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Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice
Mutation Research, 282 (1992) 147-150 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
147
MUTLET 0671
Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice Darina Chorvatovi~ov~
a
and Jana Navarov~i b
a Institute of Ecobiology and b Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava (Czechoslovakia) (Received 6 December 1991) (Revision received 24 February 1992) (Accepted 25 February 1992)
Keywords: Carboxymethylglucan; Cyclophosphamide; Antimutagenicity; Micronucleus test; (Mice)
Summary The effect of pretreatment with carboxymethylglucan (CMG) on the frequency of micronuclei induced by cyclophosphamide administration in mice was evaluated. Two doses of CMG (50 mg/kg body weight) injected either intraperitoneally 24 h or intravenously 1 h prior to two cyclophosphamide administrations (80 mg/kg) significantly decreased the frequency of micronucleated PCE in bone marrow. Of two evaluated derivatives of carboxymethylglucan, the K 3 derivative was most efficient. The results show that it is possible to achieve a suppressive effect of soluble carboxymethylglucan prepared from Saccharomyces cerevisiae against cyclophosphamide mutagenicity. The notion may be useful for glucan's effects against pharmacocarcinogenesis. Therapeutic application of glucan with cyclophosphamide therapy may provide a remarkable decrease of the secondary tumour risk. The utilization of these results for human patients needs to be considered.
Treatment with anticancer agents should be connected with the study of their adverse secondary effects. Many anticancer agents have been shown to be mutagenic, teratogenic and carcinogenic in experimental systems and second malignancies are known to be associated with several specific therapeutic treatments (Sorsa et al., 1985). Among them cyclophosphamide, one of the derivatives of nitrogen mustards, attracts much attention from research workers. Cy-
Correspondence: Dr. Darina Chorvatovi~ov~, Institute of Ecobiology, SAS, Mlynsk6 Nivy 59, 814 34 Bratislava, Czechoslovakia.
clophosphamide belongs to a class of occupational carcinogens, the handling of which should involve no unnecessary exposure (Sorsa et al., 1985). It is also a well-known mutagen for many groups of animals in chromosome aberration tests, in sister-chromatid exchange and micronucleus tests in vivo and in vitro (Goetz et al., 1975; R/Shrborn and Basler, 1977; Roszinsky-Koecher and R6hrborn, 1979; Adler and Kliesch, 1990; Madle et al., 1986). Glucan is a component of most yeast and fungal cell walls. It is a typical skeletal constituent and consists of D-glucose units mutually linked by fl-l-3- and /~-l-6-glucosidic bonds (Kogan et al., 1989). Glucans have antitumour,
148 antibacterial, antifungal and antiviral properties, a wide range of reticuloendothelial potentiating activities (Williams e t al., 1983; Song and Di Luzio, 1979; Chihara et al., 1982; Di Luzio et al., 1985). Glucan enhances the antitumour activity of cyclophosphamide, the cytolytic activity of Kupffer cells and the proliferative activity of bone marrow cells (Williams et al., 1987). Most of the results mentioned above were obtained with insoluble glucans from yeast, which are not usable for parenteral application in human medicine. We have already shown the antimutagenic effects of carboxymethylglucans against Co 6° irradiation in mice (Chorvatovi~ov~, 1991). The aim of the present experiments was to study a possible anticlastogenic effect of soluble carboxymethylglucan, prepared from Saccharomyces cerevisiae, against cyclophosphamide-induced clastogenicity in mice.
Materials and methods
boxymethylglucan, fraction II (CMG) was obtained from the Institute of Chemistry, Slovak Academy of Sciences, Bratislava (Sandula et al., 1990). It was isolated from the cell wall of Saccharomyces cerevisiae in two degrees of substitution (DS) of carboxymethyl groups, K 2 = DS 0.56 and K 3 = DS 0.89. Fetal bovine serum was obtained from the University of veterinary Medicine (Brno, Czechoslovakia).
Methods The preparation and staining of bone marrow cells were carried out according to Schmid (1975). The bone marrow cells were flushed out with fetal bovine serum, centrifuged, smeared and stained with May-Griinwald and Giemsa solutions. From each animal 1000 polychromatic erythrocytes (PCE) were examined for micronuclei (MN). The number of PCE was scored in a visual field of 2000 normochromatic erythrocytes (NCE) and the index P C E / ( P C E + NCE) was calculated.
Animals Male Swiss mice (Dobrfi Voda, Czechoslovakia) employed in the experiments were 6 - 8 weeks old and weighed between 18 and 20 g. The animals were maintained on standard mouse food and water ad libitum.
Statistical evaluation
Chemicals
Scheme of the experiment
Cyclophosphamide (Cyclostin ® 200) was from Pharmitalia, Carlo Erba G m b H (Italy). Car-
Cyclophosphamide (CP) administration consisted of two intraperitoneal (i.p.) injections of 80
K a s t e n b a u m - B o w m a n tables (1970) for determining the statistical significance of mutation frequencies were used to compare the differences between the groups.
TABLE 1 INDUCTION OF MICRONUCLEI IN CYCLOPHOSPHAMIDE-TREATEDSWISS MICE INJECTED WITH GLUCAN Application
Group characteristic
PCE with MN/1000 PCE (mean 5:SD)
PCE (%) PCE + NCE
Intravenous +
Control - saline - CMG K 2 - CMG K 3 CP CP + CMG K 2 CP + CMG K 3 CP + CMG K 2 CP+CMG K 3
1.75+ 1.04 1.88+ 1.25 1.63+ 1.19 70.25 a + 16.98 44.38 a,b + 14.85 27.25 a.c + 12.17 46.13 a,b 5:10.90 12.63 a,c 5:8.31
50.72 50.15 51.25 46.86 48.80 48.55 49.38 49:41
Intraperitoneal Intravenous Intraperitoneal
The number of animals in each group is 8. Statistical significance: a p < 0.01 compared to control; b p < 0.05 compared to CP; c p < 0.01 compared to CP.
149 m g / k g bw separated by 24 h. Cyclophosphamide and glucan were dissolved in physiological saline (Imuna, Sari~sk6 Micha~any, Czechoslovakia). Mice were divided into eight groups of 8 animals each. Two groups of mice were injected i.p. 24 h prior to each cyclophosphamide injection with 50 m g / kg bw of either the K 2 or the K 3 carboxymethylglucan derivative. Another two groups of mice were injected intravenously (i.v.) with the same dose of glucan derivatives 1 h prior to cyclophosphamide administration. One group was treated with CP only. Control animals consisted of one group treated with saline and two groups of animals treated either with the K 2 or the K 3 carboxymethylglucan derivative at the same time intervals as the exposed animals. The animals were killed by cervical dislocation 24 h after the second CP injection and frequencies of MN in PCEs were determined. Results The effects of glucan treatment on the frequency of micronuclei induced by cyclophosphamide are given in Table 1. There were statistically significant differences ( P < 0.01) between the control groups and all groups treated with cyclophosphamide. The frequencies of micronuclei significantly decreased in all glucan-treated groups in comparison to the cyclophosphamide group without glucan. Different degrees of suppression were observed within the glucan groups. The most effective reduction of MN was observed with the K 3 derivative of carboxymethylglucan with both ways of application ( P < 0.01). Intraperitoneal application of K 3 24 h prior to CP administration resulted in the most pronounced decrease of CP-induced micronuclei in bone marrow cells of mice. The K 2 derivative also decreased the frequency of micronuclei ( P < 0.05) and no differences were observed between intraperitoneal pretreatment 24 h or intravenous pretreatment 1 h prior to CP administration. Discussion The results of our previous experiment (ChorvatoviEovfi, 1991) confirmed the suppressing effect of carboxymethylglucan on micronucleus
frequencies induced by Co 6° irradiation in mice. The possibility that glucan may radioprotect by traditional radical-scavenging mechanisms could not be excluded (Patchen et al., 1987). The ability of carboxymethyl derivatives of glucan to trap OH radicals was proved (Mi~ik et al., 1990). The efficiency of the K 1 and K 2 derivatives was practically the same and K 3 was more effective. The results presented here and in our previous paper (ChorvatoviEovfi, 1991) confirm that the K 3 derivative has the highest efficiency. As we can see from the results of the present experiment, pretreatment of mice with carboxymethylglucan has decreased the clastogenic effect of the strong mutagen cyclophosphamide. Cyclophosphamide is metabolized by the mixed-function oxidase system and thus likely to induce these enzymes. The dose and duration (acute or chronic) of CP exposure may then determine whether the net effect on the mixed-function oxidase enzymes will be induction or detoxification. Several of the metabolites of CP have - O H functional group (Newman et al., 1990). Scavenging of reactive molecules represents one of the most promising approaches in antimutagenesis and anticarcinogenesis (De Flora and Ramel, 1988). The hydroxyl radical is highly reactive and is regarded as the most important with respect to biological consequences (Yasukawa et al., 1989). The possible mechanism of glucan protection against the clastogenic effects of cyclophosphamide could be the scavenging ability of carboxymethylglucan to trap hydroxyl radicals. The results lead us to conclude that carboxymethylglucan pretreatment of mice suppressed the induction of micronuclei in polychromatic erythrocytes of bone marrow cells. Both ways of application of glucan (i.v. and i.p.) were efficient, only the time of pretreatment prior to cyclophosphamide administration was different. Glucan had to be injected intraperitoneally 24 h prior to CP injection. The results of our study could be useful in the therapy of malignant diseases when cyclophosphamide is applied. Cyclophosphamide treatment alone is associated with an increased risk of leukaemia in case-control studies (Sorsa et al., 1985). These increased risk ratios are obtained when patients are treated with total doses rising up to grammes. A thera-
150
peutic application of carboxymethylglucan with cyclophosphamide administration may provide a remarkable decrease of secondary tumor risk. The clinical significance of these findings for human patients undergoing cyclophosphamide treatment needs to be considered. References Adler, I.-D., and U. Kliesch (1990) Comparison of single and multiple treatment regimens in the mouse bone marrow micronucleus assay for hydroquinone (HQ) and cyclophosphamide (CP), Mutation Res., 234, 115-123. Chihara, G., Y.Y. Maeda and J. Hamuro (1982) Current status and perspectives of immunomodulators of microbial origin, Int. J. Tissue React., 4, 207-225. Chorvatovi~.ovL D. (1991) Suppressing effects of glucan on micronuclei induced by Co 6° in mice, Strahlenther. Onkol., 167, 612-614. De Flora, S., and C. Ramel (1988) Mechanisms of inhibitors of mutagenesis and carcinogenesis. Classification and overview, Mutation Res., 202, 285-306. Di Luzio, N.R., D.L. Williams, E.R. Sherwood and I.W. Browder (1985) Modification of diverse experimental immunosuppressive states by glucan, Surv. lmmunol. Res., 4, 160-167. Goetz, P., R.J. Srfim and J. Dohnalov~i (1975) Relationship between experimental results in mammals and man. I. Cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamide, Mutation Res., 31,247254. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Kogan, G., L. Masler, J. Sandula, J. Navarovfi and T. Trnovec (1989) Recent results on the structural and immunomodulating activities of yeast glucan, in: V. Crescenzi, I.C.M. Dea, S. Paoletti, S.S. Stivala and I.W. Sutherlands (Eds.), Biomedical and Technological Advances in Industrial Polysaccharides, Gordon and Breach, New York, pp. 251258. Madle, E., A. Korte and B. Beek (1986) Species differences in mutagenicity testing. II. Sister-cbromatid exchange and micronucleus induction in rats, mice and Chinese hamster treated with cyclophosphamide, Mutagenesis, 1, 419-422.
Mi~ik, V., D. Gerge! and K. Ondrifi~ (1990) About possible mechanisms of radioprotective effect of carboxymethylglucans, in: Glucans and Other Immunomodulating Polysaccharides, Book of Abstracts of Symposium in High Tatra, p. 43. Newman, M.A., S.S. Quehee, R.S. Schoeny and L. Lowry (1990) Biological monitoring screening of patients provided antineoplastic drug adriamycin, cyclophosphamide, 5-fluorouracil, methotrexate and vincristine, Cancer Res., 50, 3357-3366. Patchen, M.L., M.M. D'Alessandro, I. Brook, W.F. Blakely and T.J. MacVinie (1987) Glucan: Mechanisms involved in its 'radioprotective' effect, J. Leucocyte Biol., 42, 95-105. R6hrborn, G., and A. Basler (1977) Cytogenetic investigations of mammals. Comparison of the genetic activity of cytostatics in mammals, Arch. Toxicol., 38, 35-43. Roszinsky-Koecher, G., and G. R6hrborn (1979) Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations in Chinese hamster bone marrow cells, Hum. Genet., 46, 51-55. Sandula, J., G. Kogan and L. Masler (1990) Structure and some characteristics of the yeast /3-o-glucans, Voprosy Med. Chimiji, 36, 39-42. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Song, M., and N.R. Di Luzio (1979) Yeast glucan and immunotherapy of infectious diseases, in: J.T. Dingle, P.J. Jacques and I.B. Shaw (Eds.), Lysosomes in Biology and Pathology, 6, Elsevier North-Holland, Amsterdam, pp. 533-547. Sorsa, M., K. Hemminki and H. Vainio (1985) Occupational exposure to anticancer drugs: potential and real hazard, Mutation Res., 154, 135-149. Williams, D.L., J.W. Browder and N.R. Di Luzio (1983) lmmunotherapeutic modification of Escherichia coli-induced experimental peritonitis and bacteremia by glucan, Surgery, 93, 448-454. Williams, D.L., E.R. Sherwood, R.B. McNamee, E.L. Jones, I.W. Browder and N.R. Di Luzio (1987) Chemoimmunotherapy of experimental hepatic metastases, Hepatology, 7, 1296-1304. Yasukawa, M., T. Terasima and M. Seki (1989) Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid, Radiat. Res., 120, 456-467. Communicated by I.-D. Adler
147
MUTLET 0671
Suppressing effects of glucan on micronuclei induced by cyclophosphamide in mice Darina Chorvatovi~ov~
a
and Jana Navarov~i b
a Institute of Ecobiology and b Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava (Czechoslovakia) (Received 6 December 1991) (Revision received 24 February 1992) (Accepted 25 February 1992)
Keywords: Carboxymethylglucan; Cyclophosphamide; Antimutagenicity; Micronucleus test; (Mice)
Summary The effect of pretreatment with carboxymethylglucan (CMG) on the frequency of micronuclei induced by cyclophosphamide administration in mice was evaluated. Two doses of CMG (50 mg/kg body weight) injected either intraperitoneally 24 h or intravenously 1 h prior to two cyclophosphamide administrations (80 mg/kg) significantly decreased the frequency of micronucleated PCE in bone marrow. Of two evaluated derivatives of carboxymethylglucan, the K 3 derivative was most efficient. The results show that it is possible to achieve a suppressive effect of soluble carboxymethylglucan prepared from Saccharomyces cerevisiae against cyclophosphamide mutagenicity. The notion may be useful for glucan's effects against pharmacocarcinogenesis. Therapeutic application of glucan with cyclophosphamide therapy may provide a remarkable decrease of the secondary tumour risk. The utilization of these results for human patients needs to be considered.
Treatment with anticancer agents should be connected with the study of their adverse secondary effects. Many anticancer agents have been shown to be mutagenic, teratogenic and carcinogenic in experimental systems and second malignancies are known to be associated with several specific therapeutic treatments (Sorsa et al., 1985). Among them cyclophosphamide, one of the derivatives of nitrogen mustards, attracts much attention from research workers. Cy-
Correspondence: Dr. Darina Chorvatovi~ov~, Institute of Ecobiology, SAS, Mlynsk6 Nivy 59, 814 34 Bratislava, Czechoslovakia.
clophosphamide belongs to a class of occupational carcinogens, the handling of which should involve no unnecessary exposure (Sorsa et al., 1985). It is also a well-known mutagen for many groups of animals in chromosome aberration tests, in sister-chromatid exchange and micronucleus tests in vivo and in vitro (Goetz et al., 1975; R/Shrborn and Basler, 1977; Roszinsky-Koecher and R6hrborn, 1979; Adler and Kliesch, 1990; Madle et al., 1986). Glucan is a component of most yeast and fungal cell walls. It is a typical skeletal constituent and consists of D-glucose units mutually linked by fl-l-3- and /~-l-6-glucosidic bonds (Kogan et al., 1989). Glucans have antitumour,
148 antibacterial, antifungal and antiviral properties, a wide range of reticuloendothelial potentiating activities (Williams e t al., 1983; Song and Di Luzio, 1979; Chihara et al., 1982; Di Luzio et al., 1985). Glucan enhances the antitumour activity of cyclophosphamide, the cytolytic activity of Kupffer cells and the proliferative activity of bone marrow cells (Williams et al., 1987). Most of the results mentioned above were obtained with insoluble glucans from yeast, which are not usable for parenteral application in human medicine. We have already shown the antimutagenic effects of carboxymethylglucans against Co 6° irradiation in mice (Chorvatovi~ov~, 1991). The aim of the present experiments was to study a possible anticlastogenic effect of soluble carboxymethylglucan, prepared from Saccharomyces cerevisiae, against cyclophosphamide-induced clastogenicity in mice.
Materials and methods
boxymethylglucan, fraction II (CMG) was obtained from the Institute of Chemistry, Slovak Academy of Sciences, Bratislava (Sandula et al., 1990). It was isolated from the cell wall of Saccharomyces cerevisiae in two degrees of substitution (DS) of carboxymethyl groups, K 2 = DS 0.56 and K 3 = DS 0.89. Fetal bovine serum was obtained from the University of veterinary Medicine (Brno, Czechoslovakia).
Methods The preparation and staining of bone marrow cells were carried out according to Schmid (1975). The bone marrow cells were flushed out with fetal bovine serum, centrifuged, smeared and stained with May-Griinwald and Giemsa solutions. From each animal 1000 polychromatic erythrocytes (PCE) were examined for micronuclei (MN). The number of PCE was scored in a visual field of 2000 normochromatic erythrocytes (NCE) and the index P C E / ( P C E + NCE) was calculated.
Animals Male Swiss mice (Dobrfi Voda, Czechoslovakia) employed in the experiments were 6 - 8 weeks old and weighed between 18 and 20 g. The animals were maintained on standard mouse food and water ad libitum.
Statistical evaluation
Chemicals
Scheme of the experiment
Cyclophosphamide (Cyclostin ® 200) was from Pharmitalia, Carlo Erba G m b H (Italy). Car-
Cyclophosphamide (CP) administration consisted of two intraperitoneal (i.p.) injections of 80
K a s t e n b a u m - B o w m a n tables (1970) for determining the statistical significance of mutation frequencies were used to compare the differences between the groups.
TABLE 1 INDUCTION OF MICRONUCLEI IN CYCLOPHOSPHAMIDE-TREATEDSWISS MICE INJECTED WITH GLUCAN Application
Group characteristic
PCE with MN/1000 PCE (mean 5:SD)
PCE (%) PCE + NCE
Intravenous +
Control - saline - CMG K 2 - CMG K 3 CP CP + CMG K 2 CP + CMG K 3 CP + CMG K 2 CP+CMG K 3
1.75+ 1.04 1.88+ 1.25 1.63+ 1.19 70.25 a + 16.98 44.38 a,b + 14.85 27.25 a.c + 12.17 46.13 a,b 5:10.90 12.63 a,c 5:8.31
50.72 50.15 51.25 46.86 48.80 48.55 49.38 49:41
Intraperitoneal Intravenous Intraperitoneal
The number of animals in each group is 8. Statistical significance: a p < 0.01 compared to control; b p < 0.05 compared to CP; c p < 0.01 compared to CP.
149 m g / k g bw separated by 24 h. Cyclophosphamide and glucan were dissolved in physiological saline (Imuna, Sari~sk6 Micha~any, Czechoslovakia). Mice were divided into eight groups of 8 animals each. Two groups of mice were injected i.p. 24 h prior to each cyclophosphamide injection with 50 m g / kg bw of either the K 2 or the K 3 carboxymethylglucan derivative. Another two groups of mice were injected intravenously (i.v.) with the same dose of glucan derivatives 1 h prior to cyclophosphamide administration. One group was treated with CP only. Control animals consisted of one group treated with saline and two groups of animals treated either with the K 2 or the K 3 carboxymethylglucan derivative at the same time intervals as the exposed animals. The animals were killed by cervical dislocation 24 h after the second CP injection and frequencies of MN in PCEs were determined. Results The effects of glucan treatment on the frequency of micronuclei induced by cyclophosphamide are given in Table 1. There were statistically significant differences ( P < 0.01) between the control groups and all groups treated with cyclophosphamide. The frequencies of micronuclei significantly decreased in all glucan-treated groups in comparison to the cyclophosphamide group without glucan. Different degrees of suppression were observed within the glucan groups. The most effective reduction of MN was observed with the K 3 derivative of carboxymethylglucan with both ways of application ( P < 0.01). Intraperitoneal application of K 3 24 h prior to CP administration resulted in the most pronounced decrease of CP-induced micronuclei in bone marrow cells of mice. The K 2 derivative also decreased the frequency of micronuclei ( P < 0.05) and no differences were observed between intraperitoneal pretreatment 24 h or intravenous pretreatment 1 h prior to CP administration. Discussion The results of our previous experiment (ChorvatoviEovfi, 1991) confirmed the suppressing effect of carboxymethylglucan on micronucleus
frequencies induced by Co 6° irradiation in mice. The possibility that glucan may radioprotect by traditional radical-scavenging mechanisms could not be excluded (Patchen et al., 1987). The ability of carboxymethyl derivatives of glucan to trap OH radicals was proved (Mi~ik et al., 1990). The efficiency of the K 1 and K 2 derivatives was practically the same and K 3 was more effective. The results presented here and in our previous paper (ChorvatoviEovfi, 1991) confirm that the K 3 derivative has the highest efficiency. As we can see from the results of the present experiment, pretreatment of mice with carboxymethylglucan has decreased the clastogenic effect of the strong mutagen cyclophosphamide. Cyclophosphamide is metabolized by the mixed-function oxidase system and thus likely to induce these enzymes. The dose and duration (acute or chronic) of CP exposure may then determine whether the net effect on the mixed-function oxidase enzymes will be induction or detoxification. Several of the metabolites of CP have - O H functional group (Newman et al., 1990). Scavenging of reactive molecules represents one of the most promising approaches in antimutagenesis and anticarcinogenesis (De Flora and Ramel, 1988). The hydroxyl radical is highly reactive and is regarded as the most important with respect to biological consequences (Yasukawa et al., 1989). The possible mechanism of glucan protection against the clastogenic effects of cyclophosphamide could be the scavenging ability of carboxymethylglucan to trap hydroxyl radicals. The results lead us to conclude that carboxymethylglucan pretreatment of mice suppressed the induction of micronuclei in polychromatic erythrocytes of bone marrow cells. Both ways of application of glucan (i.v. and i.p.) were efficient, only the time of pretreatment prior to cyclophosphamide administration was different. Glucan had to be injected intraperitoneally 24 h prior to CP injection. The results of our study could be useful in the therapy of malignant diseases when cyclophosphamide is applied. Cyclophosphamide treatment alone is associated with an increased risk of leukaemia in case-control studies (Sorsa et al., 1985). These increased risk ratios are obtained when patients are treated with total doses rising up to grammes. A thera-
150
peutic application of carboxymethylglucan with cyclophosphamide administration may provide a remarkable decrease of secondary tumor risk. The clinical significance of these findings for human patients undergoing cyclophosphamide treatment needs to be considered. References Adler, I.-D., and U. Kliesch (1990) Comparison of single and multiple treatment regimens in the mouse bone marrow micronucleus assay for hydroquinone (HQ) and cyclophosphamide (CP), Mutation Res., 234, 115-123. Chihara, G., Y.Y. Maeda and J. Hamuro (1982) Current status and perspectives of immunomodulators of microbial origin, Int. J. Tissue React., 4, 207-225. Chorvatovi~.ovL D. (1991) Suppressing effects of glucan on micronuclei induced by Co 6° in mice, Strahlenther. Onkol., 167, 612-614. De Flora, S., and C. Ramel (1988) Mechanisms of inhibitors of mutagenesis and carcinogenesis. Classification and overview, Mutation Res., 202, 285-306. Di Luzio, N.R., D.L. Williams, E.R. Sherwood and I.W. Browder (1985) Modification of diverse experimental immunosuppressive states by glucan, Surv. lmmunol. Res., 4, 160-167. Goetz, P., R.J. Srfim and J. Dohnalov~i (1975) Relationship between experimental results in mammals and man. I. Cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamide, Mutation Res., 31,247254. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Kogan, G., L. Masler, J. Sandula, J. Navarovfi and T. Trnovec (1989) Recent results on the structural and immunomodulating activities of yeast glucan, in: V. Crescenzi, I.C.M. Dea, S. Paoletti, S.S. Stivala and I.W. Sutherlands (Eds.), Biomedical and Technological Advances in Industrial Polysaccharides, Gordon and Breach, New York, pp. 251258. Madle, E., A. Korte and B. Beek (1986) Species differences in mutagenicity testing. II. Sister-cbromatid exchange and micronucleus induction in rats, mice and Chinese hamster treated with cyclophosphamide, Mutagenesis, 1, 419-422.
Mi~ik, V., D. Gerge! and K. Ondrifi~ (1990) About possible mechanisms of radioprotective effect of carboxymethylglucans, in: Glucans and Other Immunomodulating Polysaccharides, Book of Abstracts of Symposium in High Tatra, p. 43. Newman, M.A., S.S. Quehee, R.S. Schoeny and L. Lowry (1990) Biological monitoring screening of patients provided antineoplastic drug adriamycin, cyclophosphamide, 5-fluorouracil, methotrexate and vincristine, Cancer Res., 50, 3357-3366. Patchen, M.L., M.M. D'Alessandro, I. Brook, W.F. Blakely and T.J. MacVinie (1987) Glucan: Mechanisms involved in its 'radioprotective' effect, J. Leucocyte Biol., 42, 95-105. R6hrborn, G., and A. Basler (1977) Cytogenetic investigations of mammals. Comparison of the genetic activity of cytostatics in mammals, Arch. Toxicol., 38, 35-43. Roszinsky-Koecher, G., and G. R6hrborn (1979) Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations in Chinese hamster bone marrow cells, Hum. Genet., 46, 51-55. Sandula, J., G. Kogan and L. Masler (1990) Structure and some characteristics of the yeast /3-o-glucans, Voprosy Med. Chimiji, 36, 39-42. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Song, M., and N.R. Di Luzio (1979) Yeast glucan and immunotherapy of infectious diseases, in: J.T. Dingle, P.J. Jacques and I.B. Shaw (Eds.), Lysosomes in Biology and Pathology, 6, Elsevier North-Holland, Amsterdam, pp. 533-547. Sorsa, M., K. Hemminki and H. Vainio (1985) Occupational exposure to anticancer drugs: potential and real hazard, Mutation Res., 154, 135-149. Williams, D.L., J.W. Browder and N.R. Di Luzio (1983) lmmunotherapeutic modification of Escherichia coli-induced experimental peritonitis and bacteremia by glucan, Surgery, 93, 448-454. Williams, D.L., E.R. Sherwood, R.B. McNamee, E.L. Jones, I.W. Browder and N.R. Di Luzio (1987) Chemoimmunotherapy of experimental hepatic metastases, Hepatology, 7, 1296-1304. Yasukawa, M., T. Terasima and M. Seki (1989) Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid, Radiat. Res., 120, 456-467. Communicated by I.-D. Adler