Spore germination and gametophyte development of Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) under different pH conditions

Cyathea atrovirens (Langsd. & Fisch.) Domin, an intensely exploited tree fern, is found inside forests in several succession stages, as well as in swamps, roadsides and unused fields in the Rio dos Sinos basin, in the state of Rio Grande do Sul, southern Brazil. This study evaluated the in vitro germination and gametophyte development of C.atrovirens under different pH conditions, as well as spore viability after different storage times at 7 ºC. The lowest germination rate of spores was obtained at pH 7.0. At pH 5.0 to 6.5, laminar gametophyte development started at 20 to 30 days of culture. Antheridia and archegonia were first observed at 35 and 128 days, respectively. Storage at 7 ºC did not affect germination rates. The capability of germination at different pH levels may explain the occurrence of the species in a wide range of habitats. The present study contributes to the understanding of the full life-cycle of C. atrovirens and to the analysis of the influence of abiotic components, providing information for the cultivation, management and conservation of the species.

Cyathea atrovirens (Langsd. & Fisch.) Domin, uma samambaia arborescente alvo de exploração extrativista, é encontrada no interior de florestas em diferentes estádios de sucessão, banhados, margens de estradas e campos abandonados da bacia do rio dos Sinos, no Estado do Rio Grande do Sul, sul do Brasil. Este estudo avaliou a germinação e o desenvolvimento gametofítico in vitro de C. atrovirens sob diferentes condições de pH, bem como a viabilidade de esporos após diferentes períodos de armazenamento a 7 ºC. A menor taxa de germinação de esporos foi obtida no pH 7,0. Entre os pHs 5,0 e 6,5, o início de desenvolvimento laminar dos gametófitos ocorreu entre 20 e 30 dias de cultura. Anterídios e arquegônios foram observados a partir do 35º e do 128º dia, respectivamente. A estocagem a 7ºC não afetou as taxas de germinação. A capacidade de germinação em diferentes pHs pode explicar a ocorrência da espécie em um amplo espectro de hábitats. O presente estudo contribui para a compreensão do ciclo de vida completo de C. atrovirens e para a análise da influência de fatores abióticos, fornecendo subsídios para o cultivo, o manejo e a conservação da espécie.

Evaluation of Sinos River water genotoxicity using the comet assay in fish

The Sinos River, in southern Brazil, is polluted by industrial discharges and untreated urban wastes. Fish genotoxicity biomarkers are valuable parameters for environmental risk assessment. In this study, we used the comet assay to detect genotoxicity due to multiple sources of pollution in the peripheral blood of a native fish species (Hyphessobrycon luetkenii). In addition, we analysed possible DNA damage from aluminum, lead, chromium, copper, nickel, iron and zinc contamination. Water samples were collected seasonally from three sampling sites and the fish were assessed under laboratory conditions. Water chemical analysis showed an increased level of aluminum and iron in most of the samples at sites 2 and 3, located in the middle and lower river course, respectively. The index of DNA damage assessed by the comet assay demonstrated no significant differences in different seasons or at the different sampling sites, while the frequency of cells with DNA damage was higher in water samples collected at sites 1 and 2 during the spring season. None of the metals studied seems to be associated with the increase in the frequency of cells with DNA damage observed during the spring season. The results of this study indicate that the Sinos River is contaminated with substances that are genotoxic to fish, including the waters near the river spring.

O rio dos Sinos, no sul do Brasil, é poluído tanto por descargas industriais como por resíduos urbanos não tratados. Os biomarcadores de genotoxicidade em peixes são parâmetros valiosos para a determinação de risco ambiental. Neste estudo, utilizamos o ensaio cometa em sangue periférico de um peixe nativo (Hyphessobrycon luetkenii) para detectar a genotoxicidade devido a múltiplas fontes de poluição. Além disso, analisamos a possível influência da contaminação por alumínio, chumbo, cromo, cobre, níquel, ferro e zinco sobre o dano de DNA. Amostras de água foram coletadas sazonalmente em três pontos de coleta e os peixes foram expostos sob condições de laboratório. A análise química da água mostrou níveis elevados de alumínio e ferro na maioria das amostras dos pontos 2 e 3, localizados nos cursos médio e inferior do rio, respectivamente. O índice de dano de DNA estimado pelo ensaio cometa não demonstrou diferenças significativas tanto entre estações como entre pontos de coleta, enquanto que a frequência de células com dano de DNA apresentou valores superiores nas amostras de água coletadas durante a primavera nos pontos 1 e 2. Nenhum dos metais estudados parece estar associado com o aumento na frequência de células com dano de DNA observado durante a primavera. Os resultados deste estudo indicam que o rio dos Sinos está contaminado com substâncias genotóxicas para os peixes, incluindo as águas próximas à nascente do rio.

A multibiomarker approach in rats to assess the impact of pollution on Sinos River, Southern Brazil

The aim of this study was to determine the feasibility of combining water quality analysis with different biomarkers to characterise the relationship between anthropogenic contamination and biotic response in the Sinos River, southern Brazil. Wistar rats were studied using three biomarkers combined with physical, chemical and microbiological analysis to assess the effects of pollution at four sampling sites. The induction of oxidative stress was quantified by MDA levels in peripheral blood, lymphocyte DNA damage was determined using the comet assay, and histopathological changes were analysed in the liver. After sampling, animals were allowed to drink the river water during a 48 hours period. No increase in oxidative stress and DNA damage was observed. However, liver damage was observed in the animals exposed to water samples, indicating that the Sinos River is contaminated with hepatotoxic substances. Water analyses confirmed that water quality decreased downriver.

O objetivo deste estudo foi determinar a viabilidade de combinar a análise da qualidade da água com o estudo de biomarcadores para caracterizar a relação entre contaminação antropogênica e resposta biótica no rio dos Sinos, sul do Brasil. Ratos Wistar machos foram estudados usando três biomarcadores combinados com análises físicas, químicas e microbiológicas para determinar os efeitos da poluição em quatro locais de amostragem de água. A indução do estresse oxidativo foi quantificada pelos níveis de MDA no sangue periférico, o dano do DNA linfocitário foi determinado usando o ensaio cometa, e as alterações histopatológicas foram analisadas no fígado. As amostras da água do rio dos Sinos (dos quatro pontos selecionados) foram administradas por via oral (como água para beber) por 48 horas. Nenhum aumento do estresse oxidativo ou do dano do DNA foi observado. Entretanto, dano do tecido hepático foi verificado nos animais expostos à ingestão das amostras da água, indicando que o rio dos Sinos está contaminado com substâncias hepatotóxicas. As análises das amostras de água confirmaram a redução da qualidade da água em direção à foz do rio.

Spore germination and gametophyte development of Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) under different pH conditions.

Cyathea atrovirens (Langsd. & Fisch.) Domin, an intensely exploited tree fern, is found inside forests in several succession stages, as well as in swamps, roadsides and unused fields in the Rio dos Sinos basin, in the state of Rio Grande do Sul, southern Brazil. This study evaluated the in vitro germination and gametophyte development of C. atrovirens under different pH conditions, as well as spore viability after different storage times at 7 ºC. The lowest germination rate of spores was obtained at pH 7.0. At pH 5.0 to 6.5, laminar gametophyte development started at 20 to 30 days of culture. Antheridia and archegonia were first observed at 35 and 128 days, respectively. Storage at 7 ºC did not affect germination rates. The capability of germination at different pH levels may explain the occurrence of the species in a wide range of habitats. The present study contributes to the understanding of the full life-cycle of C. atrovirens and to the analysis of the influence of abiotic components, providing information for the cultivation, management and conservation of the species.

Evaluation of Sinos River water genotoxicity using the comet assay in fish.

The Sinos River, in southern Brazil, is polluted by industrial discharges and untreated urban wastes. Fish genotoxicity biomarkers are valuable parameters for environmental risk assessment. In this study, we used the comet assay to detect genotoxicity due to multiple sources of pollution in the peripheral blood of a native fish species (Hyphessobrycon luetkenii). In addition, we analysed possible DNA damage from aluminum, lead, chromium, copper, nickel, iron and zinc contamination. Water samples were collected seasonally from three sampling sites and the fish were assessed under laboratory conditions. Water chemical analysis showed an increased level of aluminum and iron in most of the samples at sites 2 and 3, located in the middle and lower river course, respectively. The index of DNA damage assessed by the comet assay demonstrated no significant differences in different seasons or at the different sampling sites, while the frequency of cells with DNA damage was higher in water samples collected at sites 1 and 2 during the spring season. None of the metals studied seems to be associated with the increase in the frequency of cells with DNA damage observed during the spring season. The results of this study indicate that the Sinos River is contaminated with substances that are genotoxic to fish, including the waters near the river spring.

A multibiomarker approach in rats to assess the impact of pollution on Sinos River, Southern Brazil.

The aim of this study was to determine the feasibility of combining water quality analysis with different biomarkers to characterise the relationship between anthropogenic contamination and biotic response in the Sinos River, southern Brazil. Wistar rats were studied using three biomarkers combined with physical, chemical and microbiological analysis to assess the effects of pollution at four sampling sites. The induction of oxidative stress was quantified by MDA levels in peripheral blood, lymphocyte DNA damage was determined using the comet assay, and histopathological changes were analysed in the liver. After sampling, animals were allowed to drink the river water during a 48 hours period. No increase in oxidative stress and DNA damage was observed. However, liver damage was observed in the animals exposed to water samples, indicating that the Sinos River is contaminated with hepatotoxic substances. Water analyses confirmed that water quality decreased downriver.

Imaging the permeability pore transition in single mitochondria.

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.

Nitric oxide signaling mediates stimulation of L-type Ca2+ current elicited by withdrawal of acetylcholine in cat atrial myocytes.

A perforated-patch whole-cell recording method was used to determine whether nitric oxide signaling participates in acetylcholine (ACh)-induced regulation of basal L-type Ca2+ current (ICa,L) in cat atrial myocytes. Exposure to 1 microM ACh for 2 min inhibited basal ICa,L (-21 +/- 3%), and withdrawal of ACh elicited rebound stimulation of ICa,L above control (80 +/- 13%) (n = 23). Stimulation of ICa,L elicited by withdrawal of ACh (but not ACh-induced inhibition of ICa,L) was blocked by either 50 microM hemoglobin; 30 microM ODQ or 10 microM methylene blue, inhibitors of soluble guanylate cyclase; 10 microM W-7, a calmodulin inhibitor; or 10 microM L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM L-arginine, ACh-induced rebound stimulation of ICa,L was enhanced compared with control responses. Histochemical assay (NADPH diaphorase) indicated that atrial myocytes express constitutive NOS. NO-donor, spermine/NO (SP/NO), >1 microM stimulated basal ICa,L. SP/NO-induced stimulation of ICa,L was inhibited by 50 microM hemoglobin, 30 microM ODQ, or 5 microM H-89, an inhibitor of PKA, and was unchanged by 50 microM MnTBAP, a peroxynitrite scavenger. When ICa,L was prestimulated by 10 microM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase ICa,L. In cells incubated in pertussis toxin (3.4 microg/ml for 6 h; 36 degrees C), ACh failed to affect ICa,L, but 100 microM SP/NO or 10 microM milrinone still increased basal ICa,L. These results indicate that in cat atrial myocytes NO signaling mediates stimulation of ICa,L elicited by withdrawal of ACh but not ACh-induced inhibition of basal ICa,L. NO activates cGMP-induced inhibition of phosphodiesterase (type III) activity. Upon withdrawal of ACh, this mechanism allows cAMP to recover to levels above control, thereby stimulating ICa,L. Pertussis toxin-sensitive G-proteins couple M2 muscarinic receptors to NO signaling. NO-mediated stimulation of ICa, L elicited by withdrawal of ACh may be an important mechanism that rapidly restores cardiac pacemaker and contractile functions after cholinergic suppression of atrial activity.