Effect of caffeine on radiation induced micronuclei in human lymphocytes

Shweta Shukla; KB Anjaria; NN Bhat; Kapil Shirsath; B Sreedevi

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Year : 2010 | Volume : 33 | Issue : 4 | Page : 195-198

Effect of caffeine on radiation induced micronuclei in human lymphocytes

Shweta Shukla¹, KB Anjaria², NN Bhat², Kapil Shirsath², B Sreedevi²
¹ Department of Biophysics, Government Institute of Science, Aurangabad, India
² Radiological Physics and Advisory Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Date of Web Publication

1-Dec-2011

Correspondence Address:
K B Anjaria
Radiological Physics and Advisory Division, Bhabha Atomic Research Centre, Trombay, Mumbai
India

Source of Support: None, Conflict of Interest: None

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Abstract

Present study reports the effect of caffeine on ⁶⁰ Co gamma radiation induced micronuclei in human peripheral blood lymphocytes. Lymphocytes were pre-treated with 0.1-10μM caffeine for 1h at 37°C and exposed to 2 and 4 Gy of radiation before culturing for 72h. In another set of experiments, lymphocytes were exposed to 2 and 4 Gy of radiation and subsequently cultured in the presence of caffeine. Caffeine pre-treatment significantly reduced radiation induced micronuclei (MN) in a concentration dependent manner. In contrast, post-irradiation treatment with caffeine significantly potentiated radiation induced MN in a concentration dependent manner.

Keywords: Caffeine, radiation, lymphocytes, micronuclei

How to cite this article:
Shukla S, Anjaria K B, Bhat N N, Shirsath K, Sreedevi B. Effect of caffeine on radiation induced micronuclei in human lymphocytes. Radiat Prot Environ 2010;33:195-8

How to cite this URL:
Shukla S, Anjaria K B, Bhat N N, Shirsath K, Sreedevi B. Effect of caffeine on radiation induced micronuclei in human lymphocytes. Radiat Prot Environ [serial online] 2010 [cited 2023 Jun 2];33:195-8. Available from: https://www.rpe.org.in/text.asp?2010/33/4/195/90466

1. Introduction

Caffeine is an environmental chemical to which people are exposed through food items, beverages and medicines (Timson, 1977). It is also one of the most widespread stimulants in the world (as a constituent of coffee, tea, mate and cola beverages). Caffeine has been shown to modify the carcinogenic activities of UV radiation (Zajdela and Latarjet, 1973) and cigarette smoke condensate (Rothwell, 1974). It has also been shown to modify the mutagenic activities of UV, ionizing radiation and chemical mutagens in various systems. (Van Zeeland, 1978; Ichikawa-Ryo and Kondo, 1980) Caffeine also enhances the cytotoxicity of non-ionizing radiation in mammalian cells (Cleaver, 1989). Numerous studies have shown that under specific conditions, caffeine is able to increase radiation risk (Kihlman, 1977, Streffer and Müller, 1984). Most of these conditions can be met only by using extraordinarily high caffeine concentrations (1-2 mM corresponding to several hundreds of cups of coffee or tea per day). At low concentrations no effect on radiation risk is observed because either the experimental systems are too insensitive or no such effect occurs. In this paper, we have studied the effect of pre- as well as post-treatment with low concentrations (0.1-10μM) of caffeine on gamma radiation induced MN in cytokinesis blocked human peripheral blood lymphocytes.

2. Materials and Methods

2.1 Chemicals

Caffeine, RPMI 1640 medium, lectin and colcemid were purchased from Sigma Chemical Co., St. Louis, MO, USA. Foetal bovine serum was purchased from ICN Biochemicals.

2.2 Micronucleus assay

Peripheral blood was collected from a 22 year old healthy donor in sterile vacutainer containing heparin as an anticoagulant. The stock solution of caffeine was prepared in distilled water and filter sterilized using a Millipore syringe filter (0.22μm). Caffeine was added to the lymphocyte cultures to achieve a final concentration of 0.1-10μM.

For pre-irradiation treatment, caffeine was added to lymphocyte cultures consisting of 4 ml of RPMI 1640 medium, 0.5 ml serum and 0.5 ml blood in 15 ml sterile plastic disposable tubes and incubated at 37°C in a waterbath for 1 h. Cultures were subsequently exposed to 2 or 4 Gy of ⁶⁰ Co gamma radiation in a Gamma Chamber having a dose rate of 1 Gy/min. After irradiation, caffeine was washed off by centrifugation and cultures were reconstituted by adding 4 ml of RPMI 1640 medium, 0.5 ml serum, lectin (10 μg/ml final concentration), and were incubated at 37±0.05° C in a CO ₂ incubator. Cytochalasin B (6 μg/ml) was added to the cultures at 44 h after the initiation of the cultures, which were terminated at 72 h.

For post-irradiation treatment, lymphocyte cultures were set up as above without lectin and irradiated to 2 or 4 Gy of radiation. Immediately after irradiation, lectin and caffeine were added to cultures, which were incubated as described earlier for 72 h.

At the end of 72 h, cultures were harvested by centrifugation and treated with 0.125 M KCl solution at room temperature for 6 mins. The cells were fixed first with fixative solution containing a 12:6:13 mixture of methanol, acetic acid and 0.9% NaCl solution at ice-cold temperature and allowed to stand for 6 mins and centrifuged. The cell button was fixed again with a chilled mixture of 1:4 acetic acid and methanol for 5 mins and washed 3 times with the fixative.(Wuttke et al.,1996). After 3 washes, cells were gently dropped on a wet slide and stained with 2% Giemsa in Sorenson's buffer, pH 6.8 for 10-12 mins. All slides were coded and scored for the frequency of MN in cytokinesis blocked binucleated (BN) cells with well-preserved cytoplasm under a light microscope. A total of 1000 BN cells were analyzed from each experimental sample.

3. Results

[Figure 1]a shows the effect of caffeine post-treatment on micronuclei induced by 2 Gy of radiation. The results indicated that caffeine alone at 0.1 to 10 μM did not induce any MN over and above the background frequency. However, post-treatment with caffeine resulted in potentiation of radiation induced MN in a concentration dependent manner.

Figure 1: Effect of 0.1-10 μM caffeine post-treatment on MN frequency in human lymphocytes irradiated to 2 Gy (a) and 4 Gy (b). Error bars represent SD = √X where X represents number of MN in 1000 BN cells. *: p<0.005 using Student's t-test

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[Figure 1]b shows the effect of caffeine post-treatment on MN induced by 4 Gy of radiation. Even in this case, post-treatment with caffeine increased radiation induced MN in a concentration dependent manner.

[Figure 2]a shows the effect of caffeine pre-treatment on 2 Gy of radiation. The results indicate that caffeine pre-treatment resulted in decrease in radiation induced MN frequency in a concentration dependent manner.

Figure 2: Effect of 0.1-10 μM caffeine pre-treatment on MN frequency in human lymphocytes irradiated to 2 Gy (a) and 4 Gy (b). Error bars represent SD = √X where X represents number of MN in 1000 BN cells. *: p<0.005 using Student's t-test

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[Figure 2]b shows the effect of caffeine pre-treatment on MN induced by 4 Gy of radiation. The results once again indicate that caffeine pre-treatment resulted in a decrease of radiation-induced MN more or less in a concentration dependent manner.

In summary, these results clearly indicate that in the present study, caffeine post-treatment potentiates radiation induced MN whereas caffeine pre-treatment reduces radiation induced MN at both the radiation doses.

4. Discussion

With ionizing radiation, caffeine has been shown to exert both protection as well as sensitizing effects. In the present study, we have observed that caffeine pre-treatment entailed radioprotection for the induction of MN. In general, when present during irradiation, caffeine has been shown to offer protection in a wide range of systems, such as rat liver microsome model system (Devasagayam, T.P.A., 1996), mammalian cells in vitro (Kesavan and Natarajan 1985), plant seeds (Raghu and Kesavan, 1986) and bacterial spores (Kesavan and Powers, 1985). It has also been shown to exhibit radioprotection for the induction of chromosomal aberrations in bone-marrow of mice given whole body gamma radiation following pre as well as post-irradiation treatment (Farooqi and Kesavan, 1992). Caffeine also protects against whole-body lethal doses of gamma radiation (George et al., 1999). Radioprotection by caffeine pre-treatment in most of the studies was suggested to be due to it's ability to scavenge reactive oxygen species generated by radiation on the basis of it's antioxidant property. Our results are thus in agreement with those reported by others.

However, caffeine has also been shown to act as a sensitizer in some other studies. At high concentrations, caffeine potentiates radiation effects in pre-implantation mouse embryos (Müller et al. 1985). In mammalian cells, caffeine potentiates cell lethality, mutagenic and clastogenic effects of ionizing radiation (Bhattacharjee et al. 1987;Natarajan et al., 1980; Waldren and Rasko, 1978). This may be due to the direct inhibition of DNA repair (Busse et al., 1978). It can also occur due to the reduction of either the G2 block (Zapetti-Bosseler and Scott, 1985), or the reduction of radiation induced depression of DNA synthesis, which decreases the time available for repair before the damage becomes fixed by replication (Painter, 1980). However, it has also been demonstrated that these effects do not directly correlate with increased radiosensitivity (Musk and Steel, 1990).

It has been shown that caffeine post-treatment results in enhancement in radiation induced chromosomal aberrations in human lymphocytes (Natarajan et al.,1980). In the present study, caffeine enhanced radiation induced MN in the same lymphocyte system. However, these results are contrary to those reported by us in our earlier studies with yeast Saccharomyces cerevisiae, wherein we had observed reduction in radiation induced various genetic events like back mutation, gene conversion and aberrant colony formation as well as cell killing following caffeine post-treatment (Anjaria et al.1998).

Thus, the demonstrated modifying effects of caffeine are very diverse, ranging from radioprotection, to absence of any effect to even radiosensitization. In conclusion, results obtained in the present study together with the review of literature indicate that caffeine may act by more than one mechanisms and the manner in which it modifies the radiation effects depends on a number of factors, viz., test system, order of treatment, concentration of caffeine, the effect under consideration etc.

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