Toxicology Letters, 25 (1985) 137-141
MUTAGENIC EFFECT HUMAN CELLS
and A. CAPUCCI
Istituto di Cancerologia, Universitic di Bologna, Viale Filopanti, 22, 40126 Bologna (Italy) (Received
SUMMARY The mutagenic
cell line (EUE) using selection
doses of the chemical
was effective in inducing at 2 x 10m6 M.
(2 x lo-’
toxin (DT) resistance.
(NTA) was evaluated Cultures
- 2 x 1O-8 M) for 24 h, and cytotoxicity
were treated determined.
at all concentrations
on with NTA
Chemical agents known as ‘builders’ are added to detergents to improve their cleaning efficiency. Pentasodium triphosphate is used widely, and its presence in aquatic systems can cause serious disturbances of the ecological balance [ 11. Since the late 1960’s, nitrilotriacetic acid, a synthetic aminopolycarboxylic acid, has been used as a substitute for polyphosphate in household detergents. Recently its use was suspended, because of suspected dangerous health effects. NTA leads to the development of neoplasia in the urinary tract  and increases the number and size of tumors in rats previously treated with a carcinogen . NTA shows no mutagenic
7,12-dimethylbenz(a)anthracene; unit; NTA,
DT diphtheria acid trisodium
chromatid of human tetraploidy
 or in mice , and does not induce
exchanges in human or hamster cells . However, 3-5 days’ exposure peripheral blood lymphocytes to semitoxic doses of NTA induces and endoreduplication, in addition to chromosome aberrations . At
high doses, NTA causes aberrations in bean root-tips and in rat kangaroo cells in culture . Furthermore, it shows a weak transforming potential when tested in a Balb/c 3T3 transformation assay . In order to further explore the possible effect of NTA on cell genoma, we studied its ability to induce mutations in a human cell line, using selection for diphtheria toxin (DT)-resistance. The test has proved to be a useful tool in determining correlations between the mutagenic potential and the carcinogenic effect of different substances [IO- 121. MATERIALS
Chemicals NTA (chemical purity > 99%) was purchased from Aldrich Chemie; 7,12-dimethylbenz[a]anthracene (DMBA) from SIGMA Chemical Co., St. Louis, MO, U.S.A. and DT from Connaught Medical Research Laboratories, Toronto, Canada (2000 flocculating units (Lf) per ml; 1 Lf contained 57 guinea pig minimum lethal doses and 3 pg protein). DMBA was dissolved in acetone, NTA in phosphatebuffered saline (PBS). All solutions were diluted with culture medium to the desired final
Cells EUE, a line of human heteroploid grown in Eagle’s minimal essential
epithelial-like cells , was used. Cells were medium with Hank’s salts (Gibco, Glasgow,
U.K.) supplemented with 10% newborn calf serum (Gibco, Glasgow, U.K.) and 2 mM L-glutamine (Gibco), at 37°C in humidified air supplemented with 5% COz. A 0.25% solution of trypsin (Gibco) in PBS free from calcium and magnesium ions was used to remove cells from plastic surfaces and to make single-cell suspensions.
Cytotoxicity was measured by evaluating the plating efficiency of seeded cells (070 forming colonies). 500 Viable cells were seeded in 60-mm plastic Nunclon petri dishes with complete medium. After 24 h, NTA (2 x 10e2 - 2 x lo-* M) was added, and cells were exposed for one day, after which the medium was changed and the cultivation continued for one week. The cell colonies were then fixed in methanol, stained with Giemsa and counted. Each dose was tested in 6 parallels. Control dishes received normal medium throughout.
Mutagenesis assay 3 x lo6 Viable cells were seeded in complete
in SO-cm’ flasks (Nunclon)
and left to attach following
were then added 1 x 10m6; NTA,
1.1 x 10m2, 2 x 10p3,
2 x 10e5, 1.1 x 10m5, and 2 x 10e6 M. The cell cultures were treated with the chemicals for 24 h, then washed once with PBS, trypsinized and counted. To determine plating efficiency (PE), i.e., the number of colonies formed as a percentage of the cells seeded, 500 viable cells were plated in 60-mm plastic petri dishes (Nunclon), and cultured for a week. The cell colonies were fixed in methanol, stained with Giemsa and counted. The remaining cells were seeded (5 x lo5 viable cells/flask, 6 samples of each dose) and allowed to grow for 5 days. Treated and control cultures were exposed, without replating, to a selective medium containing DT of 0.05 Lf/ml, for 48 h. The toxin-containing medium was then replaced by fresh, toxin-free medium. After 15 days of growth, the surviving colonies were fixed in methanol, stained with Giemsa and counted. RESULTS
The cytotoxicity assay was performed to evaluate suitable concentrations of NTA for the mutagenesis experiments. NTA was assayed at concentrations ranging from 2 x 10e2 - 2 x lo-’ M, for 24 h. The highest dose tolerated by EUE cells was 1 .l x 10e2 M. This dose reduced the plating efficiency to 60% of control. No toxic effect was seen at lower doses. Similar results were obtained when NTA treatment was continued for a week (data not shown). The mutagenicity assay was performed using 5 concentrations of NTA, with DMBA as positive control. NTA was effective in inducing DTR mutants in EUE at all concentrations tested, except at 2 x low6 M (Table I). This mutagenesis assay does not require an exogenous source for metabolic activation, and has been used in quantitative mutagenesis studies to determine the mutagenic potential of different kinds of carcinogenic chemicals [lo-12, 141. In our laboratories, Colacci et al. (personal communication) have performed studies on in vitro binding of [14C]NTA to DNA, mediated by enzymatic fractions. High DNA TABLE
I Plating efficiency (% colonies from cells seeded)
per 1 x lo6 viable cells 1.70 + 0.56
32 1 x 10-6M
1.1 x IO-‘M
2 x 10-3M
2 x 10-j
1.1 x 10-j M 2 x 10-6M
f 1.68 f 0.63
NTA NTA Each
values is the mean
of 6 replicates.
(P < 0.01).
due to a high chemical
per se was found.
[14C]NTA to DNA was not increased by microsomal or cytosolic fractions. This may explain the mutagenic effect of NTA on EUE cells via the reaction of NTA with cell targets. Our data agree with previous
of NTA in
in vitro experiments performed on human peripheral blood lymphocytes  in which NTA-treated cultures contained a significantly increased frequency of tetraploid and endoduplicated cells, chromosome aberrations and chromatid breaks. Similar results were obtained by Kihlman et al. in bean root-tips and in kangaroo cells, both of which showed chromosome aberrations [S]. A variety of chromosomal abnormalities, such as aneuploidy and translocation, have been identified in neoplastic cells [ 151. Most tumors in mammals, including man, contain malignant cells that are chromosomally abnormal  and there are a number of ‘chromosome breakage’ syndromes associated with high cancer incidence . In many cases, chromosome aberrations are secondary consequences of tumor development, but in others they are primary causal factors [ 171. The mechanism by which NTA exerts its damaging action on DNA is unclear. NTA does not induce sister chromatid exchanges (SCE) in hamster or human cells, suggesting that it does not affective reparative processes associated with SCE . However, the present finding, that NTA is effective in inducing DTR mutants in EUE cells, and its ability to induce chromosomal aberrations in other systems [7, 81, suggest that NTA may constitute a hazard to humans. It is therefore questionable whether this agent is an acceptable substitute for polyphosphates in household detergents.
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