Feasibility study for mercury remediation by selenium competition in Pleurotus mushrooms.

Mushrooms may incorporate significant levels of Hg making its consumption harmful to human health. Mercury remediation induced by Se competition in edible mushrooms represents a valuable alternative since Se plays effective roles against Hg uptake, accumulation, and toxicity. In this way, Pleurotus ostreatus and Pleurotus djamor were cultivated on Hg-contaminated substrate simultaneously supplemented with Se(IV) or Se(VI) under different dosages in this study. The protective role of Se was assessed taking into account morphological characteristics and Hg and Se total concentrations determined by ICP-MS, as well as proteins and protein-bound Hg and Se distribution by SEC-UV-ICP-MS, and Hg speciation studies (Hg(II) and MeHg) by HPLC-ICP-MS. Both Se(IV) and Se(VI) supplementation were able to recover the morphology mainly of Hg-contaminated Pleurotus ostreatus. The mitigation effects induced by Se(IV) stood out more than Se(VI) in terms of Hg incorporation, decreasing the total Hg concentration up to 96 %. Also, it was found that supplementation mainly with Se(IV) reduced the fraction of Hg bound to medium molecular weight compounds (17-44 kDa) up to 80 %. Finally, it was shown a Se-induced inhibitory effect on Hg methylation, decreasing MeHg species content in mushrooms exposed to Se(IV) (51.2 µg g-1) up to 100 %.

Effects of Se(IV) or Se(VI) enrichment on proteins and protein-bound Se distribution and Se bioaccessibility in oyster mushrooms.

A successful mushroom enrichment process must produce foods that have compounds potentially absorbed by the human body. In this study, Pleurotus ostreatus and Pleurotus djamor mushrooms were grown on organic substrate supplemented with different Se(IV) and Se(VI) concentrations, and evaluated in the following features: Fruiting bodies morphology; Se uptake and accumulation; Distribution of proteins and protein-bound Se; Se species identification on enzymatic extracts; Se bioaccessibility; and Distribution of bioaccessible protein-bound Se. Pleurotus djamor grown on Se(IV)-supplemented substrate showed the greatest potential to uptake and accumulate Se. For Se species screening, selenomethionine was identified in white oyster mushroom, while selenomethionine, selenocystine, and Se-methylselenocysteine in pink oyster mushrooms. In soluble fractions from in vitro gastrointestinal digestion assays, Se showed high bioaccessibility (>94%). Lastly, bioaccessible Se species were found to be mainly associated to LMW (<17 kDa) in Pleurotus ostreatus (74%) and Pleurotus djamor (68%) grown on Se(IV)-supplemented substrates.

The protective role of selenium against uptake and accumulation of cadmium and lead in white oyster (Pleurotus ostreatus) and pink oyster (Pleurotus djamor) mushrooms.

Mushrooms are bioaccumulators and have been used to produce Se-enriched foods. However, these fungi can also bioaccumulate potentially toxic metals, producing food dangerous to human health. It is known that co-exposure to Se plays a protective role against metal accumulation and toxicity in some organisms due to its antioxidant properties. Thus, this study aimed to evaluate the protective effect of Se(IV) and Se(VI) on elemental uptake and accumulation as well as proteins and protein-bound Se, Cd, and Pb distribution in Pleurotus mushrooms. Pink oyster and white oyster mushrooms showed high ability to bioaccumulate Se (19-205 µg g-1), Cd (4.5 to 18.8 µg g-1), and Pb (1.6 to 7.0 µg g-1). Growth substrate supplementation with Se(IV) or Se(VI) decreased the Cd total concentration in mushrooms by 4 to 89%, while Se(VI) increased the Pb total concentration by 9% to 187%, compared to growth in absence of Se. It was found that despite molecular weights distributions of mushrooms grown on Se(IV) and Se(VI)-supplemented substrates being similar, Se(VI) supplementation favoured Se interaction with proteins of medium molecular weight (17-44 kDa), when compared to supplementation with Se(IV). Therefore, we propose the supplementation of growth substrates with Se(VI) to reduce eventual Cd accumulation and produce Se-enriched oyster mushrooms.

Elemental imaging by Laser-Induced Breakdown Spectroscopy to evaluate selenium enrichment effects in edible mushrooms.

Mushrooms are bioaccumulating organisms commonly used in selenium (Se) enrichment studies. However, the addition of Se in the culture medium may alter the distribution of other essential elements in the mushroom fruiting body. To evaluate the effects of the Se enrichment, Ca, Mg, and K distributions in pink oyster (Pleurotus djamor) and K and Mg distributions in white oyster (Pleurotus ostreatus) mushrooms were mapped by laser-induced breakdown spectroscopy (LIBS), which can be used at room temperature and requires minimal or no sample preparation. It was verified that Se enrichment favoured the accumulation of Ca in the lower part of the pink oyster mushroom and prevented the transport of this element to the edges and tops. The Se enrichment also altered the distribution of K and Mg, decreasing the numerical correlation between the K and Mg distributions (R² = 0.5871). In the white oyster mushroom, however, despite the changes in the morphological characteristics of the fruiting bodies after enrichment, there were generally nonsignificant differences in the K and Mg distributions between the control and the Se-enriched mushrooms.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA.

OBJECTIVE: The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). MATERIAL AND METHODS: Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. RESULTS: In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. CONCLUSION: mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Influência de medicações intracanal utilizadas em procedimentos endodônticos regenerativos na sobrevivência de células da papila apical in vitro / Influence of intracanal medications used in regenerative endodontic procedures on survival of apical papilla cells in vitro

Procedimentos endodônticos regenerativos proporcionaram mudanças no tratamento de pacientes com dentes imaturos e periodontite apical possibilitando desenvolvimento radicular completo e menor incidência de fratura dental. Medicações intracanal são utilizadas para a realização da desinfecção; entretanto, o efeito delas sobre às células da papila apical é pouco elucidado. Adicionalmente, pouco se conhece a respeito do efeito destas substâncias sobre células previamente submetidas à condição pró- inflamatória. O objetivo deste estudo foi avaliar a citotoxicidade de medicações intracanal empregadas em procedimentos regenerativos em Endodontia sobre células de papila humana em cultura em condição fisiológica e ativada. Cultura de células foi estabelecida a partir da papila apical removida de um terceiro molar imaturo extraído. As substâncias estudadas foram a pasta tripla antibiótica modificada: ciprofloxacina, metronidazol e cefalosporina (1:1:1); CFC: ciprofloxacina, metronidazol e hidróxido de cálcio (1:1:2) e CFC modificado: ciprofloxacina, metronidazol e hidróxido de cálcio (2:2:1). Parte das células foram estimuladas previamente por ácido lipoteicóico (LTA) de Enterococcus faecalis por 7 dias. Após plaqueamento, células foram expostas a concentrações crescentes das medicações por 1, 3, 5 e 7 dias. Foram avaliados viabilidade celular por meio de brometo de 3-(4,5-dimetiliazol-2-il)-2,5-difeniltetrazólio (MTT) e liberação de óxido nítrico (NO) pelo método de Griess

A análise estatística foi realizada por meio de análise de variância a 1 critério (ANOVA) seguida de pós teste de Tukey, com nível de significância de 5%. O CFC modificado foi a medicação que demonstrou menor efeito citotóxico sobre a viabilidade celular nos tempos experimentais estudados, o CFC promoveu queda da viabilidade celular especialmente após 7 dias de contato. A pasta tripla antibiótica modificada resultou em comprometimento importante da viabilidade podendo ser considerada a mais citotóxica. A ativação celular por LTA resultou em níveis aumentados de atividade mitocondrial para todas as medicações sendo mais evidente nos períodos experimentais mais longos. A ativação celular também contribuiu para níveis maiores de óxido nítrico. Conclui-se que o efeito citotóxico das medicações testadas é dependente de sua concentração, tempo de contato e condição celular, sendo a pasta tripla antibiótica modificada a mais citotóxica em concentrações elevadas podendo implicar clinicamente na diminuição da viabilidade das células da papila apical podendo diminuir o sucesso dos procedimentos regenerativos.

Regenerative Endodontic procedures have provided changes in treatment of patients with immature teeth and apical periodontitis enabling full root development and lower incidence of dental fracture. Intracanal dressings are used for disinfection; however, their effect on apical papilla cells is poorly elucidated. Additionally, the effect of these substances on cells previously subjected to proinflammatory condition is still unknown. The aim of this study was to evaluate the cytotoxicity of intracanal dressings used in Endodontics regenerative procedures on cultured human apical papilla cells at physiologic and activated condition. Cell culture was established from the apical papilla removed from an extracted immature third molar. The substances studied were triple antibiotic modified paste: ciprofloxacin, metronidazole and cephalosporin (1:1:1); CFC: ciprofloxacin, metronidazole and calcium hydroxide (1:1:2) and modified CFC: ciprofloxacin , metronidazole and calcium hydroxide (2:2:1). Part of the cells was stimulated previously with lipotheichoic acid (LTA) of Enterococcus faecalis por 7 days. Once plated, cells were exposed to increasing concentration of the medications for 1, 3, 5 and 7 days. Cell viability was evaluated by means of 3-bromide (4.5-dimetiliazol-2-yl) -2.5-difeniltetraze (MTT) and Nitric Oxide (NO) release was assessed by the Griess method.

The statistical analysis was done through analysis of variance with 1 criteria (ANOVA) followed by Tukey test with 5% of significance level. Modified CFC was the medication that demonstrated the less cytotoxic effect on cell viabilityat the experimental periods studied while CFC promoted significant decrease on cell viability specially after 7 days of contact. The modified triple antibiotic paste resulted in important alteration of cell viability being considered the most citotoxic. Cellular activation by LTA resulted in increased levels of mitochondrial activity for all medications being more evident at the longer experimental periods. Cellular activation also contributed to higher levels of nitric oxide release. In conclusion, the cytotoxic effect of the tested medications is dependent on concentration, time of contact and cellular condition, being the triple antibiotic modified paste the most cytotoxic in high concentrations leading clinically in the decreased of the cells viability of the apical papilla, decreasing the success of regenerative procedures.