Effects of Maternal Cigarette Smoking on Trace Element Levels and Steroidogenesis in the Maternal-Placental-Fetal Unit.

This study evaluates the interaction of toxic elements cadmium (Cd) and lead (Pb) due to exposure from cigarette smoking, essential elements, and steroidogenesis in the maternal-placental-fetal unit. In a cohort of 155 healthy, postpartum women with vaginal term deliveries in clinical hospitals in Zagreb, Croatia, samples of maternal blood/serum and urine, placental tissue, and umbilical cord blood/serum were collected at childbirth. The biomarkers determined were concentrations of Cd, Pb, iron (Fe), zinc (Zn), copper (Cu), and selenium (Se), and steroid hormones progesterone and estradiol in maternal and umbilical cord blood and the placenta. Three study groups were designated based on self-reported data on cigarette smoking habits and confirmed by urine cotinine levels: never smokers (n = 71), former smokers (n = 48), and active smokers (n = 36). Metal(loid)s, steroid hormones, urine cotinine, and creatinine levels were analyzed by ICP-MS, ELISA, GC-MS, and spectrophotometry. Cigarette smoking during pregnancy was associated with increased Cd levels in maternal, placental, and fetal compartments, Pb in the placenta, and with decreased Fe in the placenta. In active smokers, decreased progesterone and estradiol concentrations in cord blood serum were found, while sex steroid hormones did not change in either maternal serum or placenta. This study provides further evidence regarding toxic and essential metal(loid) interactions during prenatal life, and new data on sex steroid disruption in cord serum related to cigarette smoking. The results indicate that umbilical cord sex steroid levels may be a putative early marker of developmental origins of the future burden of disease related to harmful prenatal exposure to cigarette smoke.

Stress and reproductive hormones in hair associated with contaminant metal(loid)s of European brown bear (Ursus arctos).

Environmental contaminants like arsenic (As), cadmium (Cd), mercury (Hg) or lead (Pb) may disrupt hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes due to their endocrine toxicity potential. Resulting long-term physiological stress or adverse effects on wildlife reproduction and ontogeny may cause detrimental effects at the individual and population levels. However, data on environmental metal(loid)s' impact on reproductive and stress hormones in wildlife, especially large terrestrial carnivores, are scarce. Hair cortisol, progesterone and testosterone concentrations were quantified and modelled with hair As, Cd, total Hg, Pb, biological, environmental and sampling factors to test for potential effects in free-ranging brown bears (Ursus arctos) from Croatia (N = 46) and Poland (N = 27). Testosterone in males (N = 48) and females (N = 25) showed positive associations with Hg and an interaction between Cd and Pb, but a negative association with interaction between age and Pb. Higher testosterone was found in hair during its growth phase compared to quiescent phase. Body condition index was negatively associated with hair cortisol and positively associated with hair progesterone. Year and conditions of sampling were important for cortisol variation, while maturity stage for progesterone variation (lower concentrations in cubs and yearlings compared to subadult and adult bears). These findings suggest that environmental levels of Cd, Hg and Pb might influence the HPG axis in brown bears. Hair was shown to be a reliable non-invasive sample for investigating hormonal fluctuations in wildlife while addressing individual and sampling specificities.

Spatio-temporal monitoring of mercury and other stable metal(loid)s and radionuclides in a Croatian terrestrial ecosystem around a natural gas treatment plant.

The natural gas industry bears a certain contamination risk to human and biota due to, among others, mercury, arsenic, and naturally occurring radioactive material content in gas. We tracked multiple stabile metal(loid)s and radionuclides within the natural gas treatment plant Molve, Croatia, ecosystem during the last decade through a comprehensive monitoring of soil, earthworms, moss, livestock (blood, milk, hair, urine, and feces from cows), and wildlife animals (brain, muscle, liver, and kidney of European hare and pheasant). The level of mercury and other stable metal(loid)s has shown temporal variation, but without an obvious trend. The found spatial differences in soil and earthworms were based on the differing soil characteristics of the sampled locations and exceeded the maximal allowable concentration of arsenic and zinc for agricultural soil. The status of essential copper, selenium, and zinc in cows, hares, and pheasants inclined towards deficiency. The measured stable metal(loid) levels in soil and animal samples were generally in the same range of values reported in earlier decades from the same area or non-polluted areas across Europe. The consumption of local cow and game products (e.g., milk, meat) can be considered safe for human health, although game offal is advised to be avoided as a food item due to the low risk of lead and cadmium's adverse effects. Although the activity concentrations of some radionuclides in moss were higher than reported for pristine areas, transfer from soil to moss was assessed as average (except for lead-210). Radiological risk to human and biota around the gas treatment plant Molve was estimated as negligible.

Long-term effects of melatonin and resveratrol on aging rats: A multi-biomarker approach.

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 µg/kg b.m.) that exceed usual moderate consumption in humans.

The effects of maternal cigarette smoking on cadmium and lead levels, miRNA expression and biochemical parameters across the feto-placental unit.

Several miRNAs have been previously identified to be associated with cigarette smoke and/or the toxic metals cadmium (Cd) and lead (Pb). The aim of this study was to investigate the associations of maternal cigarette smoking with cadmium (Cd) and lead (Pb) levels, candidate miRNA expression and biochemical parameters across the feto-placental unit. miRNAs were isolated according to protocols provided by manufacturer from 72 healthy postpartum women using Qiagens' kits based on phenol/guanidine samples lysis and silica-membrane purification of total RNA. Candidate miRNAs (miR-1537, miR-190b, miR-16, miR-21, and miR-146a) were quantified by real-time PCR. Biochemical parameters were analyzed in plasma samples by standardized and harmonized enzymatic methods using appropriate calibrators, while CRP was determined by immunoturbidimetric method. Concentration of Cd and Pb in whole blood and placenta samples were measured by inductively coupled plasma mass spectroscopy. Cd levels in smokers were higher in all of the analyzed compartments of the feto-placental unit, Pb in maternal blood and placenta than non-smokers. Smokers also had a higher expression of miR-16 in maternal and miR-146a in cord plasma, and lower expression of miR-21 in the placenta in comparison to non-smokers. Urate concentrations in the maternal plasma of smokers were lower than this value in non-smokers. The study has demonstrated that maternal smoking was associated with toxic metals (Cd and Pb) levels, urate concentration and alteration of miRNA expression. Given that the effects of maternal smoking on miRNA expression are inadequate, all compartments of the feto-placental unit should be analyzed to obtain a complete picture. This paper is the first to report on the results of expression of cellular and circulating miRNAs simultaneously in maternal and fetal compartments and in the placenta.

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs.

INTRODUCTION: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. MATERIALS AND METHODS: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. RESULTS: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (CT) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). CONCLUSION: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.

Difference in pesticides, trace metal(loid)s and drug residues between certified organic and conventional honeys from Croatia.

Quality and safety of food, including honey, is one of the leading priorities regarding residues of anthropogenic chemicals with proven adverse health effects. In total, 61 honey samples of known botanical origin were collected in period 2018-2019 from Croatian registered organic and conventional beekeepers (N = 16 organic and N = 45 conventional honey samples). Eleven trace metal(loid)s (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn; quantitated by ICP-MS), 24 antibiotics (LC-MS/MS, ELISA, microbiological inhibition test), six indicator PCBs (GC-MS/MS) and 121 pesticides (LC-MS/MS, GC-MS/MS) originating from environment and/or beekeeping practice were measured to assess possible differences in contaminant residues between organic and conventional honeys. All honey samples had contaminant residues below the legal maximum levels and are considered safe for consumers. However, 2/16 organic and 34/45 conventional honeys contained one or two synthetic acaricides (most frequently coumaphos), while other pesticides, antibiotics and PCBs were not quantified. Also, organic honey contained lower levels of coumaphos, amitraz and amitraz metabolite N-(2,4-dimethylphenyl) formamide than conventional honeys, on average. Higher levels of Cr (p = 0.006) were detected in organic compared to conventional chestnut honeys. This study pinpointed beehive disease control treatment as prominent pesticide residue source, which was to some extent reduced in organic honeys. Quantified pesticide and metal(loid) levels were within range or lower than reported in recent literature regarding honey of the same botanical origin.

Cigarette Smoking during Pregnancy: Effects on Antioxidant Enzymes, Metallothionein and Trace Elements in Mother-Newborn Pairs.

The effect of maternal smoking as a source of exposure to toxic metals Cd and Pb on superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, metallothionein (MT), Cd, Pb, Cu, Fe, Mn, Se and Zn concentrations were assessed in maternal and umbilical cord blood and placenta in 74 healthy mother-newborn pairs after term delivery. Sparse discriminant analysis (SDA) was used to identify elements with the strongest impact on the SOD, GPx and MT in the measured compartments, which was then quantified by multiple regression analysis. SOD activity was lower in maternal and cord plasma, and higher in the placenta of smokers compared to non-smokers, whereas GPx activity and MT concentration did not differ between the groups. Although active smoking during pregnancy contributed to higher maternal Cd and Pb concentrations, its contribution to the variability of SOD, GPx or MT after control for other elements identified by SDA was not significant. However, an impaired balance in the antioxidant defence observed in the conditions of relatively low-to-moderate exposure levels to Cd and Pb could contribute to an increased susceptibility of offspring to oxidative stress and risk of disease development later in life. Further study on a larger number of subjects will help to better understand complex interactions between exposure to toxic elements and oxidative stress related to maternal cigarette smoking.

Metallothionein 2A gene polymorphism and trace elements in mother-newborn pairs in the Croatian population.

The main source of exposure for all essential and toxic elements in the general population is diet. In smokers, the main route for cadmium (Cd) and lead (Pb) intake is the inhalation of tobacco smoke. Besides gender, age, nutrition, lifestyle, and physiological conditions such as pregnancy, specific genetic characteristics also influence individual element uptake. Metallothionein MT2 is a cysteine-rich low-weight protein found ubiquitously throughout the body. Specific gene polymorphism may influence MT2 expression and subsequent binding, transfer and organ accumulation of metals, though data on these influences are lacking, especially in human mother-newborn pairs. The objective of this study was to determine selected toxic (Cd, Pb, Hg) and essential (Fe, Zn, Cu, Se) elements in maternal blood, placenta, and cord blood (by ICP-MS), and MT2 levels in maternal serum (by ELISA) in relation to maternal MT2A -5A/G (rs28366003) polymorphism (by RFLP-PCR and electrophoresis). Study participants were healthy postpartum women in Croatia (n=268, mean age 29 years) with term vaginal childbirth in a maternity ward assigned into two study groups by self-reporting about their smoking habit (by questionnaire). Smokers vs. non-smokers had increased levels of Cd and Pb in all measured samples, Fe and Cu in cord blood, Zn in placenta, and MT2 in maternal serum. Among subjects with AG/GG genotype, placental Fe was significantly lower only among non-smokers, while MT2 levels in serum were lower, though not significantly, regardless of maternal smoking habit. There was no impact of MT2A -5A/G SNP on any element in maternal or cord blood. In conclusion, the results confirmed maternal smoking-related increases in Cd and Pb levels in the maternal-placental-foetal unit. They also provided additional data on concomitant metal concentrations in representative samples of maternal blood, placenta, and cord blood, as well as increased cord blood Fe and Cu, placental Zn, and maternal serum MT2 in smokers. New evidence is that MT2A -5A/G SNP was associated with decreased placental Fe levels in non-smokers. For a final conclusion on the influence of the MT2A -5A/G polymorphism on toxic and essential element levels in mother-newborn pairs, further research would require a larger number of participants divided across subgroups defined by the main source of particular toxic metal exposure (such as specific food intake, cigarette smoking, air pollution and/or occupational exposure).

Use of human milk in the assessment of toxic metal exposure and essential element status in breastfeeding women and their infants in coastal Croatia.

Pregnant and lactating women and infants are vulnerable population groups for adverse effects of toxic metals due to their high nutritional needs and the resultant increased gastrointestinal absorption of both, essential and toxic elements. Although breastfeeding is recommended for infants worldwide, as human milk is the best source of nutrients and other required bioactive factors, it is also a pathway of maternal excretion of toxic substances including toxic metals and thus a source of infant exposure. The aim of this research was to assess health risks in breastfeeding women in the coastal area of the Republic of Croatia and their infants (N=107) due to maternal exposure to Cd and Pb via cigarette smoking, and Hg via seafood and dental amalgam fillings, and their interaction with essential elements. Biological markers of exposure were the concentrations of main toxic metals Pb, Cd and Hg in maternal blood and three types of breast milk throughout lactation stages. Biological markers of effects were the levels of essential elements Ca, Fe, Cu, Zn and Se in maternal serum and breast milk. With regard to cigarette smoking as a source of exposure to Cd and Pb, there were effects of smoking on Cd concentration in blood and correlations between the smoking index and Cd concentrations in maternal blood (ρ=0.593; P<0.001) and mature milk (ρ=0.271; P=0.011) and Pb concentration in transitional milk (ρ=0.280; P=0.042). Regarding fish, we found correlations between weekly consumption frequency and total Hg concentrations in maternal blood (ρ=0.292; P=0.003) and mature milk (ρ=0.303; P=0.003). The number of dental amalgam fillings correlated with total Hg concentrations in colostrum (ρ=0.489; P=0.005) and transitional milk (ρ=0.309; P=0.018). As for the essential element status, only Se levels in maternal serum decreased by 10% in persons who continued smoking during pregnancy compared to non-smokers. In conclusion, the levels of main toxic metals Cd, Pb and Hg and essential elements Ca, Fe, Cu, Zn and Se in maternal blood and three types of breast milk samples in the studied area of coastal Croatia showed no risk of disrupted essential element levels with regard of toxic metal exposure in both breastfeeding women and their infants.

Selenium in brown bears (Ursus arctos) from Croatia: Relation to cadmium and mercury.

Muscle (n = 111), liver (n = 111), and kidney cortex (n = 101) samples from brown bears (Ursus arctos) were collected in the 2009 and 2010 hunting seasons in Croatia and analysed for selenium (Se), cadmium (Cd), and total mercury (Hg). The aim was to assess the levels of these elements according to age, sex, and season of collection, and to investigate possible Se/Cd and Se/Hg interactions. Median Se concentrations were 0.139 µg/g in muscle, 0.409 µg/g in liver and 1.75 µg/g wet mass in kidney cortex. Median Cd and Hg were 0.0078 and 0.0018 µg/g in muscle, 1.09 and 0.031 µg/g in liver, and 16.5 and 0.206 µg/g wet mass in the renal cortex, respectively. Se/Cd molar ratios were less than 1 in the kidney cortex, and close to or above 1 in liver and muscle, respectively. Toxic Cd and Hg correlated with Se in all of the studied tissues. Sex differences were found for all three elements (except Se in liver), with females having higher tissue concentration than males. Only Cd showed age-dependence. Bear samples collected in fall had higher Se in muscles, and Hg in muscles and liver compared to samples collected in spring. Element concentrations in brown bear tissues were within the range of previously reported studies. Bear meat is considered a rich source of Se, safe for consumption with regard to its Cd and Hg content. According to the molar ratio and correlation results, we assume that Se binding is not the primary detoxification pathway for Cd and Hg in brown bears.

Cadmium and lead in grey wolf liver samples: optimisation of a microwave-assisted digestion method.

A microwave-assisted digestion method for the determination of cadmium (Cd) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS) was optimised on certified reference material (CRM) (bovine liver, BCR-185R) and wolf liver samples. Different factors influencing digestion efficiency (temperature, time, composition of the digestion mixture, sample mass) were tested. Validation included linearity (up to 200 µg L(-1) for Cd and Pb), detection (0.003 µg L(-1) for Cd and 0.035 µg L(-1) for Pb), and quantification (0.008 µg L(-1) for Cd and 0.081 µg L(-1) for Pb) limits. Good agreement between measured and certified values was achieved in all conditions, with recoveries ranging from 94 % to 111 % for Cd and from 95 % to 105 % for Pb. The precision of the method, expressed as relative standard deviation, was up to 3 % for Cd and 8 % for Pb. The best digestion parameters (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0.1 g of CRM) based on accuracy and precision were applied on two wolf liver samples to evaluate the need for the predigestion step (freeze-drying) and appropriate mass of the sample. Freeze-drying improved precision and minimising the tissue mass to 0.1 g reduced the matrix effect. Using these optimised digestion conditions, we determined Cd and Pb in 40 wolf livers collected in Croatia, and their medians (0.055 µg g(-1) and 0.107 µg g(-1), respectively) were in the range of previously reported data for the grey wolf.

Arsenic in air and soil in the vicinity of the central gas station Molve, Croatia.

Spatial and temporal distribution of arsenic levels in soil during the 9 year monitoring period was investigated on four different soil types in the area of the gas borehole system Podravina in Croatia. Arsenic levels in the PM(10) particle fraction were measured periodically at the same locations for 3 years. Arsenic levels in soil significantly depended on soil types. Elevated levels were found on gleysol vertic, at two sampling sites, with values exceeding 30 mg/kg of arsenic in soil. Arsenic levels in air were low and they were not significantly different between sampling sites, suggesting that gas borehole activities have no influence on arsenic levels in the environment.

Effect of selenium pre-treatment on antioxidative enzymes and lipid peroxidation in Cd-exposed suckling rats.

Since there are no data about the protective role of selenium (Se) against cadmium (Cd)-induced oxidative damage in early life, we studied the effect of Se supplementation on antioxidative enzyme activity and lipid peroxidation (through thiobarbituric acid reactive substances; TBARS) in suckling Wistar rats exposed to Cd. Treated animals received either Se alone for 9 days (8 µmol, i.e., 0.6 mg Se as Na(2)SeO(3) kg(-1) b.w., daily, orally; Se group), Cd alone for 5 days (8 µmol, i.e., 0.9 mg Cd as CdCl(2) kg(-1) b.w., daily, orally; Cd group), or pre-treatment with Se for 4 days and then co-treatment with Cd for the following 5 days (Se + Cd group). Our results showed that selenium supplementation, with and without Cd, increased SOD activity in the brain and kidney, but not in the liver and GSH-Px activity across all tissues compared to control rats receiving distilled water. Relative to the Cd group, Se + Cd group had higher kidney and brain SOD and GSH-Px activity (but not the liver), while in the liver caused increased and in the brain decreased TBARS level. These results suggest that Se stimulates antioxidative enzymes in immature kidney and brain of Cd-exposed rats and could protect against oxidative damage.

The effect of dietary selenium supplementation on cadmium absorption and retention in suckling rats.

Selenium (Se) reduces cadmium (Cd) toxicity in adult animals, but its effects in newborn animals are still unknown. This study investigated Cd (as CdCl2) absorption, distribution, and retention in suckling rats receiving oral Se supplementation (as Na2SeO3) in equimolar doses (8 µmol Cd and/or Se per kg b.w./day). Selenium was given either before and during Cd exposure (Sepre + Cd group; pretreatment group) or only during Cd exposure (Se + Cd group). Rats were treated from postnatal day (PND) 6-14 as follows: controls (H2O, PND 6-14), Se (PND 10-14), Cd (PND 10-14), Sepre + Cd (Se PND 6-14 + Cd PND 10-14) and Se + Cd (Se + Cd PND 10-14). Selenium supplementation, especially pre-treatment, decreased Cd levels in the blood, brain, liver and kidney of suckling rats. Selenium levels in plasma, brain, and kidney also decreased. These findings suggest that higher Se intake could efficiently reduce Cd retention during the suckling period.

Influence of selenium dose on mercury distribution and retention in suckling rats.

It is well known that metal-metal interactions in the body are age-dependent. We studied the influence of increasing selenium (Se) doses on mercury (Hg) distribution and retention in the postnatal period in Hg-exposed suckling rats. Seven-day-old Wistar pups were pretreated with three different oral doses of Se as sodium selenite (6.45, 12.9 and 19.4 micromol Se kg(-1) b.w.) over 3 days. This was followed by simultaneous Se (as sodium selenite) and Hg (as mercury chloride) oral administration over 4 days. The molar ratio between Se and Hg given to pups was 1:1, 2:1 and 3:1, respectively. Mercury and Se were measured in brain, kidneys, liver, plasma, erythrocytes and urine of pups on the day after the last administration by atomic absorption spectrometry. Results showed that in all samples Se concentrations rose almost proportionally to the dose of Se given to pups. Mercury concentration in organs, plasma and urine decreased with higher oral doses of Se. However, Hg concentration in erythrocytes increased with increasing Se dose. There was evidently a redistribution of Hg from plasma to erythrocytes at higher ratio of Se:Hg. Approximately equimolar doses of Se and Hg are necessary to produce maximum uptake of Hg by plasma and liver and minimum retention of Hg in the kidney and erythrocytes.