Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Mutation Research, 214 (1989) 97-104 Elsevier

97

MUT 02562

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells Ingrid Emerit and Madelaine Lahoud-Maghani Laboratoire de G~nOtique, Institut biomedical des Cordeliers, CNRS and UniversitO Pierre et Marie Curie, 15, rue de l'Ecole de M~deeine, 75006 Paris (France)

(Received 4 July 1988) (Accepted 16 February 1989)

Keywords: Chinese hamster V79 cells; Clastogenic factor; 12-O-Tetradecanoylphorbol 12-acetate

Summary Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.

The action of 12-O-tetradecanoylphorbol 12acetate (TPA) at the chromosomal level was first described by Kinsella and Radman (1978) as an increased level of sister-chromatid exchanges (SCE) in Chinese hamster cells. Subsequently

Correspondence: Dr. I. Emerit, Laboratoire de Grn&ique, Institut biomrdical des Cordeliers, CNRS and Universit6 Pierre et Marie Curie, 15, rue de l'Ecole de Mrdecine, 75006 Paris (France).

TPA-induced increases in SCE frequencies were reported by several other authors (Nagasawa and Little, 1979; Gentil et al., 1980; Dzarlieva and Fusenig, 1982; Schwartz et al., 1982), while Loveday and Latt (1979), Thompson et al. (1980) and Fujiwara et al. (1980) were unable to confirm this finding. Work in our laboratory has shown that TPA is strongly clastogenic for human lymphocytes (Emerit and Cerutti, 1981). Similar observations were made by others in cultures of mouse epider°

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

98 mal cells (Dzarlieva and Fusenig, 1982). In contrast to its high efficiency as a clastogen, TPA was a weak inducer of SCE in our system. However, the increase in SCE was consistently observed and statistically significant. According to several authors, TPA is not mutagenic, but enhances chemically or UV-light-induced mutation frequencies (Lancas et al., 1977; Trosko et al., 1977; Thompson et al., 1980; Raveh and Huber, 1983; Dewdney and Soper, 1984; Manuilova et al., 1987). This finding was dependent on TPA being present throughout mutation expression and mutation selection (Dewdney and Soper, 1984). We could demonstrate that the clastogenic and SCE-inducing action of TPA is indirect. It is mediated by secondary products which are formed by the cell in response to the interaction with TPA. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocyte cultures, which were essentially free of residual TPA, as demonstrated by experiments with the radioactive compound (Emerit and Cerutti, 1982). This material causes chromosome aberrations and SCE in fresh blood cultures not exposed to TPA itself. The presence of polymorphonuclear neutrophils, monocytes and platelets seemed to be a prerequisite for the formation of CF in TPAtreated blood cultures, since supernatants of cultures set up with 'pure' lymphocytes (containing no platelets and maximally 1-2% neutrophils and monocytes) were practically not clastogenic (Emerit and Cerutti, 1983). On the other hand, addition of increasing numbers of these blood cells to 'pure' lymphocytes resulted in a linear increase of the clastogenic potency of the culture media. Since superoxide dismutase (SOD) and less regularly also catalase were anticlastogenic, we concluded that active oxygen species were involved in CF formation and CF action. Birnboim (1982) has shown that TPA stimulates an oxidative burst in phagocytes and induces DNA-strand breaks in the same phagocyte population. This could be prevented by SOD and catalase, while hydroxyl radical scavengers were not effective in preventing damage. The same author noted that TPA induced D N A damage in erythroleukemia cells only when they were co-in-

cubated with phagocytes (Birnboim, 1983). Similar observations were made by Dutton and Bowden (1985), who observed DNA-strand breakage in mouse epidermal cells only in the presence of leukocytes. Since catalase was protective, the authors concluded that tumor promoters can act indirectly on target epidermal cells by stimulating the release of a CF from leukocytes through a mechanism involving H 2 0 z. Snyder (1985), on the contrary, was able to induce DNA-strand breakage in human fibroblasts by TPA in the absence of phagocytic cells. It therefore seemed of interest to study whether CF formation was restricted to cells responding to TPA with an oxidative burst or whether CF could also be isolated from fibroblast cultures with our standard procedure. We chose Chinese hamster lung cells (V79), a cell type used by various other authors for SCE induction by TPA with discordant results. Also V79 cells seemed to be most appropriate for the study of the mutagenic effects of CF. Material and methods

Cell culture conditions Human lymphocytes and Chinese hamster cells were used for the present study. The culture conditions were the same whether these cells were used for CF production or for the study of cytoand geno-toxic effects. (a) Lymphocyte cultures Blood was drawn from healthy volunteers. Lymphocytes were separated by differential centrifugation on Isopaque/Ficoll (Nyegaard, Oslo). They were essentially free of red blood cells and neutrophils, but contained monocytes and platelets in numbers varying from one donor to the other. Cultures were set up with 2.5 x 106 mononuclear cells in 5 ml of T C M 199 (Flow Laboratories, Paris) and 1 ml of heat-inactivated fetal calf serum controlled for its SOD content. The lymphocytes were stimulated to divide with phytohemagglutinin P (Difco, Paris). For CF production, TPA from CCR Eden Prairie was dissolved in acetone and added to the medium at a final concentration of 100 ng/ml. Acetone concentrations did not exceed 0.1%.

99

(b) Chinese hamster lung fibroblasts (V79) Cells were inoculated in 75-cm2 Falcon plastic flasks containing Dulbecco's modified Eagle's medium (Flow Laboratories, Paris) with 10% fetal calf serum controlled for its SOD content. The cell replication time was about 14 h. For CF preparation, TPA was added under the conditions described for lymphocyte cultures.

CF preparation Culture media from TPA-treated lymphocyte or V79 cell cultures were collected after a cultivation period of 72 h. The same protocol was adopted for V79 cells despite the shorter replication time of these cells compared to lymphocytes. In order to be in exponential growth near confluence at the time of media collection, a small number of cells was inoculated (5 × 105 for 10 ml of medium). It is important to have a sufficient number of cells to reach detectable CF concentrations, but cells should not be in a stationary phase. The culture media from both types of cultures were centrifuged at 3000 rpm and ultrafiltrated through a Diaflo filter YM 10 (cut-off 10000 dalton). They were concentrated 10 times by a second filtration through filter YM 2 (cut-off 1000 dalton). Despite its low molecular weight, TPA is retained by filter YM 10 as shown by previous experiments with radioactive TPA (Emerit and Cerutti, 1982). Control preparations were made from acetonetreated cultures, which were handled in the same way as the supernatants from TPA-treated cultures. The preparations were frozen in small aliquots until use for the study of their cyto- and geno-toxic effects.

Study of cyto- and geno-toxic effects (a) SCE induction CF and control preparations were added to human lymphocytes or Chinese hamster cells at the beginning of the cultivation period. Bromodeoxyuridine (5 /~g/ml) was added to lymphocyte cultures 6 h later and to V79 cultures 2 h later. The total cultivation period was 72 h for lymphocyte cultures and 28 h for V79 cells. Mitoses were blocked in metaphase by the addition of colchicine (0.2/tg/ml), 1 h prior to harvesting. After

hypotonic treatment with KC1 (75 mM), the cells were fixed in methanol-acetic acid (3:1). Chromosome spreads were prepared with the air-drying technique and sister chromatids differentially stained with the fluorescence plus Giemsa (FPG) technique (Perry and Wolff, 1974). A minimum of 50 well-spread metaphase plates was scored for each experimental point on coded slides.

(b) Cell-survival curves Cell survival was determined by the colony-formation assay. After 24 h exposure to CF, the cells were trypsinized, suspended in culture medium and counted. Different numbers of cells, varying between 100 and 1000 per Falcon dish of 20 mm diameter, were incubated for 7 days, after which they were fixed with methanol and stained with 10% Giemsa.

(c) Induction of point mutations V79 cells were exposed to various aliquots of CF preparations previously tested in the cellsurvival assay for their activity and cytotoxicity. After exposure to CF during 24 h, the culture medium was changed and the ceils continued to grow for another 7 days for mutation expression. They were then detached with trypsin, counted and reseeded at a density of 105 cells per 100 nun diameter Petri dish. For each quantity of CF, 20 dishes were prepared and compared to 20 dishes receiving control preparations from acetonetreated cultures. The selective agent 6-thioguanine (Sigma, Paris) was added to the culture medium at a final concentration of 30/xg/ml (Arlett, 1977). After 14 days, the 6-thioguanine-resistant colonies were fixed, stained and counted. The mutation frequencies were calculated by dividing the total number of mutant colonies by the total number of plated cells. They were corrected according to the cloning efficiency of the cells studied in 3-4 parallel dishes (20 mm) seeded with 100 cells each. Results

SCE induction In order to confirm that TPA induces SCE in V79 cells as it did in previous work with human lymphocytes, we studied the SCE frequencies of TPA-treated and solvent-treated cultures. As can

100 TABLE 1

TABLE 3

NUMBER OF SCE IN V79 CELLS EXPOSED TO 100 n g / m l OF TPA (MEAN OF 4 EXPERIMENTS)

SCE F R E Q U E N C I E S IN V79 CULTURES EXPOSED TO CF PREPARATIONS F R O M TPA-TREATED V79 CELLS. PROTECTIVE EFFECT OF SOD

TPA

TPA + SOD a

Control

Untreated cultures

11.0_+2.4

6.7_+0.1

8.5_+2.2

5.8_+1.0

Volume (gl)

a From Boehringer Mannheim, final concentration 10/~g/ml.

be seen in Table 1, a dose of 100 n g / m l TPA induced 11 SCE/cell compared to 8.5 in acetonetreated and 5.8 in untreated cultures. This is even higher than in lymphocyte cultures, where a similar dose of TPA increased the SCE rate to 8 + 2.4/cell compared to 5.2 ___2.2 in untreated cultures. These differences are highly significant, given the high number of cells studied ( p < 0.001) (Emerit and Cerutti, 1981). When CF preparations were tested instead of TPA itself, the number of SCE increased with increasing quantities. This was true for CF preparations from human lymphocytes tested on V79 cells (Table 2) and for CF preparations from hamster cells, tested either on hamster cells or on human lymphocytes (Tables 3 and 4). In all these experiments, the SCE rates for untreated cultures were 5.7-6.0 SCE/cell and corresponded to the laboratory standard. They were slightly higher with control preparations (6.3-6.8), but significantly increased with identical quantities of CF preparations (10 SCE/cell for a quantity of 300 /~1). Higher doses did not lead to a further increase in the SCE rate (see Table 2).

CF preparations CF preparations + SOD Control preparations

200

300 a

9.2 _+0.4

9.8 _+0.7

6.9 _+0.5

6.6 + 0.6

-

6.4_+ 0.2

The SCE rate in untreated cultures was 5.8_+0.8 and corresponded to that observed in other experiments. Added to 5 ml of serum-supplemented medium.

SOD was protective in all experiments, and the frequencies observed in TPA-treated cultures pretreated with this enzyme were near control values for all CF quantities studied (see Tables 2, 3 and 4). Survival of V79 cells in the presence of CF The survival of untreated V79 cells in our system was 80-100%. As can be seen in Fig. 1, even small quantities (50 gl) of lymphocyte-derived CF influenced cell survival. A considerable decrease in cell survival was observed for 150-300 /~l of CF, while higher doses up to 900 gl did not result in a dose-dependent effect. SOD used in combination with doses of 600 and 900 gl CF protected the cells, which showed a survival similar to that of the cultures exposed to control preparations (76 and 74% respectively). As expressed by the standard deviation bars, the cytotoxicity of CF prep-

TABLE 2 SCE FREQUENCIES IN V79 CULTURES TREATED WITH CF FROM TPA-TREATED H U M A N LYMPHOCYTES. PROTECTIVE EFFECT OF SOD Volume (#1) CF preparations CF preparations + SOD Control preparations

150

200

300

500 a

8.1±1.6

9.5_+0,8

10.4_+0.1

10.8_+0.3

5.9_+1.1

6.5_+0.4

6.7_+1.6

7.5_+0,3

5.6_+0.5

6.7±0.2

6.8±0.4

7.1_+0.5

The SCE rate in untreated cultures was 5.7+ 1.2 and corresponded to that observed in other experiments. a Added to 5 ml of serum-supplemented medium.

TABLE 4 SCE F R E Q U E N C I E S IN H U M A N L Y M P H O C Y T E S TREATED W I T H CF OF V79 C U L T U R E S EXPOSED TO TPA. PROTECTIVE EFFECT OF SOD Volume (gl) CF preparations CF preparations+SOD Control preparations

150

200

300 a

8.1+0.8

8.9+0.5

10.1+0.3

6,0+0.8

6.5+0.6

6.7+0.9

5.4-/-0.2

6,4-t-0.4

6.3-+1.1

SCE rate in untreated cultures was 6.0 + 0.4. a Added to 5 ml of serum-supplemented medium.

101 lOq

Z 0 O Ix

it

O Z

m

> Ix I

I

I

I

II

,L

J.

1

10

0

0,1

0~3

0,6

Fig. 1. Cell-survival curve of V79 cells exposed to increasing amounts of TPA-induced CF. - . . . . . untreated control.

arations

from

different

donor

C F(.ML)~ 0,9

lymphocytes

SOD-treated culture; - -

resistant V79 colonies. Optimum conditions for t h e r e c o v e r y o f m u t a n t s w i t h r e s p e c t t o cell d e n sity, m u t a t i o n e x p r e s s i o n t i m e a n d s e l e c t i v e c o n centration were determined in preliminary experiments (data not shown). As mentioned earlier, the quantities to be used for the different CF prepara-

was

variable.

Induction of point mutations Mutations induced by CF at the HGPRT locus were detected by the formation of 6-thioguanine-

TABLE 5 INDUCTION OF 6-THIOGUANINE-RESISTANT MUTANTS OBTAINED FOR EXPONENTIALLY GROWING V79 CELLS AFTER TREATMENT WITH CF CF preparation (FI)

Exp. Exp. Exp. Exp.

Control preparation

Untreated cultures

Volume (/tl)

100

150

300

600

600 + SOD

600

0

1 2 3 4

10 -

19 14 19

26 3 20 53

30 15 38 64

2 6 20

6 7 13

5 3 12 20

17

26

39

9

9

10

Mean

The figures represent the number of colonies/106 survivors.

102 tions were established in preliminary experiments on cell survival. Quantities of 300-600/~1, compatible with a survival of at least 20% of the cells, yielded a mean of 26 and 39 mutants respectively. Similar quantities of control preparations resulted in mutation frequencies corresponding to those of untreated cultures (Table 5). The background level of mutations in the untreated cultures varied in 3 experiments between 3 and 12, which is the standard level observed in our laboratory for V79 cells. In the last experiment, the background was higher, but here too exposure to CF resulted in a significant increase in the mutation rate. SOD prevented the induction of point mutations, if added to the cells before the exposure to CF. Discussion

The results of the present study show that Chinese hamster lung cells release a CF in response to the tumor promoter TPA, as do human leukocytes. CF from V79 cells induced SCE not only in hamster cells but also in human lymphocytes. Vice versa, CF from human lymphocytes induced SCE in hamster cells, indicating that the CF action is not species-specific. Since CF from the supernatants of hamster cells was obtained by the same ultrafiltration procedure as that from human leukocyte cultures, it is probable that the composition of the clastogenic material is similar. SOD inhibited SCE induction in V79 cells by TPA itself and by TPA-induced CF. The cytotoxic effect of CF from human or hamster cells, expressed by reduced cell survival and cloning efficiency of V79 cells, was also prevented by SOD. TPA-induced CF yielded a 3-6-fold increase in the frequency of thioguanine-resistant mutants, and SOD was protective against this mutagenic effect. We may conclude that in this cell system also formation and action of CF were linked to superoxide radical generation. The indirect action mechanism of TPA is probably the cause of the discordant results reported in the literature for SCE as well as for break induction. Variation in media components with free radical-scavenging properties may indeed influence the results (Keck and Emerit, 1979). In a previous study on TPA-

treated leukocytes, we observed significant differences in SCE frequencies according to the culture medium used. Cultures set up with TC 199 yielded a mean of 8.7 SCE/cell, while cells cultivated in Eagle's medium or RPMI 1629 showed only 6.2 and 5.9 SCE/cell respectively for the same dose of TPA (Emerit, 1984). In addition to this 'medium effect', we have elsewhere drawn attention to the influence of variations in SOD or other antioxidants in fetal calf serum due to hemolysis during its preparation (Baret and Emerit, 1983). Oxygen free radicals are known to cause strand breakage and base damage in DNA (Morgan et al., 1976; Brawn and Fridovich, 1980). Generated extracellularly in the culture medium by xanthinexanthine oxidase or by photoreduction of flavins, they induce chromosome breakage and SCE in human leukocytes (Emerit et al., 1982) and Chinese hamster ovary cells (Weitberg et al., 1983; Phillips et al., 1984). The latter authors also observed an increase in thioguanine-resistant mutants, and, since this mutagenic effect was inhibited by catalase, they concluded that hydrogen peroxide was the principal mediator. Weitzman and Stossel (1982), who prevented phagocyte-induced mutations in bacteria by SOD, catalase and hydroxyl radical scavengers, concluded that OH" was responsible for the mutagenesis. Superoxide and secondarily formed OH" radicals cannot be considered components of TPA-induced CF, since they would not be transferable into other culture systems. Hydrogen peroxide would be transferable, but was not found in CF preparations from human leukocytes. However, oxidative damage to cell membranes may result in transferable material with genotoxic properties. Lipid peroxidation is indeed accompanied by the formation of a complex mixture of aldehydic products, among which 4-hydroxynonenal is a major component and probably the most active in inducing damage to biological structures. Cytotoxicity, DNA fragmentation and SCE have been observed in Chinese hamster cells exposed to 4hydroxynonenal (Brambilla et al., 1986). A mutagenic effect has been demonstrated in the Salmonella tester strain TA104 (Marnett et al., 1985) and in V79 Chinese hamster cells (Cajelli et al., 1987). Like other homologous aldehydes with

103 relatively long life-times, at least in comparison with free radicals, 4-hydroxynonenal is considered to be one of the products explaining 'long-distance' effects promoted by lipid peroxidation. It may derive from arachidonic acid of membranes by decomposition of 13- and 15-hydroperoxides (Esterbauer, 1982). We have searched for 4-hydroxynonenal in CF preparations from human leukocyte cultures with a technique established for direct high-performance liquid chromatography analysis of this aldehyde (Lang et al., 1985). A peak with a retention time identical to the authentic 4-hydroxynonenal standard was found in about half of the samples collected after 72 and 48 h, but not in those collected after 24 h of culture. In the negative samples, the concentration was probably lower than 0.1 ~M, which is the detection limit for this method. Synthetic 4-hydroxynonenal added to leukocyte cultures was clastogenic in a dose range between 0.1 and 1 0 / t M (Emerit et al., submitted). Previous studies had already indicated that TPA-induced CF is lipophilic. The clastogenic activity was extractable with ethyl acetate, and the classical tests for lipid peroxidation products (TBA assay, conjugated dienes) were positive in the culture media (Khan and Emerit, 1985). An increased level of free arachidonic acid (AA) was found in leukocyte cultures after labelling of the cells with the radioactive compound. AA metabolites such as prostaglandins, thromboxane A 2, H E T E S and HPETES were not increased compared to controls at 72 h, when the clastogenic activity is at its maximum. The 4-hydroxynonenal present at that sampling time was probably derived from the released AA by autoxidation, a process needing more than 24 h. This is in agreement with the appearance of clastogenic activity in the culture media, which only reaches detectable levels after 18h. Previous studies had concluded that aldehydes and carbonyl compounds are direct-acting mutagens (Marnett et al., 1985). In the present study, the increase in thioguanine-resistant mutants by CF was prevented in the presence of SOD, as were the clastogenic and SCE-inducing effects of CF. If 4-hydroxynonenal is responsible for these effects, superoxide radicals seem to be involved in its action mechanism. Preliminary results indicate that

the clastogenic effect of synthetic 4-hydroxynonenal is inhibited by SOD. We may conclude that exposure of cells to TPA results in mutagenesis via a complex mechanism, in which active oxygen species and aldehydic breakdown products of unsaturated fatty acids play a role. The latter seem to act again via the intermediacy of active oxygen species leading to a circle of events with possible importance for tumor promotion.

Acknowledgement We thank the ' G r o u p e m e n t des Entreprises Fran~aises dans la Lutte contre le Cancer' ( G E F L U C ) for providing financial support for this research.

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