bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of in vivo glutathione depletion with buthionine sulfoximine on rat embryo development
Authors: Hales Barbara F. , Brown Heather
Published: June 2005

Glutathione is an abundant endogenous nucleophile whose depletion may have adverse effects on a number of cellular processes, including protein and DNA synthesis, amino acid transport, and detoxification of reactive electrophiles. Previous studies have indicated that a certain basal level of glutathione is essential for normal development in vitro in the whole rat embryo culture system. The objective of this study was to determine the effect of glutathione depletion with buthionine sulfoximine on the development of rat embryos in vivo. Timed pregnant Sprague‐Dawley rats were treated by gavage on days 10 and 11 of gestation with saline (control) or with L‐buthionine‐(S,R)‐sulfoximine (4 or 8 mmol/kg). Pregnancy outcome was assessed on day 12 or day 20 of gestation. The glutathione content of embryos and of maternal organs was measured on day 12. Glutathione concentrations were significantly decreased in the embryos and in maternal muscle and ovary but not in maternal liver and kidney. Glutathione depletion, when assessed on day 12 of gestation, was found to result in an increase in the numbers of dead and malformed embryos. Treatment with the higher dose of buthionine sulfoximine resulted in the death of 13.2% of the total implanted embryos; of the surviving embryos, 21.7% were malformed. There was also a significant decrease on day 12 after treatment with the higher dose of buthionine sulfoximine in the embryonic total morphological score, a measure of the developmental stage of the embryos (control, 57.7±1.0; 8 mmol/kg buthionine sulfoximine, 52.8±1.8). No significant effect of treatment with either dose of buthionine sulfoximine with respect to growth parameters was observed on day 12 of gestation. Interestingly, no significant effect of treatment with either dose of buthionine sulfoximine on numbers of pups, resorptions, malformations, or fetal weight was found in the litters examined on day 20 of gestation. Thus, after glutathione depletion with buthionine sulfoximine, an increase in malformations and developmental delay is found on day 12 of gestation; however the malformations observed in day 12 embryos apparently do not persist during later development.
Review of drug‐induced limb defects in mammals
Authors: Sanders Duane D. , Stephens Trent D.
Published: June 2005

The objective of this paper was to illustrate the spectrum of possible limb malformations in mammals resulting from drug exposure. A bibliography of 171 papers from 20 journals was generated from which pertinent data (drug used, limb defects reported, predominant defect location) were tabulated. These data should provide a basis for predictions about types of defects that might be expected in further studies and for judging postulated drug‐induced human limb defects. However, direct extrapolation to humans is inappropriate. The following trends were observed: 1) Distal limb defects (autopod) are almost twice as common as proximal limb defects (stylopod and zygopod). 2) Ectrodactyly is the single most common type of limb defect, accounting for over half of the autopod defects. 3) Ectrodactyly is almost twice as common in the hindlimb as in the forelimb. 4) Postaxial ectrodactyly is over twice as common as preaxial ectrodactyly in the forelimb, but preaxial ectrodactyly is four times more common in the hindlimbs. 5) Polydactyly occurs with approximately equal frequency in forelimbs and hindlimbs, and preaxial polydactyly is most common in both fore and hindlimbs. 6) Polymelia (supernumerary limbs) occurred in one case, and may have been a spurious result. 7) Either transverse hemimelia is greatly underreported in teratology studies or it essentially does not occur. We have concluded that, at least in some cases, acetazolamide, adenine, 1,7‐dimethylxanthine, and xanthine derivative aminophylline, retinoic acid, acetoxy‐methyl‐methylnitrosamine, aspirin, and cadmium can all cause unilateral defects.
Critical period of rat development when sidedness of asymmetric body structures is determined
Authors: Fujinaga Masahiko , Baden Jeffrey M.
Published: June 2005

We recently reported that rat embryos cultured from the presomite stage in a medium containing the alpha‐1 adrenergic agonist, phenyl‐ephrine, have a high incidence of situs inversus. In the present study, we have determined more precisely the critical period of development when situs inversus is induced. Rat embryos were harvested at 8 AM on Day 9 of gestation (plug day = Day 0), and divided into different stages of development, namely, early, mid, and late primitive streak stages and early, mid, and late neural plate stages. They then were cultured in rotating bottles to which phenylephrine, 0.5 mM, was added for various durations. After 49 hr of culture, embryos were examined for general morphology including sidedness of the bulboventricular loop, tail, and chorioallantoic placenta. Phenylephrine increased the incidence of situs inversus above control when administered throughout culture from either the early neural plate stage or before, and when administered for 4 hr or more from the early neural plate stage. This increase was significant even at the mid and late primitive streak stages when the control incidence was high. Our results suggest that sidedness of asymmetric body structures is determined during the early neural plate stage. This period is well before the 6–8‐somite stage when morphological signs of body asymmetry first appear.
Heterogeneity of neural tube defects in europe: The significance of site of defect and presence of other major anomalies in relation to geographic differences in prevalence
Authors: Dolk H. , de Wals P. , Gillerot Y. , Lechat M. F. , Ayme S. , Cornel M. , Cuschieri A. , Garne E. , Goujard J. , Laurence K. M. , Lillis D. , Lys F. , Nevin N. , Owens J. , Radic A. , Stoll C. , Stone D. , Kate L. Ten
Published: June 2005

In the period 1980–1987, neural tube defects were two to three times more prevalent in populations covered by EUROCAT registries in the United Kingdom and Ireland (UKI) than in Continental Europe and Malta (CEM). 1864 NTD cases in a total population of 580,000 births in UKI and 455 cases in a population of 380,000 births in CEM were analysed to find if there were differences in the ratio of prevalence rates between UKI and CEM according to site of the defect and association with non‐central nervous system (CNS) anomalies. The prevalence rate ratio was high for anencephaly with accompanying spina bifida, iniencephaly, and upper spina bifida, and low for encephalocele, lower spina bifida, and anencephaly without other neural tube defects. There was a greater female excess for anencephaly with accompanying spina bifida, iniencephaly, and upper spina bifida than for other defects in both geographic areas. There was a female excess for encephalocele in UKI but a male excess in CEM. Certain sites (anencephaly with accompanying spina bifida, iniencephaly, and encephalocele) were more likely to have accompanying non‐CNS anomalies. The prevalence rate ratio of multiply malformed NTD was in general lower than for isolated NTD but showed the same pattern by site. The prevalence rate ratio was high for multiply malformed anencephaly with accompanying spina bifida, iniencephaly, and upper spina bifida. The sex ratio was similar between isolated and multiply malformed cases when site of the defect is taken into account. It is concluded that the geographic prevalence pattern and sex ratio differ according to site of NTD but do not differ substantially according to whether NTD is isolated or associated with non‐CNS anomalies.
Toxicity of sera from individuals with Chagas' disease to cultured rat embryos: Role of antibodies to laminin
Authors: Robbins Bertha , Klein Norman W. , Cavalcanti Hamilton
Published: June 2005

In previous studies antilaminin antibodies in the sera of immunized monkeys and rats were found to be toxic to cultured rat embryos. In order to extend these studies to humans, head‐fold stage rat embryos were cultured for 48 hours on ten different serum samples from individuals with Chagas' disease. All embryos (n = 20) cultured on these sera were found to be abnormal. Using ELISA, Western immunoblot, and indirect immunofluorescence it could be shown that antibodies in these sera reacted with laminin. That these antilaminin antibodies were, at least in part, responsible for the toxicity was indicated 1) by reduced cultured embryo toxicity for six of seven serum samples after pre‐absorption with purified laminin, 2) by demonostrating a relationship between the amount of affinity‐purified antilaminin IgG added to control serum for culture and the severity of embryo abnormalities seen at the end of culture, and 3) by the sera's failing to react with other basement membrane proteins, including type IV collagen, fibronectin, osteonectin, and heparan sulfate proteoglycan.
Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre‐spina bifida splotch mouse embryos
Authors: Trasler Daphne G. , Morriss‐Kay Gillian
Published: June 2005

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch‐delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre‐spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigation and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre‐spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/+) or + / +. Immunohistochemical localization of CSPG by means of the CS‐56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5‐, 10‐, and 15‐somite stages, whereas similar staining was observed at 16‐ and 19‐somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14‐, 15‐, 16‐, and 19‐somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal ( + / + ) embryos in CS‐56 stain intensity. Immunohistochemical staining with antibody to a basement membrane HSPG showed a more intense stain in the two Spd/Spd 10‐somite stage embryos tested than in matched +/+ embryos. It is suggested that the greater intensity of CSPG and HSPG staining in Sp and Spd mutant embryos compared to +/+ embryos could be caused by an altered sequence within the Sp gene locus that either directly disrupts the production or degradation of these and other molecules, or does so indirectly through its interaction with neural cell adhesion molecules.
Cadherin mRNAs during rat embryo development in vivo and in vitro
Authors: Chen Beiyun , Blaschuk Orest W. , Hales Barbara F.
Published: June 2005

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady‐state mRNA concentrations of a family of Ca2+ ‐dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E‐cadherin, N‐cadherin, and P‐cadherin. Although E‐cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E‐cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5‐kb E‐cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8‐fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N‐cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N‐cadherin mRNA was detected only after 45 hr in culture. Third, P‐cadherin mRNA was detected as a single 3.5‐kb species, mainly in embryos. Pcadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6‐fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E‐cadherin and N‐cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady‐state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.
Effects of isotretinoin (13‐Cis‐retinoic acid) on the development of mouse limbs in vivo and in vitro
Authors: Kwasigroch Thomas E. , Bullen Michael
Published: June 2005

Isotretinoin (13‐cis‐RA) is known to be teratogenic in humans and laboratory animals. The relatively low potency of 13‐cis‐RA in NRMI mice in comparison to the all‐trans isomer has been proposed to be due to minimal transfer across the placenta (Creech‐Kraft et al., '87). To further delineate the teratogenic potential of 13‐cis‐RA, a dose‐response, temporal study was conducted in vivo and in vitro using submerged limb culture and image analysis evaluation of development. Dose‐dependent embryotoxicity was produced by treatment on GD 7, while later treatments produced inconsistent effects on resorption rate and fetal weight. Treatment on either GD 7 or GD 8 produced a number of malformations in dose‐dependent manner. Most common were tail and cleft palate defects, which were produced by 13‐cis‐RA on each of the days tested (GD 7–GD 11), with peak malformations occurring on GD 9 and GD 10 for tail and cleft palate, respectively. Most limb defects were produced after GD 10 and GD 11 exposure. The observed frequency of defects confirmed that in ICR mice 13‐cis‐RA is about 10‐fold less potent than all‐trans‐RA as a limb teratogen (Kwasigroch and Kochhar, '80; Kochhar and Penner, '87). Effects observed via image analysis following maintenance of limbs in serum‐free culture medium were dose dependent. Low dose treatment produced occasional polydactyly. The intermediate dose caused somewhat variable region‐dependent increases in cartilaginous bone anlagen area. The high dose of 13‐cis‐RA produced irregular limb outlines, a reduction in bone anlagen area, and an inhibition of alcian blue staining of cartilage without affecting morphogenesis of bone anlagen. These results confirm that, when the effects of the administered doses are evaluated, 13‐cis‐RA is a much less potent teratogen in comparison to the all‐trans isomer. More importantly, the results show that retinoids can enhance (at low and intermediate doses), depress (at high doses), or eliminate (high dose) chondro‐genenic expression during limb morphogenesis in vitro. This indicates that retinoids such as 13‐cis‐RA can manipulate events in development in a variety of ways (i. e., produce malformations, interfere with chondrogenic expression without affecting morphogenesis, and stimulate growth) in a dose‐ and time‐dependent manner. Although the ability of RA to act as a true morphogen has recently been questioned (Wanek et al., '91; Noji et al., '91), the results presented here support the position that RA can modulate the development of the limb (and probably other organ systems) in several vertebrate species.
Parental fertility and infant hypospadias: An international case‐control study
Authors: Källén Bengt , Castilla Eduardo E. , Kringelbach Mette , Lancaster Paul A. L. , Martínez‐Frías Maria Luisa , Mastroiacovo Pierpaolo , Mutchinick Osvaldo , Robert Elisabeth
Published: June 2005

The authors have performed an international case‐control study on the significance of exogenous hormones for the origin of hypospadias (Källén et al., 1991a, b). Using data from this study on 846 infants with isolated hypospadias and equally many controls (next male infant born in the same hospital as the case), variables that might indicate an increased rate of fertility problems in couples who had a boy with hypospadias were studied. Cases had slightly fewer previous pregnancies, there was practically no difference in the rate of previous induced abortions, and no demonstrable difference in menstrual history. Infertility periods of at least 6 months were more likely among cases than controls but the difference did not reach statistical significance in a two‐tailed test. There was no demonstrable difference in the time to conceive between cases and controls. The difficulties of directly studying subfertility problems are stressed.
Teratogenicity of all‐trans retinoic acid during early embryonic development in the cynomolgus monkey (Macaca fascicularis)
Authors: Hendrickx Andrew G. , Hummler Hans
Published: June 2005

The embryotoxic and teratogenic potential of all‐trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all‐trans retinoic acid (Tretinoin, Hoffmann‐La Roche) once daily from GD 10–20 and twice daily from GD 21–24 at three different dosages, 5 (n=9), 10 (n=6) and 20 mg/kg (n=1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all‐trans retinoic acid. Maternal weight loss was observed in the 10‐ and 20‐mg/kg groups. A dose‐dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth‐retarded fetus, was observed in the 5‐mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10‐mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13‐cis‐retinoic acid during the pre‐ and early organogenic stages of development (Hummler et al., Teratology 42:263‐272, 1990), no thymic hypo‐ or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13‐cis retinoic acid treatment period, i.e., GD 10–27. However, external ear and temporal bone defects were common to both all‐trans and 13‐cis retinoic acid. The similarity observed in the malformation syndrome induced by both all‐trans and 13‐cis retinoic acid in the cynomolgus monkey and 13‐cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A‐related compounds.
Aberrant convergence of the neural folds in the mouse mutant vl
Authors: Wilson Doris B. , Wyatt Darlene P.
Published: June 2005

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuratio of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22‐28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30–35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.
The use of dysmorphology in birth defects epidemiology
Authors: Friedman J. M.
Published: June 2005

Most human teratogens have been identified by the clinical recognition of characteristic patterns of congenital anomalies among children whose mothers were exposed to a particular agent during pregnancy. Although this dysmorphologic method has been valuable, it is criticized because it is not easily amenable to statistical evaluation. Conventional birth defects epidemiological studies are designed to permit rigorous statistical assessment, but such investigations usually classify congenital anomalies without adequate consideration of their known etiological heterogeneity. It is possible to combine the best aspects of these two approaches to identifying human teratogens in a “dysmorphologic case/control study”. Instead of including all available cases with a given defect, only individuals having the anomaly in the context of a multiple congenital anomaly pattern without a recognizable cause would be selected for inclusion among the case group. The frequency of exposure to the putative teratogen would be determined among these selected cases and among appropriately chosen controls; conventional statistical analysis would then be performed. Although this design reduces the size of the case group compared to a conventional case/control study, the statistical power is unchanged or increased. In addition, biological plausibility is increased by concentrating upon a group of cases that is more likely to have a teratogenic cause.
The role of the visceral yolk sac in hyperglycemia‐induced embryopathies in mouse embryos in vitro
Authors: Hunter E. S. , Sadler T. W.
Published: June 2005

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia‐induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4‐6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S‐methionine and 35S‐cysteine‐labeled hemoglobin (35S‐Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16‐18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S‐labeled products was observed at shorter exposure periods (12‐24 and 24‐36 h). In early somite stage embryos hyperglycemia (⩾ 33 mM) reduced both VYS uptake and embryonic accumulation of 35S‐Hb during the last 12 h of a 48 h exposure period. However, there was no concentration dependent effect on the embryonic accumulation of 35S at the early somite stage. The time course for hyperglycemia‐induced effects on morphology and VYS histiotrophic function are different in the early head fold versus early somite staged embryos. Thus, at the early head fold stage malformations were induced only after a 6‐12 h exposure, while the histiotrophic function was reduced only during the 32‐44 h of exposure. Therefore, our study indicates that hyperglycemia can alter the histiotrophic function of the VYS following long exposure periods. The time course for alterations in VYS histiotrophic function is not correlated with that for induction of malformations but may be related to the growth reduction produced by long‐term exposure to hyperglycemia in vitro.
Maternal methionine supplementation promotes the remediation of axial defects in Axd mouse neural tube mutants
Authors: Essien Francine B.
Published: June 2005

The Axd (axial defects) mouse model system (Essien et al., Teratology 42:183‐194, '90) is characterized by a dominant mutation which causes posterior open neural tube defects (NTD) and a variety of tail anomalies (curly tails, or CT). Repeated backcrosses to BALB/cByJ mice have resulted in a 50% increase in Axd penetrance among neonates of heterozygous matings and loss of a correlation with maternal tail phenotype. Analysis of D12‐D18 embryos from Axd/ + × Axd/ + matings indicates that soft tissues can superficially heal over some lesions from open NTD and that some curly tails can straighten (macroscopically) as gestation proceeds. Similarly, in embryos of Axd/ + × BALB crosses, there is remediation of approximately 33% of the tail flexion defects by birth. Numerous studies show that maternal nutritional status can affect the development of the neural tube and related axial structures. One nutrient of special interest is methionine, which is required for neurulation in cultured rat embryos (Coelho et al., J. Nutr. 119:1716, '89). Thus, the major question addressed by this study was whether supplemental methionine administered to Axd/ + dams crossed to Axd/ + males would alter the prenatal expression of the gene. When given IP (70 mg/kg) on D8 and D9, methionine resulted in a 41% reduction (from 29% to 17%) in the incidence of NTD in D 14 embryos (P <0.01).
Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass‐specific cell death
Authors: Kelly Sara M. , Robaire Bernard , Hales Barbara F.
Published: June 2005

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four‐week treatment of male rats with a low dose of cyclophosphamide cause a dramatic, dose‐dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague‐Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark‐stained pyknotic nuclei. Very few or no inner cell mass‐derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass‐derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide‐treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.
Axial skeletal malformations induced by acetazolamide in rabbits
Authors: Nakatsuka Toshio , Komatsu Tetsuro , Fujii Takaaki
Published: June 2005

In order to evaluate the teratogenic potential of acetazolamide in rabbits, three groups of 18 artificially inseminated females were treated orally with 50, 100, or 150 mg/kg/day of acetazolamide on days 6‐18 of gestation. These doses induced maternal acidosis and electrolyte changes, consistent with those reported in rats and considered to be a result of carbonic anhydrase inhibition, as well as reductions in maternal body weight gain. At cesarean sections, average fetal body weights in the acetazolamide groups were dosedependently decreased compared with controls. There were no effects of acetazolamide on embryonic survival or external morphology of live fetuses. In the fetal skeletal examination, thoracic and lumbar vertebral malformations occurred in 0.7%, 3.9%, and 6.1% of fetuses in the 50, 100, and 150 mg/kg/day groups, respectively, compared with none in the control group. In addition, missing vertebra was seen in a small number of fetuses in the 100 and 150 mg/kg/day groups. These axial skeletal malformations were, in some cases, associated with costal malformations. These results indicate that acetazolamide at maternotoxic doses can produce axial skeletal malformations in the rabbit. © 1992 Wiley‐Liss, Inc.
Analysis of high dysmorphogenic activity of Ro 13‐6307, a tetramethylated tetralin analog of retinoic acid
Authors: Kochhar D. M. , Penner John D.
Published: June 2005

Certain synthetic retinoids differ widely from retinoic acid (RA) in teratogenic potency, being much more or much less effective than RA. It is assumed that the potency of a retinoid may depend on the nature of its interaction with cellular binding components (nuclear retinoic acid receptors or cytoplasmic binding proteins) and, as in the case of retinoids that are mammalian teratogens, on factors that determine its accessibility to the embryo. To investigate some of the factors that contribute to potency, we used a new synthetic retinoid Ro 13‐6307 that differs in structure from RA in having an aromatic ring inserted in its side chain along with gem dimethyl modification of the natural cyclohexenyl ring. Pregnant ICR mice were given a single oral dose (0, 1, or 10 mg/kg) on day 11 of gestation, and the resultant teratogenic outcome was monitored on day 17. Direct effects on cell differentiation were obtained by exposing high density cultures of limb bud mesenchymal cells to a range of concentrations (0.3 ng/ml‐3 μg/ml) of Ro 13‐6307 and scoring for chondrogenic suppression. Concentrations reaching the embryo after maternal administration of Ro 13‐6307 were measured by HPLC to quantify the analog for a period of 4 h after administration of the oral dose. We found that this retinoid was 40‐fold as active as RA in both inducing teratogenesis and suppressing chondrogenesis, yet its concentration in the affected embryo was only a fraction of that achieved after an equivalent dose of RA was employed in a similar protocol. Since the morphogenetic activity of Ro 13‐6307 is disproportionately in excess of its levels in the mouse embryo, obligatory mediation by the receptors or by other binding proteins, or both, is likely involved. © 1992 Wiley‐Liss, Inc.
Trends in rates of multiple vascular disruption defects, Atlanta, 1968–1989: Is there evidence of a cocaine teratogenic epidemic?
Authors: Martin M. Louise , Khoury Muin J. , Cordero José F. , Waters Grady D.
Published: June 2005

Research suggests that, perhaps through mechanisms initiated by vasoconstriction and leading to vessel thrombosis or embolism, cocaine causes vascular disruption defects, and that frequent cocaine use during early pregnancy could disrupt multiple organ systems in the fetus. We hypothesized that if cocaine is an important cause of multiple vascular disruption defects, a rising prevalence of cocaine use by mothers during pregnancy should be accompanied by rising rates of these defects in their offspring. Using data from the Metropolitan Atlanta Congenital Defects Program, we identified all infants born in Atlanta from 1968 through 1989 who had nonsyndromic, provisional vascular disruption defects affecting more than one organ system: 61 infants (78%) had gastrointestinal and genitourinary defects, 7 (9%) had gastrointestinal and abdominal wall defects, 2 (3%) had gastrointestinal and limb reduction defects, 2 (3%) had limb reduction and abdominal wall defects, 2 (3%) had central nervous system and gastrointestinal defects, 2 (3%) had genitourinary and limb reduction defects, 1 (1%) had genitourinary and abdominal wall defects, and 1 (1%) had central nervous system and genitourinary defects. The prevalence of Atlanta infants with more than one vascular disruption defect is 0.13 per 1,000 live births. Chi‐square analysis for trends showed no increase in prevalence during the study period. Our data are from one of the first population‐based studies in which trends for defects potentially caused by maternal cocaine use are examined; the results of our study show no significant change in the prevalence of multiple vascular disruption defects over time. We suspect that if cocaine is a teratogen, its teratogenicity is weak or is associated with a small subset of birth defects that are yet to be identified. © 1992 Wiley‐Liss, Inc.
Growth and differentiation schedule of mouse embryos obtained from delayed matings
Authors: Ishikawa Hitoshi , Omoe Katsuhiko , Endo Akira
Published: June 2005

We previously showed that digit formation in mouse embryos from early morning mating seemed to progress faster than those from overnight mating. In this study, to confirm this phenomenon, we examine whether the embryos from normal (0 hr from ovulation to fertilization) and delayed matings (3, 6, or 9 hr from ovulation to fertilization) respond differently to some acute teratogens when they are treated at the same time point from mating.

Five mg/kg of cytosine arabinoside (Ara‐C) was given to pregnant mice intraperitoneally at 246, 249, or 252 hr (day of gestation (dg) 10) after mating. The patterns of Ara‐C induced digit malformations in embryos from the delayed mating groups were those of more advanced stages, when compared with normal mating groups with the same time intervals from mating to Ara‐C treatment. In other words, oocytes fertilized up to 9 hr after the presumed time of ovulation could grow similarly to those of normally fertilized oocytes. This catch‐up phenomenon suggests that the ovulation clock should be used for the startpoint of the time scale of the growth and differentiation of embryos rather than fertilization clock. © 1992 Wiley‐Liss, Inc.
Further genetic studies of the cause of exencephaly in SELH mice
Authors: Gunn T. M. , Juriloff D. M. , Harris M. J.
Published: June 2005

We have developed an inbred stock of mice called SELH that has a high frequency of the neural tube defect exencephaly at birth. A previous genetic study indicated that the exencephaly is due to two to three additive loci differing between SELH and a closely related normal strain, ICR/Bc, but this analysis was not designed to detect genetic maternal effects. Recently, we demonstrated that there is genetic polymorphism among normal mouse strains leading to differences in site of initiation of closure of the cranial neural tube. In the present study, an inbred substrain of SELH mice, with 24% exencephaly among embryos, was crossed with an unrelated normal strain, SWV/Bc, and the frequency of exencephaly in subsequent generations used to extend our understanding of the genetic cause of exencephaly in SELH mice.

The purposes of the genetic studies reported here were twofold. First, based on the influence of genetic maternal effects on other genetically complex birth defects in mice, we hypothesized that the exencephaly of SELH mice would exhibit strong genetic maternal effects. This hypothesis was tested by comparisons among the four possible reciprocal backcrosses to SELH. The result was an overall frequency of 2.3% exencephaly in first backcross embryos with no difference among the four crosses and no evidence of genetic maternal effects. Second, the frequency of exencephaly recovered in the backcross and F1 embryos was compared with the previous genetic study and with various genetic models. The frequencies were similar to those obtained from the cross to ICR/Bc mice and were compatible with a hypothesis of additive gene action at a few loci. The similarity of results between a wide outcross to SWV/Bc and a narrow outcross to ICR/Bc suggests that the exencephaly‐causing genes in SELH are mutations, not polymorphisms. © 1992 Wiley‐Liss, Inc.
Effects of a 48 hour continous intravenous infusion of CGS 13080‐primagrel, a selective thromboxane synthetase inhibitor, on the perinatal and early postnatal period in the guinea pig
Authors: Keith J. , Rowles T. , Warwick K. , Yau E.
Published: June 2005

CGS 13080, imidazo[1,5‐a]pyridine‐5‐hexanoic acid, was evaluated for perinatal and postnatal effects in third trimester pregnant guinea pigs and their offspring. The compound was administered via 48 hour continous intravenous infusion to a group of pregnant guinea pigs (n = 16) at a dose of 3 mg/kg/hr starting on gestational day 52 (via chronically implanted indwelling jugular venous cannulas). A saline control group (n = 12) received equivalent volumes of normal saline 0.5 ml/kg/hr throughout the dosing period. A third group (surgery‐sham, n = 16) was subjected to cannulation but not infused. A gross examination of each dam and piglets was conducted at necropsy on day 5 of lactation. The neonatal brains and all gross lesions (maternal and neonatal) were removed and fixed for histopathological examination. Compound‐related clinical signs were noted in dams during the dosing phase of gestation. Six guinea pigs developed cephalic lymphatic swelling during the infusion. This observation may be correlated to the reported redistribution of fluid volume to the thorax of guinea pigs given intravenous injections of CGS 13080. There were no compound‐induced effects on labor, delivery, or any of the examined reproductive parameters. There were no compound‐related clinical signs, or effects on survival, body weight and developmental parameters in the F1 generation. Histopathological examination of the brains and other organs did not reveal any compound‐related abnormalities. Based on these results, it was concluded that CGS 13080 did not elicit adverse perinatal and postnatal effects in guinea pigs. © 1992 Wiley‐Liss, Inc.
A case of agnathia, situs inversus, and a normal central nervous system
Authors: Stoler Joan M. , Holmes Lewis B.
Published: June 2005

We reprot here a premature female infant with agnathia, lowset but normally formed ears, a downward eye slant, choanal atresia and a cleft palate. She had severe respiratory distress and died despite maximum intervantion at 5 days of age. Autopsy revealed situs inversus totalis; crossed fused renal ectopia; agnathia; normal thyroid, larynx, trachea, and bronchi; incomplete lobation of the lungs; immature pulmonary development with early hyaline membranes; and a normal central nervous system. This lack of significant central nervous system abnormalities distinguishes this infant from the majority of previously reported infants with agnathia and situs inversus. © 1992 Wiley‐Liss, Inc.
Ischemia/reperfusion: A new hypothesis for the developmental toxicity of cocaine
Authors: Fantel Alan G. , Barber Charles V. , Mackler Bruce
Published: June 2005

It has been shown that multiple exposures of gravid rats to cocaine during late gestation result in significant incidences of severe malformations. Hind limb reduction defects were frequent findings in this study. Other studies have shown that comparable abnormalities can be induced in experimental animals by various procedures including vascular clamping, direct fetal exposure to epinephrine, uterine handling following laparotomy, as well as by exposure to hyperbaric oxygen. This paper reviews these and other studies, and presents a novel mechanistic hypothesis that explains their common findings. It is proposed that in each instance, conceptual hypoxia results from hypoperfusion caused by transient vasoconstriction. Following the resumption of normal perfusion, reactive oxygen species are generated by the ischemia/reperfusion mechanisms thought to underlie many pathobiologic lesions. It is proposed that the conceptus is particularly vulnerable to the toxicity of oxygen radicals because of its low antioxidant activities and the highly reduced state of its undifferentiated cells. Sensitivity to cocaine and uterine handling appears to be enhanced during late gestation and it is hypothesized that this results from changes in oxygenation and iron content that increase both the substrate and catalyst for generation of reactive oxygen species. © 1992 Wiley‐Liss, Inc.
Accumulation of heat shock protein 72 (hsp 72) in postimplantation rat embryos after exposure to various periods of hyperthermia (40°–43°c) in vitro: Evidence that heat shock protein 72 is a biomarker of heat‐induced embryotoxicity
Authors: Mirkes Philip E. , Doggett Barbara
Published: June 2005

A monoclonal antibody to the 72 kDa heat shock protein and Western blot analysis blot analysis were used to determine the induction, accumulation and turnover of hsp 72 after day 10 rat embryos were exposed to elevated temperatures (40°–43°C) for various lengths of time (2.5 minutes to 18 hours). Embryos exposed to temperatures that exceed the normal culture temperature (37°C) by 4°C or more for as little as 2.5 minutes (43°C) or 15 minutes (41, 42°C) synthesized and accumulated detectable amounts of heat‐inducible hsp 72. Hsp 72 could not be detected by Western blot analysis of proteins from embryos cultured at 40°C or below. Once induced, hsp 72 can be detected in embryos for 24–48 hours after they are removed from the hyperthermic conditions and returned to normothermic conditions. Our results also indicate that hsp 72 is induced by all hyperthermic exposures that induce alterations in rat embryo growth and development; therefore, hsp 72 is a potential biomarker for heat‐induced embryotoxicity. © 1992 Wiley‐Liss, Inc.
Embryotoxicity studies of norfloxacin in cynomolgus monkeys. II. Role of progesterone
Authors: Cukierski M. A. , Hendrickx A. G. , Prahalada S. , Tarantal A. F. , Hess D. L. , Lasley B. L. , Peter C. P. , Tarara R. , Robertson R. T.
Published: June 2005

Norfloxacin, an orally active fluoroquinolone antimicrobial, has been reported to be embryolethal but not teratogenic when administered to pregnant cynomolgus macaques prior to gestational day (GD) 36 at doses ≤200 mg/kg/day. Additional studies have been performed in an effort to examine the mechanism responsible for this effect, particularly regarding the role of progesterone (P). The first study (Study I) investigated the effect of norfloxacin administration during early pregnancy (200 mg/kg/day; daily GD 20–30) in the absence of a functional corpus luteum (CL). The CL was surgically removed from 16 gravid females on GD 19 in order to focus on placental‐derived P; ten were dosed with norfloxacin and six received vehicle only. Embryolethality was observed for 7/10 (70%) of the treated animals during GD 25–31 versus 0/6 (0%) for controls. A reduction in serum P was noted prior to embryonic loss, although no significant effects on chorionic gonadotropin (CG), 17β‐estradiol (E2), or P or E urinary metabolites were observed. A second study (Study II) was performed in order to evaluate the capacity of norfloxacin (200 mg/kg) to reduce CL‐derived P in both normally cycling and CG‐stimulated nonpregnant females (ten treated, ten controls; daily for 8 days). No effects on P production or on luteal phase or menstrual cycle lengths were observed. The third study (Study III) was designed to examine the effect of norfloxacin on the metabolism and excretion of P in nonpregnant females. Silastic P implants were placed subcutaneously in order to maintain constant P levels during a 10 day treatment regimen (200 mg/kg/day; ten controls, nine treated). Five of the controls and four of the norfloxacin‐treated females also received 14C‐P intravenously within 1 hr of the last dose of norfloxacin in order to study excretory patterns. No significant differences between control and treated groups were observed. The results of these studies combined suggest that the developmental toxic effects observed in prior studies and Study I are specific to pregnancy and directly related to placental‐derived P production. © 1992 Wiley‐Liss, Inc.
Histochemical and immunohistochemical distribution of glycosaminoglycans, type II collagen, and fibronectin in developing fetal cartilage of congenital osteochondrodysplasia rat (ocd/ocd)
Authors: Kikukawa Keiichiro , Suzuki Katsushi
Published: June 2005

The osteochondrodysplasia rat (ocd/ocd) is a lethal dwarfism. The ocd/ocd shows histological abnormalities of the epiphysis, characterized by a decrease in amount of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). The present study describes histochemical and immunohistochemical distributions of GAGs, type II collagen, and fibronectin (FN) in abnormal humeral cartilage of the ocd/ocd fetuses on days 16–21 of gestation. A wide‐spread region with severe necrosis was observed in the cartilage on days 20 and 21. The affected cartilage has small amounts of ECM, irregular columnizations, thinner hypertrophic zones, and expanded and pyknotic chondrocytes on days 16–21 of gestation. The severely expanded chondrocytes did not have cytoplasmic glycogens on days 19–21. Reactions for chondroitin sulfate (CS) and hyaluronic acid (HA) in the ECM were consistently lower in ocd/ocd than in +/+ during the entire period of observation, although there were granules immunoreactive to CS within the chondrocytes of ocd/ocd. The distribution of type II collagen seemed normal in relatively normal regions in the affected cartilage. Strong reactions for CS, HA, type II collagen, and FN were present in the necrotic region on days 20 and 21 of gestation. These findings suggest that the affected chondrocyte may have some defects in releasing ECM substances, which may be released by the process of cell rupture. We hypothesize that some defects in releasing processes inherent to the ocd/ocd cartilage may relate to cellular differentiation and cell death. © 1992 Wiley‐Liss, Inc.