Mutation Research, 122 (1983) 355-360 355 Elsevier

MRLett 0501

Evaluation of the mutagenicity of formaldehyde in mam- malian cytogenetic assays in vivo and vitro

A . T . N a t a r a j a n t'2, F. D a r r o u d i 1, C . J . M . Bussman ~ and A .C . van

Kes te ren-van Leeuwen ~

~Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands) and 2J.A. Cohen Institute of

Radiopathology and Protection, Leiden (The Netherlands)

(Accepted 7 September 1983)

Formaldehyde (FA) is now a recognized mutagen to which the human population is exposed both environmentally and occupationally. FA is prevalent in several in- dustries (textile, paper, chemical synthesis, synthetic resin etc.), horticultural and agricultural operations (chemical seed disinfection, use of fertilizers, insecticides and pesticides) as well as in the general environment (automobile exhausts, tobacco smoke etc.). Though the mutagenicity of FA was reported by Rapoport as early as 1946, most of the further work has been concentrated in Drosophilla (Auerbach et al., 1977). FA is not effective in inducing chromosomal aberrations in Vicia faba root-tip cells (Loveless, 1951; Rieger and Michaelis, 1960), but it is mutagenic in bacteria and fungi (Auerbach et al., 1977). Studies on the mutagenic effects of FA in mammals are few. Epstein and co-workers (1977), as well as Fontignie- Houbrechts (1981), could not detect any effect of FA in a dominant lethal assay or, in vivo, in the cytogenetics assay of male germ cells of mice. The present study is an attempt to evaluate FA in a cytogenetic assay in vitro by using chromosomal aberrations and SCEs in Chinese hamster ovary cells as well as in assays in vivo, in mice by using induction of micronuclei and chromosomal aberrations in bone- marrow cells and chromosomal aberrations in spleen cells as end-points. While FA was very active in conditions in vitro, it was not active in vivo.

Material and methods

Paraformaldehyde from Aldrich was dissolved in distilled water, and NaOH was added to clear the solution. The solution was adjusted to pH 7.2 before use.

Studies in vitro CHO cells were grown in Ham's F10 medium supplemented with 15070 new-born

calf serum and antibiotics. For detection of both chromosomal aberrations and

0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.

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TABLE 1

INDUCTION OF CHRO MO SO MA L ABERRATIONS AND SCEs IN CHO CELLS TREATED

WITH FORMALDEHY D E (IN THE PRESENCE AND ABSENCE OF METABOLIC ACTIVATION)

Concen- $9 mix Total Aberrat ions/100 cells SCEs/

tration cells celt ± S.E. b Gaps Breaks and Exchanges

~ l / m l ) scored a fragments

Control - 200 6 5.5 0 9.40 + 1.42

Control + 200 5.5 4 0 8.70 +_ 1.27

0.003 - 50 13 10 0 11.89 ± 1.62

0.003 + 50 10 5 0 11.57 ± 1.49

0.006 - 150 26.6 24 4 18.98 + 2.13

0.006 + 150 12.6 13.3 2.6 13.71 + 1.59

0.012 - 300 41 103 102 32.94 ___ 2.55

0.012 + 200 23.5 42 51 23.14 _+ 2.58

0.024 - 100 64 185 169 53.60 ± 4.73

0.024 + 100 51 149 123 44.10 + 4.18

"Results from 2 Expts. were pooled.

bResults from 2 Expts. were pooled, and, in total, 60 cells were scored for each treated concentration.

TABLE 2

INDUCTION OF MICRONUCLEI IN P O L Y C H R O M A T I C ERYTHROCYTES OF

MOUSE BONE-MARROW CELLS TREATED WITH FORMALDEHYDE

Dose (mg/kg Number of Sex Fixation time Number of micronuclei /

b.w.) mice (h) 1000 polychromatic _+ S.E.

erytbrocytes

0 (saline) 5 M 16 0.8 ± 0.45

0 5 F 16 0.4 -t 0.55

6.25 3 M 16 0.6 + 0.58

6.25 3 F 16 0.6 +_ 0.58

12.50 5 M 16 1.6 _+ 0.55

12.50 5 F 16 1.2 + 0.45

25.00 5 M 16 1.6 _+ 0.55

25.00 5 F 16 1.6 + 0.55

0 (saline) 5 M 40 0.8 ± 0.45

0 5 F 40 0.8 _+ 0.84

6.25 3 M 40 1.3 ± 0.84

6.25 3 F 40 1.3 ± 0.84

12.50 5 M 40 1.4 ± 0.45

12.50 5 F 40 1.4 + 0.84

25.00 5 M 40 1.6 + 1.14

25.00 5 F 40 1.6 ± 0.89

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SCEs, a one-cycle protocol was used (Natarajan and van Kesteren-van Leeuwen, 1981). The cells were first grown for 12 h (one cycle) in medium containing 5/zM 5-bromodeoxyuridine (BrdUrd). The medium was removed, and the cells were treated with various concentrations of FA for 2 h in the presence or absence of rat- liver metabolizing system ($9 fraction, with cofactors) according to the protocol of Natarajan et al. (1976). After the treatment, the cells were rinsed with PBS and restored in normal medium with the addition of thymidine (4 #M, end concentra- tion) for 16 to 20 h. Chromosomal preparations were made by routine procedures. Slides were stained with 2°70 Giemsa solution, or with Fluorochrome plus Giemsa, for the analysis of chromosomal aberrations or SCEs, respectively.

Studies in vivo

Male and female CBA mice were 10-12 weeks old. At the time of treatment, male mice were 25-30 g and females were 20-25 g in weight. Formaldehyde solution, 0.4 ml, was injected intraperitoneally; the concentration of the solution had been ad- justed to give the desired final dose. Control mice received 0.4 ml of isotonic saline. Two injections were given with an interval of 24 h, and cells were sampled 16 and 40 h after the second injection. Colcemid was injected 2 h before the mouse was kill- ed by cervical dislocation.

One femur was used for determining the frequencies of micronuclei in the polychromatic erythrocytes (Schmid, 1975) and the other femur was processed for determination of chromosomal aberrations (Tates and Natarajan, 1976). Suspen- sions of spleen cells were obtained from the same animal and these cells were col- lected by removing the spleen and cleaning it of fat. Then one end of the spleen was cut, balanced salt solution was injected into the spleen with the needle and the free spleen cells were collected (Palitti et al., 1982). 1000 polychromatic erythrocytes were analysed for the presence of micronuclei. 75-100 cells were scored for the presence of chromosomal aberrations in bone-marrow and spleen cells from each animal. The results were analysed by Student's t test.

Results

Studies in vitro

The frequencies of chromosomal aberrations and SCEs induced by FA in CHO cells are shown in Table 1. FA increased the frequencies of chromosomal aberra- tions and SCEs, with increasing dose. All classes of aberration, i.e. gaps, breaks and exchanges, were induced by FA. All the aberrations were chromatid-type, indicating that FA acts as an S-dependent agent. The efficient induction of SCEs by FA, sup- ports this conclusion. The addition of mammalian metabolic activation system reduced the frequencies of FA-induced aberrations at all doses, probably owing to deactivation of FA. Similarly, there was also a reduction in the frequencies of SCEs induced by FA, if the treatment was done in the presence of $9.

358

TABLE 3

INDUCTION OF CHROMOSOMAL ABERRATIONS IN BONE-MARROW CELLS OF MICE TREATED WITH FORMALDEHYDE

Dose Number Sex Fixation Number Abnormal Total (mg/kg b.w.) of mice time (h) of cells cells ± S.E. number of

analysed (%) breaks

0 (saline 5 M 16 500 3.0 + 0.7 15 0 5 F 16 500 2.8 + 0.5 14

6.25 3 M 16 300 3.6 ± 0.7 11 6.25 3 F 16 300 3.3 ± 0.6 10

12.50 5 M 16 500 3.0 _ 0.4 16 12.50 3 F 16 280 3.6 ± 0.5 I1

25.00 5 M 16 405 3.2 ± 0.4 14 25.00 5 F 16 385 3.3 ± 0.6 15

0 (saline) 5 M 40 485 2.7 ± 0.5 13 0 5 F 40 476 2.9 :t: 0.7 14

6.25 3 M 40 288 4.1 ± 0.8 12 6.25 3 F 40 290 4.1 + 0.7 13

12.50 4 M 40 380 3.7 +_ 0.9 15 12.50 4 F 40 369 3.8 ± 0.7 14

25.00 5 M 40 479 2.9 + 0.6 16 25.00 4 F 40 370 3.7 ± 0.8 15

Studies in vivo

The results o f the micronuc leus test are summar ized in Table 2. None o f the con-

cent ra t ions used increased the f requencies o f micronucle i over the cont ro l level. The

frequencies o f c h r o m o s o m a l aber ra t ions in b o n e - m a r r o w and spleen cells are shown

in Tab le 3 and 4. FA was not effect ive in inducing c h r o m o s o m a l aberra t ions in these

two types o f ceil. Thus, all results ob ta ined in vivo show that FA is not active in

mice in vivo.

Discussion

The results presented here show that FA is eff ic ient in inducing c h r o m o s o m a l

aberra t ions and SCEs in m a m m a l i a n cells under t r ea tment condi t ions in vitro. The

abili ty o f F A to induce c h r o m o s o m a l aberra t ions in human f ibroblasts and SCEs

in h u m a n lymphocytes has recently been repor ted (Levy et al., 1983; Kreiger and

Garry , 1983). T h o u g h FA is known to induce D N A single-strand breaks and

D N A - p r o t e i n cross-l inks (Magaf ia -Schwencke et al., 1978a; Magaf ia -Schwencke

and Kert , 1978b), the cytological effects observed appear to be due to cross-l inking.

FA induced only ch romat id - type aber ra t ions even when the t rea tment was done in

GI stage (as the cells were t reated 20 h before fixation), an observa t ion point ing

TABLE 4

INDUCTION OF CHROMOSOMAL ABERRATIONS

WITH FORMALDEHY D E

IN SPLEEN

359

CELLS OF MICE TREATED

Dose Number Sex Fixation Number Abnormal Total (mg/kg b.w.) of mice time (h) of cells cells ± S.E. number of

analysed (070) breaks

0 (saline) 5 M 16 500 2.4 ± 0.6 12

0 5 F 16 500 2.2 _+ 0.7 11

6.25 3 M 16 280 3.5 ± 0.7 10

6.25 3 F 16 276 4.0 _+ 0.8 11

12.50 5 M 16 485 2.8 ± 0.8 15

12.50 4 F 16 378 3.4 ± 0.7 13

25.00 5 M 16 458 3.0 ± 0.6 14

25.00 5 F 16 492 2.6 _+ 0.7 13

0 (saline) 5 M 40 469 2.5 ± 0.8 12

0 5 F 40 478 2.5 + 0.7 12

6.25 3 M 40 265 3.4 + 0.7 9

6.25 3 F 40 275 3.6 ± 0.7 10

12.50 4 M 40 356 3.0 ± 0.8 I1

12.50 4 F 40 348 3.1 _+ 0.6 11

25.00 4 M 40 340 3.5 ± 0.8 12

25.00 4 F 40 360 3.0 _+ 0.7 12

towards the S-dependent mode of action of this agent, similar to that of all other cross-linking agents. Like other S-dependent agents, FA increases SCEs effectively. The ability of FA to induce single-strand breaks appears to be a secondary process occurring during DNA repair, and such single-strand breaks have been reported to occur less efficiently in yeast cells defective in excision-repair (Magafia-Schwencke et al., 1978a). CHO cells are not efficient in excision-repair, and by analogy one would expect less induction of SSBs in these cells due to FA treatment. Experiments are in progress at present in our laboratory to verify this conclusion. FA also induces mutations in cultured human cells (Goldmacher and Thiily, 1983). FA turned out to be negative in the experiments done by us in vivo. Earlier experiments with FA in vivo had been done to evaluate the effects in the germ cells, and the present ex- periments were done with somatic cells. The probable reason for these negative results in vivo may be the inability of FA to reach target cells in sufficient quantity to induce biological effects. The reduced effect by rat-liver homogenate in tests in vitro may indicate a possible detoxication of FA, which may also occur in vivo.

FA induces squamous-cell carcinomas of the rat nasal cavity (Swenberg et al., 1980). However, these tumours appear after inhalation of FA vapour, and is therefore not subjected to detoxication by the liver, as FA injected into the animal would be. Chronic exposure studies in animals with FA should be done before a

360

d e f i n i t e c o n c l u s i o n c a n b e d r a w n a b o u t t h e i n e f f i c i e n c y o f F A to b e ac t i ve , in v i v o

in i n d u c i n g c h r o m o s o m a l a b e r r a t i o n s .

Acknowledgement

W e t h a n k P r o f . F . H . S o b e l s f o r h is w a r m e n c o u r a g e m e n t . T h i s s t u d y was f i n a n -

c ia l ly s u p p o r t e d b y t h e K o n i n g i n W i l h e l m i n a F o n d s (SG 81-91) , a n d t h e E E C

C h e m i c a l M u t a g e n P r o g r a m m e .

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