Comparative toxicity of coal and coal ash: Assessing biological impacts and potential mechanisms through in vitro and in vivo testing.

BACKGROUND: Coal and coal ash present inorganic elements associated with negative impacts on environment and human health. The objective of this study was to compare the toxicity of coal and coal ash from a power plant, assess their inorganic components, and investigate the biological impacts and potential mechanisms through in vitro and in vivo testing. METHODS: Particle-Induced X-ray Emission method was used to quantify inorganic elements and the toxicity was evaluated in Caenorhabditis elegans and Daphnia magna in acute and chronic procedures. The genotoxic potential was assessed using alkaline and FPG-modified Comet assay in HepG2 cells and mutagenicity was evaluated using Salmonella/microsome assay in TA97a, TA100, and TA102 strains. RESULTS: Inorganic elements such as aluminum (Al) and chromium (Cr) were detected at higher concentrations in coal ash compared to coal. These elements were found to be associated with increased toxicity of coal ash in both Caenorhabditis elegans and Daphnia magna. Coal and coal ash did not induce gene mutations, but showed genotoxic effects in HepG2 cells, which were increased using the FPG enzyme, indicating DNA oxidative damage. CONCLUSIONS: The combined findings from bioassays using C. elegans and D. magna support the higher toxicity of coal ash, which can be attributed to its elevated levels of inorganic elements. The genotoxicity observed in HepG2 cells confirms these results. This study highlights the need for continuous monitoring in areas affected by environmental degradation caused by coal power plants. Additionally, the analysis reveals significantly higher concentrations of various inorganic elements in coal ash compared to coal, providing insight into the specific elemental composition contributing to its increased toxicity.

Differences in MCF-7 response to endocrine disruptors in waste, superficial, and treated water from Southern Brazil.

The aim of this study was to evaluate the effect of possible endocrine disruptors in surface and wastewater using a cell proliferation assay in an estrogen-responsive cell line (MCF-7). This study was conducted in the Sinos River (Brazil). The residual water was collected from a Pilot Treatment Plant (using Typha domingensis) and surface waters of the Luis Rau stream, the Sinos River, and the Water Treatment Station (WTS). After exposures (24-120 h), a Sulforhodamine B assay was performed to determine the proliferation rate. The higher increase in proliferation rate was observed with the Luiz Rau stream and the sewage treated by macrophytes in a flotation filter. The results from WTS water remained with a proliferation rate similar to the negative control at all times, suggesting that the conventional treatment is partially effective for the withdrawal of endocrine-disrupting agents. The study demonstrated the efficiency of the MCF-7 line in assessing endocrine disruption caused by wastewater and surface water samples. Our results indicate that conventional water treatment can partially remove the polluting load of endocrine disruptors, minimizing their environmental and public health impacts. Besides, it demonstrates the need to expand sanitary services to improve the population's quality of life.

Permitted daily exposure from preclinical studies of Ginkgo biloba L. dry extract

Abstract Resolution 658/2022 of the Brazilian Regulatory Agency requires the determination of the permitted daily exposure (PDE) of pharmaceutical agents. Ginkgo biloba L. is used therapeutically to treat memory deficits and other brain diseases. However, published results indicate that more studies are needed to confirm the safety of Ginkgo biloba. This study aimed to evaluate the dry extract of Ginkgo biloba L. leaves PDE as an ingredient in an oral pharmaceutical product in preclinical studies using mice. Acute oral toxicity and repeated dose experiments were performed based on OECD guidelines, as well as genotoxicity tests. The results indicate that Ginkgo biloba L. has low acute toxicity, no liver toxicity, and does not alter blood glucose levels. No changes in weight gain were observed, but food intake decreased in males during the first week of treatment at the highest dose. Hematological parameters were not altered in males, whereas females presented lower leukocyte and lymphocyte counts and higher neutrophil counts at the highest dose. The lipid profile was not altered in males, whereas total cholesterol was increased in females. The estimated PDE was 0.1 mg/day and, when related to the maximum residual concentration, indicates that the cleaning process used is safe and does not require reassessment.

Study of the acute and repeated dose 28-day oral toxicity in mice treated with PT-31, a molecule with a potential antipsychotic profile.

Schizophrenia is a psychiatric disorder that affects 1% of the world population and is treated with antipsychotics, which may induce important biochemical and hematological alterations. Since it is necessary to verify the safety of new molecules with antipsychotic potential, the present study aimed to evaluate the oral toxicity of PT-31, a putative α2-adrenoreceptor agonist, after acute (2000 mg/kg) and repeated doses (28 days) gavage treatment, in three different doses: minimum effective dose in animal models (10 mg/kg), twice the dose (20 mg/kg), and four times the dose (40 mg/kg), as recommended by the OECD guidelines. Balb/C female adult mice were used, and biochemical, hematological, and histopathological analyses were performed. PT-31 10 and 20 mg/kg did not cause biochemical alterations related to hepatic and renal toxicity, and neither altered glycemic and lipid profiles. The preclinical dose of PT-31 also did not promote mice histopathological changes in the liver, kidney, and brain. In the hematimetric parameters, PT-31 only increased HGB at 20 mg/kg, and MCH and MCHC at 40 mg/kg. However, all the tested doses of PT-31 showed platelet increase, which must be better investigated. Therefore, further studies are needed to investigate the safety of PT-31 as a potential antipsychotic drug.

Cytotoxic and genotoxic effects induced by associated commercial glyphosate and 2,4-D formulations using the Allium cepa bioassay.

Studies assessing the toxicity of glyphosate and 2,4-dichlorophenoxyacetic acid mixture are scarce. The aim of this study was to evaluate the cytotoxicity and genotoxicity of the mixture of these herbicides using Allium cepa. Roots were exposed to glyphosate (1.56 and 11.66 mg mL-1), 2,4-D (0.28 and 17.5 mg mL-1) and mixture for 24 h, based on the average concentration applied in the field and the acute reference dose (ARfD) established in Brazil. Both isolated and associated herbicides induced a significative decrease in mitotic index (MI) (P < 0.0001) in all tested concentrations. Regarding the genotoxicity results, 2,4-D and the mixture showed, at concentrations applied in the field, a significative increase of chromosomal anomalies (CA) index compared to control (P < 0.0001) and glyphosate (P = 0.024 and P = 0.0002, respectively). All tested groups from the ARfD showed a significative difference compared to the control group (P < 0.0001), as well as glyphosate and 2,4-D isolated compared to the mixture (P = 0.0005 and P < 0.0001, respectively). The most observed CA were apoptotic bodies, giant cells, and nuclear erosions. We emphasize the need for further studies assessing the toxicity of these herbicides' mixture due to the distinct effects caused in different organisms.

Repeated dose of meloxicam induces genotoxicity and histopathological changes in cardiac tissue of mice.

Meloxicam is the non-steroidal anti-inflammatory drug most used in small animals; however, studies on genotoxicity, oxidative stress, and histopathologic alterations in cardiac tissue are limited, especially at therapeutical doses used in these animals. This study evaluated the toxic effects caused by the treatment involving repeated low at higher doses of meloxicam in mice, by genotoxicity, oxidative stress, and histopathological parameters. Mice (CF1, male) received, by gavage, meloxicam at the therapeutic dose indicated for small animals (0.1 mg/kg) and at higher doses (0.5 and 1 mg/kg) for 28 days. Later, they were euthanized for blood and organ analysis. Oxidative stress was analyzed by the plasma ferric reduction capacity (FRAP) and catalase, and genotoxicity, by the comet assay and the micronucleus test. Heart, liver, lung, and kidney tissues were analyzed by the histology, and stomach and duodenum were analyzed with a magnifying glass. The relative weight of organs did not present significant alterations. However, congestion of duodenum vessels was observed at the three tested doses and caused hyperemia of stomach mucosa at 1 mg/kg. In the heart histology there was a reduction in the number of cardiomyocytes, accompanied by an increase in cell diameter (possible cell hypertrophy) dose-dependent. The highest tested dose of meloxicam also increased the DNA damage index, without alterations in the micronucleus test. Meloxicam did not affect the catalase activity but increased the FRAP (1 mg/kg). Meloxicam at the dose prescribed for small animals could potentially cause cardiac histopathologic alterations and genotoxic effects.

Combination of Water Quality Parameters and Bioassays for the Assessment of Two Rivers, Southern Brazil.

In the present study, water physicochemical and microbiological parameters, as well as bioassays using Allium cepa L. seeds and the fish species Astyanax jacuhiensis were used to assess the water quality of two rivers - Ilha River and Paranhana River -, located in southern Brazil. Water samples were collected at the source and mouth of the rivers and then, laboratory experiments were performed. The results evidenced high levels of aluminum and iron in water samples collected at the four sampling sites. The micronucleus (MN) test in fish showed significant difference in the frequencies of nuclear abnormalities (NA) in the mouth of the Paranhana River in comparison to control group in one sampling period, whereas the A. cepa test evidenced significant spatial differences in cytotoxicity between the source and mouth of both rivers. Therefore, these data evidence the poor water quality of the rivers studied as well as the potential toxicity to the aquatic organisms.

Potential use of distinct biomarkers (trace metals, micronuclei, and nuclear abnormalities) in a heterogeneous sample of birds in southern Brazil.

The analysis of metal concentrations in bird feathers and genotoxicity tests are tools used to evaluate anthropogenic impacts on ecosystems. We investigated the response of birds, used as bioindicators, to disturbances observed in three areas with distinctive environmental characteristics (natural, agricultural, and urban) in southern Brazil. For this purpose, we quantified metals (Mn, Cu, Cr, and Zn) in feathers and determined the number of micronuclei (MN) and other nuclear abnormalities (NA) in 108 birds from 25 species and 17 families captured in the study area. No significant differences was found in the metal concentrations and the number of MN and NA between the sampling areas. Zn and Cu concentrations were significantly higher in insectivorous than those in omnivorous birds. The Zn concentration was significantly different between some species, and the Cu concentration was significantly higher in juveniles than that in adults. The best generalized linear models showed that omnivorous birds had more MN and NA and that juveniles and birds with better body condition index had increased NA numbers. This study demonstrates that the analyzed variables contribute in different ways to the result of each biomarker, mainly due to particular ecological and physiological characteristics of each species. We conclude that wild birds have the potential to be used as environmental bioindicators in the study area, but future studies should focus on one or a few species whose ecological and physiological habits are well known.

Evaluation of transgenerational effects caused by metals as environmental pollutants in Daphnia magna.

The present study aimed to evaluate the acute and chronic toxicity of environmentally relevant concentrations of metals (Mn, Al, Fe, and Pb) in Daphnia magna and the generational transposition of reproductive and morphological damages. The effective concentration for 10% of the organisms from each metal was obtained by the acute toxicity test (96 hours); then, another five concentrations lower than this one were defined for the chronic experimentation (21 days), in which the number of neonates generated by each individual was checked daily. At the end of the exposition, the lengths and number of morphological damages were recorded in each adult daphnid. During this, the molt generated on the 14th and 21st days were collected and cultivated for posterior evaluation of the same parameters. Alterations in the reproductive performance were observed in the organisms exposed to manganese and aluminum (4.0 and 0.5 mg L-1, respectively). Organisms exposed to aluminum (0.05 mg L-1) and iron (0.27 mg L-1) showed a reduction in body length. It is also noteworthy that the molt of these adults and their respective offspring also presented reproductive alterations, especially the molt from the 14th day of lead exposure (0.02 mg L-1) and the 21st day of manganese exposure (4.0 mg L-1). Such effects allow us to conclude that environments polluted by metals can reduce the ability of the species to maintain themselves in the ecosystem. In addition, there is a need to increase the control and monitoring of metals, such as aluminum, which present risks even in low concentrations.

Histopathological, genotoxic, and behavioral damages induced by manganese (II) in adult zebrafish.

Manganese is a metal often found as an environmental pollutant and very associated with neurological disorders when in high concentrations. However, little is known about the effects that this contaminant can cause when in environmentally relevant concentrations and occurrence, that is, much lower than those commonly studied. So, the aim of the study was to evaluate the effects that environmentally relevant concentrations of this metal would cause in different zebrafish organs (brain, liver, and blood). Acute 96-h and chronic 30-day exposures were performed using the manganese chloride salt as a pollutant. Behavioral alterations of anxiogenic type were observed in the animals after chronic exposures to 4.0 mg L-1 MnCl2, which traveled a greater distance at the bottom of the aquarium. This may be associated with neuronal damages in the telencephalic region responsible for motor and cognitive activity of the fish, observed in animals from the same exposure. In addition, hepatic histopathological damage as vacuolization of hepatocytes and genotoxic damage, identified by comet assay and micronucleus test, was also observed after acute and chronic exposure, especially at the highest pollutant concentrations (8.0 and 16.0 mg L-1 in acute exposure, and 4.0 mg L-1 in chronic exposure. The study reinforces the risk that environmental pollutants pose to the ecosystem, even in low concentrations.

Selective brain neuronal and glial losses without changes in GFAP immunoreactivity: Young versus mature adult Wistar rats.

Normal ageing results in brain selective neuronal and glial losses. In the present study we analyze neuronal and glial changes in Wistar rats at two different ages, 45 days (young) and 420 days (mature adult), using Nissl staining and glial fibrillary acidic protein (GFAP) immunohistochemistry associated to the Sholl analysis. Comparing mature adults with young rats we noted the former present a decrease in neuronal density in the cerebral cortex, corpus callosum, pyriform cortex, L.D.D.M., L.D.V.L., central medial thalamic nucleus and zona incerta. A decrease in glial density was found in the dorsomedial and ventromedial hypothalamic nuclei. Additionally, the neuron/glia ratio was reduced in the central medial thalamic nucleus and increased in the habenula. No changes were found in the neuronal and glial densities or neuron/glia ratio in the other studied regions. The number of astrocytic primary processes and the number of intersections counted in the Sholl analysis presented no significant difference in any of the studied regions. Overall, neither GFAP positive astrocytic density nor GFAP immunoreactivity showed alteration.

Impact of Anthropogenic Activities on Water Quality of The Paranhana River, Southern Brazil

Abstract: The Paranhana River, located in Southern Brazil, is one of the major tributaries of the Sinos River basin and receives mainly industrial and domestic effluents. In the present study, water physicochemical and microbiological analyses, condition factor, micronucleus test, gill histopathology and metal bioaccumulation in the muscle of the native fish Bryconamericus iheringii collected at two sites (S1 and S2) of the Paranhana River under different degrees of anthropogenic pressures were assessed in four sampling campaigns. Data from water quality parameters, condition factor, mucous cells proliferation in fish gills and bioaccumulation of chromium and manganese in muscle evidenced higher impacts at S2, whereas a higher genotoxic potential was observed at S1. Gill histopathological alterations were found in fish captured at both sampling sites. Temporal variations in all biomarkers analyzed and bioaccumulation of manganese and nickel were observed at S1, whereas only variations in condition factor, gill alterations and bioaccumulation of manganese and aluminum were found at S2. Our study evidences that S1 is under minor anthropogenic impacts and that the high urbanization at S2 reflects in a poor water quality. Nonetheless, the human consumption of fish from the Paranhana River should be avoided given the high concentrations of cadmium, chromium and lead.

Analysis of histopathological abnormalities in the gills of Astyanax jacuhiensis (Characidae) for assessment of water quality in the Ijuí River, southern Brazil / Análisis de anormalidades histopatológicas en las branquias de Astyanax jacuhiensis (Characidae) para la evaluación de la calidad del agua del Río Ijuí, sur de Brasil

The aim of this study was to assess histopathological alterations in the gills of Astyanax jacuhiensis from different points along the Ijuí River and to determine if these abnormalities can be used as biomarkers in biomonitoring studies. Fish specimens were collected from three sites on the Ijuí River in winter and summer and examined histologically for abnormalities of the secondary lamellae of their gills. For each fish, estimates were made of the frequencies of lamellae with edema, hyperplasia, hypertrophy, epithelium lifting, lamellar fusion or deformation, in addition to the overall frequency of abnormal lamellae and the number of abnormalities per lamella. No differences were observed between sampling points during the winter. In summer the frequency of lamellae with hypertrophy was significantly higher at site 3 (Pirapó) than at site 2 (Santo Ângelo) and the frequency of abnormal lamellae was increased at site 1 (Ijuí) in comparison to site 3. Additionally, all three sample points had a significantly higher value in the winter than in the summer for one of the histological parameters analyzed. These results indicate temporal and spatial variation in the level of contamination of the Ijuí River and also shows that fish gill histopathology can be used for in situ biomonitoring studies.

El agua de la cuenca del río Ijuí, en el sur de Brasil, se utiliza principalmente para irrigar los cultivos y para el abastecimiento público de agua. El objetivo del presente estudio fue evaluar alteraciones histopatológicas en las branquias de Astyanax jacuhiensis en diferentes puntos del Río Ijuí y determinar si tales anormalidades pueden ser usadas como biomarcadores en estudios de biomonitoramiento. Los especímenes fueron recolectados en tres puntos del río durante el invierno y el verano y luego las lamelas secundarias de las branquias fueron examinadas histológicamente para la presencia de anormalidades. Para cada pez se estimaron las frecuencias de lamelas con edema, hiperplasia, hipertrofia, desprendimiento del epitelio, fusión y deformación lamelar. Además, se obtuvieron las frecuencias de lamelas alteradas, así como el número medio de cambios por lamela. No se observaron diferencias entre los puntos de recolección durante el invierno. En el verano, la frecuencia de lamelas con hipertrofia fue significativamente mayor en el punto 3 (Pirapó) que en el punto 2 (Santo Angelo), y el punto 1 (Ijuí) presentó mayor frecuencia de lamelas alteradas que el punto 3. Además, en los tres puntos muestreados, el período de invierno presentó valores superiores para uno de los parámetros histológicos analizados. Estos resultados indican variación espacial y temporal en los niveles de contaminación del Río Ijuí y demuestran que el análisis histológico de las branquias de peces puede ser utilizado en estudios de monitoreo in situ.

In situ monitoring of the Sinos River, southern Brazil: water quality parameters, biomarkers, and metal bioaccumulation in fish.

The Sinos River is an important water supply in Southern Brazil and receives industrial, agricultural, and domestic effluents which may affect aquatic biota. Water physicochemical and microbiological analyses, biomarker responses (scaled mass index (SMI), gill histopathology, and micronucleus and nuclear abnormality (MN and NA) frequencies), and metal bioaccumulation in muscle were assessed in the fish species Bryconamericus iheringii (Characidae) captured at three sampling sites (S1, S2, and S3) in four sampling periods. The mean values of five parameters (total phosphorus, thermotolerant coliforms, aluminum, iron, and lead) exceeded the limits established by the Brazilian legislation at the three sampling sites. Although physicochemical analysis indicated higher impacts at S3, in some samples, significantly higher MN frequencies and bioaccumulation of manganese in fish muscle were observed at S1, whereas low SMI and higher concentrations of aluminum and zinc in fish muscle were found at S2. Histopathological alterations in gills were observed in fish collected at the three sampling sites; however, no spatial differences were observed, indicating similar environmental conditions with respect to this biomarker. Moreover, temporal variation of biomarker responses and metal bioaccumulation were found at all sampling sites. Furthermore, the consumption of fish from the Sinos River should be avoided given the concentrations of chromium (all samples), cadmium, and lead in fish muscle above the threshold for safe human consumption.

Water quality parameters, biomarkers and metal bioaccumulation in native fish captured in the Ilha River, southern Brazil.

The Ilha River is one of the main tributaries of the Sinos River, southern Brazil, and it is located in an area characterized by low population density and presence of agricultural activities. Thus, this study aimed to assess the water quality of two sites of the Ilha River (source and mouth, S1 and S2 respectively) in five sampling periods using water physicochemical and microbiological analyses, biomarkers, such as condition factor, micronucleus test, gill histopathological analysis, and metal bioaccumulation in the native fish Bryconamericus iheringii. Mean values of BOD5, thermotolerant coliforms, aluminum, iron and lead exceeded the limits established by the Brazilian legislation for surface waters at both sampling sites. Significant higher micronucleus, nuclear abnormalities and mucous cells frequencies were found at S2 in, at least, one sampling period, whereas fish from S1 presented significant lower condition factor, higher frequencies of lamellar alterations and higher concentrations of chromium and nickel in muscle. Additionally, concentrations of cadmium, chromium and lead in fish muscle exceeded the limits considered safe for human consumption at both sites in at least one sampling period. Data from our study evidenced the mouth of the Ilha River suffers from point genotoxic effects, whereas the source is also contaminated by metals, despite being located in an area under minor anthropic activities.

Uso do bioensaio com Allium cepa L. e análises físico-químicas e microbiológicas para avaliação da qualidade do Rio da Ilha, RS, Brasil / Allium cepa L. bioassay and physicochemical and microbiological analysis to evaluate the water quality of the Ilha River, RS, Brazil

O estudo avalia a toxicidade, citotoxicidade, genotoxicidade e análises físico-químicas e microbiológicas de amostras de águas coletadas em dois pontos (nascente e foz) do Rio da Ilha - um dos principais afluentes do Rio dos Sinos, RS, Brasil - em dois períodos: inverno (2014) e verão (2015), através do bioensaio com Allium cepa que fornece esses dados através da mensuração das raízes dos bulbos, índice mitótico e presença de aberrações cromossômicas. Os resultados demonstraram níveis de citotoxicidade principalmente na foz do rio, e alguns parâmetros (DBO5, fósforo, alumínio, chumbo, ferro, níquel e coliformes termotolerantes) acima da legislação estabelecida, mesmo a região sofrendo pouco impacto de origem antrópica.

This study evaluates the toxicity, cytotoxicity, genotoxicity and physicochemical and microbiological analysis of water samples collected at sites (source and mouth) of the Ilha River -one of the main tributaries of the Sinos River, RS, Brazil - in winter (2014) and summer (2015), by Allium cepa bioassay which provided the data by measuring the roots of the bulbs, mitotic index and presence of chromosomal aberrations. The results show levels of cytotoxicity especially at the mouth of the river, and some parameters (DBO5, phosphorus, aluminum, lead, iron, nickel and fecal coliforms) above the limits established by the Brazilian legislation, despite the localization of the region in an area under minor anthropic impact.

Morphometric differences of Microgramma squamulosa (Kaulf.) de la Sota (Polypodiaceae) leaves in environments with distinct atmospheric air quality.

Plants growing in environments with different atmospheric conditions may present changes in the morphometric parameters of their leaves. Microgramma squamulosa (Kaulf.) de la Sota is a neotropical epiphytic fern found in impacted environments. The aims of this study were to quantitatively compare structural characteristics of leaves in areas with different air quality conditions, and to identify morphometric parameters that are potential indicators of the effects of pollution on these plants. Fertile and sterile leaves growing on isolated trees were collected from an urban (Estância Velha) and a rural (Novo Hamburgo) environment, in Rio Grande do Sul, Brazil. For each leaf type, macroscopic and microscopic analyses were performed on 192 samples collected in each environment. The sterile and fertile leaves showed significantly greater thickness of the midrib and greater vascular bundle and leaf blade areas in the rural environment, which is characterized by less air pollution. The thickness of the hypodermis and the stomatal density of the fertile leaves were greater in the urban area, which is characterized by more air pollution. Based on the fact that significant changes were found in the parameters of both types of leaves, which could possibly be related to air pollutants, M. squamulosa may be a potential bioindicator.

Leaf Dimorphism Of Microgramma Squamulosa (Polypodiaceae): a qualitative and quantitative analysis focusing on adaptations to epiphytism.

The epiphytic fern Microgramma squamulosa occurs in the Neotropics and shows dimorphic sterile and fertile leaves. The present study aimed to describe and compare qualitatively and quantitatively macroscopic and microscopic structural characteristics of the dimorphic leaves of M. squamulosa, to point more precisely those characteristics which may contribute to epiphytic adaptations. In June 2009, six isolated host trees covered by M squamulosa were selected close to the edge of a semi-deciduous seasonal forest fragment in the municipality of Novo Hamburgo, State of Rio Grande do Sul, Brazil. Macroscopic and microscopic analyzes were performed from 192 samples for each leaf type, and permanent and semi-permanent slides were prepared. Sections were observed under light microscopy using image capture software to produce illustrations and scales, as well as to perform quantitative analyses. Fertile and sterile leaves had no qualitative structural differences, being hypostomatous and presenting uniseriate epidermis, homogeneous chlorenchyma, amphicribal vascular bundle, and hypodermis. The presence of hypodermal tissue and the occurrence of stomata at the abaxial face are typical characteristics ofxeromorphic leaves. Sterile leaves showed significantly larger areas (14.80cm2), higher sclerophylly index (0.13g/cm2) and higher stomatal density (27.75stomata/mm2) than fertile leaves. The higher sclerophylly index and the higher stomatal density observed in sterile leaves are features that make these leaves more xeromorphic, enhancing their efficiency to deal with limited water availability in the epiphytic environment, compared to fertile leaves.

Leaf dimorphism of Microgramma squamulosa (Polypodiaceae): a qualitative and quantitative analysis focusing on adaptations to epiphytism

The epiphytic fern Microgramma squamulosa occurs in the Neotropics and shows dimorphic sterile and fertile leaves. The present study aimed to describe and compare qualitatively and quantitatively macroscopic and microscopic structural characteristics of the dimorphic leaves of M. squamulosa, to point more precisely those characteristics which may contribute to epiphytic adaptations. In June 2009, six isolated host trees covered by M. squamulosa were selected close to the edge of a semi-deciduous seasonal forest fragment in the municipality of Novo Hamburgo, State of Rio Grande do Sul, Brazil. Macroscopic and microscopic analyzes were performed from 192 samples for each leaf type, and permanent and semi-permanent slides were prepared. Sections were observed under light microscopy using image capture software to produce illustrations and scales, as well as to perform quantitative analyses. Fertile and sterile leaves had no qualitative structural differences, being hypostomatous and presenting uniseriate epidermis, homogeneous chlorenchyma, amphicribal vascular bundle, and hypodermis. The presence of hypodermal tissue and the occurrence of stomata at the abaxial face are typical characteristics of xeromorphic leaves. Sterile leaves showed significantly larger areas (14.80cm²), higher sclerophylly index (0.13g/cm²) and higher stomatal density (27.75stomata/mm²) than fertile leaves. The higher sclerophylly index and the higher stomatal density observed in sterile leaves are features that make these leaves more xeromorphic, enhancing their efficiency to deal with limited water availability in the epiphytic environment, compared to fertile leaves.

El helecho epífito Microgramma squamulosa se encuentra en el Neotrópico y tiene hojas estériles y fértiles dimorfas. El objetivo de este estudio fue describir y comparar cuantitativa y cualitativamente la organización estructural de las hojas de la M. squamulosa, investigando las características morfológicas y anatómicas, y señalando los factores que contribuyen a la adaptación al ambiente epífito. Los análisis macroscópicos y microscópicos se realizaron a partir de 192 muestras de hojas de cada tipo. Láminas permanentes y semi-permanentes fueron preparadas y las secciones fueron observadas en el microscopio de luz. Hojas fértiles y estériles no mostraron diferencias estructurales cualitativas, son hipostomáticas, presentan epidermis uniseriada, haz vascular anficrival e hipodermis. La presencia de hipodermis y aparatos estomáticos en la nsuperficie abaxial de la epidermis son típicas de hojas xeromórficas. Hojas estériles presentaran áreas significativamente mayores, tasa más alta de esclerofilia y mayor densidad de estomas que hojas fértiles. La mayor tasa de esclerofilia y mayor densidad de estomas son características que hacen las hojas estériles más xeromórficas, aumentando su eficiencia para hacer frente a la disponibilidad de agua limitada en el ambiente epífito, en comparación con las hojas fértiles.

Histological organization is similar in human vocal muscle and tongue--a study of muscles and nerves.

One of the most exciting questions about the human voice is how the vocal fold produces and modulates different sounds. One hypothesis to explain the wide range of movements found in the vocal fold is based on the variety of muscle fiber orientations in the thyroarytenoid (TA) muscle. The tongue (TO) muscle is considered the most complex structure in the body in terms of muscle fiber orientation and movements. Thus, possible similarities between these two muscles and their innervations, the recurrent laryngeal nerve (RLN) and hypoglossal nerve (XII), could explain the complex movements executed by the focal fold. Moreover, such studies help us to understand some microanatomical aspects of vocal fold reinnervation, based on XII-to-RLN anastomosis. Therefore, this study investigates the histological organization of TA and TO muscles and their innervations (n=12 subjects). The muscle fibers were classified into three categories according to their orientation (transverse, undefined, and longitudinal). To quantify the percentage of fibers in each category in the TA and TO, the shape coefficient (shape Z) was estimated. Qualitative analysis and estimation of fiber area and shape Z show that the histological organization of TA and TO muscle is similar. Both muscles present the same percentage of transversal (~72%), undefined (~15%), and longitudinal fibers (~10%). By contrast, the authors' analysis of the morphometric parameters of the RLN and XII shows that there is no correlation between these nerves. In conclusion, in humans, TA and TO muscles present similar histological organization and this finding could help to explain interesting questions about human phonation.