Water Quality and Associated Human Health Risk Assessment Related to Some Ions and Trace Elements in a Series of Rural Roma Communities in Transylvania, Romania.

This research aims to assess the content of some ions and trace elements in water sources in 24 rural Roma communities in Transylvania in order to assess the human health risk associated with exposure to such elements and ions. To this end, eight ions (F-, Cl-, Br-, NO2-, NO3-, SO42-, PO43-, NH4+) and ten trace elements (Cr, Ni, As, Pb, Cd, Mn, Cu, Zn, Fe, and Hg) were determined in 71 water samples by ion chromatography coupled with a conductivity detector for ions and atomic absorption spectrophotometry for all trace elements. General parameters were also determined. Non-conformity (as number of samples), according to the EU Drinking Water Directive, was observed as follows: pH (7), EC (7), hardness (1), oxidizability (15), Cl- (4), NO3- (30), SO42- (6), Fe (16), Mn (14), As (3), and Ni (1 sample). The incidence of ions was Cl- (71), SO42- (70), F- (67), NO3- (65), NH4+ (21), Br- (10), PO43-, and NO2- (1 sample) and for trace elements, Mn (59), Fe (50), As (38), Ni (32), Cu (29), Zn (28), Cd (12), Cr (11), and Pb (3 samples). Hg was not detected. Non-carcinogenic (HI) values exceeded one for As in 13 Roma communities, with higher values for children than for adults. For NO3-, the HI values were >1 in 12 for adults and 14 communities for children. The carcinogenic risk (CR) for As through ingestion ranged from 0.795 to 3.50 × 10-4 for adults and from 1.215 to 5.30 × 10-4 for children. CR by dermal contact was in the range of ×10-6 both for adults and children.

Assessment of heavy metals in wild and farmed tilapia (Oreochromis niloticus) on Lake Kariba, Zambia: implications for human and fish health.

The aim of this study was to assess the levels of heavy metals in both wild and farmed tilapia on Lake Kariba in Zambia and to evaluate the impact of intensive fish farming on wild tilapia. Three sites for wild fish (2 distant and 1 proximal to fish farms) and two fish farms were selected. One hundred fish (52 from distant sites; 20 near fish farms; 28 farmed fish) were sampled and muscle tissues excised for analysis of heavy metals (Mg, Fe, Zn, Al, Cu, Se, Co, Mo, As, Cr, V, Ni, Hg, Pb, Li, Cd, and Ag) by acid (HNO3) digestion and ICP-MS. All metals were found to be below the maximum limits (MLs) set by WHO/EU. Essential metals were higher in farmed tilapia, whereas non-essential metals were higher in wild tilapia. Significantly higher levels of essential metals were found in wild fish near the fish farms than those distant from the farms. Estimated weekly intake (EWI) for all metals were less than the provisional tolerable weekly intakes (PTWI). Target hazard quotients (THQ) and Hazard Indices (HI) were <1, indicating no health risks from a lifetime of fish consumption. Selenium Health Benefit Value (HBVSe) was positive for all locations, indicating protective effects of selenium against mercury in fish. Total cancer risk (CR) due to As, Cr, Cd, Ni and Pb was less than 1 × 10-4, indicating less than 1 in 10,000 carcinogenic risk from a lifetime consumption of tilapia from Lake Kariba. Hg levels (0.021 mg/kg) in wild tilapia at site 1 were higher than the Environmental quality standard (EQS = 0.020 mg/kg) set by EU, indicating possible risk of adverse effects to fish. Except for Hg, levels of metals in fish were safe for human consumption and had no adverse effects on fish.

Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression.

Ionizing radiation from natural sources or anthropogenic activity has the potential to cause oxidative stress or genetic damage in living organisms, through the ionization and excitation of molecules and the subsequent production of free radicals and reactive oxygen species (ROS). The present work focuses on radiation-induced biological effects using the zebrafish (Danio rerio) vertebrate model. Changes in developmental traits and gene expression in zebrafish were assessed after continuous external gamma irradiation (0.4, 3.9, 15 and 38 mGy/h) with corresponding controls, starting at 2.5 hours post fertilization (hpf) and lasting through embryogenesis and the early larval stage. The lowest dose rate corresponded to recommended benchmarks at which adverse effects are not expected to occur in aquatic ecosystems (2-10 mGy/day). The survival observed at 96 hours post fertilization (hpf) in the 38 mGy/h group was significantly lower, while other groups showed no significant difference compared to controls. The total hatching was significantly lower from controls in the 15 mGy/h group and a delay in hatching onset in the 0.4 mGy/h group was observed. The deformity frequency was significantly increased by prolonged exposure duration at dose rates ≥ 0.4 mGy/h. Molecular responses analyzed by RNA-seq at gastrulation (5.5 hpf transcriptome) indicate that the radiation induced adverse effects occurred during the earliest stages of development. A dose-response relationship was found in the numbers of differentially regulated genes in exposure groups compared to controls at a total dose as low as 1.62 mGy. Ingenuity Pathway Analysis identified retinoic acid receptor activation, apoptosis, and glutathione mediated detoxification signaling as the most affected pathways in the lower dose rate (0.54 mGy/h), while eif2 and mTOR, i.e., involved in the modulation of angiogenesis, were most affected in higher dose rates (5.4 and 10.9 mGy/h). By comparing gene expression data, myc was found to be the most significant upstream regulator, followed by tp53, TNF, hnf4a, TGFb1 and cebpa, while crabp2b and vegfab were identified as most frequent downstream target genes. These genes are associated with various developmental processes. The present findings show that continuous gamma irradiation (≥ 0.54 mGy/h) during early gastrula causes gene expression changes that are linked to developmental defects in zebrafish embryos.

Steroidogenic differential effects in neonatal porcine Leydig cells exposed to persistent organic pollutants derived from cod liver oil.

Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects.

Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153.

Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic.

Transcriptional profiling in burbot (Lota lota) from Lake Mjøsa--a Norwegian Lake contaminated by several organic pollutants.

Lake Mjøsa is the largest freshwater repository in Norway, receiving runoff from a wide surrounding region of urban country. As a result of industrial activity, large quantities of persistent organic pollutants (POPs) have been discharged into Lake Mjøsa during the last century. The levels of PCBs, DDTs and PBDEs in burbot from Lake Mjøsa (study population) exceed the corresponding levels in burbot from Lake Losna (reference) by a factor of 3, 6 and 113, respectively. We used shotgun and suppression subtraction hybridization (SSH) cDNA libraries followed by 454 FLX sequencing (957 303 reads sequenced in total) and RT-qPCR to study the effects of POPs in burbot from Lake Mjøsa. The gene list of putatively higher or lower expressed genes in liver of burbot from Lake Mjøsa compared to liver of burbot from Lake Losna, generated from the SSH cDNA libraries, suggest that mechanisms associated with drug metabolism and oxidative stress are enriched in burbot from Lake Mjøsa (Ingenuity Pathway Analysis (IPA) top networks). According to the IPA analyses, the top toxicity list ranking was "LXR/RXR activation", "Negative/Positive acute phase response proteins", "LPS/IL-1-mediated inhibition of RXR function" and "FXR/RXR activation". Functional analyses further identified PPAR, HNF4A, dexamethasone and ß-estradiol as potential upstream key regulator factors. Overall, the study shows that SSH cDNA libraries coupled to next-generation sequencing (RNA-Seq) may be a valuable supplement or alternative to microarray technology in toxicogenomic discovery of environmental samples.

High prevalence of infections and pathological changes in burbot (Lota lota) from a polluted lake (Lake Mjøsa, Norway).

The aim of the present study was to investigate whether exposure to high levels of persistent organic pollutants (POPs) affected a fish population in Lake Mjøsa. Lake Mjøsa is known to be contaminated by polybrominated diphenyl ethers (PBDEs), a subgroup of brominated flame retardants from local industrial activities. Fish from Lake Losna, a less contaminated lake located close to Lake Mjøsa, was used as reference (control). The sampling of burbot (Lota lota) was carried out between 2005 and 2008. Hepatic levels of POPs were analysed in burbot from the two lakes, and the fish were examined for bacterial- and parasite infection and histopathological changes. The levels of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), and PBDEs were about 10, 15 and 300 times higher in fish from Lake Mjøsa compared to fish from Lake Losna. Mycobacterium salmoniphilum was present in 7% and 35% of the fish from Lake Losna and Lake Mjøsa respectively. Significantly higher number of external and visceral macroscopic lesions, histopathological diffuse changes and granulomas were seen in fish from Lake Mjøsa compared to Lake Losna. Furthermore the parasite infection was higher and the hepatic lipid content was significantly lower in burbot from Lake Mjøsa. The results of the present study suggest that the high level of contamination in Lake Mjøsa could have a negative impact on the health status of wild fish inhabiting the lake.

Parental exposure to natural mixtures of POPs reduced embryo production and altered gene transcription in zebrafish embryos.

Determination of toxicity of complex mixtures has been proposed to be one of the most important challenges for modern toxicology. In this study we performed genome wide transcriptome profiling to assess potential toxicant induced changes in gene regulation in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POPs). The mixtures used were extracted from burbot (Lota lota) liver originating from two lakes (Lake Mjøsa and Lake Losna) belonging to the same freshwater system in Norway. The dominating groups of contaminants were polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane metabolites (DDTs). Because both mixtures used in the present study induced similar effects, it is likely that the same toxicants are involved. The Mjøsa mixture contains high levels of PBDEs while this group of pollutants is low in the Losna mixture. However, both mixtures contain substantial concentrations of PCB and DDT suggesting these contaminants as the predominant contributors to the toxicity observed. The observed effects included phenotypic traits, like embryo production and survival, and gene transcription changes corresponding with disease and biological functions such as cancer, reproductive system disease, cardiovascular disease, lipid and protein metabolism, small molecule biochemistry and cell cycle. The changes in gene transcription included genes regulated by HNF4A, insulin, LH, FSH and NF-κB which are known to be central regulators of endocrine signaling, metabolism, metabolic homeostasis, immune functions, cancer development and reproduction. The results suggest that relative low concentrations of the natural mixtures of POPs used in the present study might pose a threat to wild freshwater fish living in the lakes from which the POPs mixtures originated.

In utero and lactational exposure to PCB 118 and PCB 153 alter ovarian follicular dynamics and GnRH-induced luteinizing hormone secretion in female lambs.

The effects of in utero and lactational exposure to two structurally different polychlorinated biphenyl (PCB) congeners on follicular dynamics and the pituitary-gonadal axis in female lambs were investigated. Pregnant ewes received corn oil, PCB 118, or PCB 153, and offspring was maintained until 60 days postpartum. Ovarian follicles were quantified using stereology. Plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured using radioimmunoassay before and after administration of a gonadotropin releasing hormone (GnRH) analog. PCB 118 exposure increased numbers of transitional, secondary, and the sum of secondary, early antral, and antral (Σsecondary-antral) follicles, PCB 153 exposure only increased the number of primary follicles. GnRH-induced LH levels were significantly elevated in the PCB 153 exposure group. We conclude that PCB 153 and PCB 118 alter follicular dynamics in lambs and modulate the responsiveness of the pituitary gland to GnRH.

Mycobacterium salmoniphilum infection in burbot Lota lota.

Burbot Lota lota sampled from lakes Mjosa and Losna in southeastern Norway between 2005 and 2008 were found to be infected with Mycobacterium salmoniphilum at a culture-positive prevalence of 18.6 and 3.3%, respectively. The condition factor (CF) of mycobacteria-affected fish sampled from Mjøsa in 2008 was lower than the average CF of total sampled fish the same year. Externally visible pathological changes included skin ulceration, petechiae, exopthalmia and cataract. Internally, the infections were associated with capsulated, centrally necrotic granulomas, containing large numbers of acid-fast bacilli, found mainly in the mesenteries, spleen, heart and swim bladder. Mycobacterial isolates recovered on Middlebrook 7H10 agar were confirmed as M. salmoniphilum by phenotypical investigation and by partial sequencing of the 16S rRNA, rpoB and Hsp65genes as well as the internal transcribed spacer (ITS1) locus. This study adds burbot to the list of fish species susceptible to piscine mycobacteriosis and describes M. salmoniphilum infection in a non-salmonid fish for the first time.

Natural mixtures of POPs affected body weight gain and induced transcription of genes involved in weight regulation and insulin signaling.

Obesity is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes, cardiovascular disease, hypertension and dyslipidemias (metabolic syndrome). Commonly held causes of obesity are overeating coupled with a sedentary lifestyle. However, it has also been postulated that exposure to endocrine disrupting chemicals (EDCs) may be related to the significant increase in the prevalence of obesity and associated diseases. In the present study, developmental and reproductive effects of lifelong exposure to environmentally relevant concentrations of two natural mixtures of persistent organic pollutants (POPs) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway (Lake Mjøsa and Lake Losna). The concentration of POPs in the zebrafish ranged from levels detected in wild fish (Lake Mjøsa and Lake Losna), to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) earlier onset of puberty, (2) elevated male/female sex ratio, and (3) increased body weight at 5 months of age. Interestingly, genome-wide transcription profiling identified functional networks of genes, in which key regulators of weight homeostasis (PPARs, glucocoricoids, CEBPs, estradiol), steroid hormone functions (glucocoricoids, estradiol, NCOA3) and insulin signaling (HNF4A, CEBPs, PPARG) occupied central positions. The increased weight and the regulation of genes associated with weight homeostasis and insulin signaling observed in the present study suggest that environmental pollution may affect the endocrine regulation of the metabolism, possibly leading to increased weight gain and obesity.

Three structurally different polychlorinated biphenyl congeners (Pcb 118, 153, and 126) affect hormone production and gene expression in the human H295R in vitro model.

Polychlorinated biphenyls (PCB) are ubiquitous environmental pollutants that have been linked to adverse health effects including endocrine disruption. This study compared the mono-ortho-substituted PCB 118 and di-ortho-substituted PCB 153 with the non-ortho-substituted PCB 126, for possible effects on steroid hormone production and on the expression of 10 genes encoding proteins involved in steroidogenesis. The H295R human adenocarcinoma cell line was used as an in vitro model. Cells were exposed for 48 h to solvent control (dimethyl sulfoxide, DMSO) or 6 different concentrations ranging from 40 pM to 4 muM of one of the three test compounds. All three congeners significantly increased the production of estradiol-17beta. PCB 118 produced a rise in progesterone and cortisol in a concentration-dependent manner, similar to PCB 126. Testosterone was significantly reduced in response to PCB 153 but not PCB 118 or PCB 126. All three congeners elevated aldosterone at the highest concentration tested. A significant increase was observed in CYP11B2 mRNA levels in cells exposed to the three congeners. In addition, PCB 126 upregulated CYP19, 3beta-HSD2, StAR, and HMGR mRNA levels at the highest concentration tested, and downregulated CYP21 at 40 nM. In conclusion, all three PCB congeners are capable of modulating steroidogenesis in H295R in a concentration-dependent manner, whereby the hormone profile following PCB 118 exposure resembles that of PCB 126. Where changes in gene expression profile are concerned, exposure to PCB 126 showed the greatest effects.

Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs).

This study investigated in vitro endocrine disrupting effects of three mixtures of POPs: 'Marine mix' extracted from Atlantic cod liver, and two mixtures extracted from burbot liver, 'Mjøsa mix' and 'Losna mix'. The POP mixtures were chemically characterized. Co-culture of theca and granulosa cells, were exposed for 48h with different doses of 'Marine mix', 'Mjøsa mix' or 'Losna mix'. As an end point cell viability was determinated by LDH test, steroid analysis by EIA and caspase-3 by colorimetric substrate. Chemical characterization of the mixtures demonstrated that the 'Marine mix' contained high levels of DDTs and PCBs. In the 'Mjøsa mix', the dominant pollutants were BDEs and HBCD. The concentrations of POPs measured in the 'Losna mix' were considerably lower. All mixtures used in the present study had a stimulatory effect on testosterone and estradiol secretion with 'Marine mix'>'Mjøsa mix'>'Losna mix'. These results show that even a mixture containing background concentrations of POPs significantly affected steroid secretion. A higher steroidogenic response in low dose ranges, compared with high dose ranges indicated xenobiotic-conditioning hormesis. This could complicate predictions of effects in risk assessments.