Mutation Research, 241 (1990) 133-137 Elsevier
133
MUTGEN 01546
Mouse bone marrow cytogenetic damage produced by residues of tequila E. M a d r i g a l - B u j a i d a r , A. R o j a s C., A. R a m o s C., E. R o s a s P. a n d S. D i a z B a r r i g a - A r c e o Laboratorio de Gendtica, Depto. de Morfologia, Escuela Nacional de Ciencias Biol6gicas, I.P.N. Carpio y Plan de Ayala, Mexico City (Mexico) (Received 19 April 1989) (Revision received 20 November 1989) (Accepted 4 December 1989)
Keywords: Residues of tequila; Chromosomal aberrations; Sister-chromatid exchanges; Mouse bone marrow
Summary Five concentrations (50-860 mg/kg) of residues obtained after distillation and lyopbilization of commercial tequila were injected into mice for evaluation of chromosome aberrations, sister-chromatid exchanges, and proliferation kinetics in mouse bone marrow cells. Appropriate positive and negative controls were included. Our results showed significant dose-related increases of chromosomal aberrations starting at 50 mg/kg and for sister-chromatid exchanges at 430 mg/kg. Cellular proliferation kinetics showed no alterations. With these data we demonstrated that the residues of tequila are genotoxic in vivo.
A number of adverse health effects are associated with chronic alcohol consumption. Epidemiological studies have detected a relationship between d r i n k i n g a n d a n increased cancer incidence of the upper alimentary tract, upper respiratory tract, liver, large bowel, pancreas and breast [1-3]. With regard to mutagenic action it is known that alcoholics show elevated numbers of chromosomal aberrations and sister-chromatid exchanges (SCE) as compared to controls [4-6], while in former alcoholics SCE and chromosome aberration levels are similar to those observed in controls [6]. The mutagenicity of ethanolacetaldehyde has been tested in different systems [7], and studies using lymphocytes in vitro suggest that acetaldehyde is the main genotoxic agent [8].
Correspondence: Dr. E. Madrigal-Bujaidar, Laboratorio de Gen~tica, Depto. de Morfologla, I.P.N. Carpio y Plan de Ayala, Col. Santo Tom~s, C.P. 11340, Mexico City (Mexico).
Alcoholic beverages contain many other components besides ethanol and water, which are called congeners, and whose genotoxic potential is presently being studied. A mutagenic response has been observed using the Ames test with or without metabolic activation for evaporates of alcoholic beverages including several brands of whisky, cognac, brandy, rum, wine and rice and barley spirits [9-12]. When human lymphocyte cultures are treated in vitro a positive response has also been reported for whisky, brandy and rum [13]. There is no information regarding the genotoxic potential of residues in animals treated in vivo. Alcoholism in Mexico is increasing. In Mexico City approximately 25% of the population are consumers on a regular basis and at least 6% are heavy drinkers [14]. Alcoholic hepatic cirrhosis constitutes the primary cause of death in the population aged 40-59, and mortality due to alcoholism (including psychosis) is about 4.5/
0165-1218/90/$03.50 © 1990 Elsevier Science Pubfishers B.V. (Biomedical Division)
134
100000 inhabitants [14,15]. Tequila, a beverage obtained after fermentation and distillation of the stems of Agave tequilana, is the fourth highest in consumption. The 1975 production was 20588 milhon liters with a consumption per capita of 0.637 1, while in 1980 the figures were 41927 and 1.090 respectively [15]. Considering the high rate of consumption of this beverage we have analyzed its genotoxic capacity using several short-term tests. In this investigation we report the cytogenetic results obtained in the bone marrow cells of mice treated with the residues of tequila. Material and method
Residues We used commercial bottles of colorless tequila for the study. The beverage was initially distilled at 70 °C in vacuum and the aqueous fraction was subjected to a lyophiliration process using a 2-step displacement apparatus, 160 l/rain (Fehwelch). We obtained 54.2 mg of a yellowish residue per hter, which was dissolved in distilled water using a sonicator (Brawn Sonic 1510). The residues up to 4 g / k g were inoculated intraperitoneally into experimental animals with no lethality. The doses used for the genotoxic experiment were 50, 215, 430, 645 and 860 mg/kg. Negative controls and mice treated with 2 mg/kg of mitomycin C (MMC, Sigma) as positive controls were included.
Cytogenetic methods We used male mice, NIH (SW), with an average weight of 23 g, maintained in groups of 5, with food and water ad hbitum. A 53-rag tablet of 5-bromo-2'-deoxyuridine (BrdU) partially coated with paraffin (58-60 o C) was subcutaneously implanted in the posterolateral region of the animals [16,17]. After the assay about 75% of the tablet was absorbed. 1 h later either the test substance, distilled water (0.5 ml), or MMC was injected i.p. 21 h afterwards the mice were injected i.p. with 5 mg/kg of colchicine (Sigma) and were killed 3 h later by cervical dislocation. The bone marrow cells were dispersed in 0.075 M KC1 for 20 rain and fixed twice in methanol-acetic acid (3:1).
Slides were made by flaming after being immersed in 70% methanol. Staining was done by a modification of the method of Goto et al. [18]. Slides were treated for 30 min with Hoechst 33258 (Riedel de Haen) diluted in distilled water (100 mg/ml), washed, dried and immersed in a phosphate-citrate buffer at pH 7.0 and exposed for 30 min to black hght. Slides were again washed, dried and finally stained with a 4% Giemsa solution made in 0.01 M phosphate buffer at pH 6.8. Shdes were then coded and scored bhnd. We performed the following evaluations. (1) Sixty first-generation metaphases per mouse were scored to quantify the frequency of chromosomal aberrations (CA). (2) Thirty second-generation mitoses per mouse were evaluated to determine the frequency of sister-chromatid exchanges (SCE). (3) CeUular prohferation kinetics were analyzed in 100 cells per animal by counting the proportion of first, second, third or higher mitoses, and average generation times were calculated [19]. (4) Statistical analysis was performed using Student's t test and the chi-square test. Results and discussion
We did not find chromosome-type aberrations or chromatid exchanges. The most frequently observed aberrations were chromatid deletions (p = 0.01) compared to negative controls even at the lowest tested dose. The highest dose increased the CA basal value more than 4 times, but in MMCtreated cells this level was almost 7 times higher (Table 1). The regression line calculated by the least-squares method showed a positive dose-response relationship with a correlation coefficient of r = 0.980 (Fig. 1). SCE results also gave a positive response, the residues of tequila almost doubled the basal level at the highest dose (860 mg/kg) and showed a significant difference from the third highest dose (430 mg/kg). The analysis of SCE in the MMCtreated mice showed a 4-fold elevation over the rate of controls (Table 2). The regression line with r = 0.991 is presented in Fig. 2.
135 TABLE 1 F R E Q U E N C Y O F C H R O M O S O M A L A B E R R A T I O N S I N D U C E D BY T H E R E S I D U E S O F T E Q U I L A IN M O U S E BONE M A R R O W CELLS Dos:
Cells
Animals
Chromosomal aberrations (number) X + SD
(mg/kg) 0
300
5
50
300
5
215
300
5
430
300
5
645
300
5
860
300
5
MMC 2
300
5
Total
Gaps
Isogaps
Chromatid breaks
Isochromatid breaks
(11) 0.036 + 0.014 (17) 0.056 + 0.018 (15) 0.05 + 0.026 (26) * 0.086 + 0.021 (25) * 0.083 :t: 0.039 (37) * 0.123 + 0.030 (74) * 0.246 + 0.041
(1) 0.003 + 0.007 (1) 0.003 + 0.006 (2) 0.006 + 0.014 (1) 0.003 + 0.007 (4) 0.013 5:0.018 (4) 0.013 + 0.013 (16) * 0.02 + 0.013
(6) 0.02 + 0.007 (13) 0.043 + 0.009 (16) 0.053 + 0.017 (22) * 0.073 + 0.022 (31) * 0.103 5:0.006 (43) * 0.143 + 0.028 (127) * 0.423 + 0.056
(0) (1) 0.003 + 0.007 (0) (0) (0) (1) 0.003 + 0.007 (5) * 0.016 + 0.011
(18) 0.06 + 0.009 (32) * 0.106 + 0.021 (33) * 0.11 + 0.025 (49) * 0.163 + 0.007 (60) * 0.20 5:0.031 (85) * 0.28 + 0.054 (212) * 0.700 + 0.322
* Significant. Student's t test, p = 0.01.
Only minor variability was noted in response between animals within treatment groups for both measured endpoints. Likewise, there were no cells with multiple CA or SCE that could influence the interpretation of the results.
% 16
m
The frequency of first, second and third or higher mitoses for all tested doses did not show a significant difference from controls, indicating no effect of residues of tequila on cell-cycle duration (Table 3). The identity of the specific compound(s) in residues of tequila that are genotoxic are not known. However, it is known that evaporates of alcoholic beverages may contain hundreds of components belonging to different chemical species that are formed during the production and storage
12 TABLE 2 FREQUENCY OF SISTER-CHROMATID EXCHANGES P R O D U C E D BY R E S I D U E S O F T E Q U I L A IN M O U S E BONE M A R R O W CELLS
I
I
I
I
I
200
400
600
800
1000
Dose mg/kg Fig. 1. Regressionline of chromosomal aberrations (excluding gaps) for mouse bone marrow cells treated with residues of tequila. Y = 2.495 +0.013X, where X ffi concentration of tequila residue ( m g / k g ) injected i.p. into mice (r = 0.991).
Dose (mg/kg)
Ceils
Anireals
Range
SCE ('X + SD)
0 50 215 430 645 860 MMC 2
150 150 150 150 150 150 150
5 5 5 5 5 5 5
0-12 0-11 0-15 3-15 4-15 5-22 11-34
5.4 + 0.44 5.2 + 0.40 6.2 + 0.42 6.8 * +0.16 8.0 * +0.36 9.2 * -t-0.30 20.4 * +2.26
* Significant. Student's t test, p = 0.01.
136 IC SCE
L 50
i 200
i 400
i 600
i i 800 1000 Dose mg/kg
Fig. 2. Regression line of sister-chromatid exchanges in mouse bone marrow cells treated with residues of tequila. Y = 5.172 + 0.00453X, where X = concentration of tequila residue (mg/kg) injected i.p. into mice (r = 0.991).
processes [7,13]. A t t e m p t s to relate these chemicals with different effects to alcoholism are b e i n g performed. I n this context, G a v a l e r et al. [20,21] have shown that the phyto-estrogen b i o c h a n i n A, as well as the p l a n t sterol fl-sitosterol, could p l a y a n i m p o r t a n t role i n the e n d o c r i n e changes observed i n alcoholics. W i t h respect to genotoxiccarcinogenic agents, efforts have b e e n m a d e to d e t e r m i n e the activity of n i t r o s a m i n e s [7,22]. O t h e r p o t e n t i a l l y h a r m f u l substances have b e e n detected i n the residues of alcoholic beverages, such as acrolein, glycidol, furfural, urethane, n - n i t r o soproline a n d flavonols [9,23-26]. A t p r e s e n t there is n o definitive correlation b e t w e e n a specific substance a n d a cancer increase i n alcoholics. O u r data show that genotoxic d a m a g e is produced in vivo b y high c o n c e n t r a t i o n s of residues
TABLE 3 CELLULAR PROLIFERATION KINETICS IN MOUSE BONE MARROW CELLS TREATED WITH RESIDUES OF TEQUILA Dose (mg/kg)
Cells
Cell generation (%) First Second Third
Average generation time (h)
0 50 215 430 645 860
500 500 500 500 500 500
37.8 34.9 36.4 32.4 30.2 30.6
14.09 14.39 14.08 14.31 14.28 14.33
57.4 61.0 59.2 62.8 66.0 66.6
4.8 4.1 4.4 4.8 3.8 2.8
The results are not statistically significant. X2, p = 0.01.
of tequila. This, together with previously observed m u t a g e n i c effects of congeners of other drinks, suggests that alcoholic beverages i n a chronically d r i n k i n g p e r s o n p r o d u c e deleterious effects in add i t i o n to those m e d i a t e d b y acetaldehyde [27] or ethanol. Such effects c o u l d possibly involve activities of u n k n o w n chemicals as cocarcinogens, as i n d u c e r s of m i c r o s o m a l e n z y m e s activating procarcinogens, or b y decreasing the ability of the liver to m e t a b o l i z e n i t r o s a m i n e s [2,28], a n d also t h r o u g h the a c t i o n of other m u t a g e n s that in spite of low a m o u n t s could initiate genetic damage.
References
1 Lieber, C.S., E. Baraona, M.A. Leo and A. Garro (1987) Metabolism and metabolic effect of ethanol, including interaction with drugs, carcinogens and nutrition, Mutation Res., 186, 201-233. 2 Tuyns, A.J. (1987) Cancer risks derived from alcohol, Med. Oncol. Tumor Pharmacother., 4, 241-244. 3 Williams, R.R., ahd J.W. Horm (1977) Association of cancer sites with tobacco and alcohol consumption and socioeconomic studies of patients: interview study from Third National Cancer Survey. J. Natl. Cancer Inst., 58, 525-547. 4 0 b e , G., and H. Ristow (1979) Mutagenic, cancerogenic and teratogenic effects of alcohol, Mutation Res., 65, 229259. 5 Kucheria, K., N. Taneja and D. Mohan (1986) Chromosomal aberrations and sister chromatid exchanges in chronic male alcoholics, Indian J. Med. Res., 83, 417-421. 6 Butler, M.G., W.G. Sanger and G.E. Veomett (1981) Increased frequency of sister chromatid exchanges in alcoholics, Mutation Res., 85, 71-76. 7 0 b e , G., and O. Anderson (1987) Genetic effects of ethanol, Mutation Res., 186, 177-200. 8 0 b e , G., R. Jonas and S. Schmidt (1986) Metabolism of ethanol in vitro produces a compound which induces sister chromatid exchanges in human peripheral lymphocytes in vitro: acetaldehyde not ethanol is mutagenic, Mutation Res., 174, 47-51. 9 Loquet, C., G. Toussaint and J.Y. LeTalaer (1981) Studies on mutagenic constituents of apple brandy and various alcoholic beverages collected in western France, a high incidence area for oesophageal cancer, Mutation Res., 88, 155-164. 10 Nagao, M., Y. Takahashi, K. Wakabayashi and T. Sugimura (1981) Mutagenicity of alcoholic beverages, Mutation Res., 88, 147-154. 11 Lee, J.S.D., and L.Y.Y. Fong (1979) Mutagenicity of Chinese alcoholic spirits, Food Cosmet. Toxicol., 17, 575578. 12 Stoltz, B., D. Stavric, R. Krewsky, R. Klaussen, R. Bendall and B. Jankins (1982) Mutagenicity screening of foods. I. Results with beverages, Environ. Mutagen., 4, 477-492.
137 13 Hoeft, H., and G. Obe (1983) SCE inducing congeners in alcoholic beverages, Mutation Res., 121, 247-251. 14 Medina-Mora, M.E., C.A. De la Parra and G. Terroba (1980) El consumo de alcohol en la poblaci6n del Distrito Federal, Sal. Pub. M6x., 22, 281-288. 15 Mas, C., A. Manrique, C. Varela and H. Rosovsky (1986) Variables mb~icas y sociales relacionadas con el consumo de alcohol en Mdxico, Sal. Pub. Mdx., 28, 473-479. 16 McFee, A.F., L. Lowe and J. San Sebastian (1983) Improved sister chromatid differentiation using paraffin coated bromodeoxyuridine tablets in mice, Mutation Res., 119, 83-88. 17 Madrigal, E., and E. Rosas (1989) In vivo and in vitro genotoxic evaluation of indorenate, Mutation Res., 222, 317-321. 18 Goto, K.P., T. Komatzu and H. Shimatzu (1975) Simple differential Giemsa staining after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma, 53, 223-230. 19 Ivett, J., and R.R. Tice (1982) Average generation time: a new method of analysis and quantitation of cellular proliferation kinetics, Environ. Mutagen., 4, 358. 20 Gavaler, J.S., A.F. Imhoff, C. Pohl, E.R. Rosenblum and D.H. Van Tiel (1987) Alcoholic beverages: a source of estrogenic substances?, Alcohol Alcoholism, 1, 545-549. 21 Gavaler, J.S., E.R. Rosenblun, D.H. Vantiel, P.K. Eagon,
22
23
24
25
26
27 28
C.R. Pohl, J.M. Campbell and J. Gavaler (1987) Biologically active phyto estrogens are present in bourbon, Alcoholism, 11, 399-406. (}off, E.M., and D.H. Fine (1979) Analysis of volatile N-nitrosamines in alcoholic beverages, Food Cosmet. Toxicol., 17, 569-573. Ruoff, J., A. Laires, M. G6mez, H. Borba and M. Halpern (1984) DNA damaging activity of flavonoid containing beverages, Mutation Res., 130, 243. Nakayashu, M., H. Sakamoto, M. Terada, M. Nagao and T. Sugimura (1986) Mutagenicity of quercetin in Chinese hamster lung cells in culture, Mutation Res., 174, 79-83. Johnson, P., J. Pfab and R. Massey (1988) A method for the investigation of free and protein bound N-nitroso proline in beer, Food Addit. Contain., 5, 119-125. Cairns, T., E.G. Siegmund, M.A. Luke and G.M. Doose (1987) Residue levels of ethyl carbamate in wines and spirits by gas chromatography and mass spectrometry/mass spectrometry, Anal. Chem., 59, 2055-2059. DeLlarco, V.L. (1988) A mutagenicity assessment of acetaldehyde, Mutation Res., 195, 1-20. Swarm, P.F., R.J. Graves and R. Mace (1987) Effect of ethanol on nitrosamine metabolism and distribution. Impfications for the role of nitrosamines in human cancer and the influence of alcohol consumption on cancer incidence, Mutation Res., 186, 261-267.
133
MUTGEN 01546
Mouse bone marrow cytogenetic damage produced by residues of tequila E. M a d r i g a l - B u j a i d a r , A. R o j a s C., A. R a m o s C., E. R o s a s P. a n d S. D i a z B a r r i g a - A r c e o Laboratorio de Gendtica, Depto. de Morfologia, Escuela Nacional de Ciencias Biol6gicas, I.P.N. Carpio y Plan de Ayala, Mexico City (Mexico) (Received 19 April 1989) (Revision received 20 November 1989) (Accepted 4 December 1989)
Keywords: Residues of tequila; Chromosomal aberrations; Sister-chromatid exchanges; Mouse bone marrow
Summary Five concentrations (50-860 mg/kg) of residues obtained after distillation and lyopbilization of commercial tequila were injected into mice for evaluation of chromosome aberrations, sister-chromatid exchanges, and proliferation kinetics in mouse bone marrow cells. Appropriate positive and negative controls were included. Our results showed significant dose-related increases of chromosomal aberrations starting at 50 mg/kg and for sister-chromatid exchanges at 430 mg/kg. Cellular proliferation kinetics showed no alterations. With these data we demonstrated that the residues of tequila are genotoxic in vivo.
A number of adverse health effects are associated with chronic alcohol consumption. Epidemiological studies have detected a relationship between d r i n k i n g a n d a n increased cancer incidence of the upper alimentary tract, upper respiratory tract, liver, large bowel, pancreas and breast [1-3]. With regard to mutagenic action it is known that alcoholics show elevated numbers of chromosomal aberrations and sister-chromatid exchanges (SCE) as compared to controls [4-6], while in former alcoholics SCE and chromosome aberration levels are similar to those observed in controls [6]. The mutagenicity of ethanolacetaldehyde has been tested in different systems [7], and studies using lymphocytes in vitro suggest that acetaldehyde is the main genotoxic agent [8].
Correspondence: Dr. E. Madrigal-Bujaidar, Laboratorio de Gen~tica, Depto. de Morfologla, I.P.N. Carpio y Plan de Ayala, Col. Santo Tom~s, C.P. 11340, Mexico City (Mexico).
Alcoholic beverages contain many other components besides ethanol and water, which are called congeners, and whose genotoxic potential is presently being studied. A mutagenic response has been observed using the Ames test with or without metabolic activation for evaporates of alcoholic beverages including several brands of whisky, cognac, brandy, rum, wine and rice and barley spirits [9-12]. When human lymphocyte cultures are treated in vitro a positive response has also been reported for whisky, brandy and rum [13]. There is no information regarding the genotoxic potential of residues in animals treated in vivo. Alcoholism in Mexico is increasing. In Mexico City approximately 25% of the population are consumers on a regular basis and at least 6% are heavy drinkers [14]. Alcoholic hepatic cirrhosis constitutes the primary cause of death in the population aged 40-59, and mortality due to alcoholism (including psychosis) is about 4.5/
0165-1218/90/$03.50 © 1990 Elsevier Science Pubfishers B.V. (Biomedical Division)
134
100000 inhabitants [14,15]. Tequila, a beverage obtained after fermentation and distillation of the stems of Agave tequilana, is the fourth highest in consumption. The 1975 production was 20588 milhon liters with a consumption per capita of 0.637 1, while in 1980 the figures were 41927 and 1.090 respectively [15]. Considering the high rate of consumption of this beverage we have analyzed its genotoxic capacity using several short-term tests. In this investigation we report the cytogenetic results obtained in the bone marrow cells of mice treated with the residues of tequila. Material and method
Residues We used commercial bottles of colorless tequila for the study. The beverage was initially distilled at 70 °C in vacuum and the aqueous fraction was subjected to a lyophiliration process using a 2-step displacement apparatus, 160 l/rain (Fehwelch). We obtained 54.2 mg of a yellowish residue per hter, which was dissolved in distilled water using a sonicator (Brawn Sonic 1510). The residues up to 4 g / k g were inoculated intraperitoneally into experimental animals with no lethality. The doses used for the genotoxic experiment were 50, 215, 430, 645 and 860 mg/kg. Negative controls and mice treated with 2 mg/kg of mitomycin C (MMC, Sigma) as positive controls were included.
Cytogenetic methods We used male mice, NIH (SW), with an average weight of 23 g, maintained in groups of 5, with food and water ad hbitum. A 53-rag tablet of 5-bromo-2'-deoxyuridine (BrdU) partially coated with paraffin (58-60 o C) was subcutaneously implanted in the posterolateral region of the animals [16,17]. After the assay about 75% of the tablet was absorbed. 1 h later either the test substance, distilled water (0.5 ml), or MMC was injected i.p. 21 h afterwards the mice were injected i.p. with 5 mg/kg of colchicine (Sigma) and were killed 3 h later by cervical dislocation. The bone marrow cells were dispersed in 0.075 M KC1 for 20 rain and fixed twice in methanol-acetic acid (3:1).
Slides were made by flaming after being immersed in 70% methanol. Staining was done by a modification of the method of Goto et al. [18]. Slides were treated for 30 min with Hoechst 33258 (Riedel de Haen) diluted in distilled water (100 mg/ml), washed, dried and immersed in a phosphate-citrate buffer at pH 7.0 and exposed for 30 min to black hght. Slides were again washed, dried and finally stained with a 4% Giemsa solution made in 0.01 M phosphate buffer at pH 6.8. Shdes were then coded and scored bhnd. We performed the following evaluations. (1) Sixty first-generation metaphases per mouse were scored to quantify the frequency of chromosomal aberrations (CA). (2) Thirty second-generation mitoses per mouse were evaluated to determine the frequency of sister-chromatid exchanges (SCE). (3) CeUular prohferation kinetics were analyzed in 100 cells per animal by counting the proportion of first, second, third or higher mitoses, and average generation times were calculated [19]. (4) Statistical analysis was performed using Student's t test and the chi-square test. Results and discussion
We did not find chromosome-type aberrations or chromatid exchanges. The most frequently observed aberrations were chromatid deletions (p = 0.01) compared to negative controls even at the lowest tested dose. The highest dose increased the CA basal value more than 4 times, but in MMCtreated cells this level was almost 7 times higher (Table 1). The regression line calculated by the least-squares method showed a positive dose-response relationship with a correlation coefficient of r = 0.980 (Fig. 1). SCE results also gave a positive response, the residues of tequila almost doubled the basal level at the highest dose (860 mg/kg) and showed a significant difference from the third highest dose (430 mg/kg). The analysis of SCE in the MMCtreated mice showed a 4-fold elevation over the rate of controls (Table 2). The regression line with r = 0.991 is presented in Fig. 2.
135 TABLE 1 F R E Q U E N C Y O F C H R O M O S O M A L A B E R R A T I O N S I N D U C E D BY T H E R E S I D U E S O F T E Q U I L A IN M O U S E BONE M A R R O W CELLS Dos:
Cells
Animals
Chromosomal aberrations (number) X + SD
(mg/kg) 0
300
5
50
300
5
215
300
5
430
300
5
645
300
5
860
300
5
MMC 2
300
5
Total
Gaps
Isogaps
Chromatid breaks
Isochromatid breaks
(11) 0.036 + 0.014 (17) 0.056 + 0.018 (15) 0.05 + 0.026 (26) * 0.086 + 0.021 (25) * 0.083 :t: 0.039 (37) * 0.123 + 0.030 (74) * 0.246 + 0.041
(1) 0.003 + 0.007 (1) 0.003 + 0.006 (2) 0.006 + 0.014 (1) 0.003 + 0.007 (4) 0.013 5:0.018 (4) 0.013 + 0.013 (16) * 0.02 + 0.013
(6) 0.02 + 0.007 (13) 0.043 + 0.009 (16) 0.053 + 0.017 (22) * 0.073 + 0.022 (31) * 0.103 5:0.006 (43) * 0.143 + 0.028 (127) * 0.423 + 0.056
(0) (1) 0.003 + 0.007 (0) (0) (0) (1) 0.003 + 0.007 (5) * 0.016 + 0.011
(18) 0.06 + 0.009 (32) * 0.106 + 0.021 (33) * 0.11 + 0.025 (49) * 0.163 + 0.007 (60) * 0.20 5:0.031 (85) * 0.28 + 0.054 (212) * 0.700 + 0.322
* Significant. Student's t test, p = 0.01.
Only minor variability was noted in response between animals within treatment groups for both measured endpoints. Likewise, there were no cells with multiple CA or SCE that could influence the interpretation of the results.
% 16
m
The frequency of first, second and third or higher mitoses for all tested doses did not show a significant difference from controls, indicating no effect of residues of tequila on cell-cycle duration (Table 3). The identity of the specific compound(s) in residues of tequila that are genotoxic are not known. However, it is known that evaporates of alcoholic beverages may contain hundreds of components belonging to different chemical species that are formed during the production and storage
12 TABLE 2 FREQUENCY OF SISTER-CHROMATID EXCHANGES P R O D U C E D BY R E S I D U E S O F T E Q U I L A IN M O U S E BONE M A R R O W CELLS
I
I
I
I
I
200
400
600
800
1000
Dose mg/kg Fig. 1. Regressionline of chromosomal aberrations (excluding gaps) for mouse bone marrow cells treated with residues of tequila. Y = 2.495 +0.013X, where X ffi concentration of tequila residue ( m g / k g ) injected i.p. into mice (r = 0.991).
Dose (mg/kg)
Ceils
Anireals
Range
SCE ('X + SD)
0 50 215 430 645 860 MMC 2
150 150 150 150 150 150 150
5 5 5 5 5 5 5
0-12 0-11 0-15 3-15 4-15 5-22 11-34
5.4 + 0.44 5.2 + 0.40 6.2 + 0.42 6.8 * +0.16 8.0 * +0.36 9.2 * -t-0.30 20.4 * +2.26
* Significant. Student's t test, p = 0.01.
136 IC SCE
L 50
i 200
i 400
i 600
i i 800 1000 Dose mg/kg
Fig. 2. Regression line of sister-chromatid exchanges in mouse bone marrow cells treated with residues of tequila. Y = 5.172 + 0.00453X, where X = concentration of tequila residue (mg/kg) injected i.p. into mice (r = 0.991).
processes [7,13]. A t t e m p t s to relate these chemicals with different effects to alcoholism are b e i n g performed. I n this context, G a v a l e r et al. [20,21] have shown that the phyto-estrogen b i o c h a n i n A, as well as the p l a n t sterol fl-sitosterol, could p l a y a n i m p o r t a n t role i n the e n d o c r i n e changes observed i n alcoholics. W i t h respect to genotoxiccarcinogenic agents, efforts have b e e n m a d e to d e t e r m i n e the activity of n i t r o s a m i n e s [7,22]. O t h e r p o t e n t i a l l y h a r m f u l substances have b e e n detected i n the residues of alcoholic beverages, such as acrolein, glycidol, furfural, urethane, n - n i t r o soproline a n d flavonols [9,23-26]. A t p r e s e n t there is n o definitive correlation b e t w e e n a specific substance a n d a cancer increase i n alcoholics. O u r data show that genotoxic d a m a g e is produced in vivo b y high c o n c e n t r a t i o n s of residues
TABLE 3 CELLULAR PROLIFERATION KINETICS IN MOUSE BONE MARROW CELLS TREATED WITH RESIDUES OF TEQUILA Dose (mg/kg)
Cells
Cell generation (%) First Second Third
Average generation time (h)
0 50 215 430 645 860
500 500 500 500 500 500
37.8 34.9 36.4 32.4 30.2 30.6
14.09 14.39 14.08 14.31 14.28 14.33
57.4 61.0 59.2 62.8 66.0 66.6
4.8 4.1 4.4 4.8 3.8 2.8
The results are not statistically significant. X2, p = 0.01.
of tequila. This, together with previously observed m u t a g e n i c effects of congeners of other drinks, suggests that alcoholic beverages i n a chronically d r i n k i n g p e r s o n p r o d u c e deleterious effects in add i t i o n to those m e d i a t e d b y acetaldehyde [27] or ethanol. Such effects c o u l d possibly involve activities of u n k n o w n chemicals as cocarcinogens, as i n d u c e r s of m i c r o s o m a l e n z y m e s activating procarcinogens, or b y decreasing the ability of the liver to m e t a b o l i z e n i t r o s a m i n e s [2,28], a n d also t h r o u g h the a c t i o n of other m u t a g e n s that in spite of low a m o u n t s could initiate genetic damage.
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