Issue 2 - "Update on adverse respiratory effects of indoor air pollution" Part 1): Indoor air pollution and respiratory diseases: A general update and a Portuguese perspective.

OBJECTIVE: To quantify the impact of different air pollutants on respiratory health based on robust estimates based on international data and to summarise the evidence of associations between indoor exposure to those pollutants and respiratory morbidity in the Portuguese population. RESULTS: Several systematic reviews and meta-analyses (MA) at the world level demonstrate the impact of indoor air quality on respiratory health, with indoor particulate matter and gasses exerting a significant effect on the airways. Volatile organic compounds (VOC) have been related to asthma and lung cancer. However, only meta-analyses on biomass use allowed documentation of long-term respiratory effects. While early publications concerning Portuguese-based populations mainly focused on indoor exposure to environmental tobacco smoke, later studies relocated the attention to relevant exposure environments, such as day care buildings, schools, residences and nursing homes. Looking at the pooled effects from the reviewed studies, high levels of carbon dioxide and particulate matter in Portuguese buildings were significantly associated with asthma and wheezing, with VOC and fungi showing a similar effect in some instances. CONCLUSIONS: Despite the significant reduction of indoor air pollution effects after the 2008 indoor smoking prohibition in public buildings, studies show that several indoor air parameters are still significantly associated with respiratory health in Portugal. The country shares the worldwide necessity of standardisation of methods and contextual data to increase the reach of epidemiological studies on household air pollution, allowing a weighted evaluation of interventions and policies focused on reducing the associated respiratory morbidity.

Issue 1 - "Update on adverse respiratory effects of outdoor air pollution" Part 2): Outdoor air pollution and respiratory diseases: Perspectives from Angola, Brazil, Canada, Iran, Mozambique and Portugal.

OBJECTIVE: To analyse the GARD perspective on the health effects of outdoor air pollution, and to synthesise the Portuguese epidemiological contribution to knowledge on its respiratory impact. RESULTS: Ambient air pollution has deleterious respiratory effects which are more apparent in larger, densely populated and industrialised countries, such as Canada, Iran, Brazil and Portugal, but it also affects people living in low-level exposure areas. While low- and middle-income countries (LMICs), are particularly affected, evidence based on epidemiological studies from LMICs is both limited and heterogeneous. While nationally, Portugal has a relatively low level of air pollution, many major cities face with substantial air pollution problems. Time series and cross-sectional epidemiological studies have suggested increased respiratory hospital admissions, and increased risk of respiratory diseases in people who live in urban areas and are exposed to even a relatively low level of air pollution. CONCLUSIONS: Adverse respiratory effects due to air pollution, even at low levels, have been confirmed by epidemiological studies. However, evidence from LMICs is heterogeneous and relatively limited. Furthermore, longitudinal cohort studies designed to study and quantify the link between exposure to air pollutants and respiratory diseases are needed. Worldwide, an integrated approach must involve multi-level stakeholders including governments (in Portugal, the Portuguese Ministry of Health, which hosts GARD-Portugal), academia, health professionals, scientific societies, patient associations and the community at large. Such an approach not only will garner a robust commitment, establish strong advocacy and clear objectives, and raise greater awareness, it will also support a strategy with adequate measures to be implemented to achieve better air quality and reduce the burden of chronic respiratory diseases (CRDs).

Ecotoxicity of plant extracts and essential oils: A review.

Plant-based products such as essential oils and other extracts have been used for centuries due to their beneficial properties. Currently, their use is widely disseminated through a variety of industries and new applications are continuously emerging. For these reasons, they are produced industrially in large quantities and consequently they have the potential to reach the environment. However, the potential effects that these products have on the ecosystems' health are mostly unknown. In recent years, the scientific community started to focus on the possible toxic effects of essential oils and plant extracts towards non-target organisms. As a result, an increasing body of knowledge has emerged. This review describes the current state of the art on the toxic effects that essential oils and plant extracts have towards organisms from different trophic levels, including producers, primary consumers, and secondary consumers. The majority of the studies (76.5%) focuses on the aquatic environment, particularly in aquatic invertebrates (45.1%) with only 23.5% of the studies focusing on the potential toxicity of plant-derived products on terrestrial ecosystems. While some essential oils and extracts have been described to have no toxic effects to the selected organisms or the toxic effects were only observable at high concentrations, others were reported to be toxic at concentrations below the limit set by international regulations, some of them at very low concentrations. In fact, L(E)C50 values as low as 0.0336 mg.L-1, 0.0005 mg.L-1 and 0.0053 mg.L-1 were described for microalgae, crustaceans and fish, respectively. Generally, essential oils exhibit higher toxicity than extracts. However, when the extracts are obtained from plants that are known to produce toxic metabolites, the extracts can be more toxic than essential oils. Overall, and despite being generally considered "eco-friendly" products and safer than they synthetic counterparts, some essential oils and plant extracts are toxic towards non-target organisms. Given the increasing interest from industry on these plant-based products further research using international standardized protocols is mandatory.

Chemical characterization and bioactive potential of Thymus×citriodorus (Pers.) Schreb. preparations for anti-acne applications: Antimicrobial, anti-biofilm, anti-inflammatory and safety profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus × citriodorus (Pers.) Schreb. is an interspecific hybrid between Thymus pulegioides and Thymus vulgaris, known for its pharmacological activities as diaphoretic, deodorant, antiseptic and disinfectant, the last mostly related with its antimicrobial activity. The folk use of other extracts, as hydrolates, have also been disseminated, as regulators of oily skin with anti-acne effect. AIM OF THE STUDY: We aimed to evaluate the anti-acne potential of two Thymus x citriodorus (TC) preparations, the essential oil (EO) and the hydrolate, to be used as active ingredients for skin applications. Specifically, we intend to validate their anti-acne potential by describing their activity on acne related bacteria, bacterial virulence, anti-oxidant and anti-inflammatory potential, and biocompatibility on inflammatory cells. Additionally, we aimed to report their ecotoxicity under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), thus focusing not only on the consumer, but also on environmental safety assessment. MATERIALS AND METHODS: Minimum inhibitory concentration (MIC) against C. acnes, S. aureus and S. epidermidis was evaluated. Minimum lethal concentration (MLC) was also determined. The effect on C. acnes biofilm formation and disruption was evaluated with crystal violet staining. Anti-inflammatory activity was investigated on LPS-stimulated mouse macrophages (RAW 264.7), by studying nitric oxide (NO) production (Griess reagent) and cellular biocompatibility through MTT assay. In-vitro NO and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging potential were also evaluated. The ecotoxicity was evaluated using Daphnia magna acute toxicity assays. RESULTS: EO presented direct antimicrobial activity, with visual MICs ranging from 0.06% for S. epidermidis and C. acnes to 0.125% for S. aureus. MLCs were higher than the obtained MICs. Hydrolate revealed visual MIC only for C. acnes. TC essential oil was effective in preventing biofilm formation and disrupting preformed biofilms even at sub-inhibitory concentrations. Hydrolate showed a more modest anti-biofilm effect. Regarding anti-inflammatory activity, TC hydrolate has a higher cellular biocompatibility. Still, both plant preparations were able to inhibit at least 50% of NO production at non-cytotoxic concentrations. Both EO and hydrolate have poor anti-oxidant activities. Regarding the ecotoxicity, TC essential oil was classified under acute 3 category, while the hydrolate has proved to be nontoxic, in accordance to the GHS. CONCLUSIONS: These results support the anti-acne value of different TC preparations for different applications. TC hydrolate by presenting higher biocompatibility, anti-inflammatory potential and the ability to modulate C. acnes virulence, can be advantageous in a product for everyday application. On the other hand, EO by presenting a marked antimicrobial, anti-biofilm and anti-inflammatory activities, still with some cytotoxicity, may be better suited for application in acute flare-ups, for short treatment periods.

Frailty syndrome, biomarkers and environmental factors - A pilot study.

Frailty is an age-related syndrome expected to increase over the next decades. This syndrome has been identified to be the most common condition leading to disability, institutionalisation and death in the elderly. The aim of this pilot study is to investigate a possible link between frailty status, biomarkers and environmental exposures. A group of 71 older adults (≥65 years old) was engaged in this study. The study population was classified as 45.1% robust, 45.1% pre-frail and 9.8% frail. A significant higher prevalence of second-hand smokers was found in the pre-frail group when compared to robust. Furthermore, a higher prevalence of robust individuals was found among those consuming home-produced vegetables and water from well/springs. Significant differences were found between data collected in a lifetime exposure questionnaire (LTEQ) and the levels of genotoxicity endpoints and the mercury levels analysed regarding some exposure-related parameters, namely, smoking habits, intake of home-produced vegetables and the use of pesticides in agriculture. Understanding if the way we live(d) or worked can impact the way we age are important questions to be explored. Data obtained in this pilot study encourage further studies on this matter, exploring the role of exposures history and its impact on health.

A circular toxicity approach to isoprostanes: From markers of oxidative stress, to epidemiological warning systems and agents of aquatic toxicity.

Isoprostanes (IsoPs) are a class of oxidation products naturally formed in vivo that are indicative of endogenous oxidative stress. In individuals with chronic and oxidative stress related diseases, IsoPs are increased to pathological levels. Since they are excreted through urine into sewage systems, IsoPs can be detected in wastewater treatment plants' (WWTPs) effluents and thus can be used to evaluate the health status of a given population. The underlying principle is that higher isoprostanes WWTPs' levels correspond to populations undergoing higher levels of oxidative stress, and thus disease. However, IsoPs are not eliminated by WWTPs and will end up being released into the aquatic environment, where they will be available for uptake by aquatic species. Being bioactive molecules, it has been suggested that IsoPs in the environment may elicit oxidative stress in aquatic organisms. In this context, we have critically reviewed the available data on IsoPs as products and effectors of toxicity, and propose the new concept of "circular toxicity". In general, IsoPs excreted by humans as a consequence of oxidative stress are released into the aquatic environment where they may interact with aquatic organisms and induce the production of more IsoPs. These stress markers, in turn, will also be excreted, increasing the already high levels of stressors in the aquatic environment and thus create an escalating cycle of oxidative stress.

Acute phase proteins response in cats naturally infected by hemotropic mycoplasmas.

Information about the acute phase proteins (APP) response in cats naturally infected with hemoplasmas and in cats co-infected with different species of hemoplasmas is lacking. This study evaluated serum amyloid A (SAA), haptoglobin (Hp) and albumin in 48 cats naturally infected with hemoplasmas, including 25 with Candidatus Mycoplasma haemominutum and 23 co-infected with different hemoplasmas agents; and in 10 healthy control cats. Infected cats had significantly higher Hp and lower albumin than controls. Symptomatic cats had significantly higher SAA and Hp, and lower albumin than asymptomatic animals, and also than controls. Asymptomatic cats had significantly higher Hp than controls. Concentrations of APP were not significantly different between single infected and co-infected cats. According with these results, hemoplasmosis should be considered when alterations in APP are detected in diseased cats with compatible clinical signs. Furthermore, a subclinical infection should be considered in apparently healthy cats from endemic areas with increased Hp.

Levels of TBT and other selected organotin compounds in duplicate diet samples.

Organotin compounds (OTs) are ubiquitous contaminants with a broad range of applications ranging from biocides and pesticides to catalysts for the production of polyurethane foams and silicones. The deleterious effects of some OTs (particularly tributyltin - TBT) upon wildlife and experimental animals are well documented and include endocrine disruption, immunotoxicity, neurotoxicity, genotoxicity and metabolic dysfunction in which obesity is included. However, virtually no data on the current human exposure levels is available. In order to bridge this gap, we quantified for the first time the levels of OTs in duplicate diet samples from members of the University of Aveiro in Portugal. OTs were detected in 32% of the 28 diet samples analyzed, at relatively low levels. TBT and monobutyltin were detected only in two samples and dibutyltin was detectable in three samples. Dioctyltin was quantified in four samples and monooctyltin in three samples. Phenyltins were below the detection limit in all the diet samples analyzed. Overall, for the vast majority of the samples (89%), the estimated daily intakes (EDI) of organotins through food were much lower than the established tolerable daily intakes (TDI). Hence, for the majority of the participants the risk associated with food ingestion is low.

Lead in duplicate diet samples from an academic community.

Lead is a naturally occurring element that with the advent of the industrial era became a serious environmental and public health issue. Leaded gasoline, lead based paints, use of lead in plumbing and water pipes, ceramics with lead-containing glazes and tobacco smoke are potential sources of lead exposure for humans. Despite these multiple sources, food is still considered the most important one for the general non-smoking population. Hence, in the present study, the dietary intake of lead was determined in duplicate diet samples provided by 30 participants working or studying at University of Aveiro, Portugal. Pb was detected in all the analysed samples with values ranging between 0.009 and 0.10mgkg-1 ww which correspond to estimated daily intakes between 0.22 and 3.5µgkg-bw-1day-1. Risk estimations disclose that at least 3.3% and 26.7% of the participants might suffer cardiovascular and nephrotoxic effects, respectively.

Urine protein-to-creatinine concentration ratio in samples collected by means of cystocentesis versus manual compression in cats.

Objective-To compare urine protein-to-creatinine concentration (UPC) ratios in samples collected by means of cystocentesis versus manual compression in cats. Design-Evaluation study. Animals-43 client-owned cats requiring urinalysis. Procedures-In all cats, 5 mL of urine from the midstream phase of micturition was collected by means of manual compression and, subsequently, an additional 5 mL of urine was obtained by means of ultrasound-guided cystocentesis. A complete urinalysis was performed on all samples, and UPC ratios were determined. Results-Cats were classified on the basis of the International Renal Interest Society substaging system as being free from proteinuria (UPC ratio, < 0.2; n = 19) or as having borderline proteinuria (UPC ratio, 0.2 to 0.4; 7) or proteinuria (UPC ratio, > 0.4; 17). None of the cats had postrenal proteinuria. A significant linear correlation was identified between UPC ratios in urine samples obtained by means of manual compression and ratios in samples obtained by means of cystocentesis. For all cats, UPC ratios for samples obtained by the 2 collection methods resulted in classification in the same IRIS substage. Conclusions and Clinical Relevance-Results suggested that collection of a urine sample from the midstream phase of micturition by manual compression would be a reliable alternative to cystocentesis for the determination of UPC ratio in cats, provided that postrenal proteinuria was excluded by means of urine sediment analysis. Once postrenal proteinuria was ruled out, the method used to collect urine samples did not appear to influence the quantification of urine protein concentration.

Characterization of fungal communities in house dust samples collected from central Portugal-a preliminary survey.

House dust is a repository and concentrator of many chemical and biological agents including fungi. Considering that dust acts as a long-term reservoir of airborne fungi and that cumulative exposure is more relevant to potential health problems than single-day or short-term exposure, characterization of fungal communities in dust samples is of paramount importance. In the present study, the fungal composition of Portuguese house dust samples was determined. A total of 28 samples were obtained from vacuum cleaner deposits from households located in central Portugal. DNA was extracted from dust samples and fungal communities were analyzed using a culture-independent polymerase chain reaction (PCR)- denaturing gradient gel electrophoresis (DGGE) approach. Cultural analyses were also performed in order to identify the viable fungi species present in selected samples. Fungal diversity, reported as the number of operational taxonomic units (OTU), varied between 9 and 56 OTU. This analysis of viable fungi showed that Aspergillus was the most abundant genus, followed by Penicillium, Mucor, and Rhizomucor. Trichoderma, Chrysosporium, Fusarium, Rhizopus, and Stachybotrys were found in a limited number of houses. Our results demonstrated that dust is, in fact, home for a diverse and heterogeneous fungal community and that some of the species found are known allergic agents with severe negative impacts on human health.

Biomonitoring of several toxic metal(loid)s in different biological matrices from environmentally and occupationally exposed populations from Panasqueira mine area, Portugal.

In the Panasqueira mine area of central Portugal, some environmental media show higher metal(loid) concentrations when compared with the local geochemical background and the values proposed in the literature for these environmental media. In order to evaluate the effect of the external contamination on selected indexes of internal dose, As, Cd, Cu, Cr, Fe, Hg, Mg, Mn, Mo, Ni, Pb, S, Se, Si, and Zn were quantified by inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry in blood, urine, hair and nail samples from individuals environmentally (N = 41) and occupationally exposed (N = 41). A matched control group (N = 40) was also studied, and data from the three groups were compared. Results obtained agreed with those reported by environmental studies performed in this area, pointing to populations living nearby and working in the mine being exposed to metal(loid)s originated from mining activities. Arsenic was the element with the highest increase in exposed populations. The concentration of other elements such as Cr, Mg, Mn, Mo, Ni, Pb, S, Se, and Zn was also increased, although at a lesser extent, specifically in the individuals environmentally exposed and in females. These findings confirm the need for competent authorities to act as soon as possible in this area and implement strategies aimed to protect exposed populations and the entire ecosystem.

Metal(loid) levels in biological matrices from human populations exposed to mining contamination--Panasqueira Mine (Portugal).

Mining activities may affect the health of miners and communities living near mining sites, and these health effects may persist even when the mine is abandoned. During mining processes various toxic wastes are produced and released into the surrounding environment, resulting in contamination of air, drinking water, rivers, plants, and soils. In a geochemical sampling campaign undertaken in the Panasqueira Mine area of central Portugal, an anomalous distribution of several metals and arsenic (As) was identified in various environmental media. Several potentially harmful elements, including As, cadmium (Cd), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and selenium (Se), were quantified in blood, urine, hair, and nails (toe and finger) from a group of individuals living near the Panasqueira Mine who were environmentally and occupationally exposed. A group with similar demographic characteristics without known exposure to mining activities was also compared. Genotoxicity was evaluated by means of T-cell receptor (TCR) mutation assay, and percentages of different lymphocyte subsets were selected as immunotoxicity biomarkers. Inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis showed elevated levels of As, Cd, Cr, Mn, and Pb in all biological samples taken from populations living close to the mine compared to controls. Genotoxic and immunotoxic differences were also observed. The results provide evidence of an elevated potential risk to the health of populations, with environmental and occupational exposures resulting from mining activities. Further, the results emphasize the need to implement preventive measures, remediation, and rehabilitation plans for the region.

A feeding inhibition based prediction of the toxic effect of dissolved metal mixtures upon Echinogammarus marinus (Crustacea: Amphipoda) at field relevant concentrations across a latitudinal gradient.

Risk assessment of metals in the environment is performed mainly with toxicity evaluations on single metals, which is largely inadequate since these substances occur in mixtures. The development of models predicting combined toxic effects on the basis of the concentration-response relationships of individual compounds has emerged as an answer. In the present study, metal effects on post-exposure anorexia (the concept of FdC(50)--concentration causing 50% of feeding inhibition--is implemented) in Echinogammarus marinus, a widely distributed gammarid amphipod, were assessed and compared with modelled ones obtained through the application of the concentration addition (CA) model, which represents a reasonable worst-case scenario for the risk assessment of metal mixtures. Data were validated using in situ experiments performed along a latitudinal gradient (Iceland, Scotland and Portugal) aiming at establishing a geographic profile of autochthonous population susceptibilities to metals. For all of the metals studied concentrations in the water column at exposure sites were in good agreement with feeding inhibition levels. Models gave low to relatively high percentage agreement between predictions and experimental data. Boreal populations demonstrated higher susceptibility to single metals, but not to mixture exposures. Meridional populations denoted lower susceptibilities with higher FdC(50).

Amphipod susceptibility to metals: cautionary tales.

Heavy metals accumulated by aquatic crustaceans in environmental studies are normally investigated using the whole body burden, with little regard paid to uptake in different tissues, to potential gender of life stage differences, or to the influence of nutrition on the test organism. This is likely to give erroneous conclusions for a dose-response relationship within the toxicity test and potentially lead to wrong conclusions for the ecological risks of metals where species may have higher sensitivities with gender and life stage than indicated or that functionally metals may be sequestered into parts of the body so are not bioavailable. This could lead to under-estimation or over-estimation of the toxicity of metals, respectively, inaccuracy of metal budget calculations and evaluation of trophic transfers of metals. This study evaluated the influences of life stage, gender, and a priori nutritional state in the uptake of the metals zinc (an essential micro-nutrient; Zn) and cadmium (a non-essential element; Cd) in the amphipod Echinogammarus marinus. The study showed that life stage, and nutritional stage did significantly influence the uptake and bioaccumulation for both metals, but only Cd showed differential uptake and bioaccumulation with gender. In addition, it was concluded that there was a significant uptake and accumulation of both metals within the exoskeleton of the amphipods, which though adding to the full body burden would add little to toxicity through lack of bioavailability. These results showed that care should be taken when interpreting results from tests normally preformed on such test organisms.

Amphipod intersex, metals and latitude: a perspective.

Intersexuality has been widely reported in crustaceans with several mechanisms being directly or indirectly held responsible for its occurrence, amongst which pollution. No mechanistic relationship between metals and intersex has ever been established. Also the incidence of intersex in populations of the same invertebrate species in a latitudinal gradient has never been studied so far. Three populations (Iceland, Scotland and Portugal) of the amphipod Echinogammarus marinus were scrutinized. Intersex females from Iceland registered the highest fecundity loss. Only in Scottish samples females with two genital papillae and males with only one genital papillae were observed. Nevertheless, water, biota and sediment samples pointed to equivalent metal levels, and in consonance the prevalence of intersex was not significantly different between locations. An unequivocal relationship between metal presence and intersex induction cannot be presented, but our results advocate the potential role of metals as a direct cause of intersexuality in E. marinus.

Fucus spp. as a mercury contamination bioindicator in costal areas (Northwestern Portugal).

Mercury has been considered as one of the most important pollutants in coastal and estuarine areas. Efforts have been made to detect, as early as possible, the effects of this and other metals in several species. Macroalgae, particularly Fucus spp., have been widely used as biomonitors of metal pollution. In this study, three Fucus species (F. spiralis, F. vesiculosus and F. ceranoides) were collected from several sampling sites in Portugal. The concentrations of mercury were determined in three structural parts (holdfast, stipe and receptacles). Two different techniques were used to determine mercury concentrations. Almost all mercury concentrations (in sediments and in water) were below national and international standards. Mercury concentration in the specimens (0.012-0.061 microg g(-1) for receptacles, 0.028-0.221 microg g(-1) for stipe and 0.029-0.287 microg g(-1) for holdfast) was always higher that those obtained for the sediment (0.001-0.112 microg g(-1)). With few exceptions the contrary was found for receptacles. In general, a good agreement between concentrations of mercury in sediment and Fucus was found. The results indicate that Fucus accumulate mercury and may be a suitable species for use in risk assessment for coast and estuarine areas, by providing valuable information regarding the levels of mercury that will be available for the consumers of Fucus spp.

Acute effects of 3,4-dichloroaniline on biomarkers and spleen histology of the common goby Pomatoschistus microps.

The aromatic amine 3,4-dichloroaniline (DCA) is a model environmental contaminant, precursor for synthesis and degradation product of several herbicides, which is commonly found in European estuarine ecosystems. In this work, the possibility of using biochemical and histological markers to assess sub-lethal effects of DCA in natural populations of Pomatoschistus microps juveniles was investigated. Alterations on the activities of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferase (GST) and histological alterations on spleen were investigated after 96 h of exposure to sublethal concentrations of DCA (0.50-1.49 mg/l). At the concentrations tested, DCA had no effect on AChE activity. LDH and GST activities were significant altered in treated animals when compared to control groups. As already described for mammals, DCA induced splenic histological alterations in P. microps, including expansion of red pulp and deposition of hemosiderin granules in a concentration-dependent manner. This suggests that DCA is a xenobiotic of concern in estuaries receiving agricultural effluents.