The Relation Between Human Exposure to Mercury and Thyroid Hormone Status

The aim of this study was to investigate total mercury (THg) and methylmercury (MeHg) exposure of 75 mother-child pairs in relation to their thyroid hormone status (thyroid-stimulating hormone (TSH), triiodothyronine (T3), free triiodothyronine (fT3), thyroxine (T4), and free thyroxine (fT4)). THg and MeHg in blood samples were measured by atomic absorption spectrometry and gas chromatography-inductively coupled plasma-mass spectrometry, respectively. The median THg and MeHg levels in maternal blood, cord blood, and blood of 6-month-old children were 0.50, 0.53, and 0.32 and 0.22, 0.32, and 0.08 μg/L, respectively. There were significant correlations between paired maternal-cord blood levels for THg and MeHg, with a greater transplacental transport of MeHg compared with THg (mean cord/maternal blood ratio, 1.80 vs. 1.24). The maternal blood THg was found to be a better predictor of TSH levels in children than their current THg exposure. There was a positive correlation between maternal THg and children's TSH. T3 and fT3 levels in children were negatively related to cord blood THg in the majority (Caucasian) subgroup, whereas these associations were positive in the Roma subgroup. Mothers with dental amalgam fillings had significantly lower T4 and fT4 levels. Moreover, fT4 in the mothers of boys negatively correlated with maternal THg levels. MeHg exposure lowered T3 levels in the mothers of girls. Our results suggest that low-level exposure to Hg can affect thyroid hormone status during prenatal and early postnatal exposure depending on the form of Hg, gender, ethnicity, lifestyle, or socioeconomic status (dental amalgam fillings).     

The Tubifex tubifex assay for the determination of acute toxicity

An express (3-minute) test for acute toxicity determination by using the oligochaete annelid, Tubifex tubifex, is described. The EC50(Tubifex tubifex) [EC50(Tt)] for movement inhibition was calculated by using a concentration-response dependence. The reproducibility of the test was checked over several years and by several workers. Its applicability is limited to compounds which are soluble in water. The calculated EC50(Tt) indices correlate with LC50 values determined by using the fish, Pimephales promelas (96-hour assay), and with ICG50 values determined by using the ciliate, Tetrahymena pyriformis (48-hour assay) with high statistical significance (r = 0.822, n = 35, and r = 0.927, n = 80, respectively). The correlation between the EC50(Tt) indices and rat oral LD50 values (48-hour assay) was r = 0.519 (n = 67). The correlation within organic compounds was closer (r = 0.635, n = 60) than with the heterogeneous series of chemicals. A similar trend was noticed for the correlation with mouse oral LD50 values (r = 0.479, n = 56) with the heterogeneous series of chemicals, as compared that with the series without inorganic salts (r = 0.605, n = 42), and similarly with mouse intraperitoneal LD50 values, where r = 0.543 (n = 50) with the heterogeneous series of chemicals and r = 0.893 (n = 33) with the series of organic chemicals.     

Rhythmic clock gene expression in heart, kidney and some brain nuclei involved in blood pressure control in hypertensive TGR(mREN-2)27 rats

Hypertensive TGR(mREN-2)27 rats exerting inverted blood pressure (BP) profile were used to study clock gene expression in structures responsible for BP control. TGR and control Sprague Dawley male rats were synchronized to the light:dark cycle 12:12 with food and water ad libitum. Daily rhythm in per2, bmal1, clock and dbp expression in the suprachiasmatic nucleus (SCN), rostral ventrolateral medulla (RVLM), nucleus of the solitary tract (NTS), heart and kidney was determined in both groups. Sampling occurred in regular 4 h intervals when rats of both strains were 11-weeks-old. Blood pressure and relative heart weight were significantly elevated in TGR rats in comparison with control. Expression of bmal1 and clock was up regulated in SCN of TGR rats but daily rhythm in per2 and dbp expression was similar in both groups. Mesor of per2 expression in RVLM was significantly higher in TGR than in control rats. In NTS of TGR rats expression of per2 was phase delayed by 3.5 h in comparison with control and bmal1 did not exert rhythmic pattern. Our study provided the first evidence about modified function of central and peripheral circadian oscillators in TGR rats at the level of clock gene expression. Expression of clock genes exerted up regulation in SCN and RVLM and down regulation in NTS. Circadian oscillators in selected brain structures were influenced more than oscillators in the heart and kidney by additional renin gene. Interactions of RAS and circadian system probably contribute to the development of inverted BP profile in TGR rats.     

Enantiospecific Effects of Ketoconazole on Aryl Hydrocarbon Receptor

Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(−)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5–20× higher agonist activity (efficacy), as compared to (−)-KET; both enantiomers were AhR antagonists with equal potency (IC₅₀). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (−)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (−)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (−)-KET are weak ligands of AhR that displaced [³H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.     

Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.     

A duplication dup(4)(q28q35.2) de novo in a newborn

We report here a case of a newborn with hypotrophy and somatic stigmatization: microcephaly, facial dysmorphism, heart defect and immunodeficiency syndrome. The proband's karyotype was 46,XY,dup(4)(q28q35.2) de novo with chromosomal breaks in 4% of metaphases. We demonstrate the usefulness of a combination of physical examination, classical cytogenetics, FISH and PCR techniques in order to establish correct diagnosis because of overlap of some clinical and cytogenetic features of Nijmegen breakage syndrome (NBS) and duplication 4q in our patient. Although FISH technique detected translocation t(14q;21q) in 4 metaphases, deletion 657del5 in exon 6 of the NBS1 gene associated with NBS in Slavic population was not confirmed. We compare in this report similarity of the clinical picture of our patient, NBS cases and other patients carrying a duplication of the distal part of 4q as described in the literature.     

Extract EGb 761 Pretreatment Limits Ubiquitin Positive Aggregates in Rabbit Spinal Cord Neurons after Ischemia-Reperfusion

1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone.2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia.3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin–positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells.4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia.5. Evaluated ubiquitin–positive neurons of the L₅–L₆ segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord.     

Polymorphisms in FTO and near TMEM18 associate with type 2 diabetes and predispose to younger age at diagnosis of diabetes

Variations in the FTO gene and near the TMEM18 gene are risk factors for common form of obesity, but have also been linked with type 2 diabetes (T2D). Our aim was to investigate the contribution of these variants to risk of T2D in a population in Latvia. Four single nucleotide polymorphisms (SNP) in the first and fourth intronic regions of FTO and one close to TMEM18 were genotyped in 987 patients with T2D and 1080 controls selected from the Latvian Genome Data Base (LGDB). We confirmed association of SNPs in the first intron (rs11642015, rs62048402 and rs9939609) of FTO and rs7561317 representing the TMEM18 locus with T2D. Association between SNP in FTO and T2D remained significant after correction for body mass index (BMI). The rs57103849 located in the fourth intron of FTO and rs7561317 in TMEM18 showed BMI independent association with younger age at diagnosis of T2D. Our results add to the evidence that BMI related variants in and near FTO and TMEM18 may increase the risk for T2D not only through secondary effects of obesity. The influence of variants in the fourth intron of the FTO gene on development of T2D may be mediated by mechanisms other than those manifested by SNPs in the first intron of the same gene.     

Ixodid tick salivary gland extracts suppress human transforming growth factor-β1 triggered signalling pathways in cervical carcinoma cells

Ticks, very successful vectors and reservoirs of diverse viruses and other infectious agents, are able to counteract host defence mechanisms and wound healing processes to facilitate blood-feeding. Cutting the epidermis by the tick chelicerae and penetration of the hypostome into the dermis followed by feeding elicit a wound healing response, involving coagulation, inflammation, angiogenesis, synthesis of extracellular matrix and ground substances, as well as tissue remodelling. These repair processes of injured skin are coordinated by a wide array of cytokines, chemokines and growth factors. Tick salivary gland compounds, in addition to their vital anticoagulant and immunomodulatory functions, also possess significant anti-tumour properties and represent very promising sources of novel pharmaceuticals. Among life-threatening cancers, cervical carcinoma is still a high risk. Human transforming growth factor-beta 1 (huTGF-β1) inhibitors are significant in anti-cancer therapy. In this study, we investigated effects of salivary gland extracts (SGE) of females of two ixodid ticks, Dermacentor reticulatus (DR SGE) and Hyalomma anatolicum excavatum (HAE SGE), on signalling pathways triggered by huTGF-β1 in two cervical carcinoma cell lines, SiHa and HeLa, varying in their response to huTGF-β1. By dual luciferase reporter assay we monitored activation/inhibition of canonical SMAD or non-canonical signalling pathways in cells treated with huTGF-β1 and DR SGE/HAE SGE. Using specific monoclonal antibodies we tested effects of SGE on phosphorylation of ERK1/2 and/or AKT-1 molecules. We were the first to detect significant inhibition of huTGF-β1 induced signalling pathways, canonical as well as non-canonical, by DR and HAE SGE. Although the overall effects of both SGE on signalling pathways were similar, we revealed different patterns of effects of the two SGE on phosphorylation of the monitored signal transducers. Further investigations in this field are required.