Search

International audience; One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

The European Joint Programme HBM4EU coordinated and advanced human biomonitoring (HBM) in Europe in order to provide science-based evidence for chemical policy development and improve chemical management. Arsenic (As) was selected as a priority substance under the HBM4EU initiative for which open, policy relevant questions like the status of exposure had to be answered. Internal exposure to inorganic arsenic (iAs), measured as Toxic Relevant Arsenic (TRA) (the sum of As(III), As(V), MMA, DMA) in urine samples of teenagers differed among the sampling sites (BEA (Spain) > Riksmaten adolescents (Sweden), ESTEBAN (France) > FLEHS IV (Belgium), SLO CRP (Slovenia)) with geometric means between 3.84 and 8.47 μg/L. The ratio TRA to TRA + arsenobetaine or the ratio TRA to total arsenic varied between 0.22 and 0.49. Main exposure determinants for TRA were the consumption of rice and seafood. When all studies were combined, Pearson correlation analysis showed significant associations between all considered As species. Higher concentrations of DMA, quantitatively a major constituent of TRA, were found with increasing arsenobetaine concentrations, a marker for organic As intake, e.g. through seafood, indicating that other sources of DMA than metabolism of inorganic As exist, e.g. direct intake of DMA or via the intake of arsenosugars or -lipids. Given the lower toxicity of DMA(V) versus iAs, estimating the amount of DMA not originating from iAs, or normalizing TRA for arsenobetaine intake could be useful for estimating iAs exposure and risk. Comparing urinary TRA concentrations with formerly derived biomonitoring equivalent (BE) for non-carcinogenic effects (6.4 μg/L) clearly shows that all 95th percentile exposure values in the different studies exceeded this BE. This together with the fact that cancer risk may not be excluded even at lower iAs levels, suggests a possible health concern for the general population of Europe. HBM4EU is co-financed under Horizon 2020 (grant agreement No 733032). The authors thank all investigators of the contributing studies for their participation and contribution to the joint HBM4EU survey and the national programme owners for their financial support. Also thanks to the participating teenagers and their families, the field workers that collected the samples. The FLEHS IV study was conducted within the framework of the Flemish Center of Expertise on Environment and Health (FLEHS 2016–2020) and funded by the Flemish Government, Department of Environment & Spatial Development. We thank the teenagers and their families that participated in the study, the field workers from the Pro vincial Institute of Hygiene and VITO for the sample and data collection. All collaborators of the scientific teams of the Flemish Center of Expertise on Environment and Health (https://www.milieu-en-gezondheid.be/en/about-the-center-0) and Karen Van Campenhout and Caroline Teughels from the Flemish Department of Environment & Spatial Development for their valuable input in the field work committee. The funding of the German Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection is gratefully acknowledged. BEA study was co-funded by the Spanish Ministry of Agriculture, Fisheries and Food and the Insituto de Salud Carlos III (SEG 1321/15). In Slovenia the work was cofounded by the Slovenian Research Funding Agency – ARRS through a research programme P-0143. ESTEBAN was Funded by Sant´e Publique France and the French ministries of Health and the Environment. The study of RIKSMATEN was conducted and mainly financed by the Swedish Food Agency. Financial support was provided from the Swedish Civil Contingencies Agency and from the Swedish Environmental Pro tection Agency (SEPA). Sí

Trace elements, including zinc (Zn) and copper (Cu), are known to play important roles in human health. The present study aimed to assess the levels of Zn and Cu in cord blood and maternal milk and to analyze their association with maternal and infant characteristics and pregnancy outcomes in a Slovenian study population of mothers and their neonates recruited within the PHIME prospective cohort study. The study included 324 mothers, but the data on Zn and Cu levels in both cord blood and maternal milk was available for 243 mothers. Questionnaires were used to assess the socio-demographic and health status of the mothers, their lifestyle habits (including detailed nutritional habits), and their residential and occupational histories. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to measure Zn and Cu levels in cord blood and maternal milk. Low Zn levels in cord blood were associated with lower gestational age and birth weight and were correlated with an increased probability of the birth of small for gestational age (SGA) infants. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. Cord blood Cu levels were higher and Cu levels in maternal milk were lower in smoking compared to non-smoking mothers. Most importantly, a decreased Zn/Cu ratio in cord blood was associated with lower gestational age and lower birth weight. This indicates the overall positive effects of Zn and negative effects of Cu on pregnancy outcomes. Soavtorji: Živa Miriam Geršak, Nataša Karas Kuželički, Janja Snoj Tratnik, Darja Mazej, Ingrid Falnoga, Milena Horvat and Ksenija Geršak. Nasl. z nasl. zaslona. Opis vira z dne 4. 11. 2022. Št. članka: 4667. Bibliografija: str. 16-19. Slovenian Research Agency, P3-0124, P1-0143, EU 6th Framework Programme, Public Health Impact of Long- term Low-level Mixed Element Exposure in Susceptible Population Strata (PHIME)

The impacts of single-nucleotide polymorphisms (SNPs) in ALAD and VDR genes on Pb health effects and/or kinetics are inconclusive at low exposure levels, while studies including APOE SNPs are rare. In this study, we examined the associations of ALAD, VDR and APOE SNPs with exposure biomarkers of Pb and other trace elements (TEs) in Italian pregnant women (N = 873, aged 18-44 years) and their newborns (N = 619) with low-level mixed-element exposure through diet, the environment or endogenously. DNA from maternal peripheral venous blood (mB), sampled during the second and third trimesters, was genotyped for ALAD (rs1800435, rs1805313, rs1139488, rs818708), VDR (rs2228570, rs1544410, rs7975232, rs731236) and APOE (rs429358, rs7421) using TaqMan SNP assays. Personal and lifestyle data and TE levels (mB, maternal plasma, hair and mixed umbilical cord blood [CB]) from the PHIME project were used. Multiple linear regression models, controlling for confounding variables, were performed to test the associations between SNPs and TEs. The geometric means of mB-Pb, mB-Hg, mB-As and mB-Cd (11.0 ng/g, 2.16 ng/g, 1.38 ng/g and 0.31 ng/g, respectively) indicated low exposure levels, whereas maternal plasma Zn and Se (0.72 μg/mL and 78.6 ng/g, respectively) indicated adequate micronutritional status. Variant alleles of ALAD rs1800435 and rs1805313 were negatively associated with mB-Pb levels, whereas a positive association was observed for rs1139488. None of the VDR SNPs or their haplotypes had any association with Pb levels. Regarding APOE, the ɛ4 allele was associated with lower mB-Hg and CB-Hg, while a positive association was found with the ɛ2 allele and CB-Pb when the model included only newborn girls. The observed associations indicate possible modification effects of ALAD and APOE SNPs on Pb or Hg kinetics in women and their newborns with low exposure to non-essential TEs, as well as an adequate nutritional status of Zn and Se.

Since arsenic (As) exposure is largely due to geochemical contamination, this study focused on the remediated area of Santana do Morro, a district of Santa Barbara, Minas Gerais, Brazil, which was previously contaminated with As due to gold mining. Total As concentrations in sediment, soil and plants were determined, next to As species, anionic arsenic compounds As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), in plants samples. Total As concentrations in soil and sediments were slightly elevated (16-18 µg g -1 ) and most of the plants contained low levels of As (< 1 µg g -1 ). The exception was a native plant Eleocharis geniculata (L.) which contained elevated levels of As (4 µg g -1 ). The exposure of this plant to As under controlled conditions (hydroponics) indicated its possible tolerance to elevated As levels and suggesting its potential use in phytomonitoring of As-contaminated sites. This plant is able to metabolize arsenate to arsenite and contained MMA and DMA, both in its natural habitat and under controlled conditions.

For reason beyond the control of the authors or the editors, the article titled “Colloidal Organic Matter and Metal(loid)s in Coastal Waters (Gulf of Trieste, Northern Adriatic Sea)” by Katja Klun1 · Ingrid Falnoga2 · Darja Mazej2 · Primož Sket3 · Jadran Faganeli1 ( https://doi.org/10.1007/s10498-019-09359-6 ) was published in the regular issue Vol. 25 issue 5-6 instead of this special section, where it was originally scheduled to appear. Therefore, the full article is reprinted here.

The colloidal organic matter (COM) was isolated from the exudates of three cultured phytoplonkters, namely the chlophyte nanoflagellate Tetraselmis sp., the diatom Chaetoceros socialis and the dinoflagellate Prorocentrum minimum, from the Gulf of Trieste (northern Adriatic Sea). The isolation of COM was performed by ultrafiltration with molecular weight cut-off membranes of 5 kDa and final desalinisation by dialysis. The composition of the COM was characterised using C elemental analysis and 1H NMR spectroscopy and compared with COM isolated from a marine sample from the same area (Gulf of Trieste). By using 1H NMR spectroscopy, it was possible to semi-quantitatively determine the concentrations of the main biochemical constituents present in the COM samples. The results showed that the phytoplankton COM was predominantly composed of polysaccharides, with minor contributions from proteins and especially lipids. Therefore, the phytoplankton COM mainly contributes to the marine COM pool in the polysaccharide fraction and less in the protein and lipid fractions.

Large volumes of seawater were sampled in the Gulf of Trieste (northern Adriatic Sea) in order to study the interactions between colloidal organic matter (COM) and metal(loid)s (Me) in coastal waters. COM (> 5 kDa) was isolated by ultrafiltration and characterized using 1H NMR spectroscopy and elemental Corg. and Ntot. analyses. COM in the gulf represents about one quarter of the dissolved organic carbon (DOC), and according to 1H NMR analysis, it is composed of polysaccharides (30–45%), lipids (30–55%), proteins and carboxyl-rich alicyclic molecules (CRAM) (15–20%), and humics (

The first national human biomonitoring in Slovenia surveyed cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As), manganese (Mn), selenium (Se), copper (Cu) and zinc (Zn) in a childbearing population (18–49 years) selected from lactating primiparous women and men (N = 1084). The overall aim was to estimate trace elements' levels and geographical variations in order to identify sources of possible exposures and set the national reference values. The study population was selected evenly from 12 study areas across Slovenia, including rural, urban and known or potentially contaminated environments. Within 6–8 weeks after delivery, venous blood, spot urine, scalp hair and breast milk samples were collected to determine the selected elements. The data analysis included descriptive statistics and multiple linear regression using elemental concentrations in biological matrices, questionnaire data and environmental datasets. Essential elements showed no significant deficiencies or excessive levels in the study population and were largely determined by sex and/or the participating women's physiological status (postpartum, lactation), as well as by certain dietary sources. Toxic elements' levels were mainly below the levels considered to present increased health risk. Lifestyle and nutritional habits appeared as significant determinants of exposure to Cd (smoking and game meat consumption), Hg (seafood and amalgam fillings), As (seafood) and Pb (alcohol consumption, smoking, game meat consumption and type of water supply). A distinctive geographical pattern was confirmed, due to past mining activities combined with naturally elevated background levels in the cases of Pb (Mežica Valley), Hg (Idrija and Posocje) and As exposure (Zasavje). Increased seafood consumption in the coastal study area contributed to higher Hg and As (arsenobetaine) levels. Extensive sample size database accompanied with life-style and environmental data improved the prediction of exposure patterns, set the reference values for the childbearing population living in Slovenia, and provided a strong basis for evaluating spatial and temporal trends in exposure. To our best knowledge, this is the first study to establish reference values for lactating primiparous women.

Chemical pollution is a major environmental concern especially in coastal areas, having adverse impacts on marine organisms and ecosystem services. Macroalgae can accumulate trace elements, but available studies are restricted to a limited number of elements and species. The goal of this research was to assess, seasonally, the concentrations of 22 elements in the brown alga Padina pavonica from monitoring sampling sites in Slovenian waters. The concentration of most elements in thalli differed significantly between spring and autumn, with generally higher levels in autumn samples. However, it was not possible to correlate these concentrations with the ecological status of macroalgae. The maximum values set by European regulations for the potentially hazardous As, Cd and Hg in food and feed were never exceeded, while Pb concentrations were slightly higher. The results show that P. pavonica can act as an effective bioindicator of chemical pollution.

Since arsenic (As) exposure is largely due to geochemical contamination, this study focused on the remediated area of Santana do Morro, a district of Santa Bárbara, Minas Gerais, Brazil, which was previously contaminated with As due to gold mining. Total As concentrations in sediment, soil and plants were determined, next to As species, anionic arsenic compounds As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), in plants samples. Total As concentrations in soil and sediments were slightly elevated (16-18 µg g-1) and most of the plants contained low levels of As (1 µg g-1). The exception was a native plant Eleocharis geniculata (L.) which contained elevated levels of As (4 µg g-1). The exposure of this plant to As under controlled conditions (hydroponics) indicated its possible tolerance to elevated As levels and suggesting its potential use in phytomonitoring of As-contaminated sites. This plant is able to metabolize arsenate to arsenite and contained MMA and DMA, both in its natural habitat and under controlled conditions.

A survey of highly toxic arsenic compounds, together with some other elements was carried out on 40 polished rice samples (white, basmati and parboiled) and 10 brown rice samples from the Slovenian market. The average total As concentration was 157 ± 60 μg kg−1; highest levels were found in parboiled and brown rice and lowest in basmati. The average inorganic As concentration was 90 ± 35 μg kg−1. Dimethylarsinic acid and monomethylarsonic acid, which also exhibit high toxicity levels in some cases constitute >50% of total arsenic and might deserve more attention. Contrary to other foods, the total arsenic concentration in rice may even be a better health hazard indicator than the inorganic arsenic concentration. Elemental analysis of rice revealed large differences between polished and brown rice, especially for Mg, Mn, P, Fe and K, which were 2–4 times higher in brown rice than in polished rice.

The total activity of 210 Po was determined by alpha-spectrometry in various samples (matrices) collected in the Gulf of Trieste (northern Adriatic Sea) where fresh water inflows, especially from the Isonzo River in the northern part, affect water quality. Observed 210 Po levels were: 1) 0.56–3.75 mBq/L in the dissolved phase ( S transect in the gulf and sectioned to the depth of 20 cm) and 4) 239 (autumn) – 415 to 1800 (spring) Bq/kg (dw) in meso(zoo)plankton (>200 μm). In seawater and tributaries, up to 80% (mean 49%) of total 210 Po was found in particulate form. In sediments, slightly higher levels were encountered in the Isonzo prodelta and in the central (depocenter) part of the gulf. K D (L/kg) calculated between seawater and SPM, and seawater and sediment amounted to about 5 × 10 6 and 6 × 10 4 , respectively. Lower autumn 210 Po levels can be a consequence of biological dilution by higher mesozooplankton biomass in the autumn compared to spring. Plankton fractionation revealed in general the highest levels in the >200 μm mesoplankton fraction (239–1800 Bq/kg) followed by 50–200 μm (388–996 Bq/kg) and 20–50 μm (318–810 Bq/kg) microplankton fractions. Obtained data show higher 210 Po levels in all matrices analyzed in the Gulf of Trieste compared to other Adriatic (central Adriatic) and western Mediterranean areas. The 210 Po/ 210 Pb ratios in water, plankton and sediments were mostly below or around 1, while this ratio was much higher at higher trophic levels (up to about 50), reflecting a preferential bioaccumulation of 210 Po over 210 Pb. 210 Po accumulation between seawater and SPM and seawater and mesozooplankton amounted to 3.7 × 10 4 and 1.1 × 10 4 , respectively, similar to other Adriatic areas. Comparison of the relative importance of pelagic and benthic bioaccumulation pathways, excluding the filter feeder bivalves, suggests greater accumulation in pelagic-feeding species.

Metal(loid)s' accumulation and interactions were studied in Tetrahymena thermophila after 24 h exposure to 100 µM CuI, 20 µM CdII, 1 µM of AgI, HgII, PbII, AsIII, AsV, SbIII, SbV, SeIV, or ...

At three uranium (U) legacy sites in Kyrgyzstan, namely, Kadji Sai, Mailuu-Suu and Sumsar, an initial human bio-monitoring programme was introduced as a complementary activity to environmental impact studies in these areas. The aim was to assess trace element (TE) contents in blood and genetic susceptibility for Pb as one of the contaminants. The programme included the determination of 9 TE in blood samples from 123 residents living permanently in this environment. The analyses included U and the potentially toxic TE, lead (Pb), cadmium, mercury (Hg), and arsenic (As), together with essential elements iron (Fe), copper, selenium (Se) and manganese (Mn). TE were analysed by inductively coupled plasma mass spectrometry (ICPMS) and genetic background effect by three single nucleotide polymorphisms (SNPs) of delta-aminolevulinic acid dehydratase (ALAD; rs1805313, rs818708, rs1800435) genotyped by quantitative polymerase chain reaction (qPCR). The obtained results were generally similar to literature reference values obtained from the U non-exposed environments. However, some significant findings indicated elevated levels of certain contaminants typical of the studied environment (U, Pb). Several essential (Se, Mn) and toxic TE (Pb, Hg, As, U) in blood showed statistically significant differences among the studied areas. All areas showed diminished Fe blood levels. Altogether, this indicated specific and different environmental conditions at three industrial legacy sites for U milling and processing along with the accompanying chemical (pollutant) elements. Blood U concentrations were slightly higher at Mailuu-Suu, known for elevated technogenic and naturally occurring U. At Sumsar, the distribution of elevated blood Pb concentrations indicated an airborne source of pollution that was different from the anticipated aqueous exposure pathway. Pb blood variability was found associated with ALAD polymorphisms (SNPs rs1805313, rs1800435). Results are confirming that human data will be a useful and scientifically important additional tool for environmental impact assessment studies at industrial legacy sites in Kyrgyzstan.

Urine cadmium (Cd) and renal function biomarkers, mostly analysed in urine spot samples, are well established biomarkers of occupational exposure. Their use and associations at low environmental level are common, but have recently been questioned, particularly in terms of physiological variability and normalisation bias in the case of urine spot samples.To determine the appropriateness of spot urine and/or blood Cd exposure biomarkers and their relationships with renal function biomarkers at low levels of exposure. To this end, we used data from Slovenian human biomonitoring program involving 1081 Slovenians (548 males, mean age 31 years; 533 lactating females, mean age 29 years; 2007-2015) who have not been exposed to Cd occupationally.Geometric means (GMs) of Cd in blood and spot urine samples were 0.27ng/mL (0.28 for males and 0.33 for females) and 0.19ng/mL (0.21 for males and 0.17 for females), respectively. Differing results were obtained when contrasting normalisation by urine creatinine with specific gravity. GMs of urine albumin (Alb), alpha-1-microglobulin (A1M), N-acetyl-beta-glucosaminidase (NAG), and immunoglobulin G (IgG) were far below their upper reference limits. Statistical analysis of unnormalised or normalised urine data often yielded inconsistent and conflicting results (or trends), so association analyses with unnormalised data were taken as more valid. Relatively weak positive associations were observed between urine Cd (ng/mL) and blood Cd (β=0.11, p=0.002 for males and β=0.33, p0.001 for females) and for females between urine NAG and blood Cd (β=0.14, p=0.04). No associations were found between other renal function biomarkers and blood Cd. Associations between Cd and renal function biomarkers in urine were stronger (p0.05, β=0.11-0.63). Mostly, all of the associations stayed significant but weakened after normalisation for diuresis. In the case of A1M, its associations with Cd were influenced by current smoking and blood Pb in males and by pre-pregnancy smoking and blood Se in females (β up to 0.34, p0.001). Statistical analysis of unnormalised or normalised urine data often yielded inconsistent and conflicting results (or trends), so association analyses data with unnormalised were taken as more valid.The observed uncertainties introduced by urine normalisation, particularly by creatinine, confirm blood Cd as a superior low-Cd exposure biomarker versus urine Cd in cases when 24h urine is unattainable. Evidence that A1M can be positively related to Cd, smoking (current or pre-pregnancy), Pb, and Se status, points to the versatile biological functions of A1M.

Objectives The aim of this study was to evaluate the association of maternal blood selenium (Se) levels and cord blood Se levels with neonatal cerebellum measures and child neurodevelopment at the age of 18 months. Moreover, to investigate whether the neonatal cerebellum measures could be used as a potential biomarker for selenium homeostasis during pregnancy. Study group and methods The study population consisted of 205 mother-child pairs from Croatian Mother and Child Cohort. Maternal blood and cord blood were obtained at delivery and selenium level was analyzed by Inductively Coupled Plasma Mass Spectrometry. Cranial ultrasonography examination was performed on 49 newborns – cerebellum length and width have been measured. Neurodevelopmental assessment of cognitive, language and motor skills were conducted on 154 children, using The Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III), at the age of 18 months. Results The mean levels of selenium in maternal blood and cord blood were 92.6 ng/g and 97.0 ng/g, respectively. Maternal blood selenium levels were moderately and negatively correlated (r = −0.372; p = 0.008) with cerebellum length, while cord blood selenium levels were positively correlated with cerebellum width (r = 0.613; p = 0.007) among female children group. Maternal blood selenium levels were weakly and positively correlated (r = 0.176; p = 0.029) with child's cognitive abilities. Conclusion To the best of our knowledge, our study is the first one investigating the association between neonatal brain measures and selenium levels in mother-child pairs. Our results indicate that prenatal selenium intake correlated with cerebellum length and width measured by cranial ultrasonography. Hence, cerebellum may be used as a potential biomarker and a target “organ” for early detection of possible adverse effects of prenatal status to various micronutrients.

The Gulf of Trieste and adjacent Grado and Marano Lagoon (northern Adriatic Sea) are considered as an important “natural bioreactor” that can increase sedimentary methylmercury (MeHg) production by mobilizing the extensive mercury (Hg) pool as a result of historical (500 years) Hg mining and smelting activities in Idrija (NW Slovenia), the world’s second largest Hg mine. Even though the mine was definitely closed in 1995 the Soča/Isonzo River system still delivers about 1.5 t of Hg to the gulf annually and the MeHg is elevated in this marine environment. Research conducted by late M.E. Hines showed that Hg is readily methylated and demethylated in marine, lagoonary and freshwater sediments but the relative activities varied greatly with locality. Methylation activity increased from freshwater to the marine sites, conversely the highest demethylation was found in estuarine and lagoon sites. The methylation/demethylation ratios were consequently low in coastal sites but increased ib the gulf. Subsequent investigations in the southern, less contaminated (“pristine”) sediments of the gulf revealed that MeHg is also continuously produced especially in autumn in parallel with higher bottom water temperature and lower oxygen content. Sulphate reduction was found to control both processes. Hg methylation in sediments of coastal lagoon was controlled by rapid demethylation and the Hg bioavailability was affected by Hg adsorption and precipitation, In offshore marine sites, the sulphide produced by sulphate reduction lowers Hg methylation. Methylation of dissolved Hg, nearly 100% colloidally bonded, in the seawater column of the gulf was not detected (showing a pronounced Hg reduction potential) suggesting that sediments are the principal methylation site and the source of MeHg to the water column. It is likely that demethylation and reduction prevent the excessive accumulation of MeHg in the food webs of the Gulf of Trieste.

The relationships between inorganic arsenic (iAs) metabolism, selenium (Se) status, and genetic polymorphisms of various genes, commonly studied in populations exposed to high levels of iAs from drinking water, were studied in a Croatian- Slovenian population from the wider PHIME-CROME project. Population consisted of 136 pregnant women in the 3rd trimester and 176 non-pregnant women with their children (n = 176, 8–9 years old). Their exposure to iAs, defined by As (speciation) analyses of biological samples, was low. The sums of biologically active metabolites (arsenite + arsenate + methylated As forms) for pregnant women, non-pregnant women, and children, respectively were: 3.23 (2.84–3.68), 1.83 (1.54– 2.16) and 2.18 (1.86–2.54) ng/mLSG ; GM (95 CI). Corresponding plasma Se levels were: 54.8 (52.8– 56.9), 82.3 (80.4–84.0) and 65.8 (64.3–67.3) ng/mL ; GM (95 CI). As methylation efficiency indexes confirmed the relationship between pregnancy/childhood and better methylation efficiency. Archived blood and/or saliva samples were used for single nucleotide polymorphism (SNP) genotyping of arsenic(3+) methyltransferase - AS3MT (rs7085104, rs3740400, rs3740393, rs3740390, rs11191439, rs10748835, rs1046778 and the corresponding AS3MT haplotype) ; methylene tetrahydrofolate reductase - MTHFR (rs1801131, rs1801133) ; aquaporin - AQP 4 and 9 (rs9951307 and rs2414539) ; selenoprotein P1 - SELENOP (rs7579, rs3877899) ; indolethylamine N- methyltransferase - INMT (rs6970396) ; and metallothionein 2A - MT2A (rs28366003). Associations of SNPs with As parameters and urine Se were determined through multiple regression analyses adjusted using appropriate confounders (blood As, plasma Se, ever smoking, etc.). SNPs’ influence on As methylation, defined particularly by the secondary methylation index (SMI), confirmed the ‘protective’ role of minor alleles of six AS3MT SNPs and their haplotype only among non-pregnant women. Among the other investigated genes, the carriers of AQP9 (rs2414539) were associated with more efficient As methylation and higher urine concentration of As and Se among non-pregnant women ; poorer methylation was observed for carriers of AQP4 (rs9951307) among pregnant women and SELENOP (rs7579) among non- pregnant women ; MT2A (rs28366003) was associated with higher urine concentration of AsIII regardless of the pregnancy status ; and INMT (rs6970396) was associated with higher As and Se concentration in non-pregnant women. Among confounders, the strongest influence was observed for plasma Se ; it reduced urine AsIII concentration during pregnancy and increased secondary methylation index among non-pregnant women. In the present study of populations with low As exposure, we observed a few new As–gene associations (particularly with AQPs). More reliable interpretations will be possible after their confirmation in larger populations with higher As exposure levels.

Large volumes of seawater were sampled monthly from December 2011 to October 2012 in the southeastern part of the Gulf of Trieste (northern Adriatic Sea) in order to study the seasonal changes of colloidal organic matter (COM) concentrations, its origin and composition. Isolation of COM was performed by ultrafiltration with molecular weight cut-off membranes of 5 kDa and final desalinization by dialysis. COM was characterized using 1H NMR and FT-IR spectroscopy, elemental C and N and 13C and 15N isotope analyses and high-pressure size exclusion chromatography. COM represents about 1/4 of dissolved organic carbon (DOC) and appears to be mainly of marine origin. COM is mainly composed of polysaccharides, lipids, proteins and humics. FT-IR analysis indicated the presence of OH (carbohydrates and lipids), COOH (proteins and lipids) and NH (proteins) containing functional groups. Using the NMR technique, it was possible to semiquantitatively determine variations of four main biochemical constituents present in...

Since the environmental levels of selenium (Se) can moderate the bioaccumulation and toxicity of mercury (Hg) in marine organisms, their interactions were studied in seawater, sediments, plankton and the benthic (Bull ray Pteromylaeus bovinus, Eagle ray Myliobatis aquila) and the pelagic (Pelagic stingray Dasyiatis violacea) rays, as apex predators in the Gulf of Trieste (Northern Adriatic Sea). Male and female rays showed no difference in the Se contents in muscle tissue. Pelagic species contained higher Se levels in muscle but slightly lower levels in the livers of both genders. The Hg/Se ratios in seawater dissolved and colloidal fractions, plankton and sediment were

The environmental status of the marine environment in the NE Adriatic Sea was assessed, using as a bioindicator species the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819. Samples were collected seasonally from mariculture sites and from major Istrian ports between the years 2010 and 2013. The condition indices of mussels ranged from 13.3 to 20.9% at mariculture sites and from 14.3 to 23.3% at port locations. The seasonally δ13CDIC values of seawater varied between -10.9 to 0.7‰. Pollution by sewage sludge (based on δ15N values) was confirmed only in two ports. Tissue concentrations of Mn, Co, Ni, Cu, Zn, Se, Cd, and Pb were significantly higher in the tissue of the mussels collected from the ports (polluted sites). Arsenobetaine was the major As compound present in the samples and there was no significant difference in the levels of total As in mussel tissues from mariculture and port sites.

Meconium is formed early in gestation and it is normally not excreted until after birth. Thus it may provide a longer and cumulative record of exposure to mercury (Hg). The present study aims to speciate Hg in meconium samples (N = 488) from Slovenian and Croatian new-borns prenatally exposed to low levels of methyl-Hg (MeHg) from maternal seafood intake and to Hg0 from maternal dental amalgam fillings. We had complete data of total Hg (THg) and MeHg in meconium and THg in maternal hair (MH), while THg and MeHg in maternal blood (MB) were available only for Croatian mothers. Personal data namely maternal seafood intake, age, pre-pregnancy BMI, parity, smoking, estimated gestational age at birth, sex, and birth weight were available for the majority of participants, except the number of dental amalgams which was in most cases missing for Croatian mothers. The median THg concentration in meconium was 11.1 (range: 0.41–375.2) ng/g and inorganic Hg (Hg(II)) presented 98.8% (range: 82%–100%, CV: 2%) of THg. We observed significant correlation between meconium and MH Hg levels, with the highest correlation between hair THg and meconium MeHg. Correlation analysis including MB (available only for Croatian population) showed a significant positive correlation between THg in meconium and THg in MB (Rs = 0.642). Additionally, MeHg from MB was correlated with MeHg in meconium (Rs = 0.898), while the correlation between Hg(II) in MB and meconium was positive, but not significant. Maternal seafood intake was significantly correlated with meconium MeHg (Rs = 0.498) and Hg(II) (Rs = 0.201). Multiple linear regression (performed on the Slovenian population, N = 143) confirmed a positive association between meconium MeHg and seafood intake. Furthermore, meconium Hg(II) was positively associated with the number of maternal dental amalgam fillings, but linear regression models did not confirm correlation between seafood intake and meconium Hg(II) levels. We assume that Hg0 released from maternal dental amalgam fillings and MeHg from seafood intake were both transported through the placental barrier and portioned between different foetal compartments including meconium. Weak correlation between maternal seafood intake and Hg(II) levels in meconium suggests that there is certain evidence of MeHg demethylation. However, because this correlation was not confirmed by the multiple regression, MeHg demethylation during prenatal life cannot be neither confirmed nor excluded. Further investigations at higher level of exposure are needed to confirm this observations. We can conclude that meconium is a suitable biomarker for MeHg and Hg0 exposure during pregnancy. However, comparability of the results reported in meconium in different studies is hindered by a lack of standardized sampling protocols, storage, and analysis.

In recent years, several studies have addressed the issue of prenatal exposure to methylmercury (MeHg); however, few have actually analysed MeHg blood concentrations. Our study population included mothers and their new-borns from Slovenia (central region; N = 584) and Croatia (coastal region; N = 234). We have measurements of total Hg (THg) and MeHg in maternal hair, maternal peripheral blood, and cord blood. Cord blood Hg concentrations were low to moderate (median THg = 1.84 ng/g and MeHg = 1.69 ng/g). The proportion of THg as MeHg (%MeHg) in maternal and cord blood varied between 4% and 100% (coefficient of variation, CV = 32%) and between 8% and 100% (CV = 20%), respectively. Our data shows that variability of %MeHg was higher at lower blood THg levels. Concentrations of MeHg in maternal blood and cord blood were highly correlated (Rs = 0.943), in the case of inorganic Hg correlation was significant but weaker (Rs = 0.198). MeHg levels in maternal blood and cord blood were positively associated with seafood intake, maternal age, and negatively associated with pre-pregnancy BMI. Additionally, MeHg in maternal blood was positively associated with plasma selenium levels, and cord blood MeHg was negatively associated with parity. The results of multiple linear regression models showed that speciation analysis provides more defined estimation of prenatal exposure in association modelling. Associations between Hg exposure and cognitive performance of children (assessed using Bayley Scales of Infant and Toddler development) adjusted for maternal or child Apolipoprotein E genotypes showed higher model R2 and lower p-values when adjusted for MeHg compared to THg. This study demonstrates that Hg speciation improves the association between exposure and possible negative health effects. Bibliografija: str. 8-10. Abstract. ARRS ARRS, NEURODYS EU

Macroaggregates (macrogels) occurring episodically in the northern Adriatic, offer a rare opportunity to study interactions between marine organic macromolecules and metals and metalloids (Me). Macrogels are formed by agglomeration of dissolved heteropolysaccharides, mostly of phytoplankton origin. Filtration and centrifugation were used to discriminate between macrogel matrix and interstitial water colloids. The interstitial fraction was then used in an incubation experiment to follow the progress of microbial degradation of colloids. The colloids were ultrafiltered through membranes and each size class of colloids (> 30 kDa, 10–30 kDa and 5–10 kDa) was analyzed for metal, metalloid, organic carbon (C org ) and total nitrogen (N tot ) content. Higher Me concentrations and lower Me/C org ratios in matrix, which are an indicator of Me bound to organic matter, suggest that Me in a matrix can also be bound onto Me containing particles. All the studied Me in macroaggregate colloidal fraction, except Se, proved to be bound to large macromolecules (> 30 kDa), mainly glycoproteins and aminopolysaccharides. During the rapid degradation of all, except the 5–10 kDa fraction, the Me associated with the > 30 kDa fraction were markedly preserved. Consequently, the Me/C org ratios increased due to the loss of C org and relative increase of metal-binding sites. The observed differences between our Me/C org ratios and the Irving–Williams series can be explained by variable background concentrations of the studied Me in the northern Adriatic. Comparison between Me levels in macroaggregate colloids and dissolved marine colloids, considered to be macroaggregate precursors, revealed similar values. Distribution coefficients (K d ) between colloidal and truly dissolved Me were lower than K d for particulate matter, suggesting that organic colloids, including macroaggregates, exhibit rather weak Me binding capacity and that Me are not distributed homogenously in the various particle pools in the northern Adriatic coastal waters.

The aim of the present study was to evaluate the association between prenatal exposure to mercury (Hg) and neurodevelopment of the child, taking into account genetic polymorphism of apolipoprotein E (Apoe) and other relevant confounders. Six hundred and one mother-child pairs were recruited from the central Slovenia region and 243 from Rijeka, on the Croatian coast of the northern Adriatic. The total Hg in cord blood, Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) assessment at 18 months of age and Apoe genotyping was performed on 361 children; 237 of them were from Slovenia and 124 from Croatia. The results showed negative association between low-to-moderate Hg exposure in children with normal neurodevelopmental outcome and cognitive and fine motor scores at 18 months of age as assessed by Bayley III. The Hg-related decrease in cognitive score was observed only in children carrying at least one Apoe ε4 allele, while the decrease in fine motor scores was independent of the Apoe genotype. Adjusting for selenium (Se) and lead (Pb) levels, a positive association between Se and the language score and a negative association between Pb and the motor score was observed, but not in the subgroup of children carrying the ε4 allele.

The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO2) nanoparticles (nano-TiO2). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO2 containing 100, 1,000, 3,000, or 5,000 µg nano-TiO2 per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma–mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO2 by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO2 particles, and at higher extracellular concentrations of nano-TiO2, the nanoparticles were internalized. Environ. Toxicol. Chem. 2012; 31: 1083–1090. © 2012 SETAC

The objectives of this work were to get information on Zn species in two different specimens of edible plants (pumpkin seeds and iceberg lettuce) and simulation of their behavior in the human gastrointestinal tract. An array of analytical techniques was used to aid in this research: ultrasound-assisted variable volume extraction, size exclusion chromatography (SEC), and physiologically based extraction tests (PBETs); elemental detection was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Results show that pumpkin seeds and iceberg lettuce have different Zn species fingerprints (in water extracts) with a high (ca. 70 %) low-molecular-weight fraction (ca. 500 Da) in iceberg lettuce and a high (ca. 60 %) intermediate/high-molecular-weight fraction (10–20 kDa) in pumpkin seeds. When these Zn species are subjected to conditions simulating the human stomach (pH ~ 2) complete scrambling to their basic ionic form (Zn2+) takes place. Under conditions simulating the digestion in the intestines (pH ~ 7) formation of insoluble Zn complexes occurs, especially for pumpkin seeds, which may be related to antinutrients like naturally present phytate, leading to reduced Zn bioaccessibility in the small intestine.

Pumpkin seeds are one of the foodstuffs recommended in diets which do not contain other Zn-rich sources. The main objectives of this work were to get information on Zn and its species in pumpkin seeds, and their possible degradation in the human gastrointestinal tract, indicative of Zn bioaccessibility. A sequential analytical approach was applied, focusing on total Zn, spatial Zn distribution, extractability, speciation and bioaccessibility of Zn and its species. It was shown that water extracts of pumpkin seeds exhibit a specific Zn species fingerprint with ca. 30% of a low-MW fraction (0.5–2 kDa) and ca. 60% of an intermediate/high-MW fraction (10–20 kDa). Digestion of Zn species under simulated stomach conditions proved that Zn species identified in plant extracts were completely decomposed to Zn2+. The subsequent digestion under intestinal conditions showed that Zn becomes less accessible, indicating that antinutrients like naturally present phytate may be responsible for complex formation in the small intestines, thus reducing the potential for Zn bioavailability.

Various biomarkers were used to assess selenium (Se) status during 3 months of basic military training in a group of recruits. Samples of whole blood and plasma or serum were taken from a group of conscripts three times: at the beginning (n = 15), in the middle immediately after a severely stressful physical activity (n = 15) and at the end of military training (n = 13). Selenium was determined in diet samples, blood, plasma and plasma protein fractions as selenoprotein P (SelP) and glutathione peroxidase (eGPx). Selenium was determined by hydride generation-atomic fluorescence spectrometry and fractionation of proteins performed by affinity chromatography. Total serum glutathione, erythrocyte and serum GPx activity were followed as well. The average Se intake was calculated according to meal consumption questionnaires and Se determined in composite diet samples, giving an assessed intake of 48 ± 10 μg/day. At all three samplings, the average blood Se concentration was within the framework of adequate supply (87 ± 12, 94 ± 15 and 80 ± 13 ng/g). Plasma Se was between 70 and 80 ng/g (71 ± 10, 79 ± 9 and 76 ± 10 ng/g), which is believed to enable the full expression of plasma GPx. The average shares of plasma Se proteins were 61 ± 6%, 58 ± 6% and 54 ± 9% for SelP and 27 ± 4%, 34 ± 7% and 29 ± 5% for GPx. Although the observed tendency of the increases of serum and erythrocyte GPx activities at the second and third samplings with respect to the first was statistically insignificant, it is still indicative of some protection against oxidative stress, while the decreasing SelP levels during training suggest a slowly decreasing biologically active selenium pool.

Since a great quantity of dissolved Hg in natural waters was shown to be bonded onto colloidal organic matter (COM), Hg speciation, transport, biological availability and cycling are affected by interactions between Hg and COM. They are in large proportion dependent on the chemical composition and structure of COM. Macroaggregates (macrogels), produced episodically in the northern Adriatic by agglomeration of dissolved organic macromolecules—mostly heteropolysaccharides—of prevalently phytoplankton (diatom) origin, offer a rare opportunity to study these topics. The chemical speciation of macroaggregate Hg in the Gulf of Trieste (northern Adriatic Sea) was studied using filtration and centrifugation discriminating between matrix (water insoluble) and interstitial water (water soluble) colloidal fraction. The colloidal fraction was subsequently ultrafiltered through membranes with a nominal pore size of 30, 10 and 5 kDa cutoff sequentially (in a cascade fashion), and fractions analyzed for carbohydrate, Co...

This work presents an analytical chemist's view on the sometimes unconscious use of arsenic trioxide in (bio)medical research. Arsenic trioxide is a frequently used chemical in cancer treatment research and its action to various malignant cells has been extensively studied and published. Unfortunately some research articles show trivial errors with regards to background knowledge of the chemical, handling the chemical, experimental design and interpretation of results like e.g. in a range of articles comparing advantages of tetraarsenic oxide over arsenic trioxide (dimeric/monomeric) although the dissolution of both yields the same active compound (HAsO(2)). To fully understand the implications of these errors we will highlight some of them with the intent to harmonize future work in this field.

Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

Carbon and nitrogen stable isotope ratios of particulate organic matter (POM) were studied approximately weekly during spring and summer 2003 and 2004 in the Gulf of Trieste (northern Adriatic Sea) in order to track the temporal variations and differences between two years. In parallel, particulate organic carbon (POC) and particulate nitrogen (PN), phytoplankton biomass (chlorophyll a ), and N and P nutrients were monitored. All studied parameters, especially N and P nutrients and chlorophyll a , showed higher concentrations and larger variability in spring 2004. As a consequence the macroaggregates were produced in late spring 2004. The C and N isotope composition of POM was not directly linked to phytoplankton biomass dynamics. The δ 13 C POC values covaried with temperature. In 2004, δ 13 C POC variations followed the δ 15 N PN values as well as the δ 13 C DIC values which were probably more dependent on the photosynthetic use of 12 C. Variations in δ 15 N POM values were most probably the consequence of variations in N nutrient sources used in phytoplankton assimilation. The significant correlation between δ 15 N PN values and nitrate concentrations in 2004 implies intense nitrate assimilation in the presence of higher nitrate concentration. This suggests nitrate as the key nutrient in the »new primary production«, later producing macroaggregates with a mean δ 13 C and δ 15 N values of − 19‰ and 5‰, respectively. A low fractionation factor e

Selenium-mercury interactions were most extensively studied in relation to alleviation of Hg toxicity by added selenium. This presentation considers the influence of mercury on endogenous selenium, on its tissue and cellular "status" after lifelong or acute exposure to mercury vapor (Hg o). Discussed are data obtained from (1) humans living near or working in a mercury mine, and (2) rats experimentally exposed in the mine. Mercury vapor is unique--or similar to methylmercury--because of its ability to penetrate cell membranes and so invade all cells, where it is oxidized in the biologically active form (Hg++) by catalase. Such in situ-generated ions can react with endogenously generated highly reactive Se metabolites, like HSe-, and render a part of the selenium unavailable for selenoprotein synthesis. Data on human populations indicate that in moderate Hg exposure combined with an adequate selenium supply through diet, Se bioavailability can be preserved. On the other hand, the results of an acute exposure study emphasize the dual role of selenium in mercury detoxification. Besides the well-known Se coaccumulation through formation of nontoxic Hg-Se complexes, we observed noticeable Se (co)excretion, at least at the beginning of exposure. The higher Hg accumulation rate in the group of animals with lower basal selenium levels can also point to selenium involvement in mercury excretion. In such conditions there is a higher probability for decreased selenoprotein levels (synthesis) in some tissues or organs, depending on the synthesis hierarchy.

The total activity of

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As2O3/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15-25% of As(III), 35-55% of DMA, 25-30% of MMA and 1-5% of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2% As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4% of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40%) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.

The precise mechanisms of mercury accumulation and retention are still unclear. Generally, the association of mercury with selenium is used to explain these phenomena. It seems that the presence of coaccumulated endogenous Se can protect cells from the harmful effects of Hg. However, as speculated by some authors, this binding of Se to Hg can also result in a relative deficiency of biologically available Se needed for selenoenzyme syntheses. Deriving from the assumption that Hg deposited in tissues is bound to Se in a 1:1 ratio, the quantity of non-Hg bound Se could be calculated by the difference between the molar contents of the two elements (Se(mol)-Hg(mol)). In this study we applied such an approach to the data from our previous investigation, where Hg and Se concentrations were determined in autopsy samples of mercury exposed retired Idrija mercury mine workers, Idrija residents living in a Hg contaminated environment and a control group with no known Hg exposure from the environment. Based on these data we tried to estimate the influence of Hg exposure on the physiologically available selenium content in selected tissues, particularly endocrine glands and brain tissues. Comparing the calculated values of (Se(mol)- Hg(mol)) it was found that for Idrija residents the values were similar to those of the control group and as expected, diminished values were found in some mercury-loaded organs of retired Idrija miners. It could be speculated that in Idrija residents Hg sequestration of selenium is sufficiently compensated by increased Se levels, but that particularly in active miners and in some organs of retired miners, the activity and/or synthesis of selenoenzymes could be disturbed.

The binding of mercury (Hg) to metallothioneins (MTs) and the relation between Hg and selenium in supernatants of hepatopancreas and gill tissues of the common mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated. The mussels were exposed to different Hg concentrations in laboratory conditions: 2.5 microgHg/L, 4 d exposure (short term) and 60 microgHg/L, 33 d exposure (long term). In addition, the results were compared to those found for mussels from nature (polluted and unpolluted region). In control and short-term-exposed mussels, the level of Hg extraction (cytosol) from hepatopancreas and gill cells was very low with respect to the total Hg concentrations in the corresponding tissues, around 10% in control and around 20% after exposure. As expected, Hg exposure was followed by Se increase. For Se, the levels of extraction were higher, around 20% in control and up to 50% (heaptopacrease) of 70% (gills) after exposure. In order to study the distribution of Hg and Se in the cells of these organs, the total Hg and Se concentrations were analyzed in the subcellular fractions obtained after differential centrifugation. Although after exposure the concentrations of both element increased in all subcellular fractions, their percentages in particular fractions were lower or higher. In this study, the convincing binding of Hg to metallothionein-like proteins was perceived after long-term laboratory exposure (gills, heapatopancreas) and in wild mussels collected near industrial port (hepatopancreas). In latter case, we also detected the traces of Se bound to the MT fractions after size-exclusion chromatography.