J. RADIAT. RES.

, 43, 237245 (2002)

Drinking Beer Reduces Radiation-induced Chromosome Aberrations in Human Lymphocytes

MANAMI MONOBE1,2 and KOICHI ANDO2*
Beer / Radioprotection / Chromosome aberration / X rays / Heavy ions We here investigated and reported the effects of beer drinking on radiation-induced chromosome aberrations in blood lymphocytes. Human blood that was collected either before or after drinking a 700 ml beer was in vitro irradiated with 200 kVp X rays or 50 keV/ m carbon ions. The relation between the radiation dose and the aberration frequencies (fragments and dicentrics) was significantly (p < 0.05) lower for lymphocytes collected 3 h after beer drinking than those before drinking. Fitting the dose response to a linear quadratic model showed that the alpha term of carbon ions was significantly (p < 0.05) decreased by beer drinking. A decrease of dicentric formation was detected as early as 0.5 h after beer drinking, and lasted not shorter than 4.5 h. The mitotic index of lymphocytes was higher after beer drinking than before, indicating that a division delay would not be responsible for the low aberrations induced by beer drinking. An in vitro treatment of normal lymphocytes with 0.1 M ethanol, which corresponded to a concentration of 6-times higher than the maximum ethanol concentration in the blood after beer drinking, reduced the dicentric formation caused by X-ray irradiation, but not by carbonion irradiation. The beer-induced reduction of dicentric formation was not affected by serum. It is concluded that beer could contain non-ethanol elements that reduce the chromosome damage of lymphocytes induced by high-LET radiation. rtant role. Free radicals can be reduced by radical scavengers, including alcohols, dimetylsulfoxide (DMSO) and superoxide dismutase (SOD)14). Radioprotectors that act by scavenging free radicals for the most part nd it difcult to prevent the occurrence of direct damage. Phosphorothioates, including WR2721 and WR-151327, were the most effective class of radioprotectors against high-LET radiation5). Conventional radioprotectors generally display their radioprotective properties only at high, toxic concentrations. Radioprotectors with low toxicity have been searched for many years. Some food elements, including vitamins, squalene and garlic extract, have recently been reported to possess radioprotective activities against damage caused by low-LET radiation68). There is, however, no report on food elements that protect mammalian cells from damage caused by high-LET radia-

INTRODUCTION The search for effective radioprotectors is a major concern in the medical, military, environmental, and space sciences. Densely ionizing radiation, such as radon - particles and cosmic rays, has a characteristic of high LET (linear energy transfer), and produces lesions through direct action. Sparsely ionizing radiation (X rays and rays) induces DNA lesions mostly by indirect action, in which free radicals play an impo*Corresponding author: Phone: +81432063231, Fax: +81432064149, E-mail: ando@nirs.go.jp 1 Graduate School of Science and Technology, Chiba University, 133, Yayoi-cho, Inage-ku, Chiba 2638522, Japan 2 Division of Heavy-ion Radiobiology Research Group, National Institute of Radiological Sciences, 91, Anagawa-4-chome, Inage-ku, Chiba 263 8555, Japan

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tion. In the present study, we found that beer drinking reduced high- and low-LET radiation-induced chromosome aberrations in lymphocytes irradiated in vitro.

MATERIALS AND METHODS Irradiation Whole blood samples were irradiated in air at

Fig. 1. Dose-response of chromosome aberrations in lymphocytes exposed to either 200 kVp X-radiation or LET 50 KeV/m carbon ion either before or 3 h after beer drinking. (a) and (b), X rays; (c) and (d), carbon ions. The values represent means SEM (n=100). Data was fitted to a linear-quadratic dose response function. The open circles ( ), before beer drinking; solid square (), 3 h after beer drinking. The statistic significance was obtained between control and beer drinking (p < 0.05 by analysis of Two-way factorial ANOVA test; Statview, Macintosh).

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room temperature with either X rays (200 kVp; 20 mA; 0.5 mm Cu + 0.5 mm Al lters; focus center distance, 55 cm) at a dose rate of 1 Gy/min or carbon-12 ion beams. Carbon ions were accelerated up to 290 MeV/u by HIMAC synchrotron at the National Institute of Radiological Science (NIRS, Chiba, Japan). Lucite absorbers with 141 mm thickness were used to select a LET of 50 keV/m, which is a dose-averaged value including the contribution of fragments. X-ray irradiations were performed within 30 min after blood collection, while carbon-ion irradiations were made 4 h after blood collection with a variable dose rate of between 1 and 5 Gy/min. Blood sampling A healthy, non-smoking 28-year-old female volunteer drank a 700 ml beer (ethanol content; 40 mg/ ml) within 15 min. Blood was collected in heparinized tubes (Becton Dickinson Co., NJ, USA) either before

beer drinking or 0.5, 1, 2, 3, 4 and 4.5 h afterwards. The ethanol concentration in plasma was measured by gas chromatography at the KOTO microorganism laboratory (Tokyo, Japan). Isolation of lymphocytes and plasma Lymphocytes were isolated using LeucoPREP tubes (Becton Dickinson Co., NJ, USA). Each tube was centrifuged for 30 min at 3000 rpm, and a white layer containing mononuclear cells was collected by a pipette and transferred to a conical centrifuge tube (Becton Dickinson Co., NJ, USA). Phosphate-buffered saline (PBS) was added up to 14 ml, and the tube was centrifuged for 5 min at 1500 rpm. The resulting pellet was washed 5 once again in PBS and resuspended in either the RPMI 1640 medium (Gibco-BRL, N.Y., USA) or the plasma 3 h after beer drinking. In order to avoid dilution by the separation liquid contained in LeucoPREP, the blood plasma was isolated

Table 1. Parameters of dose-response for the induction of dicentrics and acentric fragments.
X rays

Dicentric
(102) SD(Gy1) (102) SD(Gy2)

Acentric fragment
(102) SD(Gy1) (102) SD(Gy2)

Non-treatment beer drinking 0.1 M ethanol 0.01 M ethanol

14.21.8 8.11.0* 14.04.5 24.13.0

7.10.4 5.70.2 2.60.9 2.40.6

15.9 4.1 11.74.8 11.68.4 23.67.1

14.90.8 14.91.1 9.01.7 10.41.4

carbon ions Dicentric

(102) SD(Gy1) (102) SD(Gy2)

Acentric fragment
(102) SD(Gy1) (102) SD(Gy2)

Non-treatment beer drinking 0.1 M ethanol 0.01 M ethanol

46.60.9 28.70.7* 39.40.9 38.60.7

104.43.6 67.41.7 104.62.1 102.53.6

Parameters and were derived from maximum-likelihood ts of data to linear quadratic dose response functions; Y = D + D2 For carbon ions is not signicantly different from zero. Thus, the coefcient for carbon ions was set to zero. *: p < 0.05

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Fig. 2. Time course of the chromosome aberration frequency in lymphocytes and plasma ethanol concentrations after beer drinking. Lymphocytes were exposed in vitro to 4 Gy of X rays (a) or carbon ions (b). The values of the chromosome aberration frequency represent means SEM (n=100). The statistic significance (*; p < 0.05, t-test) was obtained between control and beer drinking.

using heparinized tubes. Each tube was centrifuged for 5 min at 3000 rpm, and the contents were transferred to a conical tube (Becton Dickinson Co., NJ, USA). In vitro ethanol experiments Ninety-nine (99%) ethanol was added to whole blood samples in vitro, providing a nal concentration of 10 mM, which roughly corresponded to the blood ethanol concentration 3 h after beer drinking. Blood samples with a high concentration of 100 mM ethanol were also prepared. The samples were kept at room temperature for 1 h before irradiation. Blood cultures and chromosome analysis Either 1.0 ml of irradiated whole blood or lymphocytes was suspended in 9 ml of a RPMI 1640 medium (Gibco-BRL, N.Y., USA) supplemented with 20% (v/v) fetal bovine serum, 0.06 mg/ml kanamycin (antibiotics) (Meiji Seika, Tokyo, Japan), 1.25 mg/ml sodium bicarbonate, 90 g/ml PHA-M (Murex Biotech Ltd., UK), and 0.05 g/ml colcemid (Wako pure Chemical Industries, Ltd., Osaka, Japan). Cells were plated in T-25 asks and incubated at 37C in a humidied atmosphere containing 95% air plus 5% CO2. Colcemid was added to the medium at the beginning of the culture in order to obtain a sufcient number of cells at the metaphase in the rst cell division9). After 53 h of incubation, cultured cells were treated

with a hypotonic solution (75 mM KCl) for 20 min at 37C and xed with methanol-acetic acid (3:1). Chromosome slides were made by the conventional method of air-drying under a warm and humid condition. Chromosome slides were stained by Giemsa in phosphate buffered saline (pH 6.8), embedded in Eukitt (O. Kindler, Germany), and nally observed under a light microscope. with the highest magnication ( 1000).

Fig. 3. Mitotic index of lymphocytes after 53 h culture. Lymphocytes were collected either before beer drinking ( ) or 3 h after beer drinking ( ), and were irradiated with 4 Gy of either X rays (left) or carbon ions (right). The mitotic index was determined as the percentage of dividing cells among 10000 cells. The statistic significance (*; p < 0.01, t-test) was obtained between before drinking and beer drinking.

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One hundred metaphase gures were examined to detect unstable types of chromosome aberrations, such as dicentrics and acentric fragments, while 1000 cells

were examined for non-radiation control. The experiments were repeated twice, and all of the data were combined for analysis.

Fig. 4. Effects of ethanol on radiation-induced chromosome aberrations of lymphocytes. Ethanol of either 0.01 M ( ) or 0.1 M ( ) was added to the collected blood in vitro 1 h before irradiation with X rays (a; dicentric, b; fragment) or carbon ions (c; dicentric, d; fragment). Closed circles (), before beer drinking. Data (n =100, mean SEM) were fit to a linear-quadratic dose response function.

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Curve fit and statistical analysis Dose-response data were t to the following linear-quadratic regression: Y = D + D2, where Y is the yield of induced chromosome aberrations per cell, D is the dose in Gy, and and are t coefcients. No fragments or dicentrics were detectable for the non-radiation control. The data were analyzed by using a two-way factorial ANOVA test (Statview, SAS Institute Inc.).

RESULTS Figure 1 shows the dose-response curve of either X rays or carbon ions before and 3 h after beer drinking. The chromosome aberrations induced by radiation were signicantly lower (p < 0.05 by analysis of the two-way factorial ANOVA test; Statview, Macintosh) for lymphocytes collected from a donor 3 h after beer drinking than those without beer drinking. The alpha () components of both X rays and carbon ions were decreased (p < 0.05) after beer drinking (Table 1). The number of dicentrics per cell induced by 4 Gy X rays was signicantly (p < 0.05) decreased from 1.75, a pre-beer drinking value, to 1.24 when lymphocytes were collected as early as 30 min after beer drinking, and continued to be low up to 4.5 h after beer

drinking (Fig. 2a). The number of dicentrics per cell induced by 4 Gy of carbon ions was also signicantly (p < 0.05) decreased from 1.85 to 1.38 when lymphocytes were collected 30 min after beer drinking (Fig. 2b). During the period of 30 min through 4.5 h after beer drinking, the ethanol concentration in plasma changed ~ 8 times, and ranged from 2 to 16 mM (Fig. 2 a, b). Figure 3 shows a mitotic index of lymphocytes that received 4 Gy of either X rays or carbon ions 3 h after beer drinking. The mitotic index was signicantly (p < 0.01) higher for the post-beer drinking condition than the pre-beer drinking condition. The ratio of post- to pre-beer drinking groups was 1.65 (3.07 / 1.86) for X rays, and 2.05 (2.48 / 1.21) for carbon ions. The addition of ethanol to the blood samples before X rays signicantly reduced the chromosome aberrations (p < 0.05 by analysis of the two-way factorial ANOVA test; Statview, Macintosh) (Fig. 4 a, b). The addition of 10 mM ethanol to the blood samples before 4 Gy of X-radiation reduced chromosome aberrations; the number of dicentrics per cell decreased from 1.75 to 1.30, and the number of acentric fragments per cell decreased from 2.91 to 2.34, respectively. These numbers were similar to the values obtained for lymphocytes collected 3 h after beer drinking (Fig.

Fig. 5. Effects of plasma on dicentric formation. Lymphocytes in peripheral blood that were collected either before ( ) or 3 h after () beer drinking were separated from plasma, and were then suspend either in serum that was collected 3 h after beer drinking or in a RPMI medium. Lymphocytes were exposed in vitro to 4 Gy of X rays (a) or carbon ions (b). The values represent means SEM (n=100).

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1a, b). Ethanol also showed reduced carbon-induced dicentrics (p < 0.05 by analysis of Two-way factorial ANOVA test; Statview, Macintosh). However, an increase of the ethanol concentration from 10 to 100 mM did not increase the reduction of dicentrics (Fig. 4c). Ethanol failed to reduce any acentric fragments induced by carbon-ion radiation (Fig. 4d). A possibility that plasma could contain aberration-reducing substances was examined by separating the lymphocytes from the plasma. As shown in Fig. 5, replacing the plasma with RPMI 1640 medium did not affect the yields of chromosome aberrations. Less dicentrics were detected for the post-beer drinking lymphocytes than for the pre-beer drinking lymphocytes

DISCUSSION In the present study, we showed that beer drinking reduced the chromosome aberration in lymphocytes that were in vitro irradiated with X rays or 50 keV/m carbon ions. Beer drinking decreased the alpha component rather than the beta component of X rays as well as of carbon ions (Table 1). The alpha component is a coefcient that suggests the single track-2 hits event in DNA10). Slow electrons of 700 eV would produce densely ionizing events over a dimension of ~ 10 nm, which could be responsible for mammalian cell killing by the single-hit mechanisms of X rays and charged particles as well. A scavenger of the hydroxyl (OH) radical, DMSO, protects mammalian cell kill by reducing the alpha component of not only X rays, but also of high-LET charged particles11). The mechanism by which high-concentration DMSO suppresses highLET radiation-induced cell killing is not yet understood. Some food elements, including vitamins, squalene and garlic extract, have recently been reported to possess radioprotective activities against damage caused by low-LET radiation68). The protective effect observed in vitamins has been explained by the scavenging of oxidizing free-radicals. The mechanism of

radioprotection is, however, not clear. Ghiselli et al.12) report that beer increases the plasma antioxidant capacity in humans, and that ethanol alone dose not affect the plasma antioxidant capacity. Beer is effective to inhibit salmonella mutations caused by heterocyclic amines (HAs), 2- chloro-4-methylthiobutanoic acid (CMBA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)1315); this inhibition is not due to alcohol. However, there is no report that beer decreases chromosome aberration. Ethanol protects DNA from X rays inducing strand breaks by OH radical scavenging1). In the present study, the in vitro addition of 10 mM ethanol that corresponded to a plasma concentration of ethanol 23 h after beer drinking, reduced the X-ray-induced chromosome aberrations (Fig. 1a, b; Fig. 4a, b). The OH radical scavenging by ethanol could be responsible for the X-ray induced-aberration reduction by beer drinking. However, the plasma ethanol concentration at 4.5 h after beer drinking is around 2 mM, which is 8-times lower than the highest concentration at 0.5 h, and much lower than the ethanol in vitro used (Fig. 2). Also, ethanol showed little efcacy to protect carboninduced chromosome aberrations. An increase in the ethanol concentration from 10 to 100 mM was not effective to further reduce dicentrics (Fig. 4c). It is, therefore, unlikely that the beer-induced aberration reduction is solely due to ethanol. Figure 5 shows the possibility of an intracellular change. Schlorff et al. (1999)16) report that manganese-superoxide dismutase (Mn-SOD) activity signicantly increases in a dosedependent manner. SODs play a key role in preventing the DNA damage caused by reactive free radicals. Joksic et al (2000)17) report that Mn-SOD plays an important role in decreasing chromosome aberration induced by radiaton in lymphocytes. The intracellular changes induced by beer drinking may, in part, be due to increased SODs activity in lymphocytes. The densely-ionizing radiation induces severe cell-cycle delay1820), which can modify the shape of the dose-response curve at M-phase analysis21). The cell-cycle delay may not, however, contribute to the aberration reduction by beer drinking, because the mitotic indices rather increased upon beer drinking

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(Fig. 3). Because our cell-culture method allows only few (1.6%) lymphocytes to progress to the second mitosis9), it is not likely that the escape of cells from the colcemid block could largely contribute to the aberration reduction by beer drinking. The radiation-induced chromosome aberrations could be inuenced by a hormonal condition in women. The female estradiol concentration in serum varies depending on the sexual cycle. Kanda et al.22) have reported that estradiol in blood decreases mitosis and increases chromosome aberration in lymphocytes. Because the present studies were performed at a random period in the sexual cycle of a volunteer, the sexual cycle may not, if any, signicantly contribute to the present results. Dicentrics and translocations of lymphocytes are most often used to estimate the radiation doses received by astronauts, people involved in radiation accidents and during radiation therapy2327). Dicentrics are well associated with long-term effects caused by radiation. Similar yields of dicentrics and translocations are induced by ionizing radiation2830). Beer drinking may affect the biological dose estimation using human lymphocytes. Further studies are required to elucidate the mechanisms of chromosome aberration reduction induced by beer drinking.

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ACKNOWLEDGEMENTS
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This work was supported in part by a Grant-inAid from the Ministry of Education, Science, Sports and Culture of Japan and by the Special Coordination Funds for Research Project with Heavy Ions at the National Institute of Radiological Sciences Heavy ion Medical Accelerator in Chiba (NIRS-HIMAC). We would like to thank Drs. K. Fujitaka, H. Yamaguchi, S. Kakinuma, Y. Shimada, K. Nojima and S. Koike for their helpful comments.
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