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Revista de la Real acadèmia de medicina de les Illes Balears. Volumen 32, número 1, 2017

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VOLUM 32 NÚM. 1 GENER - ABRIL 2017 Medicina Balear PUBLICACIÓ DE LA REIAL ACADÈMIA DE MEDICINA DE LES ILLES BALEARS Effects of sex hormones disruption, after prenatal and postnatal exposure to chlordimeform, on monoaminergic neurotransmitters systems in female and male rat’s prefrontal cortex Análisis del origen y evolución de las infecciones respiratorias agudas de etiología viral en la población adulta, temporada 2015-2016 Utilidad en enfermería de diferentes índices antropométricos y analíticos para valorar la existencia de síndrome metabólico con los criterios NCEP ATPIII e IDF en población mediterránea española Programa de Cuidados Paliativos de las Islas Baleares. Evaluación del período 2013-2016 El Comitè d’Ètica de la Investigació de les Illes Balears: els primers quinze anys Varón africano de 49 años, SIDA, que acude a URGA por fiebre y convulsiones www.medicinabalear.org

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Medicina Balear www.medicinabalear.org Medicina Balear, òrgan de la Reial Acadèmia de Medicina de les Illes Balears, va aparèixer el 1986 amb l’objectiu de donar curs a les inquietuds científiques i fomentar l’esperit d’investigació dels professionals de la sanitat balear i amb la pretensió suplementària de projectar en la societat temes d’interès sanitari. Medicina Balear publica en català, castellà o anglès treballs originals, articles de revisió, cartes al director i altres escrits d’interès relacionats amb les ciències de la salut i presta particular atenció als treballs que tinguin per àmbit les Illes Balears i altres territoris de la conca mediterrània occidental. La revista sotmet els originals a la revisió anònima per al menys dos experts externs (peer review). El material científic publicat a Medicina Balear resta protegit per drets d’autor. Medicina Balear no és responsable de la informació i opinions dels autors. Aquesta obra -llevat que s’indiqui el contrari en el text, en les fotografies o en altres il·lustracions- és subjecta a la llicència de Reconeixement-NoComercial-SenseObraDerivada 3.0 Espanya de Creative Commons; http://creativecommons.org/licenses/by-nc-nd/3.0/es/. Així, doncs, s’autoritza al públic en general a reproduir, distribuir i comunicar l’obra sempre que se’n reconegui l’autoria i l’entitat que la publica i no se’n faci un ús comercial ni cap obra derivada. Medicina Balear es troba incorporada a la Biblioteca Digital de les Illes Balears, de la Universitat de les Illes Balears, i està inclosa en les bases de dades següents: Latindex (catàleg), Dialnet, Índice Médico Español, DOAJ, Imbiomed E D I TA Reial Acadèmia de Medicina de les Illes Balears www.ramib.org Campaner, 4, baixos. 07003 Palma de Mallorca Tel. 971 72 12 30 Email: info@ramib.org Pàgina web: http://www.ramib.org  Dipòsit Legal: PM 486 - 95 eISSN: 2255 - 0569 Disseny i maquetació Intelagencia Publicitat - www.intelagencia.es - intelagencia@intelagencia.es

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Medicina Balear Publicació quadrimestral de ciències de la salut de la Reial Acadèmia de Medicina de les Illes Balears Director A. Arturo López González, RAMIB, Reial Acadèmia de Medicina de les Illes Balears (RAMIB) CONSELL EDITORIAL Subdirector Joan March Noguera, RAMIB Editor científic Marta Couce Matovelle, Case Western Reserve University Assessors editorials José A. Guijarro Pastor, AEMET · Jaume Rosselló Mir, UIB Redactor en cap J. L. Olea Vallejo, RAMIB Vocals Antoni Aguiló Pons, Universitat de les Illes Balears · Bartolomé Bur- guera González, Cleveland Clinic (Ohio) · Amador Calafat Far, Socidrogalcohol · Carlos Campillo Artero, Universitat Pompeu Fabra · Valentín Esteban Buedo, Conselleria de Sanitat, Generalitat Valenciana · Carmen González Bosch, Universitat de València · Miguel A. Limon Pons, Institut Menorquí d’Estudis · Virgili Páez Cervi, Bibliosalut · Lucio Pallarés Ferreres, Hospital Son Espases, Ibsalut · Ignacio Ricci Cabello, University of Oxford · Guillermo Sáez Tormo, Universitat de València · Ma Teófila Vicente Herrero, IUNICS CONSELL CIÉNTIFIC Mª José Anadón Baselga (Universidad Complutense de Madrid), Miquel Capó Martí (Universidad Complutense de Madrid), Antonio Coca Payeras (Universitat de Barcelona), James Drane (Edinboro University), Leopoldo Forner Navarro (Universitat de València), Alexandre García-Mas, (Universitat de les Illes Balears), Antoni Gelabert Mas (Universitat Autònoma de Barcelona), Joan Grimalt Obrador (Consell Superior d’Investigacions Científiques, CSIC), Federico Hawkins Carranza (Universitat Complutense de Madrid), Joan Carles March Cerdà (Escuela Andaluza de Salud Pública, EASP), Gabriel Martí Amengual (Universitat de Barcelona), Jasone Monasterio Aspiri (Universitat Autònoma de Barcelona) Rosa Pulgar Encinas (Universidad de Granada), Ciril Rozman (Universitat de Barcelona). Amb la col·laboració de www.medicinabalear.org

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PREMIOS Y BECAS PREMIOS DE INVESTIGACIÓN 2016-17 La Fundació Patronat Científic convoca los Premios de Investigación Mateu Orfila, Damià Carbó y Metge Matas con la finalidad de premiar la trayectoria de la investigación en Ciencias de la Salud en nuestra comunidad. http://www.comib.com/wp-content/uploads/2017/01/Premios-investigacioon-2016-17-1.pdf PREMIO FUNDACIÓ MUTUAL MÈDICA AL MEJOR PROYECTO DE TESIS DOCTORAL El premio tiene como finalidad facilitar a los médicos colegiados en las Illes Balears la obtención del título de Doctor. Con el patrocinio de la Fundació Mutual Médica. http://www.comib.com/wp-content/uploads/2017/01/Poster-Becas-Mutual-2017.pdf BECAS FUNDACIÓN BANCO SABADELL DE ROTACIÓN EXTERNA PARA MÉDICOS RESIDENTES Facilitar a los médicos que realicen la residencia en hospitales de Balears la formación, al menos durante un mes, en centros hospitalarios nacionales y extranjeros, en el último año de la residencia o al terminar la misma. http://www.comib.com/wp-content/uploads/2017/01/Poster-Rotacioon-Externa-2017.pdf BECAS DE INNOVACIÓN PARA MÉDICOS COLEGIADOS, EXCEPTO MIR Tienen como objetivo facilitar a los médicos colegiados en las Illes Balears, excepto MIR, la formación, durante al menos un mes, en centros sanitarios nacionales y extranjeros en técnicas diagnósticas o terapéuticas. http://www.comib.com/wp-content/uploads/2017/01/Poster-Beca-Innovacion-2017.pdf I CERTAMEN DE CASOS CLÍNICOS PARA MÉDICOS RESIDENTES La Fundació Patronat Científic convoca el I Certamen de Casos Clínicos de cualquier especialidad médica o quirúrgica para médicos residentes. http://www.comib.com/wp-content/uploads/2017/01/I-Certamen-de-casos-clinicos-residentes.pdf PREMIO CAMILO JOSÉ CELA DE HUMANIDADES MÉDICAS Premio literario destinado a honrar la especial relación que tuvo el Nobel con los médicos a lo largo de su vida. http://www.comib.com/wp-content/uploads/2017/01/Diiptico-Premio-Camilo-Jose-Cela-16-17.pdf www.comib.com/patronatcientific

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VOLUM 32 NÚM. 1 GENER - ABRIL 2017 Medicina Balear PUBLICACIÓ DE LA REIAL ACADÈMIA DE MEDICINA DE LES ILLES BALEARS www.medicinabalear.org SUMARI EDITORIAL Medicamentos peligrosos Olga Delgado Sánchez 9 ORIGINALS Efectos de la disrupción de las hormonas sexuales, tras la exposición prenatal y posnatal al Clordimeformo, sobre los sistemas de neurotransmisores monoaminérgicos en la corteza prefrontal de ratas macho y hembra José Manuel García, Paula Moyano, María Teresa Frejo, María José Anadón, Miguel Andrés Capó, Gloria Gómez, Javier del Pino 11-19 Análisis del origen y evolución de las infecciones respiratorias agudas de etiología viral en la población adulta, temporada 2015-2016 Jordi Reina, Cristina Taboada, Joan Vidal, Melchor Riera, Javier Murillas, José Ignacio Ayestarán 20-25 Utilidad en enfermería de diferentes índices antropométricos y analíticos para valorar la existencia de síndrome metabólico con los criterios NCEP ATPIII e IDF María Gil Llinás, Pilar Estades Janer, Sheila García Agudo, Rosa González Casquero, Irene Campos González 26-34 Programa de Cuidados Paliativos de las Islas Baleares. Evaluación del período 2013-2016 Noemí Sansó, Estefania Serratusell, Joan Llobera, Eusebi Castaño, Mercé Llagostera, Carlos Serrano, Antonia Rotger, Enric Benito 35-42 ARTICLE ESPECIAL El Comitè d’Ètica de la Investigació de les Illes Balears: els primers quinze anys Joan Bargay-Lleonart, Lourdes de la Vega Llompart, Francesca M. Cifre-Gual, Joan B. Soriano i membres del CEI-IB 43-52 ESTUDI DE CASOS Varón africano de 49 años, SIDA, que acude a URGA por fiebre y convulsiones Adelaida Rey, Héctor Rodrigo, Pilar Salvà, Elena Delgado, Paula Carrillo, Manuel del Río Vizoso 53-57 eISSN 2255-0569

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¿Qué profesional puede tener 45 años de edad y 90 de experiencia? La respuesta es Banca March La experiencia de un profesional no está únicamente en su edad, sino también en la edad de la firma para la que trabaja. Y 90 años de experiencia es lo que ofrecen los profesionales de Banca March. 90 años gestionando patrimonios y demostrando entre otras cosas, que la prudencia no está reñida con la rentabilidad. www.bancamarch.es

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VOLUME 32 NUMBER 1 JANUARY - APRIL 2017 Medicina Balear SCIENTIFIC JOURNAL OF THE ROYAL ACADEMY OF MEDICINE OF THE BALEARIC ISLANDS www.medicinabalear.org CONTENTS EDITORIAL Hazardous drugs Olga Delgado Sánchez 9 ORIGINALS Effects of sex hormones disruption, after prenatal and postnatal exposure to chlordimeform, on monoaminergic neurotransmitters systems in female and male rat’s prefrontal cortex José Manuel García, Paula Moyano, María Teresa Frejo, María José Anadón, Miguel Andrés Capó, Gloria Gómez, Javier del Pino 11-19 Analysis of the origin and evolution of acute respiratory infections of viral etiology in the adult population, 2015-2016 season Jordi Reina, Cristina Taboada, Joan Vidal, Melchor Riera, Javier Murillas, José Ignacio Ayestarán 20-25 Usefulness in nursing of different anthropometric and analytical indices to assess the existence of metabolic syndrome with the NCEP ATP III and IDF criteria in Spanish Mediterranean population María Gil Llinás, Pilar Estades Janer, Sheila García Agudo, Rosa González Casquero, Irene Campos González 26-34 Palliative Care Program of the Balearic Islands. Evaluation of the period 2013-2016 Noemí Sansó, Estefania Serratusell, Joan Llobera, Eusebi Castaño, Mercé Llagostera, Carlos Serrano, Antonia Rotger, Enric Benito 35-42 ARTICLE ESPECIAL The Research Ethics Committee of the Balearic Islands: the first fifteen years Joan Bargay-Lleonart, Lourdes de la Vega Llompart, Francesca M. Cifre-Gual, Joan B. Soriano i membres del CEI-IB 43-52 ESTUDI DE CASOS 49 years old african male, AIDS, attended in the emergency department because of fever and seizures Adelaida Rey, Héctor Rodrigo, Pilar Salvà, Elena Delgado, Paula Carrillo, Manuel del Río Vizoso 53-57 Medicina Balear 2017; 32 (1); 5-6 eISSN 2255-0569

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.com Haz algo grande por tu salud En Asisa somos expertos en salud y sabemos que el sueño es vital para el buen funcionamiento de tu corazón, tu cerebro y todo tu organismo. Los especialistas determinan que una persona adulta necesita entre 7 y 9 horas diarias de sueño para estar bien. Sin embargo, se estima que el 80% de los españoles duermen menos de este tiempo, exponiéndose a sufrir hipertensión, taquicardia, depresión, pérdida de memoria, sobrepeso y diabetes, entre otros problemas. Y como sabes, en Asisa solo nos preocupa tu salud. Por eso invertimos todos nuestros recursos en cuidarte, incluido este anuncio en el que te aconsejamos que duermas una hora más todos los días. Empresa Colaboradora: Asisa Palma de Mallorca. C/ Pere Dezcallar i Net, 10 asisa.es 901 10 10 10 Nada más que tu salud Nada menos que tu salud

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EDITORIAL Medicamentos peligrosos Hazardous drugs Olga Delgado Sánchez Jefe de Servicio de Farmacia Hospital Universitari Son Espases, Palma eISSN 2255-0569 El término Medicamento Peligroso fue introducido en 1990 por la American Society of Hospital Pharmacy (ASHP) y define medicamentos con riesgo carninogénico, teratogénico o genotóxico, entre otros. El Instituto Nacional para la Seguridad y Salud Ocupacional (NIOSH) de Estados Unidos publica periódicamente un listado de Medicamentos Peligrosos, que se actualizó por última vez en 20161. NIOSH introdujo un cambio determinante en 20142 pues, hasta entonces, el listado consistía en una lista de medicamentos citostáticos, pero en este año añade un grupo de medicamentos peligrosos no antineoplásicos, citando medicamentos de uso muy generalizado en las instituciones sanitarias, que hasta ahora se han manipulado sin ninguna precaución especial. Este nuevo listado obligó a revisar el manejo de estos medicamentos en todas las organizaciones en las que se utilizaban, que, aunque ya manipulaban en condiciones de seguridad los tratamientos antineoplásicos, tuvieron que adaptarse e incluir estos medicamentos en un circuito que fuese seguro para el personal sanitario. En Estados Unidos, la U.S.Pharmacopeia Covention (USP) Chapter <800> se ha publicado específicamente para establecer los estándares que se deben seguir para el manejo de Medicamentos Peligrosos en el entorno sanitario3 y que incluyen todo el cirucuito, desde el transporte a la administración, de forma que se garanti- ce un manejo seguro con protección para los trabajadores y para el medio ambiente. En España el Instituto Nacional de Seguridad e Higiene en el Trabajo publicó en septiembre de 2016 su Documento Técnico “Medicamentos Peligrosos: medidas para su preparación y adminsitración”4 en el que se detalla un listado positivo de medicamentos peligrosos utilizados en nuestro país, lo que ha constituido un hito, siendo el primer país que adapta la publicación de NIOSH a la situación de su propio entorno. Por lo tanto, hay normas de manipulación muy concretas, lo que es necesario es información y formación, para incorporar las prácticas seguras en las organizaciones sanitarias, y no es suficiente con realizar procedimientos seguros, también es necesario tener un proceso de implantación, seguimiento y supervisión del cumplimiento de las normas. Es necesario comprender que no existen riesgos nuevos, pero sí de que estamos en un nuevo escenario, que implica nuevas prácticas, nuevos estándares y nuevos equipamientos que garanticen un manejo seguro tanto para el paciente, como para el personal sanitario y para el medio ambiente, otro concepto nuevo que hasta ahora no se había tenido en cuenta, pero que sin duda afecta a la calidad de las prácticas asistenciales sanitarias. Bibliografía 1. NIOSH 2016. List of Antineoplastic and Other Hazardous Drugs in Healthcare Settings. 2016. https://www.cdc.gov/niosh/topics/antineoplastic/pdf/hazardous-drugs-list_2016-161.pdf 2. NIOSH 2014NIOSH [2014] List of Antineoplastic and Other Hazardous Drugs in Healthcare Settings, 2014. http://www.cdc.gov/niosh/ docs/2014-138/pdfs/2014-138.pdf 3. General Chapter <800> Hazardous Drugs—Handling in Healthcare Settings. United States Pharmacopeia. USP 2016 http://www.usp. org/usp-nf/notices/general-chapter-hazardous-drugs-handlinghealthcare-settings 4. Documento Técnico Medicamentos peligrosos. Medidas de pre- vención para su preparación y administración. Instituto Nacional de Seguridad e Higiene en el Trabajo (INSHT) del Ministerio de Empresa y Seguridad Social. Septiembre 2016. http://www.insht.es/portal/site/ Insht/menuitem.1f1a3bc79ab34c578c2e8884060961ca/?vgnextoi d=37ea6cec26747510VgnVCM1000008130110aRCRD&vgnextch annel=25d44a7f8a651110VgnVCM100000dc0ca8c0RCRD Medicina Balear 2017; 32 (1): 9 9

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ORIGINAL eISSN 2255-0569 Effects of sex hormones disruption, after prenatal and postnatal exposure to chlordimeform, on monoaminergic neurotransmitters systems in female and male rat’s prefrontal cortex Efectos de la disrupción de las hormonas sexuales, tras la exposición prenatal y posnatal al Clordimeformo, sobre los sistemas de neurotransmisores monoaminérgicos en la corteza prefrontal de ratas macho y hembra José Manuel García1, Paula Moyano1, María Teresa Frejo2, María José Anadón1, Miguel Andrés Capó2, Gloria Gómez1, Javier del Pino2 1. Departamento de Toxicología y Legislación Sanitaria, Facultad de Medicina, Universidad Complutense of Madrid, Spain. 2. Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense of Madrid, Spain. Correspondencia Javier del Pino PhD Facultad de Veterinaria. Universidad Complutense de Madrid Departamento de Toxicología y Farmacología Avda. Puerta de Hierro s/n · 28040, Madrid Teléfono: 913 550 920 E-mail: jdelpino@pdi.ucm.es Recibido: 21 – XI – 2016 Aceptado: 22 – XII – 2016 doi: 10.3306/MEDICINABALEAR.32.01.11 Abstract Introduction: Chlordimeform, as well as other formamidine pesticides, has been described to induce permanent sex- and regiondependent effects on development of monoaminergic neurotransmitter systems. The mechanisms that induce these effects are not known, but it has been suggested that these effects could be related to monoamine oxidase (MAO) inhibition. However, chlordimeform is a very weak MAO inhibitor, which suggest that other mechanism should be involved. In this regard, formamidines, in general, and chlordimeform, in particular, alter the serum levels of steroid hormones, which regulate the expression of enzymes that mediate the synthesis and metabolism of monoaminergic neurotransmitters systems. Therefore, an alteration of these hormones in the brain could mediate the effects observed. Objectives and methods: In order to confirm that the formamidines produce permanent alterations of the monoamine neurotransmitter systems, through disruption of sex hormones in the brain, by alteration of the expression of the enzymes that synthesize and/ or metabolize these neurotransmitters, we evaluated, in frontal cortex of male and female rats, the effect on the levels of testosterone and estradiol at 11 days of age, as well as the expression of MAO, COMT, BDH, TH, TRH, and AD enzymes at 60 days of age after maternal exposure to chlordimeform (5 mg/kg body weight). Results: Chlordimeform induced a significant decrease in testosterone and estradiol levels in frontal cortex of rats at 11 days of age. We observed sex interaction with treatment in the content of T and E2. We determined a bigger increase in the expression of TH [35,66% (P<0,001)] and TRH [42,14% (P<0,001)] enzymes in males than in females. Chlordimeform treatment did not alter the expression of MAO, COMT, AD, BDH enzymes, but decreased the expression of the enzymes TRH TH in both males and females. Conclusions: The present findings indicate that after maternal exposure to formamidines, in general, and chlordimeform, in particular, a permanent alteration of monoaminergic neurotransmitters, through alteration of the enzymes that synthetize these neurotransmitters, mediated by sex hormones disruption in frontal cortex is induced. Keywords: Chlordimeform; formamidines; neurodevelopmental toxicity; TH; TRH; rats; human risk assessment Resumen Introducción: Se ha descrito que el clordimeformo, así como otros plaguicidas formamidínicos, induce alteraciones permanentes de los sistemas de neurotransmisores monoaminérgicos región y sexo dependiente. Los mecanismos por los que se inducen estos efectos no se conocen, pero se ha sugerido que podrían estar relacionados con la inhibición de la monoamino oxidasa (MAO). Sin embargo, el clordimeformo es un inhibidor muy débil de la MAO lo que sugiere que otro mecanismo debería estar implicado. En este sentido, se ha descrito que las formamidinas en general y el clordimeformo en particular, alteran los niéveles séricos de distintas hormonas las cuales regulan la expresión de las enzimas que sintetizan y metabolizan estos neurotransmisores. Por lo tanto, una alteración de estas hormonas a nivel cerebral podría mediar los efectos observados. Objetivos y métodos: Con el objetivo de confirmar que las formamidinas produce alteraciones permanentes de los neurotransmisores monoaminérgicos, a través de la disrupción de las hormonas sexuales a nivel cerebral por alteración de la expresión de las enzimas que sintetizan y/o metabolizan estos neurotransmisores, se evaluaron los efectos, en la corteza frontal de ratas macho y hembra, sobre los niveles de testosterona y estradiol a los 11 días de edad, así como sobre la expresión de las enzimas MAO, COMT, BDH, TH , TRH, y AD a los 60 días de edad tras la exposición maternal al clordimeformo (5 mg/kg de peso corporal). Medicina Balear 2017; 32 (1): 11-19 11

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Javier del Pino et al. Resultados: El clordimeformo indujo una disminución significativa de los niveles de testosterona y estradiol en la corteza frontal de las ratas descendientes a la edad de 11 días. Se observó una interacción por sexo con el tratamiento en el contenido de T y E2. Además se observó una mayor expresión de las enzimas TH [35,66% (P<0,001)] y TRH [42,14% (P<0,001)] en los machos que en las hembras. El tratamiento con clodimeformo no alteró la expresión de las enzimas MAO, COMT, AD, BDH, pero disminuyó la expresión de las enzimas TH y TRH tanto en machos como en hembras. Conclusiones: Los presentes resultados indican que las formamidinas, en general, y el clordimeformo, en particular, inducen, tras la exposición maternal, una alteración permanente de los sistemas de neurotransmisores monoaminergicos en la corteza frontal, a través de la alteración de las enzimas que sintetizan estos neurotransmisores, mediada por la alteración de las hormonas sexuales. Palabras clave: Clordimeformo; formamidinas; neurotoxicidad en el desarrollo; testosterona; estradiol; TH, TRH, ratas; evaluación del riesgo para el hombre Introduction Formamidine pesticides have been reported to induce permanent alteration of brain development. In this regard, the formamidine compound amitraz has been described to produce the induction of permanent alterations on the development of central nervous system (CNS) such as those that affect monoamine neurotransmitter systems1. Moreover, chlordimeform [N2-(4-chloro-o-tolyl)-N1. N1-dimethylformamidine] (Figure 1), which is another member of formamidines family, has also been reported to induce permanent alterations of serotoninergic, noradrenergic and dopaminergic systems2,3. The mechanism by which these effects occur is not known. Figure 1: Chlodimeform chemical structure (C10H13Cl N2). CH3 Cl N= CH - N CH3 CH3 droxylase (DBH), tryptophan hydroxylase (TRH), MAO, catechol-O-metyltransferase (COMT), aldehyde dehydrogenase (AD), aldehyde reductase (AR) required for synthesis and metabolism of these neurotransmitters11-18. In this regard, chlordimeform is an endocrine disruptor that alters prolactin and adrenocorticotropic hormones, among others19, and amitraz alters testosterone and estradiol serum hormone levels in rats20. Therefore, if chordimeform, as well as other formamidine pesticides, alters sex hormones in the brains, this could mediate the effect observed on monoaminergic neurotransmitters systems. According to all exposed above, we performed a study to establish if maternal exposure to formamidines during gestation and lactation induces permanent alterations on the enzymes that synthetize and metabolize 5-HT, NE and DA neurotransmitters in adult age, through sex hormones disruption. Chlordimeform was chosen because it is the most representative compound in its group, which presents a very low inhibition of MAO, allowing us to study more clearly whether the permanent changes observed on levels of these neurotransmitters are due to an alteration of the enzymes that catalyze the synthesis and metabolism of these neurotransmitter rather than inhibition of MAO. Currently, it is assumed that the monoaminergic neurotransmitters play a role during development, defined as “morphogenetic” 4-7. Any change in the levels of catecholamines during development could have a profound effect on brain development, both structural and functional8. In this sense, monoamine oxidase (MAO) inhibition was among the first biochemical actions of the formamidines to be reported9-10 and has been suggested as a possible mechanism of action, because neuronal MAO participates in metabolic inactivation of biogenic monoamines, which include the neurotransmitters serotonin (5-HT), norepinephrine (NE), and dopamine (DA). However, chlordimeform is a very weak MAO inhibitor, which suggest other mechanisms are involved in these effects. Alternatively, the changes in NE, DA and 5-HT and its metabolites levels observed in rats’ brain after formamidines exposure could be attributed to a possible effect on sex steroid hormones that modulate the expression of enzymes such as tyrosine hydroxylase (TH), dopamine-β-hy- This work focuses its interest in providing new data of formamidines induced neurotoxicity during nervous system development, because new compounds of this family are being developed with therapeutic applications for which these effects are not considered in their risk assessment, which poses a potential health hazard. Material and methods Biological material All experiments were performed in accordance with European Union guidelines (2003/65/CE) and Spanish regulations (BOE 67/8509-12, 1988) regarding the use of laboratory animals. Eight pregnant Wistar rats were housed individually in polycarbonate cages and were assigned randomly to two experimental groups: a chlordimeform treatment group (n = 4) and a control group (n = 4). Test Chemical and Treatment Chlordimeform (Sigma, Madrid, Spain) was dissolved in 12 Medicina Balear 2017; 32 (1): 11-19

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Effects of sex hormones disruption, after prenatal and postnatal exposure to chlordimeform, on monoaminergic neurotransmitters systems in female and male rat’s prefrontal cortex corn oil to provide fast and complete absorption and was administered orally by gavage in a volume of 2 mg/ml. The animals received daily chlordimeform at the dose of 5 mg/kg on days 6 to 21 of pregnancy (GD 6-21) and on days 1 to 10 of lactation (PN 1-10). Control dams received vehicle (corn oil 2.5 ml/kg) on the same schedules. Dose of chlordimeform was selected based on a previous preliminary study that indicated that this dose was the higher one that did not cause weight loss or mortality, reduction of food or water intake as well as did not induce haematological modifications of other clinical histopathological signs of overt toxicity. None of the prenatal or postnatal treatment evoked a significant change in weight of any of the brain regions on PN 60 (data not shown). Dams were examined daily throughout the gestation and lactation periods for mortality, general appearance and behaviour. The maternal body weights were measured on GD 1, GD 5, GD 6, GD 15 and GD 20. Food and water consumption during pregnancy, length of gestation, litter size and sex ratio were also assessed. On PN1, all litters were examined externally, sexed and weighed. Litters were organized in groups of twenty-four pups, twelve males and twelve females. Litters were weighed at PN 1, PN 7, PN14 and PN 21. The offspring were weaned on lactation day 21 and were maintained in appropriate conditions, housed individually and without any treatment with full access to food and water until adult age. The study was organized in treated groups of six males and six females randomly selected respectively from the dams’ litters exposed to chlordimeform, and control groups of six males and six female’s pups randomly selected respectively from the control dams’ litters. At PN11, for the analysis of testosterone and estradiol’s brain levels and at PN 60, for the analysis of MAO A, MAO B, COMT, BDH, AD, TH and TRH gene expression, male and female rats from control and treated groups (pups from control dams, and pups from dams exposed to chlordimeform, respectively) were sacrificed by decapitation. The brain was removed quickly and the frontal cortex was a rapidly dissected out at 4ºC21, since this brain region was previously describe to present sex differences in the effect observed on these neurotransmitters systems and to be one of the most affected2,3. Tissues were rapidly weighed and stored at -80ºC until analysis. All data were collected by experimenters blind to the treatment condition of the offspring. Estradiol and testosterone quantification Estradiol and testosterone content were measured in prefrontal cortex from treated animals in order to determine whether sex hormones are altered by chlordimeform exposure. Estradiol and testosterone content in the prefrontal cortex was measured using an enzyme immunoassay kit (Estradiol EIA Kit, Cayman Chemical Compa- ny, MI, USA), according to the manufacturer’s instruction. Tissues were homogenized in 300 μl of an equal mixture of ethyl acetate and 0.1 M phosphate-buffered saline. The homogenates were centrifuged at 21,000 g for 15 min at 4ºC. The resulting mixture was then incubated in a MeOH/dry ice bath to solidify the aqueous phase (bottom) and the organic phase was eluted into a new tube. The ethyl acetate portion was collected and dried. The dried material was reconstituted in 120 μl EIA buffer, and 100 μl of the sample was used for EIA at duplicate. ELISA values were obtained (pg/ml) and corrected for weigh tissue (mg/ml), producing a final unit of pg/mg and presented as a percentage of the untreated control. Real-time PCR analysis The MAOA, MAOB, COMT, AD, TH, TRH and DBH expression was measured in frontal cortex tissue from control and chlordimeform treated animals in order to determine whether chlordimeform, through sex hormones disruption, alters permanently the expression of these enzymes. Total RNA was extracted using the Trizol Reagent method (Invitrogen, Madrid, Spain). The final RNA concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Madrid, Spain), and the quality of total RNA samples was assessed using an Experion LabChip (Bio-Rad, Madrid, Spain) gel. First-strand cDNA was synthesized with 1000 ng of cRNA by using a PCR array first strand-synthesis kit (C-02; SuperArray Bioscience, Madrid, Spain) in accordance with the manufacturer’s instructions and including a genomic DNA elimination step and external RNA controls. After reverse transcription, QPCR was carried out using prevalidated primer sets (SuperArray Bioscience) for mRNAs encoding MAOA (PPR46359A), COMT (PPR06789A), AD (PPPR43520B), TH (PPR45220F), TRH (PPR48244A), DBH (PPR52652A), and ACTB (PPM02945B). ACTB was used as an internal control for normalization. Reactions were run on a CFX96 using Real-Time SYBR Green PCR master mix PA-012 (SuperArray Bioscience). The thermocycler parameters were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 72°C for 30 seconds. Relative changes in gene expression were calculated using the Ct (cycle threshold) method. The expression data are presented as actual change multiples22. Data analysis Statistical analysis of data was performed using a Statgraphics software, version Plus 4.1 for windows. Values are expressed as mean ± S.E.M. obtained from 12 animals, six males and six females, in each group (control and treated groups). For values combined for males and females, a two-way ANOVA with treatment × sex interaction was the initial test used. Where a significant treatment × sex interaction was detected, a separate Student’s t test was carried out for each sex. The results were considered significant at P<0.05. Results significantly different from controls are also presented as change from control (%). Medicina Balear 2017; 32 (1): 11-19 13

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Javier del Pino et al. Results Maternal and offspring body weight, physical and general activity development were unaffected by the exposure of dams to chlordimeform (5 mg/kg bw orally on days 6 to 21 of pregnancy and 1 to 10 of lactation). Estradiol and testosterone quantification Oral treatment with chlordimeform to dams during the gestation period from day 6 to day 21 and during lactation from day 1 to day 10 affected the content of T and E2 in the region of frontal cortex of rats offspring at the age of 11 days. The content of T (ng/g tissue) in the region of frontal cortex in the control group and treated group is presented in table I. The content of E2 (ng/g tissue) in the region of the frontal cortex of the control group and the treated group is presented in table II. The results expressed in tables I and II show that in 11 days old rats treated during gestation days 6-21 and during lactation days 1-10 through their mothers, a statistically significant loss of E2 and T content in the frontal cortex compared to control animals was pro- duced. A sex interaction with treatment in the content of T and E2 was observed (Figure 2). In frontal cortex the loss observed of E2 content was 46.53% (P <0.01) and 57.39% (P <0.001) in males and females, respectively, and the loss in the content of T was 13.44% (P <0.001) and 20.70% (P <0.001) in males and females, respectively (Figure 2). Real-time PCR analysis Oral treatment with chlordimeform to dams during the gestation period from day 6 to day 21 and during lactation from day 1 to day 10 affected the TH and TRH gene expression of rats offspring at the age of 60 days. In 60 days old rats treated during gestation days 6-21 and during lactation days 1 to 10 a decrease in the expression of TH and TRH enzymes in frontal cortex with respect to control animals was observed. No effect on gene expression of MAO, COMT, BDH and AD enzymes was observed (Figure 3). A sex difference in TH and THR gen expression was observed, being higher the expression of TH [35,66% (P<0,001)] and THR [42,14% (P<0,001)] in males than females rats (Figure 4). Table I: Tissue T (pg/ml) content determined in frontal cortex from male and female rats at 11 days of age treated with vehicle or chlordimeform (5 mg/kg bw orally on days 6 to 21 of pregnancy and 1 to 10 of lactation). Frontal Cortex Animal Control group Treated group Control group Treated group Males Males Females Females 1 2 3 4 5 6 397,56 344,56 403,76 310,56 401,45 348,62 395,91 321,65 385,87 341,67 387,76 306,75 396,74 338,59 389,56 311,39 399,50 331,99 408,75 318,95 388,71 345,81 401,64 323,84 Mean ± 394,97±2,54*** 341,87±2,43*** 397,90±3,38*** 315,52±2,81*** SEM (-13,44%) (-20,70) Values are mean ± S.E.M.; control animals (n= 6 males, n= 6 females); treated group (n= 6 males, n= 6 females). Statistical significance is reported for the ***P<0.001 levels compared with the control group. Table II: Tissue E2 (pg/ml) content determined in frontal cortex from male and female rats at 11 days of age treated with vehicle or chlordimeform (5 mg/kg bw orally on days 6 to 21 of pregnancy and 1 to 10 of lactation). Frontal Cortex Animal Control group Treated group Control group Treated group Males Males Females Females 1 2 3 4 5 6 132,56 73,98 128,65 55,44 133,65 69,44 125,74 51,76 136,59 64,32 131,78 48,87 129,54 75,93 129,75 60,76 128,65 72,59 134,87 57,53 131,72 67,62 126,64 56,89 Mean ± 132,12±1,17*** 70,65±1,76*** 129,57±1,38*** 55,21 ±1,74*** SEM (-46,53%) (-57,39%) Values are mean ± S.E.M.; control animals (n= 6 males, n= 6 females); treated group (n= 6 males, n= 6 females). Statistical significance is reported for the ***P<0.001 levels compared with the control group. 14 Medicina Balear 2017; 32 (1): 11-19

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Effects of sex hormones disruption, after prenatal and postnatal exposure to chlordimeform, on monoaminergic neurotransmitters systems in female and male rat’s prefrontal cortex Figure 2: Tissue T and E2 (pg/ml) content determined in frontal cortex from male and female rats at 11 days of age treated with vehicle or chlordimeform (5 mg/kg bw orally on days 6 to 21 of pregnancy and 1 to 10 of lactation). 150 Males Females 150 Males Females E2 levels (% Control) T levels (% Control) 100 100 *** 50 50 0 Frontal Cortex 0 Frontal Cortex T levels (% Control) 150 Control CMZ MALES 100 *** 50 0 Frontal Cortex T levels (% Control) Control CMZ 150 FEMALES 100 *** 50 0 Frontal Cortex E2 levels (% Control) 150 Control CMZ MALES 100 50 *** 0 Frontal Cortex E2 levels (% Control) 150 Control CMZ FEMALES 100 50 *** 0 Frontal Cortex Figure 3: Sex difference results from real-time PCR targeting MAO, COMT, BDH, AD, TH y TRH genes after chlordimeform treatment in male and female rats. MAO, COMT, BDH, AD, TH y TRH gene expression was compared to male rats results. Each bar represents mean ± SD of 6 samples. Levels were measured using QPCR. ACTB was used as an internal control. ***p ≤ 0.001,**p ≤ 0.01, significantly different from males. mRNA levels (% Males) 200 Males Females 150 MAO A 100 50 0 Frontal Cortex mRNA levels (% Males) 200 Males Females 150 COMT 100 50 0 Frontal Cortex mRNA levels (% Males) 200 Males Females 150 BDH 100 50 0 Frontal Cortex mRNA levels (% Males) 200 Males Females 150 AD 100 50 0 Frontal Cortex mRNA levels (% Males) 200 Males Females 150 TH 100 *** 50 0 Frontal Cortex mRNA levels (% Males) 200 Males Females 150 THR 100 *** 50 0 Frontal Cortex Medicina Balear 2017; 32 (1): 11-19 15

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