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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 11 4286-4289
Copyright © 2007 by The Endocrine Society

BRIEF REPORT

Anticlastogenic Effect of Ginkgo Biloba Extract in Graves’ Disease Patients Receiving Radioiodine Therapy

A. Dardano, M. Ballardin, M. Ferdeghini, E. Lazzeri, C. Traino, N. Caraccio, G. Mariani, R. Barale and F. Monzani

Departments of Internal Medicine (A.D., N.C., F.M.), Biology (M.B., R.B.), and Oncology, Transplants and Advanced Technologies in Medicine (E.L., G.M.), University of Pisa, 67-56126 Pisa, Italy; Department of Morphological-Biomedical Sciences (M.F.), University of Verona, 37134 Verona, Italy; and Health Physics Service (C.T.), S. Chiara Hospital, 56100 Pisa, Italy

Address all correspondence and requests for reprints to: Fabio Monzani, M.D., Department of Internal Medicine, University of Pisa, via Roma 67-56126 Pisa, Italy. E-mail: fmonzani{at}med.unipi.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Chromosomal damage, as assessed by clastogenic factors (CFs) and micronuclei (MN) appearance, after radioiodine therapy of Graves’ disease has been reported.

Objective and Methods: Our objective was to evaluate the effect of Ginkgo biloba extract (EGb 761) supplementation on the time course (up to 120 d) of CFs and MN appearance in lymphocytes from patients with Graves’ disease after iodine-131 (131I) therapy. Patients were randomly assigned to EGb 761 or placebo, in a blinded manner.

Results: In the placebo group, MN increased early (P < 0.001) after 131I, peaking at the 21st day (P = 0.0003) and declining thereafter. In EGb 761-treated patients, MN increased early (P < 0.05), while returning toward baseline value thereafter. Therefore, mean MN increment was significantly higher in the placebo group as compared with EGb 761-treated patients (P < 0.01). Moreover, an early (P < 0.0001) and sustained (up to 35 d; P < 0.001) MN increase induced by CFs was observed in the placebo group. Conversely, in EGb 761-treated patients, MN increase induced by CFs never reached the statistical significance; therefore, the mean of the MN increments was significantly lower than in placebo (P < 0.05). A significant positive correlation between MN maximum increment and the bone marrow dose was observed in the placebo group only (P = 0.03). No significant difference was observed in clinical outcome between the two groups.

Conclusions: EGb 761 supplementation neutralized genotoxic damage induced by radioiodine treatment, without affecting the clinical outcome. Although 131I therapy is generally safe, our data suggest that Gingko biloba extracts may prevent genetic effects of radioiodine therapy for hyperthyroid Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
IODINE-131 (131I) THERAPY is commonly used to treat hyperthyroidism and is becoming the treatment of choice for recurrent hyperthyroidism (1). Most studies have shown no increased mortality due to secondary malignancies in patients receiving 131I therapy for hyperthyroidism, provided that standard radioprotection procedures are fulfilled (2). However, an increased overall cancer incidence among hyperthyroid patients treated with radioiodine has been recently reported (3). Some chromosomal damage to peripheral lymphocytes has been reported in Graves’ disease patients treated with 131I (4). In this regard, ionizing radiation causes the formation of reactive oxygen species, which in turn induce lipid peroxidation of cell structures (5). In addition, increased production of superoxide radicals may induce the release of chromosome-damaging material, called clastogenic factors (CFs), in circulating plasma. These low-molecular weight substances stimulate further production of superoxide radicals, thus perpetuating chromosomal damage. This self-sustaining mechanism may exceed the DNA-repair capability of cells, thus representing a potentially dangerous condition. CFs so far identified include lipid peroxidation products, inosine nucleotides, and cytokines (4, 6).

Chromosomal abnormalities can be assessed simply by evaluating the frequency of micronuclei (MN) in dividing cells, an index of either numerical or structural chromosome alterations (7). Therefore, the yield of MN in peripheral blood lymphocytes can be considered as a real "biological dosimeter" for radiation exposure of patients undergoing radiation therapy (8). In a recent study, we demonstrated that the MN frequency of peripheral blood lymphocytes as well as the formation of CFs in Graves’ patients treated by 131I were associated with an impairment of antioxidant defenses, as demonstrated by a significant depletion of vitamin E (4).

Several studies have shown that Ginkgo biloba extracts, especially EGb 761, can weaken the deleterious effects of oxidative stress, with various mechanisms (9, 10). Indeed, extract from Ginkgo biloba leaves has been used as a therapeutic agent for some neurological disorders related to oxidant/antioxidant imbalance (11). Although the exact mechanism is still unknown, EGb 761 is able to inhibit the genotoxic effect of CFs both in vitro and in vivo (6, 12).

The aim of the study was to evaluate the effect of EGb 761 supplementation on the time course of CFs and MN appearance in lymphocytes from Graves’ disease patients after 131I therapy.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
The study was performed in 25 patients (19 women, mean age 47.9 ± 15.3 yr), all nonsmokers, affected by relapsing Graves’ disease. Exclusion criteria for 131I treatment were pregnancy or lactation, suspicion of thyroid malignancy, and severe eye symptoms. All the patients were receiving methimazole (10–15 mg/d), which was discontinued 7 d before 131I administration and resumed 3 d afterward (10 mg/d) up to 30 d. Patients were randomly assigned to EGb 761 (Tanakan; Ipsen, Paris, France) (n = 10) or placebo (n = 15) in a blinded manner. Tablets containing EGb 761 (120 mg/d) or placebo were accurately counted and given orally to each patient from 3 d before, up to 30 d after 131I therapy. Subjects with bleeding disorders and those taking oral anticoagulants were excluded from the study. Written informed consent was obtained from all patients following guidelines of the Institutional Review Board of the University of Pisa.

According to Willemsen et al. (13), administered therapeutic activities (521 ± 157 MBq, mean ± SD) were calculated based on the maximal radioiodine uptake test and thyroid volume as assessed by ultrasound, according to the ellipsoid formula ({pi}/6 x width x length x thickness). Serum TSH, free T4 (FT4), free T3 (FT3), antithyroglobulin (TgAb), antithyroperoxidase (TPOAb), and anti-TSH receptor (TRAb) antibody levels, as well as MN frequency and plasma CFs, were assessed in each patient at baseline, then 7, 14, 21, 35, 90, and 120 d after radioiodine administration.

Serum FT3 and FT4 levels were measured by RIA, and TSH by ultrasensitive IRMA (Techno-Genetics, Milan, Italy). TgAb was measured by IRMA (Biocode, Sclessin, Belgium), and TPOAb and TRAb by RIA (Sorin Biomedica, Saluggia, Italy, and B.R.A.H.M.S, Henningsdorf, Germany, respectively). Normal ranges were: FT4, 8.6–18.6 pg/ml (11.0–23.9 pmol/liter); FT3, 2.1–4.6 pg/ml (3.2–7.1 pmol/liter); TSH, 0.3–3.6 mU/liter; TgAb, less than 50 IU/ml; TPOAb, less than 10 IU/ml; and TRAb, less than 1 IU/liter.

Micronucleus assay

Two paired, independent lymphocyte cultures were set up for each blood sample as previously described (14). After 44-h cell culturing, cytochalasin B (Sigma-Aldrich, Milan, Italy) was added to block cytokinesis and make dividing lymphocytes upon phytohemagglutinin stimulation. Cells that have undergone the first mitosis are recognized as binucleated (BN) cells, which are selectively screened for the presence of MN. Cells were harvested at 72 h, and slides were prepared according to standard procedures (15). A total of 2000 BN cells for each blood sample was selectively screened for the presence of MN (14).

Assessment of CFs

At each sampling time from 0–120 d after 131I therapy, 5 ml plasma was obtained after centrifuging blood at 800 x g for 15 min, and immediately frozen at –20 C. For the isolation of CFs, plasma was ultrafiltered at 800 x g for 2 h through a 10-kDa cutoff filter, using Centriplus concentrators (Amicon, Danvers, MA).

Peripheral blood cultures obtained at various times from one healthy donor were used throughout the study. Lymphocyte cultures were set up by incubating 0.3 ml of the healthy donor’s blood with 4.7 ml TCM 199 (Life Technologies, Inc., Milan, Italy) plus 0.4 ml plasma ultrafiltrate (15). The net BN-MN increase induced by CFs was estimated by subtracting the spontaneous frequency observed in the reference donor lymphocytes from the frequency of BN MN observed after treatment with the plasma ultrafiltrate of patients.

Dosimetry

The committed dose to the thyroid was assessed as previously described (16). The blood absorbed dose was assessed on blood samples by a germanium-gallium detector. According to Stabin (17), bone marrow absorbed dose (mGy/MBq) was calculated through the medical internal radiation dose formalism.

Statistical analysis

Data are expressed as mean ± SEM unless otherwise stated. The time course of MN increase and the MN increment induced by CFs with respect to baseline were expressed as {Delta}MN and {Delta}CFs, respectively.

Student’s t test, {chi}2, and one-way ANOVA were used as appropriate. Correlations among continuous variables were computed by Pearson’s correlation coefficient. A general linear mixed (GLM) model approach was used to analyze the repeated measures data. The time effect was tested by the Wald {chi}2 test. Dunnett’s test was used to analyze differences between each time point and the baseline level. Statistical significance was assumed for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
At baseline, demographic and biochemical characteristics, as well as MN and CFs levels, did not differ between the two groups of patients. Moreover, the administered 131I activity and bone marrow dose were comparable in EGb 761 and placebo-treated patients (Table 1Go). No clinically relevant side effects to EGb 761 were observed during the entire treatment period.


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  		TABLE 1. Clinical features of 131I treated Graves’ patients receiving Ginkgo biloba extract (EGb 761; 120 mg/d) or placebo

 
The efficacy of 131I therapy in patients receiving EGb 761 or placebo was similar. In detail, the cure rate was 100% in the EGb 761 group (60% developing permanent hypothyroidism) and 93% in the placebo group (73% developing permanent hypothyroidism). No significant difference was found in the median time interval of occurrence of hypothyroidism (4 months, range 3–6 for both) as well as in volume reduction (11.7 ± 4.8 and 8.0 ± 7.1 ml, respectively).

MN induction

In the placebo group, a significant increase of MN was observed 7 d after radioiodine administration (12.5 ± 1.7 vs. 5.2 ± 1.0; P < 0.001 vs. baseline), reaching a peak at the third week (24.7 ± 3.6; P = 0.0003 vs. baseline) and declining to pretreatment values at 3-month follow-up (7.5 ± 1.0). EGb 761 administration significantly modified the time course of MN induction. Indeed, an early increase of mean MN count was observed (14.3 ± 2.2 vs. 8.7 ± 1.8; P < 0.05; 7 d vs. baseline), followed by a no longer significant plateau value (Fig. 1AGo). MN increase was significantly different from placebo also considering the bone marrow dose, as well as patients’ age, gender, and thyroid hormone profile (GLM analysis, P < 0.005). In detail, both the peak of {Delta}MN (19.5 ± 3.2 vs. 3.8 ± 2.2; P < 0.01) and the mean of {Delta}MN (10.5 ± 1.8 vs. 5.3 ± 1.5; P < 0.01) were significantly higher in the placebo than EGb 761 group (Fig. 2AGo).


Figure 1
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  		FIG. 1. Time course of MN increase after radioiodine therapy in lymphocytes from Graves’ disease patients receiving placebo (filled triangles) or EGb 761 (filled squares) (A), and of the variation of MN induced by CFs from Graves’ disease patients receiving placebo or EGb 761 (B). *, P < 0.001. **, P = 0.0003. °, P = 0.002 vs. baseline (placebo group). #, P < 0.05 vs. baseline (EGb 761 group).

 

Figure 2
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  		FIG. 2. A, Mean of time-by-time MN increase after radioiodine therapy in the group of Graves’ disease patients receiving placebo or Ginkgo biloba extract (EGb 761) (mean {Delta}MN). §, P < 0.01 vs. placebo. B, Mean of time-by-time MN increase induced by CFs from Graves’ disease patients receiving placebo or EGb 761 (mean {Delta}CFs). °, P < 0.05 vs. placebo.

 
A positive correlation (P = 0.03; {rho} = 0.68) between the maximum {Delta}MN and the bone marrow dose was found in the placebo group only.

CF formation

In the placebo group, the effect of radioiodine on MN induced by CFs was observed from the first sampling time (P < 0.0001 vs. baseline) onward, decreasing gradually to the baseline level 90 d after radioiodine administration, whereas in EGb 761-treated patients never reached the statistical significance (Fig. 1BGo). As a consequence, the time course of {Delta}CFs was quite different between the two groups, also considering the bone marrow dose, as well as patients’ age, gender, and thyroid hormone profile (GLM analysis, P = 0.001). Therefore, the mean of {Delta}CFs was significantly higher in the placebo group compared with EGb 761-treated patients (4.4 ± 0.5 vs. 2.1 ± 1.3, respectively; P < 0.05) (Fig. 2BGo).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
To the best of our knowledge, the present work demonstrates for the first time that Ginkgo biloba extracts’ supplementation is effective in reducing radiation-induced genotoxic damage in hyperthyroid patients receiving 131I therapy. It is noteworthy that this protective effect was elicited without any adverse modification of the clinical outcome. In detail, our data confirm that 131I therapy induces a significant, although transient, genetic damage characterized by the increase of MN and clastogenic activity in patients’ lymphocytes. The concomitant administration of EGb 761, at a dosage currently used in clinical practice, significantly blunted MN induction. Indeed, EGb 761-treated patients did not show the progressive increase of MN count. Moreover, the induction of MN by CFs never reached the statistical significance, showing a time-course pattern quite different from the placebo group of Graves’ patients. In agreement with previous data in irradiated rats (18), the reduction of the clastogenic scores was likely due to the EGb 761 treatment. Indeed, the protective effect of Ginkgo biloba extracts was still present when correcting data by clinical and biochemical parameters (i.e. bone marrow dose, age, gender, and thyroid hormone profile). Although methimazole has been postulated to act as free radicals’ scavenger (19), in our protocol all patients received an identical dose of methimazole with the same time course. Therefore, it is likely that decreased MN formation was independent from methimazole antioxidant activity and essentially related to the effect of EGb 761 administration.

Although the exact mechanism is still unknown, several studies have highlighted the ability of EGb 761 and its constituents to act as antioxidants and scavenger of free radicals, such as NO•, OH•, O2•–, and peroxyl radicals (20). Moreover, EGb 761 is able to inhibit the genotoxic effect of CFs both in vitro and in vivo (6, 12). However, further studies are needed to identify the compound responsible for the anticlastogenic effect, by using isolated components of the extract or various combinations of them.

In conclusion, the current study shows that EGb 761 supplementation may neutralize genotoxic damage induced by radioiodine treatment, without affecting the clinical outcome. Although 131I therapy is generally safe, our data suggest that Gingko biloba extracts may prevent genetic effects of radioiodine therapy for hyperthyroid Graves’ disease.


   Footnotes
 
This work was supported in part by a grant from Ministero Università and Ricerca Scientifica, Rome, Italy.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 21, 2007

Abbreviations: BN, Binucleated; CF, clastogenic factor; FT3, free T3; FT4, free T4; GLM, general linear mixed; 131I, iodine-131; MN, micronuclei; TgAb, antithyroglobulin antibody; TPOAb, antithyroperoxidase antibody; TRAb, anti-TSH receptor.

Received March 15, 2007.

Accepted August 10, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Streetman DD, Khanderia U 2003 Diagnosis and treatment of Graves disease. Ann Pharmacother 37:1100–1199[Abstract/Free Full Text]
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  3. Metso S, Auvinen A, Huhtala H, Salmi J, Oksala H, Jaatinen P 2007 Increased cancer incidence after radioiodine treatment for hyperthyroidism. Cancer 109:1972–1979[CrossRef][Medline]
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  5. Wolfram RM, Palumbo B, Chene F, Palumbo R, Budinsky AC, Sinzinger H 2004 (Iso) Prostaglandins in saliva indicate oxidation injury after radioiodine therapy. Rev Esp Med Nucl 23:183–188[CrossRef][Medline]
  6. Liu TZ, Stern A, Emerit I 1999 Clastogenic factors: biomarkers of oxidative stress of potential utility in the clinical chemistry laboratory. Ann Clin Lab Sci 29:134–139[Medline]
  7. Fenech M, Morley AA 1986 Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat Res 161:193–198[Medline]
  8. Watanabe N, Yokoyama K, Kinuya S, Shuke N, Shimizu M, Futatsuya R, Michigishi T, Tonami N, Seto H, Goodwin DA 1998 Radiotoxicity after iodine-131 therapy for thyroid cancer using the micronucleus assay. J Nucl Med 39:436–440[Abstract/Free Full Text]
  9. Rong Y, Geng Z, Lau BHS 1996 Ginkgo biloba attenuates oxidative stress in macrophages and endothelial cells. Free Radic Biol Med 20:121–127[CrossRef][Medline]
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  13. Willemsen UF, Knesewitsch P, Kreisig T, Pickardt CR, Kirsch CM 1993 Functional results of radioiodine therapy with a 300-Gy absorbed dose in Graves’ disease. Eur J Nucl Med 20:1051–1055[Medline]
  14. Fenech M 1993 The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 285:35–44[Medline]
  15. Fenech M, Morley AA 1985 Measurement of micronuclei in lymphocytes. Mutat Res 147:29–36[CrossRef][Medline]
  16. Traino AC, Di Martino F, Lazzeri M, Stabin MG 2001 Study of the correlation between administered activity and radiation committed dose to the thyroid in 131I therapy of Graves’ disease. Radiat Prot Dosimetry 95:117–124[Abstract]
  17. Stabin MG 1996 MIRDOSE: personal computer software for internal dose assessment in nuclear medicine. J Nucl Med 37:538–546[Free Full Text]
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  19. Ferreira AC, de Carvalho Cardoso L, Rosenthal D, de Carvalho DP 2003 Thyroid Ca2+/NADPH-dependent H2O2 generation is partially inhibited by propylthiouracil and methimazole. Eur J Biochem 270:2363–2368[Medline]
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