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Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice
Mutation Research, 246 (1991) 123-127
123
Elsevier MUT 04921
Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice Penka M. Blagoeva, Roumen M. Balansky and Tzvetana J. Mircheva Laboratory of Chemical Mutagenesis and Carcinogenesis, National Center of Oncology, Sofia 1156 (Bulgaria) (Received 6 February 1990) (Revision received 29 June 1990) (Accepted 2 July 1990)
Keywords: Caffeine; Mitomycin C; Cyclophosphamide; Micronudei; Clastogenicity
Summary Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57B1 or BDF 1 (C57B1 x DBA2) mice with caffeine (1 or 3 x 50 m g / k g and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57B1 and BDF 1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57B1 compared to BDF 1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57B1 mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57B1 and BDF~ mice but not in fetal mouse livers.
Huge population groups are daily exposed to caffeine as a dietary and drug ingredient. Some preliminary epidemiological studies have suggested a possible role for caffeine in human carcinogenesis (Somani and Gupta, 1988). However, despite the intensive studies carried out no
Correspondence: Dr. P.M. Blagoeva, Laboratory of Chemical Mutagenesis and Carcinogenesis, National Center of Oncology, Str. Plovdivsko pole 6, Sofia 1156 (Bulgaria).
clear correlation between caffeine consumption and cancer incidence has so far been established. On the other hand, caffeine has been found to interact with D N A bringing to life a broad spectrum of genetic consequences that are being thoroughly investigated (especially in vitro) as well as to interfere with the genetic activities of other compounds (to mention only the well-documented caffeine potentiation of cytotoxicity and mutagenicity of alkylating agents many of which are strong carcinogens) (Kihlman, 1977; Pohle and Fritzsche,
0027-5107/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
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1981; Levin, 1982; Hansson et al., 1984; Lialiaris et al., 1988). That is why the possible involvement of caffeine in chemical mutagenesis and carcinogenesis is still a matter of debate and needs further clarification. It should also be mentioned that according to Aeschbacher et al. (1986) caffeine by itself when applied at a dose of 100 m g / k g could cause chromosomal damage in vivo, as shown by elevated micronucleus formation in outbred Swiss CD-1 mice or in inbred M S / A e mice, thus increasing the necessity for additional assessment of the genotoxic potential of caffeine. Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenic activity of caffeine as well as its influence on the DNA-damaging activity of mitomycin C (MMC) and cyclophosphamide (CP) were studied.
(i.p.). Some mice were treated repeatedly with caffeine (100 mg/kg, s.c.) during 3 consecutive days (for pregnant mice days 15-17 were used for this purpose). In several experiments a combined treatment of mice with caffeine (triple treatment) followed by MMC or CP was used (Tables 1 and 2).
Processing of bone marrow The animals were killed by cervical dislocation 30 h after the i.p. injection of MMC or CP or 30 h after the last caffeine administration. The bone marrow smears were prepared and stained with M a y - G r f i n w a l d - G i e m s a according to Schmid (1975). From each mouse (10 mice per group, the number of pregnant mice per group and the litters obtained are indicated in Table 2), 1000 polychromatic erythrocytes (PCEs) were screened for micronuclei.
Materials and methods
Processing of fetal liver Treatment of animals Male, female and pregnant (on day 17 of gestation) female mice of C57B1 and BDF 1 (C57B1 × DBA2) strains 7-10 weeks of age were injected s.c. with caffeine (Merck, F.R.G.) at doses of 50 and 100 m g / k g body weight o r / a n d with MMC (Kyowa, Japan) at a dose of 2 m g / k g (i.p.) o r / a n d with CP (Sigma, U.S.A.) at a dose of 30 m g / k g
Single-cell suspensions of fetal livers were prepared in fetal calf serum by pipetting. The smears were air-dried and stained with M a y - G r t i n w a l d Giemsa (Schmid, 1975; Cole et al., 1979, 1981, 1982). At least 2000 PCEs per fetus were examined. Statistical analysis of the data obtained was performed according to Student's t-test.
TABLE 1 F R E Q U E N C Y OF M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S (MN P C Es ) P ER 1000 PCEs IN BONE M A R R O W OF MICE, 30 h A F T E R T R E A T M E N T W I T H C A F F E I N E , M I T O M Y C I N C O R C Y C L O P H O S P H A M I D E Group a
Control (untreated) Caffeine, 1 × 50 m g / k g , s.c. Caffeine, 1 × 100 m g / k g , s.c. Caffeine, 3 × 100 m g / k g , s.c. b Mitomycin C, 2.0 m g / k g , i.p. Cyclophosphamide, 30 m g / k g , i.p. Caffeine b + mitomycin C Caffeine b + cyclophosphamide
C57BI
BDF 1
male
female
male
female
7.2 + 0.69 7.4_+0.58 7.4 _+0.61 7.6 + 0.55 81.4 .-+4.47 53.1 _+3.39 101.2_+7.12 * 73.4_+4.32 *
6.8 + 0.57 7.1 _+0.42 6.9 _+0.53 7.3 -+ 0.61 74.8 -+ 5.03 51.2 _+3.06 98.2_+6.58 * 70.5_+4.71 *
1.76 + 0.24 1.6_+0.37 1.7 _+0.31 1.5 + 0.25 58.4 _+4.23 44.2 _+3.96 87.1_+5.40 * 63.9_+4.53 *
1.3 + 0.41 1.4_+0.22 1.5 _+0.33 1.3 -+ 0.29 56.6 _+5.11 45.9 _+2.64 83.2_+5.79 * 66.7_+4.03 *
Results are expressed as means + SE. 10 mice per group. b 3 injections, once per day, 3 consecutive days in total. * P < 0.05, compared to only MMC- or CP-treated animals.
125
Results and discussion
The possible clastogenic activity of caffeine was investigated in bone marrow of young male and female mice of C57B1 and BDF 1 strains as well as in the fetal liver. According to Cole et al. (1979, 1981, 1982) the transplacental micronucleus test could be very useful in detecting weak clastogens. The data summarized in Tables 1 and 2 clearly indicate that at the experimental conditions presented no signs of caffeine-induced clastogenicity in mice were established. Even the triple treatment of animals with caffeine (100 mg/kg, s.c.) was ineffective in inducing micronuclei in fetal and maternal mouse erythroblasts. The results obtained are in line with the literature data showing in general a lack of genotoxic activity of caffeine in vivo. The slightly increased level of MNPCEs in bone marrow of Swiss CD-1 outbred mice treated with a huge dose of caffeine (1 or 2 x 100 mg/kg, p.o.) reported by Aeschbacher et al. (1986) might be linked to the specifically high erythroblasts sensitivity to clastogenic agents of this particular mouse strain as indicated by the authors. The data presented added a new
test, namely the transplacental micronucleus assay, that gives negative results when testing caffeine for clastogenicity. In addition, significant strain differences were also established in the present experiments when the spontaneous MNPCEs rates in C57B1 and BDF 1 (C57B1 × DBA2) mice were compared (Table 1). The number of MNPCEs in bone marrow cells of C57B1 mice was about 4-fold higher than that established in BDF~ mice. The same conclusion might be drawn concerning the MNPCEs frequency in fetal livers obtained from litters of the 2 strains used (Table 2). The high spontaneous bone marrow MNPCEs level in C57B1 compared to most other mouse strains investigated so far has been known for some time (Heddle et al., 1983; Styles et al., 1983; Aeschbacher, 1986; Sato et al., 1987). The present study reveals that a similar tendency could be found when screening fetal liver PCEs for micronuclei. Obviously, the genetic reasons that are of importance for the increased bone marrow level of MNPCEs in C57B1 mice were abolished when this particular strain was crossed with DBA2 mice. In addition, no significant sex differences in
TABLE 2 F R E Q U E N C Y O F M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S ( M N PCEs) IN BONE M A R R O W OF P R E G N A N T MICE A N D IN T H E LIVER O F T H E I R FETUSES, 30 h A F T E R T R E A T M E N T W I T H C A F F E I N E , M I T O M Y C I N C OR C Y C L O P H O S P H A M I D E Group
C57B1
BDF1
mice/ fetuses
MNPCEs/ 1000 PCEs
Bone marrow of pregnant mice Controls (untreated) Caffeine, 3 × 100 mg,/kg, s.c. a Mitomycin C, 2.0 m g / k g , i.p. Cyclophosphamide, 30 rng/kg, i.p. Caffeine + mitomycin C Caffeine + cyclophosphamide
10 7 7 7 6 6
6.3-t-0.52 6.2 + 0.41 61.6 + 3.48 45.5 + 2.31 82.1 + 3.73 ( + 33%) b 61.2+4.07 (+35%) b
7 7 6 6 5 5
1.6+0.23 1.5+0.21 50.7 + 3.17 39.9 + 2.89 74.0 + 4.32 (+46%) b 54.8 + 3.06 ( + 37%) b
Fetal liver Controls (untreated) Caffeine Mitornycin C Cyclophosphamide Caffeine + mitomycin C Caffeine + cyclophosphamide
31 34 39 42 33 36
6.1 + 0.37 6.4 + 0.42 21.5 + 2.02 12.8 + 0.77 25.4 + 1.85 ( + 14%) b 11.4+1.33 (--11%)b
41 45 43 40 36 33
1.9+0.17 1.8+0.21 12.9+0.53 10.7 -I-0.62 16.6 + 1.06 ( + 29%) b 9.8 + 0.67 (-- 8%) b
a 3 injections, once per day. b Compared to M M C - or CP-treated animals.
mice/ fetuses
MNPCEs/ 1000 PCEs
126
spontaneous MNPCEs frequency were detected (Table 1). No significant sex differences were established either in the sensitivity of the 2 strains used to MMC- and CP-induced clastogenicity in mouse bone marrow, the clastogenic response of C57B1 being better expressed in this case than in BDF~ mice (Table 1). The last conclusion is in force predominantly for the damaging activity of MMC. The pregnancy of C57B1 and DBF 1 female mice had no significant effect on spontaneous or MMCand CP-induced MNPCEs levels although in the latter case a slight tendency to a less expressed clastogenic response was found in bone marrow cells of pregnant compared to virgin female mice. The pretreatment of mice of the 2 strains used with caffeine during 3 consecutive days caused about 24-49% potentiation of MMC-induced clastogenicity and a similar 35-45% potentiation of the CP-induced elevation of bone marrow MNPCEs levels in male, female and pregnant female C57B1 and BDF 1 mice. In general, similar results have already been published revealing the ability of caffeine to potentiate the chromosomedamaging activity of some alkylating agents (namely thiotepa, methyl methanesulfonate and N-methyl-N-nitrosourea) in vivo in mouse bone marrow cells (Frei and Vennitt, 1975; Hansson, 1978). Caffeine's ability to increase the cell killing and chromosome-damaging effects of alkylating agents, probably due to its interference with DNA-repair processes and with cell-cycle progression of damaged cells because it removes the m u t a g e n - i n d u c e d G 2 block, has been investigated in several studies and is well documented (Ceccherini et al., 1988; Peliccia et al., 1988). However, it should be mentioned that simultaneous treatment of mice with caffeine and one of the above-mentioned substances as well as posttreatment (6 h) of mice with caffeine were used by the authors cited (Frei and Vennitt, 1975; Hansson, 1978). In contrast, pretreatment during 3 consecutive days (the last injection given simultaneously with MMC or CP) of mice with caffeine was chosen in the present experiments. Taking into account that methylxanthines might influence different physiological processes in cells not only linked to D N A repair, it seems likely that multiple caffeine pretreatment of animals might better re-
veal the biological consequences when the methylxanthine is given in combination with other pharmacological agents, alkylating substances included. In the present experiments no signs of caffeine potentiation of MMC- and CP-induced clastogenicity were established in fetal livers of either strain. A slight tendency to a diminution of fetal liver MNPCEs was even found in mouse fetuses treated transplacentally with caffeine and CP compared to only CP-treated animals. It should be mentioned that the clastogenic effects of MMC were 3-5-fold better expressed in bone marrow cells of pregnant female mice compared to the number of MNPCEs detected in the livers of their fetuses. Similar trends have been reported by Barale et al. (1987) for treatment of C57BI and CD-1 mice as well as F 1 hybrids with CP. It seems likely that the transplacental micronucleus test is less sensitive than the bone marrow assay when testing the clastogenic activity of MMC and CP. An opposite tendency has been established with other mutagens/carcinogens also requiring metabolic activation like diethylnitrosamine, which could be detected as a clastogen employing the micronucleus test in fetal mouse liver but not in bone marrow cells (Cole et al., 1982). The decreased response of fetal liver erythroblasts to MMC- and CP-induced clastogenic effects might be due to differences in biotransformation of the drugs used, their effective concentrations in the target tissues, their halflife and elimination rates, etc., which are at least partially under genetic control. Obviously, the metabolic competence and protective role of the placenta and fetal liver should be taken into account as factors which might be of crucial importance in the realization of MMC and CP DNA-damaging activity in mouse fetal fiver erythroblasts. It should also be mentioned that in vitro caffeine significantly inhibits the mutagenic effect of MMC in S. typhimurium TA92 (Kim and Levin, 1986), probably due to putative interference with an error-prone repair process as suggested by the authors. Another possible explanation of these data was put forward by MacPhee (1987) who suggested that caffeine might inhibit the excision repair of D N A thus increasing the death rate of bacterial cells with D N A damaged by MMC. If
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such processes could indeed occur in vivo in mice, they would obviously result in a caffeine-induced potentiation of MMC clastogenicity. Bearing in mind that caffeine consumption is a fact of life for millions of human beings, all aspects of its possible genotoxicity when applied alone or in combination with other compounds should be carefully investigated, especially with respect to its use in population groups with a high cancer risk or increased sensitivity to external mutagenic/ carcinogenic stimuli, pregnant women included. References Aeschbacher, H.U. (1986) Rates of micronuclei induction in different mouse strains, Mutation Res., 164, 109-115. Aeschbacher, H.U., H. Meier and E. Jaccaud (1986) The effect of caffeine in the in vivo SCE and micronucleus mutagenicity tests, Mutation Res., 174, 53-58. Barale, R., L. Migliore, D. Zucconi, R. Ciranni, D. Casini, A. Carducci and N. Loprieno (1987) Experimental model for evaluating the effects of genetic and exogenous factors on transplacental mutagenesis, Mutagenesis, 2, 173-177. Ceccherini, I., N. Loprieno and I. Sbrana (1988) Caffeine posttreatment causes a shift in the chromosome aberration types induced by mitomycin C, suggesting a caffeine-sensitive mechanism of DNA repair in G2, Mutagenesis, 3, 39-44. Cole, R., N. Taylor, J. Cole and C. Arlett (1979) Transplacental effects of chemical mutagens detected by the micronucleus test, Nature (London), 277, 317-318. Cole, R., N. Taylor, J. Cole and C. Arlett (1981) Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts, Mutation Res., 80, 141-157. Cole, R., N. Taylor, J. Cole, L. Henderson and C. Arlett (1982) Short-term tests for transplacentally active carcinogens. II. Sensitivity of the transplacental micronucleus test to diethylnitrosamine, Mutation Res., 104, 165-171. Frei, J., and S. Vennitt (1975) Chromosome damage in the bone marrow of mice treated with the methylating agents methyl methanesulfonate and N-methyl-N-nitrosourea in the presence of absence of caffeine, and its relationship with thymoma induction, Mutation Res., 29, 89-96.
Hansson, K. (1987) Caffeine enhancement of chromosomal aberrations induced by thiotepa in bone marrow cells of mice, Hereditas, 89, 129-131. Hansson, K., B. Kihlman, C. Tanzarella and F. Palitti (1984) Influence of caffeine and 3-aminobenzamide in G2 on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine in human lymphocytes, Mutation Res., 126, 251-258. Heddle, J., M. Hite, B. Kirkhart, K. Mavournin, J. MacGregor, G. Newell and M. Salamone (1983) The induction of micronuclei as a measure of genotoxicity, Mutation Res., 123, 61-118. Kihlman, B. (1977) Caffeine and Chromosomes, Elsevier, Amsterdam. Kim, J. and R. Levin (1986) Influence of caffeine on mitomycin C induced mutagenesis, Microbios, 46, 15-20. Levin, R. (1982) Influence of caffeine on mutations induced by nitrosoguanidine in Salmonella typhimurium tester strains, Environ. Mutagen., 4, 689-694. Lialiaris, T., D. Mourelatis and J. Dozi-Vassiliades (1988) Enhancement of cytogenetic damage by chloropromazine in human lymphocytes treated with alkylating antineoplastics and caffeine, Mutation Res., 206, 361-365. MacPhee, D. (1987) Influence of caffeine on mitomycin C induced mutagenesis: a simple explanation, Microbios, 50, 109. Pelliccia, F., G. Belloni, A. Bosi, A. Micheli and G. Olivieri (1988) Studies on chromosome aberrations induced by incorporated tritium: effect of post-treatment with hydroxyurea and caffeine in G2, Mutation Res., 199, 139-144. Pohle, W., and H. Fritzsche (1981) DNA-caffeine interactions. Evidence of different binding models by infrared spectroscopy, Studia Biophys., 82, 81-96. Sato, S., H. Kitajiama, S. Konishi, H. Takizama and N. Inui (1987) Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons, Mutation Res., 192, 185-189. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-46. Somani, S., and P. Gupta (1988) Caffeine: a new look at an age-old drug, Int. J. Clin. Pharmacol. Ther. Toxicol., 26, 521-533. Styles, J., C. Richardson and B. Burlinson (1983) A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyclophosphamide or hexamethyl-phosphoramide, Mutation Res., 122, 143147.
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Elsevier MUT 04921
Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice Penka M. Blagoeva, Roumen M. Balansky and Tzvetana J. Mircheva Laboratory of Chemical Mutagenesis and Carcinogenesis, National Center of Oncology, Sofia 1156 (Bulgaria) (Received 6 February 1990) (Revision received 29 June 1990) (Accepted 2 July 1990)
Keywords: Caffeine; Mitomycin C; Cyclophosphamide; Micronudei; Clastogenicity
Summary Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57B1 or BDF 1 (C57B1 x DBA2) mice with caffeine (1 or 3 x 50 m g / k g and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57B1 and BDF 1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57B1 compared to BDF 1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57B1 mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57B1 and BDF~ mice but not in fetal mouse livers.
Huge population groups are daily exposed to caffeine as a dietary and drug ingredient. Some preliminary epidemiological studies have suggested a possible role for caffeine in human carcinogenesis (Somani and Gupta, 1988). However, despite the intensive studies carried out no
Correspondence: Dr. P.M. Blagoeva, Laboratory of Chemical Mutagenesis and Carcinogenesis, National Center of Oncology, Str. Plovdivsko pole 6, Sofia 1156 (Bulgaria).
clear correlation between caffeine consumption and cancer incidence has so far been established. On the other hand, caffeine has been found to interact with D N A bringing to life a broad spectrum of genetic consequences that are being thoroughly investigated (especially in vitro) as well as to interfere with the genetic activities of other compounds (to mention only the well-documented caffeine potentiation of cytotoxicity and mutagenicity of alkylating agents many of which are strong carcinogens) (Kihlman, 1977; Pohle and Fritzsche,
0027-5107/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
124
1981; Levin, 1982; Hansson et al., 1984; Lialiaris et al., 1988). That is why the possible involvement of caffeine in chemical mutagenesis and carcinogenesis is still a matter of debate and needs further clarification. It should also be mentioned that according to Aeschbacher et al. (1986) caffeine by itself when applied at a dose of 100 m g / k g could cause chromosomal damage in vivo, as shown by elevated micronucleus formation in outbred Swiss CD-1 mice or in inbred M S / A e mice, thus increasing the necessity for additional assessment of the genotoxic potential of caffeine. Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenic activity of caffeine as well as its influence on the DNA-damaging activity of mitomycin C (MMC) and cyclophosphamide (CP) were studied.
(i.p.). Some mice were treated repeatedly with caffeine (100 mg/kg, s.c.) during 3 consecutive days (for pregnant mice days 15-17 were used for this purpose). In several experiments a combined treatment of mice with caffeine (triple treatment) followed by MMC or CP was used (Tables 1 and 2).
Processing of bone marrow The animals were killed by cervical dislocation 30 h after the i.p. injection of MMC or CP or 30 h after the last caffeine administration. The bone marrow smears were prepared and stained with M a y - G r f i n w a l d - G i e m s a according to Schmid (1975). From each mouse (10 mice per group, the number of pregnant mice per group and the litters obtained are indicated in Table 2), 1000 polychromatic erythrocytes (PCEs) were screened for micronuclei.
Materials and methods
Processing of fetal liver Treatment of animals Male, female and pregnant (on day 17 of gestation) female mice of C57B1 and BDF 1 (C57B1 × DBA2) strains 7-10 weeks of age were injected s.c. with caffeine (Merck, F.R.G.) at doses of 50 and 100 m g / k g body weight o r / a n d with MMC (Kyowa, Japan) at a dose of 2 m g / k g (i.p.) o r / a n d with CP (Sigma, U.S.A.) at a dose of 30 m g / k g
Single-cell suspensions of fetal livers were prepared in fetal calf serum by pipetting. The smears were air-dried and stained with M a y - G r t i n w a l d Giemsa (Schmid, 1975; Cole et al., 1979, 1981, 1982). At least 2000 PCEs per fetus were examined. Statistical analysis of the data obtained was performed according to Student's t-test.
TABLE 1 F R E Q U E N C Y OF M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S (MN P C Es ) P ER 1000 PCEs IN BONE M A R R O W OF MICE, 30 h A F T E R T R E A T M E N T W I T H C A F F E I N E , M I T O M Y C I N C O R C Y C L O P H O S P H A M I D E Group a
Control (untreated) Caffeine, 1 × 50 m g / k g , s.c. Caffeine, 1 × 100 m g / k g , s.c. Caffeine, 3 × 100 m g / k g , s.c. b Mitomycin C, 2.0 m g / k g , i.p. Cyclophosphamide, 30 m g / k g , i.p. Caffeine b + mitomycin C Caffeine b + cyclophosphamide
C57BI
BDF 1
male
female
male
female
7.2 + 0.69 7.4_+0.58 7.4 _+0.61 7.6 + 0.55 81.4 .-+4.47 53.1 _+3.39 101.2_+7.12 * 73.4_+4.32 *
6.8 + 0.57 7.1 _+0.42 6.9 _+0.53 7.3 -+ 0.61 74.8 -+ 5.03 51.2 _+3.06 98.2_+6.58 * 70.5_+4.71 *
1.76 + 0.24 1.6_+0.37 1.7 _+0.31 1.5 + 0.25 58.4 _+4.23 44.2 _+3.96 87.1_+5.40 * 63.9_+4.53 *
1.3 + 0.41 1.4_+0.22 1.5 _+0.33 1.3 -+ 0.29 56.6 _+5.11 45.9 _+2.64 83.2_+5.79 * 66.7_+4.03 *
Results are expressed as means + SE. 10 mice per group. b 3 injections, once per day, 3 consecutive days in total. * P < 0.05, compared to only MMC- or CP-treated animals.
125
Results and discussion
The possible clastogenic activity of caffeine was investigated in bone marrow of young male and female mice of C57B1 and BDF 1 strains as well as in the fetal liver. According to Cole et al. (1979, 1981, 1982) the transplacental micronucleus test could be very useful in detecting weak clastogens. The data summarized in Tables 1 and 2 clearly indicate that at the experimental conditions presented no signs of caffeine-induced clastogenicity in mice were established. Even the triple treatment of animals with caffeine (100 mg/kg, s.c.) was ineffective in inducing micronuclei in fetal and maternal mouse erythroblasts. The results obtained are in line with the literature data showing in general a lack of genotoxic activity of caffeine in vivo. The slightly increased level of MNPCEs in bone marrow of Swiss CD-1 outbred mice treated with a huge dose of caffeine (1 or 2 x 100 mg/kg, p.o.) reported by Aeschbacher et al. (1986) might be linked to the specifically high erythroblasts sensitivity to clastogenic agents of this particular mouse strain as indicated by the authors. The data presented added a new
test, namely the transplacental micronucleus assay, that gives negative results when testing caffeine for clastogenicity. In addition, significant strain differences were also established in the present experiments when the spontaneous MNPCEs rates in C57B1 and BDF 1 (C57B1 × DBA2) mice were compared (Table 1). The number of MNPCEs in bone marrow cells of C57B1 mice was about 4-fold higher than that established in BDF~ mice. The same conclusion might be drawn concerning the MNPCEs frequency in fetal livers obtained from litters of the 2 strains used (Table 2). The high spontaneous bone marrow MNPCEs level in C57B1 compared to most other mouse strains investigated so far has been known for some time (Heddle et al., 1983; Styles et al., 1983; Aeschbacher, 1986; Sato et al., 1987). The present study reveals that a similar tendency could be found when screening fetal liver PCEs for micronuclei. Obviously, the genetic reasons that are of importance for the increased bone marrow level of MNPCEs in C57B1 mice were abolished when this particular strain was crossed with DBA2 mice. In addition, no significant sex differences in
TABLE 2 F R E Q U E N C Y O F M I C R O N U C L E A T E D P O L Y C H R O M A T I C E R Y T H R O C Y T E S ( M N PCEs) IN BONE M A R R O W OF P R E G N A N T MICE A N D IN T H E LIVER O F T H E I R FETUSES, 30 h A F T E R T R E A T M E N T W I T H C A F F E I N E , M I T O M Y C I N C OR C Y C L O P H O S P H A M I D E Group
C57B1
BDF1
mice/ fetuses
MNPCEs/ 1000 PCEs
Bone marrow of pregnant mice Controls (untreated) Caffeine, 3 × 100 mg,/kg, s.c. a Mitomycin C, 2.0 m g / k g , i.p. Cyclophosphamide, 30 rng/kg, i.p. Caffeine + mitomycin C Caffeine + cyclophosphamide
10 7 7 7 6 6
6.3-t-0.52 6.2 + 0.41 61.6 + 3.48 45.5 + 2.31 82.1 + 3.73 ( + 33%) b 61.2+4.07 (+35%) b
7 7 6 6 5 5
1.6+0.23 1.5+0.21 50.7 + 3.17 39.9 + 2.89 74.0 + 4.32 (+46%) b 54.8 + 3.06 ( + 37%) b
Fetal liver Controls (untreated) Caffeine Mitornycin C Cyclophosphamide Caffeine + mitomycin C Caffeine + cyclophosphamide
31 34 39 42 33 36
6.1 + 0.37 6.4 + 0.42 21.5 + 2.02 12.8 + 0.77 25.4 + 1.85 ( + 14%) b 11.4+1.33 (--11%)b
41 45 43 40 36 33
1.9+0.17 1.8+0.21 12.9+0.53 10.7 -I-0.62 16.6 + 1.06 ( + 29%) b 9.8 + 0.67 (-- 8%) b
a 3 injections, once per day. b Compared to M M C - or CP-treated animals.
mice/ fetuses
MNPCEs/ 1000 PCEs
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spontaneous MNPCEs frequency were detected (Table 1). No significant sex differences were established either in the sensitivity of the 2 strains used to MMC- and CP-induced clastogenicity in mouse bone marrow, the clastogenic response of C57B1 being better expressed in this case than in BDF~ mice (Table 1). The last conclusion is in force predominantly for the damaging activity of MMC. The pregnancy of C57B1 and DBF 1 female mice had no significant effect on spontaneous or MMCand CP-induced MNPCEs levels although in the latter case a slight tendency to a less expressed clastogenic response was found in bone marrow cells of pregnant compared to virgin female mice. The pretreatment of mice of the 2 strains used with caffeine during 3 consecutive days caused about 24-49% potentiation of MMC-induced clastogenicity and a similar 35-45% potentiation of the CP-induced elevation of bone marrow MNPCEs levels in male, female and pregnant female C57B1 and BDF 1 mice. In general, similar results have already been published revealing the ability of caffeine to potentiate the chromosomedamaging activity of some alkylating agents (namely thiotepa, methyl methanesulfonate and N-methyl-N-nitrosourea) in vivo in mouse bone marrow cells (Frei and Vennitt, 1975; Hansson, 1978). Caffeine's ability to increase the cell killing and chromosome-damaging effects of alkylating agents, probably due to its interference with DNA-repair processes and with cell-cycle progression of damaged cells because it removes the m u t a g e n - i n d u c e d G 2 block, has been investigated in several studies and is well documented (Ceccherini et al., 1988; Peliccia et al., 1988). However, it should be mentioned that simultaneous treatment of mice with caffeine and one of the above-mentioned substances as well as posttreatment (6 h) of mice with caffeine were used by the authors cited (Frei and Vennitt, 1975; Hansson, 1978). In contrast, pretreatment during 3 consecutive days (the last injection given simultaneously with MMC or CP) of mice with caffeine was chosen in the present experiments. Taking into account that methylxanthines might influence different physiological processes in cells not only linked to D N A repair, it seems likely that multiple caffeine pretreatment of animals might better re-
veal the biological consequences when the methylxanthine is given in combination with other pharmacological agents, alkylating substances included. In the present experiments no signs of caffeine potentiation of MMC- and CP-induced clastogenicity were established in fetal livers of either strain. A slight tendency to a diminution of fetal liver MNPCEs was even found in mouse fetuses treated transplacentally with caffeine and CP compared to only CP-treated animals. It should be mentioned that the clastogenic effects of MMC were 3-5-fold better expressed in bone marrow cells of pregnant female mice compared to the number of MNPCEs detected in the livers of their fetuses. Similar trends have been reported by Barale et al. (1987) for treatment of C57BI and CD-1 mice as well as F 1 hybrids with CP. It seems likely that the transplacental micronucleus test is less sensitive than the bone marrow assay when testing the clastogenic activity of MMC and CP. An opposite tendency has been established with other mutagens/carcinogens also requiring metabolic activation like diethylnitrosamine, which could be detected as a clastogen employing the micronucleus test in fetal mouse liver but not in bone marrow cells (Cole et al., 1982). The decreased response of fetal liver erythroblasts to MMC- and CP-induced clastogenic effects might be due to differences in biotransformation of the drugs used, their effective concentrations in the target tissues, their halflife and elimination rates, etc., which are at least partially under genetic control. Obviously, the metabolic competence and protective role of the placenta and fetal liver should be taken into account as factors which might be of crucial importance in the realization of MMC and CP DNA-damaging activity in mouse fetal fiver erythroblasts. It should also be mentioned that in vitro caffeine significantly inhibits the mutagenic effect of MMC in S. typhimurium TA92 (Kim and Levin, 1986), probably due to putative interference with an error-prone repair process as suggested by the authors. Another possible explanation of these data was put forward by MacPhee (1987) who suggested that caffeine might inhibit the excision repair of D N A thus increasing the death rate of bacterial cells with D N A damaged by MMC. If
127
such processes could indeed occur in vivo in mice, they would obviously result in a caffeine-induced potentiation of MMC clastogenicity. Bearing in mind that caffeine consumption is a fact of life for millions of human beings, all aspects of its possible genotoxicity when applied alone or in combination with other compounds should be carefully investigated, especially with respect to its use in population groups with a high cancer risk or increased sensitivity to external mutagenic/ carcinogenic stimuli, pregnant women included. References Aeschbacher, H.U. (1986) Rates of micronuclei induction in different mouse strains, Mutation Res., 164, 109-115. Aeschbacher, H.U., H. Meier and E. Jaccaud (1986) The effect of caffeine in the in vivo SCE and micronucleus mutagenicity tests, Mutation Res., 174, 53-58. Barale, R., L. Migliore, D. Zucconi, R. Ciranni, D. Casini, A. Carducci and N. Loprieno (1987) Experimental model for evaluating the effects of genetic and exogenous factors on transplacental mutagenesis, Mutagenesis, 2, 173-177. Ceccherini, I., N. Loprieno and I. Sbrana (1988) Caffeine posttreatment causes a shift in the chromosome aberration types induced by mitomycin C, suggesting a caffeine-sensitive mechanism of DNA repair in G2, Mutagenesis, 3, 39-44. Cole, R., N. Taylor, J. Cole and C. Arlett (1979) Transplacental effects of chemical mutagens detected by the micronucleus test, Nature (London), 277, 317-318. Cole, R., N. Taylor, J. Cole and C. Arlett (1981) Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts, Mutation Res., 80, 141-157. Cole, R., N. Taylor, J. Cole, L. Henderson and C. Arlett (1982) Short-term tests for transplacentally active carcinogens. II. Sensitivity of the transplacental micronucleus test to diethylnitrosamine, Mutation Res., 104, 165-171. Frei, J., and S. Vennitt (1975) Chromosome damage in the bone marrow of mice treated with the methylating agents methyl methanesulfonate and N-methyl-N-nitrosourea in the presence of absence of caffeine, and its relationship with thymoma induction, Mutation Res., 29, 89-96.
Hansson, K. (1987) Caffeine enhancement of chromosomal aberrations induced by thiotepa in bone marrow cells of mice, Hereditas, 89, 129-131. Hansson, K., B. Kihlman, C. Tanzarella and F. Palitti (1984) Influence of caffeine and 3-aminobenzamide in G2 on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine in human lymphocytes, Mutation Res., 126, 251-258. Heddle, J., M. Hite, B. Kirkhart, K. Mavournin, J. MacGregor, G. Newell and M. Salamone (1983) The induction of micronuclei as a measure of genotoxicity, Mutation Res., 123, 61-118. Kihlman, B. (1977) Caffeine and Chromosomes, Elsevier, Amsterdam. Kim, J. and R. Levin (1986) Influence of caffeine on mitomycin C induced mutagenesis, Microbios, 46, 15-20. Levin, R. (1982) Influence of caffeine on mutations induced by nitrosoguanidine in Salmonella typhimurium tester strains, Environ. Mutagen., 4, 689-694. Lialiaris, T., D. Mourelatis and J. Dozi-Vassiliades (1988) Enhancement of cytogenetic damage by chloropromazine in human lymphocytes treated with alkylating antineoplastics and caffeine, Mutation Res., 206, 361-365. MacPhee, D. (1987) Influence of caffeine on mitomycin C induced mutagenesis: a simple explanation, Microbios, 50, 109. Pelliccia, F., G. Belloni, A. Bosi, A. Micheli and G. Olivieri (1988) Studies on chromosome aberrations induced by incorporated tritium: effect of post-treatment with hydroxyurea and caffeine in G2, Mutation Res., 199, 139-144. Pohle, W., and H. Fritzsche (1981) DNA-caffeine interactions. Evidence of different binding models by infrared spectroscopy, Studia Biophys., 82, 81-96. Sato, S., H. Kitajiama, S. Konishi, H. Takizama and N. Inui (1987) Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons, Mutation Res., 192, 185-189. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-46. Somani, S., and P. Gupta (1988) Caffeine: a new look at an age-old drug, Int. J. Clin. Pharmacol. Ther. Toxicol., 26, 521-533. Styles, J., C. Richardson and B. Burlinson (1983) A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyclophosphamide or hexamethyl-phosphoramide, Mutation Res., 122, 143147.