A guide to mycetisms: A toxicological and preventive overview.

Fungi are often considered a delicacy and are primarily cultivated and harvested, although numerous species are responsible for intoxication due to toxin content. Foodborne diseases are a significant public health concern, causing approximately 420 000 deaths and 600 million morbidities yearly, of which mushroom poisoning is one of the leading causes. Epidemiological data on non-cultivated mushroom poisoning in individual countries are often unrepresentative, as intoxication rarely requires emergency intervention. On the other hand, the lack of specialist knowledge among medical personnel about the toxicological manifestations of mushroom consumption may result in ineffective therapeutic interventions. This work aims to provide an easy-to-consult and wide-ranging tool useful for better understanding the variability of mushroom intoxications, the associated symptoms, and the main treatments for the most severe cases, given the absence of a complete species mapping tool toxic. Moreover, we establish an effective collection network that describes the incidence of mushroom poisonings by reporting the species and associated toxicological manifestations for each case. In conclusion, we highlight the need to establish appropriate primary prevention interventions, such as training the affected population and increasing consultancy relationships between mycological experts and specialised healthcare personnel.

We propose a review of the literature that describes the main syndromes resulting from the consumption of toxic fungal species, reporting symptoms and clinical manifestations, latency times and, where possible, diagnostic tools for recognising the species involved and interventions to be carried out.

MicroRaman spectroscopy detects the presence of microplastics in human urine and kidney tissue.

There is a growing concern within the medical community about the potential burden of microplastics on human organs and tissues. In this study, we investigated by microRaman spectroscopy the presence of microplastics in human kidneys and urine. Moreover, an open-access software was developed and validated for the project, which enabled the comparison between the investigated spectra and a self-created spectral database, thus enhancing the ability to characterize polymers and pigments in biological matrices. Healthy portions of ten kidneys obtained from nephrectomies, as well as ten urine samples from healthy donors were analyzed: 26 particles in both kidney and urine samples were identified, with sizes ranging from 3 to 13 µm in urine and from 1 to 29 µm in kidneys. The most frequently determined polymers are polyethylene and polystyrene, while the most common pigments are hematite and Cu-phthalocyanine. This preclinical study proves the presence of microplastics in renal tissues and confirms their presence in urine, providing the first evidence of kidney microplastics deposition in humans.

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.

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.

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.

Micronuclei in Fish Erythrocytes as Genotoxic Biomarkers of Water Pollution: An Overview.

Freshwater and marine water bodies receive chemical contaminants from industrial, agricultural, urban, and domestic wastes. Eco-genotoxicity assays are useful tools to assess the cumulative genotoxicity of these pollutants. Fish are suitable indicators for biomonitoring of mutagenic and carcinogenic pollution.In this review, we present a complete overview of the studies performed so far using the micronucleus test in peripheral erythrocytes of fish exposed to polluted water. We have listed all the species of fish used and the geographical distribution of the investigations. We have analyzed and discussed all technical aspects of using this test in fish, as well as the advantages and disadvantages of the different experimental protocols. We have reported the results of all studies. This assay has become, for years, one of the simplest, fastest, and most cost-effective for assessing genotoxic risk in aquatic environments. However, there are still several factors influencing the variability of the results. Therefore, we have given indications and suggestions to achieve a standardization of experimental procedures and ensure uniformity of future investigations.

Clastogenic effects of cigarette smoke and urethane and their modulation by olive oil, curcumin and carotenoids in adult mice and foetuses.

In spite of the overwhelming epidemiological evidence for cigarette smoke (CS) carcinogenicity, less attention has been paid to the effects of CS as a complex mixture. As assessed in a series of experiments in murine models, the whole-body exposure to mainstream CS induced significant increases of micronucleated cells in the respiratory tract, bone marrow and peripheral blood of adult mice as well as in the liver and peripheral blood of foetuses whose mothers had been exposed throughout pregnancy. Urethane was potently clastogenic in the same cells when injected intraperitoneally. The daily administration of extra-virgin olive oil by gavage produced evident and consistent protective effects in all monitored experimental systems. In contrast, sunflower oil exhibited some adverse effects. Curcumin did not produce any significant effect in the bone marrow of both CS-exposed adults and foetuses but it elicited a dose-dependent protective effect traceable in blood erythrocytes. However, the higher curcumin dose further increased the frequency of micronucleated pulmonary alveolar macrophages. The apparent protective effects produced by lycopene and by a carotenoid mix were overwhelmed by those produced by olive oil, and lycopene even exhibited a worsening effect on the frequency of micronucleated erythroblasts in the bone marrow of urethane-treated adult mice.

Dispersion of Natural Airborne TiO2 Fibres in Excavation Activity as a Potential Environmental and Human Health Risk.

Titanium is the ninth most abundant element, approximately 0.7% of the Earth crust. It is used worldwide in large quantities for various applications. The IARC includes TiO2 in Group 2B as possibly carcinogenic to humans suggesting that pathological effects correlate to particle size and shape. This study case quantifies the release of natural TiO2 particles during mining activity, involving meta-basalt and shale lithologies in the Ligurian Alps, during excavation of the Terzo Valico as part of the Trans-European Transport Network. Type, width, length, aspect ratio, and concentration of TiO2 particles in needle habit were determined. The different samplings have reported that airborne concentrations in meta-basalt were 4.21 ff/L and 23.94 ff/L in shale. In both cases, the concentration never exceeds the limits established by various organizations for workers health protection. Nevertheless, TiO2 elongated particles, recognized as rutile, showed the dimensional characteristic of fibres, as reported by WHO. These fibres deserve particular attention because they can reach the alveolar space and trigger inflammation and chronic diseases. The results indicate that monitoring the TiO2 in both working environments and Ti-rich geological formations, associated with epidemiological studies, may represent a useful tool to determine the exposure risk of workers and the general population.

Growth and decline of the COVID-19 epidemic wave in Italy from March to June 2020.

March 21, 2020 was the ridgeline between the growth of the coronavirus disease 2019 (COVID-19) epidemic wave in Italy and the start of its decline. We analyzed the epidemic patterns from March 1 to June 30. There was a progressive drop of cases from March (104,710) to April (94,888), May (25,705) and June (8110). Likewise, after a slight increase of deaths in April (14,804) compared to March (12,396), a considerable decline occurred in May (5170) and June (1464). Doubling times of cumulative cases grew from 2 to 6 days until March 20 to 2 weeks up to April 5, and thereafter no further doubling occurred until June 30. There was a striking North-South gradient of both cases and deaths. At the end of June, the nine Northern Italian regions or provinces, five central regions, and seven southern regions had contributed to the 81.1%, 12.4%, and 6.5% of the 240,578 national cases, respectively. Lombardy, the most populous region, was by far the most heavily affected one, accounting for the 39.0% of the national cases occurring over the analyzed 4-month period. However, in relative terms, it was preceded by Aosta Valley, the least populous region, less than 1% of the population of both regions having been affected by cases of COVID-19. The curves showing the ratio of daily cumulative cases and deaths to those of the previous day tended to flatten with time by approaching the zero growth but without reaching it, which documents a persisting circulation of severe acute respiratory syndrome coronavirus 2 in the Italian territory.

Rationale for the use of N-acetylcysteine in both prevention and adjuvant therapy of COVID-19.

COVID-19 may cause pneumonia, acute respiratory distress syndrome, cardiovascular alterations, and multiple organ failure, which have been ascribed to a cytokine storm, a systemic inflammatory response, and an attack by the immune system. Moreover, an oxidative stress imbalance has been demonstrated to occur in COVID-19 patients. N- Acetyl-L-cysteine (NAC) is a precursor of reduced glutathione (GSH). Due to its tolerability, this pleiotropic drug has been proposed not only as a mucolytic agent, but also as a preventive/therapeutic agent in a variety of disorders involving GSH depletion and oxidative stress. At very high doses, NAC is also used as an antidote against paracetamol intoxication. Thiols block the angiotensin-converting enzyme 2 thereby hampering penetration of SARS-CoV-2 into cells. Based on a broad range of antioxidant and anti-inflammatory mechanisms, which are herein reviewed, the oral administration of NAC is likely to attenuate the risk of developing COVID-19, as it was previously demonstrated for influenza and influenza-like illnesses. Moreover, high-dose intravenous NAC may be expected to play an adjuvant role in the treatment of severe COVID-19 cases and in the control of its lethal complications, also including pulmonary and cardiovascular adverse events.

Inhalation exposure to cigarette smoke and inflammatory agents induces epigenetic changes in the lung.

Smoking-related lung tumors are characterized by profound epigenetic changes including scrambled patterns of DNA methylation, deregulated histone acetylation, altered gene expression levels, distorted microRNA profiles, and a global loss of cytosine hydroxymethylation marks. Here, we employed an enhanced version of bisulfite sequencing (RRBS/oxRRBS) followed by next generation sequencing to separately map DNA epigenetic marks 5-methyl-dC and 5-hydroxymethyl-dC in genomic DNA isolated from lungs of A/J mice exposed whole-body to environmental cigarette smoke for 10 weeks. Exposure to cigarette smoke significantly affected the patterns of cytosine methylation and hydroxymethylation in the lungs. Differentially hydroxymethylated regions were associated with inflammatory response/disease, organismal injury, and respiratory diseases and were involved in regulation of cellular development, function, growth, and proliferation. To identify epigenetic changes in the lung associated with exposure to tobacco carcinogens and inflammation, A/J mice were intranasally treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the inflammatory agent lipopolysaccharide (LPS), or both. NNK alone caused minimal epigenetic alterations, while exposure either to LPS or NNK/LPS in combination led to increased levels of global cytosine methylation and formylation, reduced cytosine hydroxymethylation, decreased histone acetylation, and altered expression levels of multiple genes. Our results suggest that inflammatory processes are responsible for epigenetic changes contributing to lung cancer development.

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.

Epidemiological trends of COVID-19 epidemic in Italy over March 2020: From 1000 to 100 000 cases.

The coronavirus disease 2019 epidemic started in Italy by the end of January 2020 and, after 1 month, it affected 1049 persons. Based on the Italian Ministry of Health data, we reconstructed the daily course of virus-positive cases and deaths over March 2020 for the whole of Italy, 19 regions and 2 provinces. From 29 February to 31 March, there was a 100.9-fold increase in the cumulative number of cases and a 428.6-fold increase in the number of deaths in Italy. When plotted on a semilogarithmic scale, the curves tended to diverge from linearity with 23%, 16%, and 7% average daily increases during the three decades of March. Similarly, the number of deaths decreased from an average daily growth of 19% over the second decade to 10% over the third decade. The correlation coefficients relating the days to cases or deaths over each one of the three decades approached unity. As inferred from the equations of the regression lines relative to the three decades, the doubling times of cases were 3.4, 5.1, and 9.6 days, respectively. The doubling times of deaths over the second and third decades were 4.9 and 7.0 days, respectively. There was a broad geographic variability, with a striking gradient from the North, where 40.8% of cases and 57.9% of deaths occurred in Lombardy, to the South. On the whole, over March there was a trend to epidemic growth decline but the time for the end of the epidemic will depend on a variety of factors and, at present, it is unpredictable.

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.

Carbon nanotubes and central nervous system: Environmental risks, toxicological aspects and future perspectives.

Due to their morphological and physicochemical properties, carbon nanotubes (CNTs) enhance the structural properties of several materials and are produced in great volumes. The production and the manufacturing of CNTs-incorporated products can lead to the potential environmental release of CNTs. For these reasons, CNTs can represent a serious concern for human health. Humans are exposed to nanoparticles through inhalation, ingestion and skin uptake. After their entrance, the particles can reach the Central Nervous System (CNS) through three different pathways: the systemic, olfactory and trigeminal pathways. In the first, through systemic blood circulation, nanoparticles cross both the blood-brain and blood-spinal cord barriers, which are highly selective semipermeable barriers that protect the CNS compartments. The second is the step from the nose to brain route and occurs along axons and via nerve bundles that cross the cribriform plate to the olfactory bulb. In the third, the compounds diffuse through the nasal cavity mucosa to reach the branches of the trigeminal nerve in the olfactory and respiratory regions, and they reach brain stem via axonal transport. After their entrance, CNTs reach the CNS where they may cause cytotoxicity of selected neurons in several CNS regions, impairing molecular pathways and contributing to the onset and progression of chronic brain inflammation, microglia activation and white matter abnormalities with an increased risk for autism spectrum disorders, lower IQ in children, neurodegenerative diseases and stroke. The large surface area to mass ratio of CNTs greatly increases surface reactivity. Despite this property considerable contributes to their toxicological profile in biological systems, also makes CNTs very attractive in the medical field, where they can be used as carriers of bioactive molecules, contrast agents, biological platforms and for many other applications in medicine.

Modulation of genomic and epigenetic end-points by celecoxib.

Celecoxib, a nonsteroidal anti-inflammatory drug that selectively targets cyclooxygenase-2, is a promising cancer chemopreventive agent. However, safety concerns have been raised in clinical trials evaluating its ability to prevent colorectal adenomas. The rationale for the herein reported studies was to analyze genomic and epigenetic end-points aimed at investigating both the chemopreventive properties of celecoxib towards cigarette smoke-associated molecular alterations and its possible adverse effects. We carried out three consecutive studies in mice treated with either smoke and/or celecoxib. Study 1 investigated early DNA alterations (DNA adducts, oxidative DNA damage, and systemic genotoxic damage) and epigenetic alterations (expression of 1,135 microRNAs) in lung and blood of Swiss H mice; Study 2 evaluated the formation of DNA adducts in lung, liver, and heart; and Study 3 evaluated the expression of microRNAs in 10 organs and 3 body fluids of ICR (CD-1) mice. Surprisingly, the oral administration of celecoxib to smoke-free mice resulted in the formation of DNA adducts in both lung and heart and in dysregulation of microRNAs in mouse organs and body fluids. On the other hand, celecoxib attenuated smoke-related DNA damage and dysregulation of microRNA expression. In conclusion, celecoxib showed pleiotropic properties and multiple mechanisms by counteracting the molecular damage produced by smoke in a variety of organs and body fluids. However, administration of celecoxib to non-smoking mice resulted in evident molecular alterations, also including DNA and RNA alterations in the heart, which may bear relevance in the pathogenesis of the cardiovascular adverse effects of this drug.

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.

Release of MicroRNAs into Body Fluids from Ten Organs of Mice Exposed to Cigarette Smoke.

Purpose: MicroRNAs are small non-coding RNAs that regulate gene expression, thereby playing a role in a variety of physiological and pathophysiological states. Exposure to cigarette smoke extensively downregulates microRNA expression in pulmonary cells of mice, rats, and humans. Cellular microRNAs are released into body fluids, but a poor parallelism was previously observed between lung microRNAs and circulating microRNAs. The purpose of the present study was to validate the application of this epigenetic biomarker by using less invasive collection procedures. Experimental design: Using microarray analyses, we measured 1135 microRNAs in 10 organs and 3 body fluids of mice that were either unexposed or exposed to mainstream cigarette smoke for up to 8 weeks. The results obtained with selected miRNAs were validated by qPCR. Results: The lung was the main target affected by smoke (190 dysregulated miRNAs), followed by skeletal muscle (180), liver (138), blood serum (109), kidney (96), spleen (89), stomach (36), heart (33), bronchoalveolar lavage fluid (32), urine (27), urinary bladder (12), colon (5), and brain (0). Skeletal muscle, kidney, and lung were the most important sources of smoke-altered microRNAs in blood serum, urine, and bronchoalveolar lavage fluid, respectively. Conclusions: microRNA expression analysis was able to identify target organs after just 8 weeks of exposure to smoke, well before the occurrence of any detectable histopathological alteration. The present translational study validates the use of body fluid microRNAs as biomarkers applicable to human biomonitoring for mechanistic studies, diagnostic purposes, preventive medicine, and therapeutic strategies.

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.