Test for induction of chromosomal aberrations in Chinese hamster ovary cells (in vitro) by 2-(2′,4′-diaminophenoxy)ethanol

Test for induction of chromosomal aberrations in Chinese hamster ovary cells (in vitro) by 2-(2′,4′-diaminophenoxy)ethanol

Mutation Research, 102 (1982) 351-355 351 Elsevier BiomedicalPress Test for induction of chromosomal aberrations in Chinese hamster ovary cells (in...

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Mutation Research, 102 (1982) 351-355

351

Elsevier BiomedicalPress

Test for induction of chromosomal aberrations in Chinese hamster ovary cells (in vitro) by 2-(2',4'-diaminophenoxy) ethanol F. Darroudi, A.C. van Kesteren-van Leeuwen and A.T. Natarajan Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72, Leiden (The Netherlands)

(Received25 May 1981) (Revisionreceived7 October 1981) (Accepted 15 April 1982)

Among the short-term tests for the detection of mutagenicity by chemicals, the test for the induction of chromosomal aberrations in vitro has several advantages. In addition to the simplicity of the technique, higher concentrations can be used for treatment than in tests in vivo. Moreover, the introduction of metabolic activation by rat-liver homogenate ($9) in this test in vitro (Natarajan et al., 1976) has increased its value, as indirectly acting mutagens/carcinogens can now also be easily evaluated. Existing evidence shows that this test can detect most mutagens or carcinogens that are genotoxic (Natarajan and van Kesteren-van Leeuwen, 1981). Results of experiments aimed at testing the mutagenic potential of 2-(2',4'-diaminophenoxy)ethanol with this system in vitro are presented in this paper.

Materials and methods

An established CHO line, with a modal chromosome number of 2n = 21-22, and a generation time of about 12h was used. The cells were grown in Ham's F10 medium, supplemented with 15% newborn calf serum. Before treatment, cells (CHO) were trypsinized and centrifuged, and the pellet was suspended in about 1-2 ml complete medium (F10 medium with 15% calf serum), to provide about 1-2 × 107 cells/ml. Then the cell suspension was added to the $9 mix (2.0 ml 20 mM Hepes buffer, pH 7.2), 1.3 ml 50 mM MgC12, 1 ml 330 mM KC1, 1.0 ml 50 mM glucose 6-phosphate, 2.7 ml F10 medium (without serum), 1.0 ml $9 fraction, 1.0 ml 40 mM NADP, totalling I0 ml). Each treatment tube contained 0.8 ml of $9 mix, 0.1 ml of the test compound [2-(2',4'-diaminophenoxy)ethanol] adjusted to the desired concentration and 0.1 ml of the cell suspension. One set of tubes was oxygenated for a few seconds, and for the others nitrogen was used. The tubes were tightly dosed. A parallel treatment was also done without $9 mix. The treatment was done at 37°C, 0165-1218/82/0000-0000/$02.75 © ElsevierBiomedicalPress

352

TABLE

1

CHROMOSOMAL

ABERRATIONS

IN

CHO

CELLS

TREATED

WITH

2-(2',4'-DIAMINO-

PHENOXY)ETHANOL A v e r a g e o f 2 0 0 cells. Compound

$9

(mg/ml)

mix

0 2

N2

Fixation

Chromosomal

aberrations/100

cells

time (h)

Gaps

Breaks

Exchanges

Total

Control

-

-

+

6

2.5

2.5

0

5.0

Control

-

+

-

6

3.0

2.0

0

5.0

Control

+

-

+

6

3.0

2.0

0

5.0

Control

+

+

-

6

3.5

2.5

0

6.0

0.6

-

-

+

6

3.0

3.0

0

6.0

0.6

-

+

-

6

4.5

3.5

0

8.0

0.6

+

-

+

6

3.0

3.5

0

6.5

0.6

+

+

-

6

3.5

3.0

0

6.5

1.2

--

--

+

6

4.0

3.0

0

7.0

1.2

--

+

--

6

4.5

3.5

0

8.0

1.2

+

--

+

6

4.0

3.0

0

7.0

8.0

1.2

+

+

--

6

4.0

4.0

0

Control

-

-

+

12

4.5

5.0

0

9.5

Control

--

+

-

12

5.5

6.0

0

11.5

Control

+

-

+

12

4.0

4.5

0

8.5

Control

+

+

-

12

6.0

4.5

0

10.5

0.6

-

-

+

12

3.5

5.5

0

9.0

0.6

--

+

--

12

6.0

6.5

0

12.5

0.6

+

--

+

12

5.0

3.5

0

8.5

0.6

+

+

--

12

5.5

5.0

1

11.5

1.2

--

--

+

12

6.5

6.0

0

12.5

1.2

--

+

--

12

7.0

7.0

3

17.0

1.2

+

--

+

12

6.5

6.5

1

14.0

1.2

+

+

--

12

7.0

7.0

1

15.0

Control

-

-

+

16

4.0

2.5

0

6.5

Control

-

+

-

16

4.0

3.0

0

7.0

Control

+

-

+

16

4.5

3.5

0

8.0

Control

+

+

-

16

4.0

3.0

0

7.0

0.6

-

-

+

16

5.0

4.5

0

9.5

0.6

-

+

-

16

6.0

6.0

0

12.0

0.6

+

-

+

16

4.0

5.0

0

9.0

0.6

+

+

-

16

6.0

6.0

0

12.0

1.2

--

--

+

16

5.0

5.5

0

10.5

1.2

--

+

--

16

6.5

6.5

0.5

13.5

1.2

+

--

+

16

5.5

6.0

0

11.5

1.2

+

+

--

16

6.0

5.0

0

11.0

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355 with continuous shaking for 1 h, after which the cells were centrifuged, washed in complete medium and plated in petri dishes. The cells were allowed to recover, at 37°C, in 5% CO 2. Fixations were done at different times, namely 6, 12 and 16 h after the end of the treatment. A 2-h colcemid (0.00028%) treatment preceded trypsinization, hypotonic shock 0 % sodium citrate) and fixation with acetic acid-methanol (1:3). Air-dried preparations were made on ice-cold wet slides and stained with 2% aqueous solution of Giemsa for 10 min. The slides were mounted with a cover glass b y using Depex. 100 cells were scored for each point for the presence of chromosomal aberrations; i.e. gaps, breaks, exchanges. Experiments were conducted twice. Positive control experiments with the same protocol as above were carried out by using 2,4-diaminoanisole dihydrochloride. All test chemicals were supplied in pure form by l'Or~al, Paris.

Results and conclusions The results are presented in Tables 1 and 2. The 3 different fixation times chosen, i.e. 6, 12 and 16h, should sample cells that had been in G2, S and G l stages, respectively, during treatment, taking into account the mitotic delay due to both plating and treatment with the test chemical. The results indicate that (a) there was no concentration-dependent increase in the frequencies of chromosomal aberrations induced; (b) the presence of oxygen or nitrogen did not have an influence on the frequencies of aberrations; and (c) the inclusion of a metabolic activation system had no influence on the frequencies of chromosomal aberrations. These results lead to the conclusion that the test compound, 2-(2',4'-diaminophenoxy)ethanol, is not mutagenic, as judged by this test. On the other hand, the positive control, i.e. 2,4-diaminoanisole (2-4D), increased the frequencies of all types of chromosomal aberration (mainly chromatid type) at all concentrations tested, as well as at all fixation times, independently of metabolic activation. Interestingly, 2-4D increased the frequencies of endoreduplicated cells, indicating impairment of splitting of sister chromatids.

References Natarajan, A.T., and A.C. van Kesteren-vanLeeuwen (1981) Mutagenic activity of 20 coded compounds in chromosome aberrations/sister chromatid exchanges assay using Chinese hamster ovary (CHO) cells, in: F.J. de Serres and J. Ashby (Eds.), Progress in Mutation Research, I. Evaluation of Short Term Tests for Carcinogens, Elsevier/North-Holland, pp. 551-559. Natarajan, A.T., A.D. Tates, P.P.W. van Buul, M. Meijers and N. de Vogel (1976) Cytogeneticeffects of mutagens/carcinogens after activation in a microsomalsystem in vitro, Mutation Res., 37, 83-90.

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