The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens.

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Mechanistic Interrogation of Cell Transformation In Vitro: The Transformics Assay as an Exemplar of Oncotransformation.

The Transformics Assay is an in vitro test which combines the BALB/c 3T3 Cell Transformation Assay (CTA) with microarray transcriptomics. It has been shown to improve upon the mechanistic understanding of the CTA, helping to identify mechanisms of action leading to chemical-induced transformation thanks to RNA extractions in specific time points along the process of in vitro transformation. In this study, the lowest transforming concentration of the carcinogenic benzo(a)pyrene (B(a)P) has been tested in order to find molecular signatures of initial events relevant for oncotransformation. Application of Enrichment Analysis (Metacore) to the analyses of the results facilitated key biological interpretations. After 72 h of exposure, as a consequence of the molecular initiating event of aryl hydrocarbon receptor (AhR) activation, there is a cascade of cellular events and microenvironment modification, and the immune and inflammatory responses are the main processes involved in cell response. Furthermore, pathways and processes related to cell cycle regulation, cytoskeletal adhesion and remodeling processes, cell differentiation and transformation were observed.

Source-related components of fine particulate matter and risk of adverse birth outcomes in Northern Italy.

BACKGROUND/AIM: The aim of the present study was to assess the association between PM2.5, its sources, and preterm birth (PTB), low birth weight (LBW), and small for gestational age (SGA) in a large open residential cohort (Supersito Project in the Emilia-Romagna Region - Northern Italy). METHODS: We collected 2012-2014 pregnancy and childbirth data from Birth Assistance Certificates and selected the pregnancies of interest. PTBs (gestational age < 37 weeks), LBW (weight < 2500 g), and SGA (newborns weighing ≤ 10th age and pregnancy week-specific percentile) were considered. Three-year measurements of daily concentrations and constituents of PM2.5 were available at four sites and were analyzed through a source apportionment approach identifying 6 sources (traffic, biomass burning, oil combustion, anthropogenic mix, and two secondary factors). Exposure to PM2.5 and sources was calculated at address level. Using logistic regression models, associations between exposure and outcomes were derived, applying single-pollutant and two-pollutant models, to verify the independent effect of each source. RESULTS: The study included 23,708 neonates born to 23,415 women, among whom 1,311 PTB, 424 LBW, and 1,354 SGA occurred. PTB was the only outcome associated with PM2.5 mass (OR 1.03, 95% CI 1.002-1.058 per 1 µg/m3). Traffic, oil combustion and secondary sulfates and organics showed independent effects on PTB. Exposure to secondary nitrates was associated with a lower risk of PTB. There was no association between LBW or SGA and source-specific PM2.5 components or the residual PM2.5 related to all other sources. CONCLUSION: This study found an association between PTB and PM2.5. Traffic, secondary sulfates, and organic and oil combustion were the sources with most consistent association.

Chemical carcinogen safety testing: OECD expert group international consensus on the development of an integrated approach for the testing and assessment of chemical non-genotoxic carcinogens.

While regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests (which include mutagenicity assays). If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, but under most chemical regulations (except plant protection, biocides, pharmaceuticals), this is rare. The decision to conduct further testing based on genotoxicity test outcomes creates a regulatory gap for the identification of non-genotoxic carcinogens (NGTxC). With the objective of addressing this gap, in 2016, the Organization of Economic Cooperation and Development (OECD) established an expert group to develop an integrated approach to the testing and assessment (IATA) of NGTxC. Through that work, a definition of NGTxC in a regulatory context was agreed. Using the adverse outcome pathway (AOP) concept, various cancer models were developed, and overarching mechanisms and modes of action were identified. After further refining and structuring with respect to the common hallmarks of cancer and knowing that NGTxC act through a large variety of specific mechanisms, with cell proliferation commonly being a unifying element, it became evident that a panel of tests covering multiple biological traits will be needed to populate the IATA. Consequently, in addition to literature and database investigation, the OECD opened a call for relevant assays in 2018 to receive suggestions. Here, we report on the definition of NGTxC, on the development of the overarching NGTxC IATA, and on the development of ranking parameters to evaluate the assays. Ultimately the intent is to select the best scoring assays for integration in an NGTxC IATA to better identify carcinogens and reduce public health hazards.

The transformics assay: first steps for the development of an integrated approach to investigate the malignant cell transformation in vitro.

The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.

Disruptive chemicals, senescence and immortality.

Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated 'selection and succession' of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of 'driver mutations' enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes.

Metabolic reprogramming and dysregulated metabolism: cause, consequence and/or enabler of environmental carcinogenesis?

Environmental contributions to cancer development are widely accepted, but only a fraction of all pertinent exposures have probably been identified. Traditional toxicological approaches to the problem have largely focused on the effects of individual agents at singular endpoints. As such, they have incompletely addressed both the pro-carcinogenic contributions of environmentally relevant low-dose chemical mixtures and the fact that exposures can influence multiple cancer-associated endpoints over varying timescales. Of these endpoints, dysregulated metabolism is one of the most common and recognizable features of cancer, but its specific roles in exposure-associated cancer development remain poorly understood. Most studies have focused on discrete aspects of cancer metabolism and have incompletely considered both its dynamic integrated nature and the complex controlling influences of substrate availability, external trophic signals and environmental conditions. Emerging high throughput approaches to environmental risk assessment also do not directly address the metabolic causes or consequences of changes in gene expression. As such, there is a compelling need to establish common or complementary frameworks for further exploration that experimentally and conceptually consider the gestalt of cancer metabolism and its causal relationships to both carcinogenesis and the development of other cancer hallmarks. A literature review to identify environmentally relevant exposures unambiguously linked to both cancer development and dysregulated metabolism suggests major gaps in our understanding of exposure-associated carcinogenesis and metabolic reprogramming. Although limited evidence exists to support primary causal roles for metabolism in carcinogenesis, the universality of altered cancer metabolism underscores its fundamental biological importance, and multiple pleiomorphic, even dichotomous, roles for metabolism in promoting, antagonizing or otherwise enabling the development and selection of cancer are suggested.

Environmental immune disruptors, inflammation and cancer risk.

An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis.

One of the important 'hallmarks' of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

Wastewater-Based Epidemiology for SARS-CoV-2 in Northern Italy: A Spatiotemporal Model.

The study investigated the application of Wastewater-Based Epidemiology (WBE) as a tool for monitoring the SARS-CoV-2 prevalence in a city in northern Italy from October 2021 to May 2023. Based on a previously used deterministic model, this study proposed a variation to account for the population characteristics and virus biodegradation in the sewer network. The model calculated virus loads and corresponding COVID-19 cases over time in different areas of the city and was validated using healthcare data while considering viral mutations, vaccinations, and testing variability. The correlation between the predicted and reported cases was high across the three waves that occurred during the period considered, demonstrating the ability of the model to predict the relevant fluctuations in the number of cases. The population characteristics did not substantially influence the predicted and reported infection rates. Conversely, biodegradation significantly reduced the virus load reaching the wastewater treatment plant, resulting in a 30% reduction in the total virus load produced in the study area. This approach can be applied to compare the virus load values across cities with different population demographics and sewer network structures, improving the comparability of the WBE data for effective surveillance and intervention strategies.

In vitro evaluation of the carcinogenic potential of perfluorinated chemicals.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the major components of long-chain per- and polyfluorinated alkyl substances (PFAS), known for their chemical stability and environmental persistence. Even if PFOA and PFOS have been phased out or are limited in use, they still represent a concern for human and environmental health. Several studies have been performed to highlight the toxicological behavior of these chemicals and their mode of action (MoA). Data suggested the causal association between PFOA or PFOS exposure and carcinogenicity in humans, but the outcomes of epidemiological studies showed some inconsistency. Moreover, the hypothesized MoA based on animal studies is considered not relevant for human cancer. In order to improve the knowledge on PFAS toxicology and contribute to the weight of evidence for the regulatory classification of PFAS, we used the BALB/c 3T3 cell transformation assay (CTA), an in vitro model under consideration to be included in an integrated approach to testing and assessment for non-genotoxic carcinogens (NGTxCs). PFOS and PFOA were tested at several concentrations by using a validated experimental protocol. Our results demonstrated that PFOA is not able to induce cell transformation, whereas PFOS exposure led to a concentration-related increase of type-III foci. Malignant foci formation is triggered at PFOS concentrations equal to or higher than 50 ppm. It is not directly associated with cytotoxicity or proliferation induction. The divergent CTA outcomes suggest that different molecular events could be responsible for the toxicological profiles of PFOS and PFOA, which were not completely captured in our study.

This study explored PFOS and PFOA, common PFAS chemicals, to understand their potential harm and cancer risk. PFAS are known for their durability and resistance to heat, water and oil. They are persistent in the environment and may pose health risks despite decreased use. To better understand how PFOS and PFOA might be harmful, we conducted an in vitro test that can resemble the carcinogenesis process in experimental animals. Testing these chemicals on lab cells revealed PFOS can cause cancer-like changes, at levels of 50 parts per million or higher, but not PFOA. This difference suggests PFAS chemicals affect cells differently, but we need more research to understand exactly how they work and how they might cause cancer. Understanding this could help regulate and reduce PFAS harmful effects. Moreover, this research aligns with 3R principles by using cell-based tests as an alternative to animal testing, promoting ethical research practices.

The micronucleus assay as a biological dosimeter in hospital workers exposed to low doses of ionizing radiation.

The health risk associated with low levels of ionizing radiation is still a matter of debate. A number of factors, such as non-target effects, adaptive responses and low-dose hypersensitivity, affect the long-term outcome of low-dose exposures. Cytogenetic bio-dosimetry provides a measure of the absorbed dose, taking into account the individual radiation sensitivity. The aim of the present study is to evaluate the value of the micronucleus (MN) test as a bio-dosimeter in hospital workers exposed to low doses of ionizing radiation. Blood samples were obtained from 30 subjects selected among workers exposed to X- and gamma-radiation, and 30 controls matched for sex, age and smoking from the same hospital. Micronucleus frequencies were analyzed by use of the cytokinesis-block method. The MN frequency was compared among the groups considering the confounding factors and the length of employment. No increase in the number of bi-nucleated cells with MN (BNMN), but a significant increase in the number of mono-nucleated cells with micronuclei (MOMN) was observed in exposed subjects compared with the controls. The relationship between MN frequency and accumulated dose (mSv) was evaluated. The length of employment did not affect the extent of MN frequency, but an increase of BNMN and MOMN cells was observed based on the accumulated radiation dose. Our study shows the sensitivity of the MN test in the detection of cytogenetic effects of cumulative exposure levels, suggesting the potential usefulness of this assay in providing a biological index in medical surveillance programs.

Effectiveness of a Protocol to Reduce Children's Exposure to Particulate Matter and NO2 in Schools during Alert Days.

Reducing children's exposure to air pollutants should be considered a primary goal, especially for the most vulnerable subjects. The goal of this study was to test the effectiveness of applying a protocol in the event of alert days, i.e., days with forecasted PM10 levels above the EU limit value (50 µg/m3). The test was conducted, before the onset of SARS-CoV-2 restrictions, in a classroom of a primary school in Parma (Italy)-a highly polluted area in Northern Italy. The protocol included indications for the frequency of opening windows and doors, as well as the activation of an air purifier. Teachers and students were asked to apply the protocol only in the event of alert days, while no indications were provided for non-alert days. A monitoring system measuring PM1, PM2.5, PM10, CO2, and NO2 was deployed in the classroom. Measurements of the same parameters were also performed outdoors near the school. The application of the protocol reduced the indoor/outdoor (I/O) ratio for all toxic pollutants. The reduction was also remarkable for PM10-the most critical air quality parameter in the study area (1.5 and 1.1 for non-alert and alert days, respectively). Indoor concentrations of PM10-especially during non-alert days-were often higher than outdoors, showing a major contribution from resuspension due to the movement of people and personal cloud. The protocol did not cause any increase in indoor CO2 levels. Our findings showed that the application of a ventilation protocol together with the contribution of an air purifier may represent an effective way to reduce children's exposure to air pollution during severe air pollution episodes. Considering the onset of COVID-19 and the airborne transmission of pathogens, this protocol now has more meaningful implications for children's welfare, and can be integrated with protocols designed as measures against the spread of SARS-CoV-2.

A nationwide study of air pollution from particulate matter and daily hospitalizations for respiratory diseases in Italy.

BACKGROUND/AIM: The relationship between air pollution and respiratory morbidity has been widely addressed in urban and metropolitan areas but little is known about the effects in non-urban settings. Our aim was to assess the short-term effects of PM10 and PM2.5 on respiratory admissions in the whole country of Italy during 2006-2015. METHODS: We estimated daily PM concentrations at the municipality level using satellite data and spatiotemporal predictors. We collected daily counts of respiratory hospital admissions for each Italian municipality. We considered five different outcomes: all respiratory diseases, asthma, chronic obstructive pulmonary disease (COPD), lower and upper respiratory tract infections (LRTI and URTI). Meta-analysis of province-specific estimates obtained by time-series models, adjusting for temperature, humidity and other confounders, was applied to extrapolate national estimates for each outcome. At last, we tested for effect modification by sex, age, period, and urbanization score. Analyses for PM2.5 were restricted to 2013-2015 cause the goodness of fit of exposure estimation. RESULTS: A total of 4,154,887 respiratory admission were registered during 2006-2015, of which 29% for LRTI, 12% for COPD, 6% for URTI, and 3% for asthma. Daily mean PM10 and PM2.5 concentrations over the study period were 23.3 and 17 µg/m3, respectively. For each 10 µg/m3 increases in PM10 and PM2.5 at lag 0-5 days, we found excess risks of total respiratory diseases equal to 1.20% (95% confidence intervals, 0.92, 1.49) and 1.22% (0.76, 1.68), respectively. The effects for the specific diseases were similar, with the strongest ones for asthma and COPD. Higher effects were found in the elderly and in less urbanized areas. CONCLUSIONS: Short-term exposure to PM is harmful for the respiratory system throughout an entire country, especially in elderly patients. Strong effects can be found also in less urbanized areas.