Impact of the environment on the health: From theory to practice.

The Erice 56 Charter titled "Impact of the environment on the health: from theory to practice" was unanimously approved at the end of the 56th course of the "International School of Epidemiology and Preventive Medicine G. D'Alessandro" held from 3rd to November 7, 2019 in Erice - Sicily (Italy) and promoted by the Study Group of "Environment and Health" of the Italian Society of Hygiene, Preventive Medicine and Public Health. The course, that included lectures, open discussions and guided working groups, was aimed to provide a general training on epidemiological and toxicological aspects of the environmental health impact, to be used by public health professionals for risk assessment, without forgetting the risk communications. At the end of the course 12 key points were agreed among teachers and students: they underlined the need of specific training and research, in the perspective of "One Health" and "Global Health", also facing emerging scientific and methodological issues and focusing on communication towards stakeholders. This Discussion highlight the need to improve knowledge of Health and Environment topic in all sectors of health and environmental prevention and management.

The fine tuning of metabolism, autophagy and differentiation during in vitro myogenesis.

Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.

Dietary style and acid load in an Italian population of calcium kidney stone formers.

BACKGROUND AND AIMS: Animal protein intake may cause an acid load that predisposes individuals to stones by influencing calcium and citrate excretion. These associations were not confirmed in recent studies. Therefore the present study was aimed to compare acid load of diet in stone formers and controls. METHODS AND RESULTS: Participants to the study were 157 consecutive calcium stone formers and 144 controls. Diet was analyzed in these subjects using a software that evaluated nutrient intake from a three-day food intake diary. This software also estimated the potential renal acid load (PRAL, mEq/day). Twenty-four-hour urine excretion of ions and citrate was measured in stone formers. Stone former diet had lower intake of glucose, fructose, potassium and fiber and higher PRAL in comparison with controls. The multinomial logistic regression analysis showed that stone risk decreased in association with the middle and the highest tertiles of fiber intake and increased in association with the highest tertile of PRAL. The linear multiple regression analysis showed that calcium excretion was associated with the sodium excretion and that citrate excretion was associated with the PRAL and animal protein intake in stone formers. CONCLUSION: Our findings suggest that stone formers may undergo a greater dietary acid load sustained by a low vegetable intake and base provision. Dietary acid load does not appear as the main determinant of calcium excretion, but may promote stone risk by decreasing citrate excretion. Sodium intake may predispose to stones by stimulating calcium excretion.

Functional and molecular defects of hiPSC-derived neurons from patients with ATM deficiency.

Loss of ataxia telangiectasia mutated (ATM) kinase, a key factor of the DNA damage response (DDR) pathway, causes the cancer predisposing and neurodegenerative syndrome ataxia-telangiectasia (A-T). To investigate the mechanisms of neurodegeneration, we have reprogrammed fibroblasts from ATM-null A-T patients and normal controls to pluripotency (human-induced pluripotent stem cells), and derived from these neural precursor cells able to terminally differentiate into post-mitotic neurons positive to >90% for ß-tubulin III+/microtubule-associated protein 2+. We show that A-T neurons display similar voltage-gated potassium and sodium currents and discharges of action potentials as control neurons, but defective expression of the maturation and synaptic markers SCG10, SYP and PSD95 (postsynaptic density protein 95). A-T neurons exhibited defective repair of DNA double-strand breaks (DSBs) and repressed phosphorylation of ATM substrates (e.g., γH2AX, Smc1-S966, Kap1-S824, Chk2-T68, p53-S15), but normal repair of single-strand breaks, and normal short- and long-patch base excision repair activities. Moreover, A-T neurons were resistant to apoptosis induced by the genotoxic agents camptothecin and trabectedin, but as sensitive as controls to the oxidative agents. Most notably, A-T neurons exhibited abnormal accumulation of topoisomerase 1-DNA covalent complexes (Top1-ccs). These findings reveal that ATM deficiency impairs neuronal maturation, suppresses the response and repair of DNA DSBs, and enhances Top1-cc accumulation. Top1-cc could be a risk factor for neurodegeneration as they may interfere with transcription elongation and promote transcriptional decline.

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity.

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.

Calcium sensing receptor and renal mineral ion transport.

Calcium sensing receptor (CaSR) is a component of the C family of the G protein-coupled receptors. It is ubiquitously expressed in human and mammal cells but is more expressed in parathyroid glands and kidney cells. It is located on the cell plasma membrane and senses the changes of extracellular calcium concentrations. Thus, it may modify cell functions according to serum calcium levels. CaSR has a key role in calcium homeostasis because it allows parathyroid glands and kidney to regulate PTH secretion and calcium reabsorption in order to keep serum calcium concentration within the normal range. CaSR appears as an important player in the regulation of renal calcium handling and body calcium metabolism. Thus, CaSR may protect human tissues against calcium excess. In kidneys, its protective effect includes the stimulation of diuresis and phosphate retention, along with the potential prevention of calcium precipitation and deposition in kidney tubules and interstitium.

Role of nucleotide excision repair proteins in oxidative DNA damage repair: an updating.

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage. Base excision repair (BER) has an important role in preventing the deleterious effects of oxidative DNA damage, but recent evidence points to the involvement of several repair pathways in this process. Oxidative damage may arise from endogenous and exogenous sources and may target nuclear and mitochondrial DNA as well as RNA and proteins. The importance of preventing mutations associated with oxidative damage is shown by a direct association between defects in BER (i.e. MYH DNA glycosylase) and colorectal cancer, but it is becoming increasingly evident that damage by highly reactive oxygen species plays also central roles in aging and neurodegeneration. Mutations in genes of the nucleotide excision repair (NER) pathway are associated with diseases, such as xeroderma pigmentosum and Cockayne syndrome, that involve increased skin cancer and/or developmental and neurological symptoms. In this review we will provide an updating of the current evidence on the involvement of NER factors in the control of oxidative DNA damage and will attempt to address the issue of whether this unexpected role may unlock the difficult puzzle of the pathogenesis of these syndromes.

Mechanisms of dealing with DNA damage in terminally differentiated cells.

To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

Renal osteodystrophy and vascular calcification.

Chronic kidney disease (CKD) is characterized by phosphate retention and reduced synthesis of 1.25(OH)2-vitamin D stimulating parathyroid hyperplasia. These changes cause a complex osteopathy, defined as renal osteodystrophy, and vascular calcification. Renal osteodystrophy increases the risk of fracture and causes deformities and disability. Vascular calcification occurs in a large proportion of hemodialysis patients and is a marker of arteriopathy. Calcifying arteriopathy induces arterial stiffness and contributes to the high cardiovascular mortality and morbidity among CKD patients. Vascular calcification results from a process of local bone formation induced by osteoblast-like cells developing in the vascular wall from resident cells. Osteoblast differentiation of resident vascular cells may be mediated by metabolic factors and may be induced by high concentrations of phosphate. Therefore, phosphate retention appears as the most detrimental factor affecting arteries in CKD patients. There is no specific therapy to revert soft tissue calcification, but calcification must be prevented in the early stages of CKD.

[What do we know after ten years of genetic research into calcium kidney stones?]. / Quali insegnamenti dopo dieci anni di ricerca genetica sulla calcolosi renale di calcio?

Genetic studies of calcium kidney stones have so far assessed single candidate genes by testing linkage disequilibrium or association between a locus and stone disease. They showed the possible involvement of the calciumsensing receptor gene, vitamin D receptor gene, and bicarbonate-sensitive adenylate cyclase gene. In addition to research in humans, the study of different strains of knock-out mice let us include the gene of phosphate reabsorption carrier NPT2, caveolin-1, protein NHERF-1 modulating calcium and urate reabsorption, osteopontin and Tamm-Horsfall protein among the possible determinants. However, the interactions between genes and also between environmental factors and genes are generally considered fundamental in calcium stone formation. Thus, the genetic studies carried out to date have not led to a significant growth of the knowledge about the causes of calcium kidney stones, even though they have allowed us to assess the size of the problem and define criteria to address it. Further knowledge of the causes of calcium stones may be obtained using the instruments that modern biotechnology and bioinformatics have made available to researchers.

The role of CSA in the response to oxidative DNA damage in human cells.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Interleukin-1 gene polymorphisms and gastric cancer risk in a high-risk Italian population.

OBJECTIVES: Host genetic factors, including the IL1 gene cluster, play a key role in determining the long-term outcome of Helicobacter pylori infection. The aim of the study was to investigate the relationship between selected IL1 loci polymorphisms and gastric cancer risk in an Italian population. METHODS: In a case-control study we compared the IL1B-31 and IL1B+3954 biallelic and IL1RN pentaallelic variable number of tandem repeats (VNTR) polymorphisms in 185 gastric cancer patients and 546 controls randomly sampled from the general population of an area at high gastric cancer risk (Tuscany, Central Italy). RESULTS: Genotype frequencies of the IL1B-31 T/C, IL1B+3954 C/T, and IL1RN polymorphisms among our population controls were in Hardy-Weinberg equilibrium. In multivariate analyses, no increase in gastric cancer risk was observed for the IL1B-31*C- and IL1B+3954*T- carriers; a significant 50% increase emerged for IL1RN*2 allele carriers (OR = 1.49; 95% CI: 1.01-2.21). Analyses based on combined genotypes showed also that the association with IL1RN*2 allele was limited to two-variant allele carriers who were also homozygous for the IL1B-31*T allele (OR = 2.23; 95% CI: 1.18-4.23) with a statistically significant interaction between these two genotypes (p= 0.043). Haplotype analysis showed an increased risk for the haplotype IL1RN*2/IL1B-31*T. CONCLUSIONS: Our results suggest that host genetic factors (such as the IL1RN and the IL1B-31 polymorphisms) interact in the complex process of gastric carcinogenesis in this high-risk Italian population. Overall, this effect appears more modest than previously reported in other populations, supporting the hypothesis that other still-to-be-defined factors are important in gastric carcinogenesis. These findings might be due to a haplotype effect.

Characterization of the ultraviolet B and X-ray response of primary cultured epidermal cells from patients with disseminated superficial actinic porokeratosis.

BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is the most common porokeratosis and is characterized by multiple keratotic lesions which tend to occur at sun-exposed sites. A mild hypersensitivity to X-rays has been reported for DSAP-derived fibroblasts and frequent over-expression of p53 has been found in lesional epidermis. OBJECTIVES: In order to clarify whether genome maintenance mechanisms might be compromised in this disease the following approaches were undertaken: (i) primary cultured keratinocytes and fibroblasts from DSAP patients were characterized for ultraviolet (UV) B and X-ray response; (ii) 15 lesions were studied for p53 mutations, and (iii) the differentiation status of DSAP-derived keratinocytes was evaluated. METHODS: Primary cultures of keratinocytes and fibroblasts were established from lesional and nonlesional skin biopsies of two subjects with DSAP. p53 mutations were analysed by DNA sequencing of the conserved region of the TP53 gene. Differentiation was evaluated both in stratified epithelial sheets from confluent keratinocyte cultures and in organotypic skin cultures. RESULTS: The cytotoxic and apoptotic response to UVB or X-irradiation was similar in DSAP-derived keratinocytes and fibroblasts when compared with normal cells. Two of 15 lesions examined presented p53 mutations located at nondipyrimidine sites. A strikingly decreased expression of filaggrin was observed both in reconstructed epidermis and in reconstructed skin. CONCLUSIONS: The UVB and X-ray response of DSAP-derived keratinocytes and fibroblasts indicates that the actinic character of this skin pathology is not due to radiation hypersensitivity. In agreement with this finding, mutations in the p53 gene, which are often associated with UV-related skin carcinogenesis, were rarely detected in DSAP lesions and were not UV-specific. Reconstructed epidermis and reconstructed skin models successfully reproduced the main features of this genodermatosis, showing that DSAP-derived keratinocytes bear an inherent defect in the terminal differentiation programme.

8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways.

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein.

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.

The base excision repair: mechanisms and its relevance for cancer susceptibility.

Base damage or loss occurs at high frequency in the cells (almost 10(4) bases are damaged and hydrolysed per cell per day). DNA repair is fundamental to maintain genomic integrity. Base excision repair (BER) is the main mechanism by which cells correct various types of damaged DNA bases generated either by endogenous or exogenous factors. The widely accepted model for BER mechanism involves five sequential reactions: (i) base removal; (ii) incision of the resulting abasic site; (iii) processing of the generated termini at the strand break; (iv) DNA synthesis, and (v) ligation. In this review, we will briefly summarise the biochemistry of each BER step and will concentrate on the biological relevance of BER as inferred from in vitro and in vivo studies. This information will be the basis for speculation on the potential role of malfunction of BER in human pathology.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

Risk factors for basal cell carcinoma in a Mediterranean population: role of recreational sun exposure early in life.

OBJECTIVE: To investigate the role of pigmentary traits, different patterns of sun exposure, artificial sources of UV radiation, and lifestyle-related factors on the risk of basal cell carcinoma (BCC) in a Mediterranean population from central-southern Italy. DESIGN: Hospital-based case-control study. SETTING: A referral dermatological hospital in Rome, Italy. PATIENTS: A convenience sample of 166 case patients with histologically confirmed BCC and 158 cancer-free control subjects with minor dermatological conditions observed between March 1995 and June 1997. RESULTS: In the multivariate analysis, the mean number of weeks per year spent at the beach before the age of 20 years was significantly associated with BCC. A dose-response trend was found for subjects who had spent 3 to 4 (odds ratio, 1.8; 95% confidence interval, 0.8-4.4), 5 to 8 (odds ratio, 3.7; 95% confidence interval, 1.5-9.0), or more than 8 (odds ratio, 4.5; 95% confidence interval, 1.9-10.5) weeks per year at the beach (P =.01 for trend). There was a significant association with the presence of actinic keratoses or solar lentigines, whereas no effect was found for skin type, history of sunburns, exposure to nonsolar UV radiation, and lifestyle-related habits such as cigarette smoking, alcohol consumption, and coffee drinking. Subjects reporting a family history of skin cancer had an extremely increased risk of BCC. CONCLUSION: The definite association with recreational sun exposure during childhood and adolescence and the strong relation with family history of skin cancer suggest that genetic predisposition and peculiar exposure patterns to UV radiation are key independent risk factors for the development of BCC in a southern European population.

The mechanism of switching among multiple BER pathways.

To preserve genomic beta DNA from common endogenous and exogenous base and sugar damage, cells are provided with multiple base excision repair (BER) pathways: the DNA polymerase (Pol) beta-dependent single nucleotide BER and the long-patch (2-10 nt) BER that requires PCNA. It is a challenge to identify the factors that govern the mechanism of switching among these pathways. One of these factors is the type of DNA damage induced in DNA. By using different model lesions we have shown that base damages (like hypoxanthine and 1, N6-ethenoadenine) excised by monofunctional DNA glycosylases are repaired via both single-nucleotide and long-patch BER, while lesions repaired by a bifunctional DNA glycosylase (like 7,8-dihydro-8-oxoguanine) are repaired mainly by single-nucleotide BER. The presence of a genuine 5' nucleotide, as in the case of cleavage by a bifunctional DNA glycosylase-beta lyase, would then minimize the strand displacement events. Another key factor in the selection of the BER branch is the relative level of cellular polymerases. While wild-type embryonic mouse fibroblast cell lines repair abasic sites predominantly via single-nucleotide replacement reactions (80% of the repair events), cells homozygous for a deletion in the Pol beta gene repair these lesions exclusively via long-patch BER. Following treatment with methylmethane sulfonate, these mutant cells accumulate DNA single-strand breaks in their genome in keeping with the fact that repair induced by monofunctional alkylating agents goes predominantly via single-nucleotide BER. Since the long-patch BER is strongly stimulated by PCNA, the cellular content of this cell-cycle regulated factor is also extremely effective in driving the repair reaction to either BER branch. These findings raise the interesting possibility that different BER pathways might be acting as a function of the cell cycle stage.