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A fully automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos
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Abstracts / Reproductive Toxicology 64 (2016) 29–49
to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole), the obtained gene expression patterns could lead us to a better classification of these compounds regarding the spectrum of their potency in conjunction with the regulated gene-groups. We also aimed to individually define the pharmacological and toxicological potency of each tested compound by evaluating in which level of sensitivity these azoles affected essential biological pathways during embryonic development. To this end, rat embryos were exposed to flusilazole in a range of concentrations for either 4 or 24 h. The other five compounds were tested in embryos, which were exposed for 4 h at each compound’s ID10 concentration, calculated after former embryonic morphological assessment. After 4- and 24-h exposure, we identified significant gene expression changes that were linked to the morphological outcome observed after prolonged exposure for 48 h (0–48 h). More precisely, the early exposure (0–4 h) of rat embryos to the tested azoles revealed commonly significant expression changes of Cyp26, Dhrs3, Gbx2, Rarb and Hox, which were involved in the general embryonic development and retinoic acid (RA) pathways. These gene expression alterations were preceded by commonly observed malformations (craniofacial and axial defects) in the 48-h exposed rat WEC to azoles, such as flusilazole, triadimefon, ketoconazole and miconazole. Additionally, a comparison between 4- and 24-h exposures to flusilazole revealed a significant embryonic response of genes, such as Cyp51, Pcsk9 and Scd1, that participated in the sterol pathway. Given that embryotoxic responses are also highly dependent on the time of exposure, after comparing the gene regulation differences among 4- and 24-h of exposure to flusilazole, we detected specific pathways that were more sensitive during embryonic development, related also to the tested azole. To conclude, the quantification of the significantly regulated genes in embryos exposed to azoles during 0–4 h revealed that pathways involved in embryonic development were more sensitive compared to the sterol biosynthesis pathway. Therefore, in terms of early-induced developmental toxicity, flusilazole, triadimefon and ketoconazole were the three most potent compounds, while in terms of disturbed sterol function difenoconazole and ketoconazole were the compounds with the most pronounced effect. http://dx.doi.org/10.1016/j.reprotox.2016.06.063 P-3 A fully automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos Stefan Scholz 1,∗ , Sergio Jarque 2 , Eva Fetter 1,3 1 Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany 2 Masaryk University, Faculty of Science, RECETOX, Kamenice 5, CZ-62500 Brno, Czech Republic 3 Present address: Federal Environment Agency – UBA, Wörlitzer Platz 1, 06844 Dessau-Roßlau, Germany
Thyroid hormones play an important role in development of vertebrates and hence, detection of a goitrogenic potential, i.e. disruption of the thyroid gland function, can be important for the screening of potential adverse effects of chemicals. We present a method for the screening of goitrogens based on the automatic orientation of embryos of the transgenic zebrafish line tg(tg:mCherry) in a glass capillary and the subsequent imaging
of reporter gene fluorescence in the thyroid gland. Fluorescence intensities were automatically extracted from images using a KNIME workflow. The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to thyroxine, a thyroid receptor agonist, a negative control compound (3,4-dichloroaniline), and a concentration series of known goitrogenic compounds (resorcinol, KClO4 , 6-propyl-2-thiouracil, sulfamethoxazole, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on determination of mortality and/or solubility. Exposure to thyroxine decreased the fluorescence signal, while 3,4-dichloroaniline did not provoke any induction. Most of the selected goitrogenic compounds exhibited a reproducible and clear concentration-dependent induction of reporter fluorescence. The concentration-dependent expression was described by different non-linear models and used to calculate goitrogenic potencies. The new automated method offers an unsupervised and efficient screening approach for goitrogenic activity that can also be combined with other endpoints. http://dx.doi.org/10.1016/j.reprotox.2016.06.064 P-4 Trends in maternal mortality in the United States Yasmin H. Neggers Department of Human Nutrition, University of Alabama, Tuscaloosa, AL 35487, USA Introduction: Maternal mortality is a major global concern. Although a notable decline in maternal mortality in the United States occurred during the mid-20th century, this progress stalled during the late 20th century. Furthermore, maternal mortality rates have increased during the early 21st century. Around the year 2000 the maternal mortality rate began to rise and has since nearly doubled. Methods: Maternal deaths are identified in the United States via Vital Statistics and Pregnancy Mortality Surveillance System (PMSS). Counts of maternal deaths and the maternal mortality ratio (MMR) are reported by the National Center for Health Statistics and are considered as the official maternal death statistics for domestic trends and international comparisons. Pregnancy-related mortality is defined as the death of women while pregnant or within 1 year of termination, irrespective of the duration or site of the pregnancy, from any cause related to or aggravated by her pregnancy or its management. Results: Since the Pregnancy Mortality Surveillance System (PMSS) was implemented, the numbers of deaths in the U.S. have steadily increased from 7.2 deaths per 100,100 live births in 1987 to a high of 17.8 deaths per 100,000 live births in 2011, the latest year data is available. The annualized rate of change in maternal mortality ratio (MMR) from 1990 to 2013 was +1.7% for the U.S., while this overall annualized rate of change was negative worldwide (−1.3%) and for developed (−3.1%) and developing countries (−1.4%) Given that at least half of maternal deaths in the U.S. are preventable, the rise in maternal deaths in the U.S. is historic and worrisome. The reason for this disturbing trend is not clear. It is uncertain whether the degree to which this increase in maternal mortality in the U.S. can be attributed to enhanced identification of maternal deaths or actual increase in risks. Also, there are persistent disparities in maternal mortality; particularly the 3 to 4 fold increased risk of death due to pregnancy complications among African–American women compared to Caucasian women.
Abstracts / Reproductive Toxicology 64 (2016) 29–49
to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole), the obtained gene expression patterns could lead us to a better classification of these compounds regarding the spectrum of their potency in conjunction with the regulated gene-groups. We also aimed to individually define the pharmacological and toxicological potency of each tested compound by evaluating in which level of sensitivity these azoles affected essential biological pathways during embryonic development. To this end, rat embryos were exposed to flusilazole in a range of concentrations for either 4 or 24 h. The other five compounds were tested in embryos, which were exposed for 4 h at each compound’s ID10 concentration, calculated after former embryonic morphological assessment. After 4- and 24-h exposure, we identified significant gene expression changes that were linked to the morphological outcome observed after prolonged exposure for 48 h (0–48 h). More precisely, the early exposure (0–4 h) of rat embryos to the tested azoles revealed commonly significant expression changes of Cyp26, Dhrs3, Gbx2, Rarb and Hox, which were involved in the general embryonic development and retinoic acid (RA) pathways. These gene expression alterations were preceded by commonly observed malformations (craniofacial and axial defects) in the 48-h exposed rat WEC to azoles, such as flusilazole, triadimefon, ketoconazole and miconazole. Additionally, a comparison between 4- and 24-h exposures to flusilazole revealed a significant embryonic response of genes, such as Cyp51, Pcsk9 and Scd1, that participated in the sterol pathway. Given that embryotoxic responses are also highly dependent on the time of exposure, after comparing the gene regulation differences among 4- and 24-h of exposure to flusilazole, we detected specific pathways that were more sensitive during embryonic development, related also to the tested azole. To conclude, the quantification of the significantly regulated genes in embryos exposed to azoles during 0–4 h revealed that pathways involved in embryonic development were more sensitive compared to the sterol biosynthesis pathway. Therefore, in terms of early-induced developmental toxicity, flusilazole, triadimefon and ketoconazole were the three most potent compounds, while in terms of disturbed sterol function difenoconazole and ketoconazole were the compounds with the most pronounced effect. http://dx.doi.org/10.1016/j.reprotox.2016.06.063 P-3 A fully automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos Stefan Scholz 1,∗ , Sergio Jarque 2 , Eva Fetter 1,3 1 Department of Bioanalytical Ecotoxicology, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany 2 Masaryk University, Faculty of Science, RECETOX, Kamenice 5, CZ-62500 Brno, Czech Republic 3 Present address: Federal Environment Agency – UBA, Wörlitzer Platz 1, 06844 Dessau-Roßlau, Germany
Thyroid hormones play an important role in development of vertebrates and hence, detection of a goitrogenic potential, i.e. disruption of the thyroid gland function, can be important for the screening of potential adverse effects of chemicals. We present a method for the screening of goitrogens based on the automatic orientation of embryos of the transgenic zebrafish line tg(tg:mCherry) in a glass capillary and the subsequent imaging
of reporter gene fluorescence in the thyroid gland. Fluorescence intensities were automatically extracted from images using a KNIME workflow. The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to thyroxine, a thyroid receptor agonist, a negative control compound (3,4-dichloroaniline), and a concentration series of known goitrogenic compounds (resorcinol, KClO4 , 6-propyl-2-thiouracil, sulfamethoxazole, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on determination of mortality and/or solubility. Exposure to thyroxine decreased the fluorescence signal, while 3,4-dichloroaniline did not provoke any induction. Most of the selected goitrogenic compounds exhibited a reproducible and clear concentration-dependent induction of reporter fluorescence. The concentration-dependent expression was described by different non-linear models and used to calculate goitrogenic potencies. The new automated method offers an unsupervised and efficient screening approach for goitrogenic activity that can also be combined with other endpoints. http://dx.doi.org/10.1016/j.reprotox.2016.06.064 P-4 Trends in maternal mortality in the United States Yasmin H. Neggers Department of Human Nutrition, University of Alabama, Tuscaloosa, AL 35487, USA Introduction: Maternal mortality is a major global concern. Although a notable decline in maternal mortality in the United States occurred during the mid-20th century, this progress stalled during the late 20th century. Furthermore, maternal mortality rates have increased during the early 21st century. Around the year 2000 the maternal mortality rate began to rise and has since nearly doubled. Methods: Maternal deaths are identified in the United States via Vital Statistics and Pregnancy Mortality Surveillance System (PMSS). Counts of maternal deaths and the maternal mortality ratio (MMR) are reported by the National Center for Health Statistics and are considered as the official maternal death statistics for domestic trends and international comparisons. Pregnancy-related mortality is defined as the death of women while pregnant or within 1 year of termination, irrespective of the duration or site of the pregnancy, from any cause related to or aggravated by her pregnancy or its management. Results: Since the Pregnancy Mortality Surveillance System (PMSS) was implemented, the numbers of deaths in the U.S. have steadily increased from 7.2 deaths per 100,100 live births in 1987 to a high of 17.8 deaths per 100,000 live births in 2011, the latest year data is available. The annualized rate of change in maternal mortality ratio (MMR) from 1990 to 2013 was +1.7% for the U.S., while this overall annualized rate of change was negative worldwide (−1.3%) and for developed (−3.1%) and developing countries (−1.4%) Given that at least half of maternal deaths in the U.S. are preventable, the rise in maternal deaths in the U.S. is historic and worrisome. The reason for this disturbing trend is not clear. It is uncertain whether the degree to which this increase in maternal mortality in the U.S. can be attributed to enhanced identification of maternal deaths or actual increase in risks. Also, there are persistent disparities in maternal mortality; particularly the 3 to 4 fold increased risk of death due to pregnancy complications among African–American women compared to Caucasian women.