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Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model
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Mutation Research 634 (2007) 126–134
Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model Guillermo M. Z´un˜ iga-Gonz´alez a,∗ , Cecilia M. Batista-Gonz´alez a , Belinda C. G´omez-Meda a , Mar´ıa L. Ramos-Ibarra a , Ana L. Zamora-Perez b , Tereza Mu˜noz-Magallanes c , Carmen Ramos-Vald´es c , Martha P. Gallegos-Arreola d a
c
Laboratorio de Mutag´enesis, Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico b Laboratorio de Farmacogen´ omica y Biomedicina Molecular, Centro Interdisciplinario de Investigaci´on para el Desarrollo Integral Regional, Instituto Polit´ecnico Nacional, Durango, Durango, Mexico Servicio de Endocrinolog´ıa, Unidad M´edica de Alta Especialidad, Hospital de Especialidades, Centro M´edico Nacional de Occidente “Lic. Ignacio Garc´ıa T´ellez”, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico d Laboratorio de Gen´ etica Molecular, Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico Received 9 March 2007; received in revised form 19 June 2007; accepted 23 June 2007 Available online 28 June 2007
Abstract Diabetes mellitus (DM) is associated with a high risk of health complications, mainly due to excessive free radical (FRs) production that could result in an increased frequency of micronuclei. The consumption of antioxidants, like folic acid (FA), may mitigate the effects of the FRs. In the present study, micronucleated polychromatic erythrocyte (MNPCE) frequencies were determined in blood sampled weekly from the tails of pregnant female Wistar rats and pregnant Wistar rats with experimental diabetes that were given unsupplemented diets and diets supplemented with FA. At birth, the pups were sampled to analyze micronucleated erythrocyte (MNE) and MNPCE frequencies. Moreover micronucleated cells (MNCs) were evaluated in buccal mucosa samples taken from 81 healthy adult subjects, 48 patients with DM, and 30 DM patients who were sampled before and after FA treatment. Increases in MNPCE frequencies were significant only at the first sampling (P < 0.01 and P < 0.03) in pregnant rats with experimental diabetes. In addition, the pups from the diabetic group and from diabetic group treated with FA had higher frequencies of MNEs (P < 0.03 and P < 0.001, respectively) and MNPCEs (P < 0.009 and P < 0.05, respectively) than the controls. No differences were found in diabetic rats and newborn rats born to diabetic mothers treated with FA compared with untreated animals. Patients with DM had a higher frequency of MNCs compared with healthy subjects (P < 0.001). Also FA reduced the frequency of MNCs in DM patients (P < 0.001). The results of this study indicate that diabetes results in elevated frequencies of micronuclei, and that, at least in humans, FA can protect against the elevation. © 2007 Elsevier B.V. All rights reserved. Keywords: Micronuclei; Diabetes; Folic acid; Free radicals; Human; Rat
∗
Corresponding author at: Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada 800, Col. Independencia, C.P. 44340, Guadalajara, Jalisco, Mexico. Tel.: +52 33 36189410; fax: +52 33 36181756. E-mail address: [email protected] (G.M. Z´un˜ iga-Gonz´alez). 1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2007.06.006
G.M. Z´un˜ iga-Gonz´alez et al. / Mutation Research 634 (2007) 126–134
1. Introduction Diabetes mellitus (DM) is characterized by an elevation in blood glucose concentration. The disease is progressive and is associated with the development of complications, like atherosclerosis, renal and neuronal damage, and blindness [1–3]. Experimental evidence indicates that these complications are due mainly to the production of excessive concentrations of free radicals (FRs), which result in oxidative damage to biomolecules [1,4–9]. Oxidative damage to the genetic material could cause DNA strand breaks [5,8,10–13] and micronuclei (MN), and these types of damage could have teratogenic or carcinogenic consequences [14–17]. Elevated frequencies of micronucleated erythrocytes (MNEs) have been measured in premature children born to mothers with pathologies related to oxidative stress [16], like arterial hypertension and DM. Also, increases in micronucleated cells (MNCs) have been observed in buccal mucosa of patients with other pathologies characterized by increases in FRs production, like rheumatoid arthritis [18,19]. The damage caused by FRs can be mitigated by antioxidant defence systems, which in the case of DM, become overwhelmed by FRs generated by the disease processes [4,7,8]. Antioxidant support systems can in theory be supplemented by using antioxidants like folic acid (FA) [20] that have the capacity to resist (or to neutralize) the effects of FRs [12,21–25]. FA deficiency increases spontaneous chromosomal damage by massive incorporation of uracil within DNA, which produces chromosomal breakage and MN formation [26], and can influence the genotoxic responses to other compounds [27,28]. The use of supplemental FA by women preand post-conception diminishes the occurrence of neural tube defects in their offspring [29]. Observations such as these illustrate the health advantages produced by the intake of this vitamin [30]. Previous studies indicate that FA supplementation decreases the frequency of MN in humans and experimental systems [14,20,31]. In the present study, we have evaluated the effect of DM on MN frequency, and the effect of FA supplementation on DM-associated MN.
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Care Committee. All experiments were performed according to the guidelines for the care and use of experimental animals at the Centro de Investigaci´on Biom´edica de Occidente, which are in compliance with those given in national (M´exico; NOM062-ZOO-2001) and international guidelines for the humane treatment of research animals. Twenty-one 3.5-month-old female Wistar rats were housed individually in polycarbonate cages, and given water and food ad libitum. All animals used in the study were supplied by the laboratory animal facility of the Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, M´exico. In addition, 48 pups of undetermined sex, born to these rats, were used during the course of the experiments. 2.1.2. Adults rats 2.1.2.1. Study groups. Three groups of seven adult female rats were formed. Group 1 (negative control) received a single intraperitoneal (i.p.) injection of 0.3 ml injectable distilled water, and then were mated with males of the same strain. Pregnancy was determined by the presence of sperm in a vaginal smear [14], and when pregnancy was confirmed, the rats were given daily orally by gavages doses of 0.5 ml water until delivery. Group 2 and 3 rats were first made diabetic as described below. Group 2 rats (diabetes without FA) were mated and treated with water as above. Group 3 animals (diabetes with FA) underwent the same procedure as Group 2, except that they received daily 0.5 ml doses of 0.7 mg of FA/kg (Sigma, St. Louis, MO; CAS No. 59-30-3) orally by gavages (in water) from the first day of pregnancy until the birth. The dose of FA was based on the therapeutic dose recommended for pregnant women (5 mg/day), using an average human body weight of 70 kg and multiplying by 10, because is established that on a body weight basis, humans are generally more vulnerable than are experimental animals, probably by a factor of about 10 [32]. 2.1.2.2. Experimental diabetes induction in rats. Fourteen rats (Groups 2 and 3) received i.p. injections of 65 mg streptozotocin (STZ)/kg (Sigma, St. Louis, MO; CAS No. 18883-66-4) to induce DM [33,34], at least 3 days previously to be mating. The diabetes was confirmed with a glucometer (One Touch Ultra; Reg. Not 1691E2002, S.S.A. Johnson & Johnson, M´exico, S.A. of C.V.). To consider that an experimental diabetes in the rat was established, it was required that the rat maintains values greater than 250 mg/dl of blood glucose after the induction. In order to assure that the hyperglycaemic state was maintained, glucose levels were monitored every 7 days during the experiment.
2. Materials and methods 2.1. Rat study 2.1.1. Animals The study was approved by our Institutional Research Committee (register number 2002249018) and by a local Animal
2.1.2.3. Sample preparation and MNE analysis. A total of five samples of blood were taken of each rat during the experiment. The basal sample was taken previously to mating and the next samples were taken every 7 days during the pregnancy at days 7, 14 and 21 of gestation, and at 7 days after the delivery, and two smears were made of each sample time on pre-cleaned
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and pre-coded microscope slides. The smears were air-dried, fixed in absolute ethanol for 10 min, and stained with acridine orange [35]. The slides were scored manually using an Olympus BX51 microscope equipped with epifluorescence and a 100× objective. The number of micronucleated polychromatic erythrocytes (MNPCEs) was counted in 3000 polychromatic erythrocytes (PCEs), as well as the proportion of PCEs in 1000 total erythrocytes (TEs). 2.1.3. Newborn rats 2.1.3.1. Study groups. At birth, four pups were selected randomly from each of four dams from the three experimental groups. The pup groups were designated based on the treatment of the mother: Group 4: negative control; Group 5: diabetes without FA; Group 6: diabetes with FA. 2.1.3.2. Sample preparation and MNE analysis. A drop of peripheral blood was taken from the tip of the tail of each newborn rat pup immediately after birth, and smears were prepared on pre-coded glass slides. The smears were air-dried, fixed in absolute ethanol for 10 min, and stained with acridine orange [35]. The slides were scored manually using an Olympus BX51 epifluorescence microscope and a 100× objective. The number of MNEs was determined in 10,000 TEs and the number of MNPCEs was determined in 1000 PCEs. The number of PCEs in 1000 TEs also was counted. 2.2. Human study The study was approved by our Institutional Research Committee (register number 2002249018). The study group consisted of 159 individuals divided into three subgroups: Group I (n = 81) healthy subjects; Group II (n = 48) diabetic patients; Group III (n = 30) diabetic patients under FA treatment. All individuals who agreed to participate in the study signed a letter of informed consent and answered a detailed questionnaire about personal information as gender, age, alimentary habits, consumption of drugs or antioxidants, smoking habit, illness. 2.2.1. Healthy subjects group Eighty-one adult volunteers without diabetes were sampled. The subjects had blood glucose levels of <110 mg/dl and had not used supplemental antioxidants for approximately 1 month prior to the study. 2.2.2. Diabetic patient groups Adult diabetic patients from the Endocrinology Service of the Centro M´edico Nacional de Occidente, Instituto Mexicano del Seguro Social, were included in the study. Patients with controlled diabetes had glycosylated hemoglobin (HbA1c) levels of <7%, while patients with uncontrolled diabetes had HbA1c levels >7%. None of the patients included in the study consumed supplemental antioxidants for approximately 1 month prior to the study.
Forty-eight diabetic patients were sampled immediately to evaluate the effect of DM on MN frequency. Another group of 30 diabetic patients took 5 mg of FA (Lab. Valdecasas, S.A., lot 051033; Reg. No. 82231, S.S.A.) three times a day for 30 days p.o. 2.2.3. Sample preparation and MN analysis Buccal mucosal samples were collected from each subject. In the case of diabetic patients who took FA, samples were taken before starting the FA supplementation (basal sample) and after 30 days of FA intake. Subjects were asked to rinse their mouth with water, then a polished slide was used to collect cells from the buccal mucosa of the right and left cheeks; the samples were spread directly into two separate pre-cleaned and pre-coded slides. The smears were air-dried and fixed in 80% methanol for 48 h [15], and then stained with acridine orange [35]. MNCs were counted in 2000 cells at 100× magnifications using an Olympus BX51 microscope equipped with epifluorescence. The guidelines for identifying MNCs in oral epithelial cells were done according to the criterion established by Tolbert et al. [36]. 2.3. Statistical analysis All results are expressed as mean ± standard deviation. The results were evaluated using the Statistical Program for Social Sciences (SPSS v11.0) for Windows® medical pack (SPSS, Chicago, IL). A P-value of <0.05 was considered statistically significant. 2.3.1. Adult rats Differences in MNPCE and PCE frequencies between groups were tested by means of a one-way ANOVA for intergroup comparisons, followed by Dunnett’s T3 test for multiple post hoc comparisons versus the appropriate control. A Pearson correlation was performed to test the relationship between blood glucose levels and MNPCE frequencies increases. 2.3.2. Newborn rats The litter was used as the experimental unit (n = 4/group). The MNPCE, MNE, and PCE frequencies were evaluated by means of a one-way ANOVA. Dunnett’s T3 test was employed to correct the significance values for multiple post hoc comparisons. 2.3.3. Healthy subjects and diabetic patients The differences in the MNC frequencies between groups were evaluated using a Mann–Whitney U-test for independent and Wilcoxon test for related samples. To evaluate if variables as gender or smoking habit influenced in the MNC frequencies a Mann–Whitney U-test was done.
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Table 1 MNPCE and blood glucose values in rats experimentally made diabetic before and during pregnancy Sample
Basal 1◦ 2◦ 3◦ 4◦
Group 1: negative control (n = 7)
Group 2: diabetes without FA (n = 7)
Group 3: diabetes with FA (n = 7)
MNPCE
MNPCE
Glucose
MNPCE
Glucose
1.71 ± 0.95, NS 2.00 ± 1.15, P < 0.01 2.42 ± 1.61, NS 4.00 ± 3.36, NS 2.43 ± 0.97, NS
109.00 460.42 444.14 378.85 461.42
1.28 ± 1.49, NS 1.71 ± 1.25, P < 0.03 1.57 ± 1.39, NS 3.14 ± 1.77, NS 3.28 ± 1.60, NS
116.85 423.00 485.00 428.42 441.14
0.71 0.42 1.57 1.42 1.71
± ± ± ± ±
1.25 0.53 1.90 1.13 1.70
Glucose 110.57 104.85 78.71 100.14 104.28
± ± ± ± ±
6.62 10.51 6.07 6.20 6.36
± ± ± ± ±
9.20 78.08 82.67 70.02 82.13
± ± ± ± ±
6.54 55.06 67.73 85.32 92.73
The basal sample was taken previously to mating and the samples 1◦ –4◦ were taken every 7 days during the pregnancy at days 7, 14 and 21 of gestation, and at 7 days after the delivery. Data are expressed as means ± standard deviations of MNPCE/3000 PCE. Blood glucose levels are given as mg/dl. MNPCE, micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; FA, folic acid; NS, not significant; n, sample size. Comparisons were made between diabetes groups vs. negative control values.
3. Results 3.1. Animal experiments 3.1.1. Adult rats Means, standard deviations, and the significance of comparisons between the MNPCE frequencies of the experimental groups versus their corresponding control values are shown in Table 1. Compared to the control group (Group 1), the mean frequency of MNPCEs was significantly greater for the first sampling of both Group 2 (P < 0.01) and Group 3 (P < 0.03); there was no significant difference in the PCE frequency for these data points. Diabetic rats maintained blood glucose level greater than 350 mg/dl during the experimental procedures. A significant correlation was not seen between MNPCE frequencies and the blood glucose levels in Groups 2 and 3 (Table 1). 3.1.2. Newborn rats Means and standard deviations, as well as statistical differences between the MNE, MNPCE and PCE frequencies of treated and control newborn rats are shown in Table 2. Intergroup comparisons revealed differences in the mean frequencies of MNE and MNPCE in Group 5 (MNE: P < 0.03; MNPCE: P < 0.009) and Group 6 (MNE: P < 0.001; MNPCE: P < 0.05) compared with
Group 4. In addition, increased proportions of PCEs were found for Groups 5 and 6 compared with Group 4 (P < 0.001). No significant differences were found between pups born to diabetic mothers with or without FA treatment. 3.2. Human analysis 3.2.1. Healthy subjects and patients with DM The main characteristics of all individuals that participated in the human study are given in Table 3. The mean frequencies of MNCs for the healthy subjects and the patients with DM group, as well as the statistical evaluation of these frequencies, are shown in Table 4. The MNC frequencies in patients with DM were higher (P < 0.001) than in healthy subjects (Table 4). According to the level of disease control among the DM patients, both DM patient groups had MNC frequencies greater than that healthy subjects group (controlled: P < 0.004; uncontrolled: P < 0.001). When comparisons were made taking into account the control by type of DM, uncontrolled DM type 1 patients present significant increases in MNC number compared with controlled (Table 3; P < 0.002). Gender did not influence in the MNC number in the healthy subjects and DM patients, and no differences were found for type of DM. When comparison
Table 2 MNE, MNPCE, and PCE from newborn rats born to mothers of the studied groups Groups
n
MNE/10,000 TE
MNPCE/1000 PCE
PCE/1000 TE
4 (Negative control) 5 (Diabetes without FA) 6 (Diabetes with FA)
16 16 16
15.06 ± 5.01 26.81 ± 15.05, P < 0.03a 23.87 ± 6.61, P < 0.001a , NSb
1.81 ± 1.42 4.06 ± 2.32, P < 0.009a 3.37 ± 1.96, P < 0.05a , NSb
854.56 ± 65.71 960.43 ± 15.68, P < 0.001a 943.00 ± 29.02, P < 0.001a , NSb
Data are expressed as means ± standard deviations. MNE, micronucleated erythrocytes; TE, total erythrocytes; MNPCE, micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; FA, folic acid; n, sample size; NS, not significant. Comparisons were made a between diabetes groups vs. negative control values, and b between diabetes without FA vs. diabetes with FA groups.
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Table 3 Characteristics of subjects from the study Characteristics
n (%)
MNC
Significance
159 (100) 35.16 ± 13.48
1.78 ± 1.45
Gender Female Average age in years
88 (55) 33.94 ± 12.82
1.77 ± 1.57
Male Average age in years
71 (45) 36.66 ± 14.21
1.79 ± 1.31
NSa
Smoking status Smokers Non-smokers
77 (48) 82 (52)
1.81 ± 1.53 1.76 ± 1.37
NSb
Type and control of diabetes DM patients Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
78 (49* ) 24 (31) 54 (69)
2.41 ± 1.43 1.96 ± 1.20 2.61 ± 1.48
NSc
Diabetes Type 1 Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
55 (71) 18 (33) 37 (67)
2.35 ± 1.35 1.50 ± 0.71 2.76 ± 1.40
P < 0.002e
Diabetes Type 2 Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
23 (29) 6 (26) 17 (74)
2.57 ± 1.62 3.33 ± 1.37 2.29 ± 1.65
NSf
All individuals Average age in years
NSd
MNC are expressed as means ± standard deviations/2000 cells. MNC, micronucleated cells from buccal mucosa; n, sample size; NS, no significant; DM, diabetes mellitus; FA, folic acid; HbA1c, glycosylated hemoglobin. * Percentage from total individuals of the study. Comparisons were as follow: MNC frequency between a gender; b smoking status; c DM control: controlled vs. uncontrolled; d type of DM: type 1 vs. type 2; e DM type 1: controlled vs. uncontrolled; f DM type 2: controlled vs. uncontrolled.
Table 4 Characteristics of healthy subjects and patients with DM according to smoking status and gender
Average age in years MNC
Healthy subjects (n = 81)
DM patients (n = 78)
35.15 ± 11.55 1.17 ± 1.20 (P < 0.001)
35.17 ± 15.32 2.41 ± 1.43
Gender
MNC Average age in years per gender
Female (n (%))
Male (n (%))
Female (n (%))
Male (n (%))
47 (58%) 1.02 ± 1.22, NS 33.19 ± 10.14
34 (42%) 1.38 ± 1.15 37.85 ± 12.92
41 (53%) 2.56 ± 1.48, NS 34.80 ± 15.43
37 (47%) 2.26 ± 1.37 35.57 ± 15.39
Smoking status
MNC Intragroup Intergroup
Smokers (n (%))
Non-smokers (n (%))
Smokers (n (%))
Non-smokers (n (%))
53 (65%) 1.34 ± 1.31 NSa P < 0.001b
28 (35%) 0.86 ± 0.89 NSa P < 0.001b
24 (31%) 2.83 ± 1.52
54 (69%) 2.22 ± 1.35
MNC are expressed as means ± standard deviations/2000 cells. MNC, micronucleated cells from buccal mucosa; n, sample size; NS, no significant; DM, diabetes mellitus. Comparisons from smoking status were a intragroups: smokers vs. non-smokers from each group, and b intergroup: smokers vs. smokers or non-smokers vs. non-smokers from the different groups.
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Table 5 MNC frequency of healthy subjects and patients with DM with and without FA intake Groups Patients with DM Patients without FA intake (n = 48)
MNC
2.38 ± 1.44 P < 0.001a
Healthy subjects (n = 81) Patients with FA intake (n = 30) Before FA intake
After FA intake
2.47 ± 1.43 P < 0.001a
0.67 ± 0.55 P < 0.001a
1.17 ± 1.20
P < 0.001b Data are expressed as means ± standard deviations of MNC/2000 cells. DM, diabetes mellitus; FA, folic acid; MNC, micronucleated cells from buccal mucosa; n, sample size. Comparisons were made a between patient values vs. healthy subjects; b between before and after FA intake in patient group with FA.
was made according to smoking status, differences were observed only between healthy smokers and DM smokers (P < 0.001), and the same for non-smokers (Table 4). 3.2.2. Diabetic patients with FA The mean frequencies of MNCs for the 30 patients before and after FA consumption are shown in Table 5. Patients displayed a significantly lower MNC frequency following 30 days of FA intake (P < 0.001). 4. Discussion In the present study, we evaluated the genotoxicity of DM in humans and in an experimental model using the MN assay; we also evaluated the possible mitigation of DM genotoxicity by the consumption of FA. 4.1. Rat study 4.1.1. Adult rats The reticuloendothelial system of the adult rat removes MNEs from the circulation [37]; therefore, the genotoxicity evaluation in rats was made using PCEs. The MNPCE frequencies in Group 1, the control, did not vary significantly in samples taken from pregnant female rats over the course of the gestational period, suggesting that the handling of the experimental animals did not alter their MNPCE frequencies. Significant increases in MNPCE frequencies were observed for Groups 2 and 3 only at the first sampling period, after about 7 days of gestation. However the increase observed in the rats with experimental diabetes, could be explain by many physiological changes caused by the pancreatic -cell destruction induced by SZT. This damage may trigger inflammatory processes, as well as hyperglycaemia-induced oxidative stress [38], that may be sufficient to increase the MNPCE frequency.
In the present study, the evolution of experimental diabetes induced in rats was for a short period previous to the experimental procedures. Perhaps a prolonged diabetic state of rats before starting the sampling could have produced variable amounts of additional damage in the diabetic rats due to metabolic alterations that occurred as a result of diabetes, as happens in humans with longterm hyperglycaemia who acquire diverse vascular and renal dysfunctions. These pathologies may cause inflammatory reactions that also are associated with increased FRs production, and, as a consequence, with increased frequencies of MN [16,19]. Even though FA had no significant effect on MNPCE frequency in the diabetic rat model, we speculate that possibly the FA treatment schedule and/or the short-term FA treatment may limit our ability to demonstrate an effect for FA on MNPCEs induction in the diabetic rat model. In the present study, rats received a single daily dose of FA, and considering that the highest drug concentration occurs 30–60 min after administration [39], it could be that this administration scheme was not optimum for diminishing the MNPCE frequency. Possibly, administering FA two or three times a day would provide FA in the circulation for longer periods of time and give more protection against the DNA damage. Another complicating factor to be considered is that the rats used in this experiment were pregnant and pregnancy is considered by some authors to protect against DNA damage, perhaps via altered hormone concentrations [19]. This could have obscured any effect of FA on diabetes-induced MNPCE frequency. In the present, no significant correlation was observed between blood glucose levels and MNPCE frequency. This could indicated that blood glucose levels it is not the direct cause of the genetic damage seen as increases in the MNPCE frequency, but rather to the compli-
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cations that results from the DM itself may be the responsible. Also, the PCE frequency data suggested that the experimental conditions had not effect on cytotoxicity. 4.1.2. Newborn rats The MNE and MNPCE frequencies in the offspring of rats with experimental diabetes were higher than in offspring from the negative control group. This result is consistent with previous observations of increased MN frequencies in preterm infants born to mothers with pathologies that involved an increase in FRs production, such as systemic arterial hypertension, vaginal infection, and DM [16]. These findings suggest that the intrauterine environment in the diabetic rat during pregnancy is genotoxic and this genotoxicity may have teratogenic consequences [14]. This could explain the increased incidence of congenital defects in children born to mothers with DM [40]. As in the adult rats, the proportion of PCEs was not diminished in newborn rats born to diabetic mothers. As was the case in the diabetic rats groups, FA supplementation not reduced the MNE or MNPCE frequencies in the pups born to diabetic mothers (Table 2), similarly as was seen in newborn rats born to mothers without diabetes [14]. This could be explained also because the folate intake might have been still too low to maintain folate levels into circulation that permits to observe a beneficial effect against DNA damage. 4.2. Human study 4.2.1. Effect of DM on MNC frequencies DM patients had a higher frequency of MNCs than healthy subjects (Table 4). Similarly elevated MN frequencies have been measured in patients with cancer, and rheumatoid arthritis [19,41,42], and these increased MN frequencies may be associated with the collateral complications in these patients. In the present study, the frequency of MNCs in DM patients was approximately two-fold higher than in healthy subjects (P < 0.001), indicating that the DM patients had a greater amount of damage in their DNA than the spontaneous level of damage that occurs in subjects without DM. It has been observed that smoking increases the frequency of MN [43]; but in the present work, no differences in the MNC number was observed when comparisons were made intragroup between smokers and non-smokers into both groups, however differences in the MNC number were observed when comparisons were made between healthy subjects smokers and
DM patients smokers (P < 0.001), as well as in healthy subjects non-smokers versus DM patients non-smokers (P < 0.001), and this increase was clearly due to DM itself. Further analyzes of the data indicate there were no significant intragroup or intergroup differences in MNC frequency related to gender or type of DM. When the level of disease control in DM patients was considered, either controlled or uncontrolled DM patients had MNC frequencies greater than that healthy subjects group (controlled: P < 0.004; uncontrolled: P < 0.001), but among the DM patients this variable did not influence on the MNC frequency (controlled versus uncontrolled DM patients; Table 3). However, in comparison according DM type, uncontrolled patients with DM type 1 display increases in the MNC number compared with controlled patients with the same type of DM (Table 3; P < 0.002). This shows the benefits of a balanced diet and the intake of prescribed hypoglycaemic drugs. 4.2.2. Effect of FA supplementation on MNC frequency in DM patients The 30 DM patients who took FA supplementation for 30 days displayed a remarkable decrease in the frequency of MNCs (P < 0.001; Table 5) at the end of the treatment, and even when compared with the healthy subjects group (P < 0.001). The significant reduction in MN observed in humans may be due to the FA treatment protocol. The FA three-times per day regimen may have maintained more consistent FA levels over time, and diminished the amount of DNA damage caused by FRs. Highly reactive FRs can start a chain reaction that damages cells and tissue [4,6]. FRs-induced oxidative DNA damage can occur by many routes, including the oxidative modification of the nucleotide bases, sugars, or by forming crosslinks. Such modifications can lead to mutations, cancer, loss in the expression of some genes, DNA strand breaks, and MN formation [4,5,44]. The increase in blood glucose levels in DM patients produces an oxidative state and excess FRs production, which results in cellular imbalances; these factors may account for the early collateral complications that DM patients develop [5,8]. Ramos-Remus et al. [19] demonstrated that rheumatoid arthritis patients have increased MNC frequencies and hypothesized that this increase in DNA damage could be related to the high incidence of leukaemia in these patients. Some reports suggest that DM patients are also at increased risk for some types of cancer [45,46], but other reports indicate that diabetic patients are at reduced risk for tumor development [47,48]. Our results are more consistent with the former observations, since the increase in MNC frequency could indicate that DM
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patients are at increase cancer risk, as was seen previously in other studies of MN in human lymphocytes [17]. FRs are formed under normal physiological conditions, and are controlled by cellular defence mechanisms. In pathological situations like in DM, the production of FRs is increased and may result in a state of oxidative stress in the organism [1,4]. In the present study, we found that DM patients taking FA supplements for 30 days had reduced frequencies of MNCs. The protective effects of FA in reducing MN frequencies have been observed previously [14,20]. These observations raise the question of whether or not the side effects associated with the genotoxicity of DM can be reduced by suitable doses of FA. Acknowledgments The authors would like to acknowledge Dr. Robert H. Heflich who review this manuscript and supplied valuable suggestions that enhanced its readability. References [1] S.P. Wolff, Diabetes mellitus and free radicals. Free radicals, transition metals and oxidative stress in the aetiology of diabetes mellitus and complications, Br. Med. Bull. 49 (1993) 642–652. [2] C. Pavia, I. Ferrer, C. Valls, R. Artuch, C. Colom´e, M.A. Vilaseca, Total homocysteine in patients with type 1 diabetes, Diabetes Care 23 (2000) 84–87. [3] A. Khan, S.S. Lasker, T.A. Chowdhury, Are spouses of patients with type 2 diabetes at increased risk of developing diabetes? Diabetes Care 26 (2003) 710–712. [4] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [5] P. Dandona, K. Thusu, S. Cook, B. Snyder, J. Makowski, D. Armstrong, T. Nicotera, Oxidative damage to DNA in diabetes mellitus, Lancet 347 (1996) 444–445. [6] R.W. Gracy, J.M. Talent, Y. Kong, C.C. Conrad, Reactive oxygen species: the unavoidable environmental insult? Mutat. Res. 428 (1999) 17–22. [7] J.W. Baynes, Chemical modification of proteins by lipids in diabetes, Clin. Chem. Lab. Med. 41 (2003) 1159–1165. [8] V. Pitozzi, L. Giovannelli, G. Bardini, C.M. Rotella, P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes, Mutat. Res. 529 (2003) 129–133. [9] F. Astaneie, M. Afshari, A. Mojtahedi, S. Mostafalou, M.J. Zamani, B. Larijani, M. Abdollahi, Total antioxidant capacity and levels of epidermal growth factor and nitric oxide in blood and saliva of insulin-dependent diabetic patients, Arch. Med. Res. 36 (2005) 376–381. [10] M.V. Lafleur, J. Retel, Contrasting effects of SH-compounds on oxidative DNA damage: repair and increase of damage, Mutat. Res. 295 (1993) 1–10. [11] Y. Yoshie, H. Ohshima, Synergistic induction of DNA strand breakage by cigarette tar and nitric oxide, Carcinogenesis 18 (1997) 1359–1363.
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Mutation Research 634 (2007) 126–134
Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model Guillermo M. Z´un˜ iga-Gonz´alez a,∗ , Cecilia M. Batista-Gonz´alez a , Belinda C. G´omez-Meda a , Mar´ıa L. Ramos-Ibarra a , Ana L. Zamora-Perez b , Tereza Mu˜noz-Magallanes c , Carmen Ramos-Vald´es c , Martha P. Gallegos-Arreola d a
c
Laboratorio de Mutag´enesis, Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico b Laboratorio de Farmacogen´ omica y Biomedicina Molecular, Centro Interdisciplinario de Investigaci´on para el Desarrollo Integral Regional, Instituto Polit´ecnico Nacional, Durango, Durango, Mexico Servicio de Endocrinolog´ıa, Unidad M´edica de Alta Especialidad, Hospital de Especialidades, Centro M´edico Nacional de Occidente “Lic. Ignacio Garc´ıa T´ellez”, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico d Laboratorio de Gen´ etica Molecular, Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico Received 9 March 2007; received in revised form 19 June 2007; accepted 23 June 2007 Available online 28 June 2007
Abstract Diabetes mellitus (DM) is associated with a high risk of health complications, mainly due to excessive free radical (FRs) production that could result in an increased frequency of micronuclei. The consumption of antioxidants, like folic acid (FA), may mitigate the effects of the FRs. In the present study, micronucleated polychromatic erythrocyte (MNPCE) frequencies were determined in blood sampled weekly from the tails of pregnant female Wistar rats and pregnant Wistar rats with experimental diabetes that were given unsupplemented diets and diets supplemented with FA. At birth, the pups were sampled to analyze micronucleated erythrocyte (MNE) and MNPCE frequencies. Moreover micronucleated cells (MNCs) were evaluated in buccal mucosa samples taken from 81 healthy adult subjects, 48 patients with DM, and 30 DM patients who were sampled before and after FA treatment. Increases in MNPCE frequencies were significant only at the first sampling (P < 0.01 and P < 0.03) in pregnant rats with experimental diabetes. In addition, the pups from the diabetic group and from diabetic group treated with FA had higher frequencies of MNEs (P < 0.03 and P < 0.001, respectively) and MNPCEs (P < 0.009 and P < 0.05, respectively) than the controls. No differences were found in diabetic rats and newborn rats born to diabetic mothers treated with FA compared with untreated animals. Patients with DM had a higher frequency of MNCs compared with healthy subjects (P < 0.001). Also FA reduced the frequency of MNCs in DM patients (P < 0.001). The results of this study indicate that diabetes results in elevated frequencies of micronuclei, and that, at least in humans, FA can protect against the elevation. © 2007 Elsevier B.V. All rights reserved. Keywords: Micronuclei; Diabetes; Folic acid; Free radicals; Human; Rat
∗
Corresponding author at: Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada 800, Col. Independencia, C.P. 44340, Guadalajara, Jalisco, Mexico. Tel.: +52 33 36189410; fax: +52 33 36181756. E-mail address: [email protected] (G.M. Z´un˜ iga-Gonz´alez). 1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2007.06.006
G.M. Z´un˜ iga-Gonz´alez et al. / Mutation Research 634 (2007) 126–134
1. Introduction Diabetes mellitus (DM) is characterized by an elevation in blood glucose concentration. The disease is progressive and is associated with the development of complications, like atherosclerosis, renal and neuronal damage, and blindness [1–3]. Experimental evidence indicates that these complications are due mainly to the production of excessive concentrations of free radicals (FRs), which result in oxidative damage to biomolecules [1,4–9]. Oxidative damage to the genetic material could cause DNA strand breaks [5,8,10–13] and micronuclei (MN), and these types of damage could have teratogenic or carcinogenic consequences [14–17]. Elevated frequencies of micronucleated erythrocytes (MNEs) have been measured in premature children born to mothers with pathologies related to oxidative stress [16], like arterial hypertension and DM. Also, increases in micronucleated cells (MNCs) have been observed in buccal mucosa of patients with other pathologies characterized by increases in FRs production, like rheumatoid arthritis [18,19]. The damage caused by FRs can be mitigated by antioxidant defence systems, which in the case of DM, become overwhelmed by FRs generated by the disease processes [4,7,8]. Antioxidant support systems can in theory be supplemented by using antioxidants like folic acid (FA) [20] that have the capacity to resist (or to neutralize) the effects of FRs [12,21–25]. FA deficiency increases spontaneous chromosomal damage by massive incorporation of uracil within DNA, which produces chromosomal breakage and MN formation [26], and can influence the genotoxic responses to other compounds [27,28]. The use of supplemental FA by women preand post-conception diminishes the occurrence of neural tube defects in their offspring [29]. Observations such as these illustrate the health advantages produced by the intake of this vitamin [30]. Previous studies indicate that FA supplementation decreases the frequency of MN in humans and experimental systems [14,20,31]. In the present study, we have evaluated the effect of DM on MN frequency, and the effect of FA supplementation on DM-associated MN.
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Care Committee. All experiments were performed according to the guidelines for the care and use of experimental animals at the Centro de Investigaci´on Biom´edica de Occidente, which are in compliance with those given in national (M´exico; NOM062-ZOO-2001) and international guidelines for the humane treatment of research animals. Twenty-one 3.5-month-old female Wistar rats were housed individually in polycarbonate cages, and given water and food ad libitum. All animals used in the study were supplied by the laboratory animal facility of the Centro de Investigaci´on Biom´edica de Occidente, Instituto Mexicano del Seguro Social, M´exico. In addition, 48 pups of undetermined sex, born to these rats, were used during the course of the experiments. 2.1.2. Adults rats 2.1.2.1. Study groups. Three groups of seven adult female rats were formed. Group 1 (negative control) received a single intraperitoneal (i.p.) injection of 0.3 ml injectable distilled water, and then were mated with males of the same strain. Pregnancy was determined by the presence of sperm in a vaginal smear [14], and when pregnancy was confirmed, the rats were given daily orally by gavages doses of 0.5 ml water until delivery. Group 2 and 3 rats were first made diabetic as described below. Group 2 rats (diabetes without FA) were mated and treated with water as above. Group 3 animals (diabetes with FA) underwent the same procedure as Group 2, except that they received daily 0.5 ml doses of 0.7 mg of FA/kg (Sigma, St. Louis, MO; CAS No. 59-30-3) orally by gavages (in water) from the first day of pregnancy until the birth. The dose of FA was based on the therapeutic dose recommended for pregnant women (5 mg/day), using an average human body weight of 70 kg and multiplying by 10, because is established that on a body weight basis, humans are generally more vulnerable than are experimental animals, probably by a factor of about 10 [32]. 2.1.2.2. Experimental diabetes induction in rats. Fourteen rats (Groups 2 and 3) received i.p. injections of 65 mg streptozotocin (STZ)/kg (Sigma, St. Louis, MO; CAS No. 18883-66-4) to induce DM [33,34], at least 3 days previously to be mating. The diabetes was confirmed with a glucometer (One Touch Ultra; Reg. Not 1691E2002, S.S.A. Johnson & Johnson, M´exico, S.A. of C.V.). To consider that an experimental diabetes in the rat was established, it was required that the rat maintains values greater than 250 mg/dl of blood glucose after the induction. In order to assure that the hyperglycaemic state was maintained, glucose levels were monitored every 7 days during the experiment.
2. Materials and methods 2.1. Rat study 2.1.1. Animals The study was approved by our Institutional Research Committee (register number 2002249018) and by a local Animal
2.1.2.3. Sample preparation and MNE analysis. A total of five samples of blood were taken of each rat during the experiment. The basal sample was taken previously to mating and the next samples were taken every 7 days during the pregnancy at days 7, 14 and 21 of gestation, and at 7 days after the delivery, and two smears were made of each sample time on pre-cleaned
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and pre-coded microscope slides. The smears were air-dried, fixed in absolute ethanol for 10 min, and stained with acridine orange [35]. The slides were scored manually using an Olympus BX51 microscope equipped with epifluorescence and a 100× objective. The number of micronucleated polychromatic erythrocytes (MNPCEs) was counted in 3000 polychromatic erythrocytes (PCEs), as well as the proportion of PCEs in 1000 total erythrocytes (TEs). 2.1.3. Newborn rats 2.1.3.1. Study groups. At birth, four pups were selected randomly from each of four dams from the three experimental groups. The pup groups were designated based on the treatment of the mother: Group 4: negative control; Group 5: diabetes without FA; Group 6: diabetes with FA. 2.1.3.2. Sample preparation and MNE analysis. A drop of peripheral blood was taken from the tip of the tail of each newborn rat pup immediately after birth, and smears were prepared on pre-coded glass slides. The smears were air-dried, fixed in absolute ethanol for 10 min, and stained with acridine orange [35]. The slides were scored manually using an Olympus BX51 epifluorescence microscope and a 100× objective. The number of MNEs was determined in 10,000 TEs and the number of MNPCEs was determined in 1000 PCEs. The number of PCEs in 1000 TEs also was counted. 2.2. Human study The study was approved by our Institutional Research Committee (register number 2002249018). The study group consisted of 159 individuals divided into three subgroups: Group I (n = 81) healthy subjects; Group II (n = 48) diabetic patients; Group III (n = 30) diabetic patients under FA treatment. All individuals who agreed to participate in the study signed a letter of informed consent and answered a detailed questionnaire about personal information as gender, age, alimentary habits, consumption of drugs or antioxidants, smoking habit, illness. 2.2.1. Healthy subjects group Eighty-one adult volunteers without diabetes were sampled. The subjects had blood glucose levels of <110 mg/dl and had not used supplemental antioxidants for approximately 1 month prior to the study. 2.2.2. Diabetic patient groups Adult diabetic patients from the Endocrinology Service of the Centro M´edico Nacional de Occidente, Instituto Mexicano del Seguro Social, were included in the study. Patients with controlled diabetes had glycosylated hemoglobin (HbA1c) levels of <7%, while patients with uncontrolled diabetes had HbA1c levels >7%. None of the patients included in the study consumed supplemental antioxidants for approximately 1 month prior to the study.
Forty-eight diabetic patients were sampled immediately to evaluate the effect of DM on MN frequency. Another group of 30 diabetic patients took 5 mg of FA (Lab. Valdecasas, S.A., lot 051033; Reg. No. 82231, S.S.A.) three times a day for 30 days p.o. 2.2.3. Sample preparation and MN analysis Buccal mucosal samples were collected from each subject. In the case of diabetic patients who took FA, samples were taken before starting the FA supplementation (basal sample) and after 30 days of FA intake. Subjects were asked to rinse their mouth with water, then a polished slide was used to collect cells from the buccal mucosa of the right and left cheeks; the samples were spread directly into two separate pre-cleaned and pre-coded slides. The smears were air-dried and fixed in 80% methanol for 48 h [15], and then stained with acridine orange [35]. MNCs were counted in 2000 cells at 100× magnifications using an Olympus BX51 microscope equipped with epifluorescence. The guidelines for identifying MNCs in oral epithelial cells were done according to the criterion established by Tolbert et al. [36]. 2.3. Statistical analysis All results are expressed as mean ± standard deviation. The results were evaluated using the Statistical Program for Social Sciences (SPSS v11.0) for Windows® medical pack (SPSS, Chicago, IL). A P-value of <0.05 was considered statistically significant. 2.3.1. Adult rats Differences in MNPCE and PCE frequencies between groups were tested by means of a one-way ANOVA for intergroup comparisons, followed by Dunnett’s T3 test for multiple post hoc comparisons versus the appropriate control. A Pearson correlation was performed to test the relationship between blood glucose levels and MNPCE frequencies increases. 2.3.2. Newborn rats The litter was used as the experimental unit (n = 4/group). The MNPCE, MNE, and PCE frequencies were evaluated by means of a one-way ANOVA. Dunnett’s T3 test was employed to correct the significance values for multiple post hoc comparisons. 2.3.3. Healthy subjects and diabetic patients The differences in the MNC frequencies between groups were evaluated using a Mann–Whitney U-test for independent and Wilcoxon test for related samples. To evaluate if variables as gender or smoking habit influenced in the MNC frequencies a Mann–Whitney U-test was done.
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Table 1 MNPCE and blood glucose values in rats experimentally made diabetic before and during pregnancy Sample
Basal 1◦ 2◦ 3◦ 4◦
Group 1: negative control (n = 7)
Group 2: diabetes without FA (n = 7)
Group 3: diabetes with FA (n = 7)
MNPCE
MNPCE
Glucose
MNPCE
Glucose
1.71 ± 0.95, NS 2.00 ± 1.15, P < 0.01 2.42 ± 1.61, NS 4.00 ± 3.36, NS 2.43 ± 0.97, NS
109.00 460.42 444.14 378.85 461.42
1.28 ± 1.49, NS 1.71 ± 1.25, P < 0.03 1.57 ± 1.39, NS 3.14 ± 1.77, NS 3.28 ± 1.60, NS
116.85 423.00 485.00 428.42 441.14
0.71 0.42 1.57 1.42 1.71
± ± ± ± ±
1.25 0.53 1.90 1.13 1.70
Glucose 110.57 104.85 78.71 100.14 104.28
± ± ± ± ±
6.62 10.51 6.07 6.20 6.36
± ± ± ± ±
9.20 78.08 82.67 70.02 82.13
± ± ± ± ±
6.54 55.06 67.73 85.32 92.73
The basal sample was taken previously to mating and the samples 1◦ –4◦ were taken every 7 days during the pregnancy at days 7, 14 and 21 of gestation, and at 7 days after the delivery. Data are expressed as means ± standard deviations of MNPCE/3000 PCE. Blood glucose levels are given as mg/dl. MNPCE, micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; FA, folic acid; NS, not significant; n, sample size. Comparisons were made between diabetes groups vs. negative control values.
3. Results 3.1. Animal experiments 3.1.1. Adult rats Means, standard deviations, and the significance of comparisons between the MNPCE frequencies of the experimental groups versus their corresponding control values are shown in Table 1. Compared to the control group (Group 1), the mean frequency of MNPCEs was significantly greater for the first sampling of both Group 2 (P < 0.01) and Group 3 (P < 0.03); there was no significant difference in the PCE frequency for these data points. Diabetic rats maintained blood glucose level greater than 350 mg/dl during the experimental procedures. A significant correlation was not seen between MNPCE frequencies and the blood glucose levels in Groups 2 and 3 (Table 1). 3.1.2. Newborn rats Means and standard deviations, as well as statistical differences between the MNE, MNPCE and PCE frequencies of treated and control newborn rats are shown in Table 2. Intergroup comparisons revealed differences in the mean frequencies of MNE and MNPCE in Group 5 (MNE: P < 0.03; MNPCE: P < 0.009) and Group 6 (MNE: P < 0.001; MNPCE: P < 0.05) compared with
Group 4. In addition, increased proportions of PCEs were found for Groups 5 and 6 compared with Group 4 (P < 0.001). No significant differences were found between pups born to diabetic mothers with or without FA treatment. 3.2. Human analysis 3.2.1. Healthy subjects and patients with DM The main characteristics of all individuals that participated in the human study are given in Table 3. The mean frequencies of MNCs for the healthy subjects and the patients with DM group, as well as the statistical evaluation of these frequencies, are shown in Table 4. The MNC frequencies in patients with DM were higher (P < 0.001) than in healthy subjects (Table 4). According to the level of disease control among the DM patients, both DM patient groups had MNC frequencies greater than that healthy subjects group (controlled: P < 0.004; uncontrolled: P < 0.001). When comparisons were made taking into account the control by type of DM, uncontrolled DM type 1 patients present significant increases in MNC number compared with controlled (Table 3; P < 0.002). Gender did not influence in the MNC number in the healthy subjects and DM patients, and no differences were found for type of DM. When comparison
Table 2 MNE, MNPCE, and PCE from newborn rats born to mothers of the studied groups Groups
n
MNE/10,000 TE
MNPCE/1000 PCE
PCE/1000 TE
4 (Negative control) 5 (Diabetes without FA) 6 (Diabetes with FA)
16 16 16
15.06 ± 5.01 26.81 ± 15.05, P < 0.03a 23.87 ± 6.61, P < 0.001a , NSb
1.81 ± 1.42 4.06 ± 2.32, P < 0.009a 3.37 ± 1.96, P < 0.05a , NSb
854.56 ± 65.71 960.43 ± 15.68, P < 0.001a 943.00 ± 29.02, P < 0.001a , NSb
Data are expressed as means ± standard deviations. MNE, micronucleated erythrocytes; TE, total erythrocytes; MNPCE, micronucleated polychromatic erythrocytes; PCE, polychromatic erythrocytes; FA, folic acid; n, sample size; NS, not significant. Comparisons were made a between diabetes groups vs. negative control values, and b between diabetes without FA vs. diabetes with FA groups.
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Table 3 Characteristics of subjects from the study Characteristics
n (%)
MNC
Significance
159 (100) 35.16 ± 13.48
1.78 ± 1.45
Gender Female Average age in years
88 (55) 33.94 ± 12.82
1.77 ± 1.57
Male Average age in years
71 (45) 36.66 ± 14.21
1.79 ± 1.31
NSa
Smoking status Smokers Non-smokers
77 (48) 82 (52)
1.81 ± 1.53 1.76 ± 1.37
NSb
Type and control of diabetes DM patients Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
78 (49* ) 24 (31) 54 (69)
2.41 ± 1.43 1.96 ± 1.20 2.61 ± 1.48
NSc
Diabetes Type 1 Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
55 (71) 18 (33) 37 (67)
2.35 ± 1.35 1.50 ± 0.71 2.76 ± 1.40
P < 0.002e
Diabetes Type 2 Controlled (HbA1c < 7%) Uncontrolled (HbA1c > 7%)
23 (29) 6 (26) 17 (74)
2.57 ± 1.62 3.33 ± 1.37 2.29 ± 1.65
NSf
All individuals Average age in years
NSd
MNC are expressed as means ± standard deviations/2000 cells. MNC, micronucleated cells from buccal mucosa; n, sample size; NS, no significant; DM, diabetes mellitus; FA, folic acid; HbA1c, glycosylated hemoglobin. * Percentage from total individuals of the study. Comparisons were as follow: MNC frequency between a gender; b smoking status; c DM control: controlled vs. uncontrolled; d type of DM: type 1 vs. type 2; e DM type 1: controlled vs. uncontrolled; f DM type 2: controlled vs. uncontrolled.
Table 4 Characteristics of healthy subjects and patients with DM according to smoking status and gender
Average age in years MNC
Healthy subjects (n = 81)
DM patients (n = 78)
35.15 ± 11.55 1.17 ± 1.20 (P < 0.001)
35.17 ± 15.32 2.41 ± 1.43
Gender
MNC Average age in years per gender
Female (n (%))
Male (n (%))
Female (n (%))
Male (n (%))
47 (58%) 1.02 ± 1.22, NS 33.19 ± 10.14
34 (42%) 1.38 ± 1.15 37.85 ± 12.92
41 (53%) 2.56 ± 1.48, NS 34.80 ± 15.43
37 (47%) 2.26 ± 1.37 35.57 ± 15.39
Smoking status
MNC Intragroup Intergroup
Smokers (n (%))
Non-smokers (n (%))
Smokers (n (%))
Non-smokers (n (%))
53 (65%) 1.34 ± 1.31 NSa P < 0.001b
28 (35%) 0.86 ± 0.89 NSa P < 0.001b
24 (31%) 2.83 ± 1.52
54 (69%) 2.22 ± 1.35
MNC are expressed as means ± standard deviations/2000 cells. MNC, micronucleated cells from buccal mucosa; n, sample size; NS, no significant; DM, diabetes mellitus. Comparisons from smoking status were a intragroups: smokers vs. non-smokers from each group, and b intergroup: smokers vs. smokers or non-smokers vs. non-smokers from the different groups.
G.M. Z´un˜ iga-Gonz´alez et al. / Mutation Research 634 (2007) 126–134
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Table 5 MNC frequency of healthy subjects and patients with DM with and without FA intake Groups Patients with DM Patients without FA intake (n = 48)
MNC
2.38 ± 1.44 P < 0.001a
Healthy subjects (n = 81) Patients with FA intake (n = 30) Before FA intake
After FA intake
2.47 ± 1.43 P < 0.001a
0.67 ± 0.55 P < 0.001a
1.17 ± 1.20
P < 0.001b Data are expressed as means ± standard deviations of MNC/2000 cells. DM, diabetes mellitus; FA, folic acid; MNC, micronucleated cells from buccal mucosa; n, sample size. Comparisons were made a between patient values vs. healthy subjects; b between before and after FA intake in patient group with FA.
was made according to smoking status, differences were observed only between healthy smokers and DM smokers (P < 0.001), and the same for non-smokers (Table 4). 3.2.2. Diabetic patients with FA The mean frequencies of MNCs for the 30 patients before and after FA consumption are shown in Table 5. Patients displayed a significantly lower MNC frequency following 30 days of FA intake (P < 0.001). 4. Discussion In the present study, we evaluated the genotoxicity of DM in humans and in an experimental model using the MN assay; we also evaluated the possible mitigation of DM genotoxicity by the consumption of FA. 4.1. Rat study 4.1.1. Adult rats The reticuloendothelial system of the adult rat removes MNEs from the circulation [37]; therefore, the genotoxicity evaluation in rats was made using PCEs. The MNPCE frequencies in Group 1, the control, did not vary significantly in samples taken from pregnant female rats over the course of the gestational period, suggesting that the handling of the experimental animals did not alter their MNPCE frequencies. Significant increases in MNPCE frequencies were observed for Groups 2 and 3 only at the first sampling period, after about 7 days of gestation. However the increase observed in the rats with experimental diabetes, could be explain by many physiological changes caused by the pancreatic -cell destruction induced by SZT. This damage may trigger inflammatory processes, as well as hyperglycaemia-induced oxidative stress [38], that may be sufficient to increase the MNPCE frequency.
In the present study, the evolution of experimental diabetes induced in rats was for a short period previous to the experimental procedures. Perhaps a prolonged diabetic state of rats before starting the sampling could have produced variable amounts of additional damage in the diabetic rats due to metabolic alterations that occurred as a result of diabetes, as happens in humans with longterm hyperglycaemia who acquire diverse vascular and renal dysfunctions. These pathologies may cause inflammatory reactions that also are associated with increased FRs production, and, as a consequence, with increased frequencies of MN [16,19]. Even though FA had no significant effect on MNPCE frequency in the diabetic rat model, we speculate that possibly the FA treatment schedule and/or the short-term FA treatment may limit our ability to demonstrate an effect for FA on MNPCEs induction in the diabetic rat model. In the present study, rats received a single daily dose of FA, and considering that the highest drug concentration occurs 30–60 min after administration [39], it could be that this administration scheme was not optimum for diminishing the MNPCE frequency. Possibly, administering FA two or three times a day would provide FA in the circulation for longer periods of time and give more protection against the DNA damage. Another complicating factor to be considered is that the rats used in this experiment were pregnant and pregnancy is considered by some authors to protect against DNA damage, perhaps via altered hormone concentrations [19]. This could have obscured any effect of FA on diabetes-induced MNPCE frequency. In the present, no significant correlation was observed between blood glucose levels and MNPCE frequency. This could indicated that blood glucose levels it is not the direct cause of the genetic damage seen as increases in the MNPCE frequency, but rather to the compli-
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cations that results from the DM itself may be the responsible. Also, the PCE frequency data suggested that the experimental conditions had not effect on cytotoxicity. 4.1.2. Newborn rats The MNE and MNPCE frequencies in the offspring of rats with experimental diabetes were higher than in offspring from the negative control group. This result is consistent with previous observations of increased MN frequencies in preterm infants born to mothers with pathologies that involved an increase in FRs production, such as systemic arterial hypertension, vaginal infection, and DM [16]. These findings suggest that the intrauterine environment in the diabetic rat during pregnancy is genotoxic and this genotoxicity may have teratogenic consequences [14]. This could explain the increased incidence of congenital defects in children born to mothers with DM [40]. As in the adult rats, the proportion of PCEs was not diminished in newborn rats born to diabetic mothers. As was the case in the diabetic rats groups, FA supplementation not reduced the MNE or MNPCE frequencies in the pups born to diabetic mothers (Table 2), similarly as was seen in newborn rats born to mothers without diabetes [14]. This could be explained also because the folate intake might have been still too low to maintain folate levels into circulation that permits to observe a beneficial effect against DNA damage. 4.2. Human study 4.2.1. Effect of DM on MNC frequencies DM patients had a higher frequency of MNCs than healthy subjects (Table 4). Similarly elevated MN frequencies have been measured in patients with cancer, and rheumatoid arthritis [19,41,42], and these increased MN frequencies may be associated with the collateral complications in these patients. In the present study, the frequency of MNCs in DM patients was approximately two-fold higher than in healthy subjects (P < 0.001), indicating that the DM patients had a greater amount of damage in their DNA than the spontaneous level of damage that occurs in subjects without DM. It has been observed that smoking increases the frequency of MN [43]; but in the present work, no differences in the MNC number was observed when comparisons were made intragroup between smokers and non-smokers into both groups, however differences in the MNC number were observed when comparisons were made between healthy subjects smokers and
DM patients smokers (P < 0.001), as well as in healthy subjects non-smokers versus DM patients non-smokers (P < 0.001), and this increase was clearly due to DM itself. Further analyzes of the data indicate there were no significant intragroup or intergroup differences in MNC frequency related to gender or type of DM. When the level of disease control in DM patients was considered, either controlled or uncontrolled DM patients had MNC frequencies greater than that healthy subjects group (controlled: P < 0.004; uncontrolled: P < 0.001), but among the DM patients this variable did not influence on the MNC frequency (controlled versus uncontrolled DM patients; Table 3). However, in comparison according DM type, uncontrolled patients with DM type 1 display increases in the MNC number compared with controlled patients with the same type of DM (Table 3; P < 0.002). This shows the benefits of a balanced diet and the intake of prescribed hypoglycaemic drugs. 4.2.2. Effect of FA supplementation on MNC frequency in DM patients The 30 DM patients who took FA supplementation for 30 days displayed a remarkable decrease in the frequency of MNCs (P < 0.001; Table 5) at the end of the treatment, and even when compared with the healthy subjects group (P < 0.001). The significant reduction in MN observed in humans may be due to the FA treatment protocol. The FA three-times per day regimen may have maintained more consistent FA levels over time, and diminished the amount of DNA damage caused by FRs. Highly reactive FRs can start a chain reaction that damages cells and tissue [4,6]. FRs-induced oxidative DNA damage can occur by many routes, including the oxidative modification of the nucleotide bases, sugars, or by forming crosslinks. Such modifications can lead to mutations, cancer, loss in the expression of some genes, DNA strand breaks, and MN formation [4,5,44]. The increase in blood glucose levels in DM patients produces an oxidative state and excess FRs production, which results in cellular imbalances; these factors may account for the early collateral complications that DM patients develop [5,8]. Ramos-Remus et al. [19] demonstrated that rheumatoid arthritis patients have increased MNC frequencies and hypothesized that this increase in DNA damage could be related to the high incidence of leukaemia in these patients. Some reports suggest that DM patients are also at increased risk for some types of cancer [45,46], but other reports indicate that diabetic patients are at reduced risk for tumor development [47,48]. Our results are more consistent with the former observations, since the increase in MNC frequency could indicate that DM
G.M. Z´un˜ iga-Gonz´alez et al. / Mutation Research 634 (2007) 126–134
patients are at increase cancer risk, as was seen previously in other studies of MN in human lymphocytes [17]. FRs are formed under normal physiological conditions, and are controlled by cellular defence mechanisms. In pathological situations like in DM, the production of FRs is increased and may result in a state of oxidative stress in the organism [1,4]. In the present study, we found that DM patients taking FA supplements for 30 days had reduced frequencies of MNCs. The protective effects of FA in reducing MN frequencies have been observed previously [14,20]. These observations raise the question of whether or not the side effects associated with the genotoxicity of DM can be reduced by suitable doses of FA. Acknowledgments The authors would like to acknowledge Dr. Robert H. Heflich who review this manuscript and supplied valuable suggestions that enhanced its readability. References [1] S.P. Wolff, Diabetes mellitus and free radicals. Free radicals, transition metals and oxidative stress in the aetiology of diabetes mellitus and complications, Br. Med. Bull. 49 (1993) 642–652. [2] C. Pavia, I. Ferrer, C. Valls, R. Artuch, C. Colom´e, M.A. Vilaseca, Total homocysteine in patients with type 1 diabetes, Diabetes Care 23 (2000) 84–87. [3] A. Khan, S.S. Lasker, T.A. Chowdhury, Are spouses of patients with type 2 diabetes at increased risk of developing diabetes? Diabetes Care 26 (2003) 710–712. [4] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [5] P. Dandona, K. Thusu, S. Cook, B. Snyder, J. Makowski, D. Armstrong, T. Nicotera, Oxidative damage to DNA in diabetes mellitus, Lancet 347 (1996) 444–445. [6] R.W. Gracy, J.M. Talent, Y. Kong, C.C. Conrad, Reactive oxygen species: the unavoidable environmental insult? Mutat. Res. 428 (1999) 17–22. [7] J.W. Baynes, Chemical modification of proteins by lipids in diabetes, Clin. Chem. Lab. Med. 41 (2003) 1159–1165. [8] V. Pitozzi, L. Giovannelli, G. Bardini, C.M. Rotella, P. Dolara, Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes, Mutat. Res. 529 (2003) 129–133. [9] F. Astaneie, M. Afshari, A. Mojtahedi, S. Mostafalou, M.J. Zamani, B. Larijani, M. Abdollahi, Total antioxidant capacity and levels of epidermal growth factor and nitric oxide in blood and saliva of insulin-dependent diabetic patients, Arch. Med. Res. 36 (2005) 376–381. [10] M.V. Lafleur, J. Retel, Contrasting effects of SH-compounds on oxidative DNA damage: repair and increase of damage, Mutat. Res. 295 (1993) 1–10. [11] Y. Yoshie, H. Ohshima, Synergistic induction of DNA strand breakage by cigarette tar and nitric oxide, Carcinogenesis 18 (1997) 1359–1363.
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