Evaluación in vitro de la inducción proliferativa de organoclorados y de sus mezclas, mediante la aplicación del ensayo E-Screen

Los compuestos tóxicos, persistentes y bioacumulables son de gran interés en la evaluación de riesgos para la salud del ser humano y de las especies animales. Entre ellos, destaca el interés por los pesticidas organoclorados y los bifenilospoliclorados (PCB). Su presencia ha sido continua durante décadas en todos los niveles de las cadenas tróficas; sin embargo, el interés es creciente en las especies ubicadas en las partes más altas de las cadenas alimentarias (incluido el ser humano), pues son integradores de la exposición en los eslabones inferiores. Las grandes especies de rapaces se ubican en las partes más altas de sus redes tróficas, al mismo nivel que el ser humano, siendo así consideradas muchas de ellas, ideales centinelas de riesgos ambientales. Estudios previos de biomonitorización de pesticidas organoclorados y PCB realizados en diversas especies de rapaces por el grupo de investigación de Toxicología de la Universidad de Murcia habían detectado de forma continua presencia este tipo de contaminantes (entre otros) en sangre y otras muestras de estas aves. Sin embargo, la presencia de un compuesto en un ser vivo no es suficiente para explicar efectos deletéreos o indeseables en ellos. Por otro lado, estos compuestos han sido ampliamente estudiados por su potencial efecto perturbador endocrino; quedando aún lagunas sobre ciertos efectos y las condiciones de exposición (dosis, mezclas, etc). El objetivo de este trabajo es el estudio in vitro de la alteración de la proliferación celular, parámetro vinculado a la actividad estrogénica y englobada en la función endocrina, sobre la línea celular MCF-7 por la exposición a concentraciones subletales de 14 pesticidas y 4 PCB y 8 de sus mezclas más frecuentemente observadas en estudios de biomonitorización en rapaces. Para ello, se aplicó el ensayo E-Screen, ensayo in vitro de proliferación celular sobre la línea MCF-7 (Soto et al., 1994, 1995) para la exposición a dichos organoclorados frecuentemente monitorizadas en grandes rapaces. El E-Screen es un ensayo de 6 días de duración que emplea la línea celular MCF-7, capaz de expresar endógenamente receptores estrogénicos sobre los que pueden actuar ciertos perturbadores endocrinos. Se estudió representantes de los siguientes grupos: difenilalifáticos (p,p’-DDT, p,p’-DDE, p,p’-DDD y dicofol), ciclodiénicos (aldrín, dieldrín, endrín, endrín aldehído, endosulfánαβ, endosulfán sulfato, heptacloro y heptacloro epóxido), hexaclorociclohexanos o HCH (γ-HCH y lindando técnico) y PCB (PCB# 118, PCB# 126, PCB# 153, y PCB# 180). En cuanto a las ocho mezclas diseñadas en base a los estudios de campo previos, se agrupan en mezclas binarias[MB h/c/: lindano técnico+ endosulfán αβ (50:50); MB d/d/: p,p’-DDT+p,p’-DDE (50:50); MBc/d/1/: endosulfán αβ+p,p’-DDE (75:25); MB c/d/2/:endosulfán αβ +p,p’-DDT (75:25)] mezclas ternarias [MT c/d/d/: endosulfán αβ + p,p’-DDT + p,p’-DDE (75:12,5:12,5); MT h/d/d/ :lindano técnico + p,p’-DDT + p,p’-DDE (75:12,5:12,5)], una mezcla cuaternaria [MC c/c/d/d/: aldrín + dieldrín + p,p’-DDT + p,p’-DDE (25:25:25:25)] y una senaria [{MS c/c/c/d/d/h/: endosulfán αβ+aldrín + dieldrín + p,p’-DDT + p,p’-DDE + lindano técnico (50:12,5:12,5:6,25:6,25:12,5)]. Se abarcó un rango de 7 concentraciones (desde 1,0-5M hasta 1,0E-11M para los compuestos individuales, variando en las mezclas hasta 3,0E-13M en algunos casos), empleando como control positivo el 17β-estradiol y DMSO como solvente. En función del efecto proliferativo relativo (RPE) obtenido, se clasificó cada compuesto y mezcla según la categorización pre-establecida en la bibliografía como débilmente estrogénicos, parcial y fuertemente estrogénicos (RPE al 75%, respectivamente). Los análisis de los compuestos evaluados individualmente, revelaron que todos ellos dieron lugar a una intensa proliferación celular, puesto que no se detectaron efectos débilmente estrogénicos. Las concentraciones 1,0E-9M y 1,0E-8M fueron las responsables del máximo efecto observado en la mayoría de los análisis. El ciclodiénicoaldrín fue el OC que indujo mayor proliferación, tanto de los ensayos individualizados como de las mezclas. Por otro lado, al menos una concentración de cada uno de los difenilalifático se catalogó como fuertemente estrogénico, en contraposición con los HCH, ya que tan sólo el lindano técnico demostró ser fuertemente estrogénico. Los PCB se caracterizaron por la diversidad de respuesta, pudiendo aparecer valores por debajo del control negativo y fuertemente estrogénicos en el mismo producto. En cuanto a las mezclas, destaca el menor RPE respecto a los análisis individuales. Entre las mezclas binarias se encuentra la mezcla más proliferativa y la de menor efecto. Por su parte, la mezcla más compleja (conformada por 6 compuestos) dio lugar a un RPE máximo del 33%. Asimismo, se comprobó que el efecto de las mezclas, no siempre se igualaba al de sus integrantes a pesar de que pudieran estar compuestos mayoritariamente por OC fuertemente estrogénicos. Con carácter general, se observó una variación del efecto sin un patrón definido a lo largo del rango analizado, por lo que un compuesto/mezcla puede ser catalogado de distinta manera en función de la concentración escogida. Este hecho, dificulta la comparación de datos entre estudios, ya que no siempre se dispone de toda la iformación deseable. Los resultados de este trabajo aportan luz sobre el efecto estrogénico inducido por la exposición a organoclorados y diferentes mezclas ambientalmente posibles; sin embargo, es preciso profundizar aún más en los potenciales efectos estrogénicos de las mezclas y los mecanismos que los explican. Bioaccumulable, persistent, toxic compounds are of particular interest when assessing risks to the health of humans and animal species. Of these compounds, organochloride pesticides and polychlorinated biphenyls (PCB) stand out as especially interesting. They have been continuously present for decades in all levels of the trophic chain, although there is a growing interest in species in the upper links of the food chain (including human beings), as they integrate the exposure of the lower links. The large species of birds of prey are at the top of their food webs, on the same level as human beings, and many of them are therefore considered to be ideal sentinels of environmental risks. Previous bio-monitoring studies of organochloride pesticides and PCBs carried out on different species of birds of prey by the research group of the University of Murcia Toxicology Department had detected a continuous presence of this type of contamination (among others) in blood and other samples taken from these birds. However, the mere presence of a compound in a living organism is not enough to explain any harmful and undesirable effects in it. These compounds have also been widely studied in relation to their potential endocrine-disrupting effect; and there are still some gaps in our understanding of certain effects and the conditions of exposure (dose, mixtures, etc.). The aim of this thesis is the in vitro study of the alteration of cell proliferation, a parameter linked to oestrogenic activity and encompassed by the endocrine function, on the MCF-7 cell line due to exposure to sub-lethal concentrations of 14 pesticides, 4 PCB and 8 mixtures of them most commonly observed in bio-monitoring studies of birds of prey. To do this, we used the E-Screen assay, an in vitro assay of the cell proliferation on the MCF-7 cell line for the exposure to the aforementioned organochlorides frequently monitored in large birds of prey. The E-Screen is a 6-day assay that employs the MCF-7 cell line, which is able to endogenously express the oestrogen receptors on which certain endocrine-disruptors act. Representatives of the following groups were studied: Diphenyl aliphatics (p,p’-DDT, p,p’-DDE, p,p’-DDD and dicofol), cyclodienes (aldrin, dieldrin, endrin, endrin aldehyde, endosulfan αβ, endosulfan sulphate, heptachlor and heptachlor epoxide), hexachlorocyclohexane or HCH (γ-HCH and technical lindane) and PCB (PCB# 118, PCB# 126, PCB# 153, and PCB# 180). The eight mixtures designed as a result of the prior field studies are grouped in binary mixtures [MB h/c/: technical lindane + endosulfan αβ (50:50); MB d/d/: p,p’-DDT+p,p’-DDE (50:50); MBc/d/1/: endosulfan αβ+p,p’-DDE (75:25); MB c/d/2/:endosulfan αβ +p,p’-DDT (75:25)] ternary mixtures [MT c/d/d/: endosulfan αβ + p,p’-DDT + p,p’-DDE (75:12,5:12,5); MT h/d/d/:technical lindane + p,p’-DDT + p,p’-DDE (75:12,5:12,5)], a quaternary mixture [MC c/c/d/d/:aldrin + dieldrin + p,p’-DDT + p,p’-DDE (25:25:25:25)] and a senary one [{MS c/c/c/d/d/h/: endosulfan αβ+aldrin + dieldrin + p,p’-DDT + p,p’-DDE + technical lindane (50:12,5:12,5:6,25:6,25:12,5)]. A range of 7 concentrations was covered (from 1.0-5M to 1.0E-11M for the individual compounds, with the mixtures varying up to 3.0E-13M in some cases), employing as a positive control 17β-oestradiol and DMSO as a solvent. Depending on the relative proliferative effect (RPE) obtained, each compound and mixture was classified according to the categories pre- established in the literature as weakly oestrogenic, partially oestrogenic and fully oestrogenic (RPE < 25%, between 25 and 75 %, and > 75%, respectively). The analysis of the compounds assessed individually revealed that all of them led to an intense cell proliferation, as no weak oestrogenic effects were detected. The 1.0E-9M and 1.0E-8M concentrations were responsible for the maximum effect observed in most of the analyses. The aldrin cyclodiene was the OC that induced the greatest proliferation, both in the individual assays and the mixtures. At least one concentration of each of the diphenyl aliphatics was categorised as fully oestrogenic, unlike the HCH, as only the technical lindane was demonstrated to be fully oestrogenic. The PCBs were characterised by the diversity of their responses – the values could be below the negative control and be fully oestrogenic. As regards the mixtures, we can highlight the lower RPE in relation to the individual analyses. Among the binary mixtures we find the most proliferative mixture and the least effect. The most complex mixture (comprising 6 compounds) resulted in a maximum RPE of 33%. It was also observed that the effect of the mixtures was not always equal to that of their integral parts, in spite of the fact they could be mostly made up of fully oestrogenic OCs. In general, we observed a variation of the effect without a defined pattern throughout the range analysed, which means a compound/mixture can be categorised differently depending on the chosen concentration. This fact makes it more difficult to compare the data of different studies, as the information required is not always available. The results of this study throw light on the oestrogenic effect induced by exposure to organochlorides and different possible environmental mixtures. However, a more in-depth study needs to be made of the potential oestrogenic effects of mixtures and the mechanisms that explain them

HTLV-1 infection in solid organ transplant donors and recipients in Spain

HTLV-1 infection is a neglected disease, despite infecting 10-15 million people worldwide and severe illnesses develop in 10% of carriers lifelong. Acknowledging a greater risk for developing HTLV-1 associated illnesses due to immunosuppression, screening is being widely considered in the transplantation setting. Herein, we report the experience with universal HTLV testing of donors and recipients of solid organ transplants in a survey conducted in Spain. All hospitals belonging to the Spanish HTLV network were invited to participate in the study. Briefly, HTLV antibody screening was performed retrospectively in all specimens collected from solid organ donors and recipients attended since the year 2008. A total of 5751 individuals were tested for HTLV antibodies at 8 sites. Donors represented 2312 (42.2%), of whom 17 (0.3%) were living kidney donors. The remaining 3439 (59.8%) were recipients. Spaniards represented nearly 80%. Overall, 9 individuals (0.16%) were initially reactive for HTLV antibodies. Six were donors and 3 were recipients. Using confirmatory tests, HTLV-1 could be confirmed in only two donors, one Spaniard and another from Colombia. Both kidneys of the Spaniard were inadvertently transplanted. Subacute myelopathy developed within 1 year in one recipient. The second recipient seroconverted for HTLV-1 but the kidney had to be removed soon due to rejection. Immunosuppression was stopped and 3 years later the patient remains in dialysis but otherwise asymptomatic. The rate of HTLV-1 is low but not negligible in donors/recipients of solid organ transplants in Spain. Universal HTLV screening should be recommended in all donor and recipients of solid organ transplantation in Spain. Evidence is overwhelming for very high virus transmission and increased risk along with the rapid development of subacute myelopathy

All-cause mortality in the cohorts of the Spanish AIDS Research Network (RIS) compared with the general population: 1997Ł2010

Abstract Background: Combination antiretroviral therapy (cART) has produced significant changes in mortality of HIVinfected persons. Our objective was to estimate mortality rates, standardized mortality ratios and excess mortality rates of cohorts of the AIDS Research Network (RIS) (CoRIS-MD and CoRIS) compared to the general population. Methods: We analysed data of CoRIS-MD and CoRIS cohorts from 1997 to 2010. We calculated: (i) all-cause mortality rates, (ii) standardized mortality ratio (SMR) and (iii) excess mortality rates for both cohort for 100 personyears (py) of follow-up, comparing all-cause mortality with that of the general population of similar age and gender. Results: Between 1997 and 2010, 8,214 HIV positive subjects were included, 2,453 (29.9%) in CoRIS-MD and 5,761 (70.1%) in CoRIS and 294 deaths were registered. All-cause mortality rate was 1.02 (95% CI 0.91-1.15) per 100 py, SMR was 6.8 (95% CI 5.9-7.9) and excess mortality rate was 0.8 (95% CI 0.7-0.9) per 100 py. Mortality was higher in patients with AIDS, hepatitis C virus (HCV) co-infection, and those from CoRIS-MD cohort (1997. Conclusion: Mortality among HIV-positive persons remains higher than that of the general population of similar age and sex, with significant differences depending on the history of AIDS or HCV coinfection

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p &lt; 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Segureño ovine transhumance: an opportunity for veterinary innovation teaching

La importancia de la producción de cordero en el Sureste, la existencia de un núcleo de trashumantes de ovino en la Sierra de Segura, y la consideración de la trashumancia como manifestación representativa del patrimonio cultural inmaterial nos ha llevado a explorar el potencial docente de la trashumancia con ovino segureño para estudiantes de veterinaria. Entre 2016 y 2019, cuatro profesores y dieciocho estudiantes de veterinaria de las Universidades de Murcia y Cardenal Herrera (UCH-CEU) de Valencia, hemos realizado diecinueve jornadas de trashumancia. Entre las acciones de innovación docente generadas destaca la exposición fotográfica “La Vereda de los Pontoneros” y el documental “Trashumancia, la Última Frontera”. Proponemos la integración de una semana de trashumancia en el periodo de las dos semanas de prácticas tuteladas con la Asociación Nacional de Criadores de Ovino Segureño (ANCOS), discutimos su integración en el grado de Veterinaria de Murcia y evidenciamos el interés docente de esta actividad inmersiva, que supera los propios contenidos del grado de veterinariaThe importance of lamb production in the Southeast, the existence of ovine trashumants in Sierra de Segura and the consideration of transhumance as a representative manifestation of the intangible cultural heritage has leaded us to explore the teaching potential of transhumance with segureño sheep for veterinary students. Bet-ween 2016 and 2018, four professors and eighteen veterinary students from the University of Murcia and the University CEU Cardenal Herrera of Valencia participated during nineteen days in transhumance. Within the generated teaching innovation activities highlights the photographic exposition “La Vereda de los Pontoneros” and the documentary “Trashumancia, la última frontera”. We propose the integration of a week of transhuman-ce within the practical period with the Asociación Nacional de Criadores de Ovino Segureño (ANCOS), we discuss its inclusion in the Veterinary Degree program of the University of Murcia and we evidence the teaching interest of this immersive activity, which widely approach others fields than the veterinary