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Open access

Matej Skocaj, Metka Filipic, Jana Petkovic and Sasa Novak

Titanium dioxide in our everyday life; is it safe?

Background. Titanium dioxide (TiO2) is considered as an inert and safe material and has been used in many applications for decades. However, with the development of nanotechnologies TiO2 nanoparticles, with numerous novel and useful properties, are increasingly manufactured and used. Therefore increased human and environmental exposure can be expected, which has put TiO2 nanoparticles under toxicological scrutiny. Mechanistic toxicological studies show that TiO2 nanoparticles predominantly cause adverse effects via induction of oxidative stress resulting in cell damage, genotoxicity, inflammation, immune response etc. The extent and type of damage strongly depends on physical and chemical characteristics of TiO2 nanoparticles, which govern their bioavailability and reactivity. Based on the experimental evidence from animal inhalation studies TiO2 nanoparticles are classified as "possible carcinogenic to humans" by the International Agency for Research on Cancer and as occupational carcinogen by the National Institute for Occupational Safety and Health. The studies on dermal exposure to TiO2 nanoparticles, which is in humans substantial through the use of sunscreens, generally indicate negligible transdermal penetration; however data are needed on long-term exposure and potential adverse effects of photo-oxidation products. Although TiO2 is permitted as an additive (E171) in food and pharmaceutical products we do not have reliable data on its absorption, distribution, excretion and toxicity on oral exposure. TiO2 may also enter environment, and while it exerts low acute toxicity to aquatic organisms, upon long-term exposure it induces a range of sub-lethal effects.

Conclusions. Until relevant toxicological and human exposure data that would enable reliable risk assessment are obtained, TiO2 nanoparticles should be used with great care.

Open access

Valerija Zager, Maja Cemazar, Irena Hreljac, Tamara Lah, Gregor Sersa and Metka Filipic

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

Background. Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major down-stream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.

Methods. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay.

Results. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability.

Conclusions. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.

Open access

Metka Filipič, Bojana Žegura, Bojan Sedmak, Irena Horvat-Žnidaršic, Aleksandra Milutinovič and Dušan Šuput

Subchronic exposure of rats to sublethal dose of microcystin-YR induces DNA damage in multiple organs

Background. Microcystins (MCs) are cyclic heptapeptides that are considered to be liver specific toxins. They are potent tumour promoters and recent studies indicate that they are also genotoxic. In this study we measured DNA damage in lymphocytes, liver, kidney (cortex and medulla), lung, spleen and brain cells of male Fisher F344 rats that were exposed to sublethal dose (every second day 10 μg/kg b.w.; i.p) of micro-cystin-YR (MCYR) for one month.

Methods. At the end of exposure the animals were sacrificed, the lymphocytes were isolated from blood taken from jugular vein, liver cells were obtained by perfusion with collagenase A and the cells from other organs were isolated by incubating small tissue pieces with collagenase A. The DNA damage in isolated cells was measured with the single cells gel electrophoresis (SCGE) also called the comet assay.

Results. A significant increase of the % tail DNA in MCYR-exposed animals compared to the nonexposed control ones was observed in brain (2.5 fold), liver (2.1 fold), kidney medulla (1.9 fold), kidney cortex (1.8 fold) and lung (1.7 fold) cells, while the DNA from lymphocytes and spleen cells was not affected.

Conclusion. This study demonstrated that subchronic exposure to sublethal doses of MCs can induce systemic genotoxicity in mammals, and it affects not only the liver but also other vital organs.

Open access

Anja Fic, Bojana Žegura, Marija Sollner Dolenc, Metka Filipič and Lucija Peterlin Mašič

Environmental oestrogen bisphenol A (BPA) and its analogues are widespread in our living environment. Because their production and use are increasing, exposure of humans to bisphenols is becoming a significant issue. We evaluated the mutagenic and genotoxic potential of eight BPA structural analogues (BPF, BPAF, BPZ, BPS, DMBPA, DMBPS, BP-1, and BP-2) using the Ames and comet assay, respectively. None of the tested bisphenols showed a mutagenic effect in Salmonella typhimurium strains TA98 and TA100 in either the presence or absence of external S9-mediated metabolic activation (Aroclor 1254-induced male rat liver). Potential genotoxicity of bisphenols was determined in the human hepatoma cell line (HepG2) at non-cytotoxic concentrations (0.1 μmol L-1 to 10 μmol L-1) after 4-hour and 24-hour exposure. In the comet assay, BPA and its analogue BPS induced significant DNA damage only after the 24-hour exposure, while analogues DMBPS, BP-1, and BP-2 induced a transient increase in DNA strand breaks