Search

The impact of volatile organic compounds (VOCs) on indoor air quality and on human health is widely recognized. However, VOC contamination in hospital indoor air is rarely studied and chemical compounds that singularly do not show high toxicity are not submitted to any regulation. This study aimed to compare VOC contamination in two different anatomical pathology wards in the same hospital. Hydrocarbons, alcohols, and terpenes were sampled by passive diffusive samplers. Analytical tests were performed by thermal desorption coupled with gas chromatography and mass spectrometry detector. Results highlighted a different VOC pollution in the two wards, due to the structural difference of the buildings and different organizational systems. The scarcity of similar data in the literature shows that the presence of VOCs in pathology wards is an underestimated problem. We believe that, because of the adverse effects that VOCs may have on the human health, this topic is worth exploring further.

Glaucoma is a disease known since the time of Hippocrates. Today, the term glaucoma refers to a spectrum of neurodegenerative diseases that have in common progressive optical atrophy resulting from the apoptosis of retinal ganglion cells, and axonal atrophy and degeneration extending to the visual areas of the brain cortex, finally leading to the characteristic optical-cup neuropathy and irreversible visual loss. The action of many factors, including aging, genetic predisposition, exogenous environmental and endogenous factors, is necessary for the development of glaucoma. In addition to ganglion cell loss, most glaucoma types are characterized by high intraocular pressure. This is due to the damage that occurs in the trabecular meshwork, a key region in the pathogenesis of high-pressure glaucoma. In normal-pressure glaucoma, the pathogenesis is different, with vascular factors playing a very important role.

This study evaluates effects of good burning practice and correct installation and management of wood heaters on indoor air pollution in an Italian rural area. The same study attests the role of education in mitigating wood smoke pollution. In August 2007 and winters of 2007 and 2008, in a little mountain village of Liguria Apennines (Italy), indoor and outdoor benzene, toluene, ethylbenzene, and xylene (BTEX) concentrations were measured in nine wood-heated houses. During the first sampling, several mistakes in heating plant installations and management were found in all houses. Indoor BTEX concentrations increased during use of wood burning. Low toluene/benzene ratios were in agreement with wood smoke as main indoor and outdoor pollution source. Other BTEX sources were identified as the indoor use of solvents and paints and incense burning. Results obtained during 2007 were presented and discussed with homeowners. Following this preventive intervention, in the second winter sampling all indoor BTEX concentrations decreased, in spite of the colder outdoor air temperatures. Information provided to families has induced the adoption of effective good practices in stoves and fire management. These results highlight the importance of education, supported by reliable data on air pollution, as an effective method to reduce wood smoke exposures. Implications:Information about burning practices and correct installation and management of wood heaters, supported by reliable data on indoor and outdoor pollution, may help to identify and remove indoor pollution sources. This can be an effective strategy in mitigate wood smoke pollution.

Lapatinib, a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), is prescribed for the treatment of patients with metastatic breast cancer overexpressing HER-2. Involvement of this drug in pulmonary carcinogenesis has been poorly investigated. We used murine models suitable to evaluate cigarette smoke-related molecular and histopathological alterations. A total of 481 Swiss H mice were used. The mice were exposed to mainstream cigarette smoke (MCS) during the first four months of life. After 10 weeks, MCS caused an elevation of bulky DNA adducts, oxidative DNA damage and an extensive downregulation of microRNAs in lung. After four months, an increase in micronucleus frequency was observed in peripheral blood erythrocytes. After 7.5 months, histopathological alterations were detected in the lung, also including benign tumors and malignant tumors, and in the urinary tract. A subchronic toxicity study assessed the non-toxic doses of lapatinib, administered daily with the diet after weaning. After 10 weeks, lapatinib significantly attenuated the MCS-related nucleotide changes and upregulated several low-intensity microRNAs in lung. The drug poorly affected the MCS systemic genotoxicity and had modest protective effects on MCS-induced preneoplastic lesions in lung and kidney, when administered under conditions that temporarily mimicked interventions either in current smokers or ex-smokers. On the other hand, it caused some toxicity to the liver. Thus, on the whole, lapatinib appears to have a low impact in the smoke-related lung carcinogenesis models used, especially in terms of tumorigenic response.

The anti-diabetic drug metformin is endowed with anti-cancer properties. Epidemiological and experimental studies, however, did not provide univocal results regarding its role in pulmonary carcinogenesis. We used Swiss H mice of both genders in order to detect early molecular alterations and tumors induced by mainstream cigarette smoke. Based on a subchronic toxicity study, oral metformin was used at a dose of 800 mg/kg diet, which is 3.2 times higher than the therapeutic dose in humans. Exposure of mice to smoke for 4 months, starting at birth, induced a systemic clastogenic damage, formation of DNA adducts, oxidative DNA damage, and extensive downregulation of microRNAs in lung after 10 weeks. Preneoplastic lesions were detectable after 7.5 months in both lung and urinary tract along with lung tumors, both benign and malignant. Modulation by metformin of 42 of 1281 pulmonary microRNAs in smoke-free mice highlighted a variety of mechanisms, including modulation of AMPK, stress response, inflammation, NFκB, Tlr9, Tgf, p53, cell cycle, apoptosis, antioxidant pathways, Ras, Myc, Dicer, angiogenesis, stem cell recruitment, and angiogenesis. In smoke-exposed mice, metformin considerably decreased DNA adduct levels and oxidative DNA damage, and normalized the expression of several microRNAs. It did not prevent smoke-induced lung tumors but inhibited preneoplastic lesions in both lung and kidney. In conclusion, metformin was able to protect the mouse lung from smoke-induced DNA and microRNA alterations and to inhibit preneoplastic lesions in lung and kidney but failed to prevent lung adenomas and malignant tumors induced by this complex mixture.

Our previous studies showed that nucleotide alterations, evaluated by (32)P postlabeling, are systematically detected in smooth muscle cells of atherosclerotic lesions localized in the aorta of surgical patients. The level of these molecular lesions was correlated with the occurrence of known atherogenic risk factors, among which the number of currently smoked cigarettes, and was significantly enhanced in individuals having a null GSTM1 genotype as compared to individuals carrying the GSTM1 genotype. The present study had the dual objective of evaluating the formation of DNA adducts in the whole thoracic aorta of Sprague-Dawley rats, exposed whole-body to cigarette smoke for 28 consecutive days, and of investigating the effects of chemopreventive agents given orally during the same period. High levels of (32)P postlabeled DNA adducts were formed in the aorta of smoke-exposed rats, with an overall 11 times increase over the total levels observed in sham-exposed rats, and with increases ranging between three and 63 times for seven individual DNA adducts. Supplement of the diet with either 1,2-dithiole-3-thione, phenethyl isothiocyanate or 5,6-benzoflavone had no or poor effects on the smoke-related formation of nucleotide alterations in the aorta. In contrast, oltipraz, given with the diet, N-acetyl-L-cysteine, given with drinking water and, even more potently, their combination exerted remarkable protective effects. The results of this experimental study, together with the previous findings in humans, suggest that DNA alterations may contribute to the atherogenic process, clarify a possible mechanism of cigarette smoke, a well known atherogen, and show the potential protective effects of certain drugs towards these alterations.

A combination of tobacco smoking with certain agents has been shown to exert synergistic carcinogenic effects. On the other hand, antagonism betweeen smoke and other pulmonary carcinogens has also been documented by both epidemiological and experimental data. In spite of a very large number of studies carried out for decades in workers exposed to hexavalent chromium, the influence of smoking habits on lung carcinogenesis induced by this metal has not been clarified. For this reason, we performed two studies evaluating clastogenic effects in rodents. In the first one, BDF(1) mice were exposed whole-body to mainstream cigarette smoke for 5 days and, on the last day, they received an i.p. injection of potassium dichromate. In the second study, Sprague-Dawley rats were exposed whole-body to environmental cigarette smoke for 18 consecutive days and for the same period of time they received daily intra-tracheal instillations of sodium dichromate. Individually, the two hexavalent chromium salts and cigarette smoke, either mainstream or environmental, enhanced the frequency of micronuclei in bone marrow polychromatic erythrocytes of both mice and rats. Moreover, individual exposure to either environmental cigarette smoke or sodium dichromate enhanced the frequency of micronuclei and multiple nuclei in pulmonary alveolar macrophages of rats. In both studies, combined exposure to cigarette smoke and hexavalent chromium produced less than additive clastogenic effects. These results are consistent with our previous data, showing that hexavalent chromium and either benzo[a]pyrene or cigarette smoke condensate behave antagonistically in in vitro mutagenicity test systems and that the chromium reducing capacity of human pulmonary alveolar macrophages and peripheral lung parenchyma is enhanced in smokers. Taken together, in the absence of any epidemiological evidence, these findings rule out any occurrence of synergism between cigarette smoke and hexavalent chromium, at least in certain stages of the carcinogenesis process.

The influence of airborne pollution on mortality rates has been examined since the well known severe pollution episodes of Pennsylvania (1948) and London (1952). Three main epidemiological approaches are available: transversal studies, time series studies, prospective studies. The most frequently used method is the time series analysis, which retrospectively determines daily mortality rates as compared to daily pollution levels in a defined period. The vast majority of studies applying this approach confirm the existence of a positive correlation between mortality and airborne pollution. The relative risk of death during the most severely polluted days as compared to the least polluted days ranges between 1.02 and 1.13. Airborne pollution affects mortality as related to cardiovascular and chronic obstructive pulmonary diseases mainly in subjects previously affected by these pathologies. The dose-response mortality-pollution curve shows linear increments of mortality at low pollution levels, weak increments at high pollution levels. This phenomenon is attributed to the existence of subjects highly susceptible to pollution induced adverse health effects. These subjects, dying at low pollution levels, are not available to increase mortality rates at high pollution levels. Mortality is mainly related to the concentration of single pollutants, such as suspended particle matter, sulfur oxides, nitric oxides and ozone. Elderly are highly susceptible to pollution-induced mortality increases. Therefore, the study of mortality-pollution correlation is of particular interest in cities characterized by a relative prevalence of the elderly in the population. The latency period between pollution increases and related mortality increases falls in the 1-3 days interval range. The pollution-mortality relationship is influenced by many factors, such as the occurrence of exceedingly high or low environmental temperatures, influenza epidemics, etc. Many hypotheses has been raised to support the causality of the mortality-pollution association, including a direct effect of pollutants on cardiovascular and respiratory apparatus, and the release of inflammatory mediators affecting blood viscosity and pneumocytes homeostasis. On the whole, available data suggest that pollution is able to increase mortality only if associated to other risk factors determining an increased susceptibility in the exposed population.

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.

Recently, several molecular genetic bases of polymorphic enzyme activities involved in drug activation and detoxification have been elucidated. Many molecular epidemiology studies based on these premises have sought to gather information on the association of genetically determined metabolic variants with different risks of environmentally induced cancer. While rare alterations of tumor suppressor genes dramatically raise cancer risk for the single affected subjects, far more common and less dramatic differences in genes encoding for drug metabolism enzymes can be responsible for a relatively small, but rather frequent increase of cancer risk at the population level. This increase could be especially important in specific cases of occupational, pharmacological or environmental exposure. Examination of the current literature reveals that the most extensively investigated metabolic polymorphisms are those of P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases (GSTs; M1 and, to a lesser extent, M3, P1 and T1) and N-acetyltransferases (NATs; NAT1 and NAT2). Making reference to these enzymes, we have assayed the current knowledge on the relations among polymorphisms of human xenobiotic-metabolizing enzymes and cancer susceptibilities. We have found intriguing models of susceptibility toward different types of cancer. We have reviewed and commented these models on light of the complex balance among different enzyme activities that, in each individual, determines the degree of each cancer susceptibility. Moreover, we have found techniques of molecular genetic analysis, more suitable than previous ones on phenotypic expression, now allowing better means to detect individuals at risk of cancer. According to the models presently available, a systematic screening of individuals at risk seems to make sense only in situations of well defined carcinogenic exposures and when performed by the polymorphism analysis of coordinated enzyme activities concurring to the metabolism of the carcinogen(s) in question. Genetic polymorphism analysis can allow for the detection of patients more prone to some types of specific cancers, or to the adverse effects of specific pharmaceutical agents. Considering the increasingly confirmed double-edged sword nature of metabolism polymorphism (both wild-type and variant alleles can predispose to cancer, albeit in different situations of exposure), individual susceptibility to cancer should be monitored as a function of the nature, and mechanism of action, of the carcinogen(s) to which the individual under study is known to be exposed, and with reference to the main target organ of the considered type of exposure.

In spite of the epidemiological evidence supporting a synergism between alcohol consumption and cigarette smoking in the pathogenesis of cancers of the aerodigestive tract, there is a paucity of experimental studies evaluating the effects of these agents under well-controlled conditions and exploring the mechanisms involved. We exposed groups of female BD6 rats, aged 8 months, to ethanol (5% in drinking water for 8 consecutive months) and/or whole-body to mainstream cigarette smoke (1 h/day, 5 days/week for 8 months). DNA was purified from different organs and analyzed for the presence of DNA-protein crosslinks and 32P-postlabeled DNA adducts after butanol enrichment. No significant increase of DNA-protein crosslinks, compared to untreated controls, was induced by any treatment in liver, lung, or heart. 'Spontaneous' nucleotidic modifications were detected by 32P-postlabeling in organs of untreated rats, with the highest levels occurring in the heart. Ingestion of ethanol did not affect DNA adduct levels in any of the organs examined: esophagus, liver, lung, and heart. Exposure to cigarette smoke induced formation of DNA adducts in the lung and heart, but not in the esophagus or liver. The combined ingestion of ethanol resulted in a significant formation of smoke-related DNA adducts in the esophagus and in their further, dramatic increase in the heart. It thus appears that ethanol consumption increases the bioavailability of DNA binding smoke components in the upper digestive tract and favors their systemic distribution. The mechanisms responsible for the interaction between ethanol and smoke and for the selective localization of DNA alterations in different organs are discussed. Formation of DNA adducts in the organs examined may be relevant in the pathogenesis of lung and esophageal cancers as well as in the pathogenesis of other types of chronic degenerative diseases, such as chronic obstructive pulmonary diseases and cardiomyopathies.

An in vivo study was carried out with the objectives of evaluating ( a ) the localization of DNA lesions resulting from exposure to chromium(VI) by the respiratory route, ( b ) the molecular nature of DNA alterations, and ( c ) modulation of DNA damage by a known chemopreventive agent. To this purpose, Sprague-Dawley rats received intratracheal instillations of sodium dichromate (0.25 mg/kg body weight) for three consecutive days, and the day after the last treatment lung and liver were removed for DNA purification. The results showed a selective localization of DNA lesions in the lung but not in the liver, which can be ascribed to toxicokinetics and metabolic characteristics of chromium(VI). DNA alterations included DNA-protein crosslinks, DNA fragmentation, nucleotidic modifications, and 8-hydroxy-2′-deoxyguanosine. The last two endpoints were evaluated, for the first time in chromium toxicology, by means of 32 P postlabeling procedures. This methodology was adapted to the detection of the DNA damage produced by those reactive oxygen species which result from the intracellular reduction of chromium(VI). The oral administration of the thiol N -acetylcysteine completely prevented any induction of DNA lesions in lung cells.

During the last 15 years the number of studies using postlabeling techniques to detect molecular alterations in DNA exposed to genotoxicagents has been continuously growing. Detectable molecules no longer include only bulky adducts arising from environmental exposures to polycyclic aromatic hydrocarbons, heterocyclic arom atic amines, cigarette smoke, etc. Postlabeling procedures are also able to reveal small DNA adducts and small nucleotide alterations induced by genotoxic agents such as alkylating compounds and aflatoxins. DNA alterations induced by oxidizing molecules both of exogenous and endogenous source can also be revealed. Postlabeling methods have been successfully used to detect DNA damage induced by ionizing and exciting radiations. Variants of the basic procedure allow the detection of endogenous DNA modifications associated with aging, called I-compounds. Postlabeling methods are able to detect such a great variety of molecular alterations by using a negligible amount of DNA (1-5 mug) with a...

Reactive oxygen species are involved in the pathogenesis of cancer and other chronic degenerative diseases through a variety of mechanisms, including DNA damage. We investigated by 32p and 33P postlabeling analyses the nucleotidic modifications induced in vitro by treating calf thymus DNA with H2O2 and CuSO4, interacting in a Fenton type reaction. Six different enrichment procedures and three chromatographic systems were comparatively assayed. The chromatographic system using phosphate/urea, which is more suitable for detecting bulky DNA adducts, was rather insensitive. In contrast, the system using acetic acid/ammonium formate revealed high levels of mononucleotidic modifications. In terms of ratio of adduct levels in treated and untreated DNA, the enrichment procedures ranked as follows: nuclease P1 (19.6), no enrichment (18.3), digestion to trinucleotides (17.6), digestion to monophosphate mononucleotides (8.4), digestion to dinucleotides (3.4), and extraction with butanol (1.0). The system using formic acid/ammonium formate was quite efficient in detecting 8-hydroxy-2'-deoxyguanosine. Labeling with 33p further enhanced the sensitivity of the method. The oxidative damage was so intense to produce a strong DNA fragmentation detectable by agarose gel electrophoresis, and nucleotidic modifications were more intense when DNA fragmentation was greater. The DNA alterations produced by H2O2 alone were significantly lower than those produced following reaction of H2O2 with CuSO4. The thiol N-acetylcysteine (NAC) was quite efficient in inhibiting both nucleotidic modifications and DNA fragmentation produced in vitro by either H2O2 or the .OH generating system. These results support at a molecular level the findings of previous studies showing the ability of NAC to inhibit the genotoxicity of peroxides and of reactive oxygen species generated by electron transfer reactions.

Primary open-angle glaucoma is a multifactorial disease that affects the retinal ganglion cells, but currently its therapy is to lower the eye pressure. This indicates a definite involvement of the trabecular meshwork, key region in the pathogenesis of glaucoma. This is the first target of glaucoma, and its functional complexity is a real challenge to search. Its functions are those to allow the outflow of aqueous humor and not the reflux. This article describes the morphological and functional changes that happen in anterior chamber. The “primus movens” is oxidative stress that affects trabecular meshwork, particularly its endothelial cells. In these develops a real mitochondriopaty. This leads to functional impotence, the trabecular meshwork altering both motility and cytoarchitecture. Its cells die by apoptosis, losing barrier functions and altering the aqueous humor outflow. All the morphological alterations occur that can be observed under a microscope. Intraocular pressure rises and the malfunctioning trabecular meshwork endotelial cells express proteins that completely alter the aqueous humor. This is a liquid whose functional proteomics complies with the conditions of the trabecular meshwork. Indeed, in glaucoma, it is possible detect the presence of proteins which testify to what occurs in the anterior chamber. There are six classes of proteins which confirm the vascular endothelium nature of the anterior chamber and are the result of the morphofunctional trabecular meshwork decay. It is possible that, all or in part, these proteins can be used as a signal to the posterior pole.

Assessing the correlation between molecular endpoints and cancer induction or prevention aims at validating the use of intermediate biomarkers. We previously developed murine models that are suitable to detect both the carcinogenicity of mainstream cigarette smoke (MCS) and the induction of molecular alterations. In this study, we used 931 Swiss mice in two parallel experiments and in a preliminary toxicity study. The chemopreventive agents included vorinostat, myo-inositol, bexarotene, pioglitazone and a combination of bexarotene and pioglitazone. Pulmonary micro-RNAs and proteins were evaluated by microarray analyses at 10 weeks of age in male and female mice, either unexposed or exposed to MCS since birth, and either untreated or receiving each one of the five chemopreventive regimens with the diet after weaning. At 4 months of age, the frequency of micronucleated normochromatic erythrocytes was evaluated. At 7 months, the lungs were subjected to standard histopathological analysis. The results showed that exposure to MCS significantly downregulated the expression of 79 of 694 lung micro-RNAs (11.4%) and upregulated 66 of 1164 proteins (5.7%). Administration of chemopreventive agents modulated the baseline micro-RNA and proteome profiles and reversed several MCS-induced alterations, with some intergender differences. The stronger protective effects were produced by the combination of bexarotene and pioglitazone, which also inhibited the MCS-induced clastogenic damage and the yield of malignant tumors. Pioglitazone alone increased the yield of lung adenomas. Thus, micro-RNAs, proteins, cytogenetic damage and lung tumors were closely related. The molecular biomarkers contributed to evaluate both protective and adverse effects of chemopreventive agents and highlighted the mechanisms involved.

The broad application of nanotechnology in medicine, biology, and pharmacology is leading to a dramatic increase of the risk of direct contact of nanoproducts, among which gold nanoparticles (AuNP), with the human organism. The present study aimed at evaluating in vivo the genotoxicity of AuNPs with average size of 40 nm and 100 nm. A single intraperitoneal treatment of adult male and female Swiss mice (strain H) with AuNPs, at a dose of 3.3 mg/kg body weight, had no effect on the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in bone marrow. Conversely, the transplacental treatment with AuNP-100 nm, but not with AuNP-40 nm, applied intraperitoneally at a dose of 3.3 mg/kg to pregnant mice on days 10, 12, 14, and 17 of gestation, resulted in a significant increase in the frequency of MN PCEs in both liver and peripheral blood of mouse fetuses. In parallel, the same treatment with AuNP-100 nm, but not with AuNP-40 nm, produced significant changes in microRNA expression. In particular, out of 1281 mouse microRNAs analyzed, 28 were dys-regulated more than two-fold and to a statistically significant extent in fetus lung, and 5 were up-regulated in fetal liver. Let-7a and miR-183 were significantly up-regulated in both organs. The data presented herein demonstrate for the first time the transplacental size-dependent clastogenic and epigenetic effects of AuNPs in mouse fetus, thus highlighting new aspects concerning the putative genotoxicity of AuNPs during a vulnerable period of life.

Aicardi–Goutieres syndrome (AGS), described by J. Aicardi and F. Goutieres in 1984, is a rare neurological disease with onset in infancy. It is often misdiagnosed as a sequela of congenital infection or recognized later. Nowadays almost 200 cases are reported all over the world, most of them collected by the International Aicardi–Goutieres Syndrome Association (IAGSA), founded in Pavia (Italy) in 2000. AGS (MIM 225750) is a genetically-determined encephalopathy characterized by severe neurological dysfunction, acquired microcephaly associated with severe prognosis quoad valetudinem, and less frequently also quoad vitam. Some AGS children also develop some symptoms overlapping with systemic lupus erythematosus (SLE). Intracranial calcification, white matter involvement and brain atrophy revealed on MRI, lymphocytosis and elevated levels of interferon alpha (IFN-α) in the cerebrospinal fluid (CSF) are features of both AGS and congenital viral infection. No evidence of congenital infection at serological exams has ever been found. A genetic etiology was hypothesized since the first descriptions, because of the recurrence in families, and demonstrated some years ago. Nowadays five genes (AGS1-5), if mutated, can be responsible for 90% of the cases. The transmission is autosomal recessive but there are also rare “de novo” autosomal dominant cases. Even if pathogenesis is still almost unknown, it seems that responsible genes are involved in nucleic acid reparation mechanisms and consequently in a secondary activation of innate autoimmunity. The relative lack of precise information on pathogenesis and on the evolution of the disease over time has not yet allowed the creation of codified diagnostic and therapeutic models and programs.

Biphosphonate (BPN) are widely used in clinics to treat meta- static cancer and osteoporosis thus representing a problem not only for patients but also for workers involved in their prepara- tion and administration. A similar exposure occurred years ago in match-making workers undergoing bone alterations similar to those consequent to BPN exposure. Osteonecrosis of the jaw (ONJ) is a main adverse effect related to BPN administration, which is performed in millions of patients worldwide for osteopo- rosis and cancer therapy, thus representing an emerging problem in public health. In susceptible patients, BPN induce severe, pro- gressive, and irreversible degeneration of facial bones, resulting in avascular ONJ often triggered by dental surgery. BPN induced ONJ occurs in subjects depending on lifestyle factors of both environmental and endogenous origins. Exogenous risk factors include cigarette smoke, alcohol consumption, bacterial infec- tions, and cyclosporine therapy. Endogenous risk factors include systemic diseases such as diabetes or hypertension and adverse polymorphisms of genes involved in metabolism (CYPs, MTHFR), thrombosis (Factor V, Prothrombin), and detoxification (MDR). Available molecular findings provide evidence that ONJ is related to risk-factors associated with environmental mutagenesis and gene-environment interactions. This issues may be useful to iden- tify susceptible subjects by molecular analyses in order to prevent ONJ occurrence., Journal of Preventive Medicine and Hygiene, Vol 54, No 3 (2013)

Liver DNA specimens from woodchucks kept in captivity, 10 naturally infected with hepatitis virus (WHV) and five WHV-free, were examined for the presence of carcinogen-DNA adducts by 32 P-postlabeling. The number of adducts was significantly higher in WHV carriers than in uninfected animals, and the total amounts of adducts per 10 9 nucleotides were also considerably enhanced by WHV infection, when using both butanol extraction (22.2 ± 7.1 vs, 12.6 ± 2.8, means ± S.D.) and nuclease P 1 enrichment (8.5 ± 5.9 vs. 2.8 ± 1.7). Two individual adducts were also significantly higher in WHV carriers. No significant variation occurred as related to age, sex or time length of captivity. These findings are consistent with our previous studies supporting an enhanced metabolism of chemical hepatocarcinogens in both human and woodchuck hepadnavirus infections. Several significant and remarkable correlations were pointed out by relating DNA adduct data to more than 30 virological, histopathological and metabolic parameters which had been previously evaluated in the same animals. For instance, numbers and/or levels of adducts were positively related to the amounts of virus present in hepatocytes, to cell damage (γ-glutamyltranspeptidase activity), to the severity of the liver histopathological picture, and to monooxygenase activities, while they were inversely related to cellular glutathione concentrations and to detoxification of the directacting mutagen 4-nitroquinoline 1-oxide. The major adduct significantly correlated with the metabolic activation of the aromatic amine 2-aminofluorene and of the heterocyclic amines 3-amino-1- methyl -5 H -pyrido(4,3)indole (Trp-P-2) and 2-amino-3,4-dimethylimidazo(4,5-f) quinoline (MeIQ), whereas another adduct significantly correlated with the metabolic activation of the mycotoxin aflatoxin B 1 . Thus, the enhanced metabolism of chemical hepatocarcinogens and the increased formation of carcinogen-DNA adducts in the liver of WHV carriers appear to represent one of the mechanisms contributing to the association between chronic hepadnavirus infection and development of primary hepatocellular carcinoma.

Disturbances in microRNA expression by epigenetic alterations and mutations may promote not only tumorigenesis but also tumor aggressiveness, invasion, metastasis, and resistance to chemotherapy and radiotherapy. Several studies have profiled microRNA expression in normal and tumorigenic tissues, demonstrating a unique microRNA signature, which can be used as a marker for cancer diagnosis and prognosis. This review discusses the importance of microRNAs as regulatory biomolecules involved in cancer, focusing on microRNAs related to cancer invasion, metastasis, epigenetic alterations, chemoresistance, and radioresistance. The identification of both differentially expressed microRNAs in tumors and their target genes provides new tools for gene therapy; the re-expression of microRNAs silenced by cancer development or the silencing of oncogenic microRNAs can be effective in the blockade of cancer-related cell proliferation.

T lymphocytes play a major role in counteracting cancer occurrence and development. Immune therapies against cancer are focused on eliciting a cytotoxic T cell response. This anticancer activity is related to a variety of mechanisms including the activation of cytokines and proapoptotic mediators. Interferon α is an established inhibitor of cancer cell growth. A clinical situation involving the coexistence of high interferon α levels and lymphocyte activation is the Aicardi-Goutières syndrome, a progressive encephalopathy arising usually during the first year of life characterized by intracranial basal ganglia calcifications, leukodystrophy and microcephaly. Aicardi-Goutières syndrome 1 mutation silences the TREX1 gene, a major endogenous nuclease. The in vitro study presented herein evaluates the efficacy of the TREX1 mutation in potentiating the anticancer properties of T cells. A TREX1-mutated lymphocyte cell line was derived from an Aicardi-Goutières syndrome patient and co-cultured with neuroblastoma cells and vascular endothelial cells in the presence of interferon α. TREX1-mutated lymphocytes exerted marked inhibitory action on neuroblastoma cell growth. Cathepsin D was recognized by qPCR as the main mediator produced by TREX1-mutated lymphocytes involved in the inhibition of neuroblastoma cell growth. These effects were enhanced in the presence of interferon α. Similar inhibitory effects in cell growth were exerted by TREX1-mutated lymphocytes towards vascular endothelial cell angiogenesis as evaluated on Matrigel. The results obtained provide evidence that mutations of the TREX1 gene increase the capability of T-lymphocytes to inhibit growth of neoplastic neuronal cells and related angiogenesis.

The purpose of this work was to investigate the expression of glutamine synthase (GS), nitric oxide synthase (NOS) superoxide dismutase (SOD) and glutathione transferase (GST) in the aqueous humor of patients with primary open angle glaucoma and controls. Aqueous humor proteome was analyzed by antibody microarray. The expression of tested proteins was detected by protein Cy3/Cy5 labeling, column purification and hybridization on antibody-spotted glass microarray. Fluorescent signals were detected by fluorescence laser scanning. Aqueous humor levels of SOD as well as of GST were significantly lower (2.0- and 2.2-fold, p < 0.01) among patients than controls; both NOS and GS expression were significantly higher (2.2- and 2.6 fold, p < 0.01) among patients than controls. Our data showed substantial differences of GS, NOS2, SOD and GST aqueous humor levels between glaucomatous patients and controls as measured by antibody microarray technology. The overproduction of NO through inducible NOS can form toxic products and change the metabolic conditions of the TM. The GS over-expression might be related to neuronal injury or to the potential role of glutamate as a modulator in the ciliary body signaling. The reduced expression of the antioxidant enzymes SOD and GST could aggravate the unbalance between both oxygen- and nitrogen-derived free radicals production and detoxification. Based on our results, GS, NOS2, SOD and GST as measured by antibody microarray technology may be useful oxidative markers in aqueous humor of glaucomatous patients.

MicroRNAs (miRNAs) have been implicated in many biological processes, cancer, and other diseases. In addition, miRNAs are dysregulated following exposure to toxic and genotoxic agents. Here we review studies evaluating modulation of miRNAs by dietary and pharmacological agents, which could potentially be exploited for inhibition of mutagenesis and carcinogenesis. This review covers natural agents, including vitamins, oligoelements, polyphenols, isoflavones, indoles, isothiocyanates, phospholipids, saponins, anthraquinones and polyunsaturated fatty acids, and synthetic agents, including thiols, nuclear receptor agonists, histone deacetylase inhibitors, antiinflammatory drugs, and selective estrogen receptor modulators. As many as 145 miRNAs, involved in the control of a variety of carcinogenesis mechanisms, were modulated by these agents, either individually or in combination. Most studies used cancer cells in vitro with the goal of modifying their phenotype by changing miRNA expression profiles. In vivo studies evaluated regulation of miRNAs by chemopreventive agents in organs of mice and rats, either untreated or exposed to carcinogens, with the objective of evaluating their safety and efficacy. The tissue specificity of miRNAs could be exploited for the chemoprevention of site-specific cancers, and the study of polymorphic miRNAs is expected to predict the individual response to chemopreventive agents as a tool for developing new prevention strategies.

To check the pathogenic role of exposure to mutagens in cardiovascular and eye disease we examined molecular damages in affected subjects. 107 atherosclerotic patients were tested for molecular alterations in aorta specimens and molecular biomarkers evaluated for their prognostic value in a 15-year follow up. Mitochondrial damage (mitochondrial DNA common deletion 4977 bp) and oxidative DNA damage (8-oxo-dG), detected at very high level in atherosclerotic aorta, were significantly related with patients survival. Physical activity dramatically decreased these biomarkers, improving survival, although with a remarkable inter-individual variability. Indeed, preventive effects of physical activity were mainly detected in patients bearing double GSTM/T homozygous deletion, which have a 3.6-year survival only in case of being sedentary while a 9.7-year survival in case of being physically active (a 30-minute walk a day). Dealing with ocular diseases, we analyzed 73 patients affected by glaucoma, the mos...

Dysregulation of microRNAs (miRNAs) has important consequences on gene and protein expression since a single miRNA targets a number of genes simultaneously. This article provides a review of published data and ongoing studies regarding the effects of cigarette smoke (CS), either mainstream (MCS) or environmental (ECS), on the expression of miRNAs and related proteins. The results generated in mice, rats, and humans provided evidence that exposure to CS results in an intense dysregulation of miRNA expression in the respiratory tract, which is mainly oriented in the sense of downregulation. In parallel, there was an upregulation of proteins targeted by the downregulated miRNAs. These trends reflect an attempt to defend the respiratory tract by means of antioxidant mechanisms, detoxification of carcinogens, DNA repair, anti-inflammatory pathways, apoptosis, etc. However, a long-lasting exposure to CS causes irreversible miRNA alterations that activate carcinogenic mechanisms, such as modulation of oncogenes and oncosuppressor genes, cell proliferation, recruitment of undifferentiated stem cells, inflammation, inhibition of intercellular communications, angiogenesis, invasion, and metastasis. The miRNA alterations induced by CS in the lung of mice and rats are similar to those observed in the human respiratory tract. Since a number of miRNAs that are modulated by CS and/or chemopreventive agents are subjected to single nucleotide polymorphisms in humans, they can be evaluated according to toxicogenomic/pharmacogenomics approaches. A variety of cancer chemopreventive agents tested in our laboratory modulated both baseline and CS-related miRNA and proteome alterations, thus contributing to evaluate both safety and efficacy of dietary and pharmacological agents.

Chemoprevention provides an important strategy for cancer control in passive smokers. Due to the crucial role played by smoke-related chronic inflammation in lung carcinogenesis, of special interest are extensively used pharmacological agents, such as nonsteroidal anti-inflammatory drugs (NSAIDs). We evaluated the ability of aspirin and naproxen, inhibitors of both cyclooxygenase-1 and cyclooxygenase -2, to modulate environmental cigarette smoke (ECS)-induced lung carcinogenesis in A/J mice of both genders. Based on a subchronic toxicity study in 180 postweaning mice, we used 1600 mg/kg diet aspirin and 320 mg/kg diet naproxen. In the tumor chemoprevention study, using 320 mice, exposure to ECS started soon after birth and administration of NSAIDs started after weaning. At 10 weeks of life, the NSAIDs did not affect the presence of occult blood in feces. As assessed in a subset of 40 mice, bulky DNA adducts and 8-hydroxy-2'-deoxyguanosine levels were considerably increased in ECS-exposed mice and, irrespective of gender, both NSAIDs remarkably inhibited these nucleotide alterations. After exposure for 4 months followed by 5 months in filtered air, ECS induced a significant increase in the yield of surface lung tumors, the 43.7% of which were adenomas and the 56.3% were adenocarcinomas. Oct-4 (octamer-binding transcription factor 4), a marker of cell stemness, was detected in some adenocarcinoma cells. The NAIDs attenuated the yield of lung tumors, but prevention of ECS-induced lung adenomas was statistically significant only in female mice treated with aspirin, which supports a role for estrogens in ECS-related lung carcinogenesis and highlights the antiestrogenic properties of NSAIDs.

The liver S12 fractions from 23 woodchucks were assayed for the ability to activate a cigarette smoke condensate to metabolites inducing frameshift mutations in strain TA98 of S. typhimurium. At equivalent protein concentration, all samples activated this complex mixture to a similar extent, without any significant difference related to sex, hepatitis virus (WHV) infection, or primary hepatocellular carcinoma. Thus, unlike aflatoxin B1, aromatic amines and heterocyclic amines, whose metabolic activation has been shown to be stimulated by WHV infection in the same liver samples used in the present study, genotoxic components present in the particulate of mainstream cigarette smoke do not appear to be more readily biotransformed in vitro by preparations of infected hepatocytes. A significant increase of metabolism was however recorded in a small number of WHV-infected pregnant animals, which deserves attention in the light of the adverse effects of both hepadnavirus infection an cigarette smoking in pregnancy.

In this report is described a method for the measurement of the endogenous poly(ADP-ribose) polymerase (pADPRP) activity in permeabilized lymphocytes isolated from peripheral blood samples. The potential use of the data obtained by this technique is proposed for biomonitoring human exposure to genotoxic agents. This multifunctional nuclear catalytic protein is involved in basic cell functions, but a major role is associated with DNA synthesis and DNA repair activity. In fact, this enzyme is promptly stimulated by DNA strand breaks or by free ends induced by endogenous or exogenous agents. This rapid increase of the basal activity level can be used to reveal biological modifications early after exposure to physical or chemical agents. During isolation and permeabilization of blood lymphocytes, DNA structure remained unaltered. Therefore, no artificial activation of the endogenous pADPRP activity was observed. This technique combines the radiometric assay of pADPRP with the isopycnic isolation of blood lymp...

Objective. The main aim of this paper is to review the evidence available from the date of PubMed?s inception to May 2011 for a link between cancer and physical activity (PA) in both animal models and humans. Methods. We decided to select studies that comply with the scheme proposed by the American College of Sports Medicine/ American Heart Association (ACSM/AHA) that distinguish occupational physical activity (OPA) and leisure-time physical activity (LT-PA), further classified in three levels of intensity (low, moderate and heavy) based on the Metabolic Equivalent of Task (MET) index. Results. Considering animal models, there was strong evidence for an inverse association between voluntary wheel exercise and the risk of colon and breast cancer. Regarding human studies, we identified the following main results: 1) colorectum: LT-PA provided an overall colon risk reduction of 13-14%; 2) breast: significant reduction in the frequency of post-menopausal (PMP) cancers in women that practiced heavy and moderate LT-PA; 3) prostate: heavy OPA and LT-PA seemed to reduce the risk of advanced prostate cancers; 4) endometrium: strong protective effect of heavy/moderate LT-PA among overweight/ obese women; 5) lung: inverse relationship between heavy LT-PA and lung cancer in former or current smokers across all histologies. Conclusion. Increased LT-PA is associated with cancer prevention in several organs, but strong biases, such as body mass index (BMI), gender and age, make it difficult to assess which aspects of PA contribute most strongly to the reduced risk. Furthermore, we found few studies that indicated a protective role for OPA in cancer prevention when compared with LT-PA., Journal of Preventive Medicine and Hygiene, Vol 52, No 4 (2011)

The glaucomas is a group of complex and heterogeneous ocular diseases representing the second leading cause of blindness, and almost 75 million people are affected worldwide (Quigley 1999) being a major issue for public health. The prevalence of glaucoma increases with age (Friedman et al. 2006). The reported prevalence among whites in their 80s varies widely across these studies, with estimates as low as 1.9%3 to as high as 8.8%. Glaucoma is a syndrome characterized by a progressive optical atrophy resulting from the apoptosis of the retinal ganglion cells (RGCs). Growing evidence obtained from clinical and experimental studies over the past decade strongly suggests the involvement of the reactive oxygen species (ROS) in glaucoma. Free radicals can directly induce neuronal death by a protease and phosphatase-gated mechanism distinct from apoptosis (See and Loeffler 2001). In glaucoma free radicals may damage the trabecular meshwork (TM) (Sacca et al. 2005) while in the posterior segment of the eye the process of apoptotic retinal ganglion cell death starts with exposure of glial cells to elevated concentrations of free radicals (Nakazawa et al. 2006). The final neurological damage results in progressive RGCs death, axon atrophy and degeneration also extending to the brain cortex (visual areas) finally leading to the characteristic optical-cup neuropathy and irreversible visual loss (Weber and Harman 2005) (Yucel et al. 2000). In addition to the loss of the ganglion cells, the most of glaucoma types is characterized by having a high intraocular pressure (IOP). This is the most important risk factor for this disease, even if it is not yet clear what are the pathogenic events connecting IOP to glaucoma phenotype. In any case the TM damage has a key role in the increasing of IOP.

BackgroundTrabecular meshwork and Schlemm's canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.Methodology/principal findingsPrimary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4(th) and 6(th) hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.Conclusions/significanceSLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels.

a b s t r a c t Exposure to environmental mutagens results in alteration of microRNA expression mainly oriented towards down-regulation, as typically observed in cigarette smoke. However, the molecular mechanism triggering this event is still unknown. To shed light on this issue, we developed an 'in silico' analysis testing 25 established environmental mutagens (polycyclic aromatic hydrocarbons, heterocyclic com- pounds, nitrosoamines, morpholine, ethylnitrosurea, benzene derivatives, hydroxyl amines, alkenes) for their potential to interfere with the function of DICER, the enzyme involved in the cytoplasmic phase of microRNA maturation. In order to analyse the binding affinity between DICER and each mutagen, the three-dimensional bioinformatic structures of DICER-RNase III domains and of mutagens have been constructed. The binding affinity of mutagens for each DICER's RNase III domain was estimated by cal- culating the global contact-energy and the number of intermolecular contacts. These two parameters reflect the stability of the DICER-mutagen complexes. All the 25 mutagens tested form stable complexes with DICER, 20 of which form a complex with DICER A domain, that is more stable than those formed by DICER with its natural substrate, i.e. double strand short RNAs. These mutagens are benzo(a)pyrene diol epoxide, nitroimidazoles, fluorenes, naphthalene, morpholine, stilbenes, hydroxylamines, fecapentenes. In the case of exposure to mutagen mixtures (benzo(a)pyrene-diolepoxide and 4-acetylaminostilbene), synergistic or less than addictive effects occur depending on the docking order of the compounds. A group of 8 mutagens with the highest ability to interfere with this DICER function, was identified by hierarchical cluster analysis. This group included 1-ethyl-1-nitrosourea and 4-nitrosomorpholine. Herein, presented data support the view that mutagens interfere with microRNA maturation by binding DICER. This finding sheds light on a new epigenetic mechanism exerted by environmental mutagens in inducing cell damage.

This study aims at applying cDNA microarray analysis in vitro for establishing and comparing the osteogenic properties of dental implants with different surface characteristics.Saos-2 osteoblasts were cultured in bottom-cone tubes in presence of 5 different dental implants with various surface characteristics. Cells adherent to dental implants were detached and RNA purified. The expression of 18,401 genes was tested by cDNA microarray.The number and viability of cells adherent to different dental implants varied but without any significant statistical difference. Conversely, gene expression was revealed to be a more sensitive biomarker being remarkably different in cells adherent to different implants. The 5 dental implants significantly modulated the expression of 14 osteogenic activities mainly including bone morphogenetic proteins, osteomodulin, and osteoprotegerin.Despite no significant differences having been found in in vitro cell number and viability, cells adherent to 5 differently surfaced implants showed different gene expression profiles. Thus, to evaluate osteogenesis as related to dental implants, it is important to analyze not only the number of adherent cells but also the activation of genes encoding for osteogenic activities.

Intracellular RNAses are involved in various functions, including microRNA maturation and turnover. Mutations occurring in genes encoding RNAses cause Aicardi-Goutieres syndrome (AGS). AGS mutations silence RNAse activity, thus inducing accumulation of endogenous RNAs, mainly consisting of short RNAs and microRNAs. Overload of intracellular RNA triggers Toll like receptor-dependent interferon-alpha production in the brain, which in turn activates neurotoxic lymphocytes and inhibits angiogenesis thus inducing the typical clinical phenotype of AGS. However, these pathogenic mechanisms are attenuated after three years of age by the endogenous production of DNAJP58IPK and Cystatin F, which arrest AGS progression. Because RNAses are involved in microRNA turnover, we evaluated the expression of 957 microRNAs in lymphocytes from AGS patients and control patients. Our results indicate that microRNA overload occurs in AGS patients. This upregulation inhibits microRNA turnover impeding the synthesis of the novel microRNAs required for the differentiation and myelination of the brain during the initial period of postnatal life. These pathogenic mechanisms result in AGS, a neurological syndrome characterized by irritability, mild hyperpyrexia, pyramidal and extrapyramidal signs, and spastic-dystonic tetraplegia. Typical cerebrospinal fluid alterations include lymphocytosis and elevated interferon-alpha levels. Brain imaging demonstrates cerebral calcifications, white matter abnormalities, and progressive cerebral atrophy.Thus, evidence exists that mutations silencing intracellular RNases affect microRNA turnover resulting in the severe clinical consequences in the brain characterizing the clinical feature of AGS.

Objective To test the ability of dorzolamide hydrochloride and timolol maleate to display antioxidant effects. Methods Antioxidant activity was tested in whole trabecular meshwork (TM) tissue as collected from corneal donors' biopsy specimens, young (third passage) and old (10th passage) human TM cells, and acellular systems composed of pure DNA and subcellular fractions containing or devoid of mitochondria. Oxidative stress was induced by hydrogen peroxide. Monitored end points included DNA fragmentation as evaluated by the halo test, oxidative DNA damage in terms of 8-hydroxy-2′-deoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide test. Results The antioxidant effect of dorzolamide and timolol were observed on TM biopsy specimens and human TM cells exposed to hydrogen peroxide. As evaluated in cell subfractions, timolol displays antioxidant activity regardless of mitochondria presence. Conversely, the antioxidant activity of dorzolamide was maximized in the presence of mitochondria-containing subcellular fractions and in young human TM cells with functional mitochondria. Conclusions The antioxidant effect of timolol was direct. The antioxidant effect of dorzolamide involves mitochondria and is likely to be exerted mainly during the early glaucoma phases when the mitochondrial damage in the TM tissue still occurs at low levels. Clinical Relevance Timolol has an antioxidant effect on the entire cell, whereas dorzolamide exerts protective activity toward oxidative stress only in the presence of intact mitochondria (ie, in endothelial cells that are younger when the cellular damage is still limited). The important role of mitochondrial damage in primary open- angle glaucoma is supported by the finding that mutant myocilin impairs mitochondrial functions in human TM meshwork cells.

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be involved in breast and prostate carcinogenesis, which share similar estrogen-related mechanisms. We previously demonstrated that administration of BPA to female mice results in the formation of DNA adducts and proteome alterations in the mammary tissue. Here, we evaluated the ability of BPA, given with drinking water, to induce a variety of biomarker alterations in male Sprague-Dawley rats. In addition, we investigated the formation of DNA adducts in human prostate cell lines. In BPA-treated rats, no DNA damage occurred in surrogate cells including peripheral blood lymphocytes and bone marrow erythrocytes, where no increase of single-strand DNA breaks was detectable by comet assay and the frequency of micronucleated cells was unaffected by BPA. Liver cells were positive at transferase dUTP nick end labeling assay, which detects both single-strand and double-strand breaks and early stage apoptosis. BPA upregulated clusterin expression in atrophic prostate epithelial cells and induced lipid peroxidation and DNA fragmentation in spermatozoa. Significant levels of DNA adducts were formed in prostate cell lines treated either with high-dose BPA for 24 h or low-dose BPA for 2 months. The BPA-related increase of DNA adducts was more pronounced in PNT1a nontumorigenic epithelial cells than in PC3 metastatic carcinoma cells. On the whole, these experimental findings support mechanistically the hypothesis that BPA may play a role in prostate carcinogenesis and may, potentially, affect the quality of sperm.

The study aims to investigate the effect of age and socio-economic status (SES) on breast cancer relative survival at 5 years from diagnosis (RS-5%). The Liguria Region Cancer Registry collected clinico-pathological information for 1081 patients resident in Genoa city diagnosed in 1996 and 2000. Patients were divided into three age groups (0-49, 50-69 and ≥70 years) and into quintiles of SES, measured according to a synthetic regional deprivation index built by Census tract variables and validated by comparison with the national deprivation index. The association of prognostic factors and RS-5% was evaluated by bivariate and multivariate analyses. RS-5% was very high for the first two age groups (91%) and decreased in patients aged 70 or older (82%) (unadjusted HR. =2.7, P=0.001). The first four SES quintiles had homogeneous RS-5% (89-92%) and only the last very deprived group had lower rates (77%) (unadjusted HR. =2.3, P=0.011). Very deprived elderly patients showed a much lower RS-5% (58%). Elderly and very deprived women were more likely to have large tumours, positive lymph nodes and less likely to receive conserving surgery, axillary dissection and adjuvant therapies. After adjusting for tumour characteristics and treatment modalities the increased risk of dying in the elderly disappeared completely, while the higher risk of very deprived women was eliminated only after correction for all the covariates simultaneously. The survival observed for breast cancer patients resident in Genoa was very high, however elderly and very low SES women have to be monitored. These variations are probably due to inequity in healthcare access and to a difficult taken in charge. © 2010 Elsevier Ireland Ltd.

Epidemiological studies have demonstrated that it is possible to prevent lung cancer and other smoke-related diseases by avoiding exposures to tobacco smoke. A complementary strategy is chemoprevention, which is based on the administration of dietary and pharmacological agents, which is addressed to (a) addicted active smokers, who are unable to quit smoking, (b) ex-smokers, who are still at risk for several years, and (c) involuntary smokers, including passively exposed individuals as well as transplacentally exposed individuals. The biological effects of cigarette smoke (CS) as a complex mixture, either mainstream (MCS) or sidestream (SCS) or environmental (ECS), have been poorly explored. We showed that MCS and ECS induce a broad variety of alterations of intermediate biomarkers in animal models, including adducts to nuclear DNA and mtDNA, oxidatively generated DNA damage, proliferation, apoptosis, alterations of oncogenes and tumor suppressor genes, multigene expression, microRNA and proteome profiles as well as cytogenetic damage in the respiratory tract, bone marrow and peripheral blood. CS-altered end-points were variously modulated by chemopreventive agents of natural or pharmacological origin, such as N-acetyl-L-cysteine (NAC), 1,2-dithiole-3-thione, oltipraz, 5,6-benzoflavone, phenethyl isothiocyanate (PEITC), indole-3-carbinol, sulindac, and budesonide. Combinations of agents were also assayed. Since it is difficult to assess the efficacy of chemopreventives in clinical trials, it is essential to understand the mechanisms by which certain agents are expected to prevent smoke-related cancer. Preclinical studies are also useful to demonstrate the potential efficacy of chemopreventive agents. Unfortunately, until recently a suitable animal model for evaluating CS carcinogenicity and its chemoprevention was not available. We demonstrated that ECS and especially MCS become potently carcinogenic when exposure of mice starts at birth, as shown by very short latency times, high incidence and multiplicity of benign lung tumors, early occurrence of malignant lung tumors, and lesions in other organs. This mouse model was successfully used to demonstrate the ability of NAC, PEITC, and budesonide to prevent smoke-induced lung cancer, according to protocols mimicking the situation either in current smokers or in ex-smokers. Other dietary or pharmacological agents, including curcumin, anthocyanins, myo-inositol, SAHA, bexarotene and pioglitazone, are now under study. NAC was even successful to prevent lung cancer induced by MCS after birth when it was administered during the prenatal life. Therefore, it is now possible to investigate in vivo not only alterations of intermediate biomarkers but also the modulation of CS carcinogenesis by chemopreventive agents working with different mechanisms.

Transition metals in fine particulate matter generated by combustion induce oxidative DNA damage and inflammation. However, there is remarkable inter-individual variability in susceptibility to these damages. To assess this variability, an ex vivo study was performed using lymphocytes of 47 Caucasian healthy subjects. Cell samples were exposed to a water solution of oil fly ash (OFA). This was formed by the distinctive transition metals vanadium, iron, and nickel. Oxidative DNA damage was evaluated by testing cell viability, intracellular ROS production and 8-oxo-dG. DNA fragmentation and DNA repair capacity were assessed by using the Alkaline-Halo assay. GSTM1, GSTT1, hOGG1, and C677T and A1298C MTHFR gene polymorphisms were tested. Demographic and behavioral factors, collected by questionnaire, were also considered. OFA induced damages showed: (a) a 20-fold variation in range among different subjects in ROS production, (b) a 7-fold variation in range of 8-oxo-dG, and (c) a 25-fold variation in range in DNA repair capacity. A significant increase in DNA damage was detected in GSTT1-deficent subjects compared with wild type genotype carriers. Increases in cytoplasmic ROS and decreases in DNA repair capacity (P0.05) were observed in C677T and A1298C variants of MTHFR. A remarkable protective effect of high fruits and vegetable intake was observed for ROS production and DNA damage. Conversely, an adverse effect of meat intake was observed on ROS increase, DNA damage and repair capacity, probably due to the increased intake of bioavailable iron. Smoking decreased DNA repair capacity, while age increased OFA-induced DNA damage. The wide comparative analysis of the complex interactions network, between genetic and behavioral factors provides evidence of the remarkable role of several lifestyle factors. In comparison to genetic polymorphisms they seem to have a higher weight in determining individual susceptibility to the adverse effects of airborne pollutants as transition metals.

We have investigated alterations of microRNA expression profiles in the apparently healthy lung of mice and rats as an early response to exposure to cigarette smoke, either mainstream (MCS) or environmental, and/or to treatment with chemopreventive agents. Further on, we evaluated microRNA alterations at a later stage, when lung tumors were detectable in MCS-exposed mice. Lung samples were available from previous studies, in which strain H mice had been exposed to MCS for 4 months, starting immediately after birth, and then kept in filtered air for an additional 3 months. Some samples were from MCS-exposed mice treated either with N-acetyl-l-cysteine during pregnancy or with phenethyl isothiocyanate after weaning. The analysis of 576 mouse microRNAs showed that MCS strongly dysregulated microRNA expression and that both chemopreventive agents efficiently attenuated this trend, especially in noncancer tissue. MicroRNA expression was affected by histopathology, with specific signatures related to occurrence of pneumonia, adenoma, or bronchoalveolar carcinoma. Within pairs of samples from individual mice, microRNA analysis discriminated adenomatous tissue and especially carcinomatous tissue from the surrounding normal appearing tissue. A series of microRNA alterations characterized the sequential stages of pulmonary carcinogenesis. The involved functions included oncogene activation, inhibition of oncosuppressor genes, recruitment of undifferentiated stem cells, inflammation, inhibition of gap-junctional intercellular communications, angiogenesis, invasiveness, and metastatization. Thus, microRNA expression profiles in lung are dysregulated by MCS along all steps of the carcinogenesis process and depend on the interplay among exposure to noxious agents, treatment with dietary and pharmacological agents, and occurrence of pulmonary diseases.

As the only nourishment and scavenging source for most of the anterior and posterior chamber tissues in the eye, the aqueous humor represents one of the target for glaucoma. The aim of this study is to investigate the yet unexplored relationship between aqueous humor protein content and open-angle glaucoma (POAG) pathogenesis. Aqueous humor was collected from 10 POAG patients (cases) and 14 senile cataract patients (controls), matched for age and gender, undergoing surgery for trabeculectomy and cataract, respectively. Protein samples were cyanine-labeled and hybridized with antibody microarrays. Microarray signals were revealed by laser scanner, quantified, and compared by statistical analyses. Total protein amounts were not significantly different in patients versus controls. Conversely, a proteome cluster significantly modified in patients as compared to controls was identified as highly predictive for disease status. Selected proteins underwent dramatic variation, which was correlated to pathogenetic events characterizing POAG, including oxidative damage, mitochondrial damage, neural degeneration, and apoptosis. The results obtained indicate that proteomic analysis of aqueous humor is a new tool for POAG diagnosis in the case of otherwise uncertain disease recognition. Furthermore, this study allows a better understanding of mechanisms involved in the pathogenesis of POAG, the main cause of irreversible blindness worldwide.

BackgroundOpen-angle glaucoma appears to be induced by the malfunction of the trabecular meshwork cells due to injury induced by oxidative damage and mitochondrial impairment. Here, we report that, in fact, we have detected mitochondrial damage only in primary open-angle glaucoma and pseudo-exfoliation glaucoma, among several glaucoma types compared.Methodology/principal findingsMitochondrial damage was evaluated by analyzing the common mitochondrial DNA deletion by real-time PCR in trabecular meshwork specimens collected at surgery from glaucomatous patients and controls. Glaucomatous patients included 38 patients affected by various glaucoma types: primary open-angle, pigmented, juvenile, congenital, pseudoexfoliative, acute, neovascular, and chronic closed-angle glaucoma. As control samples, we used 16 specimens collected from glaucoma-free corneal donors. Only primary open-angle glaucoma (3.0-fold) and pseudoexfoliative glaucoma (6.3-fold) showed significant increases in the amount of mitochondrial DNA deletion. In all other cases, deletion was similar to controls.Conclusions/significancedespite the fact that the trabecular meshwork is the most important tissue in the physiopathology of aqueous humor outflow in all glaucoma types, the present study provides new information regarding basic physiopathology of this tissue: only in primary open-angle and pseudoexfoliative glaucomas oxidative damage arising from mitochondrial failure play a role in the functional decay of trabecular meshwork.

To analyze the frequency of mitochondrial DNA (mtDNA) damage in patients with primary open-angle glaucoma. Oxidative damage plays a major role in glaucoma pathogenesis. Since no environmental risk factor for glaucoma is recognized, we focused our attention on mitochondria, the main endogenous source of reactive oxygen species.Mitochondrial damage was evaluated analyzing a common mtDNA deletion by real-time polymerase chain reaction in trabecular meshwork collected at surgery from 79 patients with primary open-angle glaucoma and 156 unaffected matched controls. In the same samples, polymorphisms of genes encoding for antioxidant defenses (GSTM1), repair of oxidative DNA damage (OGG1), and apoptosis (FAS) were tested.Mitochondrial DNA deletion was dramatically increased (5.32-fold; P = .01) in trabecular meshwork of patients with glaucoma vs controls. This finding was paralleled by a decrease in the number of mitochondria per cell (4.83-fold; P.001) and by cell loss (16.36-fold; P.01). Patients with glaucoma bearing the GSTM1-null genotype showed increased amounts of mtDNA deletion and a decreased number of mitochondria per cell as compared with GSTM1-positive subjects. Patients bearing a FAS homozygous mutation showed only a decreased number of mitochondria per cell.Obtained results indicate that mitochondrion is targeted by the glaucomatous pathogenic processes. Some subjects bearing adverse genetic assets are more susceptible to this event. Clinical Relevance Oxidative damage to the trabecular meshwork exerts a pathogenic role in glaucoma inducing mitochondrial damage and triggering apoptosis and cell loss. This issue may be useful to develop new glaucoma molecular biomarkers and to identify high-risk subjects.

Although microRNAs (miRNA) have extensively been investigated in cancer research, less attention has been paid to their regulation by carcinogens and/or protective factors in early stages of the carcinogenesis process. The present study was designed to evaluate the modulation of mRNA expression as related to exposure of neonatal mice to environmental cigarette smoke (ECS) and to treatment with chemopreventive agents. Exposure to ECS started immediately after birth and for 2 weeks after weaning. Thereafter, groups of mice received daily either budesonide (BUD) or phenethyl isothiocyanate (PEITC) with the diet. The expression of 576 miRNAs was evaluated by miRNA microarray in liver and lung. In sham-exposed mice, the expression of miRNAs tended to be higher in liver than in lung. ECS downregulated the expression of a number of miRNAs in lung, whereas mixed alterations were observed in liver. PEITC and BUD did not substantially affect the physiological situation in lung, whereas both agents caused intense variations in liver, reflecting the occurrence of damage mechanisms, such as inflammation, DNA and protein damage, cellular stress, proliferation and apoptosis. PEITC and BUD protected the lung from ECS-induced alterations of miRNA expression but exhibited some adverse effects in liver.

Background and objective: An important issue in human biomonitoring is determining how exposure duration affects the kinetics of molecular biomarkers. In this study we compare the influence of exposure variables on DNA adducts.Methods: DNA adducts were analysed by 32P-postlabelling in lympho/monocytes of 677 Caucasian subjects.Results: After correction for other variables, DNA adducts increased depending on the length of occupational and smoke exposures. Higher DNA adducts were detected in workers with more than 14 years of exposure than in workers with shorter exposures (RR = 1.19, p = 0.049) and in smokers with more than 10 years of exposure than in smokers with shorter exposure (RR = 1.21, p