weight gain during gestation, food consumption, and food efficiency were similar in all groups except for a significantly reduced weight gain in the 10% erythritol group in Week 2 of gestation. Reproductive performance was not affected by the treatment but the fertility index was generally rather low (69% in both control and high-dose group). Examination of the fetuses for external, visceral, and skeletal alterations did not reveal any fetotoxic, embryotoxic, or teratogenic effects. The slightly lower maternal body weight in the high-dose group was interpreted as a trivial result of the consumption of a low-calorie test substance in high amounts. In conclusion, no adverse effects were observed at erythritol doses of up to about 6.6 g/kg body wt/day, i.e., the highest dose tested.

Two-generation reproduction study of erythritol in rats.

Erythritol was fed at dietary concentrations of 0, 2.5, 5, or 10% to Crl:(WI) WU BR rats of both sexes through two successive generations (F0 and F1). Twenty-four rats of each sex were mated in each group. For each generation one litter was reared until the pups were 21 days old. In the 10% erythritol group, food consumption among F0-males and -females was initially significantly reduced until the animals adapted to the erythritol diet during the first week of the study. Thereafter, food intake was higher than in controls. A consistently increased food intake also was seen in F1-males and-females of this dose group. This effect was considered to result from the caloric dilution of the food by erythritol, which has a low physiological energy value. The lower body weight and weight gain of the F0-animals of the 10% erythritol group were attributed to the initially reduced food consumption and occurrence of transient diarrhea until the animals had adapted to the erythritol intake. In the F1-animals of the 10% erythritol group, which were adapted to the treatment from weaning, the rate of body weight gain did not differ from controls. The F1-males and -females of this dose group did, however, have a reduced body weight from weaning, which was attributed to a reduced energy intake among the corresponding F0-dams during Weeks 2 and 3 of lactation. This effect was not seen in the F2-generation. It is concluded that under the conditions of this experiment, the intake of erythritol had no adverse effect on fertility and reproductive performance of parent rats or on the development of their progeny. Gross necropsy and microscopic examination of the parenteral reproductive organs also did not reveal treatment-related changes.

Effects of polychlorinated biphenyls (PCBs) and dioxins on growth and development.

Polychlorinated biphenyls (PCBs) and dioxins are potentially toxic compounds which occur widely in the environment. Their effects on the growth and development of infants at the levels currently found in highly industrialised western countries is not well known. This Dutch multicenter study, combining animal and human studies, tries to answer this question. Animal studies showed that PCB 169, given once during pregnancy at a dose of 1.8 g kg-1 bodyweight, has an effect on developmental parameters, dopamine regulation and fertility. Effects on thyroid hormones were also found in animals, probably due to both a competitive binding of PCB metabolites to the thyroxine binding protein and increased glucuronidation. Perhaps to compensate for this, an increased diodase activity in the brain was found. Human studies involved 400 mother-infant pairs, half of them being breast-fed, the other half were fed a formula devoid of PCBs and dioxins. PCB levels were measured in serum and dioxin and PCB levels in breastmilk. Levels were found to be as high as previously found in highly industrialised countries. Growth and development were carefully documented, but no data are as yet available. In pregnant women, a significant negative correlation was found between some dioxin and PCB congeners in milk and plasma thyroid hormones, while newborn infants showed higher thyroid stimulating hormone (TSH) at higher levels of dioxin exposure. In summary, data from this combined multicenter study involving animals and humans increases our insight into the potentially negative effects of PCBs and dioxins on growth and development.

Interference of polychlorinated biphenyls in hepatic and brain thyroid hormone metabolism in fetal and neonatal rats.

The effects of prenatal oral administration of 0.2, 0.6, and 1.8 mg/kg body wt of 3,3',4,4'5,5'-hexachlorobiphenyl (HCB) on Day 1 of gestation and a combination of 1 mg/kg 3,3',4,4'-tetrachlorobiphenyl (TCB) from Day 2 to Day 18 with 0.6 mg HCB/kg body wt on Day 1 of gestation on thyroid hormone status and peripheral thyroid metabolism were studied in pregnant Wistar rats and their fetuses and offspring. Plasma total thyroxine and free thyroxine levels were reduced by HCB in a dose-dependent fashion in pregnant rats (Days 12 and 20 of gestation) and neonates (Day 21 postpartum), while only a combined dose of HCB and TCB was effective in decreasing fetal thyroid hormone levels by 65% on Day 20 of gestation. The activity of type II thyroxine 5'-deiodinase (5'D-II), the enzyme responsible for the deiodination of thyroxine (T4) to biologically active triiodothyronine in the brain, was examined in whole brain homogenates in fetuses and neonates. Decreases in plasma thyroid hormones were accompanied by significant increases, up to 100%, in 5'D-II activity in brain homogenates from fetuses (Day 20 of gestation) and neonates (Days 7 and 21 postpartum). The glucuronidation of 125I-T4 by hepatic microsomes was increased by at least 100% relative to control levels by all treatments in fetuses (Day 20 of gestation) and increased at least 40% in neonates (Days 7 and 21 postpartum) by a dose of 0.6 and 1.8 mg HCB/kg and the combined dose. These data indicate that prenatal HCB and/or TCB administration result in increased peripheral T4 metabolism. The increase in 5'D-II activity suggests that local hypothyroidism occurs in the brains of fetal and neonatal rats exposed to HCB and/or TCB. Since these effects occur during a period in which thyroid hormones play an important role in brain maturation, they may help explain the mechanism of developmental neurotoxicity induced by polychlorinated biphenyls.

Chronic toxicity and carcinogenicity study of isomalt in rats and mice.

The chronic toxicity and possible carcinogenicity of the sugar replacer isomalt was studied in Wistar rats and Swiss mice. Groups of 50 animals of each sex were fed 0, 2.5, 5 or 10% isomalt in the diet for nearly 2.5 yr (rats) or 2 yr (mice). Control groups received either basal diet with 10% maize starch or basal diet with 10% sucrose. Additional groups of ten rats/sex were fed the same diets and were killed after 1 yr. Isomalt and sucrose were included in the diet at the expense of maize starch. Administration of isomalt was started, in rats, in utero, and in mice, at weaning age. Feeding isomalt did not affect the appearance or behaviour of rats or mice, nor did it cause diarrhoea. Mortality rate was unaffected. Body weights of rats and mice fed 10% isomalt were generally slightly lower than those of controls. Periodic examinations of rats for haematological criteria, clinical chemistry of the blood, urine composition and kidney function did not reveal any changes of toxicological significance. Periodic haematological examinations of mice were likewise negative. Caecal enlargement was observed in rats and mice of the high-dose group, but the microscopic structure of the caecal wall was unaffected. An increased number of treated male and female rats showed hyperplasia of the urothelium in the renal pelvis accompanied by mineralization, whereas the number of females showing corticomedullary mineralization was decreased in the treated groups. The incidence, type or location of neoplasia provided no evidence of a carcinogenic potential of isomalt. Feeding 10% sucrose did not induce significant differences compared with the controls fed 10% maize starch, whereas isomalt at levels of up to 10% produced some of the changes that are common to rats fed high levels of poorly digestible carbohydrates.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker.

The formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectin-coated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

6.5-mm human embryo with a single nasal placode: cyclopia or hypotelorism?

A macroscopic study on the missing elements in cyclopia (a single eye or closely approximated eyes with all intergrades in a single orbit) with or without proboscis and hypotelorism was performed on 12 human fetuses and 2 human fetal skulls. In addition, microscopic investigations were carried out on the orbital contents of the cyclops with a single eye without proboscis, crown-heel length (C-HL) 37 cm, and on the 6.5-mm-crown-rump length (C-RL) human embryo with a single nasal placode localized in front of two eye cups. In the embryo and in all 14 fetal cases the midfacial region was more-or-less deficient. In the two cyclopia cases without proboscis the nasal placode(s) had not developed at all during the embryonic period. In cases with proboscis, consisting of a single tube localized above both eyes, and in the hypotelorismic specimens, there could only have been a single nasal placode during development: a situation evident in the 6.5-mm-C-RL human embryo. In this holoprosencephalic embryo the single nasal placode was undulated, as if formed from two fused nasal placodes, and flanked by the prospective areas for the lateral nasal processes. Caudally, it was bordered by the maxillary processes. In view of the position of the single placode in this embryonic face, as described above, it is most likely that this is a preliminary stage of hypotelorism. Moreover, both medial nasal processes with the internasal groove in between, i.e., the interplacodal area, were missing.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for early in utero experiments in rat embryos: endoscopy.

The merits of in vitro and in vivo techniques for experiments in rat embryos are discussed in this paper. Time limitation of culture, which is only feasible during 48 hours, up to day 13 post coitum (p.c.) is a major draw-back in the in vitro whole embryo culture. With the in utero operation technique used to date, no controlled experiments can be performed in rat embryos of 15 days p.c. and younger due to the high mortality of the embryos. Therefore a new technique has been developed, in which successful in utero operations can be performed as early as day 12 of gestation. Controlled micro-injection with the help of an endoscope can be given in any desired embryonic organ or structure. This paper describes this technique. Endoscopy in rat embryos of 12 days p.c. onwards has proven to be a new facility for in utero operations.

The formation of mesoderm and mesectoderm in presomite rat embryos cultured in vitro, using WGA-Au as a marker.

Presomite rat embryos cultured in vitro were injected with the cell marker wheat germ agglutinin-gold in order to find out whether the ectoderm already formed mesodermal cells. These labelled so-called mesectodermal cells were found in all embryos studied, ranging in age from 8.7 to 9.3 days post coitum. In embryos younger than 9.0 days, the entire head fold ectoderm produced mesectodermal cells. From 9.0 days onwards, the neural crest and surface ectoderm placodes were recognizable as separate entities, both producing mesectodermal cells. The early onset of mesectodermal cell formation and the numerous and continuous manufacture led us to the conclusion that mesectodermal cells are deposited at their definitive location and that subsequent long-distance migration is unnecessary.

Wheat germ agglutinin-gold as a novel marker for mesectoderm formation in mouse embryos cultured in vitro.

The routes of movement of mesectoderm cells in mammalian embryos have not yet been investigated experimentally due to technical problems. However, the recent development of in vitro culture methods have made an experimental approach to this problem in mouse and rat embryos possible. We have used combined lectin and colloidal-gold (WGA-Au) probe as a nontraumatic, easily detectable mesectoderm marker. The probe is introduced into the amniotic cavity by microinjection. All of the cells lining the cavity, including the mesectoderm precursors, phagocytose the colloidal gold, which is then stored in membrane-bound vesicles. The probe remains inside the target mesectoderm cells after their migration into the mesoderm compartment. Vesicles containing gold are detectable in both ultrathin and semithin sections. The applicability of WGA-HRP as a probe was also assessed because of the many properties it shares with WGA-Au, but it proved to be unsatisfactory for this purpose because it is transferred between cells and also to the extracellular spaces.

The cell surface coat in neurulating mouse and rat embryos, studied with lectins.

Carbohydrates in the surface coat of cells are thought to have a function in cell adhesion. The surface coat of cells, located in the fusion zone of the neural walls is investigated during neural tube closure in mammalian embryos. The presence of alpha-D-mannose, alpha-D-glucose and N-acetyl-D-glucosamine is quantified with the help of the lectins concanavalin A and wheat germ agglutinin in absence or after enzymic treatment. A two-step incubation is used, in which the second step consists of a protein-gold conjugate. A high incidence of these sugar residues was found in the fusion zone, indicating a relation to the specific capacity of these cells in establishing cell contacts.

Quantitative analysis of the temporal pattern of somite formation in the mouse and rat. A simple and accurate method for age determination.

The relation between the logarithm of the volume of the embryo and the number of somites was studied in the mouse and rat in the period of about 0-43 somites, a period characterized by a very rapid volume increase. A linear relation was found, and the variability of the individual observations around the straight line was rather small. Using a previously defined age estimate, i.e. the 'developmental age' based on the embryonic volume, we also found a linear relation between this age estimate and the number of somites. With the help of this straight line, the developmental age can be estimated from the number of somites with an accuracy of less than 0.1 day. Therefore, the developmental age can be considered a better age estimate than those based on the usual methods. The time needed to form a new somite was found to be constant in both species during the period studied. The length of the period differed, and was 1.68 h in the mouse and 2.24 h in the rat. However, the volume of the embryo at the time when a specific somite was formed (for instance the 25th) appeared to be the same in both species. Comparison of the volume of embryos, embedded in paraffin and Epon and sectioned, enabled us to estimate the shrinkage in Epon embryos. The volume after processing was approximately 62% of the original volume.

The neural crest in presomite to 40-somite murine embryos.

The production of cells by the neural crest is studied light-microscopically in 10 microns and 1 micron serially sectioned mouse and rat embryos, ranging in age from presomite to 40-somite stages. In the head region, mesectoderm formation starts in a pre-neural plate stage. It continues to the 20-somite stage. This implies that the contribution of the neural crest to the head mesoderm must be considerable. In the trunk, the neural crest only produces cells after adhesion of the neural walls. Mesectoderm formation continues for a long time, slowly retreating in a caudal direction. At the 40-somite stage, mesectoderm formation still occurs in the most caudal part of the trunk. Compared to the head, the contribution of the neural crest in the trunk seems to be less important than that of the primitive streak.

Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo.

The frequencies of cell degeneration and mitosis were investigated in the rupturing buccopharyngeal membrane (BPM) and in the persistent first branchial membrane (BM). In the BPM, cell degeneration starts many hours before rupture is visible, but mitotic figures are absent. In the BM this situation is reversed: mitotic figures are regularly observed, but a degenerating cell only occasionally. It is concluded that the ratio between the numbers of degenerating and dividing cells regulates the fate of both the BPM and the BM.

Hypertelorism and the median cleft face syndrome. An embryological analysis.

A microscopical study of the early and late development of the face was performed in 77 human embryos and fetuses. After the transformation of both nasal placodes, via nasal grooves, into the nasal tubes the ectoderm of the face is closed superficially and the early development of the face (less than or equal to 17 mm crown-rump length (C-RL)) is terminated. Between the nasal tubes the internasal groove is present. Furthermore these embryos show physiologically a flat nose and hypertelorism. During the late development (greater than or equal to 17 mm C-RL) of the face the internasal groove disappears due to the outgrowth and differentiation of the nasal septum in the frontocaudal direction. Simultaneously (17-27 mm C-RL) the distance between the eyes decreases relatively, because of a relative lag in transverse growth. The differentiation of the facial mesenchyme into bone centres starts in the same period. From this embryological point of view the major anomalies of the median cleft face syndrome (hypertelorism--orbital as well as interorbital--and cranium bifidum occultum, median cleft nose, median cleft prolabium and median cleft premaxilla), can be classified as secondary or late, i.e., differentiation, defects.