Multi-biomarker approach to evaluate the toxicity of chlorpyrifos (active ingredient and a commercial formulation) on different stages of Biomphalaria straminea.

Biomphalaria straminea is a freshwater gastropod native to South America and used in toxicological assessments. Our aim was to estimate 48 h-LC50 and sub-chronic effects after the exposure to low concentrations of chlorpyrifos as commercial formulation (CF) and active ingredient (AI) on B. straminea adult, embryos and juveniles. Concentrations between 1 and 5000 µg L-1 were chosen for acute exposures and 0.1 and 1 µg L-1 for the sub-chronic one. After 14 days biochemical parameters, viability and sub-populations of hemocytes, reproductive parameters, embryotoxicity and offspring' survival were studied. Egg masses laid between day 12 and 14 were separated to continue the exposure and the embryos were examined daily. Offspring' survival and morphological changes were registered for 14 days after hatching. 48 h-LC50, NOEC and LOEC were similar between CF and AI, however the CF caused more sub-lethal effects. CF but not the AI decreased carboxylesterases, catalase and the proportion of hyalinocytes with respect to the total hemocytes, and increased superoxide dismutase and the % of granulocytes with pseudopods. Also CF caused embryotoxicity probably due to the increase of embryos' membrane permeability. Acetylcholinesterase, superoxide dismutase, hemocytes sub-populations, the time and rate of hatching and juveniles' survival were the most sensitive biomarkers. We emphasize the importance of the assessment of a battery of biomarkers as a useful tool for toxicity studies including reproduction parameters and immunological responses. Also, we highlight the relevance of incorporating the evaluation of formulations in order to not underestimate the effects of pesticides on the environment.

Lethality and Acetylcholinesterase Inhibition in a Native Invertebrate Species Exposed to Water Samples of an Impacted Stream (Reconquista River Basin, Argentina).

The study of multiple biomarkers in bioindicator species is a useful tool to evaluate water quality in addition to physicochemical analysis. The aim of this work was to study the toxicity of water samples from two sites with different anthropogenic impacts (R: near a residential area and FP: close to horticultural farms and industrial waste treatment plants) from Las Catonas sub-basin (Reconquista River basin) in the native gastropod Biomphalaria straminea. Some physicochemical parameters and chlorpyrifos concentration were measured in water samples. Snails were exposed in laboratory conditions 48 h to the water samples and neurotoxicity, behavior, lethality and acetylcholinesterase, carboxylesterase, glutathione S-transferase, glutathione reductase and catalase activities were measured. In water from FP, chlorpyrifos was detected and conductivity and pH were higher than in R. Lethality (60%) and a decrease (30%) in acetylcholinesterase were observed in snails exposed to FP indicating that water contamination causes high toxicity in B. straminea.

Aquatic Contamination in Lugano Lake (Lugano Lake Ecological Reserve, Buenos Aires, Argentina) Cause Negative Effects on the Reproduction and Juvenile Survival of the Native Gastropod Biomphalaria straminea.

Lugano Lake is located in an Ecological Reserve of Buenos Aires City. Biomonitoring of its water quality is essential due to its importance as a place for recreation and protection of native species. Biomphalaria straminea is a native hermaphrodite aquatic gastropod that inhabits different freshwater bodies of Argentina and was recently selected as a potential bioindicator. We propose this study as a first approach to assessing specific organisms' use in biomonitoring of urban wild reserves, and the usefulness of reproduction assays. B. straminea survival, behavior, reproduction success and offspring survival after the exposure to water samples from Lugano Lake (L1, L2, and L3) were evaluated. Temperature, pH, conductivity and dissolved oxygen were registered in situ. Samples were transported to the laboratory and chemical analysis and bioassays were performed using 20 snails per site. A control group with tap water was added. Egg masses were separated, exposed individually and observed daily using a stereoscopic microscope. After hatching, juveniles were placed in tap water and offspring survival was registered at the first, second, third and fourth months after the beginning of the assay. High levels of conductivity, turbidity and nutrients were obtained. Ammonium and nitrite were higher than the guideline level for the protection of aquatic life. During the bioassay 20% of the snails (L2 and L3) showed abnormally protruding of the head-food region. The number of eggs and embryonated eggs per mass did not differ between treatments. Egg masses exposed to water samples from the lake presented overlapping and abnormal eggs and arrested embryos. Besides, low % of hatching (L1: 33%, L2: 42%, and L3: 16%) and juvenile survival after the first (L1:14%; L2:78%) and second month (L1: 60%) were noted. In the control group, 85% of hatching and 100%-90% of survival were observed. Our results suggests the presence of pollutant in the lake. B. straminea seems to be a sensitive local species. Biomphalaria spp. reproduction assays can provide a valuable endpoint for toxicity and risk assessments and a usefulness tool for biomonitoring water quality.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of magnitude higher than cholinesterase. A greater efficiency (Vmax/Km) was obtained using acetylthiocholine and p-nitrophenyl butyrate. Acute exposure to azinphos-methyl did not cause inhibition of cholinesterase activity until 10 mg L(-1) used. Carboxylesterases towards p-nitrophenyl butyrate was inhibited by azinphos-methyl being the IC502.20±0.75 mg L(-1) of azinphos-methyl. Subchronic exposure to environmental concentrations of azinphos-methyl (0.02 and 0.2 mg L(-1)) produced a decrease in survival, protein content and carboxylesterases activity despite no inhibition of cholinesterase activity was observed. B. straminea cholinesterase is not a sensible biomarker. On the contrary, carboxylesterases activity was inhibited by azinphos-methyl. Carboxylesterases could be protecting cholinesterase activity and therefore, protecting the organism from neurotoxicity. This work confirms the advantages of measuring cholinesterases and carboxylesterases jointly in aquatic biomonitoring of pesticide contamination. This becomes relevant in order to find more sensitive biomarkers and new strategies to protect non-target aquatic organisms from pesticide contamination.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.

Ocorrência de microrganismos oportunistas e superinfectantes em crianças com 6, 12 e 18 meses de idade e suas mães / Occurrence of opportunistic and superinfecting microorganismsin from children with 6, 12 and 18 months of age and their mothers

A primeira infância é a fase mais importante para o estabelecimento de condições de saúde, desenvolvimento da microbiota bucal e para o estabelecimento de hábitos. Desde que a grande maioria das infecções graves que acometem as crianças está associada a microrganismos superinfectantes e multirresistentes a antimicrobianos, cujo estabelecimento na cavidade bucal de bebês não é adequadamente descrito, foi objetivo do estudo, analisar a presença de um grupo desses microrganismos em crianças com seis meses de idade e suas mães, ao longo de doze meses de acompanhamento, bem como a presença de genes de resistência aos principais antimicrobianos nas amostras clínicas oriundas das mães e seus bebês. Para tanto, 68 pares de bebês e suas mães foram selecionados. Após levantamento de dieta dos bebês e das condições socioeconômicas das famílias, realizava-se a coleta de saliva e biofilme subgengival das mães e saliva das crianças, para avaliação microbiológica. Esses procedimentos foram repetidos, em todos os pares, quando os bebês completaram doze e dezoito meses de idade, acrescentando-se nessas ocasiões a coleta do biofilme dental das crianças. A detecção dos diferentes microrganismos oportunistas era realizada pela amplificação do DNA dos microrganismos alvo empregando-se as reações em cadeia da DNA polimerase (PCR). As condições de saúde da mucosa bucal e gengiva foram satisfatórias para os bebês, enquanto a maioria das mães era portadora de gengivite ou periodontite. Crianças cujas mães tinham gengivite mostraram maior freqüência desses patógenos na saliva e no biofilme. Os principais microrganismos oportunistas detectados em mães e crianças foram os membros da família Enterobacteriaceae, além de Enterococcus spp., E. faecalis, Helicobacter spp. e Helicobacter pylori. Outros agentes infecciosos, como Citomegalovirus e vírus Epstein-Barr foram raramente detectados nas crianças, mas freqüentes nas mães com inflamação periodontal, enquanto outros como o vírus do...

Early childhood is the most important phase for the establishment of health conditions, development of oral microbiota and the establishment of habits. As the vast majority of infections that affect children is associated with superinfecting microorganisms and multi-resistant ones to antibiotics, whose establishment in the oral cavity of babies is not adequately described, the objective of this study was to analyze the presence of a group of these microorganisms in children aged six months of age and their mothers over twelve months of monitoring, as well as the presence of resistance genes to key antimicrobials in clinical samples derived from mothers and their babies. For this, 68 pairs of infants and their mothers were selected. After surveying the diet of babies and families’ socioeconomic conditions, it was performed the collection of the mothers’ saliva and subgingival biofilm and their children’s saliva for microbiological evaluation. These procedures were repeated in all pairs, when babies completed twelve and eighteen months of age, adding on these occasions the collection of their dental biofilm. The detection of different opportunistic microorganisms was performed by amplification of target DNA of microorganisms through the chain reactions of DNA polymerase (PCR). Health conditions of oral mucosa and gingiva were satisfactory for the babies, while most of the mothers was a carrier of gingivitis or periodontitis. Children whose mothers had gingivitis showed increased frequency of these pathogens in saliva and biofilm. The main opportunistic microorganisms detected in mothers and children were members of the family Enterobacteriaceae, and Enterococcus spp., E. faecalis, Helicobacter spp. and Helicobacter pylori. Other infectious agents such as cytomegalovirus and Epstein-Barr virus were rarely detected in these children but common among the mothers with periodontal inflammation, while others such as herpes simplex virus type 1, Pseudomonas spp…

Ocorrência de microrganismos oportunistas e superinfectantes em crianças com 6, 12 e 18 meses de idade e suas mães / Occurrence of opportunistic and superinfecting microorganismsin from children with 6, 12 and 18 months of age and their mothers

A primeira infância é a fase mais importante para o estabelecimento de condições de saúde, desenvolvimento da microbiota bucal e para o estabelecimento de hábitos. Desde que a grande maioria das infecções graves que acometem as crianças está associada a microrganismos superinfectantes e multirresistentes a antimicrobianos, cujo estabelecimento na cavidade bucal de bebês não é adequadamente descrito, foi objetivo do estudo, analisar a presença de um grupo desses microrganismos em crianças com seis meses de idade e suas mães, ao longo de doze meses de acompanhamento, bem como a presença de genes de resistência aos principais antimicrobianos nas amostras clínicas oriundas das mães e seus bebês. Para tanto, 68 pares de bebês e suas mães foram selecionados. Após levantamento de dieta dos bebês e das condições socioeconômicas das famílias, realizava-se a coleta de saliva e biofilme subgengival das mães e saliva das crianças, para avaliação microbiológica. Esses procedimentos foram repetidos, em todos os pares, quando os bebês completaram doze e dezoito meses de idade, acrescentando-se nessas ocasiões a coleta do biofilme dental das crianças. A detecção dos diferentes microrganismos oportunistas era realizada pela amplificação do DNA dos microrganismos alvo empregando-se as reações em cadeia da DNA polimerase (PCR). As condições de saúde da mucosa bucal e gengiva foram satisfatórias para os bebês, enquanto a maioria das mães era portadora de gengivite ou periodontite. Crianças cujas mães tinham gengivite mostraram maior freqüência desses patógenos na saliva e no biofilme. Os principais microrganismos oportunistas detectados em mães e crianças foram os membros da família Enterobacteriaceae, além de Enterococcus spp., E. faecalis, Helicobacter spp. e Helicobacter pylori. Outros agentes infecciosos, como Citomegalovirus e vírus Epstein-Barr foram raramente detectados nas crianças, mas freqüentes nas mães com inflamação periodontal, enquanto outros como o vírus do...

Early childhood is the most important phase for the establishment of health conditions, development of oral microbiota and the establishment of habits. As the vast majority of infections that affect children is associated with superinfecting microorganisms and multi-resistant ones to antibiotics, whose establishment in the oral cavity of babies is not adequately described, the objective of this study was to analyze the presence of a group of these microorganisms in children aged six months of age and their mothers over twelve months of monitoring, as well as the presence of resistance genes to key antimicrobials in clinical samples derived from mothers and their babies. For this, 68 pairs of infants and their mothers were selected. After surveying the diet of babies and families’ socioeconomic conditions, it was performed the collection of the mothers’ saliva and subgingival biofilm and their children’s saliva for microbiological evaluation. These procedures were repeated in all pairs, when babies completed twelve and eighteen months of age, adding on these occasions the collection of their dental biofilm. The detection of different opportunistic microorganisms was performed by amplification of target DNA of microorganisms through the chain reactions of DNA polymerase (PCR). Health conditions of oral mucosa and gingiva were satisfactory for the babies, while most of the mothers was a carrier of gingivitis or periodontitis. Children whose mothers had gingivitis showed increased frequency of these pathogens in saliva and biofilm. The main opportunistic microorganisms detected in mothers and children were members of the family Enterobacteriaceae, and Enterococcus spp., E. faecalis, Helicobacter spp. and Helicobacter pylori. Other infectious agents such as cytomegalovirus and Epstein-Barr virus were rarely detected in these children but common among the mothers with periodontal inflammation, while others such as herpes simplex virus type 1, Pseudomonas spp…

Avaliação da atividade inibitória de extratos de plantas da savana brasileira sobre bactérias associadas às periodontopatias / Evaluation of inhibiting activity of plant extracts from the Brazilian savanna on bacteria associated with periodontopathies

O objetivo deste artigo foi avaliar a atividade antimicrobiana de plantas do cerrado brasileiro sobre cepas de referência de espécies bacterianas associadas às periodontopatias. Foram usados extratos vegetais, aquosos e hidroalcoólicos de 17 plantas típicas do cerrado, preparados e submetidos a testes de atividade antimicrobiana sobre Fusobacterium nucleatum ATCC 25586, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 2564 e Aggregatibacter actinomycetemcomitans ATCC 33384. Para a triagem inicial utilizou-se o método de difusão em ágar Wilkins-Chalgren acrescido de 0,5% de extrato de levedura, hemina, menadiona e 5% de sangue desfibrinado de cavalo inoculado previamente com 108 UFC da cepa bacteriana a ser testada, adicionando-se discos de papel de filtro impregnados com 20µl do extrato testado. Determinou-se a presença ou não de halo de inibição do crescimento bacteriano. Todos os extratos que apresentaram a mínima capacidade de inibir o crescimento bacteriano na triagem inicial foram submetidos a testes para avaliar a máxima diluição inibitória dos mesmos. Os resultados mostraram que os extratos das folhas de araçá, aroeira-do-sertão, guajuvira e de casca de jacarandá tiveram atividade inibitória frente a todos os microrganismos testados, embora o tempo de contato necessário para exercer essa atividade tenha variado significativamente de acordo com o extrato testado, destacando-se o extrato de araçá e a aroreira-do-sertão.

This article aims to evaluate the antimicrobial activity of plants from the Brazilian savanna on sample strains of bacterial species associated with periodontopathies. Vegetable, aqueous and hydroalcoholic extracts from 17 typical plants of the Brazilian savanna were prepared and subjected to antimicrobial activity tests on Fusobacterium nucleatum ATCC 25586, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 2564 and Aggregatibacter actinomycetemcomitans ATCC 33384. For the initial triage stage, the Wilkins-Chalgren agar dilution procedure was used, with the addition of 0.5% yeast extract, hemin, menadione and 5% defibrinated horse blood previously inoculated with 108 CFU of the bacterial strain to be tested, adding filter-paper disks soaked with 20µl of the tested extract. The presence or absence of inhibition halos of bacterial growth was determined. All extracts that presented at least minimum capacity to inhibit bacterial growth in the initial triage stage were subjected to tests in order to evaluate their maximum inhibitory dilution. The results showed that leaf extracts from the araçá (Psidium cattleianum), aroeira-do-sertão (Myracrodruon urundeuva) and guajuvira (Patagonula americana L.) plants, and the jacarandá tree bark showed inhibiting activity on all of the tested microorganisms, although the contact time needed to accomplish it varied significantly, depending on the extract tested. The best results were obtained with the araçá and aroreira-do-sertão extracts.

Dentigerous cysts in primary dentition: report of 2 cases.

Dentigerous cysts are benign odontogenic cysts associated with the crowns of permanent teeth. They are usually single in occurrence and located in the mandible. The purpose of this case report was to describe the management of 2 dentigerous cysts in children. The treatments instituted were the extraction of the deciduous tooth involved followed by marsupialization in the first case and enucleation in the second one. Both treatments allowed rapid healing of the lesion and eruption of the permanent teeth without the need for orthodontic treatment.

Odontomas in pediatric dentistry: report of two cases.

Odontomas are developmental disturbances which manifest in the form of denticles or amorphous informes masses comprising all dental tissues, especially enamel and dentin, with variable amounts of pulp and cement. We describe here two clinical cases of odontomas in children, focusing on diagnostic means and the importance of early treatment of these lesions. The standard treatment for the two present cases was surgical removal.