Modulation of smoke-induced DNA and microRNA alterations in mouse lung by licofelone, a triple COX-1, COX-2 and 5-LOX inhibitor.

Chronic inflammation plays a crucial role in the carcinogenesis process and, in particular, in smoking-related carcinogenesis. Therefore, anti-inflammatory agents provide an interesting perspective in the prevention of smoking-associated cancers. Among nonsteroidal anti-inflammatory drugs (NSAIDs), licofelone is a triple inhibitor of both cyclooxygenases (COX-1 and COX-2) and of 5-lipooxygenase (5-LOX) that has shown some encouraging results in cancer prevention models. We previously showed that the dietary administration of licofelone, starting after weanling, to Swiss H mice exposed for 4 months to mainstream cigarette smoke since birth attenuated preneoplastic lesions of inflammatory nature in both lung and urinary tract, and had some effects on the yield of lung tumors at 7.5 months of age. The present study aimed at evaluating the early modulation by licofelone of pulmonary DNA and RNA alterations either in smoke-free or smoke-exposed H mice after 10 weeks of exposure. Licofelone protected the mice from the smoke-induced loss of body weight and significantly attenuated smoke-induced nucleotide alterations by decreasing the levels of bulky DNA adducts and 8-hydroxy-2'-deoxyguanosine in mouse lung. Moreover, the drug counteracted dysregulation by smoke of several pulmonary microRNAs involved in stress response, inflammation, apoptosis, and oncogene suppression. However, even in smoke-free mice administration of the drug had significant effects on a broad panel of microRNAs and, as assessed in a subset of mice used in a parallel cancer chemoprevention study, licofelone even enhanced the smoke-induced systemic genotoxic damage after 4 months of exposure. Therefore, caution should be paid when administering licofelone to smokers for long periods.

Carcinogenic response and other histopathological alterations in mice exposed to cigarette smoke for varying time periods after birth.

In spite of the outstanding role of tobacco smoking in human carcinogenesis, it is difficult to reproduce its effects in experimental animals. Based on the knowledge that a variety of mechanisms account for a higher susceptibility to carcinogens early in life, we have developed a murine model in which mainstream cigarette smoke becomes convincingly carcinogenic. The standard model involves exposure to smoke for 4 months, starting after birth, followed by an additional 3-4 months in filtered air. We evaluated herein the time- and dose-dependent response, at 7.5 months of life, of Swiss H mice that had been exposed to smoke for either 1, 2 or 4 months after birth. A one-month exposure, corresponding to a period of intense alveolarization, was sufficient to induce most inflammatory, degenerative and preneoplastic pulmonary lesions, including emphysema and alveolar epithelial hyperplasia, blood vessel proliferation and hemangiomas, reflecting an early proangiogenic role of smoking, and microadenomas bearing ki-67-positive proliferating cells as well as urinary bladder epithelial hyperplasia. Two months of exposure were needed to induce pulmonary adenomas and urinary bladder papillomas in males only, which highlights a protective role of estrogens in urinary bladder carcinogenesis. Four months, which in humans would correspond to the postnatal period, puberty, adolescence and early adulthood, were needed to induce other lesions, including tubular epithelial hyperplasia of kidney, bronchial epithelial hyperplasia and especially pulmonary malignant tumors. These findings highlight the concept that preneoplastic and neoplastic lesions occurring in adulthood can be induced by exposure to smoke early in life.

Aspirin abrogates impairment of mammary gland differentiation induced by early in life second-hand smoke in mice.

Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.

Reduction of hexavalent chromium by fasted and fed human gastric fluid. I. Chemical reduction and mitigation of mutagenicity.

Evaluation of the reducing capacity of human gastric fluid from healthy individuals, under fasted and fed conditions, is critical for assessing the cancer hazard posed by ingested hexavalent chromium [Cr(VI)] and for developing quantitative physiologically-based pharmacokinetic models used in risk assessment. In the present study, the patterns of Cr(VI) reduction were evaluated in 16 paired pre- and post-meal gastric fluid samples collected from 8 healthy volunteers. Human gastric fluid was effective both in reducing Cr(VI), as measured by using the s-diphenylcarbazide colorimetric method, and in attenuating mutagenicity in the Ames test. The mean (±SE) Cr(VI)-reducing ability of post-meal samples (20.4±2.6µgCr(VI)/mL gastric fluid) was significantly higher than that of pre-meal samples (10.2±2.3µgCr(VI)/mL gastric fluid). When using the mutagenicity assay, the decrease of mutagenicity produced by pre-meal and post-meal samples corresponded to reduction of 13.3±1.9 and 25.6±2.8µgCr(VI)/mL gastric fluid, respectively. These data are comparable to parallel results conducted by using speciated isotope dilution mass spectrometry. Cr(VI) reduction was rapid, with >70% of total reduction occurring within 1min and 98% of reduction is achieved within 30min with post-meal gastric fluid at pH2.0. pH dependence was observed with decreasing Cr(VI) reducing capacity at higher pH. Attenuation of the mutagenic response is consistent with the lack of DNA damage observed in the gastrointestinal tract of rodents following administration of ≤180ppm Cr(VI) for up to 90days in drinking water. Quantifying Cr(VI) reduction kinetics in the human gastrointestinal tract is necessary for assessing the potential hazards posed by Cr(VI) in drinking water.

Selective inhibition by aspirin and naproxen of mainstream cigarette smoke-induced genotoxicity and lung tumors in female mice.

The role of nonsteroidal anti-inflammatory drugs (NSAIDs) in smoke-related lung carcinogenesis is still controversial. We have developed and validated a murine model for evaluating the tumorigenicity of mainstream cigarette smoke (MCS) and its modulation by chemopreventive agents. In the present study, the protective effects of the nonselective cyclooxygenase inhibitors aspirin and naproxen were investigated by using a total of 277 Swiss H neonatal mice of both genders. Groups of mice were exposed whole-body to MCS during the first 4 months of life, followed by an additional 3.5 months in filtered air in order to allow a better growth of tumors. Aspirin (1600 mg/kg diet) and naproxen (320 mg/kg diet) were given after weanling until the end of the experiment. After 4 months of exposure, MCS significantly enhanced the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of mice, and naproxen prevented such systemic genotoxic damage in female mice. After 7.5 months, exposure of mice to MCS resulted in the formation of lung tumors, both benign and malignant, and in several other histopathological lesions detectable both in the respiratory tract and in the urinary tract. Aspirin and, even more sharply, naproxen significantly inhibited the formation of lung tumors in MCS-exposed mice, but this protective effect selectively occurred in female mice only. These results lend support to the views that estrogens are involved in smoke-related pulmonary carcinogenesis and that NSAIDs have antiestrogenic properties. The two NSAIDs proved to be safe and efficacious in the experimental model used.

Modulation by aspirin and naproxen of nucleotide alterations and tumors in the lung of mice exposed to environmental cigarette smoke since birth.

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.

Assay of lapatinib in murine models of cigarette smoke carcinogenesis.

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.

Rationale and approaches to the prevention of smoking-related diseases: overview of recent studies on chemoprevention of smoking-induced tumors in rodent models.

Tobacco smoke plays a dominant role in the epidemiology of lung cancer, cancer at other sites, and a variety of other chronic diseases. It is the leading cause of death in developed countries, and the global burden of cancer is escalating in less developed regions. For a rational implementation of strategies exploitable for the prevention smoking-related diseases, it is crucial to elucidate both the mechanisms of action of cigarette smoke and the protective mechanisms of the host organism. The imperative primary prevention goal is to avoid any type of exposure to smoke. Epidemiological studies have shown that a decrease in the consumption of cigarettes can be successful in attenuating the epidemic of lung cancer in several countries. Chemoprevention by means of dietary and/or pharmacological agents provides a complementary strategy aimed at decreasing the risk of developing smoking-associated diseases in addicted current smokers, who are unable to quit smoking, and especially in involuntary smokers and ex-smokers. The availability of new animal models that are suitable to detect the carcinogenicity of cigarette smoke and to assess the underlying molecular mechanisms provides new tools for evaluating both safety and efficacy of putative chemopreventive agents.

DNA damage in exfoliated cells and histopathological alterations in the urinary tract of mice exposed to cigarette smoke and treated with chemopreventive agents.

Cigarette smoke (CS) is convincingly carcinogenic in mice when exposure starts at birth. We investigated the induction and modulation of alterations in the kidney and urinary bladder of CS-exposed mice. A total of 484 strain H Swiss mice were either sham-exposed or exposed since birth to mainstream CS (MCS) for 4 months. Dietary agents, including myo-inositol, suberoylanilide hydroxamic acid, bexarotene, pioglitazone and a combination of bexarotene and pioglitazone, were administered after weaning. Comet analyses showed that, after 2 and 4 months, MCS causes DNA damage in exfoliated urothelial cells, which can be prevented by myo-inositol and the peroxisome proliferator-activated receptor-γ ligand pioglitazone. After 7 months, the 17.6% of MCS-exposed male mice exhibited lesions of the urinary tract versus the 6.1% of sham-exposed mice, which emphasizes the role of sex hormones in urinary tract carcinogenesis. Myo-inositol and the RXR-specific retinoid bexarotene did not affect these alterations. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (Vorinostat) increased the incidence of kidney epithelium hyperplasia. Pioglitazone significantly enhanced the incidence of kidney lesions as compared with mice exposed to MCS only, indicating possible adverse effects of this antidiabetic drug, which were lost upon combination with bexarotene according to a combined chemoprevention strategy. RXR is a heterodymeric partner for peroxisome proliferator-activated receptor-γ, thereby modulating the expression of multiple target genes. In conclusion, there is contrast between the ability of pioglitazone to inhibit DNA damage in exfoliated cells and the alterations induced in the urinary tract of MCS-exposed mice, suggesting the occurrence of non-genotoxic mechanisms for this drug.

Relationships between pulmonary micro-RNA and proteome profiles, systemic cytogenetic damage and lung tumors in cigarette smoke-exposed mice treated with chemopreventive agents.

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.

Oxidative stress in the lung of mice exposed to cigarette smoke either early in life or in adulthood.

Birth and early life stages are critical periods characterized by severe alterations of the redox balance and by "physiological" genomic changes in lung cells, which may be responsible for cancer and other diseases in adulthood. Oxidative stress is a major mechanism accounting for the carcinogenicity of cigarette smoke (CS), which becomes more potently carcinogenic in mice when exposure starts at birth and continues early in life. We compared herewith a variety of end-points related to oxidative stress, mitochondrial alterations, and cell turnover in the lung of Swiss H mice, either sham-exposed or CS-exposed for 4 weeks, starting either at birth or at 4 months of age. The results showed that the physiological levels of certain end-points are affected by age. In fact, the baseline proportion of hypodiploid cells and the mitochondrial potential and mass were higher in adults, whereas 8-hydroxy-2'-deoxyguanosine (8-oxo-dGuo) levels, the proportion of necrotic cells, and the extent of autophagy were higher early in life. Adult mice were more responsive to CS by increasing the proportion of necrotic cells and of cells in S/G2 phase, whereas young mice maintained a high extent of autophagy, exhibited a greater increase of lipid peroxidation products and 8-oxo-dGuo levels, and had a higher frequency of micronucleated cells. In addition, exposure to CS affected the mitochondrial potential/mass, especially in young mice. In conclusion, these data provide evidence that oxidative stress and the resulting DNA damage provide a major contribution to the high susceptibility of mice to CS early in life.

Comet-FISH analysis of urothelial cells. A screening opportunity for bladder cancer?

INTRODUCTION: Bladder cancer (BCa) is the most frequent cancer of the urinary tract, with more than 500,000 reported cases and nearly 200,000 related deaths yearly. Cystoscopy is the standard examination used for the initial diagnosis and follow-up of BCa in the noninvasive stage. However, the American Cancer Society does not include BCa screening in its list of recommended cancer screenings. AREAS COVERED: Recently, several urine-based bladder tumor markers (UBBTMs) that identify genomic, transcriptomic, epigenetic, or protein alterations have been introduced, some of which have been approved by the Food and Drug Administration (FDA) to improve its diagnosis and surveillance. Several biomarkers have been found in the tissues and blood of individuals with BCa or predisposed to develop the disease, further enriching our information. EXPERT OPINION: From a prevention perspective, alkaline Comet-FISH could be a valuable tool with broad potential for clinical application. Furthermore, a comet assay could be more beneficial for diagnosing and monitoring bladder cancer and determining individual susceptibility. Thus, we recommend further studies to understand the potential of this combined assay in the general population as a potential screening test and in patients initiated into the diagnostic process.

Dose-related cytogenetic damage in pulmonary alveolar macrophages from mice exposed to cigarette smoke early in life.

The micronucleus test detects both structural and numerical chromosomal aberrations caused by environmental agents. However, this test is poorly sensitive to detect the clastogenicity of cigarette smoke (CS) in human peripheral blood lymphocytes. At variance with peripheral blood lymphocytes and other cells outside the lower respiratory tract, pulmonary alveolar macrophages (PAM) are selectively affected by inhalable carcinogens and have been used to evaluate the modulation of CS-related cytogenetic alterations in vivo. The present study was aimed at evaluating (1) the cytogenetic response in PAM isolated from the lung of mice exposed to CS during the first 4 weeks of life and (2) the dose dependence of MN and polynucleated (PN) PAM formation in CS-exposed mice. To this purpose, ICR(CD-1) mice were exposed whole body to mainstream CS for 4 weeks, starting immediately after birth. Bronchoalveolar lavage (BAL) was performed to evaluate the cellularity of this fluid and the frequency of PN and MN PAM. At the doses of 119, 292, and 438 mg/m(3) total particulate matter, CS significantly increased both the proportion of PAM in the BAL fluid and the frequencies of PN and MN PAM. The cytogenetic effects were significantly correlated with the CS dose. In conclusion, PAM are suitable to detect induction by CS of clastogenic and aneugenic effects in mice during a developmental period corresponding to infancy, childhood, and early adolescence in humans. These surrogate cells, providing an important defense mechanism of the respiratory tract, are proposed as indicators of CS-related DNA damage in youngsters.

Inhibition of the Cell Uptake of Delta and Omicron SARS-CoV-2 Pseudoviruses by N-Acetylcysteine Irrespective of the Oxidoreductive Environment.

The binding of SARS-CoV-2 spikes to the cell receptor angiotensin-converting enzyme 2 (ACE2) is a crucial target both in the prevention and in the therapy of COVID-19. We explored the involvement of oxidoreductive mechanisms by investigating the effects of oxidants and antioxidants on virus uptake by ACE2-expressing cells of human origin (ACE2-HEK293). The cell uptake of pseudoviruses carrying the envelope of either Delta or Omicron variants of SARS-CoV-2 was evaluated by means of a cytofluorimetric approach. The thiol N-acetyl-L-cysteine (NAC) inhibited the uptake of both variants in a reproducible and dose-dependent fashion. Ascorbic acid showed modest effects. In contrast, neither hydrogen peroxide (H2O2) nor a system-generating reactive oxygen species (ROS), which play an important role in the intracellular alterations produced by SARS-CoV-2, were able to affect the ability of either Delta or Omicron SARS-CoV-2 pseudoviruses to be internalized into ACE2-expressing cells. In addition, neither H2O2 nor the ROS generating system interfered with the ability of NAC to inhibit that mechanism. Moreover, based on previous studies, a preventive pharmacological approach with NAC would have the advantage of decreasing the risk of developing COVID-19, irrespective of its variants, and at the same time other respiratory viral infections and associated comorbidities.

[Mechanistic issues and prevention strategies targeting occupational carcinogenesis]. / Aspetti meccanicistici e linee di prevenzione in cancerogenesi professionale.

Carcinogenesis can be visualized either as a multistep process (initiation, promotion, progression, invasion, and metastasis) or as a continuum of mutagenic and mitogenic events, with the contribution of epigenetic mechanisms. The exponential growth of the neoplastic mass explains the importance of secondary prevention (early diagnosis) and of tertiary prevention. Primary prevention, which was successful in controlling occupational cancers, aims at minimizing exposures to carcinogens in healthy subjects and at favoring the intake of chemopreventive agents with dietary and pharmacological agents. Besides chemical carcinogens, often in the form of complex mixtures, the workplace may involve exposures to physical agents, such as sunlight and artificial illumination systems delivering UV radiation, or to biological agents, such as chronic viral infections (HBV, HCV, and HIV) associated with cancers. A controversial issue is the occurrence of threshold doses for carcinogens in the workplace and the environment.

Microbial-based cleaning products as a potential risk to human health: A review.

Microbial-based cleaning products (MBCPs) have been introduced, on the market, as an alternative to traditional chemical cleaning. In addition to traditional detergents, MBCPs can perform their cleaning function, digesting the smallest particles of dirt and mitigating odours generated by environmental bacterium metabolic processes. Nevertheless, several aspects remain to be clarified and assessed, requiring further studies and new regulations to ensure safety. The particular composition of MBCPs makes it difficult to include these products in a specific class, making the European legal context incomplete and unclear. Moreover, MBCPs effects on human health are poorly documented. Exposure risks can be obtained indirectly by studies conducted in both microorganisms exposure and their metabolic products, such as enzymes, especially in workers. A further limiting factor for the accurate human health risk assessment due to MBCPs use is an incomplete indication about the MBCPs compositions. Moreover, additional factors such as host microorganisms, frequency and space of use, subject health condition, and age can determine different illness scenarios. The findings from the broad range of studies we have reviewed in this paper confirm the necessity of integrative investigation and regulation to address the use of MBCPs.

Attenuation of oxidative stress and chromosomal aberrations in cultured macrophages and pulmonary cells following self-sustained high temperature synthesis of asbestos.

Inhalation of asbestos fibres can cause lung and pleural diseases in humans and constitutes a severe public health threat worldwide. The aim of the present study was to assess the biological effects induced in both pulmonary cells (A549) and monocyte/macrophage (RAW 264.7) cell lines by combustion slags obtained from asbestos through a self-sustained high-temperature synthesis (SHS) reaction. The SHS reaction involves rapid thermal treatment and displays great ability to neutralise asbestos. Cytotoxicity, redox status imbalance, lipid peroxide production, DNA strand breaks (comet assay) and chromosomal aberrations (cytokinesis block micronucleus test) were evaluated in cells exposed either to untreated asbestos fibres or to grinded SHS-generated slags of different granulometry, tested in cultured cells at varying doses and for varying exposure times. Our results show that asbestos fibres cause redox status imbalance, especially in monocyte/macrophage cell lines. Moreover, they promote lipid peroxidation and trigger genomic alterations. When the cells were exposed to slag powders, which are the products of SHS asbestos treatment, generation of lipid peroxides and induction of DNA strand breaks still persisted, due to the high content in iron and other metals detected in these samples. However, there was an attenuation of redox status imbalance and an absence of chromosomal aberrations, which probably reflects the loss of the asbestos fibrous structure following SHS reaction, as demonstrated by electron microscopy analyses. In conclusions, SHS-treated asbestos wastes can potentially have deleterious health effects due to the oxidative stress induced by inhaled powders but they loose the asbestos ability to induce chromosomal alterations.

Brain microglia activation induced by intracranial administration of oligonucleotides and its pharmacological modulation.

Oligonucleotide overloading results in type I interferonopathies such as the Aicardi-Goutiéres Syndrome, a progressive encephalopathy determined by an immune response against endogenous DNA/RNA molecules. No therapy targeting pathogenic mechanisms is available for affected patients. Accordingly, we set up an in vitro/in vivo experimental model aimed at reproducing the pathogenic mechanisms of type I interferonopathies, in order to develop an effective pharmacological modulation and toxicological alterations caused by intracranial delivery of encapsulated CpG. The in vitro model used Aicardi-Goutiéres Syndrome immortalized lymphocytes activated by interferon I and co-cultured with human astrocytes; lymphocyte neurotoxicity was attenuated by the calcineurin-inhibitor Tacrolimus and by the anti-interferon monoclonal antibody Sifalimumab. The in vivo model was set up in mice by subcutaneous injection of encapsulated CpG oligonucleotides; the immune-stimulating activity was demonstrated by cytometric analysis in the spleen. To mime pathogenesis of type I interferonopathies in the central nervous system, CpG oligonucleotides were administered intracranially in mice. In the brain, CpG overload induced a rapid activation of macrophage-like microglial cells and focal accumulation mononuclear cells. The subcutaneous administration of Tacrolimus and, more potently, Sifalimumab attenuated CpG-induced brain alterations. These findings shed light on molecular mechanisms triggered by oligonucleotides to induce brain damage. Monoclonal antibodies inhibiting interferon seem a promising therapeutic strategy to protect brain in type I interferonopathies.