Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation : An Alternative Model System to Study FASD

The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development

Prenatal and Postnatal Exposure to DDT by Breast Milk Analysis in Canary Islands

The use of p,p' -dichlorodiphenyltrichloroethane (DDT) has been banned since the late 1970s due to its toxicity. However, its long half-life makes it persistent in the environment and, consequently, almost everyone has DDT residues in the body. Human milk constitutes an ideal non-conventional matrix to investigate environmental chronic exposure to organochlorine compounds (OCs) residues. The study aimed to identify potential population risk factors of exposure to DDT due to the proximity to countries where it is still used. Seventy-two consecutive lactating women were prospectively included in Tenerife, Canary Islands (Spain). A validated questionnaire was used to obtain socioeconomic, demographics data, and daily habits during pregnancy. DDT levels in breast milk were measured by gas chromatography with-electron capture detector (GC-ECD). Anthropometrics measurements in newborns were obtained. Thirty-four out of 72 (47.2%) of the analysed milk samples presented detectable levels of DDT (mean: 0.92 ng/g), ranging between 0.08 to 16.96 ng/g. The socio-demographic variables did not significantly differ between detectable DDT and non-detectable DDT groups. We found positive association between DDT levels and vegetables (OR (95%CI): 1.23 (1.01-1.50)) and poultry meat (OR (95%CI): 2.05 (1.16-3.60)) consumption, and also between the presence of DDT in breast milk and gestational age (OR (95%CI): 0.59 (0.40-0.90)). DDT is present in breast milk of women at the time of delivery. Residual levels and the spread from countries still using DDT explain DDT detection from vegetables and from animal origin food. The presence of this compound in breast milk represents a pre- and postnatal exposure hazard for foetuses and infants due to chronic bioaccumulation and poor elimination, with possible deleterious effects on health. This data should be used to raise awareness of the risks of OCs exposure and to help establish health policies in order to avoid its use worldwide and thus, to prevent its propagation

Acute heroin intoxication in a baby chronically exposed to cocaine and heroin: a case report

<p>Abstract</p> <p>Introduction</p> <p>Acute intoxication with drugs of abuse in children is often only the tip of the iceberg, actually hiding chronic exposure. Analysis using non-conventional matrices such as hair can provide long-term information about exposure to recreational drugs.</p> <p>Case presentation</p> <p>We report the case of a one-month-old Caucasian boy admitted to our pediatric emergency unit with respiratory distress and neurological abnormalities. A routine urine test was positive for opiates, suggesting an acute opiate ingestion. No other drugs of misuse, such as cocaine, cannabis, amphetamines or derivatives, were detected in the baby's urine. Subsequently, hair samples from the baby and the parents were collected to evaluate the possibility of chronic exposure to drug misuse by segmental analysis. Opiates and cocaine metabolites were detected in hair samples from the baby boy and his parents.</p> <p>Conclusions</p> <p>In light of these and previous results, we recommend hair analysis in babies and children from risky environments to detect exposure to heroin and other drug misuse, which could provide the basis for specific social and health interventions.</p

Hair analysis following chronic smoked-drugs-of-abuse exposure in adults and their toddler: a case report

<p>Abstract</p> <p>Introduction</p> <p>Over the past two decades, the study of chronic cocaine and crack cocaine exposure in the pediatric population has been focused on the potential adverse effects, especially in the prenatal period and early childhood. Non-invasive biological matrices have become an essential tool for the assessment of a long-term history of drug of abuse exposure.</p> <p>Case report</p> <p>We analyze the significance of different biomarker values in hair after chronic crack exposure in a two-year-old Caucasian girl and her parents, who are self-reported crack smokers. The level of benzoylecgonine, the principal metabolite of cocaine, was determined in segmented hair samples (0 cm to 3 cm from the scalp, and > 3 cm from the scalp) following washing to exclude external contamination. Benzoylecgonine was detectable in high concentrations in the child's hair, at 1.9 ng/mg and 7.04 ng/mg, respectively. Benzoylecgonine was also present in the maternal and paternal hair samples at 7.88 ng/mg and 6.39 ng/mg, and 13.06 ng/mg and 12.97 ng/mg, respectively.</p> <p>Conclusion</p> <p>Based on the data from this case and from previously published poisoning cases, as well as on the experience of our research group, we conclude that, using similar matrices for the study of chronic drug exposure, children present with a higher cocaine concentration in hair and they experience more serious deleterious acute effects, probably due to a different and slower cocaine metabolism. Consequently, children must be not exposed to secondhand crack smoke under any circumstance.</p

Transient exposure to ethanol during zebrafish embryogenesis results in defects in neuronal differentiation: An alternative model system to study FASD

Background: The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings: In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion: Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.This study was supported by grants from Fondo de Investigaciones Sanitarias (FIS) (PI10/02593; PI13/01135) and Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003) from the Instituto Carlos III (Spain), a grant from Mutua Madrileña (AP150572014), intramural funding of the Neuroscience Program at IMIM (Institut Hospital del Mar ' Investigacions Mèdiques), partially supported by Generalitat de Catalunya (Spain) (2009SGR1388; 2014SGR584) and from Spanish Ministry of Economy and Competitiveness (BFU2012-31994)

Transient exposure to ethanol during zebrafish embryogenesis results in defects in neuronal differentiation: An alternative model system to study FASD

Background: The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings: In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion: Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development.This study was supported by grants from Fondo de Investigaciones Sanitarias (FIS) (PI10/02593; PI13/01135) and Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003) from the Instituto Carlos III (Spain), a grant from Mutua Madrileña (AP150572014), intramural funding of the Neuroscience Program at IMIM (Institut Hospital del Mar ' Investigacions Mèdiques), partially supported by Generalitat de Catalunya (Spain) (2009SGR1388; 2014SGR584) and from Spanish Ministry of Economy and Competitiveness (BFU2012-31994)

Analysis of cell proliferation and apoptotic cells in control and ethanol-treated embryos.

<p>(A-B) anti-pH3 immunofluorescence at 24hpf in order to analyse cell proliferation. (C-J) Cell death visualization using TUNEL assay in 36hpf embryos (C-D), and 5dpf larvae (E-J). Tg[Mü4127:mCherry] embryos were use for landmarks of r3 and r5 in red. (E,H) Apoptotic activity was visualized in whole-mount embryos at 5dpf. Note that ethanol-treated embryos have more apoptotic figures. (F-I) TUNEL analysis (green) in 5dpf embryos displaying red in the primary sensory neurons (F-F′, I-I′) or in motoneurons (G,J). All are lateral views with anterior to the left.</p

Effects of ethanol in the population of motoneurons.

<p>Analysis of Tg[Isl1:GFP] non-exposed embryos (A-C) and ethanol-exposed (D-F). (G) Quantification of GFP-positive motoneurons in specific hemisegments and (H) measurement of axonal lenght. Note that there is not an overall change in the pattern of motoneurons or number of them in treated embryos, although axonal length is diminished.</p

Effects of ethanol in specific neuronal populations.

<p>Analysis of Tg[Isl3:GFP] non-exposed embryos (A-B) and exposed to ethanol (C-D) expressing GFP in the primary sensory neurons. (E-G) Tg[neurog1:GFP] embryos treated with ethanol display a graded phenotype: (E) Phenotype I embryos showed a normal development with high levels of GFP along the CNS, (F) Phenotype II embryos showed a delay in their development but displayed normal levels of GFP, and (G) Phenotype III, which has a very short body axis and GFP levels not grossly affected. (H) Quantification of GFP-positive sensory neurons in specific hemisegments. Note the decrease in the number of Isl3:GFP neurons per hemisegment in the ethanol-treated embryos. (I) Quantification of the penetrance of the phenotype in Tg[neurog1:GFP] embryos. ** p<0.005 vs. control group.</p

Transient ethanol exposure does not alter either hindbrain patterning or MHB formation.

<p>(A,D,E) Control and (B,F,G) ethanol-exposed embryos display no differences in expression of mCherry in r3 and r5 at 36hpf. (C) Quantification of r3 and r5 area, showing no differences upon ethanol exposure. (D-G) In situ hybridization of <i>pax2a</i> in 24hpf embryos. (D-E) Control and (F-G) ethanol-exposed embryos showed no differences in the expression of <i>pax2a</i> in the MHB and otic placode, however in the ethanol-treated embryos <i>pax2a</i> was downregulated in the optic stalks. Mid-Hindbrain Boundary (MHB); optic stalk (os); otic vesicle (ov).</p