[Studies on markers of exposure and early effect in areas with arsenic pollution: methods and results of the project SEpiAs. Epidemiological surveillance in areas with environmental pollution by natural or anthropogenic arsenic]. / Studi su marcatori di esposizione ed effetto precoce in aree con inquinamento da arsenico: metodi e risultati del progetto SEpiAs. Sorveglianza epidemiologica in aree interessate da inquinamento ambientale da arsenico di origine naturale o antropica (SEpiAS CCM 2010).

INTRODUCTION: Arsenic and its inorganic compounds are classified as carcinogenic to humans. Exposures to inorganic arsenic (iAs) in drinking water are associated with both carcinogenic and non-carcinogenic effects. The risk assessment of exposures to low-moderate levels of environmental arsenic (As) is a challenging objective for research and public health. The SEpiAs study, funded by the Italian Ministry of Health (CCM), was carried out in four areas with arsenic pollution prevalently of natural origin, Amiata and Viterbo areas, or of industrial origin, Taranto and Gela. MATERIALS AND METHODS: 271 subjects (132 men) aged 20-44, were randomly sampled stratifying by area, gender and age classes. Individual data on residential history, socio-economic status, environmental and occupational exposures, lifestyle and dietary habits, were collected through interviews using questionnaire. In urine samples of recruited subjects, the concentration of inorganic arsenic (iAs) and methylated species (MMA, DMA) was measured using inductively coupled mass spectrometer (DRCICP- MS), after chromatographic separation (HPLC). Molecular biomarkers and biomarkers of DNA damage, as well as markers of cardiovascular risk were measured The distributions of iAs and iAs+MMA+DMA were described by area and gender, geometric mean (GM), percentiles and standard deviation (SD). The associations between As species and variables collected by questionnaire were evaluated by multiple regression analysis. RESULTS: Results showed a high variability of As species within and among areas. Gela and Taranto samples showed higher iAs concentration compared to Viterbo and Amiata. Subjects with iAs>1,5 µg/L or iAs+MMA+DMA>15 µg/L (thresholds suggested by the Italian Society of Reference Values), are 137 (50,6%) and 68 (25,1%), respectively. A positive association between iAs and use of drinking water emerged in the Viterbo sample, between iAs and occupational exposure in the Gela and Taranto samples. Fish consumption was associated with higher iAs concentration in the whole sample, and particularly in men of the Gela sample. Similar results were observed for iAs+MMA+DMA. Subjects with iAs or iAs+MMA+DMA values higher than the 95th percentile were 15 (6Taranto, 5 Gela, 3Viterbo, 1 Amiata). The relationships between iAs and organic species (methylation efficiency ratios) were different between sex in the four areas. The relevance of polymorphisms AS3MT Met287Thr, GST-T1, GST-M1, OGG1 was confirmed. The analysis of carotid intima-media-thickness showed normal values, but higher among man of Viterbo, Taranto and Gela areas. CONCLUSIONS: Results are informative of exposure to inorganic and organic As in large or at least non-negligible quotas of the samples. The SEpiAs results suggest a further deepening on routes of exposure to arsenic species, and support the recommendation to implement primary prevention measures to reduce population exposure.

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: a new target for osteogenesis imperfecta pharmacological therapy.

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.