Treatment of STING-associated vasculopathy with onset in infancy in patients carrying a novel mutation in the TMEM173 gene with the JAK3-inhibitor tofacitinib.

Objectives: This study aimed to reveal the genetic background of patients in the two-generation family suffering from rheumatoid arthritis, psoriatic arthropathy pain, scratches, and bruises. Patients and methods: A clinical exome sequencing analysis was performed in 10 individuals in the same family using the Sophia Genetics clinical exome solution kit. Results: A novel V194L mutation in the TMEM173 gene was identified in three members of the family. Two of the family members were treated with the JAK3 inhibitor tofacitinib and recovered completely one month after the treatment. Conclusion: The V194L mutation was reported for the first time in this study, and a positive response was achieved with tofacitinib.

Computational and Bioinformatics Methods for MicroRNA Gene Prediction.

MicroRNAs (miRNAs) are 20-24-nucleotide-long noncoding RNAs that bind to the untranslated region (3' UTR) of their target mRNAs. The importance of miRNAs in medicine has grown rapidly in the 20 years since the discovery of them. As the regulatory function of miRNAs on biological processes was discovered, they were advocated to play a role in the underlying mechanisms of human pathogenesis. Functional studies have confirmed that miRNAs are promising in preclinical development through deregulation of genes targeted by miRNAs in many cancer cases. In this chapter, we summarize the miRNAs identified for some specific types of cancer and their functions. Besides, miRNAs function as cancer biomarker and their benefits to diagnosis and treatment of cancer are also discussed.

The dicyano compound induces autophagic or apoptotic cell death via Twist/c-Myc axis depending on metastatic characteristics of breast cancer cells.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease with various subtypes, therefore, the illumination of distinctive mechanisms between subtypes for the development of novel treatment strategies is important. Here, we revealed the antiproliferative effects of our customized dicyano compound (DC) on BC cells. METHODS AND RESULTS: We determined the antiproliferative effect of the DC on non-metastatic MCF-7 and metastatic MDA-MB-231 cell lines by MTT. We evaluated protein levels of LC3BI-II and p62 to detect effects of the DC on autophagy. Furthermore, we examined whether the DC induce apoptosis in MCF-7 and MDA-MB-231 cells by performing TUNEL and western blotting. We showed that the DC induces autophagic cell death in MDA-MB-231 while it leads to apoptosis in MCF-7, demonstrating that DC can induce different cell death mechanisms in BC cells according to what they represent subtypes. To understand the reason of different cell response to the DC, we evaluated the expressions of several regulator proteins involved in survival, cell arrest and proliferation. All findings revealed that c-Myc expression is directly correlated with autophagy induction in BC cells and it could be a marker for the selection of cell death mechanism against anti-cancer drugs. Interestingly, we showed that the overexpression of Twist, responsible for metastatic features of BC cells, imitates the effects of autophagy on c-Myc expression in MCF-7 cells, indicating that it is implicated in both the regulation of c-Myc as a upstream factor and subsequently the selection of cell death mechanisms. CONCLUSION: Taken together, we suggest that Twist/c-Myc axis may have a role in different response to the DC-induced cell death pathways in BC subtypes with different invasive characteristics.

Whole Genome Sequencing and Phylogenetic Analysis of SARS­CoV­2 strains in Turkey.

INTRODUCTION: Coronaviruses which are single-stranded RNAs, are members of a large family of viruses that may be important pathogens for humans. SARS-CoV-2 was found to cause the severe respiratory syndrome, and on January 22, 2020 first human-to-human transmission was reported. We aimed to reveal the complete genomes of 19 SARS-CoV-2 isolates from Denizli province and identify Turkish patients' genetic similarities. METHODOLOGY: 15 samples with the highest viral loads resulting from RT-PCR were selected for NGS analysis. Fifteen SARS-CoV-2 complete genome sequences were then subjected to phylogenetic analysis and uploaded to the GISAID database. Phylogenetic trees were constructed by the Neighbor-Joining method using MEGAX software. RESULTS: Whole-genome sequencing of the viral RNA samples revealed 32 missense, 21 synonymous, and 4 non-coding alleles. In all samples c.1-25C>T (5'UTR), c.14144C>T (ORF1ab), c.2772C>T (ORF1ab) and c.1841A>G(S) mutations were detected. Phylogenetic analysis revealed that most of the present study's genomes are in 20B clade while the two are in 20A. The phylogenetic tree constructed with all complete SARS-CoV-2 genomes of Turkey showed that the viruses were spread nearly homogenous on eastern (around Kars) and western (around Istanbul) sides. CONCLUSIONS: Here, we reported the viral genomes in Denizli comprehensively for the first time. We identified 11 rare missense mutations in the virus compared to the reference genome. Phylogenetic analysis revealed that while most of our isolates were similar to European sequences, some had different sublineages depending on their genomic variants.

lncRNAs as Potential Targets in Small Cell Lung Cancer: MYC -dependent Regulation.

BACKGROUND: Small Cell Lung Cancer (SCLC) is a highly aggressive malignancy. MYC family oncogenes are amplified and overexpressed in 20% of SCLCs, showing that MYC oncogenes and MYC regulated genes are strong candidates as therapeutic targets for SCLC. c-MYC plays a fundamental role in cancer stem cell properties and malignant transformation. Several targets have been identified by the activation/repression of MYC. Deregulated expression levels of lncRNAs have also been observed in many cancers. OBJECTIVE: The aim of the present study is to investigate the lncRNA profiles which depend on MYC expression levels in SCLC. METHODS: Firstly, we constructed lentiviral vectors for MYC overexpression/inhibition. MYC expression is suppressed by lentiviral shRNA vector in MYC amplified H82 and N417 cells, and overexpressed by lentiviral inducible overexpression vector in MYC non-amplified H345 cells. LncRNA cDNA is transcribed from total RNA samples, and 91 lncRNAs are evaluated by qRT-PCR. RESULTS: We observed that N417, H82 and H345 cells require MYC for their growth. Besides, MYC is not only found to regulate the expressions of genes related to invasion, stem cell properties, apoptosis and cell cycle (p21, Bcl2, cyclinD1, Sox2, Aldh1a1, and N-Cadherin), but also found to regulate lncRNAs. With this respect, expressions of AK23948, ANRIL, E2F4AS, GAS5, MEG3, H19, L1PA16, SFMBT2, ZEB2NAT, HOTAIR, Sox2OT, PVT1, and BC200 were observed to be in parallel with MYC expression, whereas expressions of Malat1, PTENP1, Neat1, UCA1, SNHG3, and SNHG6 were inversely correlated. CONCLUSION: Targeting MYC-regulated genes as a therapeutic strategy can be important for SCLC therapy. This study indicated the importance of identifying MYC-regulated lncRNAs and that these can be utilized to develop a therapeutic strategy for SCLC.

Relation of ADRB3, GEF, ROCK2 gene polymorphisms to clinical findings in overactive bladder.

PURPOSE: Adrenergic and cholinergic pathways play an important role in contraction-relaxation harmony in human bladder. Functional changes in any proteins in these pathways may result in overactive bladder. We aimed to investigate whether single gene polymorphisms affecting adrenergic and cholinergic pathways are associated with OAB syndrome. METHODS: 60 patients with idiopathic OAB and 60 healthy controls were included in the study. A validated OAB-V8 questionnaire was given to all patients. Polymorphisms of ADRB3, ROCK2, and GEF gene were detected by PCR from whole blood samples. Genotypic structures of patients and controls were compared. The relationship between genotypic structures and OAB symptom scores were investigated. RESULTS: We found no significant difference in the genotype and allele frequencies between the patients and controls for all three SNP. While there was no relationship between ADRB3 and GEF gene polymorphisms and OAB scores in OAB patients, the OAB score in heterozygous polymorphic individuals was significantly higher than in homozygous polymorphic individuals in the ROCK2 gene (p = 0.039). CONCLUSION: The polymorphisms of the ADRB3, ROCK2, and GEF genes were present in both OAB group and healthy controls, but were not associated with OAB syndrome.

MYC-driven regulation of long non-coding RNA profiles in breast cancer cells.

AIM: MYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. MATERIALS AND METHODS: Cells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. RESULTS: Our results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. CONCLUSION: We observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

The triazole linked galactose substituted dicyano compound can induce autophagy in NSCLC cell lines.

As seen in other types of cancer, development of drug resistance in NSCLC treatment causes adverse effects on disease fighting process. Recent studies have shown that one of the drug resistance development mechanisms is that cancer cells may acquire the ability to escape from cell death. Therefore, development of anticancer drugs which have the strategy to redirect cancer cells to any cell death pathways may provide positive results for cancer treatments. Autophagy may be a target mechanism of alternative cancer treatment strategy in cases of blocked apoptosis. There is also a complex molecular link between autophagy and apoptosis, has not been fully understood yet. The dicyano compound which we used in our study caused cell death in NSCLC cell lines. When we analyzed the cells which were treated with dicyano compound by transmission electron microscope, we observed autophagosome structures. Upon this result, we investigated expression levels of autophagic proteins in the dicyano compound-treated cells by immunoblotting and observed that expression levels of autophagic proteins were increased significantly. The TUNEL assay and qRT-PCR for pro-apoptotic and anti-apoptotic gene expression, which we performed to assess apoptosis in the dicyano compound-treated cells, showed that the cell death does not occur through apoptotic pathway. We showed that the dicyano compound, which was developed in our laboratories, may play a role in molecular link between apoptosis and autophagy and may shed light on development of new anticancer treatment strategies.

Exploring biomarkers in the overactive bladder: Alterations in miRNA levels of a panel of genes in patients with OAB.

AIMS: It has been demonstrated that there are abundant stable microRNAs (miRNAs) in plasma, which is potentially disease-specific. Adrenergic and muscarinic pathways play an important role in voiding physiology. Alterations in the levels of miRNAs are thought to influence the regulation of these pathways at the molecular level. The aim of this study was to investigate the relationship of miRNAs with overactive bladder pathogenesis and to provide a new perspective to treatment approaches. METHODS: This study included patients with an overactive bladder (OAB) diagnosis and a healthy control group. All patients completed a validated OAB-V8 questionnaire. The relative expression levels of 12 miRNAs were examined in plasma by PCR. Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic qualification of miRNAs. RESULTS: The relative expression levels of let-7b-5p, miR-92a-3p, miR-98-5p, miR-142-3p, and miR-200c-3p were significantly upregulated and miR-139-5p was significantly downregulated in patients with OAB and no correlation was determined between the levels of miRNAs with OAB symptom score. Among the miRNAs, miR-98-5p provided the highest diagnostic accuracy alone (area under curve [AUC] = 0.79) in ROC analysis. The combination of miR-98-5p + miR-139-5p was seen to be a good indicator (AUC = 0.839). CONCLUSIONS: These results suggest that alteration of the miRNA levels can be used as auxiliary parameters to explain the pathophysiology of OAB syndrome and could shed light on new treatment options.

NF-κB-Induced Upregulation of miR-548as-3p Increases Invasion of NSCLC by Targeting PTEN.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is an aggressive cancer type due to high metastatic capacity. Nuclear Factor Kappa B (NF-κB) is a consistently active transcription factor in malignant lung cancer cells and has crucial significance in NSCLC progression. It is also implicated in the transcriptional regulation of many genes including microRNAs (miRNAs) that function as tumor suppressor or oncogene. It has been increasingly reported that several miRNAs defined as gene members are induced by NF-κB. The present study aimed to find novel miRNAs that are regulated by NF-κB. METHODS: Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiment and bioinformatic analysis were used to determine NF-κB-dependent miRNAs. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), luciferase reporter gene assays were carried out to investigate the target genes of miRNAs. To determine biologic activity, transwell invasion and MTT assay were carried out on H1299 NSCLC cell line. miRNA expression level was evaluated in metastatic and non-metastatic tissue samples of NSCLC patients. RESULTS: ChIP-Seq and qRT-PCR experiments showed that miR-548as-3p is transcriptionally regulated by NF- κB in response to Tumor Necrosis Factor-α (TNF-α) treatment. Then, we found that tumor suppressor Phosphatase and Tension homolog (PTEN) is a direct target of miR-548as-3p. Furthermore, miR-548as-3p mediates phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway and NF-κB-implicated genes including Matrix Metalloproteinases 9 (MMP9), Slug and Zeb1. We further showed that miR-548as-3p increased invasiveness of NSCLC cells and was upregulated in metastatic tumor tissues compared to non-metastatic ones. CONCLUSION: All these findings provide a miRNAs-mediated novel mechanism for NF-κB signaling and that miR-548as-3p could be a biomarker for NSCLC metastasis.

Caffeine prevents bilirubin-induced cytotoxicity in cultured newborn rat astrocytes.

OBJECTIVE: Unconjugated bilirubin (UCB) may cause neurotoxicity in preterm neonates due to immaturity of UGT1A1 leading to bilirubin accumulation in the brain. Caffeine used in the treatment of apnea of prematurity was reported to decrease mechanical ventilation requirement, the frequencies of bronchopulmonary dysplasia, patent ductus arteriosus, cerebral palsy and neurodevelopmental disorders in very low birth weight infants. However, the effect of caffeine on hyperbilirubinemia was not yet clarified. METHODS: We used astrocyte cell cultures obtained from 2-day-old Wistar albino rats via modified Cole and de Vellis method. UCB concentration toxic to 50% of astrocytes, and caffeine concentration increasing cell viability 100% were used in experiments. While no medication was applied to the control group, UCB (50 µM) and caffeine (100 µM) were applied to the bilirubin and caffeine groups for 24 h. Prophylactic and therapeutic caffeine groups were treated with caffeine 4 h before and after UCB exposure. The effects of caffeine were investigated in rat astrocytes exposed to UCB in terms of cell viability, apoptosis, antioxidant defense, proinflammatory cytokines, and Toll-like receptor (TLR)s. RESULTS: Compared to the control group, UCB increased apoptosis, malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, total nitrate/nitrite, and TLR4 levels, and decreased cell viability, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) activities, glutathione, and TLR9 levels (for all p < .001). Conversely, prophylactic and therapeutic caffeine improved the detrimental effects of UCB. CONCLUSIONS: Caffeine seems encouraging for the prevention and treatment of bilirubin neurotoxicity in rats by means of its antiapoptotic, antioxidant, anti-inflammatory, anti-nitrosative, and anti-TLR-4 properties.

TGF-ß-SMAD-miR-520e axis regulates NSCLC metastasis through a TGFBR2-mediated negative-feedback loop.

Transforming growth factor-ß (TGF-ß) pathway plays crucial roles during the carcinogenesis and metastasis. TGF-ß receptor 2 (TGFBR2) is a key molecule for the regulation of TGF-ß pathway and frequently downregulated or lost in several cancer types including non-small cell lung cancer (NSCLC), and TGF-ß pathway is often regulated by negative-feedback mechanisms, but little is known about the mechanism of TGFBR2 downregulation in NSCLC. Here, we found that the expression of miR-520e is upregulated in metastatic tumor tissues compared with non-metastatic ones, and its expression is inversely correlated with that of TGFBR2 in clinical samples. We also discovered that TGF-ß dramatically increased the expression of miR-520e, which targeted and downregulated TGFBR2, and the suppression of miR-520e significantly impaired TGF-ß-induced TGFBR2 downregulation. Chromatin immunoprecipitation-PCR experiments further showed that miR-520e is transcriptionally induced by SMAD2/3 in response to TGF-ß. Our findings reveal a novel negative-feedback mechanism in TGF-ß signaling and the expression level of miR-520e could be a predictive biomarker for NSCLC metastasis.

Anti-invasive effect of Cyclamen pseudibericum extract on A549 non-small cell lung carcinoma cells via inhibition of ZEB1 mediated by miR-200c.

Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion-migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 ± 2.35 µg/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components.

The crosstalk between p38 and Akt signaling pathways orchestrates EMT by regulating SATB2 expression in NSCLC cells.

Epithelial-mesenchymal transition is a crucial event for metastasis and could be mediated by several pathways such as phosphoinositide 3-kinase/Akt, mitogen-activated protein kinases, as well as many epigenetic regulators. Special AT-rich sequence-binding protein 2 is an epigenetic regulator involved in epithelial-mesenchymal transition and osteoblastic differentiation. It has been reported that the crosstalk between several pathways is responsible for the regulation of epithelial-mesenchymal transition in cancer cells. However, crosstalks between p38 and Akt pathways involved in epithelial-mesenchymal transition are still unknown. We recently reported that there is a crosstalk between p38 and Akt pathways in non-small-cell lung carcinoma cells, and this crosstalk is associated with E-cadherin and special AT-rich sequence-binding protein 2 expressions. Therefore, we aimed to determine whether this crosstalk has a mediator role in the regulation of epithelial-mesenchymal transition in non-small-cell lung carcinoma. Our results showed that inhibition of p38 leads to the disruption of this crosstalk via decreased expression of phosphatase and tensin homolog (PTEN) and subsequently increased activation of Akt in non-small-cell lung carcinoma cells. Then, we found that p38 inhibition upregulated special AT-rich sequence-binding protein 2 expression and reversed epithelial-mesenchymal transition in non-small-cell lung carcinoma cells. Furthermore, special AT-rich sequence-binding protein 2 knockdown abolished the effect of p38 inhibition on epithelial-mesenchymal transition in non-small-cell lung carcinoma cells. In conclusion, our results strongly indicate that the crosstalk between p38 and Akt pathways can determine special AT-rich sequence-binding protein 2 expression and epithelial character of non-small-cell lung carcinoma cells, and special AT-rich sequence-binding protein 2 is a critical epigenetic regulator for epithelial-mesenchymal transition mediated by p38 pathway in non-small-cell lung carcinoma. Our findings will contribute to illuminate the molecular mechanisms of the epithelial-mesenchymal transition process that has a critical significance for lung cancer metastasis.

Design of a Lentiviral Vector for the Inducible Expression of MYC: A New Strategy for Construction Approach.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.

miR-181b-5p, miR-195-5p and miR-301a-3p are related with treatment resistance in schizophrenia.

The aim of the study was to determine the differences between expression levels of certain miRNAs, as their association with schizophrenia has been well presented in the literature, and to investigate their relation to treatment resistance in schizophrenic patients. Three groups were formed: 1) treatment-resistant group, 2) treatment responsive group and 3) healthy control group. Expression levels of miRNAs from peripheric blood samples were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We investigated the roles of 29 schizophrenia-related miRNAs in schizophrenia treatment and their potentials to be considered as indicators. Among these miRNAs, only miR-181b-5p, miR-195-5p and miR-301a-3p expressions were found to be significantly different between the treatment-resistant group and the group responding well to the treatment. miRNAs may cause resistance by silencing the receptor genes of the drugs used for schizophrenia treatment. miR-181b-5p, miR-195-5p and miR-301a-3p may be candidate indicators that can be used to reveal resistance against schizophrenia treatment.

Downregulation of SATB2 is critical for induction of epithelial-to-mesenchymal transition and invasion of NSCLC cells.

OBJECTIVES: The epithelial-to-mesenchymal transition (EMT) is considered as a key step in invasion of cancer cells. There are several regulator proteins responsible for induction of EMT, but underlying mechanisms are still unclear. SATB2 is an epigenetic regulator involved in osteoblastic differentiation. The role of SATB2 in EMT and invasion of NSCLC cells is unknown. Therefore, we aimed to explain roles of SATB2 with underlying molecular mechanisms in EMT and invasion of NSCLC cells. MATERIALS AND METHODS: We used A549 and NCI-H1650 cells as a model to evaluate the effects of SATB2 in EMT and invasion of NSCLC cells. Cell culture, western blot analysis, siRNA-mediated gene knockdown, and invasion assay were performed in this study. RESULTS AND CONCLUSION: In this study, we investigated the regulatory role of SATB2 expression in TGF-ß-induced EMT and invasion of NSCLC cells, and found that SATB2 is downregulated in A549 cells and TGF-ß can induce EMT in these cells, however, TGF-ß can not induce EMT in SATB2 expressing cells such as H1650, PC3, II-18, Hcc78 and Hcc193. Our results demonstrated that SATB2 knockdown is sufficient to induce generation of fibroblast-like morphology, EMT and invasion of NSCLC cells by upregulating the expressions of Slug, Twist and Zeb1. Moreover, SATB2 knockdown promotes TGF-ß-induced EMT and invasion in NSCLC cells. These results strongly suggest that SATB2 prevents induction of EMT by suppressing expression of EMT-inducing transcription factors in NSCLC cells. Furthermore, SATB2 could inhibit tumour initiation by suppressing stemness marker genes such as CD44, Nanog, Oct-4A and Sox-2. Consequently, our results clearly indicate that SATB2 plays pivotal role in EMT, invasion and stemness of NSCLC cells.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles.

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.