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BACKGROUND-AIM Metallothioneins (MT) are cysteine-rich multifunctional metal-binding proteins. In humans, the MT2A gene comprises24 SNPs. Three of them, MT2A −5A/G, −209A/G and +838G/C were found to be possibly associated with tissue concentrations of trace elements in the body. We evaluated the association of these maternal MT2A polymorphisms and the levels of selected essential and toxic trace elements in maternal and cord blood and the placenta. METHODS The study included 424 postpartum women who gave birth at term in the maternity ward of the hospitals in Zagreb and Zadar, Croatia. DNA was isolated from blood using the QIAamp Blood Mini Kits from Qiagen. MT2A gene polymorphisms were determined by PCR-RFLP methods using BsgI (– 5A/G) and SmaI (–209A/G) restriction enzymes(New England BioLabs) or by qPCR (+838G/C) using Taqman assay (Applied Biosystems). Elements were analyzed by ICP-MS. Multiple linear regression analysis was used to evaluate the relationship between MT2A gene polymorphisms and trace elements in maternal blood, placenta and cord blood together with the factors contributing to trace element concentrations (maternal smoking and fish consumption) and other potential confounding factors (age, education, pregnancy weight gain and parity). RESULTS We found negative associations between MT2A – 209A/G SNP and Pb in maternal blood as well as with Hg in maternal and cord blood and the placenta. MT2A +838G/C was negatively associated with Pb in cord blood and positively with As in maternal and cord blood. Negative associations were also found between combinations of MT2A –5A/G and –209A/G polymorphisms and As in cord blood, MT2A –5A/G and +838G/C and Cu in cord blood as well as between a combination of all three studied MT2A SNPs and Fe in the placenta. CONCLUSIONS This study provides new evidence on the association between MT2A gene polymorphisms and trace element levels invulnerable population groups of mother-infant pairs. The studied MT2A gene polymorphisms, single or combined, were negatively associated with several trace elements in maternal, placental and fetal compartments, with the exception of the MT2A +838G/C SNP and As positive association.

Supovi su dugoživuće velike ptice grabljivice, koje se hrane leševima velikih i srednjih sisara te često mogu biti izložene olovu (Pb) poreklom iz municije. Uz pesticide i nesteroidne antiinflamatorne lekove, Pb je najčešća antropogena hemikalija za koju je u svetu utvrđeno da dovodi do smrti i bolesti kod slobodnoživećih supova. Posebna fiziološka stanja, kao što su neuhranjenost, nezrelost i istovremena izloženost drugim štetnim hemikalijama mogu pogoršati štetne efekte Pb. Oni se mogu ispoljiti na hematopoetskom, nervnom, kardiovaskularnom, bubrežnom, jetrenom, imunološkom i reproduktivnim sistemima organa. Hrvatska populacija beloglavih supova (Gyps fulvus) danas se još jedino gnezdi na kvarnerskim ostrvima, na stenama iznad mora. Trenutni napori da se populacija očuva usmereni su ka smanjenju broja trovanja i istraživanju uzroka trovanja supova. Cilj ove studije bio je da se procene nivoi Pb metodom induktivno spregnute plazme masene spektrometrije (ICP-MS). Uzorak za analizu je puna krv, većinom mladih beloglavih supova, kvarnerske populacije, koja je uzimana u periodu 2018.-2022. godine, tokom oporavka u Centru za pos etitelje i oporavilištu za beloglave supove “Beli”. Prosečan nivo Pb u krvi (KPb) iznosio je 20, 9±10, 5 μg/dL (N=25) i odgovara uobičajenim nivoima za red Accipitriformes (50 μg/dL), koja je ukazivala na kliničko trovanje. Za beloglave supove se smatra da su otporniji na Pb od drugih ptica grabljivica, te su za donošenje konačnih zaključaka o toksičnosti Pb neophodna dodatna istraživanja uticaja na njihovo zdravlje.

Poznata je povezanost ekspresije nekoliko miRNA i pušenja cigareta. Cilj ovog istraživanja je bio procijeniti utjecaj majčinog pušenja na ekspresiju miRNA (miR-1537, miR-190b, miR-16, miR21 i miR- 146a) u tri odjeljka majčinog i fetalnog podrijetla. Relativna ekspresija 5 analiziranih miRNA izražena kao fold change (2-ΔCt) u pušačica i nepušačica je pokazala statistički značajnu razliku između različitih odjeljaka fetalno- placentalne jedinice (P < 0, 001). Ekspresija miR- 1537, miR-190b, miR-16 i miR-146a je bila značajno viša u tkivu posteljice u odnosu na plazmu majke ili pupkovine (P < 0, 001). Ekspresija miR-16 u nepušačica je bila značajno veća u plazmi pupkovine u odnosu na plazmu majke (P = 0, 001) dok je ekspresija miR-146a u pušačica bila značajno veća u plazmi majke u odnosu na plazmu pupkovine (P = 0, 006). Pušačice su imale najveću ekspresiju miR-21 u plazmi majke sa statistički značajnom razlikom između plazme majke i posteljice (P < 0, 001). Relativna ekspresija analiziranih miRNA pokazala je značajnu razliku između različitih odjeljaka fetalno-placentalne jedinice u pušačica i nepušačica. Pušačice u odnosu na nepušačice imaju veću ekspresiju miR-16 u plazmi majke i miR-146a u plazmi pupkovine te nižu ekspresiju miR-21 u posteljici. Obzirom da je promjena relativne ekspresije ispitivanih miRNA u različitim odjeljcima povezana s majčinim pušenjem, potrebno je analizirati sve odjeljke fetalno-placentalne jedinice kako bi se dobila potpuna slika. Rezultati su dobiveni u sklopu projekta METALORIGINS (HRZZ-IP-2016-06-1998).

Rice (Oryza sativa L.) is one of the most important and widely grown staple crop. It represents a major part of the diet for more than a half of the world’s population. Due to its high carbohydrate content (mainly starch) it represents important source of energy, but it is also a valuable source of micronutrients, including minerals. It`s usually recommended as wheat alternative for persons who suffer from various allergies, intolerances and diseases such as celiac disease. Despite its high nutritional value, rice may contain undesirable levels of toxic elements, mainly As and Cd. In this study, aim was to determine the elemental composition of 58 rice samples (20 brown, 31 white and 7 parboiled) purchased from the Croatian market using ICP-MS method, and to investigate effects of processing type, cultivation method and the origin on element levels. Additionally, the consumers benefit and health risks from element exposure were quantified based on mean consumption of one serving of cooked rice [1]. Elements in rice were estimated in the order: P > K > Mg > Ca > Fe > Mn > Zn > Cu > Mo > As > Se > Cd > Co. Brown rice contained 2 to 4-fold higher levels of essential elements Mg, P, K, Fe, Mn, Co, Zn and Se compared to white rice. Mean toxic element level in all samples were 0.142 ± 0.057 mg/kg for As and 0.038 ± 0.035 mg/kg for Cd, with highest values observed in conventionally produced integral rice sample (As: 0.259 mg/kg ; Cd: 0.207 mg/kg). Only one sample exceeded the European Commission limit for Cd of 0.20 mg/kg. Rice originated from the European Union had higher As but lower Se than those from Asia. Among integral rice, non-organically grown rice contained more P, K, Mg, Fe and Zn compared to organic ones. Moreover, negative relationship (r=-0, 47, p=0.0002) between As and Se in rice was found. According to data obtained in this study, rice, especially integral one, may be considered as an important source of P, Mg and Cu. Observed low toxic element levels in all rice samples along with the latest recorded consumption rate of 2.7 kg rice per capita among Croatian population [2] indicate that rice is, with regard to toxic element, safe for consumption. References: [1] M. Rogić, R. Kovačević, B. Horvat, in Dietary reccomendations for people with diabetes, 11th ed., Zagreb, Tiskara Varteks, 2003. [2] Statistic reports for rice consumption per capita in Croatia, https://www.helgilibrary.com/indicators/rice-consumption-per-capita/croatia/ (accessed 21 April 2022).

Mechanisms of aging, and cancer as one of main associated pathology are only on verge of understanding. Even in healthy aging gradually loss of function in excretion organs kidneys and liver appears. Previously, we did not find expected elevated tissue concentrations of reactive oxidative species (ROS), that are assumed to be present in old humans and experimental animals and that they can be reversed by antioxidants (AO) treatment. The new hypothesis was that the expression of ROS inducible, potent antioxidant and metal scavenging proteins, metallothioneins Mt1 and Mt2 (MTs), among others, increases in old age to maintain ROS levels and that they can be further modulated with AO. To that aim we studied male and female Wistar rats regularly aged together with these treated with melatonin and resveratrol. Starting from 3 months of age, for the next 9 or 21 months treated rats were drinking AO in water (~1 mg/kg b.w./day), whereas the control animals were drinking water or AO vehicle (0.01% ethanol). Trace elements (TE) were analysed by ICP-MS and the expression of MTs mRNA and proteins in kidneys and liver were determinate by end-point RT-PCR and immunochemical methods, respectively. AO did not change TE accumulation and expression of MTs in both sexes, but known iron accumulation in aged animals was followed by copper and toxic metal cadmium with known female domination. After 1 year, aging increased MTs expression in both sexes that did only slightly further increased after 2 years. We conclude that Fenton reactive metals accumulated in aged animals may influence MTs mRNA and protein expression to help retain physiological ROS level in old rats. (Croatian Science Foundation project IP-2013-11-1481).

Eating fish has known benefits for human health, especially for heart function and lowering the risk of obesity in adults whereas during pregnancy it is necessary for child neurodevelopment as an important source of omega-3 fatty acids, fat-soluble vitamins and various essential elements.[1] However, toxic trace elements that accumulate in fish meat, primarily Hg, are a potential risk for adverse health effects. Guidelines on fish consumption for sensitive population groups, such as women during reproductive period and children have been to date focused on large, predatory fish species and widely vary among countries. We evaluated the effect of fish body length on the levels of essential (Mn, Cu, Zn, Se) and toxic elements (Hg, Cd, Pb, As) in edible muscle tissue of wild European hake (Merluccius merluccius) that is the most commonly consumed fish species in Croatian population.[2] Fish specimens were collected between 2016 and 2021. They were caught by pelagic trawl net at several locations in the eastern part of the Adriatic Sea (n = 11 ; within EU project MEDIAS) and bought at fish market in Zagreb (n = 17). The samples of skinless dorsal muscle tissue were freeze dried and homogenised in the mixer mill using tungsten balls (Retsch, Germany). Approximately 0.25 g of sample was then wet digested in a high-pressure microwave system UltraClave IV (Milestone, Italy). Multielement analysis was performed using 7500cx ICP-MS (Agilent Technologies, Japan) and accuracy of the method was checked using commercially available certified reference materials IAEA 407 fish homogenate and ERM BB422 fish muscle. Concentrations of measured elements were (range, mg/kg wet wt): 0.08-0.61 for Mn ; 0.08-0.36 for Cu ; 2.4-5.1 for Zn ; 0.24-0.48 for Se ; 4.5-25 for As ; 0.05-0.97 for Hg, and (range, µg/kg wet wt) 0.03-0.89 for Cd ; 0.08-9.9 for Pb. Calculated molar ratio of Se:Hg and Se health benefit value were on average 8.1 and 3.9, respectively, indicating benefits for human nutrition. Fish specimens were categorized by fork length into three groups (G):11.0-20.9 cm (G1), 21.0-30.9 cm (G2), and 31.0-50.0 cm (G3). In G3 vs. G1 were approximately 3-fold higher Hg and 1.2-fold lower both Cu and Zn levels. Only the longest specimen of 50 cm had concentration 0.97 mg Hg/kg, which exceeded the EU regulatory limit for Hg in fish meat (0.50 mg Hg/kg). Correlations between fish length and element concentrations were positive for Hg (rs=0.65, p

The homeostasis of metals is altered in the brain of Alzheimer’s disease (AD) patients. It was proposed that changes in metal homeostasis are related to AD pathology. Additionally, metals that are not present in the brain under normal circumstances (such as mercury, lead and cadmium) were associated with AD pathogenesis. The aim of this study was to test if macro and microelements (Li, B, Na, Mg, Al, S, K, Ca, Cr, Fe, Co, Mn, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Ba, Tl, Pb, and Hg) measured in cerebrospinal fluid (CSF) and plasma of patients with dementia are associated with CSF biomarkers of AD. CSF AD biomarkers reflect pathological changes in AD brain, while the involvement of different metals in AD pathogenesis is still the matter of debate. Macro and microelements were determined by inductively coupled plasma mass spectroscopy (ICP-MS), while CSF biomarkers were measured by enzyme-linked immunosorbent assays (ELISA). Macro and microelements were measured in CSF of 194 subjects and in plasma of 144 subjects (study included overall 125 AD patients, 50 patients with mild cognitive disorder and 19 healthy controls). We used three different statistical methods to test the association of macro and microelements with CSF biomarkers of AD. All three methods (simple correlation and two machine learning algorithms ; redescription mining and principal component analysis [PCA]) demonstrated the association of macro and microelements with CSF biomarkers of AD. Possible explanations for association of macro and microelements with CSF biomarkers of AD await elucidation of their environmental sources or detection of their release from brain tissue due to cell death. Also, these results should be further validated on larger cohorts.

Lead as an ubiquitous environmental pollutant of natural and anthropogenic origin enters a bear’s organism mostly via food and water. It is initially found in the bloodstream bound to red blood cells and reflects recent exposure. As a non-essential metal, lead causes adverse health effects on a developing nervous system (maternal transfer), renal, cardiovascular, haematological, immunological and reproductive system. Unlike in humans or domestic animals, there is no threshold for ursid blood Pb levels. Information about the lead exposure in this predator species is of high conservation interest. Our aim was to compare lead levels across captive and free-ranging brown bears from two European populations (Dinara-Pindos and Carpathian) sampled between 2011 and 2019 using the inductively coupled plasma mass spectrometry (ICP-MS) method. The median (range) level of lead in 32 bears from Croatia and 15 from Poland was 64.0 (5.08 ̶ 187 µg/L) and did not differ between countries. Levels were similar to Scandinavian and Canadian brown bears, three-times higher than in the Yellowstone black bears and three times lower than reported for giant pandas. Free-ranging bears had higher Pb (med 67.8 µg/L) compared to captive individuals (med 32.8 µg/L), although not significantly (t(47) = -1.94, p = 0.058). Blood lead also displayed gender differences (t(47) = -2.26, p = 0.028) with higher levels in female (med 80.6 µg/L) than in male (med 45.1 µg/L) bears. Eleven of the 47 individuals had blood levels over the threshold for an adult human (100 µg/L), but none surpassed the levels considered toxic for large domestic animals.

Fish is a highly valuable staple food and also a main source of mercury (Hg) exposure in the general population. Among trace elements contained in fish, selenium (Se) has potential to sequester Hg and reduce its toxicity. Our study aimed to determine concentrations of total Hg and Se, as well as their molar ratios in muscle tissue of commonly consumed marine fish species in Croatia to evaluate consumer health risk. Based on the EUROFISH survey (2017), six species of fresh and frozen (pilchard, hake), wild and farmed (gilthead seabream, seabass) and canned fish (pilchard, mackerel, tuna) were selected for analysis. Fresh wild specimens from the Adriatic Sea (n = 97) were purchased in fish market, and farmed fish (n = 24), frozen (n = 8) and canned (n = 37) products in supermarkets. Elements were analyzed by ICP-MS. Concentration (in mg/kg wet wt) ranges were 0.009–1.98 for Hg and 0.050–1.09 for Se, with highest Hg obtained in wild gilthead seabream and highest Se in canned tuna. Only in wild gilthead seabream, mean Hg exceeded the European regulatory limit (0.50 mg/kg). Element levels significantly differed between species. In all fish species was obtained mean Se:Hg molar ratio >1, which is considered safe. Given the common intake of two fish servings per week and mean Hg of 0.192 mg/kg, there is no risk of increased dietary Hg exposure except from wild gilthead seabream, which requires systematic Hg monitoring. This study was partially funded by the Croatian Science Foundation (grant HRZZ-IP-2016-06-1998).