Mutation Research, 171 (1986) 139-143 139 Elsevier

MTR01089

Cytotoxic and genotoxic effects of ethanol and acetaldehyde in root-meristem cells of A llium cepa

Felipe Cort6s 1,2, Santiago Mateos 1 and Pablo Escalza 1 1 Departamento de Citolog}a e Histologia Vegetaly Animal, Facultad de Biologla de Sevilla, 41012 Sevilla (Spain) and

: Department of Genetics, University of Uppsala, Box 7003, S-750 07 Uppsala (Sweden)

(Received 3 January 1986) (Revision received 4 April 1986)

(Accepted 17 April 1986)

Summary

The current view is that ethanol itself is not a clastogenic agent or an inducer of sister-chromatid exchanges (SCE) but is mutagenic only when ~transformed metabolically into acetaldehyde. We have analyzed the cytotoxic effects of ethanol and acetaldehyde, as well as their effectiveness in the induction of chromosomal aberrations and SCEs in root-tip cells of Allium cepa. The results obtained show that the chemicals differ in their action. For all the parameters analyzed, acetaldehyde treatments resulted in a stronger effect on meristematic cells. These results seem to support the above-mentioned hypothesis that acetaldehyde is the actual mutagenic agent and, on this basis, it can be stated that the mode of metabolism of ethanol in a given system is very important as regards its effectiveness in inducing chromosome damage.

Ethanol has been reported to be harmful to man, owing to its mutagenlc, carcinogenic and teratogenic effects (for review, see Obe and Ristow, 1979). Alcoholics show significant increases of chromosomal aberrations (Obe and Herha, 1975) and sister-chromatid exchanges (SCE) (Butler et al., 1981), two cytogenetic endpoints that are con- sidered to be an expression of genetic damage induced by physical or chemical agents. In spite of this, the results of the extensive studies performed with mammalian cells both in vitro and in vivo have been contradictory (Obe and Ristow, 1979; Obe et al., 1979; Tates et al., 1980; Korte et al., 1981; Takehisa et al., 1982; de Raat et al., 1983), and so, a direct relationship between alcohol con- sumption and genetic damage has not yet been established.

Since the first metabolite of ethanol, acetalde- hyde, induces a significant increase of SCEs in

CHO cells (Obe and Ristow, 1977) and in human lymphocytes (Ristow and Obe, 1978), it was con- cluded by Obe and Ristow (1979) that ethanol itself is not a clastogenic and SCE-inducing agent but is mutagenic only when transformed into acetaldehyde. On this basis, it can be stated that the mode of metabolism of ethanol in a given system is very important as concerns the effective- ness of this chemical in inducing chromosome damage.

In order to test this, Takehisa et al. (1982) added extracts from Vicia faba roots previously exposed to ethanol to cultured CHO cells and, as a result, SCEs were induced. So did Vicia faba root S10 and rat-liver $9 activating systems (Takehisa and Kanaya, 1983), and similar results were obtained by de Raat et al. (1983) after metabolic activation of CHO cells by rat-liver homogenate.

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Plant chromosomes have also been shown to be sensitive to ethanol and acetaldehyde. In Vicia faba, ethanol treatment induces aberrations of the chromatid type (Michaelis et al., 1959; Rieger and Michaelis, 1960a) as well as SCEs (Schubert et al., 1979). On the other hand, Rieger and Michaelis (1960b) reported that acetaldehyde also induces chromatid aberrations in this system.

In Allium cepa root tips from germinating seeds, Sax and Sax (1966) found a chromosome-breaking activity of ethanol. Nevertheless, in ethanol-treated roots growing on bulbs, Barthelmess (1957) did not observe chromosomal aberrations, although multipolar ana- and telo-phases had been induced. This has been interpreted as being due to meta- bolic differences between A. cepa roots from bulbs and from seedlings (Obe and Ristow, 1979).

The first aim of the work reported here was to re-investigate the question whether ethanol is clastogenic in roots grown from bulbs. A second aim was to compare the effectiveness of ethanol and acetaldehyde in inducing chromosomal aber- rations and SCEs in our system.

Materials and methods

The material consisted of root meristems of A. cepa. The onion bulbs, 15-30 g in weight, were incubated in the dark on cylindrical glass recepta- cles with about 70 ml of tap water at 25 + 0.5°C. Air was bubbled through continuously at a rate of 10-20 ml per rain and the tap water was renewed every 24 h. All the treatments with ethanol and acetaldehyde began when the roots were 15-20 mm in length. The experiments were done at least twice, using 2 bulbs for every dose assayed.

According to Rieger and Michaelis (1960a) ethanol results in the highest frequencies of chro- matid structural changes in Vicia faba roots when the treatments are performed for 24 h at 30°C in deionized water. We chose this treatment time and the same temperature for both ethanol and acetaldehyde and, in addition, treatments for 6 h at 30°C followed by recovery periods in tap water for 24 h at 25°C were carried out. Immersion of roots in the treatment solutions was performed without aeration, while for the recovery periods the roots were aerated.

After treatment, root tips were fixed in a mix-

ture of ethanol : acetic acid (3 : 1) at 5°C overnight for the analysis of mitotic index and chromosome damage detectable as abnormal ana-telophases (ana-telophases containing fragments and /or bridges) and cells showing micronuclei. For the calculation of mitotic index, at least 1000 cells were scored in each case, while 500 cells were analyzed for chromosome damage. When the aim was to study any disturbance of mitosis induced by the chemicals tested, some roots were fixed after 2 h in the treatment solutions.

5-Bromodeoxyuridine (BrdU) substitution of DNA was carried out for two cell cycles by main- taining the roots in 10 -4 M BrdU for a total time of 40 h (renewed after first 20 h) and the treat- ments with ethanol (for the two complete cell cycles) and acetaldehyde (for 2 h just before the second S period) were performed in order to analyze their effect on the frequency of SCE. During the BrdU experiments the roots were protected from light. Finally, the roots, still at- tached to the bulbs, were treated with 0.05 % colchicine for 3 h prior to fixation.

After fixation, roots were squashed in acetic orcein or processed by the FPG technique (Schvartzman and Cortts, 1977) modified by Gonzhlez-Gil and Navarrete (1982).

Results and discussion

Effects on mitosis As shown in Table 1, different concentrations

of both ethanol and acetaldehyde were tested for continuous 24 h treatments as well as for 6-h treatments followed by 24-h recovery periods. For both experimental schedules, it is evident that acetaldehyde has a stronger cytotoxic effect than ethanol. A dose of 1.7 × 10 -2 M acetaldehyde resulted in a complete inhibition of mitosis both for continuous and pulse treatments, while for ethanol a concentration 10 times higher had only a slight inhibitory effect when the treatment was followed by a recovery period.

On the other hand, we have not observed any spindle disturbances at the concentrations of ethanol tested, while for 1.7 × 10 -2 M acetalde- hyde a high percentage of C-mitoses were observed after 2 h of treatment (data not shown). In addition, acetaldehyde at the highest concentra-

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T A B L E 1

C Y T O T O X I C A N D C L A S T O G E N I C E F F E C T S O F E T H A N O L A N D A C E T A L D E H Y D E

A g e n t D o s e (M) T r e a t m e n t R e c o v e r y Mi to t i c A b e r r a t i o n s

t ime t ime i n d e x a A n o m a l o u s

a n a - t e l o p h a s e s b

M i c r o n u c l e i c

N o n e - - - 11.9 0.4

E t h a n o l 1.7 x 1 0 - 4 24 - 7.38 0.8 1.7 x 10 - 3 24 - 8.52 1.0

1.7 x 10 - 2 24 - 5.98 0.8

1.7 x 1 0 - 1 24 - 3.42 1.4

1.7 x 10 - 4 6 24 10.16 0.4

1.7 x 1 0 - 3 6 24 13.22 0.8

1.7 × 10 - 2 6 24 12.17 0.6

1.7 x 1 0 - 1 6 24 8.87 1.2

A c e t a l d e h y d e 1.7 x 1 0 - 4 24 - 5.41 3.2 1.7 x 1 0 - 3 24 - 5.49 3.6

1.7 x 1 0 - 2 24 - 0.2 -

1 . 7 × 1 0 - 4 6 24 13.17 0.6

1.7 x 1 0 - 3 6 24 11.74 6.4

1.7 × 1 0 - 2 6 24 0.4 -

2

1 3

2

2

5

2

4

5

3

4

2

17

a A t leas t 1000 cells were a n a l y z e d in e a c h case.

b % 500 cells were scored .

c M i c r o n u c l e i in 1000 cells.

tion assayed also exerted an inhibitory effect on cytokinesis and, as a result, binucleate cells were induced.

Barthelmess (1957) reported that ethanol, in the range of 0.15-1 M, induces multipolar ana- and telo-phases in A. cepa root-tip cells. As stated above, we have not observed any effect of ethanol on the spindle at concentrations up to 1.7 x 10-1 M, and so, the threshold for these disturbances seems to be between the highest concentration used by us and the lowest used by Barthelmess (1957). As acetaldehyde is able to induce C-mito- sis and binucleate cells at a concentration of 1.7 × 10 -2 M, it can be concluded that, as seen previ- ously for cytotoxicity, this agent is more efficient than ethanol in modifying the normal develop- ment of mitosis.

Chromosomal aberrations The data for chromosomal aberrations are pre-

sented in Table 1. As can be seen, ethanol failed to induce either anomalous ana-telophases or mi- cronuclei under our experimental conditions.

While Sax and Sax (1966) observed aberrant anaphases in root tips growing from A. cepa seeds after a long exposure (2-5 days) to ethanol, no chromosomal aberrations had been observed in root tips growing on bulbs (Barthelmess, 1957; the present paper). The current view is that not al- cohols, but only the corresponding aldehydes are actually mutagenic (Obe and Ristow, 1979; Obe and Beek, 1979; G6mez-Arroyo and Souza, 1985).

On this basis it could be proposed that dif- ferences in metabolism of ethanol between A. cepa roots grown from seeds or bulbs could exist. Nevertheless, since the chromosome-breaking ef- fect of ethanol reported by Sax and Sax (1966) was observed after very long treatment times, this could also be interpreted as an accumulation of sufficient acetaldehyde in the cells to exert its clastogenic action, and not due to a more efficient metabolism. A chromosome-breaking effect of ethanol has been reported in Vicia faba by Rieger and his coworkers (Rieger and Michaelis, 1960a, 1970; Rieger et al., 1975). Since our experimental design has been similar to that proposed by these

142

authors as efficient in inducing high frequencies of chromatid structural changes in Vicia faba roots, the lack of an effect of ethanol in A. cepa seems to indicate metabolic differences between both plant species or, alternatively, differences in their sensitivity to chromosome breakage by this chem- ical or its metabolite. In this connection, it must be pointed out that acetaldehyde has shown only a moderate clastogenic action in our material at the concentrations that did not drastically inhibit cell proliferation (Table 1).

Sister-chromatid exchanges In a first study we analyzed the effect of ethanol

on the frequency of SCEs. Different concentra- tions of ethanol were administered to roots simul- taneously with BrdU for two consecutive cell cycles. As can be seen in Fig. 1, ethanol treat- ments induced SCEs in a dose-dependent manner (p < 0.001, Student's t test). Nevertheless, the highest value obtained was only approximately 2-fold that found in non-treated controls.

~ 6 0

0

0

4

cn

• r 2

Acetaldeh~

i I I

0.017 0.17 1.7 17 Dose (X 10 -2 14)

Fig. 1. The effect of various concentrations of ethanol and acetaldehyde on the frequency of SCE. Ethanol was present for the two cell cycles, simultaneously with BrdU, while acetalde- hyde was given for 2 h between the two cell cycles in BrdU. Range bars represent 95% confidence limits of the mean.

As acetaldehyde had proved to be more cyto- toxic than ethanol (Table 1) we chose a different schedule to study its effectiveness as an inducer of SCE. The treatments with acetaldehyde were car- ded out for just 2 h after a first cell cycle in the presence of BrdU and before the second S period in this nuc leos ide . .Under these conditions, acetaldehyde also induced SCEs, its effectiveness depending on the dose employed (Fig. 1). While a continuous treatment with 1.7 x 10 -1 M ethanol had resulted in only a doubling of the baseline SCEs frequency, a dose of acetaldehyde 10 times lower was able nearly to triplicate t h e baseline SCEs, in spite of the much shorter treatment carried out.These results have shown that ex- posure to ethanol results in SCEs at concentra- tions unable to induce chromosomal aberrations in A. cepa. On the other hand, acetaldehyde proved to be more effective than ethanol as an inducer of SCEs, just as it was more effective in inducing cytotoxic and clastogenic effects. The clastogenic effects and the ability of acetaldehyde to induce SCEs have been related to its capacity to induce cross-links in DNA (Ristow and Obe, 1978; Lam- bert et al., 1985)

Acknowledgement

This work was aided by a Grant from the Monte de Piedad y Caja de Ahorros de Sevilla, Spain.

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