Exposure to phenanthrene and depuration: Changes on gene transcription, enzymatic activity and lipid peroxidation in gill of scallops Nodipecten nodosus.

Understanding the mechanism of phenanthrene (PHE) biotransformation and related cellular responses in bivalves can be an important tool to elucidate the risks of polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms. In the present study it was analyzed the transcriptional levels of 13 biotransformation genes related to cytochrome P450 (CYP), glutathione S-transferase (GST), sulfotransferase (SULT), flavin-containing monooxygenase and fatty acid-binding proteins by qPCR in gill of scallops Nodipecten nodosus exposed for 24 or 96h to 50 or 200µgL(-1) PHE (equivalent to 0.28 and 1.12µM, respectively), followed by depuration in clean water for 96h (DEP). Likewise, it was quantified the activity of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), GST and levels of lipid peroxidation. Increased transcriptional levels of CYP2UI-like, CYP2D20-like, CYP3A11-like, GSTomega-like, SULT1B1-like genes were detected in organisms exposed to PHE for 24 or 96h. In parallel, GR and GPX activities increased after 96h exposure to 200µgL(-1) PHE and G6PDH activity increased after 24h exposure to 50µgL(-1) PHE. This enhancement of antioxidant and phase I and II biotransformation systems may be related to the 2.7 and 12.5 fold increases in PHE bioaccumulation after 96h exposure to 50 and 200µgL(-1) PHE, respectively. Interestingly, DEP caused reestablishment of GPX and GR activity, as well as to the transcript levels of all upregulated biotransformation genes (except for SULT1B1-like). Bioaccumulated PHE levels decreased 2.5-2.9 fold after depuration, although some biochemical and molecular modifications were still present. Lipid peroxidation levels remained lower in animals exposed to 200µgL(-1) PHE for 24h and DEP. These data indicate that N. nodosus is able to induce an antioxidant and biotransformation-related response to PHE exposure, counteracting its toxicity, and DEP can be an effective protocol for bivalve depuration after PHE exposure.

Sexual stages of the female portion in the scallop Nodipecten nodosus (Linné, 1758) and astaxanthin quantity in each stage / Estágios sexuais da porção feminina da gônada da vieira Nodipecten nodosus (Linné, 1758) e a quantidade de astaxantina em cada estágio

This work describes the gametogenic cycle of the scallop Nodipecten nodosus kept in a culture system. To this end, during one year, samples were taken from the broodstocks every 30 days to be submitted to macroscopic and microscopic analyses and to measure the amount of astaxanthin. To perform the microscopic evaluation, 5 μ slices from the median portion of the female part of the gonad were submitted to the pattern methodology for histological analyses with paraffin and HE coloration. The remaining portion of the female gonad was lyophilised to extract and quantify the levels of astaxanthin using HPLC. The microscopic analyses revealed four well defined stages for the reproductive cycle. Analyses of data taken throughout the year indicated preferential spawning periods from December to January and from July to September. The astaxanthin analyses showed higher amounts of this carotenoid during the advanced pre-spawning and the initial spawning periods than during gametogenesis, initial pre-spawning, advanced spawning, and the spent stages. According to these results, it was possible to establish a descriptive table of the sexual stages of the female portion of the gonad and the amount of astaxanthin in the sexual stage of the scallop Nodipecten nodosus.

Este trabalho descreve o ciclo gametogênico da vieira Nodipecten nodosus mantida em ambiente de cultivo. Para isto, durante um ano, amostras de indivíduos reprodutores foram coletadas a cada 30 dias e submetidas à avaliação macroscópica e microscópica e à quantificação de astaxantina. Para a avaliação microscópica, secções de 5 μ da porção mediana feminina da gônada foram submetidas à metodologia de análise histológica padrão em parafina e coloração HE. O restante da porção feminina da gônada foi liofilizado para extração e quantificação de astaxantina em HPLC. A avaliação microscópica permitiu a descrição de quatro estágios bem definidos para o ciclo reprodutivo. Na análise ao longo do ano, foram observados períodos preferenciais de desova em dezembro e janeiro e de julho a setembro. A análise da quantidade de astaxantina, mostrou, nos estádios de pré-desova avançada e de desova inicial, uma maior quantidade desse carotenoide em comparação aos estádios de gametogênese, pré-desova inicial, desova avançada e repouso. Em função desses resultados, foi possível estabelecer um quadro descritivo dos estágios sexuais da porção feminina da gônada e quantidade de astaxantina em cada estágio sexual da vieira Nodipecten nodosus.

Carotenoid extraction from the gonad of the scallop Nodipecten nodosus (Linnaeus, 1758) (Bivalvia: Pectinidae) / Extração de carotenóides da gônada da vieira Nodipecten nodosus (Bivalvia: Pectinidae)

In marine bivalve mollusks, unsaturated molecules called carotenoids are present in the natural diet and play an important role in different biological process, especially in reproduction. In order to gain more insights into these compounds in Nodipecten nodosus it was necessary to develop a suitable protocol for extraction of carotenoids from the gonads. Female gonads of cultured scallops (75 mm length) were lyophilized and macerated in liquid N2. To verify the effect of composition in organosolvents on the extracting solutions, two organic solvents were tested: acetone and hexane (Ac = O:Hex) at four ratios, 1:1, 1:3, 1:5, and 2:3, in four static extraction times: 0, 5, 10, and 15 minutes. Total carotenoids and astaxanthin contents were determined in the crude extracts by UV-visible spectrophotometry and high performance liquid chromatography (HPLC), respectively. Triplicate aliquots of 50 mg were used for each treatment. The results indicated that the best single extraction (0.312 ± 0.016 µg carotenoids/mg) was attained with Ac = O: Hex 1:3, for 15 minutes. Through exhaustive extraction methodology (10x), a superior yield (0.41 ± 0.001 µg carotenoids/mg) was obtained from a gonad sample in comparison to the highest value found for a single extraction. Astaxanthin content was reduced by 8.6 percent in carotenoid extract preservation assay, i.e., -18 °C, 26 days incubation, under N2 atmosphere.

Em moluscos bivalves marinhos, carotenóides insaturados estão presentes na dieta natural, com um importante papel em diversos processos biológicos, em especial na reprodução. A elucidação dos efeitos destes compostos em Nodipecten nodosus requer o desenvolvimento de um protocolo adequado para a extração de carotenóides das gônadas desses animais. Para isso, gônadas de vieiras cultivadas (75 mm de comprimento) foram liofilizadas e maceradas em N2 líquido. Amostras em triplicata com 50 mg foram coletadas para a utilização em cada tratamento. Os conteúdos de carotenóides totais e astaxantina foram determinados via espectrofotometria de luz UV-visível e cromatografia líquida de alta eficiência (CLAE), respectivamente. O efeito da composição em organosolventes das soluções de extração foi testado utilizando-se acetona (Ac = O) e hexano (Hex) em quatro proporções (Ac = O:Hex): 1:1, 1:3, 1:5, e 2:3; em quatro tempos de extração: 0, 5, 10, e 15 minutos. Os resultados mostraram que o melhor rendimento de extração (0,312 ± 0,016 µg carotenóides/mg) foi obtido com Ac = O:Hex, 1:3, por 15 minutos. Com a utilização de protocolo de extração exaustiva (10x), uma quantidade superior (0,41 ± 0,001 µg de carotenóides/mg) foi obtida de amostras de gônada, comparativamente aos valores obtidos em extrações únicas. O conteúdo de astaxantina foi reduzido em 8,6 por cento em testes de preservação deste metabólito em extratos crus (-18 °C, 26 dias de incubação em atmosfera de N2).