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Anticlastogenic effects of Ginkgo biloba extract (EGb 761) and some of its constituents in irradiated rats
Mutation Research 445 Ž1999. 99–104 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres
Anticlastogenic effects of Ginkgo biloba extract žEGb 761 / and some of its constituents in irradiated rats A. Alaoui-Youssefi a , I. Lamproglou b, K. Drieu c , I. Emerit a
a,)
´ Institut Sante´ et DeÕeloppement, UniÕersite´ Paris VI, 15-21 Rue de l’Ecole de Medecine, 75006 Paris, France ´ ´ b Biophysics Laboratory, XaÕier Bichat School of Medicine, Paris, France c IHB-IPSEN, 24, Rue Erlanger 75015 Paris, France Received 31 March 1999; received in revised form 8 July 1999; accepted 21 July 1999
Abstract In a previous study we reported that radiation-induced clastogenic factors ŽCF. are found in the plasma of Chernobyl accident recovery workers and that their chromosome damaging effects are inhibited by antioxidant treatment with a Ginkgo biloba extract ŽEGb761.. In the present study, we induced CF in rats with a radiation dose of 4.5 Gy. The protective effects of the complete extract were compared to those obtained with the extract devoid of its terpene fraction ŽCP205., with isolated ginkgolides A q B and bilobalide at the concentrations present in EGb761. The pretreatment samples were taken at day 22 postirradiation, the posttreatment samples the day following arrest of the 3-week treatment. The adjusted clastogenic score ŽACS. were reduced from 11.71 " 3.55 to 2.00 " 2.83 after treatment with 100 mgrkg and from 13.43 " 2.23 to 4.29 " 2.14 with 50 mgrkg of the complete extract Ž p - 0.0001.. Similar protective effects were observed with CP205, ginkgolides and bilobalide Ž p - 0.001., while the reduction of ACS in placebo-treated rats was not statistically significant Ž12.80 " 1.79 and 9.20 " 2.68.. However, if the efficacy of the treatment was compared to placebo, only the complete extract was significantly protective. While all components exerted anticlastogenic effects at the concentrations present in the complete extract, the comparison of the different groups by analysis of variance did not reveal significant differences. This may be due to to the small number of animals available in each treatment group. The complete extract reduced the ACS by 83% at the dose of 100 mgrkg, while the lower dose of 50 mgrkg and the three components reached only 66%–68% reduction. The better protection provided by the complete extract is due to synergistic rather than to additive effects. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Clastogenic factor; Chernobyl; Antioxidant; Ginkgo biloba extract; Ginkgolides; Bilobalide
1. Introduction Chromosomal aberrations resulting from ionizing radiation are well documented. While a large body of ) Corresponding author. Tel.: q33-01-43-29-99-39; fax: q3301-43-29-99-39
evidence supports a direct effect of ionizing radiation on the chromosomes of exposed cells, several observations suggest indirect radiation effects due to the presence of plasma factors able of inducing chromosomal aberrations in unexposed cells. Such chromosomal breakage or clastogenic factors ŽCF. were first described in the plasma of patients, who had been
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A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
irradiated accidentally w1x or therapeutically w2x. They were also observed in A-bomb survivors, where they persisted for many years after irradiation w3x. In a previous study, we reported that clastogenic activity was present in the plasma of Chernobyl accident recovery workers w4x and in children exposed to radiation as the consequence of this disaster w5x. We were also able to show that the damaging effects of these CF can be inhibited in vitro and in vivo by treatment with an extract from Ginkgo biloba leaves ŽEGb761. w6,7x. In the present study, we tried to determine which component of this extract was responsible for the protective effects. For this purpose, we choose an animal model, previously shown to respond to X-rays with CF formation w8x.
2. Materials and methods 2.1. Animals Male Wistars rats ŽIFFA CREDO, Les Oncins France., 3 months of age were used for this study. They weighed approximately 400 g when they were irradiated. 2.2. Radiation exposure Whole body irradiation was administered by a telecobalt unit to deliver a dose of 4.5 Gy in one fraction using two parallel opposed equally weighted lateral fields Ž8 = 8 cm at source axis distance, 80 cm.. Blood samples were taken on day 22 and 44 after irradiation and immediately centrifuged for plasma preparation. During the 3-week period between days 22 and 44, the irradiated animals were treated with EGb761, its components or water as placebo. A group of 14 animals was studied at day 8 after irradiation. In addition, a group of 10 control animals was studied in order to exclude that rat plasma exerts chromosome-damaging effects in human cell cultures. These animals were handled in the same manner as the radiation-exposed animals. They were sham-irradiated and received water from day 22 to
day 44 according to the same treatment schedule. The plasma samples for cytogenetic testing were taken on day 22 after sham-irradiation. This study was conducted in conformity with the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human services N.I.H. Publication No. 85-23, revised Ž1985.. 2.3. CF preparation and cytogenetic test system The cytogenetic procedures for detection of CF in rat plasma followed the assay described previously for human samples w4,5,7,9x. Since it is known that the clastogenic activity is in the small molecular weight fraction, the plasma is ultrafiltrated through a Millipore or Amicon ultrafiltration filter with a cut off at 30,000 Da. The ultrafiltration step is useful for elimination of all high molecular weight materials, which might disturb culture growth due to blood group incompatibilities. Regular blood cultures were set up with whole blood from a healthy donor, to which 100 ml of the plasma ultrafiltrate were added. Since the clastogenic effects are related to oxyradical production w9x, a culture medium poor in free radical scavengers has to be used. In the present study TCM199 ŽFlow Laboratories, Paris. Ž5 mlrculture tube. was supplemented with fetal calf serum Ž1 ml. ŽGibco, France., previously tested for lack of anticlastogenic properties. Lymphocyte proliferation was stimulated by the addition of phytohemagglutinin ŽWellcome Diagnostics, Dartford, UK.. After 72 h of incubation at 378C, the mitoses were arrested in metaphase by the addition of colchicine 2 h before harvesting. Microscopic slides were prepared for chromosomal analysis according to standard procedures. The chromosomes of 50 well-spread and complete metaphases Ž10 on each of five coded slides. were examined for the presence of breaks, fragments, exchanges, rings, dicentrics and other morphological abnormalities. Culture preparations with low mitotic index were eliminated, and the culture was repeated. A series of ultrafiltrates was tested the same day on cultures set up with the blood of the same donor. Two additional control cultures without ultrafiltrate served for the establishment of the spontaneous chromosomal aberration rate of the donor’s lymphocytes. This background
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
level of aberrations was subtracted from the aberration rate in ultrafiltrate-treated cultures of the same blood donor. The difference between the two values is called the ACS, which is given as a percentage for convenience w4,5,7x. This way of treating results is necessary for comparison of clastogenic activity of samples collected at subsequent dates and tested on cultures with different background level of aberrations. When background levels were studied on 10 parallel cultures set up with the same blood, the variation did not exceed "3 aberrations per 50 cells or "6 aberrations per 100 cells studied. This range was the same for two independent observers. Therefore a plasma ultrafiltrate is considered to be clastogenic, if it induces more than 3 aberrations per 50 cells. In agreement herewith, the increase in aberrations induced by ultrafiltrates from a series of 96 healthy blood donors did not exceed 4% additional aberrations for the majority of them. Only with five of these normal samples, the increase represented q6%, while increases of q8% or higher were not observed w7x. On the basis of these control data, rat plasma inducing q8% aberrations in the test system were considered as abnormal and called CFq. 2.4. Treatment EGb761 is a standardized extract of Ginkgo biloba leaves. It contains 24% flavonol glycosides and 6% terpene trilactones Ž3.1% ginkgolides and 2.9% bilobalide. w10,11x. The complete extract EGb761, an extract from which terpene trilactones had been removed ŽCP205., ginkgolides A and B and bilobalide were provided by IHB-IPSEN ŽParis, France.. All drugs were administered orally. Ginkgolides were used in their water soluble formulation Ži.e., an equilibrium of closed and open lactone forms at pH 8.75.. Bilobalide was administered in suspension in 0.5% Carboxy methyl cellulose ŽCMC.. Extracts ŽEGb761 and CP205. were dissolved in water. Treatment was given orally Žgavage. once a day during a period of 3 weeks. The complete extract EGb761 was tested at two doses Ž50 and 100 mgrkg.. Another series of rats received CP205 Ž100 mgrkg.. Ginkgolides A q B and bilobalide were studied at a dose Ž4 mgrkg., which is slightly higher than their
101
concentration in the complete extract EGb761. Five irradiated rats received water as placebo.
3. Results and discussion The present study confirms the findings of Faguet et al. w8x, who reported that the plasma of irradiated rats has clastogenic properties in cultures set up with blood of unexposed rats. The clastogenic activity was demonstrable immediately after irradiation, diminished therafter, but remained significantly increased until the end of their study period Ž10 weeks.. We studied a group of 14 animals at day 8 after irradiation and noted clastogenic activity in all samples, with a mean ACS of 13.33 " 2.37 Ž p - 0.0001 compared to plasma ultrafiltrates from unexposed rats.. The mean values of ACS with plasma from nonirradiated rats was 1.2 " 2.1, similar to values observed with plasma ultrafiltrates from healthy humans Ž0.8 " 1.0. w7x. The clastogenic activity was lower in the plasma of rats studied at day 22 postirradiation Ž9.87 " 4.17., but was still significantly increased compared to control plasma Ž p - 0.0001.. At that time point, only 72% of the animals were positive for CF ŽACS q 8% and higher.. Despite the significant decrease in clastogenic activity between day 8 and day 22 postirradiation Ž p - 0.01., the later date was chosen in the following experiments comparing the efficacy of the complete extract with that of its components. The reason herefore was the intention to treat cancer patients with EGb761, the treatment starting 3 weeks after radiation therapy. The results obtained for the 37 CFq animals are shown in Table 1. The comparison of ACS before and after treatment showed significant differences for all treatment groups except for the ‘‘placebo’’ group, i.e., the group of animals receiving water according to the same treatment schedule postirradiation. The percentage of reduction Ž28%. observed for this group may represent the spontaneous regression of CF between day 22 and day 44. If the ACS of the placebo group are considered for comparison at day 44, the ACS of all treatment groups are significantly lower ŽTable 1.. Therefore, the reduction of the clastogenic scores is not explained by the spontaneous regression of the clastogenic activity, but is due in large part to the
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
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Table 1 Clastogenic activity in the plasma of irradiated rats before and after the 3-week treatment with EGb761 or its components All animals are CFq before treatment. Comparison: Ž1. ‘‘Before’’r‘‘After’’ ŽStudent’s t-paired test. U p - 0.01; UU p - 0.001; UUU p - 0.0001; Ž2. With placebo-group ŽStudent’s t-test. 8 p - 0.05; 88 p - 0.01; 888 p - 0.001; Ž3. Analysis of variance: no difference between treatment groups for the efficacy of the protection.
Before
After
Animals CFq after treatment
5
12.80 " 1.79
9.20 " 2.68 NS
4
28%
7
11.71 " 3.55
2.00 " 2.83UUU 888
1
83%
7 8
13.43 " 2.23 11.75 " 1.67
4.29 " 2.14UUU 88 4.00 " 4.00UUU 8
1 3
68% 66%
5
11.20 " 1.79
3.60 " 2.61UU 88
1
68%
5
12.80 " 4.15
4.40 " 1.67U 88
0
66%
n
Placebo-treated rats Ždist. water. Complete extract 100 mgrkg 50 mgrkg Extract without terpenoids 100 mgrkg Gingkolides A q B 4 mgrkg Bilobalide 4 mgrkg
ACS
treatment. All animals showed reduced ACS after treatment compared to the pretreatment values for all treatment groups. While all components exerted anticlastogenic effects at the concentrations present in the complete extract, the comparison of the different groups by analysis of variance did not reveal significant differences. This may be due to the small number of animals available in each treatment group. The complete extract reduced the ACS by 83% at the dose of 100 mgrkg, while the lower dose of 50 mgrkg and
Percentage of reduction
the three components reached only 66%–68% reduction. Since all components were able to reduce the aberrations rates by 66%, the better protection provided by the complete extract was not additive, but rather due to synergistic effects. Only results observed in the 37 animals positive for CF at day 22 postirradiation are shown in Table 1. The other 13 animals negative in the CF-test at that time point were also treated, since the results were not known, when the treatment was started. No increase in chromosomal aberration rates were observed in these
Table 2 Nature of chromosomal aberrations induced with plasma from irradiated rats. Effect of treatment Without treatment n s 32 Ž%. Telomeric extrusions Chromatid breaks Isochromatid breaks FragmentsU Dots and double minutes DicentricsU Tri- and quadriradials Total Number of mitoses studied Total number of aberrations Percentage U
26.1 50.0 14.3 5.4 2.1 1.4 0.7 100 1600 280 17.6
With treatment n s 32 Ž%. 30.6 47.6 20.4 0.7 0.7
100 1600 147 9.3
Incubation time 72 h: the presence of dicentrics possibly due to a second division, on the other hand, fragments may have been lost.
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
animals at day 44, indicating that neither the complete extract nor its components has chromosome damaging effects. No adverse side effects were observed in the animals with the different treatments. As already stressed by the first reports w1,2,12x and confirmed by the report of Faguet et al. in rats, as well as by our previous work in irradiated populations w4x, CF-induced chromosome damage is mainly of the chromatid type. This was also the case in the present study ŽTable 2.. There were rare dicentrics, fragments and tri- or quadriradial configurations. Their respective frequencies were 1.7% in 1600 mitoses for cultures exposed to plasma ultrafiltrates of irradiated animals and 0.13% in 1600 mitoses exposed to plasma ultrafiltrates of animals treated with EGb761 or its components after irradiation Ž p 0.001.. In addition to the reduction of aberration frequencies, these differences in the nature of aberrations may be interpreted in favor of protective effects by EGb761 and its components. After 72 h of incubation, a certain number of cells is at their second division in culture, and certain aberrations may arise or be lost during replication. Therefore, most researchers interested in the nature of aberrations use a 48-h incubation time. However, the number of mitoses available at 48 h with TCM 199 medium is low. Nevertheless, this medium was adopted for the study since it has been found previously to be optimal because of its low antioxidant content w9x. The CF-test intends to demonstrate breakage effects, and the nature of aberrations was not the aim of this study. Ginkgo biloba extracts influence the mitotic index only at high doses, which were not reached in the plasma of animals by the treatment w6x. Therefore, variations in cell cycle conditions should not have influenced the outcome of the experiments. CF-induced clastogenic effects are superoxidemediated, as discussed in more detail elsewhere w13x. A previous study using SOD in comparison to EGb761 against CF-induced chromosome damage in vitro showed that EGb761 provided protective effects similar to those obtained with SOD w6x. It is therefore probable that the anticlastogenic effects of EGb761 are related to its antioxidant properties. EGb761 has been reported to scavenge superoxide anions in a dose-dependent manner, as indicated by electron spin resonance at high concentrations Ž125
103
and 500 mgrml. w14x. The antioxidant properties of CP205 are probably due to its content in flavonoids, known for their superoxide scavenging properties w15–17x. The antioxidant properties of ginkgolides at very low concentrations were shown in a model of ischemia reperfusion injury w18x. The protective effects were more pronounced in vivo than expected after the in vitro experiments. The authors hypothesized that the protective effects might be due to diminished oxyradical release by cells rather than to extracellular free radical scavenging. The other terpene compound, bilobalide, was found to react with superoxide by EPR and UVrVIS spectroscopy in an aprotic environment w19x. The doses used for the present study in rats are known to provide blood concentrations of active principles close to those reached in humans with the complete extract or its components. These concentrations of EGb761 were protective in our previous studies with Chernobyl-exposed persons w6x. The present study indicates that anticlastogenic effects are exerted in this model of superoxide-mediated clastogenesis by all components, but that the complete extract appears to provide the best protection. Similar observations were obtained with other plant extracts w20x. The mechanisms of these synergistic effects of the various components remain unexplained. Further studies have to be conducted with the principal components using higher concentrations than those present in EGb761.
Acknowledgements We are grateful to Professor F. Baillet and the Department of Radiotherapy, La Pitie-Salpetriere ´ ´ ` Hospital, Paris, where the animals were irradiated.
References w1x K.O. Goh, H. Summer, Breaks in normal human chromosomes: are they induced by a transferable substance in the plasma of irradiated persons exposed to total body irradiation?, Radiat. Res. 6 Ž1968. 51–60. w2x J.G. Hollowell, L.G. Littlefield, Chromosome damage induced by plasma from irradiated patients. An indirect effect of X-ray, Proc. Soc. Exp. Biol. Med. 129 Ž1968. 240–244. w3x G.S. Pant, N. Kamada, Chromosome aberrations in normal
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w18x
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Recent Results in Pharmacology and Clinic. Springer-Verlag, Berlin, pp. 32–36. D. Scott, The effect of irradiated plasma on normal human chromosomes and its relevance to the long-lived lymphocyte hypothesis, Cell Tissue Kinet. 2 Ž1969. 295–305. I. Emerit, F. Garban, J. Vassy, A. Levy, P. Filipe, J. Freitas, Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 12799–12804. J. Pincemail, M. Dupuis, C. Nasr, P. Hans, M.H. Berrurier, R. Anton, C. Deby, Experentia 45 Ž1989. 708–711. J. Robak, R.J. Gryglewski, Flavonoids are scavengers of superoxide anions, Biochem. Pharmacol. 37 Ž1988. 837–842. W. Bors, M. Saran, Radical scavenging by flavonoid antioxidants, Free Radical Res. Commun. 2 Ž1987. 289–295. W. Bors, W. Heller, C. Michel, M. Saran, 1990. Radical chemistry of flavonoid antioxidants. In: I. Emerit, L. Packer, C. Auclair ŽEds.., Antioxidants in Therapy and Preventive Medicine. Plenum, New York. S. Pietri, E. Maurelli, K. Drieu, M. Culcasi, Cardioprotective and antioxidant effects of the terpenoid constituents of Ginkgo biloba extract ŽEGb761., J. Mol. Cell. Cardiol. 29 Ž1997. 733–742. H. Scholtyssek, W. Damereau, R. Wessel, I. Schimke, Antioxidative activity of ginkgolides against superoxide in an aprotic environment, Chemico-Biological Interactions 106 Ž1997. 183–190. I. Emerit, N. Oganesian, R. Arutyunian, A. Pogossian, T. Sarkisian, L. Cernjavski, A. Levy, J. Feingold, Oxidative stress-related clastogenic factors in plasma from Chernobyl liquidators: protective effects of antioxidant plant phenols, vitamins and oligoelements, Mutat. Res. 377 Ž1997. 239–246.
Anticlastogenic effects of Ginkgo biloba extract žEGb 761 / and some of its constituents in irradiated rats A. Alaoui-Youssefi a , I. Lamproglou b, K. Drieu c , I. Emerit a
a,)
´ Institut Sante´ et DeÕeloppement, UniÕersite´ Paris VI, 15-21 Rue de l’Ecole de Medecine, 75006 Paris, France ´ ´ b Biophysics Laboratory, XaÕier Bichat School of Medicine, Paris, France c IHB-IPSEN, 24, Rue Erlanger 75015 Paris, France Received 31 March 1999; received in revised form 8 July 1999; accepted 21 July 1999
Abstract In a previous study we reported that radiation-induced clastogenic factors ŽCF. are found in the plasma of Chernobyl accident recovery workers and that their chromosome damaging effects are inhibited by antioxidant treatment with a Ginkgo biloba extract ŽEGb761.. In the present study, we induced CF in rats with a radiation dose of 4.5 Gy. The protective effects of the complete extract were compared to those obtained with the extract devoid of its terpene fraction ŽCP205., with isolated ginkgolides A q B and bilobalide at the concentrations present in EGb761. The pretreatment samples were taken at day 22 postirradiation, the posttreatment samples the day following arrest of the 3-week treatment. The adjusted clastogenic score ŽACS. were reduced from 11.71 " 3.55 to 2.00 " 2.83 after treatment with 100 mgrkg and from 13.43 " 2.23 to 4.29 " 2.14 with 50 mgrkg of the complete extract Ž p - 0.0001.. Similar protective effects were observed with CP205, ginkgolides and bilobalide Ž p - 0.001., while the reduction of ACS in placebo-treated rats was not statistically significant Ž12.80 " 1.79 and 9.20 " 2.68.. However, if the efficacy of the treatment was compared to placebo, only the complete extract was significantly protective. While all components exerted anticlastogenic effects at the concentrations present in the complete extract, the comparison of the different groups by analysis of variance did not reveal significant differences. This may be due to to the small number of animals available in each treatment group. The complete extract reduced the ACS by 83% at the dose of 100 mgrkg, while the lower dose of 50 mgrkg and the three components reached only 66%–68% reduction. The better protection provided by the complete extract is due to synergistic rather than to additive effects. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Clastogenic factor; Chernobyl; Antioxidant; Ginkgo biloba extract; Ginkgolides; Bilobalide
1. Introduction Chromosomal aberrations resulting from ionizing radiation are well documented. While a large body of ) Corresponding author. Tel.: q33-01-43-29-99-39; fax: q3301-43-29-99-39
evidence supports a direct effect of ionizing radiation on the chromosomes of exposed cells, several observations suggest indirect radiation effects due to the presence of plasma factors able of inducing chromosomal aberrations in unexposed cells. Such chromosomal breakage or clastogenic factors ŽCF. were first described in the plasma of patients, who had been
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 3 9 - 4
100
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
irradiated accidentally w1x or therapeutically w2x. They were also observed in A-bomb survivors, where they persisted for many years after irradiation w3x. In a previous study, we reported that clastogenic activity was present in the plasma of Chernobyl accident recovery workers w4x and in children exposed to radiation as the consequence of this disaster w5x. We were also able to show that the damaging effects of these CF can be inhibited in vitro and in vivo by treatment with an extract from Ginkgo biloba leaves ŽEGb761. w6,7x. In the present study, we tried to determine which component of this extract was responsible for the protective effects. For this purpose, we choose an animal model, previously shown to respond to X-rays with CF formation w8x.
2. Materials and methods 2.1. Animals Male Wistars rats ŽIFFA CREDO, Les Oncins France., 3 months of age were used for this study. They weighed approximately 400 g when they were irradiated. 2.2. Radiation exposure Whole body irradiation was administered by a telecobalt unit to deliver a dose of 4.5 Gy in one fraction using two parallel opposed equally weighted lateral fields Ž8 = 8 cm at source axis distance, 80 cm.. Blood samples were taken on day 22 and 44 after irradiation and immediately centrifuged for plasma preparation. During the 3-week period between days 22 and 44, the irradiated animals were treated with EGb761, its components or water as placebo. A group of 14 animals was studied at day 8 after irradiation. In addition, a group of 10 control animals was studied in order to exclude that rat plasma exerts chromosome-damaging effects in human cell cultures. These animals were handled in the same manner as the radiation-exposed animals. They were sham-irradiated and received water from day 22 to
day 44 according to the same treatment schedule. The plasma samples for cytogenetic testing were taken on day 22 after sham-irradiation. This study was conducted in conformity with the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human services N.I.H. Publication No. 85-23, revised Ž1985.. 2.3. CF preparation and cytogenetic test system The cytogenetic procedures for detection of CF in rat plasma followed the assay described previously for human samples w4,5,7,9x. Since it is known that the clastogenic activity is in the small molecular weight fraction, the plasma is ultrafiltrated through a Millipore or Amicon ultrafiltration filter with a cut off at 30,000 Da. The ultrafiltration step is useful for elimination of all high molecular weight materials, which might disturb culture growth due to blood group incompatibilities. Regular blood cultures were set up with whole blood from a healthy donor, to which 100 ml of the plasma ultrafiltrate were added. Since the clastogenic effects are related to oxyradical production w9x, a culture medium poor in free radical scavengers has to be used. In the present study TCM199 ŽFlow Laboratories, Paris. Ž5 mlrculture tube. was supplemented with fetal calf serum Ž1 ml. ŽGibco, France., previously tested for lack of anticlastogenic properties. Lymphocyte proliferation was stimulated by the addition of phytohemagglutinin ŽWellcome Diagnostics, Dartford, UK.. After 72 h of incubation at 378C, the mitoses were arrested in metaphase by the addition of colchicine 2 h before harvesting. Microscopic slides were prepared for chromosomal analysis according to standard procedures. The chromosomes of 50 well-spread and complete metaphases Ž10 on each of five coded slides. were examined for the presence of breaks, fragments, exchanges, rings, dicentrics and other morphological abnormalities. Culture preparations with low mitotic index were eliminated, and the culture was repeated. A series of ultrafiltrates was tested the same day on cultures set up with the blood of the same donor. Two additional control cultures without ultrafiltrate served for the establishment of the spontaneous chromosomal aberration rate of the donor’s lymphocytes. This background
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
level of aberrations was subtracted from the aberration rate in ultrafiltrate-treated cultures of the same blood donor. The difference between the two values is called the ACS, which is given as a percentage for convenience w4,5,7x. This way of treating results is necessary for comparison of clastogenic activity of samples collected at subsequent dates and tested on cultures with different background level of aberrations. When background levels were studied on 10 parallel cultures set up with the same blood, the variation did not exceed "3 aberrations per 50 cells or "6 aberrations per 100 cells studied. This range was the same for two independent observers. Therefore a plasma ultrafiltrate is considered to be clastogenic, if it induces more than 3 aberrations per 50 cells. In agreement herewith, the increase in aberrations induced by ultrafiltrates from a series of 96 healthy blood donors did not exceed 4% additional aberrations for the majority of them. Only with five of these normal samples, the increase represented q6%, while increases of q8% or higher were not observed w7x. On the basis of these control data, rat plasma inducing q8% aberrations in the test system were considered as abnormal and called CFq. 2.4. Treatment EGb761 is a standardized extract of Ginkgo biloba leaves. It contains 24% flavonol glycosides and 6% terpene trilactones Ž3.1% ginkgolides and 2.9% bilobalide. w10,11x. The complete extract EGb761, an extract from which terpene trilactones had been removed ŽCP205., ginkgolides A and B and bilobalide were provided by IHB-IPSEN ŽParis, France.. All drugs were administered orally. Ginkgolides were used in their water soluble formulation Ži.e., an equilibrium of closed and open lactone forms at pH 8.75.. Bilobalide was administered in suspension in 0.5% Carboxy methyl cellulose ŽCMC.. Extracts ŽEGb761 and CP205. were dissolved in water. Treatment was given orally Žgavage. once a day during a period of 3 weeks. The complete extract EGb761 was tested at two doses Ž50 and 100 mgrkg.. Another series of rats received CP205 Ž100 mgrkg.. Ginkgolides A q B and bilobalide were studied at a dose Ž4 mgrkg., which is slightly higher than their
101
concentration in the complete extract EGb761. Five irradiated rats received water as placebo.
3. Results and discussion The present study confirms the findings of Faguet et al. w8x, who reported that the plasma of irradiated rats has clastogenic properties in cultures set up with blood of unexposed rats. The clastogenic activity was demonstrable immediately after irradiation, diminished therafter, but remained significantly increased until the end of their study period Ž10 weeks.. We studied a group of 14 animals at day 8 after irradiation and noted clastogenic activity in all samples, with a mean ACS of 13.33 " 2.37 Ž p - 0.0001 compared to plasma ultrafiltrates from unexposed rats.. The mean values of ACS with plasma from nonirradiated rats was 1.2 " 2.1, similar to values observed with plasma ultrafiltrates from healthy humans Ž0.8 " 1.0. w7x. The clastogenic activity was lower in the plasma of rats studied at day 22 postirradiation Ž9.87 " 4.17., but was still significantly increased compared to control plasma Ž p - 0.0001.. At that time point, only 72% of the animals were positive for CF ŽACS q 8% and higher.. Despite the significant decrease in clastogenic activity between day 8 and day 22 postirradiation Ž p - 0.01., the later date was chosen in the following experiments comparing the efficacy of the complete extract with that of its components. The reason herefore was the intention to treat cancer patients with EGb761, the treatment starting 3 weeks after radiation therapy. The results obtained for the 37 CFq animals are shown in Table 1. The comparison of ACS before and after treatment showed significant differences for all treatment groups except for the ‘‘placebo’’ group, i.e., the group of animals receiving water according to the same treatment schedule postirradiation. The percentage of reduction Ž28%. observed for this group may represent the spontaneous regression of CF between day 22 and day 44. If the ACS of the placebo group are considered for comparison at day 44, the ACS of all treatment groups are significantly lower ŽTable 1.. Therefore, the reduction of the clastogenic scores is not explained by the spontaneous regression of the clastogenic activity, but is due in large part to the
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
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Table 1 Clastogenic activity in the plasma of irradiated rats before and after the 3-week treatment with EGb761 or its components All animals are CFq before treatment. Comparison: Ž1. ‘‘Before’’r‘‘After’’ ŽStudent’s t-paired test. U p - 0.01; UU p - 0.001; UUU p - 0.0001; Ž2. With placebo-group ŽStudent’s t-test. 8 p - 0.05; 88 p - 0.01; 888 p - 0.001; Ž3. Analysis of variance: no difference between treatment groups for the efficacy of the protection.
Before
After
Animals CFq after treatment
5
12.80 " 1.79
9.20 " 2.68 NS
4
28%
7
11.71 " 3.55
2.00 " 2.83UUU 888
1
83%
7 8
13.43 " 2.23 11.75 " 1.67
4.29 " 2.14UUU 88 4.00 " 4.00UUU 8
1 3
68% 66%
5
11.20 " 1.79
3.60 " 2.61UU 88
1
68%
5
12.80 " 4.15
4.40 " 1.67U 88
0
66%
n
Placebo-treated rats Ždist. water. Complete extract 100 mgrkg 50 mgrkg Extract without terpenoids 100 mgrkg Gingkolides A q B 4 mgrkg Bilobalide 4 mgrkg
ACS
treatment. All animals showed reduced ACS after treatment compared to the pretreatment values for all treatment groups. While all components exerted anticlastogenic effects at the concentrations present in the complete extract, the comparison of the different groups by analysis of variance did not reveal significant differences. This may be due to the small number of animals available in each treatment group. The complete extract reduced the ACS by 83% at the dose of 100 mgrkg, while the lower dose of 50 mgrkg and
Percentage of reduction
the three components reached only 66%–68% reduction. Since all components were able to reduce the aberrations rates by 66%, the better protection provided by the complete extract was not additive, but rather due to synergistic effects. Only results observed in the 37 animals positive for CF at day 22 postirradiation are shown in Table 1. The other 13 animals negative in the CF-test at that time point were also treated, since the results were not known, when the treatment was started. No increase in chromosomal aberration rates were observed in these
Table 2 Nature of chromosomal aberrations induced with plasma from irradiated rats. Effect of treatment Without treatment n s 32 Ž%. Telomeric extrusions Chromatid breaks Isochromatid breaks FragmentsU Dots and double minutes DicentricsU Tri- and quadriradials Total Number of mitoses studied Total number of aberrations Percentage U
26.1 50.0 14.3 5.4 2.1 1.4 0.7 100 1600 280 17.6
With treatment n s 32 Ž%. 30.6 47.6 20.4 0.7 0.7
100 1600 147 9.3
Incubation time 72 h: the presence of dicentrics possibly due to a second division, on the other hand, fragments may have been lost.
A. Alaoui-Youssefi et al.r Mutation Research 445 (1999) 99–104
animals at day 44, indicating that neither the complete extract nor its components has chromosome damaging effects. No adverse side effects were observed in the animals with the different treatments. As already stressed by the first reports w1,2,12x and confirmed by the report of Faguet et al. in rats, as well as by our previous work in irradiated populations w4x, CF-induced chromosome damage is mainly of the chromatid type. This was also the case in the present study ŽTable 2.. There were rare dicentrics, fragments and tri- or quadriradial configurations. Their respective frequencies were 1.7% in 1600 mitoses for cultures exposed to plasma ultrafiltrates of irradiated animals and 0.13% in 1600 mitoses exposed to plasma ultrafiltrates of animals treated with EGb761 or its components after irradiation Ž p 0.001.. In addition to the reduction of aberration frequencies, these differences in the nature of aberrations may be interpreted in favor of protective effects by EGb761 and its components. After 72 h of incubation, a certain number of cells is at their second division in culture, and certain aberrations may arise or be lost during replication. Therefore, most researchers interested in the nature of aberrations use a 48-h incubation time. However, the number of mitoses available at 48 h with TCM 199 medium is low. Nevertheless, this medium was adopted for the study since it has been found previously to be optimal because of its low antioxidant content w9x. The CF-test intends to demonstrate breakage effects, and the nature of aberrations was not the aim of this study. Ginkgo biloba extracts influence the mitotic index only at high doses, which were not reached in the plasma of animals by the treatment w6x. Therefore, variations in cell cycle conditions should not have influenced the outcome of the experiments. CF-induced clastogenic effects are superoxidemediated, as discussed in more detail elsewhere w13x. A previous study using SOD in comparison to EGb761 against CF-induced chromosome damage in vitro showed that EGb761 provided protective effects similar to those obtained with SOD w6x. It is therefore probable that the anticlastogenic effects of EGb761 are related to its antioxidant properties. EGb761 has been reported to scavenge superoxide anions in a dose-dependent manner, as indicated by electron spin resonance at high concentrations Ž125
103
and 500 mgrml. w14x. The antioxidant properties of CP205 are probably due to its content in flavonoids, known for their superoxide scavenging properties w15–17x. The antioxidant properties of ginkgolides at very low concentrations were shown in a model of ischemia reperfusion injury w18x. The protective effects were more pronounced in vivo than expected after the in vitro experiments. The authors hypothesized that the protective effects might be due to diminished oxyradical release by cells rather than to extracellular free radical scavenging. The other terpene compound, bilobalide, was found to react with superoxide by EPR and UVrVIS spectroscopy in an aprotic environment w19x. The doses used for the present study in rats are known to provide blood concentrations of active principles close to those reached in humans with the complete extract or its components. These concentrations of EGb761 were protective in our previous studies with Chernobyl-exposed persons w6x. The present study indicates that anticlastogenic effects are exerted in this model of superoxide-mediated clastogenesis by all components, but that the complete extract appears to provide the best protection. Similar observations were obtained with other plant extracts w20x. The mechanisms of these synergistic effects of the various components remain unexplained. Further studies have to be conducted with the principal components using higher concentrations than those present in EGb761.
Acknowledgements We are grateful to Professor F. Baillet and the Department of Radiotherapy, La Pitie-Salpetriere ´ ´ ` Hospital, Paris, where the animals were irradiated.
References w1x K.O. Goh, H. Summer, Breaks in normal human chromosomes: are they induced by a transferable substance in the plasma of irradiated persons exposed to total body irradiation?, Radiat. Res. 6 Ž1968. 51–60. w2x J.G. Hollowell, L.G. Littlefield, Chromosome damage induced by plasma from irradiated patients. An indirect effect of X-ray, Proc. Soc. Exp. Biol. Med. 129 Ž1968. 240–244. w3x G.S. Pant, N. Kamada, Chromosome aberrations in normal
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