Multispecies and multibiomarker assessment of fish health from Iguaçu River reservoir, Southern Brazil.

This study investigated the impact of micropollutants on fish health from Segredo hydroelectric reservoir (HRS) along the Iguaçu River, Southern Brazil, contaminated by urban, industrial, and agricultural activities. This is the first comprehensive study assessment in the river after the severe drought in the 2020s in three fish species from different trophic levels Astyanax spp. (water column depth/omnivorous), Hypostomus commersoni (demersal/herbivorous), and Pimelodus maculatus (demersal/omnivorous). Animals, water, and sediment samples were collected from three distinct sites within the reservoir: Floresta (upstream), Iratim (middle), and Station (downstream). The chemical analysis revealed elevated concentrations of metals (Al, Cu, Fe) and the metalloid As in water, or Cu, Zn, and As in sediment, surpassing Brazilian regulatory limits, while the organic pollutants as DDT, PAHs, PCBs, and PBDEs were found under the Brazilian regulatory limits. The metal bioaccumulation was higher in gills with no significant differences among sites. The species Astyanax spp. and H. commersoni displayed variations in hepatosomatic index (HSI) and P. maculatus in the condition factor index (K) between sites, while adverse effects due to micropollutants bioaccumulation were observed by biochemical, genotoxic, and histopathological biomarkers. The principal component analysis and integrated biomarker response highlighted the upstream site Floresta as particularly inhospitable for biota, with distinctions based on trophic level. Consequently, this multifaceted approach, encompassing both fish biomarkers and chemical analyses, furnishes valuable insights into the potential toxic repercussions of micropollutant exposure. These findings offer crucial data for guiding management and conservation endeavors in the Iguaçu River.

Pollutant bioaccumulation in sentinel fish chronically exposed in Iguaçu river reservoirs (Southern Brazil) and human health risk of fish consumption.

Bioaccumulation studies in fish mark the initial phase of assessing the risk of chemical exposure to biota and human populations. The Iguaçu River boasting a diverse endemic ichthyofauna, is grappling with the repercussions of human activities. This study delved into the bioaccumulation of micropollutants, the early-warning effects on Rhamdia quelen and Oreochomis niloticus in the Segredo Reservoir (HRS) and the potential risk of human exposure. Two groups of caged fish in three sites of the reservoir were exposed during the autumn-winter and spring-summer, while a third group (O. niloticus) underwent a twelve-month exposure, and inorganic and organic chemicals analysis in water, sediment, and biota. Additionally, metallothionein expression and genotoxicity were employed as biomarkers. PAHs, PCBs, Al, Cu, Fe, and As in water and DDTs, Cu, Zn, and As in sediment surpassed the thresholds set by Brazilian regulations, where DDT exhibited bioaccumulation in muscle, alongside metals in liver, kidney, gills, and muscle tissues. R. quelen showed metallothionein expression whereas DNA damage and NMA frequencies were elevated in target organs and in brain and erythrocytes of O. niloticus during summer. In this species the DNA damage in liver was remarkable after twelve months. Target Hazard Quotients and Cancer Risk values shedding light on the vulnerability of both children and adults. The reservoir's conditions led to heightened sensitivity to micropollutants for R. quelen species. The data presented herein provides decision-makers with pertinent insights to facilitate effective management and conservation initiatives within the Iguaçu Basin.

Assessing the pollution and ecotoxicological status of the Iguaçu River, southern Brazil: A review.

The degradation of water resources available for human consumption is increasing with the continuous release of chemicals into aquatic environments and their inefficient removal in wastewater treatment. Several watersheds in Brazil, such as the Iguaçu River, are affected by multiple sources of pollution and lack information about their pollution status. The Iguaçu River basin (IRB) has great socioeconomic and environmental relevance to both the supply of water resources and its considerable hydroelectric potential, as well as for the high rate of endemism of its ichthyofauna. Also, the IRB is home to large conservation units, such as the Iguaçu National Park, recognized by UNESCO as a natural World Heritage Site. Thus, this article discusses the chemical pollution in the IRB approaching: (i) the main sources of pollution; (ii) the occurrence of inorganic and organic micropollutants; (iii) the available ecotoxicological data; and (iv) the socioeconomic impacts in three regions of the upper, middle, and lower IRB. Different studies have reported relevant levels of emerging contaminants, persistent organic pollutants, toxic metals, and polycyclic aromatic hydrocarbons detected in the water and sediment samples, especially in the upper IRB region, associated with domestic and industrial effluents. Additionally, significant concentrations of pesticides and toxic metals were also detected in the lower IRB, revealing that agricultural practices are also relevant sources of chemicals for this watershed. More recently, studies indicated an association between fish pathologies and the detection of micropollutants in the water and sediments in the IRB. The identification of the main sources of pollutants, associated with the distribution of hazardous chemicals in the IRB, and their potential effects on the biota, as described in this review, represent an important strategy to support water management by public authorities for reducing risks to the local endemic biodiversity and exposed human populations. Integr Environ Assess Manag 2024;00:1-26. © 2023 SETAC.

Physicochemical and bioinformatic characterization of Oreochromis niloticus vitellogenin as an endocrine disruption biomarker.

Aquatic biota is increasingly being exposed to chemical pollutants due to human activities and the relationship between the level of environmental pollution and fish reproduction is a continuously ongoing issue. The vitellogenin (Vtg) protein synthesis can be induced in the liver of juvenile and male fish after stimulation of the estrogen receptor and therefore, Vtg has been used as a biomarker of xenoestrogen exposure in several fish species. The current study reported the first physicochemical characterization of Vtg from Oreochromis niloticus. Adult male fish were exposed to 17α-ethinylestradiol for Vtg induction. Purified vitellogenin from plasma showed low stability at 25 and 4 °C in saline conditions, and good stability in acidic (low pH) or in heated conditions. The 3D modeling provided useful information on the structure of O. niloticus Vtg showing conserved structural features. According to bioinformatics and experimental results, there are important structural differences between the two chemical forms of Vtg (VtgAb and VtgC) in a phylogenetic context. The present results add information about the development of ecotoxicological immunoassays to study the endocrine disruption in O. niloticus improving the Vtg performance as a biomarker of reproduction in fish.

The effects of stretching on muscle morphometry of ovariectomized rats / Efeitos do alongamento na morfometria do músculo em ratas ovarectomizadas / Los efectos del ejercicio de estiramiento en la morfometria muscular de las ratas ovariectomizadas

Abstract Introduction: Ageing is responsible for structural alterations, declining of all physiological variables, including range of motion and skeletal muscle function, known as sarcopenia. Objective: The aim of the study was to evaluate the effects of stretching on muscle morphometry in ovariectomized rats. Method: 21 female Wistar rats (12 weeks, 218 ± 22 g) were divided into 4 groups: control (CONTROL, n = 3) intact; ovariectomized and hysterectomized (OH, n = 6); Stretching (STRET, n = 6); ovariectomized and hysterectomized and stretching (OHS, n = 6). The rats were subjected to ovariectomy and hysterectomy. The stretching protocol of the soleus muscle lasted 10 repetitions of 1 minute with 45s interval between each repetition performed 3 times a week for 3 weeks. After 3 weeks, the rats were weighed and the muscles of both hind limbs were removed weighed and analyzed at muscle length; serial sarcomere number; sarcomere length; muscle fiber cross-sectional area (MFCSA) and percentage of connective tissue. Results: The final body weight increased in all groups. The serial sarcomere number of STRET was greater than the OH. The muscle fibers' cross-sectional area of OHS was higher than CONTROL. Conclusion: It can be concluded that ovariectomy and hysterectomy prevented sarcomerogenesis even when stretching was applied. However, the stretching protocol enhanced muscle trophismof ovariectomized and hysterectomized rats. It might be suggested that longitudinal growth (serial sarcomeres) and radial (ASTFM) are differently regulated by stretching in intact and/or estrogen depleted (ovariectomy and hysterectomy) skeletal muscle.

Resumo Introdução: O envelhecimento é responsável por alterações estruturais, com declínio de todas as variáveis fisiológicas, incluindo amplitude de movimento e função muscular esquelética. Objetivo: O objetivo do estudo foi avaliar os efeitos do alongamento na morfometria muscular de ratas ovariectomizadas. Método: Assim, 21 ratas Wistar (12 semanas, 218 ± 22 g) foram divididas em 4 grupos: Controle (CONTROL, n = 3) intacto; Ovariectomizadas e histerectomizadas (OH, n = 6); Alongamento (ALONG, n = 6); Ovariectomizadas e histerectomizadas e Alongamento (OHA, n = 6). As ratas foram submetidas a ovariectomia e histerectomia. O alongamento do músculo sóleo foi composto de 10 repetições de 1 minuto com intervalo de 45s entre cada repetição, realizado 3 vezes por semana, durante 3 semanas. Após 3 semanas, as ratas foram pesadas e os músculos sóleos de ambas as patas foram retirados, pesados e analisados: comprimento do músculo; número de sarcômeros em série; comprimento do sarcômero; área da seção transversal das fibras musculares (ASTFM) e porcentagem de tecido conjuntivo. Resultados: O peso corporal final aumentou em todos os grupos. O número de sarcômeros em série do ALONG foi maior que o OH. A área de secção transversa das fibras musculares do OHA foi superior ao CONTROL. Conclusão: Conclui-se que a ovariectomia e histerectomia impediram a sarcomerogênese mesmo quando realizado alongamento. Porém, o protocolo de alongamento incrementou o trofismo muscular em ratas ovarietomizadas e histerectomizadas. Sugere-se que o crescimento longitudinal (número sarcômeros em série) e radial (ASTFM) respondem diferentemente ao alongamento em músculo esquelético intacto e/ou com depleção estrogênio (ovarietomia e histerectomia).

Resumen Introducción: El envejecimiento es responsable de cambios estructurales del organismo, con disminución de todas las variables fisiológicas, incluyendo la amplitud de movimiento y función muscular esquelética, conocida como sarcopenia. Objetivo: El objetivo del estudio fue evaluar los efectos de los ejercicios de estiramiento en la morfometria muscular de ratas ovarectomizadas. Método: Fueron seleccionadas 21 ratas Wistar (12 semanas, 218 ± 22g) que fueron divididas en 4 grupos: control (CONTROL, n = 3) intacto; ovarectomizadas y histerectomizadas (OH, n = 6); estiramiento (ALONG, n = 6); ovarectomizadas y histerectomizadas y estiramiento (OHA, n = 6). Las ratas han sido sometidas a ovariectomía y histerectomía. El protocolo de estiramiento del músculo sóleo fue realizado en 10 repeticiones de 1 minuto con intervalos de 45 segundos entre cada repetición, realizada 3 veces por semana, durante 3 semanas. Después de 3 semanas, las ratas fueron pesadas y los músculos de ambas patas fueron retirados para analizar el peso muscular; la longitud del músculo; número de sarcómera en serie; longitud de sarcómera; área de la sección transversal de las fibras musculares (ASTFM) y porcentaje del tejido conjuntivo. Resultado: El peso corporal final aumentó en todos los grupos. El número de sarcómera en serie de ALONG fue mayor que el OH. El área de la sección transversal de las fibras musculares del OHA fue superior al de CONTROL. Conclusión: En conclusión, ovariectomía y histerectomía impiedieron la sarcomerogénesis mismo cuando se realizó el estiramiento. Sin embago, el estiramiento aumentó el trofismo muscular de las ratas ovarectomizadas y histerectomizadas. Sugiere que el crescimiento longitudinal (número sarcômeros) y radial (área de sección transversal) responden diferentemente al estiramiento en músculo esqueletico intacto y/o con depleción estrógeno (ovariectomía y histerectomía).

Time does matter! Acute copper exposure abolishes rhythmicity of clock gene in Danio rerio.

The circadian clock is a key cellular timing system that coordinates physiology and behavior. Light is a key regulator of the clock mechanism via its activation of Per and Cry clock gene expression. Evidence points to a key role of reactive oxygen species (ROS) in resetting this process. In this context, the aim of the present study was to explore copper as a ROS generator, using an innovative approach investigating its effects on circadian timing. Liver and brain from Danio rerio specimens exposed to 0, 5, 25 and 45 µg/L copper concentrations were obtained. Daily oscillations of superoxide dismutase (SOD) and catalase (CAT) enzymatic activity and their correlations both with clock genes (per1, per2, and cry1a) and with organism energy cost were determined. CAT expression correlates with per2 and cry1a and, thus, provides data to support the hypothesis of hydrogen peroxide production by a phototransducing flavin-containing oxidase. Higher SOD activity is correlated with higher intracellular ATP levels. Copper disturbed the daily oscillation of antioxidant enzymes and clock genes, with disturbed per1 rhythmicity in both the brain and liver, while cry1a rhythmicity was abolished in the liver at 25 µg/L copper. Coordination between the SOD and the CAT enzymes was lost when copper concentrations exceeded the limits established by international laws. These results indicate that organism synchronization with the environment may be impaired due to acute copper exposure.

Environmental risk assessment in five rivers of Parana River basin, Southern Brazil, through biomarkers in Astyanax spp.

In the current study, water quality of five river sites in Parana River basin (Brazil), utilized for public water supply, was assessed through a set of biomarkers in fish Astyanax spp. Population growth and inadequate use of land are challenges to the preservation of biodiversity and resources such as water. Some physicochemical parameters as well as somatic indexes, gills and liver histopathology, genotoxicity, and biochemical biomarkers were evaluated. The highest gonadosomatic index (GSI) and antioxidant parameters (catalase and glutathione S-transferase activities, non-protein thiols), as well as the lowest damage to biomolecules (lipid peroxidation, protein carbonylation, DNA damage) were observed in site 0 (Piava River), which is located at an environmental protected area. Site 1, located in the same river, but downstream site 0 and outside the protection area, presents some level of impact. Fish from site 2 (Antas River), which lack of riparian forest and suffer from silting, presented the highest micronucleus incidence and no melanomacrophages. Differently, individuals from site 3 (Xambrê River) and site 4 (Pinhalzinho River) which receive surface runoff from Umuarama city, urban and industrial sewage, have the highest incidences of liver and gill histopathological alterations, including neoplasia, which indicated the worst health conditions of all sites. In particular, site 4 had high levels of total nitrogen and ammonia, high turbidity, and very low oxygen levels, which indicate important chemical impact. Comparison of the biomarkers in fish allowed classification of the five sites in terms of environmental impact and revealed that sites 3 and 4 had particular poor water quality.

How and where to perform biomonitoring studies: different levels of toxic metal pollution are detected in the Alagados Reservoir in Southern Brazil.

The Alagados Reservoir located in the Brazilian city of Ponta Grossa is used to supply water for human consumption but is impacted by toxic metals. The current study combined chemical, biochemical, and multivariate analyses to determine the bioavailability of toxic metals at three sites (AL-A, AL-B, and AL-C) within the Alagados Reservoir. Metal bioaccumulation was analyzed in the liver, gills, and muscle tissue of a native fish species (Geophagus brasiliensis), and neurotoxicity, xenobiotic metabolism, and oxidative stress were evaluated using biochemical biomarkers. Additionally, histopathological studies were performed on the gills and the liver using scanning electron microscopy (SEM) and conventional light microscopy (LM), respectively. Overall, the bioaccumulation of metals, biomarkers of oxidative stress, and melanomacrophage counts indicate that the AL-C and the AL-A sites are the most and least affected by metals, respectively. The AL-B site presented the lowest acetylcholinesterase enzyme activity, a finding which was probably associated with the agricultural activities around this area of the reservoir. The biomarkers clearly revealed that toxic metals negatively affect all three sites studied herein and that human activity is the major source of pollutants. Despite the existence of different pollution levels within the Alagados Reservoir, it is still used as a human water supply.

Toxicological interactions of silver nanoparticles and non-essential metals in human hepatocarcinoma cell line.

Toxicological interaction represents a challenge to toxicology, particularly for novel contaminants. There are no data whether silver nanoparticles (AgNPs), present in a wide variety of products, can interact and modulate the toxicity of ubiquitous contaminants, such as nonessential metals. In the current study, we investigated the toxicological interactions of AgNP (size=1-2nm; zeta potential=-23mV), cadmium and mercury in human hepatoma HepG2 cells. The results indicated that the co-exposures led to toxicological interactions, with AgNP+Cd being more toxic than AgNP+Hg. Early (2-4h) increases of ROS (DCF assay) and mitochondrial O2- levels (Mitosox® assay) were observed in the cells co-exposed to AgNP+Cd/Hg, in comparison to control and individual contaminants, but the effect was partially reverted in AgNP+Hg at the end of 24h-exposure. In addition, decreases of mitochondrial metabolism (MTT), cell viability (neutral red uptake assay), cell proliferation (crystal violet assay) and ABC-transporters activity (rhodamine accumulation assay) were also more pronounced in the co-exposure groups. Foremost, co-exposure to AgNP and metals potentiated cell death (mainly by necrosis) and Hg2+ (but not Cd2+) intracellular levels (ICP-MS). Therefore, toxicological interactions seem to increase the toxicity of AgNP, cadmium and mercury.

Toxicological interactions of silver nanoparticles and organochlorine pesticides in mouse peritoneal macrophages.

Nanotechnology occupies a prominent space in economy and science due to the beneficial properties of nanomaterials. However, nanoparticles may pose risks to living organisms due to their adsorption and pro-oxidative properties. The aim of the current study was to investigate the effects of polymer-coated silver nanoparticles (AgNPs) and organochlorine pesticides (OCPs), as well as their combined effects on mouse peritoneal macrophages. Macrophages were isolated and exposed to three concentrations of AgNPs (groups: N1 = 30, N2 = 300 and N3 = 3000 ng.ml(-1)), two concentrations of OCPs (groups: P1 = 30 and P2 = 300 ng.ml(-1)) and the six possible combinations of these two contaminants for 24 h. AgNPs had irregular shape, Feret diameter of 8.7 ± 7.5 nm and zeta potential of -28.7 ± 3.9 mV in water and -10.7 ± 1.04 mV in culture medium. OCP mixtures and the lower concentrations of AgNPs had no detectable effects on cell parameters, but the highest AgNPs concentration showed high toxicity (trypan blue and MTT assays) resulting in morphological changes, increase of nitric oxide levels and phagocytic index. Foremost, the association of N3 and P2 led to distinct effects from those observed under single exposure.

The applied indicators of water quality may underestimate the risk of chemical exposure to human population in reservoirs utilized for human supply-Southern Brazil.

The knowledge concerning associations between chronic chemical exposure and many disorders with complex etiology involving gene-environment interactions is increasing, and new methods must be developed to improve water quality monitoring. The complexity of chemical mixtures in polluted aquatic environments makes the evaluation of toxic potential in those sites difficult, but the use of biomarkers and bioindicators has been recognized as a reliable tool to assess risk of exposure to biota and also the human population. In order to evaluate the use of fish and biomarkers to assess toxic potential and bioavailability of chemicals in human-related hydric resources, an in situ experiment was accomplished in two water reservoirs designated for human supply, which were previously evaluated by the local environmental regulatory agency through a set of physical, chemical, and classical biological parameters. Molecular, biochemical, and morphological biomarkers were performed in caged Oreochromis niloticus kept for 6 months in the studied reservoirs to assess potentially useful biomarkers to evaluate the quality of water for human supply. Chemical analysis of toxic metals in liver and muscle and polycyclic aromatic hydrocarbons (PAHs) in bile was considered to assess the bioavailability of pollutants and highlight human activity impact. The reservoir previously classified by a governmental agency as less impacted presented more risk of exposure to biota. These results were supported by chemical analysis, vitellogenin expression, histopathological findings (gonads, liver, and gills), as well as indicators of neurotoxic effects and oxidative stress in liver. The inclusion of some biomarkers as parameters in regulatory monitoring programs in reservoirs designated for human supply is strongly suggested to evaluate the risks of exposure to the human population. Thus, a revision of the traditional biological and physicochemical analysis utilized to establish the conditions of water quality is necessary.

Low concentrations of cylindrospermopsin induce increases of reactive oxygen species levels, metabolism and proliferation in human hepatoma cells (HepG2).

Human hepatoma cells (HepG2) were exposed to purified cylindrospermopsin (CYN), a potent toxicant for eukaryotic cells produced by several cyanobacteria. CYN was not toxic at concentrations up to 10 µgl(-1), leading to increased viability and metabolism in cells cultured with 10% fetal bovine serum (FBS). Reduction of FBS concentration to 2% and induction of cytochrome P450 (CYP) isoforms were performed in order to make xenobiotic-metabolizing capacity of HepG2 cells closest to that of 'normal' cells. HepG2 cells proliferated less after CYPs-induction, and this induction has lead to similar results of non-induced cells, except for few individual parameters such lipid peroxidation. Foremost, low concentrations of CYN (below or equal 10 µgl(-1)) have induced HepG2 cells proliferation and metabolism increase, which was not expected.

Bioconcentration and bioaccumulation of metal in freshwater Neotropical fish Geophagus brasiliensis.

From the concentration in water and sediments, bioconcentration and bioaccumulation of copper (Cu), manganese (Mn), zinc (Zn), iron (Fe), cobalt (Co), cadmium (Cd), chrome (Cr), silver (Ag), lead (Pb), nickel (Ni), aluminum (Al), and arsenic (As) were determined in the gills, liver, and muscles of Geophagus brasiliensis in the Alagados Reservoir, Ponta Grossa, Paraná, Brazil. Metals were quantified through AAS, and a study was carried out on the existing relations between metal and body weight, size, and genre of this species. The level of metal in the water of the reservoir was lower than the maximum set forth in the legislation, except for that of Cd and Fe. In sediments, Cu, Cd, Cr, and Ni presented concentrations above the threshold effect level (TEL). Pb and Cr were above the limits for the G. brasiliensis. The tendency of metals present in the muscles of G. brasiliensis was Al > Cu > Zn > Fe > Co > Mn > Cr > Ag > Ni > Pb > Cd > As. In the gills, it was Al > Fe > Zn > Mn > Co > Ag > Cr > Ni > Cu > As > Pb > Cd, and the liver presented Al > Cu > Zn > Co > Fe > Mn > Pb > Ag > Ni > Cr > As > Cd. The bioconcentration and bioaccumulation of metal in the tissues follow the global tendency liver > gills > muscle. The statistical analysis did not point to significant differences in the metal concentration and body weight, size, and gender of the species in the three tissues under analysis.

Aprendizagem através de role-playing games: uma abordagem para a educação ativa / Learning through role-playing games: an approach for active learning and teaching

This study evaluates the use of role-playing games (RPGs) as a methodological approach for teaching cellular biology, assessing student satisfaction, learning outcomes, and retention of acquired knowledge. First-year undergraduate medical students at two Brazilian public universities attended either an RPG-based class (RPG group) or a lecture (lecture-based group) on topics related to cellular biology. Pre- and post-RPG-based class questionnaires were compared to scores in regular exams and in an unannounced test one year later to assess students' attitudes and learning. From the 230 students that attended the RPG classes, 78.4% responded that the RPG-based classes were an effective tool for learning; 55.4% thought that such classes were better than lectures but did not replace them; and 81% responded that they would use this method. The lecture-based group achieved a higher grade in 1 of 14 regular exam questions. In the medium-term evaluation (one year later), the RPG group scored higher in 2 of 12 questions. RPG classes are thus quantitatively as effective as formal lectures, are well accepted by students, and may serve as educational tools, giving students the chance to learn actively and potentially retain the acquired knowledge more efficiently.

Avaliamos o uso dos role-plying games (RPGs) como uma metodologia de ensino e aprendizagem em Biologia Celular. Alunos do primeiro ano de Medicina participaram de aula expositiva tradicional (grupo referência) ou de aula com RPG (grupo RPG). Comparações foram feitas pela análise de questionários pré- e pós-aula com RPG, das notas nas provas regulares, e das notas obtidas em um teste aplicado um ano após a conclusão da disciplina. Dos 230 alunos que participaram das aulas com RPG, 78,4% responderam que o RPG é uma ferramenta eficaz na aprendizagem; 55,4% acharam que essas aulas são melhores que as aulas tradicionais, mas não as substituem; e 81% responderam que usariam essa metodologia. Os alunos do grupo referência tiveram nota maior em uma de 14 questões das provas regulares; os alunos do grupo RPG tiveram notas maiores em duas de 12 questões aplicadas um ano depois. Portanto, aulas com RPG foram tão eficientes quanto as aulas tradicionais (quantitativamente), são bem aceitas pelos estudantes, e podem ser uma metodologia que lhes dá a chance de aprender ativamente e, potencialmente, levar a uma melhor retenção do conhecimento aprendido.

Enzymatic inhibition and morphological changes in Hoplias malabaricus from dietary exposure to lead(II) or methylmercury.

Neotropical fish traíra (Hoplias malabaricus) were used to investigate the effects of dietary doses of metals through individual exposures to either inorganic lead(II) or methylmercury, respectively, 21 microg Pb2+g(-1) w.w. and 75 ng H(3)C-Hg+g(-1) w.w., every 5 days, for 70 days (14 doses). The erythrocyte delta-aminolevulinic acid dehydratase (ALAd) activity was inhibited after 14 doses of Pb2+ and H(3)C-Hg+. The muscle cholinesterase (ChE) activity was inhibited after 14 doses of H(3)C-Hg+. Damage in cytoskeleton and nuclei were observed after exposure to inorganic lead. Individuals exposed to H(3)C-Hg+ showed the presence of atypical granules and vesicles, cytoplasm disorganization, and mitochondria damages in hepatocytes also after 14 doses. The present results demonstrate that erythrocyte ALAd and muscle ChE activities can be used as long-term biomarkers of sublethal, subchronic, and trophic exposures to Pb2+, and H(3)C-Hg+ in fish. Also the morphological aspects described in the present work confirm the toxicity of both studied metals.

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis.

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.

Vacuolar system of ungerminated Colletotrichum graminicola conidia: convergence of autophagic and endocytic pathways.

Vacuoles of ungerminated Colletotrichum graminicola conidia engulf cytoplasmic structures by a process analogous to microautophagy, demonstrated by using a vacuolar membrane acid phosphatase marker. Fusion of vesicles with vacuoles, without deposition of the acid phosphatase reaction product has been observed, suggesting other pathways of material delivery to vacuoles than microautophagy. Plasma membrane invaginations, multivesicular bodies and retention of neutral red into small vesicles, which were internalized by the vacuole, were verified. These results provided evidence for endocytosis and an active endosomal system. Together, our findings with C. graminicola demonstrated that vacuoles are very dynamic compartments, playing roles in autophagy and endocytic processes.