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.

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.

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.

[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.

DNA polymerase beta is required for efficient DNA strand break repair induced by methyl methanesulfonate but not by hydrogen peroxide.

The most frequent DNA lesions in mammalian genomes are removed by the base excision repair (BER) via multiple pathways that involve the replacement of one or more nucleotides at the lesion site. The biological consequences of a BER defect are at present largely unknown. We report here that mouse cells defective in the main BER DNA polymerase beta (Pol beta) display a decreased rate of DNA single-strand breaks (ssb) rejoining after methyl methanesulfonate damage when compared with wild-type cells. In contrast, Pol beta seems to be dispensable for hydrogen peroxide-induced DNA ssb repair, which is equally efficient in normal and defective cells. By using an in vitro repair assay on single abasic site-containing circular duplex molecules, we show that the long-patch BER is the predominant repair route in Pol beta-null cell extract. Our results strongly suggest that the Pol beta-mediated single nucleotide BER is the favorite pathway for repair of N-methylpurines while oxidation-induced ssb, likely arising from oxidized abasic sites, are the substrate for long-patch BER.

Multiple mutations and frameshifts are the hallmark of defective hPMS2 in pZ189-transfected human tumor cells.

Two HeLa variants defective in the mismatch repair protein hPMS2 were isolated by selection for methylation tolerance. Neither variant expressed detectable hPMS2 protein as determined by western blotting. Cell extracts were defective in correcting a single base mispair and were unable to perform mismatch repair-dependent processing of a methylated DNA substrate. Correction of the repair defect and restoration of sensitivity to a methylating agent was achieved by introducing a wild-type copy of chromosome 7 on which the hPMS2 gene is located. Loss of hPMS2 function in the HeLa variants was associated with a 5-fold increase in mutation frequency in the supF gene of the pZ189 shuttle vector. Wild-type levels of mutagenesis were restored by the transferred chromosome 7. Comparisons of mutational spectra identified multiple base substitutions, frameshifts and, to a lesser extent, single base pair changes as the types of mutation which are selectively increased in a hPMS2-defective background. The location of multiple mutations and frameshifts indicates that misalignment-mediated mutagenesis could underlie most of these events. Thus the mutator phenotype associated with loss of hPMS2 most likely arises because of the failure to correct replication slippage errors. Our data also suggest that a considerable fraction of mutagenic intermediates are recognized by the hMutSbeta complex and processed via the hMLH1/hPMS2 heterodimer.

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.

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.

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.

Expression of the endogenous O6-methylguanine-DNA-methyltransferase protects Chinese hamster ovary cells from spontaneous G:C to A:T transitions.

We have investigated whether the presence of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), affects the nature of spontaneous mutations in a mammalian cell line. We compared spontaneous mutations in the adenine phosphoribosyl transferase gene of a Chinese hamster ovary (CHO) cell line that expressed 14,000 MGMT molecules/cell with those in the parental CHO cells lacking this DNA repair activity. The mutation rate/cell/generation of the two CHO cell lines did not differ significantly. However, DNA sequence analysis of spontaneous mutations in the MGMT-proficient CHO cell line revealed a complex picture. No significant difference from the parental CHO cells was found in the number or type of deletions, frameshifts, multiple substitutions, or insertions. The frequency of G:C to T:A transversions was elevated in MGMT-proficient CHO cells. Expression of the enzyme considerably reduced G:C to A:T transitions (25% versus 8.3%). This latter result is the first evidence that this protein is active on an endogenous source of O6-methylguanine that is normally responsible for spontaneous G:C to A:T transition mutations.

Mutagenic processing of ethylation damage in mammalian cells: the use of methoxyamine to study apurinic/apyrimidinic site-induced mutagenesis.

The aldehyde reagent methoxyamine is able to interact with apurinic/apyrimidinic sites formed in vivo within cells and displays both an anti-cytotoxic and an antimutagenic activity on N-ethyl-N'-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spectra induced by N-ethyl-N'-nitro-N-nitrosoguanidine alone and in the presence of methoxyamine in the hypoxanthine-guanine phosphoribosyltransferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions decreased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by O6-ethylguanine adducts and to a minor extent by O4-ethylthymine and (b) apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve O6-ethylguanine and O2-ethylthymine adducts.

Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation, and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1 cell line.

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Tolerance to O6-methylguanine and 6-thioguanine cytotoxic effects: a cross-resistant phenotype in N-methylnitrosourea-resistant Chinese hamster ovary cells.

The biochemical and genetic characteristics of a clone of Chinese hamster ovary cells displaying resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (6-TG) were analyzed. The initial level of 7-methylguanine, 3-methyladenine, and O6-methylguanine formation and the repair rates for these alkylated bases were the same in the resistant and in the parental cell line, indicating that the resistance to alkylation damage is not due to differences in DNA alkylation. After exposure for 24 or 48 h to 6-TG (0.6 micrograms/ml) in culture medium, the resistant clone in contrast to them, was able to replicate the DNA containing the base analogue during the following 24 h. These data are in agreement with the hypothesis that resistant cells tolerate both O6-methylguanine and 6-TG present in DNA. The tolerance to MNU and 6-TG also included chromosomal damage induced by these two agents, and MNU-resistant cells incurred less sister chromatid exchanges after treatment with either MNU or 6-TG. 6-TG-resistant cells, selected by growth in 6-TG, exhibited cross-resistance to MNU but not to methyl methanesulfonate, confirming that a common pathway of tolerance is responsible for resistance to 6-TG and O6-methylguanine.

p53 mutations and chromosome instability in basal cell carcinomas developed at an early or late age.

Tumor DNA from 45 primary basal cell carcinoma (BCC) biopsies was screened for p53 gene mutations, chromosome 9 allele loss, and microsatellite instability. p53 mutation frequency increased significantly as a function of the age at BCC onset ranging from 6% (1/16) in early BCC (before age 40 years) to 35% (10/29) in late BCC. All p53 mutations found implicated sunlight as the mutagen. Chromosome 9 instability (allele loss or microsatellite instability) was detected at high frequency (38%) independently of age at tumor onset. Allelic loss was confined to chromosome 9q, whereas microsatellite instability was observed prevalently on chromosome 9p often in association with a replication error (RER+) phenotype. Most of our late BCC patients reported occupational sun exposure, while early BCC patients recalled childhood (0-20 years) recreational sun exposure. These data suggest that chronic exposure to sunlight is responsible for accumulation of p53 mutations and thus for late BCC appearance, whereas acute UV exposure in childhood and adolescence leads to early skin cancer development in genetically susceptible individuals via a p53-independent pathway.

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.

Mammalian base excision repair by DNA polymerases delta and epsilon.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38-50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.