Efeitos do crómio-cobre-arsenato (CCA) em ratinhos : estudos morfológicos e analíticos

Mestrado em ToxicologiaO Crómio-Cobre-Arsenato (CCA) é um protector da madeira, contendo crómio, cobre e arsénio. Apesar de em 2002, a agência de protecção ambiental (EPA) ter limitado a utilização da madeira tratada com CCA, a contaminação do ambiente e o perigo para a saúde pública permanece, devido à grande durabilidade desta madeira. O principal objectivo deste trabalho foi analisar a toxicidade aguda do CCA em ratinhos. O desenvolvimento e optimização de uma metodologia de quantificação do arsénio e do crómio total nos rins de ratinhos foi também um outro objectivo. Realizaram-se experiências para caracterizar os efeitos causados pelo CCA e pelos compostos individuais (pentóxido de arsénio – As2O5) e (trióxido de crómio – CrO3) em vários órgãos de ratinhos. Os animais foram divididos em dezasseis grupos, correspondendo a diferentes tempos de exposição. Os grupos de tratamento receberam uma injecção subcutânea (0,3 mL) de CCA, CrO3 e As2O5. Aos animais controlo foi administrado o veículo (soro fisiológico). Após 14h, 24h, 48h e 96h procedeu-se ao abate dos animais e à remoção dos rins, timo, fígado, baço, testículo e epidídimo. Realizaram-se análises histológicas e histoquímicas dos rins de animais provenientes de todos os grupos. Procedeu-se a estudos de citometria de fluxo (FCM) para a análise do ciclo celular. Efectuou-se ainda um estudo preliminar para determinação da actividade da catalase (CAT) e da glutationa – S – transferase (GST) em amostras de rim. Por fim, realizou-se uma análise quantitativa destes compostos por espectrometria de absorção atómica com atomização electrotérmica (GFAAS), após a digestão das amostras pela tecnologia de microondas. O estudo histológico revelou necrose tubular aguda (ATN) nos animais expostos ao CCA e ao CrO3. Contudo, não se observaram quaisquer lesões significativas nos outros órgãos. Por esta razão, o estudo foi direccionado para o rim. A análise por citometria de fluxo (FCM) evidenciou a existência de diferenças significativas no ciclo celular dos grupos expostos ao As2O5 e ao CCA. Os ensaios bioquímicos revelaram uma tendência para a diminuição da actividade das enzimas CAT e GST, embora sem diferenças estatísticas significativas. A análise quantitativa demonstrou uma diminuição progressiva da acumulação de crómio e de arsénio nos rins. Por outro lado, observou-se que a distribuição de crómio e de arsénio era diferente para os diferentes grupos de tratamento. Por fim, o presente trabalho permite concluir que a nefrotoxicidade provocada pelo CCA nos ratinhos é muito mais acentuada do que a induzida pelo As2O5 e pelo CrO3, e alerta para o potencial tóxico do CCA para a saúde pública. ABSTRACT: Chromated copper arsenate (CCA) is a chemical wood preservative protecting timber from natural deterioration, containing chromium, copper and arsenic. Although on 2002, Environmental Protection Agency (EPA) had limited the use of CCA-treated wood, the environment contamination and the hazard to the public health will pursue due to the durability of this wood for 60 years. The main aim of the present work was the assessment of the acute toxicity of CCA, using mice as models. The development of a quantification methodology for chromium and arsenic in mice kidneys was another goal. In these studies, it was designed a set of experiments to investigate the effects caused by CCA, and the individual effects induced by arsenic pentoxide (As2O5) and chromium trioxide (CrO3) in thymus, liver, spleen, testis, epididymis and kidney. Sixteen groups of five animals each were considered in these studies, corresponding to different times of exposure. Treated groups received a single subcutaneous injection (0,3mL) of CCA, CrO3 and As2O5. In addition, control animals were administrated with the vehicle only. Following 14h, 24h, 48h and 96h animals were sacrificed, for organs removal. Afterwards, body and organs weights were also recorded. Subsequently, histology and histochemistry analysis was done. Furthermore, flow cytometry (FCM) approach was also considered for determination of cell cycle. Additionally, a set of preliminary enzyme assays was done to study the oxidative stress stimulated by these treatments. At last, the quantification of these compounds was also performed in kidney samples by graphite furnace atomic absorption spectrometry (GFAAS), after the digestion of the samples by microwave technology. The histology results, as confirmed by histochemistry, revealed acute tubular necrosis (ATN) induced by CCA and CrO3. Moreover, while FCM analysis revealed differences in cell cycle, enzyme assays showed a tendency to the decrease of the activities of CAT and GST. Finally, quantitative assays demonstrated a progressive decrease of the accumulation of CCA, arsenic and chromium during de exposure time, although no changes were observed within the other organs. For this reason, kidneys were carefully investigated. These results indicated that CCA-induced nephrotoxicity was more prominent than those caused by As2O5, and CrO3, which is an alert for its potential toxic effects on human health

PWP1 promotes nutrient-responsive expression of 5S ribosomal RNA

Peer reviewe

Comparative Cr, As and CCA induced Cytostaticity in mice kidney: a contribution to assess CCA toxicity

CCA (Chromium Copper Arsenate) treated wood, widely used in outdoor residential structures and playgrounds, poses considerable dangers of leaching of its components to the environment. In this study, mouse kidney samples were used to evaluate the effects of CCA, chromium trioxide (CrO3) and arsenic pentoxide (As2O5) on cell pathophysiology by flow cytometry. Samples were collected after 14, 24, 48 and 96 h of animal exposure. While Cr had no statistically significant cytostatic effects, As2O5 induced a S-phase delay in animals exposed for 24 h, and over time a G0/G1 phase blockage. The effects of CCA in S-phase were similar, but more severe than those of As2O5. Since environmental and public health hazards due to the long durability of CCA-treated wood products, these data confirm that CCA has profoundly toxic effects on cell cycle, distinct from the compounds themselves. These cytostatic effects support cell cycle dynamics as a valuable endpoint to assess the toxicity of remaining CCA-treated infrastructures, and the expected increased waste stream over the coming decades.publishe

Using Drosophila melanogaster to find new mechanisms of gene regulation in vivo

The survival of any organism depends on many factors, including their ability to adapt to novel environments. Nutrient accessibility in the environment is crucial for the development, growth, fertility, metabolism, and other biological functions of animals. Nutrients such as sugars, amino acids, and lipids are not only essential building blocks for the maintenance of a typical bioenergetic and cellular homeostasis of animals, but they also serve as signals to regulate cellular functions. The integration of such signals sent by the availability or scarcity of nutrients provided by the environment are sensed by nutrient sensors. Different nutrient sensors are activated or inactivated by specific nutrients, and ultimately mediate the physiological function of an animal through regulation of gene expression. The regulation of gene expression implicated in nutrient sensing can be studied using Drosophila melanogaster as a model organism. The fruit fly has several advantages for studying nutrient sensing and the impact of regulatory gene expression on the physiology of reproduction, growth, and metabolism. Examples of such advantages are reflected in the publicly available genetic toolkit, and the highly conserved molecular mechanisms to humans. This thesis used Drosophila melanogaster as a model to study novel regulatory mechanisms in gene expression. The first study demonstrated the physiological role of the heterodimeric basic-helix-loop-helix zipper (BHLH/Zip) transcription factor, Mondo-Mlx, in the adult reproductive system of fruit flies. Specifically, the impact of its loss of function in the fertility of female flies was shown for the first time. This result led to the discovery of several defects in the oogenesis progression. Finally, ribosome and oogenesis gene clusters were identified as target genes of Mlx. In the second study, evidence was shown for the role of the chromatin binding periodic tryptophan protein 1 homolog (PWP1) as a direct regulator of RNA Polymerase III (Pol III) target genes in a nutrient-dependent manner. Specifically, it was shown that PWP1 directly binds to the 5S rRNA gene, a well-known Pol III target, and induces its expression in a nutrient-dependent manner. By conducting this study, both in Drosophila melanogaster larvae and mammalian cells, it was shown that this biological role of PWP1 was conserved among organisms. The third study was the first to demonstrate the physiological role of actin in the reproductive system of adult flies and its requirement for Drosophila melanogaster oogenesis progression through direct regulation of RNA Polymerase II (Pol II) transcribed genes. In conclusion, using Drosophila melanogaster as a model, these three studies identified previously unknown mechanisms in gene expression that ultimately affect biological functions such as fertility and growth of the fruit fly.Kaiken elämän perustana on kyky sopeutua uusiin olosuhteisiin. Ravintoaineiden saatavuus vaikuttaa eläimen kehitykseen, kasvuun, lisääntymiskykyyn, aineenvaihduntaan, sekä moneen muuhun fysiologiseen prosessiin. Ravintoaineet, kuten sokerit, aminohapot ja rasvat, ovat tärkeitä eläimen solutoimintojen ja energiatalouden ylläpidossa, mutta lisäksi ne toimivat signaaleina solun toimintojen säätelyssä. Ympäristön ravintoaineiden saatavuudesta kertovia signaaleja aistitaan ns. ravintosensoreilla. Kukin ravintosensori aktivoituu tai estyy tietyillä ravintoaineilla ja ne säätelevät edelleen fysiologisia toimintoja, mm. vaikuttamalla eläimen geenien ilmentymiseen. Ravintoaineiden aiheuttamaa geenisäätelyä voidaan tutkia mallieläimillä kuten banaanikärpäsellä, Drosophila melanogaster. Banaanikärpänen tarjoaa useita etuja ravintoaineiden aiheuttaman geenisäätelyn tutkimukseen ja sen vaikutusten selvittämiseen lisääntymiseen, kasvuun ja aineenvaihduntaan. Näihin lukeutuu laaja valikoima erilaisia geneettisiä työkaluja, sekä evolutiivisesti säilyneet molekyylitason mekanismit. Tässä väitöskirjassa Drosophila melanogaster toimi mallieläimenä uusien geenisäätelymekanismien tutkimuksessa. Ensimmäinen tutkimus osoittaa transkriptiotekijä Mondo-Mlx (heterodimeerinen basic-helix-loop-helix zipper [BHLH/Zip]) roolin banaanikärpäsen lisääntymisen säätelyssä. Tutkimus osoittaa ensimmäistä kertaa, miten Mondo-Mlx puutos heikentää naaraiden lisääntymiskykyä. Tulokset osoittivat geenipuutoksen johtavan oogeneesin häiriintymiseen. Lopuksi tutkimuksessa selvisi Mlx geenin säätelevän suoraan ribosomien tuotantoon ja oogeneesiin liittyviä geeniklustereita. Toinen työ keskittyi kromatiiniin sitoutuvan periodic tryptophan protein 1 homolog (PWP1) geenin rooliin RNA-polymeraasi III:n (Pol III) ravinnosta riippuvaan säätelyyn. Erityisesti tutkimuksessa selvisi, miten PWP1 säätelee ravinnosta riippuvalla tavalla Pol III:a sitoutumalla suoraan sen tunnettuun kohdegeeniin, joka koodaa ribosomaalista 5S rRNA:ta. Tutkimuksessa käytettiin niin banaanikärpäsen toukan kuin nisäkkään soluja, jotka paljastivat PWP1 toiminnan konservoituneeksi lajien välillä. Kolmas tutkimus käsitteli aktiinin fysiologista roolia banaanikärpäsen lisääntymisen säätelyssä ja sen suoraa vaikutusta RNA-polymeraasi II:n (Pol II) kohdegeeneihin oogeneesin aikana. Käyttämällä Drosophila melanogasteria mallieläimenä, nämä kolme tutkimusta paljastavat aiemmin tuntemattomia geenisäätelymekanismeja, jotka vaikuttavat banaanikärpäsen lisääntymiskykyyn ja kasvuun

Toxicity of chromated copper arsenate: a study in mice

Chromated copper arsenate (CCA) was widespread used as a chemical wood preservative with application in the construction of playground equipment, fences, jetties, and naval. Environmental protection agency (EPA) had limited the use of CCA-treated wood on 2002, due to probable implications on both human and environmental health. Although this fact, several industries pursue the use of this product within their manufactories. In addition, the durability of this wood for 60 years, makes these treated products an hazard to the public health. In the present work, studies were explored exposing mice to CCA, during 14, 24, 48, and 96 h for the assessment of acute toxicity of CCA. Kidney and liver were removed, prepared for histology and for metalloid, and copper content evaluation by high resolution inductively coupled plasma mass spectroscopy. The histological results evidenced apparently normal structures for control animals and group exposed to As2O5. On the contrary, the renal sections of the animals treated with CCA revealed epithelium cells desquamation, hyaline, and granular casts in renal tubules lumen. Furthermore, high levels of arsenic were detected in the kidney of animals treated with CCA over 14 and 48 h, being significantly greater than controls. Although this approach underlines the potential hazard of CCA on some vital organs, further testing may be required to establish the impacts on other functions

Impairment of mice spermatogenesis by sodium arsenite

In order to assess the effect of arsenic on the male reproductive impairment in mice, 7-week-old animals were exposed to 7.5 mg sodium arsenite (NaAsO(2))/kg body weight, during 35 days (one spermatogenic cycle). One group of animals was sacrificed after exposure, while another group received distilled water for an additional period of 35 days, in order to study the spermatoxic effect and the recovery of spermatogenesis. In mice sacrificed after NaAsO(2) exposure, a decrease in testis/body weight ratio and reduction of tubular diameter were observed. Both groups of NaAsO(2)-exposed animals showed remarkable histopathological changes, such as sloughing of immature germ cells. Animals sacrificed after NaAsO(2) exposure showed decreased sperm motility, increased abnormal sperm morphology and decreased sperm viability. The effects of NaAsO(2) on sperm motility recovered to normal values after one spermatogenic cycle, while increased sperm abnormality was maintained. However, at this period, a decrease in acrosome integrity was detected. Concerning oxidative stress parameters, animals sacrificed after NaAsO(2) exposure showed a decreased selenium-dependent glutathione peroxidase activity, which was not detected after the recovery. Conversely, at this period, total glutathione peroxidase activity increased in exposed animals. These results demonstrate the toxic effects of NaAsO(2) on mice spermatogenesis and show the lack of recovery after one spermatogenic cycle.publishe

Nephrotoxicity effects of the wood preservative chromium copper arsenate on mice: histopathological and quantitative approaches

Chromium copper arsenate(CCA)was used for the protection of wood building material suntil the restriction by EPA in2002. During a short period of time 14–24h,a comparative nephrotoxicity study was performed regarding the effects of CCA and its compounds per se. Histopathological and histochemical features were correlated with the concentration of the total arsenic and chromium in mice kidney. Animals were subcutaneously injected with CCA(7.2mg/kg arsenic and 10.2mg/kg chromium per body weight), CrO3 (10.2 mg/kg),As2O5 (7.2 mg/kg)andNaCl(0.9%) per se. The histopathological examination of the renal sections evidenced acute tubular necrosis in the groups of animals exposed to CCA(in both periods of time). Although the same contents of pentavalent arsenic and hexavalent chromium were injected in treated animals with CCA and with the prepared solutions of As2O5 and CrO3, the arsenic concentration on kidneys of CCA-exposed animals was much higher than those in animals exposed to As2O5 (32- and28-fold higher at 14 and 24h,respectively). However,the elimination of chromium seems to occur similarly in the kidneys of animals treated with CCA and CrO3 per se. Interactions among the components of CCA result in a marked decrease of the ability of kidney to eliminate simultaneously both analytes.The nephrotoxicity of CCA was higher than its components per se, evidencing a possible synergetic effect

Nephrotoxicity of CCA-treated wood: a comparative study with As2O5 and CrO3 on mice

The purpose of this work was to assess the acute toxicity on male mice to a chromated copper arsenate (CCA) solution, a widespread wood preservative used in building industry until 2002. Animals were subcutaneously injected with CCA (7.2 mg/kg arsenic and 10.2 mg/kg chromium per body weight), CrO3 (10.2 mg/kg), As2O5 (7.2 mg/kg) and NaCl (0.9%) per se, during 48 h and 96 h, for histopathology, histochemistry, chromium and arsenic analysis. The results showed some histopathological changes within renal tubules lumen of CCA exposed animals (during 48 h, and 96 h), and CrO3 (for the period of 96 h). Furthermore, the renal levels of arsenic and chromium in treated animals were statistically more evident than controls. Although, the same contents of pentavalent arsenic and hexavalent chromium were injected into treated animals with CCA and with the prepared solutions of As2O5 and CrO3, a different distribution of the pattern of these compounds was observed in kidneys

Nuclear actin is required for transcription during Drosophila Oogenesis

Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor (Mrtf), to chromatin remodelling and RNA polymerase (Pol) function. However, whether actin controls transcription of only specific genes or has a global role in gene expression has remained elusive. Our genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies majority of gene promoters together with Pol II, and on highly expressed genes these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies and manipulation of nuclear transport factors for actin leads to decreased expression of egg shell genes. Collectively, these results uncover a global role for actin in transcription, and demonstrate the in vivo importance of balanced nucleo-cytoplasmic shuttling of actin in transcriptional control of a developmental process.Peer reviewe

Mechanisms of kidney toxicity for chromium- and arsenic-based preservatives: Potential involvement of a pro-oxidative pathway

Metals have been extensively used for the preservation of wood. Among metallic conservatives, mixtures of chromated copper arsenate (CCA) were thoroughly used. However, the release and consequent mobilization of such compounds by biota, may culminate in the exertion of toxic chemical effects. The present study intended to show the toxicological effects caused by arsenic (7.2 mg/kg body weight), chromium (10.2 mg/kg Cr body weight) and the commercial mixture CCA (7.2 mg/kg As body weight and 10.2 mg/kg Cr body weight) in mice, namely the oxidative stress response (catalase - CAT, glutathione peroxidase - GPx, and glutathione-S-transferases - GSTs), in kidney tissues. The determination of the tested parameters was performed after exposure; organisms were exposed, and then sacrificed at two distinct periods, namely 14 and 96 h after the administration of toxicants. Exposure to chromium and arsenic induced significant modifications in the redox state of the test organisms, evidenced by significant alterations in GSTs and GPx activities. No alterations were found concerning the activity of catalase. These findings showed that the chemical mixture used as household product may exert significant toxicological outcomes in exposed animals, such as rodents, conditioning their redox homeostasis and antioxidant response. (C) 2013 Published by Elsevier B.V