Odor emissions: A public health concern for health risk perception.

The olfactory nuisance, due to the emissions of active molecules, is mainly associated with unproperly managed waste disposal and animal farming. Volatile compounds e.g., aromatics, organic and inorganic sulfide compounds, as well as nitrogen and halogenated compounds are the major contributor to odor pollution generated by waste management plants; the most important source of atmospheric ammonia is produced by livestock farming. Although an odorous compound may represent a nuisance rather than a health risk, long-term exposure to a mixture of volatile compounds may represent a risk for different diseases, including asthma, atopic dermatitis, and neurologic damage. Workers and communities living close to odor-producing facilities result directly exposed to irritant air pollutants through inhalation and for this reason the cumulative health risk assessment is recommended. Health effects are related to the concentration and exposure duration to the odorants, as well as to their irritant potency and/or biotransformation in hazardous metabolites. The health effects of a single chemical are well known, while the interactions between molecules with different functional groups have still to be extensively studied. Odor emissions are often due to airborne pollutants at levels below the established toxicity thresholds. The relationship between odor and toxicity does not always occurs but depends on the specific kind of pollutant involved. Indeed, some toxic agents does not induce odor nuisance while untoxic agents do. Accordingly, the relationship between toxicity and odor nuisance should be always analyzed in detail evaluating on the characteristics of the airborne mixture and the type of the source involved.

Long term mortality and morbidity of Italian soldiers after deployment in Iraq as related to biomarkers assessment: Results of the SIGNUM study.

INTRODUCTION: The health profile of military veterans deployed in foreign operative theatres was assessed by several international studies because of potential exposure to depleted uranium and other pollutants. Here we reported results of 15-year epidemiological surveillance assessing long-term health effects in a cohort of Italian soldiers deployed in Iraq in 2004-2005 and participating in a biomonitoring campaign to identify potential genotoxic exposure to environmental xenobiotics before and after deployment (n = 981, SIGNUM cohort). METHODS: We evaluated mortality and hospitalization risks of the SIGNUM cohort retrospectively until 2016 and 2018 respectively. A wide cohort of military personnel never deployed abroad (n = 114,260) and the general Italian population were used as control populations in risk assessment. Causes of death and diagnoses of hospitalization were derived through deterministic record linkage with official national databases of mortality and hospital discharge. Standardized Mortality Ratio (SMR) and Standardized Hospitalization Ratio (SHR) were computed adjusting according to sex, age, area of birth, and calendar year. Differential pre-post deployment in xenobiotics concentrations and early effect biomarkers (oxidative DNA alterations and micronuclei) measured in blood serum were analysed in relation to cancer hospitalization. RESULTS: Mortality risk due to pathologies was more than halved compared to the general population (SMR = 0.41, 95% CI 0.11-1.05) and not significantly different compared to soldiers never deployed abroad (SMR = 0.69, 95% CI 0.19-1.68). Similarly overall hospitalization risk due to pathologies was decreased with respect to the general population (SHR = 0.86, 95% CI 0.80-0.92) and comparable to the control military group (SHR = 0.99, 95% CI: 0.93-1.06). For haematological cancers a decreased hospitalization risk compared to the Italian general population was observed (SHR = 0.38, 95% CI 0-0.92). No statistically significant differences emerged in the patterns of biomarkers in association with cancer hospitalization. CONCLUSION: The study confirms the so called 'healthy warrior' effect for the SIGNUM veterans and showed no correlation between cancer occurrence and biomonitoring markers measured on field.

Development of an integrated environmental monitoring protocol for SARS-CoV-2 contamination. Applications at the IRCSS San Martino Polyclinic Hospital in Genoa, Italy.

SCIENTIFIC BACKGROUND: Environmental sampling of SARS-CoV-2 is a fundamental tool for evaluating the effectiveness of non-specific prophylaxis measures in counteracting virus spread. The purpose of our work was to evaluate the effectiveness of the different sampling methods in the hospital setting to assess their correlation with the structural, functional, and operational situation of the monitored departments and to define the dynamics of the spread of the virus in indoor environments. METHODS: The monitoring (air bubbling sampling, surface wipe test) was carried out at the San Martino Polyclinic Hospital (Genoa, Italy) in the period since April 2020 to June 2021. The presence of viral RNA in the collected samples was evaluated by qPCR. The infection capacity of the samples collected was also evaluated by an in vitro challenge test on cells sensitive to SARS-CoV-2 infection. RESULTS: The percentage of positivity with respect to the number of tests performed (sensitivity) were air bubbler 50%, wipe test 17%, and challenge test 11%. Only 20% of the samples tested positive in the wipe test and 43% of the samples tested positive in the bubbler sampling were also positive in the challenge test. All the positivity obtained was detected at a distance of less than 2 m and height of less than 1.5 from COVID-19 patients. CONCLUSIONS: Environmental contamination from SARS-CoV-2 detected at the San Martino Polyclinic Hospital is found lower than similar assessments performed in other hospitals both in Italy and abroad. Our study predicted that environmental monitoring of SARS-CoV-2 must be carried out in an integrated way by not using a single sampling method, as each individual test has a different biological significance and performance. However, the virus detected by wipe test only is often a degraded viral fragment and not an intact infecting virion.

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.

Microarray expression in peri-implant tissue next to different titanium implant surfaces predicts clinical outcomes: a split-mouth study.

OBJECTIVES: This split-mouth study evaluated miRNA expression of tissues around implants with different surface treatments. MATERIAL AND METHODS: Each patient of the sample (five men and five women) received two implants (one control and one test) into an edentulous quadrant to support fixed partial dentures. The control implants (Osseotite) had a dual acid-etched (DAE) surface in the apical portion and a machined coronal part, test implants (Full Osseotite, FOSS) were completely DAE. Machined healing abutments were placed on control implants and DAE abutments on test ones. All implants were assigned codes for blinding. Standardized periapical radiographs were taken at baseline, 2 and 6 months, and 1 year after surgery. Plaque index (PI), bleeding on probing (BOP), and probing depth (PD) were recorded at 3 and 6 weeks, and 2, 3, 6, and 12 months post-implant placement. After 3 months, a mini-invasive sample of soft tissue was collected from seven patients (four women and three men) for miRNA microarray analysis. RESULTS: Control implants showed greater bone resorption (BR) and lower PI: this was not statistically significant. No statistically significant differences in BOP and PD appeared. miRNA modulated by implant surfaces as well as by other clinical conditions has been identified. miRNA microarray analysis revealed that: (i) implant sites with low PI and absence of BOP had a miRNA expression profile similar to those with plaque and absence of BOP; sites with high PI and high BOP had a different profile. (ii) Implant sites with BOP presented similar profiles independently from implant surface. (iii) Implant sites with high PI and normal BR differed from others for miRNA expression profile. (iv) Implant sites with normal BR despite high BOP differed from others. This profile resembled that of FOSS implants. (v) Implant surface affected BR; groups having similar BR clusterized differently according to the implant type. CONCLUSIONS: DAE surfaces induced lower BR and more plaque accumulation: This did not affect the health of soft tissues. miRNA analysis indicated that soft tissue inflammation is more related to gene expression profile than to plaque or to implant surface. Specific miRNA profile can protect implant sites from bleeding and BR irrespective of plaque accumulation.

Molecular fingerprints of environmental carcinogens in human cancer.

Identification of specific molecular changes (fingerprints) is important to identify cancer etiology. Exploitable biomarkers are related to DNA, epigenetics, and proteins. DNA adducts are the turning point between environmental exposures and biological damage. DNA mutational fingerprints are induced by carcinogens in tumor suppressor and oncogenes. In an epigenetic domain, methylation changes occurs in specific genes for arsenic, benzene, chromium, and cigarette smoke. Alteration of specific microRNA has been reported for environmental carcinogens. Benzo(a)pyrene, cadmium, coal, and wood dust hits specific heat-shock proteins and metalloproteases. The multiple analysis of these biomarkers provides information on the carcinogenic mechanisms activated by exposure to environmental carcinogens.

Indoor pollution and burning practices in wood stove management.

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 ofsolvents andpaints 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 ofeducation, supported by reliable data on air pollution, as an effective method to reduce wood smoke exposures.

Inhibition of the de-myelinating properties of Aicardi-Goutières syndrome lymphocytes by cathepsin D silencing.

Molecular mechanisms relating interferon-alpha (IFN-alpha) to brain damage have recently been identified in a microarray analysis of cerebrospinal fluid lymphocytes from patients with Aicardi-Goutières Syndrome (AGS). These findings demonstrate that the inhibition of angiogenesis and the activation of neurotoxic lymphocytes are the major pathogenic mechanisms involved in the brain damage consequent to elevated interferon-alpha levels. Our previous study demonstrated that cathepsin D, a lysosomal aspartyl endopeptidase, is the primary mediator of the neurotoxicity exerted by AGS lymphocytes. Cathepsin D is a potent pro-apoptotic, neurotoxic, and demyelinating protease if it is not properly inhibited by the activities of leukocystatins. In central nervous system white matter, demyelination results from cathepsin over-expression when not balanced by the expression of its inhibitors. In the present study, we used RNA interference to inhibit cathepsin D expression in AGS lymphocytes with the aim of decreasing the neurotoxicity of these cells. Peripheral blood lymphocytes collected from an AGS patient were immortalized and co-cultured with astrocytes in the presence of interferon alpha with or without cathepsin D RNA interference probes. Cathepsin D expression was measured by qPCR, and neurotoxicity was evaluated by microscopy. RNA interference inhibited cathepsin D over-production by 2.6-fold (P<0.01) in AGS lymphocytes cultured in the presence of interferon alpha. AGS lymphocytes treated using RNA interference exhibited a decreased ability to induce neurotoxicity in astrocytes. Such neurotoxicity results in the inhibition of astrocyte growth and the inhibition of the ability of astrocytes to construct web-like aggregates. These results suggest a new strategy for repairing AGS lymphocytes in vitro by inhibiting their ability to induce astrocyte damage and leukodystrophy.

Biphosphonates-associated osteonecrosis of the jaw: the role of gene-environment interaction.

Biphosphonate (BPN) are widely used in clinics to treat metastatic cancer and osteoporosis thus representing a problem not only for patients but also for workers involved in their preparation 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 osteoporosis and cancer therapy, thus representing an emerging problem in public health. In susceptible patients, BPN induce severe, progressive, 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 infections, 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 identify susceptible subjects by molecular analyses in order to prevent ONJ occurrence.

Aqueous humor oxidative stress proteomic levels in primary open angle glaucoma.

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.

Analysis of nestin protein in the aqueous humor as biomarker of open angle glaucoma.

Primary open angle glaucoma (POAG) is a progressive optic nerve degeneration, leading to irreversible visual damage. Alterations of the aqueous humor (AH), the biological fluid filling both the anterior and the posterior chambers of the eye, play a pathogenic role in POAG. AH protein composition is altered during glaucoma progression. Nestin protein was found to be differentially expressed in the AH of glaucomatous patients compared to unaffected matched controls. METHODS: Nestin was analyzed by an open quartz crystal microbalance (QCM) in the AH of 21 glaucomatous patients compared to nine unaffected controls. The surface of the electrode used in the QCM was coated with an analyte-specific recognition layer. RESULTS: Positive nestin values were recorded in the AH collected from POAG patients; negative values of nestin detection were obtained by analyzing the AH collected from non-POAG glaucomatous patients and unaffected controls. CONCLUSION: The present study proposes and validates a new clinically applicable approach to analyze biological markers in AH for POAG diagnosis.

The Aicardi-Goutières syndrome. Molecular and clinical features of RNAse deficiency and microRNA overload.

Intracellular RNAses are involved in various functions, including microRNA maturation and turnover. Mutations occurring in genes encoding RNAses cause Aicardi-Goutiéres 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.

Physical activity and cancer prevention: a review of current evidence and biological mechanisms.

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.

Proteome alterations in primary open angle glaucoma aqueous humor.

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.

Degenerative periodontal-diseases and oral osteonecrosis: the role of gene-environment interactions.

Chronic-degenerative dentistry diseases, including periodontal diseases and oral osteonecrosis, are widespread in human populations and represent a significant problem for public health. These diseases result from pathogenic mechanisms created by the interaction between environmental genotoxic risk-factors and genetic assets conferring individual susceptibility. Osteonecrosis occurs in subjects undergoing exposure to high doses of DNA-damaging agents for chemo- and radiotherapy of neoplastic diseases. In susceptible patients, ionizing radiation and biphosphonate-chemotherapy induce severe, progressive, and irreversible degeneration of facial bones, resulting in avascular necrosis of the jaw. This may also occur in patients receiving biphosphonate for osteoporosis therapy. Periodontal diseases include chronic, aggressive, and necrotizing periodontitis, often resulting in severe alteration of periodontal tissues and tooth loss. Cigarette smoking and chronic inflammation caused by specific bacteria are the main risk factors for periodontitis. Oxidative damage plays a fundamental pathogenic role, as established by detection of mitochondrial DNA damage in the gingival tissue of patients with periodontitis. Endogenous risk factors in dental diseases include polymorphisms for metabolic enzymes such as glutathione transferases M1 and T1, N-acetyl transferase 2, and CYP 1A1. Other genetic polymorphisms that confer susceptibility to dentistry diseases affect genes encoding metalloproteases (involved in periodontal tissue remodeling and degradation), cytokines (involved in inflammation), prothrombin, and DNA repair activities. These findings provide evidence that dentistry diseases are related to risk factors associated with environmental mutagenesis. This issue warrants future investigations aimed at improving oral health and preventing oral degenerative diseases using molecular and experimental approaches currently utilized in mutagenicity studies.

Gene-environment interactions in ocular diseases.

Degenerative ocular diseases are widespread in the population and represent a major cause of reversible and irreversible blindness. Scientific evidences have been accumulating supporting the role of genotoxic damage and gene environment interactions in the pathogenesis of these diseases mainly including glaucoma, age-related macular degeneration, and cataract. Glaucoma, in its degenerative form, is characterized by the degeneration of the trabecular meshwork, the tissue of the anterior chamber of the eye devoted to aqueous-humour outflow. Such a degenerative process results in intra-ocular pressure increase and progressive damage of optic nerve head. Oxidative stress and DNA damage play an important role in inducing the degeneration of these well differentiated target tissues in which DNA damage results in a progressive cell loss. Macular degeneration is a common age-related disease affecting the central regions of the retina inducing progressive accumulation of oxidized lipoproteins and neovascularization. Environmental genotoxic risk factors include diet, light, and cigarette smoke paralleled by individual susceptibility as determined by adverse genetic assets. Cataract is a progressive opacity of the crystalline lens resulting from molecular damages induced by various risk factors including UV-containing light. This disease has been related to a failure in antioxidant defences. Experimental study provides evidence that cataract patients possess higher basal level of DNA damage, as evaluated by Comet test, in lymphocytes than controls. This finding is paralleled by the higher susceptibility to oxidative stress observed in the same patients. These novel experimental data further support the role of DNA damage as a main factor contributing to cataract onset. In conclusion, the examined degenerative ocular diseases recognise environmental risk factors often displaying genotoxic attitudes. Whenever these factors target individuals who are susceptible due their genetic assets the results is the onset of a specific eye disease depending on the affected ocular tissue.

Extracellular vesicles in biological fluids. A biomarker of exposure to cigarette smoke and treatment with chemopreventive drugs.

Extracellular vesicles (EVs) are released from cells and enter into body fluids thereby providing a toxicological mechanism of cell-cell communication. The present study aimed at assessing (a) the presence of EVs in mouse body fluids under physiological conditions, (b) the effect of exposure of mice to cigarette smoke for 8 weeks, and (c) modulation of smoke-related alterations by the nonsteroidal anti-inflammatory drug celecoxib, a selective cyclooxygenase-2 inhibitor. To this purpose, ICR (CD-1) mice were either unexposed or exposed to cigarette smoke, either treated or untreated with oral celecoxib. EVs, isolated from bronchoalveolar lavage fluid (BALF), blood serum, and urines, were analyzed by nanoparticle tracking analysis and flow cytometry. EVs baseline concentrations in BALF were remarkably high. Larger EVs were detected in urines. Smoking increased EVs concentrations but only in BALF. Celecoxib remarkably increased EVs concentrations in the blood serum of both male and female smoking mice. The concentration of EVs positive for EpCAM, a mediator of cell-cell adhesion in epithelia playing a role in tumorigenesis, was much higher in urines than in BALF, and celecoxib significantly decreased their concentration. Thus, the effects of smoke on EVs concentrations were well detectable in the extracellular environment of the respiratory tract, where they could behave as delivery carriers to target cells. Celecoxib exerted both protective mechanisms in the urinary tract and adverse systemic effects of likely hepatotoxic origin in smoke-exposed mice. Detection of EVs in body fluids may provide an early diagnostic tool and an end-point exploitable for preventive medicine strategies.

Induction by carcinogens and chemoprevention by N-acetylcysteine of adducts to mitochondrial DNA in rat organs.

Damage to mitochondrial DNA (mtDNA) has been postulated to be associated with aging, cancer, and other chronic degenerative diseases which are the predominant causes of death in the population. We used molecular dosimetry techniques, i.e., 32P postlabeling and synchronous fluorescence spectrophotometry, in order to evaluate the formation of adducts to both mtDNA and nuclear DNA (nDNA) in different organs of rats exposed to genotoxic carcinogens. Adducts to mtDNA were detected following administration of benzo(a)pyrene i.p. or 2-acetylaminofluorene by gavage as well as following exposure of animals to cigarette smoke. mtDNA adduct levels were consistently higher than those to nDNA in the same cells, and qualitative differences were also pointed out in the case of the aromatic amine. The oral administration of the thiol N-acetylcysteine, one of the most promising cancer chemopreventive agents endowed with nucleophilic and antioxidant properties, produced a significant decrease of mtDNA adducts in the liver of 2-acetylaminofluorene-treated rats and, sharply, in the lung and liver of rats exposed to cigarette smoke.

Patterns of DNA adduct formation in liver and mammary epithelial cells of rats treated with 7,12-dimethylbenz(a)anthracene, and selective effects of chemopreventive agents.

7,12-Dimethylbenz(a)anthracene (DMBA) is a prototype carcinogen that induces a high yield of mammary tumors in rats after a single feeding. We investigated the induction and chemoprevention of DNA adducts in female Sprague Dawley rats receiving DMBA by gavage according to a variety of treatment schedules. The patterns of 32P-postlabeled DNA adducts in liver and mammary epithelial cells were similar to those produced by the in vitro reaction of metabolically activated DMBA with calf thymus DNA. There was a high and statistically significant correlation between dose of DMBA administered to rats (0, 0.6, 2.4, and 12 mg/kg body weight) and levels of DNA adducts in both types of cells. The regression lines relating DMBA doses to total DNA adduct levels were significantly divergent and crossed at 1.5 mg/kg body weight, indicating that, at lower doses, the formation of DNA adducts is more intense in target mammary cells, whereas at higher doses, DNA adduct levels are more elevated in liver cells, presumably due to the greater metabolic capacity of this organ. When the rats were sacrificed 7 days rather than 2 days after DMBA administration, DNA adduct levels were approximately halved in both liver and mammary cells. The observed patterns can be interpreted based on toxicokinetic factors, local and distant metabolism, removal of DNA adducts by excision repair, and cell proliferation rate. Of three chemopreventive agents given with the diet to rats treated with 12 mg of DMBA, 5,6-benzoflavone (1650 ppm) was the most effective, inhibiting DNA adduct formation in liver and mammary cells by 96.5 and 83.5%, respectively. Feeding of 1,2-dithiole-3-thione (600 ppm) inhibited this biomarker by 68.5 and 50.2%, whereas butyl hydroxyanisole (BHA; 5000 ppm) showed a significant inhibition in the liver (46.5%) but was ineffective in mammary cells (29.0%, not significant). These data correlate nicely with the results of a parallel study in which 5,6-benzoflavone, 1,2-dithiole-3-thione, and BHA inhibited formation of hemoglobin adducts by 80.0, 44.0, and 0%, respectively; the incidence of mammary tumors by 82.4, 47.1, and 5.9%, respectively; and their multiplicity by 92.6, 80.0, and 7.4%, respectively. Therefore, biomarkers of biologically effective dose are highly predictive of the efficacy of chemopreventive agents in the DMBA rat mammary model. The selective inhibition by BHA of DNA adducts in the liver but not in mammary cells is consistent with the finding that this phenolic antioxidant stimulated phase II activities in the liver but not in the mammary gland (L. L. Song et al., manuscript in preparation). In any case, the broad-spectrum inducer 5,6-BF appears to be more effective than the two monofunctional phase II inducers, presumably because an enhanced activation of DMBA to reactive metabolites is coordinated with their blocking, detoxification, and excretion.

Biomarker alterations produced in rat lung by intratracheal instillations of air particulate extracts and chemoprevention with oral N-acetylcysteine.

Organic matter extracts were obtained from particulates recovered from 10,000-m3 air samples collected in Sicily (Italy). The overall concentrations of acenaphthene, benzo(a)pyrene, phenanthrene, anthracene, fluoranthene, and pyrene were 526 ng/m3 air in a highly polluted urban area and 48 ng/m3 in a rural area affected by motor vehicle traffic pollution. After metabolic activation, both samples were mutagenic in Salmonella typhimurium his(-) strains of the TA and YG series, with potencies in TA100 of 140.7 and 11.8 revertants/m3 air, respectively. The samples, resuspended in tricaprylin, were instilled intratracheally in Sprague-Dawley rats for 5 consecutive days, accounting for a cumulative dose in each animal of the organic fractions extracted from 400 m3 air, which corresponds approximately to the volume of air inhaled by a man in 1 month. Treatment with the rural area sample and, at higher levels, with the urban area sample resulted in the formation of adducts to lung DNA, as assessed both by synchronous fluorescence spectrophotometry and by 32P postlabeling, which showed the appearance of up to six individual adducts emerging from diffuse diagonal radioactive zones. The adducts were more efficiently detected by extraction with butanol than by digestion with nuclease P1. DNA binding of air particulate extracts was followed by alterations of early damage biomarkers only in the rats treated with the urban area sample. Repair of DNA damage in lung cells was inferred from a significant stimulation of the nuclear enzyme poly(ADP-ribose) polymerase compared with that in sham-exposed rats. Among the cells recovered by bronchoalveolar lavage, an increase in polymorphonucleate leukocytes and cells of the ciliated respiratory epithelium was accompanied by a relative decrease in pulmonary alveolar macrophages. The frequency of micronuclei was significantly enhanced both in epithelial cells and alveolar macrophages, and binucleated macrophages were also more frequent in treated rats. The thiol N-acetylcysteine, one of the most promising cancer chemopreventive agents, was administered with drinking water to a group of animals receiving the air particulate polycyclic aromatic hydrocarbon fraction from the urban area. N-acetylcysteine prevented or considerably attenuated the alterations of all monitored parameters. These findings provide evidence that, even under outstandingly high exposure conditions, it is possible to protect the respiratory tract from DNA-binding and DNA-damaging air particulate carcinogens.