PURPOSE:Fanconi anemia (FA) is a complex tumor-prone disease defined by an entangled genotype and phenotype. Despite enormous efforts in the last 20 years, a comprehensive and integrated view of the disease is still missing. The aim of this pilot study was to establish whether a global microRNA (miRNA) analysis approach could be helpful in defining aspects in FA phenotype, which might deserve future attention with the perspective to develop miRNA-based therapies.METHODS:miRNA array were employed to characterize the global miRNA (miRNoma) profile of FA RNA samples with respect to normal samples.RESULTS:We report and compare miRNA profile from two FA established cell lines and three FA patients. This analysis reveals that 36 and 64 miRNAs, respectively, are found differentially expressed (>2-fold variation and P < 0.05) in the samples from FA cell lines and FA patients. Overlap of these data results in 24 miRNAs as shared in the two sample populations. Available bioinformatics methods were used to predict target genes for the differentially expressed miRNAs and to perform pathway enrichment analysis.CONCLUSIONS:Seven pathway results associated with the FA phenotype. It is interesting to note that some of these pathways were previously unrelated to FA phenotype. It might be important to focus on these pathways not previously emerged as dysfunctional in FA to better define the pathophysiological context of this disease. This is the first report of a global miRNA analysis in FA.

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.

We recently showed that nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the lung tumors induced by cigarette smoke, either mainstream (MCS) or environmental (ECS), in female mice. We used subsets of mice to analyze the expression of 1135 microRNAs in both lung and blood serum, as related to the whole-body exposure to smoke and/or oral administration of either aspirin or naproxen. In a first study, we evaluated early microRNA alterations in A/J mice exposed to ECS for 10 weeks, starting at birth, and/or treated with NSAIDs for 6 weeks, starting after weaning. At that time, when no histopathological change were apparent, ECS caused a considerable downregulation of pulmonary microRNAs affecting both adaptive mechanisms and disease-related pathways. Aspirin and naproxen modulated, with intergender differences, the expression of microRNAs having a variety of functions, also including regulation of cyclooxygenases and inflammation. In a second study, we evaluated late microRNA alterations in Swiss H mice exposed to MCS during the first 4 months of life and treated with NSAIDs after weaning until 7.5 months of life, when tumors were detected in mouse lung. Modulation of pulmonary microRNAs by the two NSAIDs was correlated with their ability to prevent preneoplastic lesions (microadenomas) and adenomas in the lung. In both studies, exposure to smoke and/or treatment with NSAIDs also modulated microRNA profiles in the blood serum. However, their levels were poorly correlated with those of pulmonary microRNAs, presumably because circulating microRNAs reflect the contributions from multiple organs and not only from lung.

Cigarette smoke (CS) is known to dysregulate microRNA expression profiles in the lungs of mice, rats, and humans, thereby modulating several pathways involved in lung carcinogenesis and other CS-related diseases. We designed a study aimed at evaluating (a) the expression of 1135 microRNAs in the lung of Swiss H mice exposed to mainstream CS during the first 4 months of life and thereafter kept in filtered air for an additional 3.5 months, (b) the relationship between lung microRNA profiles and histopathological alterations in the lung, (c) intergender differences in microRNA expression, and (d) the comparison with microRNA profiles in blood serum. CS caused multiple histopathological alterations in the lung, which were almost absent in sham-exposed mice. An extensive microRNA dysregulation was detected in the lung of CS-exposed mice. Modulation of microRNA profiles was specifically related to the histopathological picture, no effect being detected in lung fragments with non-neoplastic lung diseases (emphysema or alveolar epithelial hyperplasia), whereas a close association occurred with the presence and multiplicity of preneoplastic lesions (microadenomas) and benign lung tumors (adenomas). Three microRNAs regulating estrogen and HER2-dependent mechanisms were modulated in the lung of adenoma-bearing female mice. Blood microRNAs were also modulated in mice affected by early neoplastic lesions. However, there was a poor association between lung microRNAs and circulating microRNAs, which can be ascribed to an impaired release of mature microRNAs from the damaged lung. Studies in progress are evaluating the feasibility of analyzing blood microRNAs as a molecular tool for lung cancer secondary prevention.

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

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

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.

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

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

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

Aicardi-Goutières syndrome is a rare encephalopathy of mutational origin characterized by increased levels of interferon alpha in cerebrospinal fluid. The aim of this study was to explore the influence of different Aicardi-Goutières syndrome genotypes on the clinical course of patients, seeking to identify specific gene expression profiles able to explain Aicardi-Goutières syndrome phenotype differences. We detected the occurrence of Aicardi-Goutières syndrome mutations in 21 patients and compared microarray gene-expression data of cerebrospinal fluid lymphocytes with clinical variables. The levels of interferon alpha in cerebrospinal fluid were high in all patients; we found differences in the expression of genes encoding for Toll-like receptor, endogenous RNases, T lymphocyte activation, angiogenesis inhibition, and peripheral interferon alpha production. These results indicate that further to interferon alpha production in the central nervous system, a variety of other pathogenic mechanisms is activated in Aicardi-Goutières syndrome to various degrees in different patients, thus explaining the interindividual difference in Aicardi-Goutières syndrome course.

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

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

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

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

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

Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m3 of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) were measured by 32P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) is of particular interest due to the very high production and widespread environmental contamination. We recently demonstrated that the oral administration of BPA to mice results in the formation of DNA adducts not only in liver but also in mammary tissue. The present study aimed at evaluating the modulation of BPA-related DNA adducts and proteome alterations by the chemopreventive agents budesonide (BUD) and phenethyl isothiocyanate (PEITC). Swiss ICR (CD-1) mice received, for 8 days, BPA with the drinking water and either chemopreventive agent with the diet. We measured DNA adducts by (32)P postlabeling and 656 proteins by antibody microarray. BPA induced the formation, with similar patterns, of DNA adducts in liver and in mammary tissue. Moreover, BPA dysregulated 13 proteins in mammary tissue, mostly in the sense of upregulation, including estrogen receptor-beta and proteins involved in cell proliferation, inhibition of apoptosis, tissue remodeling, inflammation, stress response, and glutathione synthesis. PEITC significantly inhibited the formation of BPA-induced DNA adducts, but only at the highest dose tested, and BUD was totally ineffective. The chemopreventive agents modulated a variety of BPA-induced changes in proteome profiles. However, as shown by both hierarchical cluster analysis and principal component analysis, BUD and especially PEITC were not able to restore the physiological situation in BPA-treated mice. Therefore, the in vivo use of proteome analysis proves to be a sensitive tool for the early prediction not only of protective effects but also of adverse effects of chemopreventive agents.

We previously showed that exposure to environmental cigarette smoke (ECS) for 28 days causes extensive downregulation of microRNA expression in the lungs of rats, resulting in the overexpression of multiple genes and proteins. In the present study, we evaluated by microarray the expression of 484 microRNAs in the lungs of either ECS-free or ECS-exposed rats treated with the orally administered chemopreventive agents N-acetylcysteine, oltipraz, indole-3-carbinol, 5,6-benzoflavone, and phenethyl isothiocyanate (as single agents or in combinations). This is the first study of microRNA modulation by chemopreventive agents in nonmalignant tissues. Scatterplot, hierarchical cluster, and principal component analyses of microarray and quantitative PCR data showed that none of the above chemopreventive regimens appreciably affected the baseline microRNA expression, indicating potential safety. On the other hand, all of them attenuated ECS-induced alterations but to a variable extent and with different patterns, indicating potential preventive efficacy. The main ECS-altered functions that were modulated by chemopreventive agents included cell proliferation, apoptosis, differentiation, Ras activation, P53 functions, NF-κB pathway, transforming growth factor–related stress response, and angiogenesis. Some microRNAs known to be polymorphic in humans were downregulated by ECS and were protected by chemopreventive agents. This study provides proof-of-concept and validation of technology that we are further refining to screen and prioritize potential agents for continued development and to help elucidate their biological effects and mechanisms. Therefore, microRNA analysis may provide a new tool for predicting at early carcinogenesis stages both the potential safety and efficacy of cancer chemopreventive agents. Cancer Prev Res; 3(1); 62–72