Non-invasive vessel examinations in carriers of LDL-receptor defective gene versus non-carriers with newly detected asymptomatic severe hypercholesterolemia.

UNLABELLED: The results of the research of early vascular alterations in LDL-R carriers in comparison with those in non-carriers with severe hypercholesterolemia are controversial. AIM: To investigate the difference between severe hypercholesterolemia patients that carry LDL-R defective gene and those that do not have it, in their functional (flow-mediated vasodilation) and structural (intima-media thickness of carotid artery and ankle-brachial index) characteristics of arterial wall. PATIENTS AND METHODS: The study included 120 hypercholesterolemic patients. Biochemistry parameters were studied by routine methods. The flow-mediated vasodilation (%FMD), ankle-brachial index (ABI) and intima-media thickness (IMT) of common carotid artery were determined using Hewlett Packard Sonos 5 500; MedicaSoft. IMT.lab was the software programme used in the study. RESULTS: There was no significant difference between the groups with respect to total cholesterol, LDL, HDL, Apo-B, Apo-A1, cellular adhesion molecules (sICAM-1, sVCAM-1, sP- and sE-selectine). The Apo-B/Apo A1 index differed significantly (t = 11.23, p < 0.001) between the two groups; there was difference even after adjustment for age. There was no significant difference in the endothelial dependent and independent vasodilatation between the examined groups (p > 0.05). We found a significantly greater carotid IMT and lower ABI in the carriers than the respective parameters in the non-carriers. This significant difference was confirmed after adjustment for age. CONCLUSION: Our data show that LDL-R carriers have a higher carotid IMT and lower ABI than non-carriers, whereas no difference between the groups was found with respect to the level of lipid parameters and %FMD.

No evidence for glutathione S-transferases GSTA2, GSTM2, GSTO1, GSTO2, and GSTZ1 in breast cancer risk.

Breast cancer is a complex disease and in recent years a number of breast cancer susceptibility genes have been identified, but the role of low penetrance susceptibility genes has not been completely resolved. Glutathione S-transferases (GSTs) are phase II xenobiotic metabolizing enzymes involved in the detoxification of chemical carcinogens and environmental pollutants and play an important role in cell defense mechanisms against oxidative stress. They have been in the spot light for the investigation of a potential association with breast cancer risk but so far, sparse or even no data for a potential contribution of GSTA2, GSTM2, GSTO, and GSTZ to breast cancer risk are available. We genotyped GSTA2_448_C > G (rs2180314), GSTA2_742_A > C (rs6577), GSTM2_-832_T > C (rs638820), GSTO1_-1242_G > A (rs2164624), GSTO1_419_A > C (rs4925), GSTO2_-183_A > G (rs2297235), GSTO2_342_A > G (rs156697), GSTZ1_-4378_A > G (rs1046428), and GSTZ1_94_G > A (rs3177427) by MALDI-TOF MS in the German GENICA breast cancer case-control collection of 1021 cases and 1015 controls and performed breast cancer risk association in general and with respect to the stratifications: menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index, and smoking as well as histopathological tumor characteristics including hormone receptor status, grade, histology, and node status. We did not observe any breast cancer risk associations and conclude that it is unlikely that glutathione S-transferases GSTA2, GSTM2, GSTO1, GSTO2, and GSTZ1 participate in breast cancer susceptibility.

Aged human thymus hassall's corpuscles are immunoreactive for IGF-I and IGF-I receptor.

Although Hassall's corpuscles have been proposed to act in both maturation of developing thymocytes and removal of apoptotic cells, their function remains an enigma. The involvement of insulin-like growth factor I (IGF-I) in the local autocrine and paracrine control of T-cell development in human thymus is still unclear. In this study, we investigated the structure and distribution of IGF-I and IGF-I receptor (IGF-IR)-immunopositive Hassall's corpuscles in aged human thymus using bright-field immunohistochemistry and immunoelectron microscopy. We report new immunocytochemical data for the presence of IGF-I/IGF-IR double-immunopositive Hassall's corpuscles in structurally preserved regions of age-involuted thymus and discuss the involvement of these unique thymic components in the local regulation of T-cell development and thymus plasticity during aging by IGF-I/IGF-IR-mediated cell signaling pathway.

Critical evaluation of environmental exposure agents suspected in the etiology of Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN), a kidney disease that occurs in rural villages in Bosnia, Bulgaria, Croatia, Romania, and Serbia, is thought to be linked to an environmental toxin. The authors review literature on proposed environmental exposure agents, report the results of field sampling and analysis studies to evaluate potentials for exposure to proposed agents, and propose criteria for future testing. They used these criteria to evaluate the evidence for suggested hypotheses, concluding that several proposed agents can be eliminated or considered unlikely based on apparent inconsistencies between clinical or epidemiologic evidence related to BEN and toxicologic or exposure evidence related to the agents. Mycotoxins and aristolochic acid are the primary targets of current toxicologic investigations, and while the evidence on exposures for both is potentially consistent, it is insufficient.

Evaluation of the hypothesis that Balkan endemic nephropathy is caused by drinking water exposure to contaminants leaching from Pliocene coal deposits.

Balkan endemic nephropathy (BEN) is a kidney disease that has been reported in only certain rural villages in Serbia, Bulgaria, Romania, Croatia and Bosnia. The cause of BEN remains a mystery, but researchers seem to agree that exposure to one or more environmental agents is at least partially responsible. The Pliocene lignite hypothesis suggests the disease is due to long-term exposure to polycyclic aromatic hydrocarbons (PAHs) or other toxic organic compounds that have leached into drinking water supplies from low-rank coals. Although this hypothesis has been promoted by some researchers, efforts to substantiate it have been inconclusive due to limitations in sample size and methodology. The present study was designed to further examine this hypothesis by analyzing PAHs, which were implicated in the original hypothesis, in a larger number of water samples from endemic and nonendemic villages in Bulgaria and for other chemical differences between the villages. Results show that levels of all PAHs were low, with none exceeding the drinking water standard for benzo-[a]-pyrene, the most toxic PAH, and the only one for which a maximum contaminant level (MCL) has been set for drinking water. Comparison of additional unidentified chromatographic peaks from high-pressure liquid chromatography (HPLC) technique designed to detect dissolved organic compounds (DOCs) that leach from coal failed to show higher levels in BEN villages. This study finds no basis to connect PAHs or other unknown DOCs to the etiology of BEN, and suggests that the evidence in support of the Pliocene lignite hypothesis is limited to the spatial association originally proposed.

Nitrogen species in drinking water indicate potential exposure pathway for Balkan Endemic Nephropathy.

This study explored two hypotheses relating elevated concentrations of nitrogen species in drinking water and the disease Balkan Endemic Nephropathy (BEN). Drinking water samples were collected from a variety of water supplies in both endemic and non-endemic villages in the Vratza and Montana districts of Bulgaria. The majority of well water samples exceeded US drinking water standards for nitrate + nitrite. No statistically significant difference was observed for any of the nitrogen species between villages classified as endemic and non-endemic. Other constituents (sodium, potassium and chloride) known to be indicators of anthropogenic pollution were also found at elevated concentrations and all followed the order wells > springs > taps. This ordering coincides with the proximity of human influences to the water sources. Our results clearly establish an exposure pathway between anthropogenic activity and drinking water supplies, suggesting that the causative agent for BEN could result from surface contamination.

Balkan endemic nephropathy and genetic variants of glutathione S-transferases.

BACKGROUND: Balkan endemic nephropathy (BEN) is a non-inflammatory, chronic, slow progressing kidney disease, frequently associated with urinary tract tumors. BEN displays familial clustering without an apparent Mendelian inheritance pattern. It has been suggested that environmental toxicants damage urothelial cells in genetically susceptible individuals, which could be the cause of BEN. The metabolism of some substrates that are mediated by glutathione S-transferases (GST), which are polymorphic enzymes, results in nephrotoxic products. To evaluate whether GST genetic heterogeneity could be involved in BEN, we launched a case-control study concerning the association of the most common polymorphic GST variants with BEN. METHODS: DNA was extracted from venous blood samples from 54 unrelated BEN patients and 104 controls inhabiting the same endemic region. GSTM1 and GSTT1 null deletions were identified simultaneously by a triplex polymerase chain reaction (PCR) procedure, and GSTP1 polymorphism was analyzed by PCR-restriction fragment length polymorphism (PCR-RFLP) using Alw261. RESULTS: Carriers of at least one GSTM1 wild type allele (wt-allele) were more prevalent among BEN patients compared to controls (chi2=7.92, p=0.005). The GSTT1 and GSTP1 genotype distributions did not demonstrate statistically significant differences between the groups. The carriers of at least one GSTM1 wt-allele among BEN patients were more prevalent in comparison with controls when the GSTM1 genotypes were combined in pairs with all GSTT1 (chi2=9.52, p=0.023) and GSTP1 (chi2=11.92, p=0.036) genotypes. The combined genotype distributions of the three GST genes studied among BEN patients and controls showed that the frequency of carriers of at least one GSTM1 wt-allele among BEN patients was higher or at least equal to the corresponding frequency among controls in all triple combinations. However, this difference did not reach statistical significance (chi2=14.06, p=0.170). CONCLUSIONS: GSTM1 wt-allele associates with BEN. The significantly lower prevalence of the GSTM1 deletion homozygotes among BEN patients suggests that individuals bearing the GSTM1 null genotype could be better protected.

Screening for point mutations in the LDL receptor gene in Bulgarian patients with severe hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common, autosomal dominant disorder of lipid metabolism, caused by defects in the receptor-mediated uptake of LDL (low-density lipoproteins) due to mutations in the LDL receptor gene ( LDLR). Mutations underlying FH in Bulgaria are largely unknown. The aim of the present study was to provide information about the spectrum of point mutations in LDLR in a sample of 45 Bulgarian patients with severe hypercholesterolemia. Exons 3, 4, 6, 8, 9, and 14, previously shown to be mutational hot spots in LDLR, were screened using PCR-single-strand conformation polymorphism (SSCP). Samples with abnormal SSCP patterns were sequenced. Three different, hitherto undescribed point mutations (367T>A, 377T>A, 917C>A) and two previously described mutations (858C>A and 1301C>T) in eight unrelated patients were identified; four of the detected point mutations being missense mutations and one, a nonsense mutation. One of the newly described point mutations (917C>A) is a base substitution at a nucleotide position, at which two other different base substitutions have already been reported. Thus, all three possible base substitutions at this nucleotide position have been detected, making it a hot spot for point mutations causing FH. This is the first such mutational hot spot described in exon 6 of LDLR.

Five polymorphisms of the apolipoprotein B gene in healthy Bulgarians.

Five APOB polymorphisms (I/D in the promoter region, XbaI [codon 24881, MspI [codon 3611], EcoRI [codon 41541, and 3' VNTRs) were studied in a population sample of 147 healthy normolipemic Bulgarians. For all biallelic loci, the observed genotype distributions do not deviate from Hardy-Weinberg equilibrium. In Bulgaria the insertion allele and the MspI+ allele of APOB presented the highest allelic frequencies (0.793 +/- 0.024 and 0.959 +/- 0.012, respectively) among the European population groups studied so far. The allele frequencies of the other two biallelic polymorphisms (XbaI and EcoRI) found in the Bulgarian population are similar to those previously described in other Caucasian populations. Analysis of the 3' VNTR polymorphism revealed 11 different alleles. Like studies in other Caucasian populations, this study found bimodal allele-size distribution and a high level of heterozygosity. The frequency of allele *31 (0.162 +/- 0.022) among Bulgarians is higher than that of any other European population group studied so far. Genetic distances between Bulgarians and each of six populations from southeastern Europe for which 3' VNTR allele frequencies are available showed an increase in the order: Albanians

Determination of the epigenetic effects of ochratoxin in a human kidney and a rat liver epithelial cell line.

Epidemiological studies have implicated ochratoxin A (OTA), a fungal metabolic-contaminant of animal and human food sources, in Balkan Endemic Nephropathy and renal tumors. Many environmental toxicants operate through nongenotoxic mechanisms that epigenetically control gene expression leading to a diseased state. Gap junctional intercellular communication (GJIC) plays a central role in the epigenetic control of genes in which alteration of normal GJIC has been implicated in many human pathologies, including cancer, teratogenesis, reproductive dysfunction and peripheral neuropathies. The cell proliferative stages of human diseases, such as cancer, also involves the induction of signal transduction pathways controlling the mitogenic steps, in which the mitogen activated protein kinases (MAPK), such as extracellular receptor kinase (ERK) and p38, are central to mitogenesis. We therefore determined the effects of OTA on GJIC and MAPK in a human kidney and rat liver epithelial cell line. OTA reversibly inhibited GJIC at noncytotoxic doses in the rat liver but not the human kidney cell line. Similarly, OTA was also a strong activator of MAPK, ERK and p38, in the rat liver cells but only weakly activated ERK and had no affect on p38 in the human kidney cell line. Another hallmark of human diseases is an abnormal alteration of apoptosis, also known as programmed cell death. We used our myc-transfected cell line, which exhibits higher levels of apoptosis, to test the effects of OTA on apoptosis. OTA greatly induced apoptosis in this cell line, which is contrary to the effects of most tumor promoters. In summary, OTA exhibits tumor promoting properties in the liver, but the effects of OTA on the human kidney epithelial cells suggested a lack of tumorigenic activity assuming that these epithelial cells, like the rat liver epithelial cells, are a primary target for carcinogens. These results also indicate that the nephrotoxicity of OTA either does not involve GJIC, assuming these epithelial cells play a vital role in kidney physiology, or that a more differentiated kidney cell type is the target for OTA toxicity, of which the role of GJIC remains unknown.