Mutation Research, 127 (1984) 119-122 119 Elsevier

MTR 03875

Chromosomal aberrations and SCEs in Allium cepa root-tip cells treated with caffeine and pyronin Y

Fel ipe Cort6s 1 a n d M. Jos6 H a z e n 2

Departamento de Citologla e Histologla Vegetal y Animal, Facultad de Biologia de Sevilla, Sevilla, and 2 Departamento de Citologla, Facultad de Ciencias, Universidad Autbnoma de Madrid, Madrid (Spain)

(Received 22 December 1983) (Accepted 24 February 1984)

Summary

The effectiveness of caffeine and pyronin Y in the induction of both chromosomal aberrations and sister-chromatid exchanges (SCEs) in root meristematic cells of A. cepa was studied.

The rate of SCEs proved to be increased when 5-bromo-2'-deoxyuridine- (BrdU) substituted chro- mosomes were allowed to replicate in thymidine (dT) for a second S period simultaneously with caffeine or pyronin Y. In contrast, only caffeine was able to induce aberrations in BrdU-substituted chromosomes, while pyronin Y seemed to be ineffective at the doses employed.

In recent years, many studies dealing with the possible relationship between chromosomal aber- rations and sister-chromatid exchanges (SCEs) have been carried Out. Although at first a close correlation was proposed based on the fact that agents which induce SCEs also induce, in many cases, chromosomal aberrations (Latt, 1974; Perry and Evans, 1975), both qualitative and quantita- tive differences in the production of these cyto- genetic effects have been reported in a number of cases (for review see Gebhart, 1981).

The present view is that even though both cyto- genetic endpoints are the result of direct or indi- rect damage to DNA by multiple physical and chemical agents, the mechanisms whereby they arise seem to differ (Wolff, 1982).

The repair of injuries caused to DNA is a normal event that plays an important role in order to guarantee, to some extent, cell survival under harmful conditions. For this reason, in the present study, we have analyzed the effectiveness of caf- feine and pyronin Y, two known inhibitors of DNA repair, in the induction of chromosomal

aberrations and SCEs in root meristematic cells of A. cepa.

Materials and methods

Root meristems of A. cepa were employed in this study. After removing the loose outer scales, the onion bulbs, 15-30 g in weight, were grown in the dark at a constant temperature of 25 ± 0.5 °C. The basis of the bulbs remained submerged in tap water changed daily and aerated by continuous bubbling.

In order to obtain a differential staining of sister chromatids, the cells were first allowed to replicate DNA in 10 -4 M 5-bromo-2'-deoxyuri- dine (BrdU) and 10 -7 M 5-fluorodeoxyuridine (FdU) for 20 h and then placed in 10 -4 M thymi- dine (dT) for an equal period.

The treatments with caffeine and pyronin Y took place simultaneously with dT throughout the second S period. Thereafter, the roots were fixed in a mixture of ethanol: acetic acid (3:1) at 5°C overnight for the analysis of chromosome damage

0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.

120

expressed as the percentage of abnormal ana- telophases (ana-telophases containing fragments and/or bridges) and percentage of cells with mi- cronuclei. When the effect on the frequency of SCEs was studied, roots 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 for differential staining of sister chromatids according to Schvartzman and Cort6s (1977).

Results

As shown in Table 1, different concentrations of caffeine and pyronin Y were employed after the cells had incorporated BrdU into DNA throughout the first S period after the addition of the ana- logue.

Chromosomal aberrations expressed as per- centage of abnormal ana-telophases and per- centage of cells showing micronuclei were evaluated in each case. Likewise, the frequency of SCEs detected after the different treatments was quantified. As can be seen, both caffeine and pyronin Y significantly increased (P < 0.01) the SCE frequency over the control baseline level. Nevertheless, since the values of SCEs reached

were less than twice the control frequency, these substances cannot be classified as strong inducers of SCEs.

It should be mentioned that for the higher concentration employed, both substances showed a mitodepressive effect that could perhaps account for an impediment for a number of damaged cells to reach mitosis.

In contrast, the results show that caffeine and pyronin Y differ regarding their ability to produce chromosomal aberrations. While caffeine did in- duce aberrations in BrdU-substituted chro- mosomes in a dose-related fashion, pyronin Y did not increase the values of chromosomal aberra- tions above that observed in unsubstituted and BrdU-substituted controls.

Discussion

We have analyzed the ability of caffeine and pyronin Y to induce both chromosomal aberra- tions and SCEs in BrdU-substituted chromosomes. Considering the fact that the concentrations of caffeine used were notably higher than those of pyronin Y, one could conclude that the latter substance is more efficient as an inducer of SCEs. However, since the effect observed is less than

TABLE 1

SCEs AND CHROMOSOME DAMAGE IN CONTROLS AND AFTER VARIOUS TYPES OF TREATMENT WITH CAF- FEINE AND PYRONIN Y

Treatment Dose SCEs/chromosome ~.b Chromosome damage (%) c

Abnormal ana-telophases

Micronuclei

Control (unsubstituted)

Control (BrdU-substituted)

Caffeine

Pyronin Y

- 0.4 0.8

2.8+0.17 0.6 0.8

2.5 mM 3.7 + 0.19 5.0 1.0 5 mM 5.3 + 0.25 11.4 5.0 7.5 mM 4.7+0.24 15.9 7.0

10 -6 M 3.9+0.22 0.4 0.6 5.10 -6 M 4.2+0.21 0.4 0.8 10- 5 M 4.5 + 0.24 0.5 0.7

a Mean + S.E. b 300 chromosomes were studied in each case. c 500 cells were scored.

twice the control frequency and since higher con- centrations of pyronin Y effectively inhibit cell division, we have been unable to obtain a higher frequency of SCEs with pyronin Y than with caffeine.

Kihlman (1975) found that caffeine alone markedly increased the frequency of chromosomal aberrations in BrdU-substituted chromosomes in Vicia faba, but produced only a slight, statistically non-significant, enhancement of SCEs. In con- trast, other authors have reported that caffeine per se is capable of inducing SCEs in a dose-depen- dent manner in Chinese hamster (Basler et al., 1979), rats (Granberg-Ohman et al., 1980) and in man (Ishii and Bender, 1978; Guglielmi et al., 1982). In agreement with these latter authors, we have observed a moderate but statistically signifi- cant increase in the baseline SCE frequency at the doses of caffeine employed. In our opinion, the lack of a significant effect on the frequency of SCEs reported by Kihlman (1975) with a similar experimental design could be due to the fact that the lowest concentration used by us was 5 times higher than that used by Kihlman or, alternatively, that the systems differ regarding their sensitivity to caffeine.

The present view is that chromosomal aberra- tions and SCEs are caused by different lesions in DNA (Wolff, 1982). While aberrations are likely to be the consequence of double-strand breaks, SCEs could arise from unremoved modified bases present in DNA during replication.

Our knowledge of DNA repair mechanisms in higher plants is rather poor in comparison with what we know about DNA repair in bacteria, mammalian cells etc. (for review see Veleminsk~, and Gichner, 1978). Nevertheless, in recent years it has become evident that higher plants have DNA repair mechanisms similar to that found in other organisms. Although there is no direct evidence, inhibition of post-replication repair by caffeine in plants, in a manner analogous to that encountered in mammalian cells, is suggested by studies on its interaction with radiation or alkylating agents (Kihlman, 1977).

Our results indicate that BrdU substitution per se does not result in an increase in number of chromosomal aberrations over that observed in unsubstituted controls. It is possible, therefore,

121

that in the absence of caffeine, any lesion arising in BrdU-substituted DNA is efficiently repaired before the cells enter mitosis. In contrast, pyronin Y does not seem to interfere with such a mecha- nism of repair in our system. This latter compound has been reported to inhibit strongly excision re- pair in E. coil (Grigg, 1972), and share with caf- feine the ability to bind specifically to single- stranded DNA (Grigg et al., 1971).

The most likely interpretation for the induction of SCEs by caffeine and pyronin Y is that these substances interfere directly or indirectly with DNA synthesis. Such an effect has been reported for caffeine (Cleaver, 1969; Meneghini, 1974; O'Neil, 1979), although the mechanism remains obscure. Since the treatments performed by us took place in BrdU-free medium, the enhancement of SCEs by caffeine cannot be explained as a consequence of any caffeine-induced increase in the incorporation of BrdU into DNA, as proposed by Guglielmi et al. (1982).

References

Basler, A., U. Bachmann, O. Roszinsky-K~cher and (3. R~hrborn (1979) Effects of caffeine on sister chromatid exchanges (SCE) in vivo, Mutation Res., 59, 209-214.

Cleaver, J.E. (1969) Repair replication of mammalian cell DNA: Effects of compounds that inhibit DNA synthesis or dark repair, Radiat. Res., 37, 334-348.

Oebhart, E. (1981) Sister chromatid exchange (SCE) and struc- tural chromosome aberration in mutagenicity testing Hum. Genet., 58, 235-254.

Granberg-Ohman, I., S. Johanson and A. Hjerpe (1980) Sister chromatid exchanges and chromosomal aberrations in rats treated with phenacetin, phenazone and caffeine, Mutation Res., 79, 13-18.

Grigg, G.W. (1972) Effects of coumarin, pyronin Y, 6,9- dimethyl 2-methylthiopurine and caffeine on excision repair and recombination repair in Escherichia coli, J. Gen. Micro- biol., 70, 221-230.

Grigg, G.W., M. Edwards and D. Brown (1971) Amplification of phleomycin induced death and DNA breakdown by coumarin, thiopurines and pyronin Y, J. Bacteriol., 107, 599-609.

Guglielmi, G.E., T.F. Vogt and R.R. Tice (1982) Induction of sister chromatid exchanges and inhibition of cellular pro- liferation in vitro, I. Caffeine, Environ. Mutagen., 4, 191-200.

Ishii, Y., and M.A. Bender (1978) Caffeine inhibition of pre- replication repair of mitomycin C-induced DNA damage in human peripheral lymphocytes, Mutation Res., 51,419-425.

Kihlman, B.A. (1975) Sister chromatid exchanges in Vicia faba,

122

II. Effects of thiotepa, caffeine and 8-ethoxycaffeine on the frequency of SCEs, Chromosoma, 51, 11-18.

Kihlman, B.A. (1977) Caffeine and Chromosomes, Elsevier, Amsterdam.

Latt, S.A. (1974) Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166.

Meneghini, R. (1974) Repair replication of Opossum lympho- cyte DNA: Effects of compounds that bind to DNA, Chem.-Biol. Interact., 8, 113-126.

O'Neill, F.J, (1979) Differential effects of cytochalasin B and

caffeine on control of DNA synthesis in normal and trans- formed cells, J. Cell Physiol., 101, 201-218.

Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid ex- change, Nature (London), 258, 121-124.

Schvartzman, J.B., and F. Cort6s (1977) Sister chromatid ex- changes in Allium cepa, Chromosoma, 62, 119-131.

Veleminsk~,, J., and T. Gichner (1978) DNA repair in mutagen-injured higher plants, Mutation Res., 55, 71-84.

Wolff, S. (1982) Chromosome aberrations, sister chromatid exchanges and the lesions that produce them, in: S. Wolff (Ed.), Sister Chromatid Exchange, Wiley, New York, pp. 41-57.