Use of zebrafish ( Danio rerio ) embryos as a model to assess effects of mercury on developing skeletal muscle: a morphometric and immunohistochemical study / Uso de embriones de pez cebra ( Danio rerio ) como modelo para evaluar los efectos del mercurio en el desarrollo del músculo esquelético: un estudio morfométrico e inmunohistoquímico

Exposure to mercury in the environment continues to be a significant worldwide concern, especially for developing embryos and fetuses. While extensive research effort has focused on the effects of mercury on the developing nervous system, much less is known concerning adverse effects of mercury on other organ systems, including the development of skeletal muscle. We exposed developing zebrafish embryos to a range of concentrations of mercuric chloride (100 to 400 µg/liter or ppb) and compared them to control embryos (0 µg/L mercuric chloride). Embryos were examined at 48 hours post fertilization (hpf) for morphometry and morphological deformities of skeletal muscle fibers in the trunk and tail. Embryos exposed to 400 ppb mercuric chloride showed decreased trunk and tail areas compared to control embryos. A dose-dependent reduction in muscle fiber length was observed, and exposure to all concentrations of mercuric chloride used in this study resulted in decreased muscle fiber immunohistochemical staining with anti-myosin antibodies. Irregular muscle fiber diameters, twisted muscle fibers, and degenerated muscle fibers were observed in sections of embryos stained with eosin at the higher exposure concentrations. Evidence presented in this study suggests that exposure to even low concentrations of mercuric chloride adversely affects skeletal muscle fiber development or muscle fiber integrity, or both.

La exposición al mercurio en el medio ambiente sigue siendo una preocupación mundial importante, especialmente para el desarrollo de embriones y fetos. Si bien un amplio esfuerzo de investigación se ha centrado en los efectos del mercurio en el sistema nervioso en desarrollo, se sabe mucho menos sobre los efectos adversos en otros sistemas orgánicos, incluido el desarrollo del músculo esquelético. Expusimos embriones de pez cebra en desarrollo a un rango de concentraciones de cloruro de mercurio (100 a 400 mg / l o ppb) y los comparamos con embriones de control (0 mg / L de cloruro de mercurio). Los embriones se examinaron a las 48 horas después de la fertilización (HPF) pararealizar la morfometría y verificar las deformidades morfológicas de las fibras del músculo esquelético en el tronco y la cola. Los embriones expuestos a 400 ppb de cloruro de mercurio mostraron una disminución de las áreas del tronco y la cola en comparación con los embriones de control. Se observó una reducción dependiente de la dosis en la longitud de la fibra muscular, y la exposición a todas las concentraciones de cloruro de mercurio utilizadas en este estudio, dio como resultado una tinción inmunohistoquímica de fibra muscular disminuida con anticuerpos anti-miosina. Se observaron diámetros irregulares de fibras musculares, fibras musculares retorcidas y fibras musculares degeneradas en secciones de embriones teñidos con eosina en las concentraciones de exposición más altas. La evidencia presentada en este estudio sugiere que la exposición incluso a bajas concentraciones de cloruro mercúrico afecta negativamente el desarrollo de la fibra del músculo esquelético o la integridad de la fibra muscular, o ambas.

Estudio retrospectivo sobre las posibles complicaciones maternofetales de la exposición a cabergolina / Retrospective Study of any potencial Adverse Effects of Cabergoline on Pregnancy and Embryofetal Development

Objetivos: Estimar la frecuencia de complicaciones maternofetales en mujeres que se embarazaron durante el tratamiento con cabergolina (CAB). Estimar la frecuencia de patología detectada posnacimiento en los niños producto de dichos embarazos. Material y métodos: Estudio retrospectivo y multicéntrico de 86 embarazos en 78 mujeres con hiperprolactinemia idiopática (7) o tumoral (44 micro y 27 macro), en tratamiento con CAB en el momento de la concepción. Edad: 20 a 45 años; PRL inicial: 30 a 1429 ng/ml; duración del tratamiento previo al embarazo 1 a 120 meses; dosis: 0.125 a 4 mg/semana. El rango de exposición embriofetal a la CAB fue de 3 a 27 semanas, el 96.39% de las pacientes la recibió durante el primer trimestre y el 3.61% hasta el segundo. Resultados: No hubo complicaciones mayores durante el embarazo. Se registraron 7 abortos espontáneos (8.1%) y 75 partos, de los cuales 49 fueron vaginales y 26 cesáreas. Se registraron 69 recién nacidos, 63 fueron a término y 6 pretérmino (8.8%), ninguno bajo peso para la edad gestacional. En 3 (5.2%) recién nacidos se observó: 1 malformación mayor (Síndrome de Down) y 2 menores (hernia umbilical e inguinal). Se obtuvo seguimiento de 42 recién nacidos; se diagnosticó epilepsia refractaria en uno y un trastorno generalizado del desarrollo en otro. No se halló una mayor frecuencia de complicaciones en los embarazos ni en los recién nacidos expuestos a CAB que en la población normal. Sería necesario mayor número de pacientes para concluir sobre la seguridad de CAB durante el embarazo.

Objectives: To assess the rate of any potential adverse effects on pregnancy and embryo-fetal development in women who became pregnant under treatment with cabergoline (CAB). To follow up medical data of children who were born from mothers exposed to Cab in early weeks of gestation. Material and methods: Observational, retrospective and multicenter study on 86 pregnancies in 78 women with idiopathic or tumoral hyperprolactinemia. All patients were under Cab at conception. The average age was 29 (range: 20-45). Pituitary images at diagnosis showed 44 microadenomas, 27 macroadenomas and 7 were normal. Serum PRL at baseline was between 30 and 1429 ng/ml. Duration of therapy before pregnancy ranged from 1 to 120 months. Maternal and fetal exposure to cabergoline and doses ranged from 0.125 to 4 mg/week. The mean serum PRL level under which patients achieved pregnancy was 17 ng/ml. Fetal exposure ranged from 3 to 27 weeks; 96.39% of patients received CAB during the first trimester of pregnancy and 3.61% until the second one. Results: No significant complications during pregnancy were found. Seven women (8.1%) had spontaneous abortions. Term deliveries were recorded in 63/69, preterm in six (8.8%), none of them with low weight for gestational age. Neonatal abnormalities were observed in 3 (5.2%): 1 major (Down syndrome) and 2 minor malformations (umbilical and inguinal hernia). Two out of 42, developed abnormalities during the follow- up, one of them was a refractory epilepsy during the second month of life, the other presented a Pervasive Developmental Disorder diagnosed in the third year of life. Conclusion: No significantly higher frequency of complications was found in pregnancies and/or offspring exposed to CAB than in normal population. Larger series of patients are needed to asses the safety.

Effect of antiretroviral drugs on maternal CD4 lymphocyte counts, HIV-1 RNA levels, and anthropometric parameters of their neonates

OBJETIVOS: Estudar o efeito das drogas anti-retrovirais sobre a quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV-1 e parâmetros antropométricos de seus neonatos. MÉTODOS: Estudo prospectivo avaliando 57 gestantes e seus neonatos em três grupos: Grupo AZT, gestantes portadoras do HIV utilizando zidovudina (n=20); Grupo TT, mães utilizando zidovudina+lamivudina+nelfinavir (n=25), e Grupo Controle, mulheres saudáveis (n=12). A quantificação dos linfócitos TCD4 e RNA do HIV-1 de gestantes portadoras do HIV foi analisada em dois períodos durante a gestação. O prognóstico perinatal levou em consideração as taxas de pré-termos, restrição de crescimento intra-útero, mortalidade perinatal e transmissão vertical do HIV-1. Os dados foram analisados utilizando-se testes não paramétricos de qui-quadrado, Mann-Whitney, Friedman, Kruskal-Wallys e Wilcoxon para amostras pareadas, considerando-se significativos valores associados a p<0,05. RESULTADOS: Observou-se homogeneidade entre os dados demográficos e antropométricos de realce. A carga viral, inicialmente elevada (14.370 cópias/ml), reduziu-se significativamente no grupo com tratamento tríplice , chegando a 40 cópias/ml. Quanto à contagem de linfócitos CD4, observou-se recuperação significativa nas pacientes do grupo TT, no final da gestação, sendo esse valor significativamente diferente em comparação ao grupo AZT (p = 0,0052). Não se observou diferença entre os grupos quanto à duração da gestação, aos índices de Apgar, e à classificação antropométrica neonatal. Não houve nenhum caso de transmissão vertical do HIV-1. CONCLUSÕES: Os resultados obtidos na presente casuística demonstram eficiência e sugerem segurança no uso de anti-retrovirais na gestação sobre parâmetros antropométricos dos neonatos.

Effects of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos.

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.

Valproic acid teratogenicity: a toxicogenomics approach.

Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.

Altered expression of genes related to zinc homeostasis in early mouse embryos exposed to di-2-ethylhexyl phthalate.

Numerous studies have shown that di-2-ethylhexyl phthalate (DEHP) is teratogenic in animals but the mechanism of developmental toxicity is not well understood. One hypothesis is altered zinc homeostasis. The present study has investigated the effect of DEHP exposure on several key genes in zinc metabolism (MT-I, MT-II, ZnT-1) for early mouse embryos exposed in utero. Time- and dose-dependent effects were examined using expression polymerase chain reaction (PCR) (relative to ACTB) and Western blot analysis of the maternal liver, embryonic brain, and visceral yolk sac at 9 days post-coitus (d.p.c.). Maternal exposure to 800 mg/kg DEHP increased the abundance of MT-I and MT-II transcripts in maternal liver at 3.0, 4.5 and 6.0 h after administration. MT-I and MT-II protein induction was confirmed by Western blot analysis. On the other hand, this exposure down-regulated both transcripts (MT-I, MT-II), as well as transcripts for a zinc transporter (ZnT-1), in the embryonic brain, but not the visceral yolk sac. To examine dose-response relationships, the experiment was repeated for DEHP exposures of 50, 200 and 800 mg/kg. The effect to MT-I and MT-II expression in the maternal liver became significant at the 200 mg/kg dose level. The contrasting effect to MT-I, MT-II and ZnT-1 expression in the embryo was also dose-dependent, and a benchmark computation for the dose resulting in a 5% change in the mean (BMD5) was estimated as 11.6 mg/kg for MT-I, 8.9 mg/kg for MT-II, and 6.6 mg/kg for ZnT-1. We conclude that DEHP exposure to pregnant dams at reasonably low levels during organogenesis stages can alter the expression of several key genes in embryonic zinc homeostasis.

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes.

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.

Latanoprost exposure in pregnancy.

PURPOSE: To observe pregnancies exposed to latanoprost, a prostaglandin analog administered in the treatment of glaucoma. Its prescription is limited in pregnancy, because reproduction studies in animals report a high incidence of abortion and human investigations are not adequate. As a consequence it is classified as category C drug according to the United States Food and Drug Administration's use-in-pregnancy ratings. DESIGN: Observational study. METHODS: We collected data, referred to our Teratology Information Service, relative to latanoprost exposure in pregnancy. We followed by phone interviews women treated with latanoprost during the first trimester, and we evaluated whether there had been any adverse effects on the fetus. RESULTS: Eleven cases of latanoprost exposure in pregnancy were referred to our Teratology Information Service. One case was lost to follow-up, and one case was complicated by miscarriage. Nine cases had a complete follow-up without congenital anomalies. CONCLUSIONS: Our series is too small to perform statistical significance; however, we found no evidence of adverse effects of latanoprost on pregnancy or neonatal outcomes.

A prospective study of pregravid oral contraceptive use in relation to fetal growth.

OBJECTIVE: Because oral contraceptives are so widely used, any health consequences may have substantial public health implications. Whether pregravid oral contraceptives could affect subsequent pregnancies has not been adequately studied. The study objectives were to examine whether pregravid oral contraceptive use affects fetal growth and pregnancy hormone levels. DESIGN: A prospective study of pregnant women followed through pregnancy. SETTING: A major teaching hospital in Boston, USA. POPULATION: Two hundred and sixty Caucasian pregnant women, with a mean age of 31, and a parity of no more than two. Seventy-nine percent of the women were pregravid oral contraceptive users. METHODS: Exposure and covariate data were collected through structured questionnaires. Blood was drawn for hormonal analysis during the 16th and 27th gestational week. Information on pregravid oral contraceptive use included duration and recency of use, and oral contraceptive formulation. Multivariate regression models were used to examine the effect of pregravid oral contraceptive use on birth outcomes and the studied pregnancy hormones. MAIN OUTCOME MEASURES: Birthweight, placental weight, gestational age, pregnancy hormone levels of oestriol and progesterone at 16th and 27th gestational week. RESULTS: Adjusting for confounders, pregravid oral contraceptive use increased birthweight (mean difference =+207.3 g, 95% CI =+77.6 to +337.1) and placental weight (mean difference =+64.9 g, 95% CI =+13.0 to +116.9) compared with never use. Women with prior oral contraceptive use had higher levels of serum progesterone (P= 0.002) and oestriol (P= 0.12) at the 27th gestational week measurement. The effect on birthweight, placental weight and hormones was stronger among those using oral contraceptives in the previous year and those using a high progestin/high oestrogen potency preparation. CONCLUSIONS: Pregravid oral contraceptive use is positively associated with fetal growth, and this effect may be mediated through oestriol and progesterone.

Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro.

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.

Consequences of elevated homocysteine during embryonic development and possible modes of action.

Elevated maternal homocysteine (Hcys) is a well-established risk factor for embryonic toxicity and the development of congenital defects, particularly neural tube closure defects and neurocristopathies. The mechanisms responsible are unclear but early work has focused on the role of folate metabolism because these defects are greatly reduced by folate supplementation. As a consequence, elevated Hcys is often looked upon as being an indirect consequence of faulty folate metabolism, although more recent studies show Hcys may act directly as a teratogen. Because Hcys is at the crossroads of protein and DNA metabolism, has a propensity to chemically modify proteins directly, can generate free radicals, and even perturb ligand binding to certain receptors, the developmental processes Hcys can potentially disturb are enumerable. But in recent years, investigators have begun identifying cellular and molecular targets for the direct action of Hcys. While elevating Hcys can alter a myriad of basic cellular activities needed for normal development, our current understanding as to the specific etiological mechanisms responsible for congenital defects is very speculative. Here we provide an overview of what is currently known regarding the toxicity and teratogenicity of elevated Hcys during embryonic development, paying particular attention to neural tube and neural crest cell morphogenesis.

Vitamin a requirement for early cardiovascular morphogenesis specification in the vertebrate embryo: insights from the avian embryo.

Vitamin A is required throughout the life cycle, including crucial stages of embryonic and fetal development. With the identification of retinoic acid-specific nuclear transcription factors, the retinoid receptors, considerable advances have been made in understanding the molecular function of vitamin A. The requirement for vitamin A during early embryogenesis has successfully been examined in the vitamin A-deficient avian embryo during neurulation, when in the vertebrates crucial developmental decisions take place. These studies revealed that retinoic acid is essential during these early stages of embryogenesis for the initiation of organogenesis (i.e., formation of the heart). If retinoic acid is not present at this time, abnormal development ensues, leading to early embryonic death. Though the initial insult of the absence of vitamin A appears to be on the specification of cardiovascular tissues, subsequently all development is adversely affected and the embryo dies. Molecular and functional studies revealed that retinoic acid regulates the expression of the cardiogenic transcription factor GATA-4 and several heart asymmetry genes, which explains why the heart position is random in vitamin A-deficient quail embryos. During the crucial retinoic acid-requiring developmental window, retinoic acid transduces its signals to genes for heart morphogenesis via the receptors RARalpha2, RARgamma, and RXRalpha. Elucidation of the function of vitamin A during early embryonic development may lead to a better understanding of the cardiovascular birth defects prevalent in the Western world.

Inhibition of ossification in vivo and differentiation of osteoblasts in vitro by tributyltin.

Tributyltin is ubiquitous in the environment and an endocrine disruptor for many wildlife species. However, minimal information is available regarding the effect of this chemical on bone formation. When tributyltin chloride (TBT) (1mg/kg body weight) was administered subcutaneously to pregnant mice at 10, 12, and 14 days post coitus (dpc), fetuses at 17.5 days post coitus revealed the inhibition of calcification of supraoccipital bone. In contrast, 1mg/kg body weight monobutyltin trichloride (MBT) did not affect the fetal skeleton. Therefore, we examined the effects of TBT and its metabolites (dibutyltin dichloride, DBT, and MBT) on bone metabolism using rat calvarial osteoblast-like cells (ROB cells). The viability of ROB cells was not affected by the exposure of the cells to 10(-10) to 10(-7)M TBT. However, TBT reduced the activity of alkaline phosphatase (ALPase) and the rate of deposition of calcium of ROB cells. In addition, the expression levels of mRNA for ALPase and osteocalcin, which are markers of osteoblastic differentiation, were depressed by the treatment with TBT. TBT inhibited ALPase activity and the deposition of calcium to a greater extent than did DBT. MBT had no effect on the osteoblast differentiation of ROB cells. Tributyltin is known to inhibit the activity of aromatase. However, the aromatase inhibitor aminoglutethimide did not reproduce the inhibitory effects of TBT on osteoblast differentiation. Our findings indicate that TBT might have critical effects on the formation of bone both in vivo and in vitro although its action mechanism is not clarified.

Association of in utero organophosphate pesticide exposure and fetal growth and length of gestation in an agricultural population.

Although pesticide use is widespread, little is known about potential adverse health effects of in utero exposure. We investigated the effects of organophosphate pesticide exposure during pregnancy on fetal growth and gestational duration in a cohort of low-income, Latina women living in an agricultural community in the Salinas Valley, California. We measured nonspecific metabolites of organophosphate pesticides (dimethyl and diethyl phosphates) and metabolites specific to malathion (malathion dicarboxylic acid), chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphoro-thioate], and parathion (4-nitrophenol) in maternal urine collected twice during pregnancy. We also measured levels of cholinesterase in whole blood and butyryl cholinesterase in plasma in maternal and umbilical cord blood. We failed to demonstrate an adverse relationship between fetal growth and any measure of in utero organophosphate pesticide exposure. In fact, we found increases in body length and head circumference associated with some exposure measures. However, we did find decreases in gestational duration associated with two measures of in utero pesticide exposure: urinary dimethyl phosphate metabolites [beta(adjusted) = -0.41 weeks per log10 unit increase; 95% confidence interval (CI), -0.75 -- -0.02; p = 0.02], which reflect exposure to dimethyl organophosphate compounds such as malathion, and umbilical cord cholinesterase (beta(adjusted) = 0.34 weeks per unit increase; 95% CI, 0.13-0.55; p = 0.001). Shortened gestational duration was most clearly related to increasing exposure levels in the latter part of pregnancy. These associations with gestational age may be biologically plausible given that organophosphate pesticides depress cholinesterase and acetylcholine stimulates contraction of the uterus. However, despite these observed associations, the rate of preterm delivery in this population (6.4%) was lower than in a U.S. reference population.

Prenatal insecticide exposures and birth weight and length among an urban minority cohort.

We reported previously that insecticide exposures were widespread among minority women in New York City during pregnancy and that levels of the organophosphate chlorpyrifos in umbilical cord plasma were inversely associated with birth weight and length. Here we expand analyses to include additional insecticides (the organophosphate diazinon and the carbamate propoxur), a larger sample size (n = 314 mother-newborn pairs), and insecticide measurements in maternal personal air during pregnancy as well as in umbilical cord plasma at delivery. Controlling for potential confounders, we found no association between maternal personal air insecticide levels and birth weight, length, or head circumference. For each log unit increase in cord plasma chlorpyrifos levels, birth weight decreased by 42.6 g [95% confidence interval (CI), -81.8 to -3.8, p = 0.03] and birth length decreased by 0.24 cm (95% CI, -0.47 to -0.01, p = 0.04). Combined measures of (ln)cord plasma chlorpyrifos and diazinon (adjusted for relative potency) were also inversely associated with birth weight and length (p < 0.05). Birth weight averaged 186.3 g less (95% CI, -375.2 to -45.5) among newborns with the highest compared with lowest 26% of exposure levels (p = 0.01). Further, the associations between birth weight and length and cord plasma chlorpyrifos and diazinon were highly significant (p < or = 0.007) among newborns born before the 2000-2001 U.S. Environmental Protection Agency's regulatory actions to phase out residential use of these insecticides. Among newborns born after January 2001, exposure levels were substantially lower, and no association with fetal growth was apparent (p > 0.8). The propoxur metabolite 2-isopropoxyphenol in cord plasma was inversely associated with birth length, a finding of borderline significance (p = 0.05) after controlling for chlorpyrifos and diazinon. Results indicate that prenatal chlorpyrifos exposures have impaired fetal growth among this minority cohort and that diazinon exposures may have contributed to the effects. Findings support recent regulatory action to phase out residential uses of the insecticides.

Prolonged oral treatment with an essential amino acid L-leucine does not affect female reproductive function and embryo-fetal development in rats.

L-leucine, an essential amino acid, is one of the most popular ingredients in dietary supplements. To investigate a possibility of its embryo-fetal toxicity in rats, 11- to 12-week old dams were orally administered an aqueous solution of L-leucine at doses of 300 or 1000 mg/kg body weight on gestational days 7-17. Body weight and feed intake was evaluated throughout the whole course of pregnancy (days 0-20). L-Leucine did not influence body weight, but at a dose of 1000 mg/kg, slightly enhanced feed intake on days 14 and 18 of pregnancy. Caesarean section (day 20) revealed no influences on the litter size and weight of live-born fetuses, the number of corpora lutea, implantation index or the quality of placenta, and the minor increase in feed intake was considered irrelevant to the pregnancy outcomes. Fetuses were evaluated in a battery of external, visceral and skeletal examinations. No effects of L-leucine on gender ratio and external abnormalities, and no significant treatment-related variations in visceral and skeletal pathologies were observed. These results suggested that L-leucine, administered orally during organogenesis at doses up to 1000 mg/kg body weight, did not affect the outcome of pregnancy and did not cause fetotoxicity in rats.

Embryotoxicity and teratogenicity study with alpha-cyclodextrin in rats.

The embryotoxicity/teratogenicity of alpha-cyclodextrin (alpha-CD) was examined in Wistar Crl:(WI)WU BR rats. alpha-CD was fed at dietary concentrations of 0, 1.5, 5, 10, or 20% to groups of 25 pregnant female rats from day 0 to 21 of gestation. An additional group received a diet with 20% lactose. The additions to the diet of alpha-CD and lactose were made at the expense of pregelatinized potato starch. Body weight as well as food and water intake were recorded during the treatment period. The rats were killed on day 21 and examined for standard parameters of maternal reproductive performance. The fetuses were examined for external abnormalities, body weight and crown rump length. Fetuses were examined for skeletal and visceral abnormalities. Generally, alpha-CD was well tolerated and no deaths occurred in any group. Weight gain and food consumption were similar in all groups during gestation, except for a slightly yet significantly increased food intake in the 20% alpha-CD group from day 6 to 21. Water intake was similar in all alpha-CD groups; in the lactose group, it was significantly higher than in the controls. Maternal reproductive performance was not affected by the alpha-CD treatment. Examination of the fetuses for external, visceral and skeletal changes did not reveal any fetotoxic, embryotoxic, or teratogenic effects of alpha-CD. In conclusion, no adverse effects were observed at alpha-CD intakes of up to 20% of the diet, the highest dose level tested at which the rats consumed about 13 g/kg bw/day.

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.

Prenatal nicotine exposure increases connective tissue expression in foetal monkey pulmonary vessels.

Among the many deleterious effects of maternal smoking during pregnancy on foetal development, is a higher incidence of persistent pulmonary hypertension. The recent identification of nicotinic acetylcholine receptors (nAChR) on cells of the pulmonary vessel walls suggests that maternal smoking during pregnancy may produce morphological alterations in foetal pulmonary vasculature. Timed-pregnant rhesus monkeys were treated with nicotine (1 mg x kg(-1) x day(-1)) delivered by subcutaneous osmotic mini-pumps from days 26-134 of gestation (term: 165 days). Lung sections from 134-day foetal monkeys were used for morphometric analysis, in situ hybridisation and immunohistochemical staining. Following nicotine treatment, total wall and tunica adventitia thickness of airway associated vessels (AAV) increased significantly. Nicotine exposure significantly increased collagen I and III mRNA and protein in tunica adventitia in all AAV but not in tunica media. By contrast, levels of elastin protein were significantly decreased. alpha7 nAChR were detected in AAV fibroblasts that expressed collagen mRNA. Choline acetyltransferase, the enzyme which synthesises acetylcholine, the ligand for alpha7 nAChR was also detected in endothelium and fibroblasts. These findings suggest that with smoking during pregnancy, nicotine is transported across the placenta and directly interacts with nicotinic acetylcholine receptors in pulmonary vessels to alter connective tissue expression and therefore produce vascular structural alterations.

Effects of interleukin-12 administration during the pre- and peri-implantation period on mouse embryofetal development.

PROBLEM: The immunological success of pregnancy is thought to depend upon the establishment of a balance between favorable and deleterious cytokines, the current paradigm viewing pregnancy as a T helper (Th)2 cytokine-dependent phenomenon. In this context, a particular attention should be directed to the potential role of interleukin (IL)-12, which promotes the development of Th1 responses, in the induction of adverse pregnancy-related phenomena. Indeed, very few data linked the Th1-inducer IL-12 to the event of abortion. METHODS: In this study, we have investigated the maternal and fetal effects of exogenous administration of IL-12 to CD1 (BR) ICR mice during the pre- and peri-implantation period (day 2-6 of pregnancy). Animals have been evaluated for parameters of reproductive performance, embryo and fetal developmental toxicity and maternal toxicity. RESULTS: Intraperitoneal administration of IL-12 at concentrations from 2.5 to 10 microg/kg daily did not result in an increase in the murine abortion rate. A statistically significant, although minimal, decrease in the number of somites were found in the embryos of animals treated with IL-12 at a dose of 10 microg/kg/day. However, developmental parameters at birth were similar between the two groups of animals suggesting that alteration of somites might be a transitory state during treatment. An increased body weight gains and reduced feed and water consumption were observed in the mothers treated with the cytokine. CONCLUSION: In the present experimental conditions and in this specific strain of mice, IL-12 does not exert adverse effects on reproductive performance and induces an only modest harmful action on mothers and embryos.