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Molecular and cellular responses to titanium dioxide nanoparticles
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Abstracts / Toxicology Letters 238S (2015) S56–S383
repeated exposures. Both acute and repeated exposures resulted in induction of IL-6, KC, MCP-1 and MIP-1␣. These results demonstrate that ZnO-NPs can induce pulmonary inflammation. The acute response is largely neutrophil-mediated, but repeated exposures result in a macrophage-mediated and possibly adaptive immunological induction. http://dx.doi.org/10.1016/j.toxlet.2015.08.623
P08-043 Dissolution of CuO nanoparticles and their in vitro toxicity to Caco-2 cells and bacteria Escherichia coli depends on CuO ultrasonication protocol O. Bondarenko ∗ , A. Käkinen, H. Nurmsoo, A. Kahru National Institute of Chemical Physics and Biophysics, Environmental Toxicology, Tallinn, Estonia Toxicological testing of nanoparticles (NPs) is a serious methodical challenge, partly due to unstability (agglomeration, sedimentation) of NP suspensions prior and during testing. To keep NPs in dispersion, the suspension of NPs is usually treated by ultrasound either using ultrasonication bath (low intensity) or probe sonicator (higher intensities). As there is no standardized protocol available (e.g., type of medium and intensity of sonication), NPs’ dispersions prepared in different laboratories may significantly vary and potentially lead to noisy toxicity data. Here we asked to what extent the sonication protocol affects the toxicity of NPs. In parallel, the effect of sonication on dissolution and hydrodynamic size (Dh ) of NP agglomerates was measured. For that, CuO NPs from Sigma (primary size 30 nm) and CuO from EU FP7 project NanoValid (25 nm) were differently treated and tested for the toxicity to bacterial and mammalian cells in vitro. CuO NPs were prepared either in distilled water (DI), bacterial Luria-Bertani medium (LB) or complete cell culture medium (MEM) and were treated as follows: i) no sonication; ii) 30-min sonication in water bath; iii) probe sonication with specific energy (Espec ) 5.3 × 104 kJ/m3 and iv) probe sonication with Espec = 6 × 105 kJ/m3 . Dh of CuO NPs was measured using Malvern Zetasizer, dissolution by a) Picofox spectrometer and b) Cu-ion biosensor Escherichia coli. Toxicity of CuO suspensions to Caco-2 was evaluated by reduction of viability (Resazurin assay) and to E. coli by growth inhibition (minimal inhibitory concentration; MIC). In both test media (LB and MEM) as well as in DI, the Dh of CuO agglomerates decreased ∼2-fold in the following treatment order: not sonicated > 30-min bath > Espec = 5.3 × 104 kJ/m3 probe > Espec = 6 × 105 kJ/m3 probe. In DI water dissolution of CuO was low (∼1%) and was not affected by sonication intensity. However, in LB and MEM media the higher applied sonication energy led to increased dissolution and up to 2-fold higher toxicity of CuO to E. coli and Caco-2. We assume that more intense sonication decreased Dh of CuO and enhanced dissolution, leading to increased cytotoxicity. The detailed analysis of dissolution, Dh and test medium composition on toxicity of CuO will be discussed. EU FP7 NanoValid (grant agreement No. 263147) and Estonian IUT 23-5 are acknowledged. http://dx.doi.org/10.1016/j.toxlet.2015.08.624
P08-044 Inhibition of multixenobiotic resistance (MXR) transporters by silver nanoparticles and -ions in vitro and in vivo A. Georgantzopoulou 1 , A. Gutleb 1,∗ , S. Cambier 1 , T. Serchi 1 , A. Lankoff 2,3 , M. Kruszewski 2,3 , Y.L. Balachandran 4,5 , P. Grysan 6 , J.N. Audinot 6 , J. Ziebel 1 , C. Guignard 1 , A.J. Murk 7,8 1
Luxembourg Institute of Science and Technology, ERIN, Esch-sur-Alzette, Luxembourg 2 Institute of Rural Health, Independent Laboratory of Molecular Biology, Lublin, Poland 3 Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimety, Warsawa, Poland 4 Bharathiar University, Department of Biotechnology, Coimbatore, India 5 CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing, China 6 Luxembourg Institute of Science and Technology, MRT, Esch-sur-Alzette, Luxembourg 7 Wageningen University, Sub-department of Environmental Technology, Wageningen, Netherlands 8 Wageningen Institute for Marine Resources & Ecosystem Studies, IMARES, Ijmuiden, Netherlands The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATPbinding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MDR/MXR). In this study we first evaluated the interaction of silver nanoparticles (Ag NPs, 20-200 nm) and Ag ions (AgNO3 ) with MXR efflux transporters using MDCKII and the P-gp overexpressing MDCKII-MDR1 cells and calcein-AM as a substrate. Next the in vivo modulation of MXR activity was studied in D. magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. Perfluorooctane sulfonate and bisphenol A also inhibited the efflux of calcein from daphnids. Small-sized Ag NPs and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides for the first time evidence for interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in vivo and MXR activity should be taken into account when Ag NP toxicity is assessed. http://dx.doi.org/10.1016/j.toxlet.2015.08.625
P08-046 Molecular and cellular responses to titanium dioxide nanoparticles A. Zhornik ∗ , L. Baranova, I. Volotovski Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus, Department of Molecular Biology of Cell, Minsk, Belarus The increased use of nano-sized materials in the past several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. The objective of the research is to investigate the alterations in molecular and cellular responses in culture of human lymphocytes to one of the most widely used TiO2 nanoparticles. Human lymphocytes isolated from heparinized blood samples of healthy individuals
Abstracts / Toxicology Letters 238S (2015) S56–S383
were exposed to TiO2 NPs. Viability, ROS generation, the changes in the expression of genes encoding proinflammatory mediators TNF-␣, Il-1 and Il-8 and DNA damage were assessed. To examine the toxic effects of TiO2 NPs human lymphocytes were incubated with different concentrations of nanoparticles and viability was determined 24 and 48 h after treatment. Cell viability was decreased in both a time- and concentration-dependent manner. The ability of TiO2 to induce ROS formation in lymphocytes was evaluated using DCF (2 ,7 -dichlorofluorescin) fluorescence. The fluorescence intensity of oxidized DCF was increased in cells when treated with NPs. This means that ROS generation was occurring in response to the treatment with TiO2 NPs in a concentrationand time-dependent manner, while fluorescence was insignificant in the control group. To investigate the expression level of mRNA related to the inflammation responses in lymphocytes real-time PCR was performed. The expression of Il-1, Il-8 and TNF-␣ genes was increased by the exposure to nanoparticles for 24-48 h. TiO2 nanoparticles were shown to induce the dose-dependent fragmentation of DNA strands. nMuch evidence of hazardous health effects of NPs has been reported in toxicological studies. In this study, viability was reduced under the exposure to TiO2 . Oxidative stress was elevated by the treatment with TiO2 NPs. Consequently, cytotoxicity in human lymphocytes seemed to be caused by oxidative stress. Oxidative stress may also trigger inflammation signals. Induced by exposure to NPs it may cause the translocation of transcription factors to the nucleus, which regulate pro-inflammatory genes, such as TNF-␣, Il-1, Il-8. ROS is an important factor in the apoptotic process, and the excess generation of ROS induces mitochondrial membrane permeability and damages the respiratory chain, to trigger the apoptotic process. http://dx.doi.org/10.1016/j.toxlet.2015.08.626
P08-047 The joint antibacterial effect of silver nanoparticles and antibiotics H. Wang 1,∗ , Y.-W. Wang 1 , K. Han 1 , D. Wu 1 , X. Ma 1 , D. Liu 2 , H. Tang 3 , Y. Liu 1,3 , A. Cao 1 1
Shanghai University, Institute of Nanochemistry and Nanobiology, Shanghai, China 2 University of Eastern Finland, Department of Applied Physics, Kuopio, Finland 3 Peking University, College of Chemistry and Molecular Engineering, Beijing, China Question: The extensive utilization of Ag NPs makes them most likely to be released into the environment in a great quantity and thus increase the environmental exposure. Meanwhile antibiotic contamination has become a global problem and high concentrations of antibiotics have been detected in the water environment. Then the opportunity of the coexistence of Ag NPs and antibiotics in the environment increases. Thus, the study of joint effects of Ag NPs and antibiotics on microbes becomes important for understanding the bioeffects of Ag NPs in complex systems. Methods: We studied the combined antibacterial activity of poly(N-vinyl-2-pyrrolidone) coated silver nanoparticles (Ag-PVP NPs) and antibiotics against three bacterial strains (S. aureus, E. coli and gentamicin-resistant E. coli) by measuring the fractional inhibitory concentration indices, bacterial survival rates and growth curves. Focus on Ag-PVP NPs and gentamicin, we investigated the mechanism of their synergistic effect by characterize thoroughly the properties of Ag-PVPs in the presence of gentamicin. Results: Both synergistic and antagonistic effects were observed in the joint systems. Ag-PVP NPs and gentam-
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icin possessed the synergistic antibacterial activity. The mechanism investigation indicates that gentamicin promoted the dissolution of Ag-PVP NPs and enhanced the positive charge of the NPs, which further increased the concentration of silver ions in the media and facilitated the attachment of Ag NPs onto bacteria, correspondingly. As a result, higher silver accumulation appeared in bacteria led to the bacterial growth inhibition and death. Meanwhile, gentamicin was found to inhibit the antibacterial activity of released silver ions by forming complex with silver ions. Conclusions: The joint systems of Ag-PVP NPs and antibiotics show different antibacterial activities. The significant synergistic effect of Ag-PVP NPs and gentamicin was observed. http://dx.doi.org/10.1016/j.toxlet.2015.08.627
P08-048 Effects of selected metal oxide nanoparticles on ovarian steroidogenesis: Use of whole ovary culture technique S. Scsukova 1,∗ , A. Mlynarcikova 1 , A. Kiss 1 , Z. Vecera 2 , P. Mikuska 2 , E. Rollerova 3 1
Institute of Experimental Endocrinology SAS, Bratislava, Slovakia Institute of Analytical Chemistry CAS, Brno, Czech Republic 3 Slovak Medical University, Department of Toxicology, Bratislava, Slovakia 2
Metal and metal-oxide nanoparticles (NPs) are two of the most prevalent types of manufactured NPs used in medicine, consumer products and industrial settings. Recent data show that NPs might cause adverse effects on human reproductive health. In the present study, whole ovary culture was used as an in vitro assay for analyzing ovarian steroidogenic activity following in vivo metal-oxide (cadmium oxide, CdO; lead oxide, PbO; titanium dioxide, TiO2 ) NPs exposure. Adult female mice were exposed to PbO (1.23 × 106 #/cm3 , 26.1 nm), CdO (1.25 × 106 #/cm3 , 14.4 nm) and TiO2 (5.0 × 104 and 1.5 × 106 #/cm3 , 30.0 and 29.6 nm) NPs by inhalation in whole-body inhalation chamber continuously (24 h/day) for 6 weeks. Newborn female Wistar rats were i.p. injected daily with 1% or 10% LD50 of TiO2 NPs (LD50 = 59.22 mg/kg b.w., in vitro under basal or FSH/IGF-stimulated conditions for 6 days. Every other day, culture media were collected for hormone analysis and replaced with fresh medium. Progesterone (P4) levels in the culture media were analyzed by radioimmunoassay. The presence of FSH/IGF induced stimulation in P4 secretion compared to basal secretion at each day tested. Basal P4 levels were not significantly altered by the action of tested NPs in both mice and rats, although in mice, an inhibitory trend after PbO and TiO2 NPs in vivo treatment was shown. Mice ovaries exposed in vivo to PbO NPs failed to response to FSH/IGF stimulation in P4 secretion at each time interval. Similar effect was observed in rats after 1% LD50 of TiO2 in vivo treatment. In mice, in vivo treatment by CdO NPs had no significant effect on ovarian P4 in vitro secretion. The results using cultures of whole mouse and rat ovary showed perturbation in steroidogenesis after PbO and TiO2 exposures in vivo. Possible sites of NPs action on the steroidogenic pathway should be further elucidated. This work was supported by the Slovak Research and Development Agency under the contract No APVV-0404-11; VEGA Grant 2/0172/14; the European Commission 7th Framework Programme for the QualityNano project (EC FP7, [INFRA-20101.131], Contract No. 262163) and by the Czech Science Foundation under the project “Centre for studies on toxicity of nanoparticles” (P503/12/G147). http://dx.doi.org/10.1016/j.toxlet.2015.08.628
Abstracts / Toxicology Letters 238S (2015) S56–S383
repeated exposures. Both acute and repeated exposures resulted in induction of IL-6, KC, MCP-1 and MIP-1␣. These results demonstrate that ZnO-NPs can induce pulmonary inflammation. The acute response is largely neutrophil-mediated, but repeated exposures result in a macrophage-mediated and possibly adaptive immunological induction. http://dx.doi.org/10.1016/j.toxlet.2015.08.623
P08-043 Dissolution of CuO nanoparticles and their in vitro toxicity to Caco-2 cells and bacteria Escherichia coli depends on CuO ultrasonication protocol O. Bondarenko ∗ , A. Käkinen, H. Nurmsoo, A. Kahru National Institute of Chemical Physics and Biophysics, Environmental Toxicology, Tallinn, Estonia Toxicological testing of nanoparticles (NPs) is a serious methodical challenge, partly due to unstability (agglomeration, sedimentation) of NP suspensions prior and during testing. To keep NPs in dispersion, the suspension of NPs is usually treated by ultrasound either using ultrasonication bath (low intensity) or probe sonicator (higher intensities). As there is no standardized protocol available (e.g., type of medium and intensity of sonication), NPs’ dispersions prepared in different laboratories may significantly vary and potentially lead to noisy toxicity data. Here we asked to what extent the sonication protocol affects the toxicity of NPs. In parallel, the effect of sonication on dissolution and hydrodynamic size (Dh ) of NP agglomerates was measured. For that, CuO NPs from Sigma (primary size 30 nm) and CuO from EU FP7 project NanoValid (25 nm) were differently treated and tested for the toxicity to bacterial and mammalian cells in vitro. CuO NPs were prepared either in distilled water (DI), bacterial Luria-Bertani medium (LB) or complete cell culture medium (MEM) and were treated as follows: i) no sonication; ii) 30-min sonication in water bath; iii) probe sonication with specific energy (Espec ) 5.3 × 104 kJ/m3 and iv) probe sonication with Espec = 6 × 105 kJ/m3 . Dh of CuO NPs was measured using Malvern Zetasizer, dissolution by a) Picofox spectrometer and b) Cu-ion biosensor Escherichia coli. Toxicity of CuO suspensions to Caco-2 was evaluated by reduction of viability (Resazurin assay) and to E. coli by growth inhibition (minimal inhibitory concentration; MIC). In both test media (LB and MEM) as well as in DI, the Dh of CuO agglomerates decreased ∼2-fold in the following treatment order: not sonicated > 30-min bath > Espec = 5.3 × 104 kJ/m3 probe > Espec = 6 × 105 kJ/m3 probe. In DI water dissolution of CuO was low (∼1%) and was not affected by sonication intensity. However, in LB and MEM media the higher applied sonication energy led to increased dissolution and up to 2-fold higher toxicity of CuO to E. coli and Caco-2. We assume that more intense sonication decreased Dh of CuO and enhanced dissolution, leading to increased cytotoxicity. The detailed analysis of dissolution, Dh and test medium composition on toxicity of CuO will be discussed. EU FP7 NanoValid (grant agreement No. 263147) and Estonian IUT 23-5 are acknowledged. http://dx.doi.org/10.1016/j.toxlet.2015.08.624
P08-044 Inhibition of multixenobiotic resistance (MXR) transporters by silver nanoparticles and -ions in vitro and in vivo A. Georgantzopoulou 1 , A. Gutleb 1,∗ , S. Cambier 1 , T. Serchi 1 , A. Lankoff 2,3 , M. Kruszewski 2,3 , Y.L. Balachandran 4,5 , P. Grysan 6 , J.N. Audinot 6 , J. Ziebel 1 , C. Guignard 1 , A.J. Murk 7,8 1
Luxembourg Institute of Science and Technology, ERIN, Esch-sur-Alzette, Luxembourg 2 Institute of Rural Health, Independent Laboratory of Molecular Biology, Lublin, Poland 3 Institute of Nuclear Chemistry and Technology, Centre for Radiobiology and Biological Dosimety, Warsawa, Poland 4 Bharathiar University, Department of Biotechnology, Coimbatore, India 5 CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing, China 6 Luxembourg Institute of Science and Technology, MRT, Esch-sur-Alzette, Luxembourg 7 Wageningen University, Sub-department of Environmental Technology, Wageningen, Netherlands 8 Wageningen Institute for Marine Resources & Ecosystem Studies, IMARES, Ijmuiden, Netherlands The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATPbinding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MDR/MXR). In this study we first evaluated the interaction of silver nanoparticles (Ag NPs, 20-200 nm) and Ag ions (AgNO3 ) with MXR efflux transporters using MDCKII and the P-gp overexpressing MDCKII-MDR1 cells and calcein-AM as a substrate. Next the in vivo modulation of MXR activity was studied in D. magna juveniles with the model P-gp and MRP1 inhibitors verapamil-HCl and MK571, respectively. Perfluorooctane sulfonate and bisphenol A also inhibited the efflux of calcein from daphnids. Small-sized Ag NPs and AgNO3 inhibited the MXR activity in daphnids and MDCKII-MDR1, but abcb1 gene expression remained unchanged. Both Ag NPs and dissolved ions contributed to the effects. This study provides for the first time evidence for interference of Ag NPs and AgNO3 with the MXR activity both in vitro and in vivo and MXR activity should be taken into account when Ag NP toxicity is assessed. http://dx.doi.org/10.1016/j.toxlet.2015.08.625
P08-046 Molecular and cellular responses to titanium dioxide nanoparticles A. Zhornik ∗ , L. Baranova, I. Volotovski Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus, Department of Molecular Biology of Cell, Minsk, Belarus The increased use of nano-sized materials in the past several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. The objective of the research is to investigate the alterations in molecular and cellular responses in culture of human lymphocytes to one of the most widely used TiO2 nanoparticles. Human lymphocytes isolated from heparinized blood samples of healthy individuals
Abstracts / Toxicology Letters 238S (2015) S56–S383
were exposed to TiO2 NPs. Viability, ROS generation, the changes in the expression of genes encoding proinflammatory mediators TNF-␣, Il-1 and Il-8 and DNA damage were assessed. To examine the toxic effects of TiO2 NPs human lymphocytes were incubated with different concentrations of nanoparticles and viability was determined 24 and 48 h after treatment. Cell viability was decreased in both a time- and concentration-dependent manner. The ability of TiO2 to induce ROS formation in lymphocytes was evaluated using DCF (2 ,7 -dichlorofluorescin) fluorescence. The fluorescence intensity of oxidized DCF was increased in cells when treated with NPs. This means that ROS generation was occurring in response to the treatment with TiO2 NPs in a concentrationand time-dependent manner, while fluorescence was insignificant in the control group. To investigate the expression level of mRNA related to the inflammation responses in lymphocytes real-time PCR was performed. The expression of Il-1, Il-8 and TNF-␣ genes was increased by the exposure to nanoparticles for 24-48 h. TiO2 nanoparticles were shown to induce the dose-dependent fragmentation of DNA strands. nMuch evidence of hazardous health effects of NPs has been reported in toxicological studies. In this study, viability was reduced under the exposure to TiO2 . Oxidative stress was elevated by the treatment with TiO2 NPs. Consequently, cytotoxicity in human lymphocytes seemed to be caused by oxidative stress. Oxidative stress may also trigger inflammation signals. Induced by exposure to NPs it may cause the translocation of transcription factors to the nucleus, which regulate pro-inflammatory genes, such as TNF-␣, Il-1, Il-8. ROS is an important factor in the apoptotic process, and the excess generation of ROS induces mitochondrial membrane permeability and damages the respiratory chain, to trigger the apoptotic process. http://dx.doi.org/10.1016/j.toxlet.2015.08.626
P08-047 The joint antibacterial effect of silver nanoparticles and antibiotics H. Wang 1,∗ , Y.-W. Wang 1 , K. Han 1 , D. Wu 1 , X. Ma 1 , D. Liu 2 , H. Tang 3 , Y. Liu 1,3 , A. Cao 1 1
Shanghai University, Institute of Nanochemistry and Nanobiology, Shanghai, China 2 University of Eastern Finland, Department of Applied Physics, Kuopio, Finland 3 Peking University, College of Chemistry and Molecular Engineering, Beijing, China Question: The extensive utilization of Ag NPs makes them most likely to be released into the environment in a great quantity and thus increase the environmental exposure. Meanwhile antibiotic contamination has become a global problem and high concentrations of antibiotics have been detected in the water environment. Then the opportunity of the coexistence of Ag NPs and antibiotics in the environment increases. Thus, the study of joint effects of Ag NPs and antibiotics on microbes becomes important for understanding the bioeffects of Ag NPs in complex systems. Methods: We studied the combined antibacterial activity of poly(N-vinyl-2-pyrrolidone) coated silver nanoparticles (Ag-PVP NPs) and antibiotics against three bacterial strains (S. aureus, E. coli and gentamicin-resistant E. coli) by measuring the fractional inhibitory concentration indices, bacterial survival rates and growth curves. Focus on Ag-PVP NPs and gentamicin, we investigated the mechanism of their synergistic effect by characterize thoroughly the properties of Ag-PVPs in the presence of gentamicin. Results: Both synergistic and antagonistic effects were observed in the joint systems. Ag-PVP NPs and gentam-
S211
icin possessed the synergistic antibacterial activity. The mechanism investigation indicates that gentamicin promoted the dissolution of Ag-PVP NPs and enhanced the positive charge of the NPs, which further increased the concentration of silver ions in the media and facilitated the attachment of Ag NPs onto bacteria, correspondingly. As a result, higher silver accumulation appeared in bacteria led to the bacterial growth inhibition and death. Meanwhile, gentamicin was found to inhibit the antibacterial activity of released silver ions by forming complex with silver ions. Conclusions: The joint systems of Ag-PVP NPs and antibiotics show different antibacterial activities. The significant synergistic effect of Ag-PVP NPs and gentamicin was observed. http://dx.doi.org/10.1016/j.toxlet.2015.08.627
P08-048 Effects of selected metal oxide nanoparticles on ovarian steroidogenesis: Use of whole ovary culture technique S. Scsukova 1,∗ , A. Mlynarcikova 1 , A. Kiss 1 , Z. Vecera 2 , P. Mikuska 2 , E. Rollerova 3 1
Institute of Experimental Endocrinology SAS, Bratislava, Slovakia Institute of Analytical Chemistry CAS, Brno, Czech Republic 3 Slovak Medical University, Department of Toxicology, Bratislava, Slovakia 2
Metal and metal-oxide nanoparticles (NPs) are two of the most prevalent types of manufactured NPs used in medicine, consumer products and industrial settings. Recent data show that NPs might cause adverse effects on human reproductive health. In the present study, whole ovary culture was used as an in vitro assay for analyzing ovarian steroidogenic activity following in vivo metal-oxide (cadmium oxide, CdO; lead oxide, PbO; titanium dioxide, TiO2 ) NPs exposure. Adult female mice were exposed to PbO (1.23 × 106 #/cm3 , 26.1 nm), CdO (1.25 × 106 #/cm3 , 14.4 nm) and TiO2 (5.0 × 104 and 1.5 × 106 #/cm3 , 30.0 and 29.6 nm) NPs by inhalation in whole-body inhalation chamber continuously (24 h/day) for 6 weeks. Newborn female Wistar rats were i.p. injected daily with 1% or 10% LD50 of TiO2 NPs (LD50 = 59.22 mg/kg b.w., in vitro under basal or FSH/IGF-stimulated conditions for 6 days. Every other day, culture media were collected for hormone analysis and replaced with fresh medium. Progesterone (P4) levels in the culture media were analyzed by radioimmunoassay. The presence of FSH/IGF induced stimulation in P4 secretion compared to basal secretion at each day tested. Basal P4 levels were not significantly altered by the action of tested NPs in both mice and rats, although in mice, an inhibitory trend after PbO and TiO2 NPs in vivo treatment was shown. Mice ovaries exposed in vivo to PbO NPs failed to response to FSH/IGF stimulation in P4 secretion at each time interval. Similar effect was observed in rats after 1% LD50 of TiO2 in vivo treatment. In mice, in vivo treatment by CdO NPs had no significant effect on ovarian P4 in vitro secretion. The results using cultures of whole mouse and rat ovary showed perturbation in steroidogenesis after PbO and TiO2 exposures in vivo. Possible sites of NPs action on the steroidogenic pathway should be further elucidated. This work was supported by the Slovak Research and Development Agency under the contract No APVV-0404-11; VEGA Grant 2/0172/14; the European Commission 7th Framework Programme for the QualityNano project (EC FP7, [INFRA-20101.131], Contract No. 262163) and by the Czech Science Foundation under the project “Centre for studies on toxicity of nanoparticles” (P503/12/G147). http://dx.doi.org/10.1016/j.toxlet.2015.08.628