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Mutagenesis Advance Access originally published online on July 26, 2007
Mutagenesis 2007 22(6):363-370; doi:10.1093/mutage/gem027
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations

F. Ferk, M. Misík1, C. Hoelzl, M. Uhl2, M. Fuerhacker3, B. Grillitsch4, W. Parzefall, A. Nersesyan, K. Micieta1, T. Grummt5, V. Ehrlich and S. Knasmüller*

Institute of Cancer Research, Medical University of Vienna, Austria 1Department of Botany, Comenius University, Bratislava, Slovakia 2Federal Environment Agency Austria, Vienna, Austria 3University of Natural Resources and Applied Life Sciences, Vienna, Austria 4University of Veterinary Medicine, Laboratory of Ecotoxicology, Vienna, Austria 5German Environmental Protection Agency, Bad Elster, Germany

Quaternary ammonium compounds (QACs) are cationic surfactants that are widely used as disinfectants. In the present study, we tested two important representatives, namely, benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB) in four genotoxicity tests, namely, in the Salmonella/microsome assay with strains TA 98, TA 100 and TA 102, in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes and in micronucleus (MN) assays with peripheral human lymphocytes and with root tip cells of Vicia faba. In the bacterial experiments, consistently negative results were obtained in the dose range between 0.001 and 110 µg per plate in the presence and absence of metabolic activation while significant induction of DNA migration was detected in the liver cells. With BAC, a moderate but significant effect was found with an exposure concentration of 1.0 mg/l while DDAB caused damage at lower doses (0.3 mg/l). The effects were not altered when the nuclei were treated with formamidopyridine glycosylase, indicating that they are not due to formation of oxidized purines. The MN assays with blood cells were carried out under identical conditions to the SCGE experiments and a significant increase was seen at the highest dose levels (BAC: 1.0 and 3.0 mg/l; DDAB: 1 mg/l). Both compounds also caused significant induction of MN as well as inhibition of cell division in plant cells, the lowest effective levels were 1.0 and 10 mg/l for DDAB and BAC, respectively. Our findings show that both chemicals induce moderate but significant genotoxic effects in eukaryotic cells at concentrations which are found in wastewaters and indicate that their release into the environment may cause genetic damage in exposed organisms. Furthermore, the direct contact of humans to QAC-containing detergents and pharmaceuticals that contain substantially higher concentrations than those which were required to cause effects in eukaryotic cells in the present study should be studied further in regard to potential DNA-damaging effects in man.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Quaternary ammonium compounds (QACs) are an important class of industrial chemicals with a broad spectrum of commercial and consumer uses (1Go). According to reports of the chemical industry, the annual global consumption of cationic and amphoteric surfactants increased from an estimated 1.16 to 1.97 million tons in the year 2005 (2Go). QACs are used in consumer products as well as in biocides, emulsifiers, wetting agents and processing additives (3Go). In household products, they are primarily applied in fabric softeners, hair conditioners and other hair preparations, additionally some substances are used as pesticides, in medical and veterinary products and applied by the food processing industry (for reviews, see refs 1Go,4Go–7Go).

QACs are cationic surfactants containing a tetrasubstituted ammonium salt and characterized by a positively charged quaternary nitrogen atom. Because of their positive charge, these compounds strongly adsorb to negatively charged surfaces of sludge, soil and sediments. It is also well documented that they bind to the fatty acids of cell membranes of organisms, which makes them useful as biocides (for reviews, see refs 4Go,8Go,9Go).

Two commercially and toxicologically important representatives of QACs were selected as model compounds for the present study, namely, the alkyldimethylbenzylammonium salt benzalkonium chloride (BAC) and a dialkyldimethylammonium salt (dimethyldioctadecyl-ammonium bromide, DDAB). Both groups are used in several product groups of biocides including disinfection products and preservatives. They have both been accepted for notification by the European Union according the Biocidal Product Directive (98/8/EC) and are currently being evaluated by competent authorities for their use in different product types. The acute toxic effects of QACs have been investigated extensively in rodents and in a variety of aquatic organisms (4Go,10Go) while data on their genotoxic properties are scarce. With BAC, conflicting data were obtained in bacterial assays that were conducted in the 1980s (11Go–14Go); in two recent studies with fish- and human-derived cells, clear positive results were obtained (15Go,16Go). DDAB has never been tested in regard to its genotoxic activity according to our knowledge, but a number of investigations were conducted with dimethyldioctadecyl-ammonium choride (DDAC) which is structurally equivalent and consistently negative results were obtained (for review, see ref. 17Go).

The aim of the present study was to investigate further the potential genotoxic properties of the two compounds in a panel of assays, namely, in the Salmonella/microsome test with strains TA 98, TA 100 and TA 102 (the latter strain was not used in earlier mutagenicity experiments with QACs), in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes and in micronucleus (MN) experiments with lymphocytes and root tip cells of Vicia faba. The latter assay has been used extensively for the detection of genotoxins in the aquatic environment (for reviews, see refs 18Go,19Go). Ennever et al. (20Go) have stressed that plant bioassays have a high sensitivity for the detection of carcinogens and we reported earlier than MN induction in plants is paralleled by increased rates of abortive pollen, which is associated with decreased fertility (21Go,22Go), while experiments with primary liver and peripheral blood cells that possess active phase I and phase II enzymes (23Go,24Go) reflect to a certain extent the situation in mammals and humans.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals and media
BAC (CAS-Nr 8001-54-5) and DDAB (CAS-Nr 2390-68-3) were obtained from Sigma–Aldrich (St Louis, MO).

Bacterial media were from Difco (Detroit, MI); Nutrient Agar No. 2 for overnight cultures was from Oxoid (Hampshire, UK). Inorganic salts, dimethylsulfoxide, acetone, n-hexane and HCl were from Merck (Darmstadt, Germany). nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate, methylmethanesulfonate, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), 2-aminoanthracene (2-AA), 2,4,7-trinitro-9-fluorenone, benzo(a)pyrene (B(a)P) and sodium azide (NaN3) came from Sigma–Aldrich. Aroclor 1254-induced S9 mix was purchased from MP Biomedicals (Illkirch, France).

Chemicals for the isolation and cultivation of hepatocytes [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, collagenase type IV, modified Eagle medium (MEM) and Heparin-Na] were purchased from Sigma–Aldrich. Penicillin and streptomycin came from Biochrom (Berlin, Germany); glucose was from Merck.

Low melting point agarose and normal melting point agarose were obtained from Gibco (Paisley, UK). Trisma base, Triton X, ethidium bromide and trypan blue were purchased from Sigma. Formamidopyridine glycosylase (FPG) was a gift of Dr K. J. Angelis (Laboratory of DNA Repair, Institute of Experimental Botany, Rozvojova 135, Prague, Czech Republic). To ensure optimal activities of the repair enzymes, dose response curves with lymphocytes were carried out prior to the main experiments and on the basis of the results the amounts of enzymes required to cause maximal DNA migration were established.

RPMI 1640 medium and cytochalasin B were purchased from Sigma–Aldrich (Munich, Germany); phytohaemagglutinin (PHA) was obtained from Gibco (Rockville, MD).

Acetocarmine, acetic acid, hydrochloric acid and arsenic trioxide (As2O3) which were used in experiments with Vicia were purchased from Sigma–Aldrich and CentralChem (Bratislava, Slovakia).

Bacteria tester strains
The indicator strains (TA 98, TA 100 and TA 102) were obtained from B.N. Ames (University of Berkeley, CA). The frozen permanents were stored at –80°C and new master plates were made every 6 weeks according to the protocol of Maron and Ames (25Go).

Plants (V.faba L.)
Vicia faba var. minor seeds were a gift from the National Breeding Facility, Horná Streda, Slovakia.

Animals
All experiments were carried out with Him-OFA male rats (body weight 200 ± 10 g) obtained from the breeding facility of the Medical University in Himberg (Austria). The animals were allowed to acclimatize for 1 week before the experiments at the Institute of Cancer Research and were housed in plastic cages (Macrolon type II) under standard conditions (24 ± 1°C, humidity 50 ± 5%, 12 h light/dark cycle). The animals were kept on a standard diet (ssniff R/M-H, purchased from Ssniff, Soest, Germany).

Salmonella/microsome assay
The experiments were performed as plate incorporation tests according to the standard protocol of Maron and Ames (25Go). Overnight cultures were made in Oxoid broth No. 2 and had a final density of ~1–2 x 109 viable cells per ml. Stationary phase cultures (0.1 ml), 2.5 ml overlay agar and different concentrations of test compounds were mixed and poured onto histidine-free selective agar plates in the presence and absence of metabolic activation mix (S9).

The plates were incubated at 37°C in the dark for 48 h; subsequently, the number of his+ colonies was enumerated by manual counting. Per experimental point, three plates were made in parallel.

Comet assay
The hepatocytes were isolated with two-step collagenase perfusion technique according to the protocol of Segeln (26Go) with modifications described by Parzefall et al. (27Go). The cells were cultured in MEM and incubated with different concentrations of two compounds for 60 min (37°C) and for 5 min with H2O2 (100 µM) which was used as positive control. Subsequently, the cells were washed with phosphate-buffered saline (pH 7.4). The viability was determined with the trypan blue exclusion test (28Go) and only cultures in which the viability was ≥75% were analysed for DNA migration. The comet assay with the lesion-specific enzymes FPG (1 µl, 500 µl enzyme buffer, incubation time 30 min, 37°C) was carried out according to the protocol described by Collins (29Go. DNA migration was determined with a computer-aided analysis system (30Go. The experiments were carried out according to the guidelines for comet assays published by Tice et al. (31Go). For each experimental point, three independent experiments (one animal per experiment) were carried out and from each culture 50 cells were evaluated.

MN assays with peripheral human lymphocytes
The experiments were carried out according to the protocol of Fenech et al. (32Go). Blood (50 ml) was collected by venipuncture from three healthy male non-smoking individuals (age 25–27 years) after overnight fasting. Lymphocytes were isolated by Ficoll centrifugation and cultured in triplicate in RPMI medium without foetal calf serum; each culture (740 µl per tube) contained 106 cells per ml. For each individual, two cultures were established. The cells were treated for 60 min with different concentrations of the QACs or with H2O2 (100 µM) for 30 min. Following stimulation with PHA (45 µg/ml) for 44 h, cytochalasin B was added to the cultures for 28 h, then the cells were harvested and slides were prepared. MNi were scored in binucleated cells (BNCs) according to the criteria described by Fenech et al. (33Go) and for each concentration also the nuclear division indices (NDIs) were calculated (34Go,35Go). From each culture, 1000 BNCs were evaluated.

MN experiments with V.faba
The experiments were carried out according to the protocol published by Ma et al. (36Go) with minor modifications. Briefly, seeds were soaked in aerated tap water for 24 h. After removal of the coats, the seeds were allowed to germinate between two moistened, vertically positioned filter papers for 2–3 days at 25°C until the primary roots reached a length of ~2 cm. Subsequently, the tips of the primary roots were removed, and the seedlings transferred to aerated tap water (22°C) to develop secondary roots, which were exposed to the different concentration of the test compounds for 72 h. After the exposure, the roots were fixed with acetic acid–ethanol (1:3) solutions. After 24 h, the fixative was replaced by 70% ethanol. For slide preparation and microscopic examination, the root tips were rinsed with distilled water and hydrolysed in a mix of 10 M HCl and ethanol (1:1) for 8 min. After staining (1% acetocarmine), the F1 region of the root tips was used for the determination of the MN frequencies. The cells were evaluated under 400x magnification with a light microscope (Nikon YS200).

The MN frequency was expressed as the number of micronucleated cells per 1000 cells. In each test, MNi were determined in 6 individual plants per concentration and two independent experiments were carried out. In addition, also the mitotic indices (MIs) were monitored. For this purpose, 3000 cells were examined for each experimental point (500 cells per plant).

Statistics
The results of the genotoxicity experiments were analysed with one-way analysis of variance followed by Dunnett's multiple comparison test. P-values ≤0.05 were considered as significant.

To evaluate the effect of QACs in the Vicia MN assay, Student's t-test at P ≤ 0.05 was used.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Salmonella/microsome assays
The results of the experiments with different concentrations of BAC and DDAB are summarized in Tables I and II. No indication of induction of his+ revertants was observed under any condition of test. With 11 µg per plate, the revertant numbers declined in both indicator strains as compared to the control values, while at the highest concentration (110 µg per plate), no revertant colonies were observed due to acute toxicity.


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  		Table I. Induction of his+ revertants in Salmonella tester strains by BAC

 


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  		Table II. Induction of his+ revertants in Salmonella tester strains by DDAB

 
SCGE experiments with primary rat hepatocytes
The results of representative comet experiments are shown in Figure 1. H2O2 (100 µM; exposure time 5 min) was used in this experiment as a positive control and induced significant DNA damage (tail moment of 42 ± 5 µm). The QACs were tested at three concentrations (0.11–1.0 mg/l) and induction of DNA migration was observed with both compounds. With BAC, a significant effect was detected only with the highest concentration (1.0 mg/l; Figure 1B), while DDAB caused pronounced induction of DNA migration at dose levels ≥0.33 mg/l (Figure 1D). We also conducted an additional experiment with a higher concentration of BAC (2.0 mg/l) and no further increase in DNA damage was seen under these conditions (data not shown).


Figure 1
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  		Fig. 1. Impact of BAC and DDAB on DNA migration and survival of primary rat hepatocytes. The cells were isolated with the two-stage collagenase perfusion technique and cultivated in MEM. After exposure to the test compounds for 60 min, the formation of comets was analysed by use of a computer-aided analysis system; viability of the cells was determined by use of trypan blue exclusion test. Per experimental point, three independent experiments (one animal per experiment) were carried out and from each culture 50 cells were analysed for comet formation. Bars represent means ±standard deviations. * indicate statistical significance (P≤0.05).

 
Figure 1A and C depicts the effects of the QACs on the viability of the cells, which was monitored in the same experiments. At the highest dose level (1.0 mg/l), a slight, but significant decrease (9–14%) was observed.

In order to find out if the effects of the two compounds are due to oxidative DNA damage, additional experiments were carried out in which isolated nuclei were treated with the FPG. The results are shown in Figure 2. It can be seen that DNA migration in untreated cells was significantly increased after treatment with the QACs. DNA migration attributable to the formation of oxidative purines was not clearly enhanced in nuclei from chemically treated cells while in the controls a clear-cut increase was detected.


Figure 2
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  		Fig. 2. Induction of FPG-sensitive lesions by BAC and DDAB in primary rat hepatocytes. The experiment was carried out as described in the legend of Figure 1 and the cells were exposed to 0.33 mg/l of DDAB or 0.66 mg/l of BAC for 60 min. After lysis, the nuclei were incubated with the enzyme for 30 min. Bars represent means ± standard deviations of results obtained in three independent experiments (one animal per experiment) and 50 nuclei per culture were analysed for DNA migration. * indicate statistically significant differences (P≤0.05) between untreated and FPG-treated slides.

 
MN induction in peripheral human lymphocytes
The results obtained in the MN experiments that were carried out under identical conditions to the SCGE assays are depicted in Figure 3. H2O2 (100 µM) was used as a positive control (treatment time 30 min) and induced on average 21 ± 3.0 MNi per 1000 BNCs. It can be seen that both compounds caused significant effects at a concentration of 1.0 mg/l, also with lower doses an increase of the MN numbers was observed but the effects did not reach significance.


Figure 3
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  		Fig. 3. Impact of BAC and DDAB on MN formation and on the NDI in peripheral human lymphocytes. The cells were isolated by Ficoll centrifugation and exposed to the QACs under identical conditions as in the SCGE experiments. Bars represent means ± standard deviations of results obtained in three independent experiments (one donor per experiment, two cultures per sample). * indicate statistically significant differences (P≤0.05).

 
In order to find out if the two QACs cause also effects at dose levels, that exceed those found in environmental samples, we conducted a further experiment in which higher concentrations (3, 10 and 100 mg/l) were tested. Again cells from three donors were used. In untreated control cells, the average NDI values were similar to those in the first experiment (1.82 ± 1.4) while the MN frequency was slightly lower (9.7 ± 2.2). Only the lowest dose of BAC (3 mg/l) could be evaluated for MN formation. The NDI decreased to 1.3 ± 0.3, while the MN frequency (21.3 ± 3.5) was significantly higher compared to the background. None of the other BAC concentrations could be analysed due to acute toxic effects; DDAB caused a severe reduction of the NDI already at the lowest dose (3 mg/l) and it was not possible to determine the MN frequency due to the low number of BNC on the slides.

MN induction in V.faba
The results of a representative experiment are summarized in Figure 4. It can be seen that both QACs caused significant mutagenic effects in this test system. Arsenic trioxide (As2O3 0.5 mM) was used in these experiments as a positive control and induced 5.0 ± 0.5 MNi per 1000 cells.


Figure 4
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  		Fig. 4. Impact of BAC (A and B) and DDAB (C and D) on MN formation and cell division in root tip cells of Vicia faba L. The roots were exposed to different concentrations of the two QACs for 72 h. Subsequently, they were fixed and the formation of MNs and MIs determined as described in Materials and methods. Bars represent the results (means ± standard deviations) obtained in a representative experiment with six plants per concentration; from each plant, 1000 cells were analysed for MN formation. To determine the MIs, 3000 cells (500 cells per root) were examined. * indicate statistical significance (P≤0.05).

 
With BAC, a statistically significant induction of MN was seen with the highest exposure concentration (10.0 mg/l; Figure 4A). In the same experiment, also a dose dependent, linear decline of the cell division rate was observed, which was statistically significant at dose levels ≥1.0 mg/l (Figure 4B). Also with the second compound (DDAC), induction of MN was detected; however, this effect started already at concentrations >1.0 mg/l (Figure 4C). Also the decrease of the cell division rate caused by DDAB was more pronounced (Figure 4D) than the one seen with BAC.

The positive results were confirmed in a second independent experiment which was conducted under identical experimental conditions. BAC caused again significant induction of MN at the highest dose level (2.33 ± 0.46 MNi per 1000 cells), with DDAB significant effects were seen with concentrations ≥1.0 mg/ml (1.0 mg/l: 2.62 ± 0.48 MNi per 1000; 3.0 mg/l: 2.5 ± 0.47 MNi per 1000; 5.0 mg/l: 2.5 ± 0.51 MNi per 1000; 10.0 mg/l: 2.33 ± 0.46 MNi per 1000). The frequency recorded in the negative controls was 0.72 ± 0.26 MNi per 1000, with 0.5 mM As2O3 we found 5.6 ± 0.58 MNi per 1000 cells.


   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Taken together, the results of our study show that the two QACs we tested cause DNA migration and MN formation in mammalian and plant-derived cells but no gene mutations in bacteria.

Also in earlier experiments, mainly negative results were obtained in bacterial tests. With BAC, no genotoxic activity was found in Salmonella/microsome assays (11Go,12Go) or in the umu test with Escherichia coli (13Go). The results of differential DNA-repair studies with bacterial indicators are conflicting; Lovely et al. (14Go) obtained a positive result in the E.coli and also Sagakami et al. (13Go) found induction of repairable DNA damage in experiments with Bacillus subtilis (rec– assay), while Morita et al. (11Go) found no evidence for genotoxic activity in the latter test system. According to our knowledge, no results from bacterial assays with DDAB have been published so far, but a number of bacterial studies have been conducted with DDAC, which is structurally similar, and no evidence for mutagenic properties was seen in any of these experiments (37Go).

As described above, weak but significantly increased amounts of DNA damage were found in the present study with both QACs in SCGE experiments with primary rat hepatocytes and in the MN test with lymphocytes. The findings obtained in other experiments with mammalian cells are controversial. Hikiba et al. (38Go) tested BAC in chromosomal aberration assays in SHE cells. Based on the results, the authors concluded that the compound is devoid of clastogenic activity but it is notable that at concentrations >8 µM aberrant metaphases were observed that were not seen in the control cultures. In two recent studies, clear positive results were obtained. Sánchez-Fortún et al. (16Go) tested the agent in a fish-derived cell line (RTG-2) with a method that is based on the quantification of double-strand breaks by use of a fluorescent dye and found effects with exposure concentrations >0.12 mg/ml. Also in a further study by Deutsche et al. (15Go) with a human-derived bronchial epithelial cell line, clear-cut induction of DNA migration was seen in SCGE assays. These cells are known to have a number of drug-metabolizing enzymes (39Go).

In the present study, a DDAB caused much stronger effect than BAC in the comet assay (significant DNA damage was observed already with an exposure concentration of 0.3 mg/l). This is the first report concerning the genotoxic properties of this compound in mammalian cells but a number of studies have been conducted with DDAC, which are summarized in the review of Henderson (17Go). Consistently, negative results were obtained in a number of in vitro genotoxicity tests for example in unscheduled DNA synthesis experiments with rat hepatocytes and in hypoxanthine-guanine phosphoribosyltransferase and chromosomal aberration assays with CHO cells. However, in the latter test system, a significant increase of chromosomal aberration was seen with certain dose levels but comparisons with historical control data led to the conclusion that the compound is not active (17Go). None of these reports, which were prepared for chemical industries, has been published in scientific journals. DDAC was also tested in cell transformation experiments with SCE cells and also in this test negative results were obtained (37Go).

The results obtained in V.faba root tip cells (Figure 1) show that both compounds cause also genotoxic effects in plant cells. These findings support the assumption that the lesions that are detected in SCGE assays lead to chromosome damage and MN formation. According to our knowledge, the two compounds and other QACs have not been tested before in plant bioassays that are widely used in ecogenotoxicology (40Go–42Go).

It is not known which mechanisms account for the genotoxic effects of the two QACs but the pattern of the results obtained in the present study with four different test systems allows some conclusions to be drawn. The experiments with peripheral human lymphocytes and with plant cells show that both compounds cause induction of MN which are formed as a consequence of either clastogenic effects or aneuploidy. Since positive results were also obtained in the present SCGE experiments with rat hepatocytes and also in older investigations with other cells (15Go,16Go), it is likely that the formation of MN is due to chromosome breakage since the comet assay detects single- and double-strand breaks. As described above, negative results were obtained in earlier chromosome aberration assays with non-human cell lines but the results of these investigations are not clear-cut and have never been published. The lack of an effect in the bacterial tests can be taken as an indication that the QACs do not cause gene mutations but further experiments with mammalian cells are required to confirm this.

It is unlikely that the genotoxic effects of BAC and DDAB are due to oxidative damage since negative results were obtained in the bacterial tests with strain TA 102, which is highly sensitive towards reactive oxygen species (ROS) (43Go). This assumption is also supported by the fact that no evidence for formation of oxidized DNA bases was found in SCGE experiments with FPG (Figure 2).

The test systems which we used in the present study differ not only in regard to the endpoints (gene mutations, DNA migration and MN formation) but also in regard to the representation of drug-metabolizing enzymes in the indicator cells. Phase I enzymes are not represented in bacteria; therefore, exogenous activation mixtures are routinely added; plants possess apparently only low levels of these enzymes, which are required for the detection of genotoxic procarcinogens such as polycyclic aromatic hydrocarbons, heterocyclic aromatic amines and nitrosamines (for details, see ref. 43Go); also in peripheral blood cells, the activities of most xenobiotic drug-metabolizing enzymes are quite low and high concentrations of representatives of the aforementioned groups are required to cause measurable effects in genotoxicity assays (44Go). In contrast, primary hepatocytes possess high levels of phase I and phase II enzymes and are in general highly sensitive towards genotoxic carcinogens that require metabolic activation (23Go,24Go). The fact that we obtained in the present study clear effects in plant cells, as well as in lymphocytes and in hepatocytes at similar dose levels, can be taken as an induction that the mode of action of the two QACs does not involve activation by xenobiotic drug-metabolizing enzymes that are required for different classes of promutagens.

It is likely that QACs cause DNA damage in plants in the environment and have an adverse impact on their fertility. In this context, it is notable that QACs have never been tested before in plant bioassays that are widely used in ecogenotoxicology (40Go–42Go) and that we showed in a recent investigation that the induction of MN in plants is paralleled by an increase of abortive pollen (21Go,22Go). Second, certain QACs are used for medical preparations and other products to which humans are directly exposed. For example, BAC is contained in nasal sprays and ophthalmic solutions in concentrations up to 1 g/l (45Go). It is known that these exposure levels cause acute damage in epithelial cells (for review, see ref. 46Go) but our findings indicate that direct contact of the cells to QAC-containing preparations may additionally cause genotoxic damage in exposed cells. Our results indicate that the safety of QAC-containing products as well as their release into the environment warrants further investigations in regard to their genotoxic properties.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Federal Ministry of Agriculture, Forestry, Environment and Water Management (Pfeil 5' Action Programme; Project No. 1390); Slovak Grant Agency VEGA (No. 1/3289/06).


   Notes
 
* To whom correspondence should be addressed. Tel: +43 1 427765142; Fax: +43 1 42779651; Email: siegfried.knasmueller{at}meduniwien.ac.at


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

    1. Boethling RS, Lynch DG. Quaternary ammonium surfactants. In: Anthropogenic Compounds. Detergents. The Handbook of Environmental Chemistry—Oude NT, Hutzinger O, eds. (1992) Vol. 3, Part F. Berlin, Germany: Springer. 144–177.

    2. Scheraga D. On the positive side with cationic surfactants. In: Chem. Mark. Rep. (1998) (http://www.findarticles.com/p/articles/mi_hb4250/is_199801/ai_n13242482). Accessed 20 November 2006.

    3. Madsen T, Boyd BH, Nylén D, Pedersen AR, Petersen GI, Flemming S. Environmental Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products (2001) Environmental Project No. 615, Miloprojekt, Danish Environmental Pollution Agency.

    4. Uhl M, Gans O, Grillitsch B, Fürhacker M, Kreuzinger M. Grundlagen zur Risikoabschätzung für quaternäre Ammoniumverbindungen (2005) Umweltbundesamt Bericht, BE-271.

    5. Garcia MT, Ribosa I, Guindulian T, Sanchez-Leal J, Vives-Rego J. Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment. Environ. Pollut. (2001) 111:169–175.[CrossRef][Medline]

    6. Nalecz-Jawecki G, Grabinska-Sota E, Narkiewicz P. The toxicity of cationic surfactants in four bioassays. Ecotoxicol. Environ. Saf. (2003) 54:87–91.[CrossRef][Web of Science][Medline]

    7. Petrovic M, Barcelo D. Fate and removal of surfactants and related compounds in wastewaters and sludges. In: The Handbook of Environmental Chemistry—Hutzinger O, ed. (2004) Vol. 5 Part I. Berlin, Germany: Springer. 1–28.

    8. Nagamune H, Maeda T, Ohkura K, Yamamoto K, Nakajima M, Kourai H. Evaluation of the cytotoxic effects of bis-quaternary ammonium antimicrobial reagents on human cells. Toxicol. In Vitro (2000) 14:139–147.[CrossRef][Web of Science][Medline]

    9. Onyuksel HH. Mechanistic studies on surfactant induced membrane permeability enhancement. Pharm. Res. (2000) 17:612–618.[CrossRef][Web of Science][Medline]

    10. Ostroumov SA. Biological Effects of Surfactants (2006) Boca Raton, FL: CRC Press.

    11. Morita K, Ishigaki M, Abe T. Mutagenicity of materials related with cosmetics. J. Soc. Cosmet. Chem. (1981) 15:243.

    12. Sakagami YK, Yamasaki H, Yokoyama H, Ose Y, Sato T. DNA-repair test of disinfectants by liquid rec assay. Mutat. Res. (1988a) 193:21–30.[Web of Science][Medline]

    13. Sakagami Y, Yamazaki H, Ogasawara N, Yokoyama H, Ose Y, Sato T. The evaluation of genotoxic activities of disinfectants and their metabolites by UMU test. Mutat. Res. (1988b) 209:155–160.[CrossRef][Web of Science][Medline]

    14. Lovely TJ, Levin DE, Klekowski E. Light-induced genetic toxicity of thimerosal and benzalconium chloride in commercial contact lens solutions. Mutat. Res. (1982) 10:11–19.

    15. Deutschle T, Porkert U, Reiter R, Keck T, Riechelmann H. In vitro genotoxicity and cytotoxicity of benzalkonium chloride. Toxicol. In Vitro (2006) 20:1472–1477.[Web of Science][Medline]

    16. Sánchez-Fortún S, Llorente MT, Castaño A. Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified fast micromethod assay. Aquat. Toxicol. (2005) 73:55–64.[CrossRef][Web of Science][Medline]

    17. Henderson ND. A Review of the Environmental Impact and Toxic Effects of DDAC (1992) Victoria, BC: Environmental Protection Division, BC, Ministry of Environment, Lands and Parks. 42. (www.env.gov.bc.ca/epd/epdpa/ipmp/technical_reports/antisapstain_chemicals/pdfs/DDAC.pdf). Accessed 20 November 2006.

    18. Helma C, Knasmüller S, Schulte-Hermann R. Die Belastung von Wässern mit gentoxischen Substanzen—I. Methoden zur Prüfung der Gentoxizität. UWSF (1994) 5:277–288.

    19. Helma C, Knasmüller S. Gentoxische Substanzen in Wässern—II. Industrielle Abwässer. UWSF (1997) 1:41–48.

    20. Ennever FK, Andreano G, Rosenkranz HS. The ability of plant genotoxicity assays to predict carcinogenicity. Mutat. Res. (1988) 205:99–105.[CrossRef][Web of Science][Medline]

    21. Misík M, Micieta K, Solenská M, Misíková K, Pisarcíková H, Knasmüller S. In situ biomonitoring of the genotoxic effects of mixed industrial emissions using the Tradescantia micronucleus and pollen abortion tests with wild life plants: demonstration of the efficacy of emission controls in an eastern European city. Environ. Pollut. (2007) 145:459–466.[CrossRef][Medline]

    22. Misík M, Solenská M, Micieta K, Misíková K, Knasmüller S. In situ monitoring of clastogenicity of ambient air in Bratislava, Slovakia using the Tradescantia micronucleus assay and pollen abortion assays. Mutat. Res. (2006) 605:1–6.[Web of Science][Medline]

    23. Gomez-Lechon MJ, Donato MT, Castell JV, Jover R. Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Cur. Drug Metab. (2004) 5:443–462.[CrossRef]

    24. O'Brien PJ, Chan K, Silber PM. Human and animal hepatocytes in vitro with extrapolation in vivo. Chem. Biol. Interact (2004) 150:97–114.[CrossRef][Web of Science][Medline]

    25. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. In: Handbook of Mutagenicity Test Procedures—Kilbey BJ, Legator M, Nocols W, Ramel C, eds. (1984) 2 edn. Elsevier Science Publishers BV, New York, Chapter 6. 93–140.

    26. Segeln OP. Preparation of isolated rat liver cells. In: Methods in Cell Biology—Prescott DM, ed. (1976) Vol. 13. New York, NY: Academic Press. 29–81.[Medline]

    27. Parzefall W, Monschau P, Schulte-Hermann R. Induction by cyproterone acetate of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes in serum free medium. Arch. Toxicol. (1989) 63:456–461.[CrossRef][Web of Science][Medline]

    28. Lindl T, Bauer J. Zell- und Gewebekultur (1994) Gustave Fischer Verlag, Stuttgart, Germany.

    29. Collins AR. The comet assay for DNA damage and repair. Mol. Biol. (2004) 26:249–261.

    30. Helma C, Uhl M. A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. Mutat. Res. (2000) 466:9–15.[Web of Science][Medline]

    31. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. (2000) 35:206–221.[CrossRef][Web of Science][Medline]

    32. Fenech M, Crott J, Turner J, Brown S. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis (1999) 14:605–612.[Abstract/Free Full Text]

    33. Fenech M. The cytokinesis block micronucleus technique. In: Technologies for the Detection of DNA Damage and Mutations—Pfeifer GP, ed. (1996) New York, NY: Plenum Press. 25–36.

    34. Fenech M, Bonassi S, Turner J, et al. Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project. Mutat. Res. (2003) 534:65–75.[Web of Science][Medline]

    35. Fenech M. The in vitro micronucleus technique. Mutat. Res. (2000) 455:81–95.[Web of Science][Medline]

    36. Ma T-H, Xu Z, Xu C, McConnell H, Rabago EV, Arreola GA, Zhang H. The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mutat. Res. (1995) 334:185–195.[Web of Science][Medline]

    37. Inoue K, Sunakawa T, Takayama S. Studies of in vitro cell transformation and mutagenicity by surfactants and other compounds. Food Chem. Toxicol. (1980) 18:289.

    38. Hikiba H, Watanabe E, Barrett JC, Tsutsui T. Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells. J. Pharmacol. Sci. (2005) 97:146–152.[CrossRef][Web of Science][Medline]

    39. Sheets PL, Yost GS, Carlson GP. Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells over-expressing CYP2F1. J. Biochem. Mol. Toxicol. (2004) 18:92–99.[CrossRef][Web of Science][Medline]

    40. Ma T-H. Vicia cytogenetic tests for environmental mutagens. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. (1982) 99:257–271.[Web of Science][Medline]

    41. Ma T-H, Cabrera GL, Owens E. Genotoxic agents detected by plant bioassays (review). Rev. Environ. Health (2005) 20:1–13.[Medline]

    42. Majer BJ, Grummt T, Uhl M, Knasmüller S. Use of plant bioassays for the detection of genotoxins in the aquatic environment. Acta Hydrochim. Hydrobiol. (2005) 33:45–55.[CrossRef]

    43. Levin DE, Hollstein MC, Christman MF, Schwiers EA, Ames BN. A new Salmonella tester strain (TA102) with A:T base pairs at the site of mutation detects oxidative mutagens. Proc. Natl Acad. Sci. USA (1982) 79:7445–7449.[Abstract/Free Full Text]

    44. Hölzl C, Ferk F, Ehrlich V, Knasmüller S. New endpoints for human single cell gel electrophoresis (SCGE) trials. In: In 5th International Conference on Environmental Mutagens in Human Populations (2007) Turkey. May 20–24 (abstract).

    45. Liebert MA. Final report on the safety assessment of benzalconium chloride. J. Am. Coll. Toxicol. (1989) 8:589–625.

    46. Marple B, Roland P, Benninger M. Safety review of benzalkonium chloride used as a preservative in nasal solutions: an overview of confliction data and opinions. Arch. Otolaryngol. Head Neck Surg. (2004) 130:131–141.[Free Full Text]

Received on December 6, 2006; revised on March 11, 2007; accepted on June 6, 2007.


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