Exploring the Hypocholesterolemic Potential of a Fucus vesiculosus Extract: Omic Insights into Molecular Mechanisms at the Intestinal Level.

High blood cholesterol levels are a major risk factor for cardiovascular diseases. A purified aqueous extract of Fucus vesiculosus, rich in phlorotannins and peptides, has been described for its potential to inhibit cholesterol biosynthesis and intestinal absorption. In this work, the effect of this extract on intestinal cells' metabolites and proteins was analysed to gain a deeper understanding of its mode of action on lipids' metabolism, particularly concerning the absorption and transport of exogenous cholesterol. Caco-2 cells, differentiated into enterocytes, were exposed to the extract, and analysed by untargeted metabolomics and proteomics. The results of the metabolomic analysis showed statistically significant differences in glutathione content of cells exposed to the extract compared to control cells, along with an increased expression of fatty acid amides in exposed cells. A proteomic analysis showed an increased expression in cells exposed to the extract compared to control cells of FAB1 and NPC1, proteins known to be involved in lipid metabolism and transport. To the extent of our knowledge, this study is the first use of untargeted metabolomics and a proteomic analysis to investigate the effects of F. vesiculosus on differentiated Caco-2 cells, offering insights into the molecular mechanism of the extract's compounds on intestinal cells.

Phenolic composition, antioxidant and antiacetylcholinesterase activities of Opuntia ficus-indica peel and flower teas after in vitro gastrointestinal digestion.

BACKGROUND: The flowers and peels of cactus Opuntia ficus-indica (OFI) are by-products generally discarded. Their beneficial properties have been linked to the chemical composition without taking into account its transformations during digestion. This study evaluated and compared the phenolic composition, antioxidant and antiacetylcholinesterase activities of OFI peel and flower teas before and after in vitro gastrointestinal digestion. RESULTS: Results showed that OFI peel tea was a source of natural phenolic compounds and carotenoids, while OFI flower tea was a source of flavonoids. Both OFI peel and flower teas exhibited high antioxidant activities (AAs) measured by several tests [1,1-diphenyl-2-picrylhydrazyl radical (DPPH• ), reducing power (RP), hydrogen peroxide scavenging activity (HPSA)]. Flower teas presented a higher antiacetylcholinesterase inhibition percentage than peel teas. Significant decreases of acetylcholinesterase and AAs were obtained under in vitro gastrointestinal digestion. Fourteen phenolic compounds typical of phenolic acids and flavonoids were identified in both teas. These compounds seemed to be more affected by pancreatic than by the gastric conditions. CONCLUSION: OFI peel tea has been found as a source of natural phenolic compounds and carotenoids, while OFI dry flower tea as a source of flavonoids. The OFI flower tea phytochemicals were more stable than peel tea during the digestive process. These findings suggest that peels and flowers of OFI should be considered as sources of healthy ingredients. © 2022 Society of Chemical Industry.

Untargeted metabolomic study of HepG2 cells under the effect of Fucus vesiculosus aqueous extract.

RATIONALE: Fucus vesiculosus has been described with potential to develop functional foods containing bioactive compounds against various diseases. However, more studies are needed to better understand its functioning and its previously reported bioactivities, mainly at the molecular level. METHODS: An untargeted metabolomic study was performed to analyse HepG2 cells exposed to F. vesiculosus aqueous extract, rich in phlorotannins and peptides, during 24 h. This study was carried out using liquid chromatography combined with high-resolution tandem mass spectrometry. RESULTS: This metabolomic study showed significant changes in HepG2 metabolites in the presence of the extract, standing out being the increased intensity of various fatty acid amides (oleamide, (Z)-eicos-11-enamide, linoleamide, palmitamide, dodecanamide and stearamide). This group of metabolites is reported in the literature with anticancer and hypocholesterolemic activity, bioactivities also described for F. vesiculosus. The extract induced, likewise, the expression of glutathione indicating its antioxidant effect. CONCLUSIONS: This study demonstrated the potential of the compounds present in the F. vesiculosus aqueous extract for the development of natural drugs, nutraceuticals or dietary supplements, justified at the molecular level by changes in cell metabolites related to anticancer and hypocholesterolemic activity. The results here described, using an untargeted metabolomic approach, may contribute to a better understanding of algal behaviour, when used as food, in health-promoting effects.

Bioactivities of Centaurium erythraea (Gentianaceae) Decoctions: Antioxidant Activity, Enzyme Inhibition and Docking Studies.

Centaurium erythraea is recommended for the treatment of gastrointestinal disorders and to reduce hypercholesterolemia in ethno-medicinal practice. To perform a top-down study that could give some insight into the molecular basis of these bioactivities, decoctions from C. erythraea leaves were prepared and the compounds were identified by liquid chromatography-high resolution tandem mass spectrometry (LC-MS/MS). Secoiridoids glycosides, like gentiopicroside and sweroside, and several xanthones, such as di-hydroxy-dimethoxyxanthone, were identified. Following some of the bioactivities previously ascribed to C. erythraea, we have studied its antioxidant capacity and the ability to inhibit acetylcholinesterase (AChE) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Significant antioxidant activities were observed, following three assays: free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction; lipoperoxidation; and NO radical scavenging capacity. The AChE and HMGR inhibitory activities for the decoction were also measured (56% at 500 µg/mL and 48% at 10 µg/mL, respectively). Molecular docking studies indicated that xanthones are better AChE inhibitors than gentiopicroside, while this compound exhibits a better shape complementarity with the HMGR active site than xanthones. To the extent of our knowledge, this is the first report on AChE and HMGR activities by C. erythraea decoctions, in a top-down analysis, complemented with in silico molecular docking, which aims to understand, at the molecular level, some of the biological effects ascribed to infusions from this plant.

Optimization of microbial detoxification for an aquatic mercury-contaminated environment.

Mercury (Hg) reduction performed by microorganisms is well recognized as a biological means for remediation of contaminated environment. Recently, studies demonstrated that Hg-resistant microorganisms of Tagus Estuary are involved in metal reduction processes. In the present study, aerobic microbial community isolated from a highly Hg-contaminated area of Tagus Estuary was used to determine the optimization of the reduction process in conditions such as the contaminated ecosystem. Factorial design methodology was employed to examine the influence of glucose, sulfate, iron, and chloride on Hg reduction. In the presence of several concentrations of these elements, microbial community reduced Hg in a range of 37-61% of the initial 0.1 mg/ml Hg2+ levels. The response prediction through central composite design showed that the increase of sulfate concentration led to an optimal response in Hg reduction by microbial community, while the rise in chloride levels markedly decreased metal reduction. Iron may exert antagonistic effects depending upon the media composition. These results are useful in understanding the persistence of Hg contamination in Tagus Estuary after inactivation of critical industrial units, as well as data might also be beneficial for development of new bioremediation strategies either in Tagus Estuary and/or in other Hg-contaminated aquatic environments.

In vitro digestion, antioxidant and antiacetylcholinesterase activities of two species of Ruta: Ruta chalepensis and Ruta montana.

CONTEXT: Ruta genus (Rutaceae) is abundantly used and described in the most ancient systematic records of medical practice of the Mediterranean world. In Tunisia, this genus is represented by two medicinal and aromatic shrubs: Ruta chalepensis L. and Ruta montana L. OBJECTIVE: This study investigates the antioxidant and acetylcholinesterase inhibition (AChE) activities before and after in vitro gastrointestinal metabolism of leaf decoction of R. chalepensis and R. montana. MATERIALS AND METHODS: We study, in vitro, the effect of the gastrointestinal juices gastric (1.75 mL) or pancreatic (2.5 mL) juices, on the biological activity by the measurement of the antioxidant activity and AChE inhibition during 4 h of decoction extract obtained from the leaves of the two species of Ruta. RESULTS: The results showed that the ability to inhibit the AChE enzyme was similar; being the greatest inhibitory activity exhibited by the ethanol extract (IC50 = 12 ± 1.1 µg/mL) obtained from leaves of R. chalepensis. CONCLUSION: In conclusion, we showed that there was no appreciable degradation and that the activity was kept constant after gastric and pancreatic juice digestion.

Isolation and characterization of mercury-resistant bacteria from sediments of Tagus Estuary (Portugal): implications for environmental and human health risk assessment.

Mercury (Hg) contamination of aquatic systems has been recognized as a global and serious problem affecting both human and environmental health. In the aquatic ecosystems, mercurial compounds are microbiologically transformed with methylation responsible for generation of methylmercury (MeHg) and subsequent biomagnification in food chain, consequently increasing the risk of poisoning for humans and wildlife. High levels of Hg, especially MeHg, are known to exist in Tagus Estuary as a result of past industrial activities. The aim of this study was to isolate and characterize Hg-resistant bacteria from Tagus Estuary. Mercury-resistant (Hg-R) bacteria were isolated from sediments of two hotspots (Barreiro and North Channel) and one reserve area (Alcochete). Mercury contamination in these areas was examined and bacterial susceptibility to Hg compounds evaluated by determination of minimal inhibitory concentrations (MIC). The isolates characterization was based on morphological observation and biochemical testing. Bacteria characteristics, distribution, and Hg resistance levels were compared with metal levels. Barreiro and North Channel were highly contaminated with Hg, containing 126 and 18 µg/g total Hg, respectively, and in Alcochete, contamination was lower at 0.87 µg/g total Hg. Among the isolates there were aerobic and anaerobic bacteria, namely, sulfate-reducing bacteria, and Hg resistance levels ranged from 0.16 to 140 µg/ml for Hg(2+) and from 0.02 to 50.1 µg/ml for MeHg. The distribution of these bacteria and the resistance levels were consistent with Hg contamination along the depth of the sediments. Overall, results show the importance of the characterization of Tagus Estuary bacteria for ecological and human health risk assessment.

The Hypocholesterolemic Potential of the Edible Algae Fucus vesiculosus: Proteomic and Quantitative PCR Analysis.

A brown seaweed consumed worldwide, Fucus vesiculosus, has been used to prevent atherosclerosis and hypercholesterolemia, among other uses. However, the mechanisms of action that lead to these effects are not yet fully understood. This work aims to study the in vitro effect of an aqueous extract of F. vesiculosus, previously characterized as rich in phlorotannins and peptides, on the expression of different proteins involved in the synthesis and transport of cholesterol. A proteomic analysis, Western blot, and qRT-PCR analysis were performed to identify protein changes in HepG2 cells exposed to 0.25 mg/mL of the F. vesiculosus extract for 24 h. The proteomic results demonstrated that, in liver cells, the extract decreases the expression of four proteins involved in the cholesterol biosynthesis process (CYP51A1, DHCR24, HMGCS1 and HSD17B7). Additionally, a 12.76% and 18.40% decrease in the expression of two important transporters proteins of cholesterol, NPC1L1 and ABCG5, respectively, was also observed, as well as a 30% decrease in NPC1L1 mRNA levels in the cells exposed to the extract compared to control cells. Our study reveals some of the mechanisms underlying the actions of bioactive compounds from F. vesiculosus that may explain its previously reported hypocholesterolemic effect, future prospecting its use as a functional food.

Novel, Edible Melanin-Protein-Based Bioactive Films for Cheeses: Antimicrobial, Mechanical and Chemical Characteristics.

The cheese rind is the natural food packaging of cheese and is subject to a wide range of external factors that compromise the appearance of the cheese, including color defects caused by spoilage microorganisms. First, eight films based on whey protein isolate (WPI) coatings were studied, of which IS3CA (WPI 5% + sorbitol 3% + citric acid 3%) was selected for presenting better properties. From the IS3CA film, novel films containing melanin M1 (74 µg/mL) and M2 (500 µg/mL) were developed and applied to cheese under proof-of-concept and industrial conditions. After 40 days of maturation, M2 presented the lowest microorganism count for all the microbial parameters analyzed. The cheese with M2 showed the lowest lightness, which indicates that it is the darkest cheese due to the melanin concentration. It was found that the mechanical and colorimetric properties are the ones that contribute the most to the distinction of the M2 film in cheese from the others. Using FTIR-ATR, it was possible to distinguish the rinds of M2 cheeses because they contained the highest concentrations of melanin. Thus, this study shows that the film with M2 showed the best mechanical, chemical and antimicrobial properties for application in cheese.

Edible Seaweeds Extracts: Characterization and Functional Properties for Health Conditions.

Seaweeds are popular foods due to claimed beneficial health effects, but for many there is a lack of scientific evidence. In this study, extracts of the edible seaweeds Aramé, Nori, and Fucus are compared. Our approach intends to clarify similarities and differences in the health properties of these seaweeds, thus contributing to target potential applications for each. Additionally, although Aramé and Fucus seaweeds are highly explored, information on Nori composition and bioactivities is scarce. The aqueous extracts of the seaweeds were obtained by decoction, then fractionated and characterized according to their composition and biological activity. It was recognized that fractioning the extracts led to bioactivity reduction, suggesting a loss of bioactive compounds synergies. The Aramé extract showed the highest antioxidant activity and Nori exhibited the highest potential for acetylcholinesterase inhibition. The identification of the bioactive compounds in the extracts allowed to see that these contained a mixture of phloroglucinol polymers, and it was suggested that Nori's effect on acetylcholinesterase inhibition may be associated with a smaller sized phlorotannins capable of entering the enzyme active site. Overall, these results suggest a promising potential for the use of these seaweed extracts, mainly Aramé and Nori, in health improvement and management of diseases, namely those associated to oxidative stress and neurodegeneration.

Hydroxycinnamic acid derivatives effect on hypercholesterolemia, comparison with ezetimibe: Permeability assays and FTIR spectroscopy on Caco-2 ​cell line.

High blood cholesterol levels may increase the risk of developing atherosclerosis. Since intestinal cholesterol absorption plays a major role in maintaining total body cholesterol homeostasis, the aim of the present study was to compare the effect of ezetimibe and three hydroxycinnamic acid derivatives (rosmarinic acid, chlorogenic acid and m-coumaric acid) that are present in several medicinal plants on cholesterol absorption in the intestinal Caco-2 â€‹cells. In addition to the permeability assays, studies on alteration of the biochemical properties of Caco-2 â€‹cells under the effect of ezetimibe and hydroxycinnamic acid derivatives was evaluated using FTIR accompanied with multivariate analysis by PCA. The cholesterol permeability assays showed that these compounds could decrease cholesterol permeability with a percentage ranging from 76.98 to 96.6% with the highest inhibition for ezetimibe. whereas the FTIR studies didn't show similar changes between ezetimibe and the three hydroxycinnamic acid derivatives in protein and nucleic acids region, suggesting that these compounds have hypocholesterolemic effect. Nevertheless, each compound originated a different change on Caco-2 treated cells suggesting a different mode of action.

Chromatographic behaviour of monoclonal antibodies against wild-type amidase from Pseudomonas aeruginosa on immobilized metal chelates.

The aim of this work was to devise a one-step purification procedure for monoclonal antibodies (MAbs) of IgG class by immobilized metal affinity chromatography (IMAC). Therefore, several stationary phases were prepared containing immobilized metal chelates in order to study the chromatographic behaviour of MAbs against wild-type amidase from Pseudomonas aeruginosa. Such MAbs adsorbed to Cu(II), Ni(II), Zn(II) and Co(II)-IDA agarose columns. The increase in ligand concentration and the use of longer spacer arms and higher pH values resulted in higher adsorption of MAbs into immobilized metal chelates. The dynamic binding capacity and the maximum binding capacity were 1.33 ± 0.015 and 3.214 ± 0.021 mg IgG/mL of sedimented commercial matrix, respectively. A K(D) of 4.53 × 10(-7) m was obtained from batch isotherm measurements. The combination of tailor-made stationary phases of IMAC and the correct selection of adsorption conditions permitted a one-step purification procedure to be devised for MAbs of IgG class. Culture supernatants containing MAbs were purified by IMAC on commercial-Zn(II) and EPI-30-IDA-Zn(II) Sepharose 6B columns and by affinity chromatography on Protein A-Sepharose CL-4B. This MAb preparation revealed on SDS-PAGE two protein bands with M(r) of 50 and 22 kDa corresponding to the heavy and light chains, respectively.

Molecular-level changes induced by hydroxycinnamic acid derivatives in HepG2 cell line: Comparison with pravastatin.

Hydroxycinnamic acid derivatives are an important class of polyphenols found in fruits, vegetables, and medicinal plants and widely consumed in human diet. In the present work, alterations of HepG2 cells biochemical profile under the effect of four hydroxycinnamic acid derivatives (caffeic acid, m-coumaric acid, chlorogenic acid and rosmarinic acid) relatively to the effect of pravastatin, a drug often prescribed to inhibit HMG-CoA reductase enzyme, the regulator enzyme in the cholesterol biosynthesis pathway, were reported. The application of FTIR spectroscopy in combination with multivariate analysis by PCA showed a similarity between pravastatin and the four hydroxycinnamic acid derivatives in metabolite profile modification expressed by various changes in proteins region, the phosphate region which mainly corresponds to nucleic acids as well as in lipids regions. FTIR structural analysis in the amide I region, using resolution enhancement methods, such as second derivative and amide I deconvolution method, revealed significant decrease in α-helix/random coil and intermolecular ß-sheet decreased while intramolecular ß-sheet in treated cells showed an increase. It was also noticed that the intracellular cholesterol as well as esterified ingredients such as cholesterol esters in the cell membrane decreased. Moreover, principal component analysis (PCA) of the spectral data showed that the compounds and pravastatin were well separated from untreated cells showing a different mode of action on HepG2 treated cells for each compound.

Brown Algae Potential as a Functional Food against Hypercholesterolemia: Review.

Brown algae have been part of the human diet for hundreds of years, however, in recent years, commercial and scientific interest in brown algae has increased due to the growing demand for healthier diet by the world population. Brown algae and its metabolites, such as carotenoids, polysaccharides, phlorotannins, and proteins, have been associated with multiple beneficial health effects for different diseases, such as cardiovascular diseases, one of the main causes of death in Europe. Since high blood cholesterol levels are one of the major cardiovascular risks, this review intends to provide an overview of current knowledge about the anti-hypercholesterolemic effect of different brown algae species and/or their isolated compounds.

Influence of Gender and Age of Brown Seaweed (Fucus vesiculosus) on Biochemical Activities of Its Aqueous Extracts.

Fucus vesiculosus L. is a common coastal brown seaweed associated with various benefits to human health due to its phenolic content and nutrients and is used as food through different methods of consumption. This study aims to evaluate the influence of the seaweed's gender and growth stage on different types of biological activities as well as its chemical constitution and elements present. Akin to food preparation, aqueous extracts of the seaweed were prepared at 25 °C (salad) and 100 °C (soup). Biological activities were determined by measuring total phenol content (TPC), antioxidant activity and inhibition of acetylcholinesterase (AChE). Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) was used for compound identification, and elemental analysis was carried out by using Total Reflection X-ray Fluorescence Spectrometry (TXRF). Older females and males had higher TPC compared to the new ones at 100 °C. Antioxidant activity depended on the extraction temperature but was higher for the youngest male at 100 °C. AChE inhibitory activity was higher for older males at 25 °C, but at 100 °C it was higher for older females. Primary metabolites and various phloroglucinol were the main compounds identified. Additionally, since this seaweed is often harvested in estuarine systems with high anthropogenic impacts, its safety was evaluated through the evaluation of the sample's metal content. The heavy metals detected are within the limits established by various regulating entities, pointing to a safe food source.

Melanin: Production from Cheese Bacteria, Chemical Characterization, and Biological Activities.

Pigments are compounds of importance to several industries, for instance, the food industry, where they can be used as additives, color intensifiers, and antioxidants. As the current trend around the world is shifting to the use of eco-friendly commodities, demand for natural dyes is increasing. Melanins are pigments that are produced by several microorganisms. Pseudomonas putida ESACB 191, isolated from goat cheese rind, was described as a brown pigment producer. This strain produces a brown pigment via the synthetic Müeller-Hinton Broth. This brown compound was extracted, purified, analyzed by FTIR and mass spectrometry, and identified as eumelanin. The maximum productivity was 1.57 mg/L/h. The bioactivity of eumelanin was evaluated as the capacity for scavenging free radicals (antioxidant activity), EC50 74.0 ± 0.2 µg/mL, and as an acetylcholinesterase inhibitor, with IC50 575 ± 4 µg/mL. This bacterial eumelanin did not show cytotoxicity towards A375, HeLa Kyoto, HepG2, or Caco2 cell lines. The effect of melanin on cholesterol absorption and drug interaction was evaluated in order to understand the interaction of melanin present in the cheese rind when ingested by consumers. However, it had no effect either on cholesterol absorption through an intestinal simulated barrier formed by the Caco2 cell line or with the drug ezetimibe.

Data on identification of primary and secondary metabolites in aqueous extract of Verbascum betonicifolium.

Tentative identification of primary and secondary metabolites in aqueous extracts from aerial parts of Verbascum betonicifolium Kuntze was done. This plant belongs to the Scrophulariaceae family and is used for several treatments in folk medicine. One of the processes commonly used to prepare this plant for consumption is boiling with water during approximately 20 minutes, that is, a decoction process. After filtration, this decoction was analysed in search for bioactive metabolites. The analysis was carried out by Electro-Spray Ionization (ESI) and High-Resolution Mass Spectrometry (HRMS) was done using a Quadropole Time-of-Flight (QToF, Impact II, Bruker), coupled to an Ultra High-Performance Liquid Chromatography (UHPLC, ELUTE autosampler, Bruker). The analysis was done in the negative mode (ESI-) and the identification was accomplished using the molecular formula suggestions from the Data Analysis 4.4™ software from Bruker and some databases, like Metlin and PubChem, always confirming with MS/MS results. These data can be used for finding biomarkers between Verbascum sps or to complementary medicine practitioners to get a scientific based knowledge of their results. These data are the unpublished supplementary materials related to "Bioactivities of Iridoids and flavonoids present in decoctions from aerial parts of Verbascum betonicifolium" (Fadel et al., 2020, submitted).

Effect of Food Preparations on In Vitro Bioactivities and Chemical Components of Fucus vesiculosus.

Fucus vesiculosus is a brown macroalgae used in food and generally considered safe to be consumed, according to EU Directive (EC 258/97). The aim of this study is to analyze the effect of food preparation on F.vesiculosus of different origins on what concerns its chemical constituents and final bioactivities. The aqueous extract of the seaweeds were obtained at different temperatures, similar to food preparation and then purified by SPE. The compound identification was carried out by Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) and algae extracts microstructure were observed by Scanning Electron Microscopy (SEM). The activities were determined by using antioxidant activity, inhibition of acetylcholinesterase (AChE) and 3-hidroxi-3-methyl-glutaril-CoA (HMG-CoA) reductase (HMGR) together with Caco-2 cells line simulating the intestinal barrier. The activity of AChE and the HMGR were inhibited by the extracts giving IC50 values of 15.0 ± 0.1 µg/mL and 4.2 ± 0.1 µg/mL, respectively and 45% of the cholesterol permeation inhibition. The main compounds identified were phlorotannins and peptides derivatives. The mode of preparation significantly influenced the final bioactivities. Moreover, the in vitro results suggest that the preparation of F. vesiculosus as a soup could have hypercholesterolemia lowering effect.

Action of euptox A from Ageratina adenophora juice on human cell lines: A top-down study using FTIR spectroscopy and protein profiling.

Euptox A, from Ageratina adenophora juice, is a toxin associated with the plant's resistance to infections, invasiveness and traditional use in cancer treatment. We used FTIR spectroscopy and protein profiling of cell lines to study the impact of euptox A on human cells, to clarify its mechanism of action in a top-down approach. Euptox A was extracted from the juice of A. adenophora. Its stability in the gastrointestinal tract was evaluated, as the compound/juice is generally taken orally. Cytotoxicity was determined in HeLa, Caco-2 and MCF7 cells, and the mechanism of action analyzed by protein and metabolite profiles using electrophoresis and FTIR spectroscopy. Euptox A resisted gastrointestinal digestion and was the most cytotoxic component of the extract for all cell lines tests. Euptox A-treated HeLa cells showed changes in protein profile, especially on 40S ribosomal protein S8 (RP), generally associated with cancer cells. FTIR profiles of treated cells diverged in the same metabolites as cells treated with cisplatin, both in metabolite directed analysis and in multivariate analysis (principal component analysis). In conclusion, euptox A in this top-down study showed a cellular impact that suggests a strong potential against cancer, acting on cancer targeted cellular characteristics.

Evidence of Mercury Methylation and Demethylation by the Estuarine Microbial Communities Obtained in Stable Hg Isotope Studies.

Microbial activity is a critical factor controlling methylmercury formation in aquatic environments. Microbial communities were isolated from sediments of two highly mercury-polluted areas of the Tagus Estuary (Barreiro and Cala do Norte) and differentiated according to their dependence on oxygen into three groups: aerobic, anaerobic, and sulphate-reducing microbial communities. Their potential to methylate mercury and demethylate methylmercury was evaluated through incubation with isotope-enriched Hg species (199HgCl and CH3201HgCl). The results showed that the isolated microbial communities are actively involved in methylation and demethylation processes. The production of CH3199Hg was positively correlated with sulphate-reducing microbial communities, methylating up to 0.07% of the added 199Hg within 48 h of incubation. A high rate of CH3201Hg degradation was observed and >20% of CH3201Hg was transformed. Mercury removal of inorganic forms was also observed. The results prove the simultaneous occurrence of microbial methylation and demethylation processes and indicate that microorganisms are mainly responsible for methylmercury formation and accumulation in the polluted Tagus Estuary.