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Živo srebro (Hg) in večina njegovih spojin je za naše telo toksična. Organsko živo srebro, kot je metil Hg (MeHg, CH3Hg+), in hlapi elementnega Hg (Hg0), so nevarni predvsem zaradi svoje nevrotoksičnosti, anorganske soli so nevarne zaradi kopičenja v ledvicah (nefrotoksičnosti). MeHg in hlapi Hg0 lahko prehajajo v placento in se po absorpciji lahko kopičijo v možganih zarodka. Apolipoprotein E (ApoE) je plazemski glikoprotein, ki ga uvrščamo med apolipoproteine. ApoE lahko veže toksične in endogene kovinske katione, predvsem tiste dvovalentne, med katere sodi tudi živo srebro. ApoE obstaja v treh izoformah (ApoE2, ApoE3, ApoE4), ki se med seboj razlikujejo po aminokislinskih ostankih na mestih 112 (c.334T>C rs429358) oz. 158 (c.472C>T rs7412). Genotipizacija ApoE predstavlja relativno neinvazivno, poceni in enostavno metodo, ki bi jo lahko uporabljali za odkrivanje posameznikov s povečanim tveganjem za negativne učinke Hg. Namen naše naloge je bil oceniti povezanost polimorfizmov ApoE s koncentracijami živega srebra pri materah in novorojencih v različnih bioloških vzorcih in ugotoviti, ali predstavljajo posamezniki z genotipi 4/4 in 3/4 bolj ogroženo skupino za pojav neželjenih učinkov pri kronični izpostavljenosti nizkim koncentracijam živega srebra v okolju. V analizo smo vključili 385 vzorcev DNA, odvzetih s hrvaške obobalne populacije (209 vzorcev mater, 176 vzorcev novorojencev) ter 415 vzorcev DNA slovenske populacije iz centralne regije (15 vzorcev mater, 400 vzorcev novorojencev), predhodno vključenih v evropski projekt PHIME (2006–2011). Na vzorcih DNA iz materine periferne krvi in novorojenčevega popkovnega tkiva smo opravili genotipizacijo ApoE z metodo verižne reakcije s polimerazo v realnem času z uporabo alelno specifičnih sond. Koncentracije elementov Hg, MeHg, Se, Cd, Pb, Mn, Ca, Mg, Fe, Zn, Cu, As smo vzeli iz obstoječe baze podatkov, ki so jo na Institutu Jožef Stefan pripravili v okviru PHIME projekta. Statistično analizo smo opravili s programom za prediktivno analizo SPSS. Potrdili smo, da so koncentracije živega srebra v različnih vzorcih višje pri hrvaški populaciji kot pri slovenski (predvsem celokupno Hg in MeHg v laseh mater ter nekoliko manj celokupno Hg in Se v popkovni krvi). Koncentracije celokupnega Hg in MeHg pri materah so močno korelirale s koncentracijami le-teh pri njihovih novorojencih. Ugotovili smo, da genotip mater vpliva na koncentracijo MeHg (p =0.081) in Hg2+ (p =0.007) v njeni krvi. Prisotnost alela ε4 pri materah je bila povezana z bistveno povišanim THg v materinih laseh (g. sredina 486 proti 730 ng/g p =0.028), zmerno povišanim THg v materini periferni krvi (g. sredina 2.1 proti 2.6 ng/g p =0.094), s povišanim Hg2+ v mamini krvi (g. sredina 0.48 proti 1.19 ng/g p =0.006), in s povišanim THg v popkovni krvi (g. sredina 0.8 proti 1.1 ng/g p =0.082). Prisotnost alela ε4 pri otroku je bila povezana s povišanim THg v popkovni krvi (g. sredina 1.7 proti 2.8 ng/g p =0.034). Našli smo tudi pozitivno povezavo med koncentracijami živega srebra in selena ne glede na koncentracije Hg so imele matere s prisotnim alelom ε4 višje koncentracije selena v periferni krvi in plazmi (g. sredina 88.5 proti 97.1 ng/g p =0.029 in 53.9 ng/g proti 61.8 ng/g p =0.000). Zaključimo lahko, da je pri materah in novorojencih s prisotnim alelom ε4 lahko povečana verjetnost za pojav neželenih učinkov živega srebra pri izpostavljenosti visokim koncentracijam Hg, pri izpostavljenosti nizkim pa so ti zaščiteni s povišanimi vrednostmi selena. Ob prisotnosti genotipa 4/4 oz. 3/4 velikokrat najdemo povišane koncentracije živega srebra in selena. Za potrditev teh opažanj bi v prihodnje morali povečati število vzorcev, saj je prisotnost alela ε4 redka, hkrati pa vključiti populacije, pri katerih je prehranjevanje z ribami pogostejše in izpostavljenost živemu srebru večja (Italijani in predvsem Grki). Mercury (Hg) and most of its compounds are toxic for our body. Organic mercury, such as methyl Hg (MeHg, CH3Hg+) and vapour of elemental Hg (Hg0) are dangerous mostly because of their neurotoxcity and anorganic salts, resulting in possible accumolation in kidneys (nephrotoxicity). MeHg and vapour of Hg0 can also transmit into the placenta, and after absorbtion, it can accumulate in the brains of fetous. Apolipoprotein E (ApoE) is a plasma glycoprotein, which we classify among apolipoproteins. ApoE can bound toxic and endogenous metal cations, especially bivalent, such as Hg. ApoE is present in three isoforms (ApoE2, ApoE3, ApoE4), which differ from one another by amino acid residues in two locations: 112 (c.334T>C rs429358) and 158 (c.472C>T rs7412). Genotypisation of ApoE is a non-invasive, inexpensive, simple method, which could be used for identifying individuals with greater risks for negative effects of Hg. The purpose of our work was to establish the connection between polymorphisms in ApoE with concentrations of mercury in mothers and newborns, in different biological samples, and determine whether individuals with genotypes 4/4 and 3/4 are more at risk for negative effects when exposed to low, but chronic concentrations of mercury from environment. In our analysis we included 385 samples of DNA from coastal part of Croatia (209 samples of mothers, 176 samples of newborns) and 415 samples from central Slovenia (15 samples of mothers, 400 samples of newborns) which were previously included in European project PHIME (2006-2011). On DNA samples, from maternal peripheral blood and newborn’s cord tissue, we performed the ApoE genotypisation with polymerase chain reaction method in real time, with allel-specific probes. Concentrations of elements Hg, MeHg, Se, Cd, Pb, Mn, Ca, Mg, Fe, Zn, Cu, and As were took from an existing database, which was made on Jožef Stefan Institute, during the PHIME project. Statistical analysis was made with SPSS predictive analytics software. We confirmed that concentrations of mercury are higher in Croatian population, than in Slovenian, in different biological samples (especially total Hg and MeHg in hair, and total Hg and Se in cord blood). Concentrations of total Hg (THg) and MeHg in mothers are strongly correlated with concentrations of total Hg and MeHg in their newborns. We established that mother’s genotype has an impact on concentrations of MeHg (p =0.081) and Hg2+ (p =0.007) in their blood. The presence of allel ε4 in mothers is correlated with much higher THg in maternal hair (g. mean 486 vs 730 ng/g p =0.028), higher concentrations of THg in their blood (g. mean 2.1 vs 2.6 ng/g p =0.094), higher concentrations of Hg2+ in their blood (g.mean 0.48 vs 1.19 ng/g p =0.006), and higher concentrations of THg in cord blood (g. mean 0.48 vs 1.19 ng/g p =0.006). Presence of allel ε4 in children is correlated with higher THg in cord blood (g. mean 1.7 vs 2.8 ng/g p =0.034). We also found the positive connection between concentrations of mercury and selenium mothers with allel ε4 have higher concentrations of Se in blood and in plasma (g. mean 88.5 vs 97.1 ng/g p =0.029 and 53.9 vs 61.8 ng/g p =0.000). We can conclude that both mothers and newborns with allel ε4 have a greater risk for adverse effect of mercury when its concentrations are high in relatively low concentrations those individuals are protected with higher concentrations of selenium. We found higher concentrations of mercury and selenium in individuals with genotype 4/4 and 3/4. To confirm these findings in the future, we should increase the number of samples, as the presence of allel ε4 is rare, and also include the populations which are eatign fish more frequently and are exposed to higher concentraions of mercury, such as Italian and especially Greek population. |
