Nanofibrillated cellulose: results of in vitro and in vivo toxicological assays

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1 Nanofibrillated cellulose: results of in vitro and in vivo toxicological assays Final Conference Hannu Norppa Nanosafety Research Center

2 Hazard assessment approach for NFC Methodology In vitro assays for cytotoxicity, genotoxicity, and immunotoxicity A nematode model for systemic effects and neurotoxicity Pharyngeal aspiration study with mice for pulmonary immunotoxicity and genotoxicity Hazard assessment in vitro (cell culture) tests Cytotoxicity, Immunotoxicity Genotoxicity in vivo tests Systemic effect in a nematode model Pulmonary toxicity in mice. NFC characterization Fiber size Dissolution Viscosity Risk assessment and management Control Banding approach CB Nanotool Stoffenmanager nano Exposure assessment Exposure scenario definition Data collection about the production and application of NFCs 2

3 NFC samples studied NFC-VTT Masuko grinder (5 passages) No pre-treatment, no bioside Bacteria NFC-CTP/VTT Masuko grinder (5 passages) Enzymatic pre-treatment, bioside added to the pulp Bacteria NFC-TE/VTT Masuko grinder (3 passages) TEMPO-mediated oxidation as a pre-treatment, no bioside Bacteria and yeast 3

4 NFC-TE/CTP samples studied NFC-TE/CTP B1 Lab scale high-pressure homogenizer TEMPO-mediated oxidation as pre-treatment Dialysed to remove traces of reactant No biocide added Bacteria NFC-TE/CTP B2 ± biocide Busan 19 Pilot scale high-pressure homogenizer TEMPO-mediated oxidation as pre-treatment Washed four times but might contained residues of reagents (TEMPO, sodium bromide and sodium hypochlorite) Without biocide: bacteria, yeast and mold With biocide: some bacteria 4

5 Highest Tolerated Dose (HTD) assay Cytotoxicity Qualitative morphological changes in human cervix carcinoma HeLa cells under an inverted phase contrast microscope Comparison with untreated cells and evaluation on a scale ranging from to 4 Only marginal effects seen at top doses ( 2 mg/ml) of NFCs, probably as a result of mechanical influence Total Protein Content (TPC) assay A quantitative test measuring growth inhibition of HeLa cells, total protein content as the indicator of the cells viability Based on reaction of fluorescamine with compounds containing primary amino groups to produce fluorescent products. None of the NFCs indicated a cytotoxic effect Boar Sperm Motility Assay (BSM) A qualitative test particularly suitable for testing of suspensions Boar spermatozoa s movement inhibition indicates mitochondrial or membrane damage No toxic effects seen 5

6 Immunotoxicity pulmonary inflammation In vitro studies Macrophages exposed to several doses of nanocelluloses in cell culture Cytotoxicity assessed by photometric and luminometric methods Macrophage activation assessed by measuring the expression of essential cytokines at the mrna level by real-time quantitative PCR at the protein level by ELISA In vivo studies on NFC-TE/CTP B2 + biocide Material for in vivo studies chosen by the Consortium Mice exposed by pharyngeal aspiration Inflammatory parameters analysed 6

inflammatory processes TNF-alpha, IL-1beta, and IL-6 determined in the present study In the

7 Macrophages act as the first line of defense against pathogens and particles in the lungs ingest pathogens and particles and break them down release proinflammatory cytokines which promote inflammatory processes TNF-alpha, IL-1beta, and IL-6 determined in the present study In the immunotoxicological part of the present study, human macrophages derived from blood monocytes of were used Figures: Wikimedia Commons 7

8 Cytotoxicity in human macrophages LDH release from cells expressed as percentage of total LDH, measured photometrically ATP in cells expressed as relative luminescence (percent of unexposed control), measured luminometrically Human monocyte-derived macrophages exposed for 6 h Cell culture supernatants collected Cytotoxicity measured by lactate dehydrogenase (LDH) leakage and decrease of ATP (luminescent cell viability assay) Roridin A (RoA) and silica used as positive controls NFCs were not cytotoxic to macrophages at doses up to 3 µg/ml Ilves et al., in preparation 8

9 RoA + LPS LPS NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 RoA + LPS LPS NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 IL-1 (RQ) TNF- (RQ) mrna expression of pro-inflammatory cytokines TNF-α in exposure to NFCs IL-1β and LPS Primed and unprimed human monocyte-derived macrophages exposed for 6 h Cell culture supernatants collected, mrna measured by PCR NFC-TE/VTT slightly increased IL-1-β and TNF-α mrna (due to bacteria & yeast?) 9 Ilves et al., in preparation

10 RoA + LPS LPS NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 RoA + LPS LPS NFC-VTT 3 NFC-VTT 1 NFC-VTT 3 NFC-CTP/VTT 3 NFC-CTP/VTT 1 NFC-CTP/VTT 3 NFC-TE/VTT 3 NFC-TE/VTT 1 NFC-TE/VTT 3 NFC-TE/CTP B1 3 NFC-TE/CTP B1 1 NFC-TE/CTP B1 3 IL-1 (pg/ml) TNF- (pg/ml) 4 Secretion of pro-inflammatory cytokines IL-1β and TNF-α in exposure to NFCs LPS Primed and unprimed human monocyte-derived macrophages exposed for 6 h Cell culture supernatants collected, pro-inflammatory cytokines measured by ELISA All NFCs slightly induced TNF-α and, in LPS-primed samples, IL-1-β 1 Ilves et al., in preparation

11 Roridin A + LPS LPS NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 Roridin A + LPS LPS NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 NFC-TE/CTP B2 3 NFC-TE/CTP B2 1 NFC-TE/CTP B2 3 IL-1 (RQ) TNF- (RQ) mrna expression of pro-inflammatory cytokines IL-1β and TNF-α in exposure to NFC-TE/CTP B2 biocide Biocide Biocide LPS Primed and unprimed human monocyte-derived macrophages exposed for 6 h Cell culture supernatants collected, mrna measured by PCR No significant effects observed 11 Ilves et al., in preparation

12 Roridin A LPS NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 Roridin A LPS NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 NFC-TE/CTP 3 NFC-TE/CTP 1 NFC-TE/CTP 3 IL-1 (pg/ml) TNF- (pg/ml) Secretion of pro-inflammatory cytokines IL-1β and TNF-α in exposure to NFC-TE/CTP B2 ± biocide Primed and unprimed human monocyte-derived macrophages exposed for 6 h Cell culture supernatants collected, pro-inflammatory cytokines measured by ELISA No effects observed Biocide Biocide LPS Ilves et al., in preparation

In vitro studies Genotoxicity identifying possible carcinogens Human bronchial epithelial BEAS 2B cells exposed

damage examined by the Comet assay Oxidative DNA damage studied by the enzyme-modified Comet assay Chromosome

In vivo studies on NFC-TE/CTP B2 + biocide One NFC material chosen for in vivo studies by the Consortium Mice

13 In vitro studies Genotoxicity identifying possible carcinogens Human bronchial epithelial BEAS 2B cells exposed to several doses of nanocelluloses in cell culture Cytotoxicity (cell count) utilized for dose finding DNA damage examined by the Comet assay Oxidative DNA damage studied by the enzyme-modified Comet assay Chromosome damage assessed by the micronucleus assay Cell cycle delay studied by the cytokinesis-block proliferation index In vivo studies on NFC-TE/CTP B2 + biocide One NFC material chosen for in vivo studies by the Consortium Mice exposed by pharyngeal aspiration DNA damage and oxidative DNA damage studied in bronchoalveolar lavage (BAL) cells 13

14 Living cells (% of control) Living cells (% of control) Cytotoxicity of NFC-TE/CTP B2 ± biocide in human bronchial epithelial BEAS 2B cells Double staining: Propidium iodide stains dead and dying cells, Hoechst stains cell nuclei Cell count in fluorescence microscope NFC-TE/CTP B2 with biocide NFC-TE/CTP B2 without biocide h 24 h 48 h h 24 h 48 h 2 2 2,5 5, 15, 3, 6, 12, 25, 2,5 5, 15, 3, 6, 12, 25, Dose (µg/cm²) Dose (µg/cm²) Cytotoxicity of the NFCs in BEAS 2B cells was generally low Hannukainen et al. in preparation 14

DNA damage detection by the comet assay DNA with strand

proportion of DNA in "comet tail" reflects the amount of DNA

DNA adducts to strand breaks by a specific enzyme (FPG)

15 DNA damage detection by the comet assay DNA with strand breaks wanders out of the nucleus in electrophoresis The proportion of DNA in "comet tail" reflects the amount of DNA damage Oxidative DNA damage visualized by turning oxidative DNA adducts to strand breaks by a specific enzyme (FPG) Analysis in fluorescence microscope using a semiautomatic interactive software 15

16 DNA-tail% DNA in tail (%) DNA damage induced in vitro by NFCs NFC/VTT NFC-TE/CTP 15 1 FPG Buffer Comet 2 15 FPG Buffer Comet NFC-VTT µg/cm Dose (µg/cm 2 ) No increase in DNA damage Slight increase in oxidative DNA damage (FPG vs Buffer) Slight increase in DNA damage in the comet assay Slight Increase in oxidative DNA damage at one dose 16

17 DNA in tail (%) DNA in tail (%) DNA damage induced in vitro by NFCs NFC-CTP/VTT NFC-TE/VTT 15 1 FPG Buffer Comet 15 1 FPG Buffer Comet Dose (µg/cm 2 ) Dose (µg/cm 2 ) Slight Increase in DNA damage in the Buffer and FPG series, but not in ordinary Comet assay No increase in oxidative DNA damage Slight increase in DNA damage in the comet assay and Buffer series No increase in oxidative DNA damage 17

18 DNA in tail (%) DNA in tail (%) DNA damage by NFC-TE/CTP B2 ± biocide NFC-TE/CTP B2 without biocide NFC-TE/CTP with 5 ppm Busan FPG Buffer Comet Enzyme Buffer Comet Busan Dose (µg/cm 2 ) Dose (µg/cm 2 ) No increase in DNA damage No increase in oxidative DNA damage Increase in DNA damage in the Buffer series, but not in ordinary Comet assay No increase in oxidative DNA damage 18

Micronucleus assay with NFCs A micronucleus Micronuclei reflect structural and numerical alterations of chromosomes Acridine orange staining: nuclei (DNA) are green, cytoplasm (RNA) is red Analysis

19 Micronucleus assay with NFCs A micronucleus Micronuclei reflect structural and numerical alterations of chromosomes Acridine orange staining: nuclei (DNA) are green, cytoplasm (RNA) is red Analysis in fluorescence microscope NFC Human bronchial epithelial BEAS 2B cells in fluorescence microscope after treatment with NFC-VTT (25 µg/cm 2 ). Photo: Kati Hannukainen and Hilkka Järventaus, FIOH 19

20 MNCs/1 cells MNCs/1 cells MNCs/1 cells MNCs/1 cells No induction of micronuclei by NFCs in BEAS 2B cells 25 NFC-CTP/VTT 25 NFC-TE/VTT Dose (µg/cm 2 ) NFC-VTT Dose (µg/cm 2 ) NFC-TE/CTP B2 + 5 ppm Busan h treatment Dose (µg/cm 2 ) No effect on cell cycle length either (data not shown) Biocide Dose (µg/cm 2 ) 2 Hannukainen et al., in preparation

5 mg/ml ( biocide) Single walled carbon nanotubes used as fibre control, DMSO (5%) as a positive control 24-h follow-up for

21 Nematode toxicity assay in vivo Transgenic line (P dat-1 ::GFP) of Caenorhabditis elegans expressing Green Fluorescent Protein in its dopaminergic neurons Bright fluorescence of ganglia indicates functional nervous system NFC-TE/CTP dose.5 mg/ml ( biocide) Single walled carbon nanotubes used as fibre control, DMSO (5%) as a positive control 24-h follow-up for viability, behavior and reproduction The NFCs were not toxic to C. elegans Photo: Prof. Garry Wong, University of Eastern Finland Photo: Jadiya et al

22 In vivo study in mice Female C57BL/6 mice NFC-TE/CTP B1 tested Exposure by pharyngeal aspiration Single dose: 2, 4, 8 and 2 µg/mouse (in PBS) Biocide Busan 19 tested separately at the same dose as in the NFC Negative and positive controls Samples collected 16 h after dosing (acute effect) Bronchoalveolar lavage fluid Lungs Blood Analysis of pulmonary inflammation and DNA damage 22

23 NFC-TE/CTP B2 in mouse lungs NFC In histological samples of the lungs, NFC was seen in or near bronchioles No dramatic tissue changes were observed 23

24 RU RU RU NFC-TE/CTP B2 induced mrna of pro-inflammatory cytokine IL-6 25 IL-1beta 6 TNF-alpha 3 IL-6 *** ** neg control biocide 1 4 MWCNT 1 neg control biocide 1 4 MWCNT 1 neg control biocide 1 4 MWCNT 1 24

25 cells/hpf cells/hpf cells/hpf cells/hpf NFC-TE/CTP B2 induced influx of inflammatory cells to mouse lungs macrophages neutrophils 2 15 * ** 2 15 *** ** * neg control biocide 1 4 MWCNT 1 neg control biocide 1 4 MWCNT 1 3 lymphocytes ** *.3 eosinophils neg control biocide 1 4 MWCNT 1. neg control biocide 1 4 MWCNT 1 25

26 DNA in tail (%) DNA in tail (%) No DNA damage in mouse bronchoalveolar lavage (BAL) cells after pharyngeal aspiration of NFC-TE/CTP B2 8 DNA damage after pharyngeal aspiration of NFC DNA damage in biocide-treated mice Control Biocide NFC mg/mouse Hannukainen et al. in preparation 26

27 Conclusions toxicity of NFCs In vitro Low or no cytotoxicity Slight induction of proinflammatory cytokines in macrophages with or without LPS priming in vitro Slight DNA damage in human bronchial epithelial cells similarly to a number of other "inert" nanomaterials Marginal induction of oxidative DNA damage in vitro (some NFCs) No increase in chromosome damage (micronuclei) In vivo (NFC-TE/CTP B2, pharyngeal aspiration, mice) No DNA damage in bronchoalveolar lavage cells Pulmonary inflammation Possibly due to the particulate / bacteria in the NFC 27

28 Toxicology research group FIOH, Helsinki Harri Alenius Julia Catalán Kati Hannukainen Marit Ilves Hannu Norppa Jaana Palomäki Elina Rydman Kai Savolainen Virpi Väänänen BioSafe, Kuopio Ulla Honkalampi Stefanie Kasurinen Terhi Myöhänen Atte von Wright 28

29 Acknowledgements The research leading to these results has received funding from the European Community s Seventh Framework Programme under grant agreement n o

Maria João Silva H. Louro 1, T. Borges 2, J. Lavinha 1, J.M. Albuquerque 1

Maria João Silva H. Louro 1, T. Borges 2, J. Lavinha 1, J.M. Albuquerque 1 1 National Institute of Health Dr. Ricardo Jorge 2 General-Directorate of Health, Lisbon, Portugal Venice.,10-03-2015 The number