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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. Zúñiga-González a,∗ , Cecilia M. Batista-González a ,
Belinda C. Gómez-Meda a , Marı́a L. Ramos-Ibarra a , Ana L. Zamora-Perez b ,
Tereza Muñoz-Magallanes c , Carmen Ramos-Valdés c , Martha P. Gallegos-Arreola d
a Laboratorio de Mutagénesis, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del
Seguro Social, Guadalajara, Jalisco, Mexico
b Laboratorio de Farmacogenómica y Biomedicina Molecular, Centro Interdisciplinario de Investigación para el Desarrollo

Integral Regional, Instituto Politécnico Nacional, Durango, Durango, Mexico

c Servicio de Endocrinologı́a, Unidad Médica de Alta Especialidad, Hospital de Especialidades, Centro Médico Nacional de Occidente
“Lic. Ignacio Garcı́a Téllez”, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, Mexico
d Laboratorio de Genética Molecular, Centro de Investigación Biomédica 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 Investigación Biomédica 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: mutagenesis95@hotmail.com (G.M. Zúñiga-González).

1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2007.06.006
 G.M. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134
1. Introduction
Diabetes mellitus (DM) is characterized by an ele-
vation 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 charac-
terized 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 neu-
tralize) the effects of FRs [12,21–25]. FA deficiency
increases spontaneous chromosomal damage by mas-
sive 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 pre-
and post-conception diminishes the occurrence of neu-
ral 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 exper-
imental 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.
2. Materials and methods
2.1. Rat study
2.1.1. Animals
The study was approved by our Institutional Research Com-
mittee (register number 2002249018) and by a local Animal
127
Care Committee. All experiments were performed according
to the guidelines for the care and use of experimental animals
at the Centro de Investigación Biomédica de Occidente, which
are in compliance with those given in national (México; NOM-
062-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 sup-
plied by the laboratory animal facility of the Centro de
Investigación Biomédica de Occidente, Instituto Mexicano del
Seguro Social, México. 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 sin-
gle intraperitoneal (i.p.) injection of 0.3 ml injectable distilled
water, and then were mated with males of the same strain. Preg-
nancy 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 deliv-
ery. 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,
México, S.A. of C.V.). To consider that an experimental dia-
betes 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.
2.1.2.3. 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
128 G.M. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134
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 Olym-
pus 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 ran-
domly 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 new-
born 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 num-
ber 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 treat-
ment. 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, ali-
mentary 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 Médico Nacional de Occidente, Instituto Mexicano
del Seguro Social, were included in the study. Patients with con-
trolled 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 con-
sumed 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 sub-
ject. 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 Olym-
pus 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 relation-
ship 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 com-
parisons.
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 influ-
enced in the MNC frequencies a Mann–Whitney U-test was
done.
 G.M. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134 129

Table 1
MNPCE and blood glucose values in rats experimentally made diabetic before and during pregnancy
Sample Group 1: negative control (n = 7) Group 2: diabetes without FA (n = 7) Group 3: diabetes with FA (n = 7)

MNPCE Glucose MNPCE Glucose MNPCE Glucose

Basal 0.71 ± 1.25 110.57 ± 6.62 1.71 ± 0.95, NS 109.00 ± 9.20 1.28 ± 1.49, NS 116.85 ± 6.54
1◦ 0.42 ± 0.53 104.85 ± 10.51 2.00 ± 1.15, P < 0.01 460.42 ± 78.08 1.71 ± 1.25, P < 0.03 423.00 ± 55.06
2◦ 1.57 ± 1.90 78.71 ± 6.07 2.42 ± 1.61, NS 444.14 ± 82.67 1.57 ± 1.39, NS 485.00 ± 67.73
3◦ 1.42 ± 1.13 100.14 ± 6.20 4.00 ± 3.36, NS 378.85 ± 70.02 3.14 ± 1.77, NS 428.42 ± 85.32
4◦ 1.71 ± 1.70 104.28 ± 6.36 2.43 ± 0.97, NS 461.42 ± 82.13 3.28 ± 1.60, NS 441.14 ± 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 Group 4. In addition, increased proportions of PCEs

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 con-
trol 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 proce-
dures. 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 fre-
quencies 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
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 par-
ticipated 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 frequen-
cies 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 signifi-
cant 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 differ-
ences 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) 16 15.06 ± 5.01 1.81 ± 1.42 854.56 ± 65.71

5 (Diabetes without FA) 16 26.81 ± 15.05, P < 0.03a 4.06 ± 2.32, P < 0.009a 960.43 ± 15.68, P < 0.001a
6 (Diabetes with FA) 16 23.87 ± 6.61, P < 0.001a , NSb 3.37 ± 1.96, P < 0.05a , NSb 943.00 ± 29.02, P < 0.001a , NSb

Data are expressed as means ± standard deviations. MNE, micronucleated erythrocytes; TE, total erythrocytes; MNPCE, micronucleated polychro-
matic 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.
130 G.M. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134

Table 3
Characteristics of subjects from the study
Characteristics n (%) MNC Significance

All individuals 159 (100) 1.78 ± 1.45

Average age in years 35.16 ± 13.48
Gender
Female 88 (55) 1.77 ± 1.57
Average age in years 33.94 ± 12.82
Male 71 (45) 1.79 ± 1.31 NSa
Average age in years 36.66 ± 14.21
Smoking status
Smokers 77 (48) 1.81 ± 1.53
Non-smokers 82 (52) 1.76 ± 1.37 NSb
Type and control of diabetes
DM patients 78 (49* ) 2.41 ± 1.43
Controlled (HbA1c < 7%) 24 (31) 1.96 ± 1.20
Uncontrolled (HbA1c > 7%) 54 (69) 2.61 ± 1.48 NSc
Diabetes Type 1 55 (71) 2.35 ± 1.35 NSd
Controlled (HbA1c < 7%) 18 (33) 1.50 ± 0.71
Uncontrolled (HbA1c > 7%) 37 (67) 2.76 ± 1.40 P < 0.002e
Diabetes Type 2 23 (29) 2.57 ± 1.62
Controlled (HbA1c < 7%) 6 (26) 3.33 ± 1.37
Uncontrolled (HbA1c > 7%) 17 (74) 2.29 ± 1.65 NSf

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
Healthy subjects (n = 81) DM patients (n = 78)

Average age in years 35.15 ± 11.55 35.17 ± 15.32

MNC 1.17 ± 1.20 (P < 0.001) 2.41 ± 1.43

Gender

Female (n (%)) Male (n (%)) Female (n (%)) Male (n (%))

47 (58%) 34 (42%) 41 (53%) 37 (47%)

MNC 1.02 ± 1.22, NS 1.38 ± 1.15 2.56 ± 1.48, NS 2.26 ± 1.37
Average age in years per gender 33.19 ± 10.14 37.85 ± 12.92 34.80 ± 15.43 35.57 ± 15.39

Smoking status

Smokers (n (%)) Non-smokers (n (%)) Smokers (n (%)) Non-smokers (n (%))

53 (65%) 28 (35%) 24 (31%) 54 (69%)

MNC 1.34 ± 1.31 0.86 ± 0.89 2.83 ± 1.52 2.22 ± 1.35
Intragroup NSa NSa
Intergroup P < 0.001b P < 0.001b

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. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134
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
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 smok-
ers (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.
131
Healthy subjects (n = 81)
Patients with FA intake (n = 30)
Before FA intake After FA intake
2.47 ± 1.43 0.67 ± 0.55 1.17 ± 1.20
P < 0.001a P < 0.001a
P < 0.001b
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 dia-
betic 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 long-
term hyperglycaemia who acquire diverse vascular and
renal dysfunctions. These pathologies may cause inflam-
matory 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 concentra-
tion occurs 30–60 min after administration [39], it could
be that this administration scheme was not optimum for
diminishing the MNPCE frequency. Possibly, adminis-
tering 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 compli-
cating 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-
132 G.M. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134
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 intrauter-
ine environment in the diabetic rat during pregnancy is
genotoxic and this genotoxicity may have teratogenic
consequences [14]. This could explain the increased inci-
dence 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 fre-
quencies 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 fre-
quencies 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 sub-
jects 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 compar-
isons 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 (con-
trolled: 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 tis-
sue [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 imbal-
ances; these factors may account for the early collateral
complications that DM patients develop [5,8].
Ramos-Remus et al. [19] demonstrated that rheuma-
toid 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. Zúñiga-González et al. / Mutation Research 634 (2007) 126–134
patients are at increase cancer risk, as was seen previ-
ously 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 fre-
quencies 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 valu-
able suggestions that enhanced its readability.
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