Connection between mutations in the gene for apolipoprotein E with concentrations of mercury in the mothers and newborns

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>Crs429358) oz. 158 (c.472C>Trs7412). 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/gp =0.028), zmerno povišanim THg v materini periferni krvi (g. sredina 2.1 proti 2.6 ng/gp =0.094), s povišanim Hg2+ v mamini krvi (g. sredina 0.48 proti 1.19 ng/gp =0.006), in s povišanim THg v popkovni krvi (g. sredina 0.8 proti 1.1 ng/gp =0.082). Prisotnost alela ε4 pri otroku je bila povezana s povišanim THg v popkovni krvi (g. sredina 1.7 proti 2.8 ng/gp =0.034). Našli smo tudi pozitivno povezavo med koncentracijami živega srebra in selenane 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/gp =0.029in 53.9 ng/g proti 61.8 ng/gp =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>Crs429358) and 158 (c.472C>Trs7412). 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/gp =0.028), higher concentrations of THg in their blood (g. mean 2.1 vs 2.6 ng/gp =0.094), higher concentrations of Hg2+ in their blood (g.mean 0.48 vs 1.19 ng/gp =0.006), and higher concentrations of THg in cord blood (g. mean 0.48 vs 1.19 ng/gp =0.006). Presence of allel ε4 in children is correlated with higher THg in cord blood (g. mean 1.7 vs 2.8 ng/gp =0.034). We also found the positive connection between concentrations of mercury and seleniummothers with allel ε4 have higher concentrations of Se in blood and in plasma (g. mean 88.5 vs 97.1 ng/gp =0.029 and 53.9 vs 61.8 ng/gp =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 highin 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

Mercury speciation in meconium and associated factors

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

Mercury speciation in prenatal exposure in Slovenian and Croatian population - PHIME study

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

Trace elements and APOE polymorphisms in pregnant women and their new-borns

We investigated the relationship between lipid binding glycoprotein apolipoprotein E (apoE; gene APOE) polymorphisms (ε4 allele carriers versus no carriers = ε4+/ε4-) and trace elements (TEs) (e.g., (methyl)mercury, arsenic, lead, cadmium, selenium, manganese, copper, and zinc) in mothers (N = 223) and their new-borns (N = 213) exposed to potentially toxic metal(loid)s from seafood consumption. The apoE isoform encoded by the ε4 allele is believed to have beneficial effects in early life but represents a risk factor for age-associated diseases. Under certain conditions ε4 carriers are more susceptible to oxidative stress and metal(loid) toxicity. DNA from Croatian pregnant women (N = 223, third trimester) and their new-borns (N = 176), was genotyped for APOE by TaqMan® SNP assay - rs429358 and rs7412. Seafood intake data and TE levels in maternal urine, milk, hair, peripheral venous blood, mixed cord blood, and new-borns' urine were available from previous studies. We compared TEs between ε4+ and ε4- carriers using Mann-Whitney U tests and applied multiple linear regression models to analyse the TE's dependence on the presence of allele ε4 (genotypes ε3/ε4, ε4/ε4) in combination with other explanatory variables. We identified 17% (n = 37) and 20% (n = 35) ε4 allele carriers in mothers and new-borns, respectively. The Mann-Whitney U test showed that mothers with the ε4 allele had significantly higher mean levels of (methyl)mercury in peripheral venous blood, cord blood, and hair; arsenic in urine and cord blood; and selenium in peripheral venous blood and plasma. However, taking confounders into account, only the maternal plasma selenium remained statistically significant in the linear regression models (ε4 carriers vs non-carriers: 62.6 vs 54.9 ng/mL, p < 0.001). Literature suggestions of possible ε4 allele impact on Hg levels were not observed, while superior selenium status observed in healthy pregnant women carrying allele ε4 could be linked to the proposed APOE ε4 beneficial effects early in life

Mercury speciation in prenatal exposure in Slovenian and Croatian population - PHIME study

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 regionN = 584) and Croatia (coastal regionN = 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

Prenatal mercury exposure, neurodevelopment and apolipoprotein E genetic polymorphism

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 BayleyIII. The Hg-related decrease in cognitive score was observed only in children carrying atleastone 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