Environmental Research 134 (2014) 39–45

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Genotoxic and mutagenic potential of nitramines Lise Marie Fjellsbø a,n, Sandra Verstraelen b, Alena Kazimirova c, An R. Van Rompay b, Zuzana Magdolenova a, Maria Dusinska a a

Health Effects Laboratory, MILK, NILU – Norwegian Institute for Air Research, 2007 Kjeller, Norway Flemish Institute for Technological Research (VITO), Environmental Risk and Health Unit, 2400 Mol, Belgium c Institute of Biology, Slovak Medical University (SMU), 833 03 Bratislava, Slovakia b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 March 2014 Received in revised form 6 June 2014 Accepted 9 June 2014

Climate change is one of the major challenges in the world today. To reduce the amount of CO2 released into the atmosphere, CO2 at major sources, such as power plants, can be captured. Use of aqueous amine solutions is one of the most promising methods for this purpose. However, concerns have been raised regarding its impacts on human health and the environment due to the degradation products, such as nitrosamines and nitramines that may be produced during the CO2 capture process. While several toxicity studies have been performed investigating nitrosamines, little is known about the toxic potential of nitramines. In this study a preliminary screening was performed of the genotoxic and mutagenic potential of nitramines most likely produced during amine based CO2 capture; dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2), 2-methyl-2-(nitramino)-1-propanol (AMP-NO2) and piperazine nitramine (PZ-NO2), by the Bacterial Reverse Mutation (Ames) Test, the Cytokinesis Block Micronucleus (CBMN) Assay and the in vitro Single-Cell Gel Electrophoresis (Comet) Assay. MA-NO2 and MEA-NO2 showed mutagenic potential in the Ames test and a weak genotoxic response in the CBMN Assay. AMP-NO2 and PZ-NO2 significantly increased the amount of DNA strand breaks; however, the level of breaks was below background. Most previous studies on nitramines have been performed on DMA-NO2, which in this study appeared to be the least potent nitramine. Our results indicate that it is important to investigate other nitramines that are more likely to be produced during CO2 capture, to ensure that the risk is realistically evaluated. & 2014 Elsevier Inc. All rights reserved.

Keywords: CO2 capture Health effects Genotoxicity Mutagenicity Nitramines

1. Introduction Post combustion carbon dioxide (CO2) capture from exhaust gases at large point sources like power plants and subsequent geological storage has the potential to reduce greenhouse gas emissions significantly. Several technologies have been considered for this purpose. Use of aqueous amine solvents is currently the most advanced technology (Reynolds et al., 2012), although until now there is currently no full scale application at power plants. This technology also raises potential concerns related to health and environmental impacts. During the capture process, amines

Abbreviations: AMP-NO2, 2-methyl-2-(nitramino)-1-propanol; CBMN, cytokinesis block micronucleus; CBPI, cytokinesis-block proliferation index; comet assay, single-cell gel electrophoresis assay; DMA-NO2, dimethylnitramine; MA-NO2, methylnitramine; MEA-NO2, ethanolnitramine; MNBNC, micronucleus in binucleated cells; PZ-NO2, piperazine nitramine; RGA, relative growth activity; SBs, strand breaks n Corresponding author. Fax: þ 476 3898050. E-mail address: [email protected] (L.M. Fjellsbø). http://dx.doi.org/10.1016/j.envres.2014.06.008 0013-9351/& 2014 Elsevier Inc. All rights reserved.

can react with nitrogen oxides (NOx) in the flue gas, to form degradation products, including nitrosamines and nitramines (Dai et al., 2012). Although the effluent is treated with water wash systems to remove the amines and potential degradation products, atmospheric releases of these compounds may still occur. In addition, nitramines and nitrosamines may be formed in the atmosphere, through photo oxidation of amines emitted from the capture facility (da Silva and Booth, 2013). The most likely routes of human exposure to nitrosamines and nitramines are via air or water. These compounds are hydrophilic, and it is therefore more likely that they will partition to the water phase rather than adsorb to soils and sediments, with the possibility of reaching ground and drinking water (da Silva and Booth, 2013). A previous study by Fjellsbø et al. (2013) investigated potential acute toxic effects on human eye and skin of four nitramines, and they were all found to be irritants for eye, but none were irritants or corrosive for the skin. The concentrations used in this study were higher than what can be expected near the capture facility. The emission levels have been reported to be in the range of 2–50 ng/N m3 for both nitrosamines and nitramines

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for a pilot post combustion CO2 capture plant operating with monoethanolamine (MEA) (da Silva and Booth, 2013). Because people living in close proximity to the capture facility may be exposed to these compounds for lifetime, it is important to evaluate the mutagenic and carcinogenic potential of nitrosamines and nitramines. Several studies on the carcinogenic potency and mechanisms of action of nitrosamines have been performed (Klein et al., 1991; Lin, 1990; Magee et al., 1976; Montesano and Bartsch, 1976, Peto et al., 1991a, 1991b; Wagner et al., 2012). In contrast, very little information is available for genotoxic effects of nitramines. Dimethylnitramine (DMA-NO2) was found to be both mutagenic (Frei et al. 1984, 1986; Malaveille et al., 1983; Pool et al., 1984) and carcinogenic (Goodall and Kennedy, 1976; Mirvish et al., 1980; Pliss et al., 1982). Furthermore, methylnitramine (MA-NO2) showed carcinogenic potential (CPDB, 2007; Hassel et al., 1987; Scherf et al., 1989) but no evidence was found on mutagenic activity (Frei et al., 1984, 1986; Pool et al., 1984). Based on these studies, nitramines are considered less potent in their biological activity than nitrosamines. However, since some nitramines are more stable than nitrosamines (Låg et al., 2011) and population exposure is therefore more likely, further investigation for their genotoxic and mutagenic potential is considered as very important to determine the potential adverse health effects of amine-based CO2 capture technology. Although some knowledge exists about the nitramines that may form after degradation of methylamine and dimethylamine, other amines such as MEA, diethanolamine, methyldiethanolamine, 2-amino-2-methyl-1-propanol, diglycolamine, di-isopropanolamine, triethanolamine, and piperazine are more commonly used (Gentry et al. 2013), and MEA is currently the most widely used solvent (Reynolds et al., 2012). Until now, there are no studies available investigating the genotoxic or mutagenic potency of their corresponding nitramines or other nitramines that might occur during the capturing process. In this study, a battery of tests were performed to determine the possible genotoxic and mutagenic potency of five nitramines, i.e. DMA-NO2, MA-NO2, ethanolnitramine (MEA-NO2), 2-methyl-2(nitramino)-1-propanol (AMP-NO2), and piperazine nitramine (PZNO2) were screened in the Bacterial Reverse Mutation (Ames) Test, the Cytokinesis Block Micronucleus (CBMN) Assay and the in vitro Single-Cell Gel Electrophoresis (Comet) Assay.

2. Materials and methods 2.1. Preparation and characterisation of nitramines Five nitramines DMA-NO2 (CAS No 4164-28-7), MA-NO2 (CAS No 598-57-2), MEA- NO2 (CAS No 74386-82-6), AMP- NO2 (CAS No 1239666-60-4) and PZ- NO2 (CAS No 42499-41-2) were synthesised at University of Ås (UMB, Norway). The purity was 4 99% for all nitramines and the chemical analysis (HPLC/UV/HRMS) did not show any significant presence of degradation products in the solutions. The nitramines were dissolved in sterile water (Ames test) or RPMI 1640 (Comet Assay and CBMN), and filtered to reach sterile conditions before exposure. Dissolving the nitramines in aqueous solution was important firstly to have similar conditions for all tests performed, and also for reducing risk of workers, as the crystalline state could be explosive. Concentrations of nitramines differed for the three assays and are described in detail below.

measured by a haemocytometer during cultivation and the cells were routinely diluted to  4  105 cells/ml to prevent overgrowth ( 4 1.5  106 cells/ml). 2.3. Ames test The bacterial reverse mutation test (Ames test) was performed as described in OECD Test Guideline 471 (1997). To test for metabolic activation, a liver postmitochondrial fraction S9 (Molecular Toxicology Incorporated, USA) obtained from male Sprague Dawley rats induced with Aroclor 1254 was added to the bacteria. Treatments were carried out both in the absence and presence of a 10% S9 mixture. For the treatment without S9 mixture, 500 ml 0.1 M sodium phosphate buffer pH 7.4 was added to a tube for each plate. For the treatment with S9 mixture 500 ml is added to a tube in total also, containing sodium phosphate (Na2HPO4) buffer pH 7.4 (250 ml, 0.2 M), glucose-6-phosphate (2.5 ml, 1 M), nicotinamide adenine dinucleotide phosphate (NADPH) (20 ml, 0.1 M), kalium chloride/magnesium dichloride (KCl/MgCl2) (10 ml, 1.65 M/0.4 M), S9 (50 ml), and water (167.5 ml) Selection of an adequate range of doses was based on a toxicity range-finder experiment (plate incorporation method) with the strain Salmonella typhimurium TA100 at 10 concentrations separated by a factor 2. The highest concentration was the highest water-soluble concentration of the nitramines. Untreated, solvent (sterile water), and positive control (sodium azide: 0.005 mg/plate,  S9 and 2-aminoanthracene: 0.0025 mg/plate, þS9) plates were included. Suspensions of bacterial cells were exposed to the nitramine solutions in the presence and in the absence of S9. The test solution was mixed in triplicate with the strain TA100, the sterile buffer (  S9) or the metabolic activation system ( þ S9) and with agar containing biotin and a trace L-histidine to allow a few cell divisions. Plating was achieved by the following sequence of additions in a tube: 0.5 ml of 10% S9 mixture (þ S9) or buffer solution (–S9), 0.1 ml of nitramine solution or control, 2 ml of enriched moltenagar at 45 7 2 1C, and 0.1 ml of TA100 bacterial culture containing approximately 108 viable bacteria. The mixture was rapidly mixed and poured completely on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 71 1C in the dark for 487 4 h. Quality controls characteristics (histidine-dependence, rfa character, UvrB character, and resistance to ampicillin or ampicillin plus tetracycline) and colony forming units were performed on TA100. The spontaneous reversion rate and mean revertants of solvent control were compared with the historical control range of the laboratory. After a toxicity range-finder experiment, two independent reverse mutation tests were performed. The first was a standard plate incorporation assay and the second involved a pre-incubation stage. At least five scorable concentrations for all nitramines were obtained in the range-finding study with the strain TA100. This strain was not retested in the first Reverse Mutation Experiment, where the nitramine solutions were tested for reverse mutations in the Salmonella typhimurium strains TA98, TA102, TA1535, and TA1537. Seven concentrations, separated by a factor 2 were tested in triplicate in the absence and in the presence of S9 mixture starting from a highest concentration of 4.50 mg/plate for DMA-NO2; 15.50 mg/plate for MA-NO2; 15.90 mg/plate for MEA-NO2; 18.70 mg/plate for AMP-NO2; and 7.70 mg/ plate for PZ-NO2. Untreated, solvent, and positive controls (sodium azide (TA1535: 0.001 mg/plate,  S9), 4-nitroquinoline oxide (TA98: 0.0002 mg/plate,  S9 and TA102: 0.002 mg/plate,  S9), 9-aminoacridine (TA1537: 0.05 mg/plate,  S9) and 2-aminoanthracene (TA98, TA1535, and TA1537: 0.0025 mg/plate, þS9 and TA102: 0.0075 mg/plate, þ S9)) were included. The second Reverse Mutation Experiment included a pre-incubation step. Volumes of 0.1 ml of bacterial culture, 0.1 ml of nitramine solution or control, and 0.5 ml of 10% S9 mixture (þ S9) or buffer solution (–S9) were mixed together for 30 min at 377 1 1C, where after 2 ml of molten top agar (7 45 1C) was added. The same concentrations were applied as in the first Reverse Mutation Test. Colonies were scored automatically by using the Sorcerer Image Analysis/ Colony counting system (Perspective Instruments, UK). The plates were examined for their background layer (signs of toxicity) and revertant colonies were counted. The induction factor (IF) was obtained by dividing the mean number of revertant colonies (n¼ 3) obtained with the nitramine solutions by the mean number of revertant colonies obtained with the solvent control (n ¼3). The nitramine solutions were considered to be mutagenic towards bacteria in the Ames test if a concentration related increase in the number of mean revertant colonies was observed compared to solvent control with at least a 2-fold increase of IF with the strains TA98, TA100, and TA102, and at least a 3-fold increase of IF with the strains TA1535 and TA1537 in the absence and/or in the presence of rat liver S9 mixture. 2.4. Cytokinesis block micronucleus assay

2.2. Cell culture The TK6 human lymphoblastoid cell line obtained from the European Collection of Cell Cultures (ECACC, Lot no 05F013) was used in the Comet Assay and CBMN Assay. TK6 cells were cultured in RPMI 1640 (Sigma, Cat no R8758) supplemented with 100 μg/ml penicillin-100 μg/ml streptomycin (GIBCO by Invitrogen Corporation, Cat no 15140) and 10% (v/v) heat-inactivated (15 min at 55 1C) foetal calf serum (FCS) (GIBCO, Cat no 26140). The cells were incubated in vented upright T-75 cm2 flasks in humidified atmosphere at 37 1C, 5% CO2. Cell density was

The CBMN assay was performed based on OECD TG 487 (2010). Approximately 24 h prior to treatment with test substance, the TK6 cell stock cultures were prepared in vented upright T-75 cm2 flasks at a cell density of 5–6  105 cells/ml. Concentrations of 156, 625, and 2500 μg/ml were tested for each nitramine. Concurrent positive controls both with (cyclophosphamide, 30 μg/ml) and without metabolic activation (mitomycin C, 50 ng/ml) were included in each experiment. Untreated cells were used as negative control. Prior to exposure, 2.5 ml aliquots were dispensed into 15 ml centrifuge tubes (3 h treatments) or into T-10 cm2 tissue culture flat tubes (24 h treatment).

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The cells were centrifuged (200g, 5 min) and re-suspended in RPMI 1640 and added to 15 ml falcon tubes. The cell concentration during exposure was 5  105 cells/ml. Three concentrations of each nitramine were tested; 25, 250 and 2500 mg/ml for PZ-NO2 and AMP-NO2; 25, 250 and 1930 mg/ml for DMA-NO2; 27, 277 and 2775 mg/ml for MA-NO2 and 19, 190 and 1900 mg/ml for MEA-NO2. The nitramine solutions were added to the cell suspensions, giving a total volume of 2 ml, and incubated at 37 1C for 3 h. As negative control, unexposed cells cultivated in RPMI 1640 were used. One negative control incubated with Buffer F, and one negative control incubated with formamidopyrimidine DNA glycosylase (FPG) enzyme was added during enzyme treatment. As positive control, a directly acting mutagen, 50 mM hydrogen peroxide (H2O2) was used (5 min). As positive control for detection of oxidised purines, 1 mM photosensitiser RO19-8022 (LaRoche) and visible light (250 W, 33 cm distance, 4 min on ice) were used. The cells were washed after exposure, and approximately 104 cells were placed in eppendorf tubes and put on ice. 250 ml 1% low melting point agarose in PBS were added to the cells, and two 5 μl drops of mixture were placed in parallel on a 12 gel format glass slide. The comet assay was performed with and without incubation with FPG, to detect oxidised purines, as described earlier (Magdolenova et al., 2012). At least 10 min before image analysis, the cells were stained with SYBR Gold (INVITROGEN, Cat No S11494), (0.1 μg/ml in TE buffer (10 mM Tris–HCl, 1 m MNa2EDTA, pH 7.5–8)). For each slide, 70 μl SYBR Gold solution was placed as droplets evenly distributed across the slide, and covered with a cover slip allowing the SYBR Gold solution to spread and cover all gels. The slides were analysed by a fluorescent microscope (Leica DMI 6000 B) using an image analysis system Comet assay IV (Perceptive Instruments Ltd.). The software is linked to a closed circuit digital camera mounted on the microscope, and automatically analyses individual comet images. % DNA in tail was used as the most informative parameter. Cytotoxicity was determined by the Relative Growth Activity (RGA) Assay. Cells from each exposure condition were counted using Countess™ Automated Cell Counter (Invitrogen) immediately after exposure and after 48 h. The growth rate was compared to control, and RGA less than 80% was considered cytotoxic. The Comet Assay was performed three times with two parallels within each experiment, scoring 50 cells in each gel. The results are represented as the mean of the medians from each gel. Standard Error of the Mean (SEM) was calculated for each exposure condition based on the variability between the 6 gels. The Mann– Whitney test was performed for each concentration compared to negative control. The result was considered significant at p o 0.05.

3. Results 3.1. Ames test The results of different quality controls all were within the accepted ranges according to the OECD TG 471 requirements

20

Without S9 With S9

Induction factor (IF)

2.5. Comet Assay for detection of strand breaks and oxidatively damaged DNA

and historical control data. The potential of DMA-NO2, MA-NO2, MEA-NO2, AMP-NO2 and PZ-NO2 to induce reverse mutations was evaluated in five standard Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and in the presence of S9. In the toxicity range-finder experiment, no substantial increases in revertant colony numbers over solvent control counts were obtained with strain TA100 following exposure to all five nitramines. Furthermore, no evidence of mutagenic activity was seen at any concentration of DMA-NO2, AMP-NO2, and PZ-NO2 with any of the five Salmonella typhimurium strains in the plate incorporation test as well as in the pre-incubation test in the absence and in the presence of S9 mixture (data not shown). For MA-NO2, however, a clear mutagenic response was observed at five concentrations (0.48, 0.97, 1.94, 3.88 and 7.75 mg/plate) with the Salmonella typhimurium strain TA102 in the first and second Reverse Mutation Experiment both in the absence and in the presence of S9 mixture (Fig. 1). The highest concentrations 7.75 and 15.50 mg/plate resulted in a decrease in the reversion rate in the absence and presence of S9 mixture indicating, however not visually apparent, an onset of bacteriotoxicity. No evidence of mutagenic activity was seen at any concentration of MA-NO2 with the strains TA98, TA100, TA1535 and TA1537 in the plate incorporation test as well as in the pre-incubation test in the absence and in the presence of S9 mixture. For MEA-NO2, a clear mutagenic response was observed at all concentrations (0.25– 15.90 mg/ plate) with the Salmonella typhimurium strain TA102 in the first and second Reverse Mutation Experiment both in the absence and in the presence of S9 mixture. A clear mutagenic response was also observed at the 3 highest concentrations of MEA-NO2 with the strain TA1535 in the first and second Reverse Mutation Experiment in the absence of S9 mixture. In the presence of S9 mixture, a clear mutagenic response was observed for the four highest concentrations (1.99, 3.98, 7.95 and 15.90 mg/plate) of MEA-NO2

TA98 Without S9

15

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10

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0

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15.5

Concentration MA-NO2 (mg/plate)

20 Induction factor (IF)

First, the cells were treated in duplicate cultures with and without metabolic activation for 3 h at 37 1C. S9 mix was comprised of a rat liver homogenate fraction from male Sprague Dawley rats liver treated with Aroclor 1254 (S9, Moltox), cofactors NADPH (Sigma), glucose-6-phosphate (Sigma) in buffer salt environment (KH2PO4/Na2HPO4, MgCl2, KCl). Concentration of prepared S9 in the mix was 10% (v/v). The S9 mix was added to the cell suspension so that in treatment culture was 0.6% S9. After the treatment, the cells were centrifuged for 6 min at 200g, resuspended in 2.5 ml fresh medium and transferred to T-10 cm2 tissue culture flat tubes. Cytochalasin B (6 μg/ml) was added immediately, and the cells were harvested after 24 h. Secondly, the cells were treated in duplicate with the same concentrations of test substances for 24 h in the presence of cytochalasin B (6 μg/ ml). The cells were harvested at the end of the exposure. The cells were harvested by centrifugation (128g for 8 min) treated with cold hypotonic solution (5 ml, 0.075 M KCl), and fixed with methanol/glacial acetic acid (3:1, room temperature, 5 ml). The cells were added to pre-cleaned cold microscope slides, dried, and after 24 h stained with 5% Giemsa–Romanowski solution for 10 min. Micronucleus frequencies were analysed in 2000binucleated cells per concentration (duplicate cultures with two slides in each, 4  500). To assess cell proliferation cytokinesis-block proliferation index (CBPI) 1000 cells per concentration (duplicate cultures with two slides in each, 4  250) were used, according to the formula CBPI ¼(MNCsþ 2  BNCsþ3  multinucleated cells)/N, where MNC is mononucleate cells, BNC is binucleated cells and N is total number of cells. Two independent experiments with two parallels in each were performed without metabolic activation, and one experiment in duplicate cultures with metabolic activation. Statistical analysis for experiments without S9 mix was performed as mean value of cells with micronuclei7SD from two independent experiments and for experiments with S9 as mean value of cells with micronuclei7SD from two parallels. Significance was measured using ANOVA (for normalised data) and Mann–Whitney test (for non-normalised data). IBM SPSS statistics v21 was used for the statistical analysis. A positive result was considered as a significant (po0.05) increase of micronucleated cells in exposed cells versus control cells.

41

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15

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10

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0.5

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Fig. 1. First Reverse Mutation (a) and second Reverse Mutation (b) results of methylnitramine (MA-NO2) to induce reverse mutations in Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence and in the absence of S9. A 2-fold increase of the induction factor (IF) was considered positive for the strains TA98, TA100, and TA102 (black line). A 3-fold increase of the IF was considered positive for the strains TA1535 and TA1537 (black dotted line).

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with strain TA1535 in the first and second Reverse Mutation Experiment (Fig. 2). No evidence of mutagenic activity was seen at any concentration of MEA-NO2 with the strains TA98, TA100 and TA1537 in the plate incorporation test or in the pre-incubation test in the absence and in the presence of S9 mixture. 3.2. CBMN assay The genotoxic potential of DMA-NO2, MA-NO2, MEA-NO2, AMPNO2 and PZ-NO2 was measured with CBMN assay, for 3 h (with and without S9) and 24 h. DMA-NO2, AMP-NO2 and PZ-NO2 did not induce any increase in micronucleus in binucleated cells (MNBNC) at any concentrations or test conditions. After 24 h exposure, MA-NO2 induced a significant increase of MNBNC at the highest concentration tested (2.75 7 1.75 MNBNC/500BNC), which was more than 3 fold increase compared to the negative control. After 3 h treatment of MA-NO2 with S9, a dose response is observed, although the results are not significant. Also MEA-NO2 induced an increase of MNBNC at the highest concentration after 24 h exposure (1.75 70.89 MNBNC/500BNC), which was almost a 2 fold increase compared to the control. Results are given in Fig. 3. Cytotoxicity was measured with CBPI. At the highest concentration, significant reduction of CBPI was observed for all nitramines after 24 h exposure and for MA-NO2 also after 3 h exposure (without S9). By calculating the % cytostasis, it appears that none of the nitramines are considered cytotoxic ( 460%) at the concentrations tested (calculations not shown). 3.3. Comet assay TK6 cells were tested for increase in strand breaks and oxidised bases following exposure to the five nitramines by performing the Comet Assay. Results are shown in Fig. 4. Cells exposed to AMP-

Induction factor (IF)

30 TA100 Without S9

25

TA100 With S9

20

TA98 Without S9 TA98 With S9

15

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10

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10

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0.99

1.99

3.98

7.95

15.90

TA1537 With S9

Concentration MEA-NO2 (mg/plate)

Fig. 2. First Reverse Mutation (a) and Second Reverse Mutation (b) results of ethanolnitramine (MEA-NO2) to induce reverse mutations in Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence and in the absence of S9. A 2-fold increase of the induction factor (IF) was considered positive for the strains TA98, TA100, and TA102 (black line). A 3-fold increase of the IF was considered positive for the strains TA1535 and TA1537 (black dotted line).

NO2 revealed a significant increase in strand breaks (SBs) at 250 mg/ml compared to the control, although there was no significant increase in SBs at the highest concentration (2500 ug/ ml) tested. Similarly, PZ-NO2 exposure increased significantly number of SBs at the highest concentration tested. However, for both nitramines the level of % DNA in tail is less than 5%, which is considered to be within background damage. None of these two nitramines induced oxidised bases at significant levels, however for cells exposed to PZ-NO2, a dose response was observed. There were no significant effects observed after exposure to DMA-NO2, MA-NO2 and MEA-NO2, although for MA-NO2 and MEA-NO2 a mild dose response was seen for oxidised bases. The relative growth activity tests performed on TK6 cells together with the comet assay showed no sign of cytotoxicity at the tested concentrations, except from the highest concentration of MA-NO2 where RGA was reduced to 40% compared to control.

4. Discussion As nitramines are potential by-products of amine-based CO2 capture technology, their genotoxic and mutagenic potential should be thoroughly explored. Previous mutation studies using Ames test showed that DMA-NO2 (4 200 mmol/plate  18 mg/ plate) is a bacterial mutagen towards Salmonella typhimurium TA100 in the presence of S9, although its capability to induce mutagens is not as strong as its corresponding nitrosamine. A borderline mutagenicity was observed in TA 1535 (Frei et al. 1984, 1986; Khudoley et al., 1981; Pool et al., 1984, 1986). This mutagenic potential of DMA-NO2 was not confirmed at the tested concentrations in our study towards Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence and absence of S9 mixture. Pool et al. (1984) also found no response at 50 mmol/ plate ( 4.5 mg/plate) which is similar to the concentrations tested in our study. As DMA-NO2 had slightly lower solubility than the other nitramines, we considered the maximum concentration 4.5 mg/plate (which still gave stable solution) high enough also with respect to potential exposure. Most of the concentrations were in a range with other nitramines, thus results could be compared to those achieved for the other nitramines tested. On the other hand, our results show that MA-NO2 is mutagenic towards the Salmonella typhimurium strain TA102 and thus can induce base pair substitutions, but no evidence on mutagenic activity was observed towards the other strains TA98, TA100, TA1535 and TA1537. MA-NO2 has previously been reported as a non-mutagen by the Ames test (Malaveille et al., 1983; Pool et al., 1984, 1986). According to our knowledge, these studies only investigated the mutagenic response towards strain TA100 and TA1535, which were also negative for MA-NO2 in our study. Furthermore, MEA-NO2 showed positive mutagenic effect in our study by the Ames test, both towards the strains TA102 and TA1535 in the absence and in the presence of S9 mixture. This means that MEA-NO2 can induce base pair substitutions, but not frameshift mutations. The other two nitramines AMP-NO2 and PZNO2 were found to be not mutagenic in the Ames test. To the best of our knowledge there are no other studies published on these nitramines (MEA-NO2, AMP-NO2, and PZ-NO2). For the CBMN assay, a significant increase in MNBNC was observed in the highest concentration after 24 h exposure of MA-NO2 and MEA-NO2. At the same time CBPI was significantly reduced, but not over 60% to consider that the genotoxic effect could be a secondary effect due to cytotoxicity. By investigating the individual repeats, a dose-response relationship of MNBNC was observed for both repeats of MEA-NO2; however, none of them showed significant results. For MA-NO2 one repeat had a strong dose-response relationship with

L.M. Fjellsbø et al. / Environmental Research 134 (2014) 39–45

1.4

2

1.2

2.2

8

2.0

* *

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*

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8

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625 156 DMA-NO2 (µg/ml)

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*

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*

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NC RPMI

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Fig. 3. Number of micronucleus (MNBNC) per 500 binucleated (MNC) TK6 cells exposed to the dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2), 2-methyl-2-(nitramino)-1-propanol (AMP-NO2) and piperazine nitramine (PZ-NO2) for 3 h (with and without S9) and 24 h,7 standard deviation (SD). The cytotoxicity was measured using cytokinesis block proliferation index (CBPI). Level of significance compared with control was set at po 0.05. The positive control (mitomycin c) induced significantly more MN in all test conditions (3 h: 5.1 72, 3 h S9: 7.3 7 3.3, 24 h: 9.4 7 2.1). It also reduced the CBPI significantly (3 h: 1.6 7 0.2, 3 h S9: 1.2 7 0.1, 24 h: 1.6 7 0.0).

significant increase in MNBNC for the highest concentration, while no dose-response relationship was observed for the second repeat. The following criteria are considered for the evaluation of results (OECD, 2012): (1) the increase is dose-related, (2) at least one of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control, (3) the positive result is reproducible (e.g. between duplicates or between experiments). A test substance that meets all the above criteria in at least one experimental condition is considered able to induce chromosome breaks and/or gain or loss in this system. Based on these acceptance criteria, both MA-NO2 and MEA-NO2 can be considered able to induce chromosome damage with MEA-NO2 giving weaker response. Significant mild increase in SBs, measured by the Comet Assay, was detected after exposure to AMP-NO2 and PZ-NO2. The levels of SBs are in the low range (o5% DNA in tail) which is normally considered to be within the background level and thus these

results most likely have no biological meaning. No cytotoxicity was observed at the concentrations tested. After exposure to DMANO2, MA-NO2 and MEA-NO2 no significant effects were observed. Although no significant increase in oxidised bases were observed, MA-NO2, MEA-NO2 and PZ-NO2 seemed to induce mild dose responses of oxidised bases. There are only few previous studies on the mammalian system, but measurement of single strand breaks in hepatocytes by alkaline elution have been performed showing induction of breaks in DMA-NO2 and MA-NO2 (Pala et al., 1982; Pool et al., 1986). Using fluorometric alkaline elution method, Frei et al. (1986) found no effect of DMA-NO2 on induction of DNA breaks but its monoalkyl metabolite (MA-NO2) was positive. In our study, the Comet Assay was performed without metabolic activation of the nitramines. Previous studies indicate that metabolic activation is needed in order to convert the nitramine and nitrosamine to formaldehyde which has been considered the most toxic metabolite (Frei et al. 1984, 1986; Pool et al., 1984, 1986). In these studies, single strand breaks were

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L.M. Fjellsbø et al. / Environmental Research 134 (2014) 39–45

DNA breaks (% DNA in tail)

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Fig. 4. DNA damage measured by the Comet Assay after 3 h exposure of the dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2), 2-methyl-2-(nitramino)-1-propanol (AMP-NO2) and piperazine nitramine (PZ-NO2) to TK6 cells. The results are presented as mean of medians from three individual experiments 7 SEM. Level of significance compared with control was set at p o 0.05. Cytotoxicity is measured by the relative growth activity test, and included for each nitramine.

detected in hepatocytes, and both MA-NO2 and DMA-NO2 were found to be genotoxic. Unfortunately, there are no previous studies where nitramines have been tested by the Comet Assay. However, Liviac et al. (2011) performed Comet Assay on TK6 cells with exposure to dimethylnitrosamine (NDMA) and diethylnitrosamine. No effect was observed without metabolic activation, but with external metabolic activation an increased effect was observed in both nitrosamines. These results indicate that more research should be performed especially with metabolic activation of the nitramines. The Norwegian Institute of Public Health has recommended, based on 10-6 risk of cancer, an acceptable exposure level of 4 ng/L in drinking water, and 0.3 ng/m3 in air (Låg et al., 2011). This assessment was based on NDMA, assuming that this belongs to the most potent nitrosamines. Due to lack of toxicity data on nitramines they have assumed the same risk level for nitramines as for nitrosamines, assuming that NDMA is likely to be more potent than any nitramine (Låg et al., 2011). Our results indicate that DMA-NO2 is the least potent of the nitramines tested, showing the importance of investigating the potency of all nitramines which may be emitted from a power plant or formed from the power plant emissions. In our study, the main aim was to perform a screening of the selected nitramines, and concentrations were selected based on recommendations from regulatory guidelines for testing chemicals. Even though the concentration used in our study are much

higher than what could be expected in emissions from a capture facility, they indicate that a big safety factor may be necessary. Thus the next step is to perform a proper risk assessment, to evaluate possible impact on human health.

5. Conclusion Our work has contributed to filling the knowledge gap regarding toxicity of nitramines. By performing three in vitro tests with focus on genotoxicity and mutagenicity, the first step towards risk assessment of nitramines has been completed. The screening in this study clearly indicates that some of the tested nitramines are more potent than others to induce genotoxicity and mutagenicity. DMA-NO2 did not induce genotoxic or mutagenic response in any of the tests performed. AMP-NO2 and PZ-NO2 induced SBs as measured by the Comet Assay, though the amount of breaks was in the range of background level and the biological meaning should therefore be further explored. These nitramines were considered non-genotoxic by the CBMN assay and non-mutagens by the Ames test. MA-NO2 and MEA-NO2 were considered to be mutagens in the Ames test with a weak genotoxic response in CBMN assay. If their corresponding amines should be used for CO2 capture technology, we recommend further research to ensure human and environmental safety.

L.M. Fjellsbø et al. / Environmental Research 134 (2014) 39–45

Funding sources This work was supported by EPRI (Electric Power Research Institute, USA) (Grant no. EP-P45223/C19684) and the Norwegian state (Contract no. 257430111). The latter has been part of an extensive investigation programme with the objective of developing a set of methods and procedures for evaluating health and environmental impact of amine based solvents, under the CO2 Capture Mongstad Project (CCM), organised as a joint effort by Gassnova SF and Statoil ASA. The authors also thank NILU for additional financial support (Project no. 112108). Acknowledgments The authors are grateful to Christian Dye and Arve Bjerke for chemical analysis and support, to An Jacobs and Jef Maes for their technical expertise in this study and to Philippe Vanparys for his scientific contribution. References CPDB (Carcinogenic Potency Database Project) (2007). Available: 〈http://potency. berkeley.edu/chempages/METHYLNITRAMINE.html〉. (accessed March 2013). da Silva, E.F., Booth, A., 2013. Emissions from postcombustion CO2 capture plants. Environ. Sci. Technol. 47, 659–660. Dai, N., Shah, A.D., Hu, L., Plewa, M.J., McKague, B., Mitch, W.A., 2012. Measurement of nitrosamine and nitramine formation from NOx reactions with amines during amine-based carbon dioxide capture for postcombustion carbon sequestration. Environ. Sci. Technol. 46 (17), 9793–9801. Fjellsbø, L.M., Van Rompay, A.R., Hooyberghs, J., Nelissen, I., Dusinska, M., 2013. Screening for potential hazard effects from four nitramines on human eye and skin. Toxicol. Vitro. 27, 1205–1210. Frei, E., Pool, B.L., Plesch, W., Wiessler, M., 1984. Biochemical and biological properties of prospective N-nitrodialkylamine metabolites and their derivatives. IARC Sci. Publ. 57, 491–497. Frei, E., Pool, B.L., Glatt, H.R., Gemperlein-Mertes, I., Oesch, F., Schlehofer, J.R., Schmezer, P., Weber, H., Wiessler, M., 1986. Determination of DNA single strand breaks and selective DNA amplification by N-nitrodimethylamine and analogs, and estimation of the indicator cells' metabolic capacities. J. Cancer. Res. Clin. Oncol. 111 (2), 123–128. Gentry, P.R., House-Knight, T., Harris, A., Greene, T., Campleman, S., 2013. Potential occupational risk of amines in carbon capture for power generation. Int. Arch. Occup. Environ. Health ([Epub ahead of print, Online 3 September 2013]). Goodall, C.M., Kennedy, T.H., 1976. Mutagenicity studies in Salmonelle typhimurium on some carcinogenic N-nitramines in vitro and in the host-mediated assay in rats. Cancer. Res. 41, 3205–3210. Hassel, M., Frei, E., Scherf, H.R., Wiessler, M., 1987. Investigation into the pharmacodynamics of the carcinogen N-nitrodimethylamine. IARC Sci. Publ. 84, 150–152. Khudoley, V., Malaveille, C., Bartsch, H., 1981. Mutagenicity studies in Salmonella typhimurium on some carcinogenic N-nitramines in vitro and in the hostmediated assay in rats. Cancer. Res. 41, 3205–3210.

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