Mutagenesis, Vol. 15, No. 3, 271-276,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Inducible protective processes in animal systems VI. Cross-adaptation and the influence of caffeine on the adaptive response in bone marrow cells of mouse
1 Department of Zoology, University of Mysore, Manasagangotri, Mysore-570 006 and 2 Department of Life Science, Kuvempu University, Shankarghatta-577 451, Shimoga, India
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Abstract |
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The effect of caffeine (CAF) (a replicative DNA synthesis inhibitor) given as pre-, inter- and post-treatments on the ethyl methanesulfonate (EMS)-induced adaptive response in in vivo mouse bone marrow cells was studied in order to understand the influence of CAF on the adaptive response. The pre-treatment was given 4 h before a combined treatment with EMS (conditioning + challenge) and in another set CAF was given as a conditioning dose and 4 h later the cells were challenged with a high dose of EMS. In the inter-treatment, CAF (40 mg/kg body wt) was administered 2 or 4 h after the conditioning dose of EMS and 6 or 4 h later the cells were challenged with a high dose of EMS. Similarly, in the post-treatment experiments, CAF was injected 6, 12 or 18 h after a combined treatment with EMS. The results revealed that the pre-, inter- and post-treatments with CAF significantly reduced the frequency of chromosomal aberrations compared with the challenge and combined treatments with EMS. It is interesting to note that CAF pre-treatment resulted in a much greater reduction in chromosomal aberrations compared with the inter- and post-treatments. Thus, this is an example of cross-adaptation induced by CAF in EMS-treated in vivo mouse bone marrow cells and the results also demonstrate an influence of CAF on the adaptive response.
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Introduction |
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The presence of an adaptive response in in vitro mammalian systems has been amply proved (Kaina, 1982
; Samson and Schwartz, 1980; Sankaranarayanan et al., 1989; Olivieri and Bosi, 1990). Riaz Mahmood and Vasudev (1990, 1991, 1992, 1993) for the first time showed the occurrence of an adaptive response in in vivo sub-mammalian and mammalian cells induced by alkylating agents such as ethyl methanesulfonate (EMS) and methyl methanesulfonate. Although the occurrence of an adaptive response to low levels of various physical and chemical agents has been well documented, the underlying intracellular molecular mechanism is not yet clearly understood. However, bacterial systems have provided a considerable amount of information on the possible mechanisms of the adaptive response induced by alkylating agents (for a review see Lindahl et al., 1988).
In order to gain an insight into induction of inducible repair in eukaryotic systems, a DNA repair synthesis inhibitor, caffeine (CAF), was employed along with a standard mutagen, EMS. CAF is a known potentiator of DNA damage initiated by physical and chemical agents in various test systems (for reviews see Kihlman, 1977; Kihlman and Anderson, 1987; Harvey and Savage, 1994). In human lymphocytes CAF treatment during G2 phase enhances the frequency of X-ray and chemically induced aberrations (Natarajan et al., 1980; Kihlman et al., 1982; Hansson et al., 1984). The potentiating effect of CAF has been related to its known ability to cancel the G2 arrest induced by X-rays and chemical mutagens (Painter and Young, 1980; Lau and Pardee, 1982; Rowley et al., 1984) and to inhibit the repair pathway (Gonzalez-Fernandez et al., 1985; Lopez-Saez et al., 1988).
In the present investigations, CAF has been used as a modulator to determine its role in the repair process with the presumption that as a known inhibitor of repair it may also interrupt the adaptive response initiated by a conditioning treatment with EMS. Hence, various treatment schedules of CAF along with EMS were designed. Surprisingly, in all the treatment schedules CAF did not act as an inhibitor of repair, instead it enhanced the adaptive response initiated by a low dose of EMS. The results of these experiments are presented in this communication.
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Materials and methods |
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Animals
Inbred male Swiss albino mice, 6–8 weeks old and weighing 25–30 g, were used in all the experiments. To detect a clastogenic effect in mitotic cells, a bone marrow cytogenetic assay was employed.
Test chemicals
The monofunctional alkylating agent EMS (CAS 62-50-0) and CAF (CO750) were obtained from Sigma (St Louis, MO). The former served as the standard mutagen.
EMS administration
Two doses of EMS, 80 (conditioning, L) and 240 mg/ kg body wt (challenge, H), were selected from earlier experiments (Riaz Mahmood and Vasudev, 1993). The required concentration of EMS was prepared by dissolving the chemical in 0.7% NaCl solution. An aliquot of 0.5 ml of each concentration was given i.p. to animals.
CAF administration
A series of pilot toxicity experiments were conducted using various concentrations of caffeine ranging from 25 to 100 mg/kg body wt. A single sub-lethal dose of 40 mg/kg body wt was selected for the present experiments. The test chemical was prepared by dissolving CAF in distilled water and 0.5 ml of this was administrated i.p.
Treatment schedules
EMS combined treatment.
This schedule was again selected from the earlier observations of Riaz Mahmood and Vasudev (1993), who showed that combined treatment with conditioning and challenge doses of EMS with an 8 h time lag between them offered maximum protection against chromosome damage in bone marrow cells of the mouse, thus exhibiting a peak of repair activity compared to other time lags tested. Hence, for the present investigations an 8 h time lag was selected (L-8hTL-H).
CAF pre-treatment. Two pre-treatment schedules were followed. In one experiment, CAF was given 4 h before the combined treatment with EMS. In the other, CAF was given 4 h before the challenge dose alone, skipping EMS conditioning (CAF-4h-L-8h-H; CAF-4h-H).
CAF inter-treatment. Treatment with caffeine was given during the 8 h time lag between the conditioning and challenge doses of EMS. Two time schedules of 2 and 4 h after conditioning with EMS were used and 6 and 4 h later the challenge dose of EMS was given. Thus, the total duration of 8 h between the two EMS treatments was maintained. (L-2h-CAF-6h-H; L-4h-CAF-4h-H).
CAF post-treatment. Post-treatment of CAF was done 6, 12 and 18 h after the EMS combined treatment (L-8h-H-6h-CAF; L-8h-H-12h-CAF; L-8h-H-18h-CAF). In another experiment, EMS conditioning was avoided and CAF was given 6 h after the EMS challenge dose (H-6h-CAF).
Test procedure and evaluation
Three different recovery times of 24, 48 and 72 h were employed for all the treatment procedures. Animals were killed by cervical dislocation. An aliquot of 0.5 ml of 0.05% colchicine was injected i.p. into the animals 90 min before death. The routine air drying technique of Evans et al. (1964) was followed for the preparation of slides. Air-dried slides were stained in 1:6 Giemsa:distilled water solution. Coded slides were screened for the presence of chromosomal aberrations, i.e. breaks, exchanges, rings, deletions, triradials and minutes. For each experiment three samples were taken and for each sample 500 well-spread metaphases were scored. The data obtained were subjected to Student's t-test to determine the level of significance.
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Results |
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Chromosomal aberration frequencies observed after various treatment schedules with EMS and CAF at different recovery times, namely 24, 48 and 72 h, are shown in Tables I–III
. EMS produced mainly chromatid breaks, exchanges and deletions. The conditioning and challenge doses of EMS induced the lowest and the highest number of aberrations at all recovery times. At the 24 h recovery time, the challenge dose of EMS alone produced a frequency of 0.69 ± 0.012 aberrations, which is the greatest damage observed after any treatment. The combination of conditioning + challenge doses with an 8 h time lag induced 0.36 ± 0.012 aberrations, which is significantly less (P < 0.05) than the challenge dose. CAF, the second control, induced a very low frequency of aberrations, ranging from 0.02 ± 0.003 (24 h recovery time) to 0.03 ± 0.003 (72 h recovery time), which is almost the same frequency as the controls.
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Bearing in mind the inhibition of repair effect of CAF, this chemical was given during the peak period of the adaptive response, i.e. in the 8 h time lag between the combined treatments. CAF inter-treatment 2 h after EMS conditioning produced 0.34 ± 0.008 aberrations after 24 h recovery, which is significantly less than the frequency (P < 0.05) obtained after the challenge dose of EMS alone, even though it is insignificant compared with the combined treatment. Similar results were obtained for other recovery times tested. In the next experiment, when the duration of CAF inter-treatment was increased from 2 to 4 h after EMS conditioning, interestingly the aberration frequency was greatly reduced compared with the frequency after combined treatment with EMS (Tables I–III
). Further, the effect of CAF was so prominent that the frequency was reduced to nearly 50% of the yield observed after combined treatment with EMS without CAF (Figure 1). Hence, it can be said that CAF potentiations the adaptive response.
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In the pre-treatment experiments, CAF treatment 4 h prior to the EMS conditioning dose followed by the challenge dose of EMS yielded a low frequency of aberrations at all recovery times, compared with combined treatment (P < 0.05) (Tables I–III
). Similar results were observed in another pre-treatment experiment, where only CAF was used for conditioning and then 4 h later the cells were challenged with high dose EMS (CAF-4h-H; Tables I–III and Figure 2). The results for post-treatment with CAF, which was done after three different times, namely 6, 12 and 18 h after the combined treatment with EMS, are given in Tables I–III. It was observed that post-treatment after 12 and 18 h gave a greater reduction in chromosomal aberrations compared with 6 h. When CAF was administered 6 h after the challenge dose of EMS (H-6hTL-CAF), a reduction in the chromosome damage was again observed at all recovery times. This indicates that CAF can significantly inhibit the clastogenic effect exerted by the high dose of EMS. Thus, in all the post-treatment schedules, with or without EMS conditioning, CAF exhibited potentiation of the adaptive response (Figure 3).
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In order to determine whether the observed protection by pre-, inter- and post-treatments with CAF is due to a depression in mitotic index, slides were scanned to obtain mitotic indices (Table IV
). There was no evidence of any mitotic block induced by EMS + CAF.
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Discussion |
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The present studies indicate that i.p. injection of low and high doses of EMS into mice potently induced chromosomal aberrations in their bone marrow cells. EMS-induced chromosomal aberrations consisted mainly of chromatid-type aberrations. These results show that EMS has potent activity as an inducer of chromosomal aberrations, consistent with the well-known fact that EMS is a strong mutagen and clastogen (cf. Vogel and Natarajan, 1982). The potency of EMS in inducing chromosomal aberrations is similar to that reported earlier in the bone marrow of mice (Riaz Mahmood and Vasudev, 1993
). Furthermore, it is also clear from the data that the low dose of EMS significantly reduced induction of chromosomal aberrations induced by a high dose of the same agent (Tables I–III), which is also similar to previous reports.
The main aim of the present work was to investigate the possible role of the DNA repair inhibitor CAF in modulating the in vivo adaptive response in mice. Initially, we presumed that this chemical when administered between combined doses of EMS would significantly increase the aberration yield, possibly due to an interruption in the ongoing repair activity initiated by EMS conditioning. The results show that CAF inter-treatment significantly reduced the frequency of EMS-induced chromosomal aberrations compared with combined EMS treatment. It is interesting to see that administration of CAF 4 h after EMS conditioning considerably suppressed EMS-induced chromosomal aberrations compared with 2 h after EMS conditioning or the combined EMS treatment (Figure 1). Thus these results demonstrate potentiation of the adaptive response (Tables I–III). In an attempt to get a clear picture of this modulation, pre- and post-treatments with CAF were given to EMS-treated cells. In the pre-treatments, CAF again exerted an influence on chromosomal aberrations (Tables I–III and Figure 2). The marked decrease in aberration frequency when CAF was given as a conditioning dose suggests that CAF acts against the chromosome damaging effects of a high dose of EMS in a similar way to that of a conditioning dose of EMS. This is a clear case of cross-adaptation, similar to that observed by Vijayalaxmi and Burkart (1989) in human lymphocytes and by Rieger et al. (1985) in a plant system. In post-treatment with CAF, 6 h after the challenge dose of EMS the aberration frequency was similar to that after the combined treatment. However, after 12 and 18 h the reduction was significant compared with the combined treatment (Tables I–III and Figure 3). It can be said that although CAF did not show a very prominent potentiation of the adaptive response on the 6 h time schedule and thus did not interfere with repair activity already induced by the combined EMS treatment, on the 12 and 18 h time schedules CAF potentiated the adaptive response. The mitotic index in CAF inter- and pre-treated cells was significantly increased compared with treatment with EMS (Table IV). These values were almost equal to the controls, thereby implying that the cell cycle had not been blocked by CAF + EMS treatment. The overall data obtained after various treatment schedules reveal that CAF is a cross-adaptor (pre-treatment) and also significantly potentiates the adaptive response in mouse bone marrow cells and the order of potentiation is inter-treatment > post-treatment (Table V). This is the first time that potentiation by CAF of the EMS-induced adaptive response in an intact animal system and an action as a cross-adaptor have been demonstrated. There are, of course, reports in the literature on the protective action of CAF against chemically or physically induced somatic and genetic effects. Boage et al. (1995) reported that CAF post-treatment significantly decreased the mutation frequency in EMS-treated soybeans. Protective effects of CAF against chemically induced carcinogenesis and clastogenicity have been shown by Rothwell (1974) and Ito et al. (1989), respectively. Similarly, coffee has exhibited antimutagenic effects against various chemicals such as mitomycin C, cyclophosphamide, procarbazine and adriamycin in vivo in mice (Abraham, 1989). Furthermore, Abraham (1995) has shown that administration of coffee with dietary constituents in vivo in mice resulted in a significant enhancement of the antimutagenic effects against cyclophosphamide, MNNG, ethylnitrosourea, mitomycin C and urethane. In Drosophila, it is evident that co-administration of coffee with cyclophosphamide, diethylnitrosamine, mitomycin C and urethane can lead to a protective effect against the induction of wing spots, which are formed as a result of somatic mutation or mitotic recombination (Abraham, 1994; Abraham and Graf, 1996). Farooqi and Kesavan (1992) and Devasagayam et al. (1995) have reported a protective function of CAF against radiation-induced chromosomal aberrations in vivo in mouse bone marrow cells. All these studies highlight a novel role for CAF, offering protection against damage induced by various mutagenic agents. However, the molecular mechanism of such protection is still not clearly understood. Nonetheless, in some recent reports (Kesavan, 1992; Rao et al., 1995; Devasagayam et al., 1996) the protective effect has been attributed to the free radical scavenging ability and antioxidant activity of caffeine against radiation-induced chromosomal damage. It is well documented that EMS, being an alkylating agent, can alkylate DNA and produce various kinds of adducts, but it does not produce free radicals. Therefore, it may not be feasible to attribute the activity of caffeine against EMS-induced mutations to its free radical scavenging ability. According to Wattenberg and Lam (1984), administration of coffee beans to laboratory animals (mice and rats) can enhance glutathione S-transferase activity and inhibit carcinogen-induced neoplasia. The difficulty in pin-pointing the exact mechanism responsible for the anticlastogenic effects of CAF in EMS-treated mice is due to its multidirectional or multifaceted reactions. Since the present data clearly reveal the role played by CAF as a cross-adaptor and potentiator of the adaptive response in mouse bone marrow cells, much of our interpretation is centered on the plausible mechanisms put forward by Wattenberg and Lam (1984) and Ramel et al. (1986). However further studies are needed to clarify the molecular mechanism of action of CAF.
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Acknowledgments |
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We wish to express our gratitude to the Professor and Chairman, Department of Studies in Zoology, for providing facilities and to the University Grants Commission for awarding funding for the project (contract no. F.3-58/93 SR-II). S.K.H. is grateful to the UGC, New Delhi and K.P.G. and R.M. acknowledge the CSIR, New Delhi for financial assistance.
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Notes |
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3 Present address: Department of Applied Zoology, Kuvempu University, BR Project-577 115, Shimoga, India
* To whom correspondence should be addressed at present address. Tel: +91 08282 37263; Fax: +91 08282 37255
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Received on October 29, 1999; accepted on January 28, 2000.
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