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Genotoxic studies of vanadium pentoxide (V2O5) in male mice. II. Effects in

several mouse tissues

Article  in  Teratogenesis Carcinogenesis and Mutagenesis · February 1999

DOI: 10.1002/(SICI)1520-6866(1999)19:4<243::AID-TCM1>3.0.CO;2-J · Source: PubMed

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 Teratogenesis, Carcinogenesis, and Mutagenesis 19:243–255 (1999)

Genotoxic Studies of Vanadium Pentoxide

(V2O5) in Male Mice. II. Effects in Several
Mouse Tissues

Mario Altamirano-Lozano,2 Mahara Valverde,1 Lucia Alvarez-Barrera,2

Bertha Molina,2,3 and Emilio Rojas1*
1
Instituto de Investigaciones Biomédicas
2
Facultad de Estudios Superiores-Zaragoza, UNAM
3
Instituto Nacional de Pediatría, México

Vanadium pentoxide (V205) was tested for its ability to induce genotoxic damage in
six different organs (liver, kidney, lung, spleen, heart, and bone marrow) of mice by
using the alkaline Single Cell Gel Electrophoresis (SCGE) assay. Animals were sac-
rificed 24 h after i.p. administration of the vanadium pentoxide of 23.0, 11.5, or 5.75
µg/g (corresponding to the LD50, 1/2 LD50 and 1/4 LD50, respectively). In all tissues
and organs evaluated (except for bone marrow), V205 increased the number of cells
with damage. Our results showed that i.p. injection of V205 induced DNA damage in
different organs and tissues, and that this kind of damage can be observed even 24 h
after treatment. The analysis of DNA migration and the distribution of DNA damage
showed that there are differences in sensitivity between organs and tissues to this
compound. In addition the sensitivity of SCGE assay allows the detection of long
term DNA damage and the possibility to compare it in various tissues and target
organs. Teratogenesis Carcinog. Mutagen. 19:243–255, 1999. © 1999 Wiley-Liss, Inc.

Key words: vanadium pentoxide; V2O5; single cell gel electrophoresis assay; tissues of mice;
genotoxicity

INTRODUCTION
Several metals including cadmium, arsenic, and vanadium have been shown to
be genotoxic [1–6]. Among metal-induced DNA lesions, single (SSB) and double
strand beaks (DSB), DNA-DNA crosslinks, base modification, and SSB, eventually
leading to chromosome breakage [7], have been described.

Contract grant sponsor: CONACyT; Contract grant numbers: 318P-M9607, 25417-M, DGAPA-IN
214597.

*Correspondence to: Dr. Emilio Rojas del Castillo, Departamento de Genética y Toxicología Ambiental,
Instituto de Investigaciones Biomédicas, UNAM. A.P. 70228, México 04510, D.F., México. E-mail
address: emilior@servidor.unam.mx

© 1999 Wiley-Liss, Inc.

244 Altamirano-Lozano et al.

Studies on the biological effects of vanadium have increased greatly in recent years
due to its potential toxicological [1,8], genotoxic [4–6], and reprotoxic [4,9–11] impact
on humans and animals, and its possible role as an essential element in mammals in
addition to it’s potential pharmacological use in the treatment of diabetes [12,13].
Vanadium is widely distributed in human tissues and is toxic when ingested in
large doses [1]; the most toxic forms of vanadium to mammals are the pentavalent
form compounds [14]. Vanadium poisoning is known to occur in particular by inha-
lation of vanadium-rich dust and it may cause irritation and clinical symptoms of the
respiratory tract [15]. When vanadium is administered intravenously, the tolerance
level is very low (11.2 mg/kg leads to serious health disorders). Oral intake is less
hazardous, mainly because only 2% of vanadium is absorbed. The bones, liver, and
kidney retain vanadium with a biological half-life of 20–100 h [13].
Some studies have evaluated the genotoxic effect of vanadium in mammalian
cells in vivo. Sun [16] observed micronuclei in mouse bone marrow after i.p. vana-
dium pentoxide treatment; however, oral administration yielded negative results.
Chronic feeding or i.p. treatment with vanadium pentoxide to rats and mice did not
cause chromosome aberrations nor induce SCE in bone marrow cells but lowered the
mitotic index [9,17], and the same compound by subcutaneous injection for 3 months
to male mice did not show a positive effect in the dominant-lethal assay [16]. The
single cell gel electrophoresis or “Comet Assay” has been shown to be a sensitive
method for the detection of DNA damage (primarily single strand breaks and alkali
labile damage) in individual cells [18–20]. Furthermore, this method has been used to
evaluate the genotoxicity of diverse drugs in several mouse organs [21]. In the present
study, we report the ability of the SCGE assay to detect long-term DNA damage
induced by V2O5 in different target organs and tissues of male mice.

MATERIAL AND METHODS

Animals
Male CD-1 mice (45 days old, 30–35 g, born and bred in our laboratory) were
housed in groups of four in hanging plastic cages under controlled lighting condi-
tions (12 h light/12 h dark regime) humidity and temperature (17°C), and fed Purina
Rat Chow (Purina, México) and water ad libitum.
Vanadium Pentoxide and Treatment
Vanadium pentoxide (99.6% pure, purchased from Aldrich Chemical Co. (Mil-
waukee, WI, CAS 1314-62-1), was prepared in saline and injected intraperitoneally
(i.p.) in appropriate volumes containing either 5.75, 11.5, or 23 g of V205/g body weight.
Each treatment group consisted of four mice and these concentrations represent ¼, ½,
and the LD50, respectively, and were calculated in preliminary experiments [9,10].
Tissues and Organ Isolation
Twenty-four hours after treatment, animals were killed by cervical dislocation
and the liver, kidneys, lung, spleen, and heart were dissected and stored in 1 ml of
RPMI medium (Sigma Chemical Co., St. Louis, MO). A sample of each organ or
tissue was minced in 2 ml of cold isotonic saline solution and femur bone marrow
cells were flushed with 5 ml of RPMI medium (Sigma Chemical Co., St. Louis,
MO) and resuspended. An appropriate number of cells were pelleted by centrifuga-
 Genotoxicity of V2O5 in Mice 245

tion, mixed with 75 µl of low melting point agarose (0.5%), and loaded onto a fully-
frosted microscope slide prelayered with 200 µl of normal melting agarose (0.5%).
Viability
Cell viability was determined by using the fluorescein diacetate (FDA)/ethidium
bromide (Et-Br) assay according to Strauss [22]. A freshly prepared solution consisting
of 30 µl of FDA in acetone (5 mg/ml), 200 µl of Et-Br in phosphate-buffered saline
(200 µl/ml), and 4.8 ml PBS was used. The cellular suspension (1×106 cells/RPMI-
1640 ml) was centrifuged and 25 µl of the buffy coat was mixed with 25 µl of the
staining solution, spread on a microscope slide, and covered with a coverslip. Viable
cells appear green-fluorescent, whereas orange-stained nuclei indicate dead cells. Con-
taminating erythrocytes do not label. At least 200 cells were counted per data point.
Alkaline Single Cell Gel Electrophoresis Assay
The alkaline single cell gel electrophoresis assay was performed as described by
Tice et al. [23]. Briefly, after lysis at 4°C for 1 h [2.5M NaCl, 100 mM EDTA, 1% Na-
lauryl sarcocinate, 10 mM Tris, pH 10 (10%DMSO, 1% Triton X-100); Sigma Chemi-
cal Co., St. Louis, MO], slides were placed on a horizontal electrophoresis chamber
with running buffer solution (300 mM NaOH, 1 mM Na2EDTA, pH 13; Sigma Chemi-
cal Co., St. Louis, MO). The slides remained 20 min in the electrophoresis buffer to
allow unwinding of DNA. Electrophoresis was performed for 20 min at 25 V and 300
mA, and all technical steps were conducted using very dim indirect light. After electro-
phoresis, the slides were gently removed and rinsed with neutralization buffer (0.4 M
Tris, pH 7.5) at room temperature (15 min). Ethidium bromide (75 µl of a 20 µg/ml
solution) was added to each slide and a coverglass was placed on the gel.
Individual cells were visualized at 20× magnification on a Nikon Optiphoto-2
microscope with fluorescence attachments (excitation filter of 515–560 nm and a
barrier filter of 590 nm), and the extent of migration (i.e., image length, both head
and tail of the comet) measured with a scaled ocular. To evaluate DNA migration,
100 cells were scored per tissue per animal for each concentration. Another criterion
for evaluation was the assignment of cells into three categories according to the
value of nucleus ratio. Where ratio r = measurement of the tail/measurement of the
head. If r was ≤ 1, cells were considered in low migration, medium migration if r
was ≤ 2, and high migration when r was > 2. In this latter category we included a
number of cells in which virtually all of the DNA had migrated out the head region
[24]. The shape factor was calculated as the ratio of length to diameter. Migration
was calculated as the difference between length and diameter.
Statistical Analysis
All the statistical analysis were performed with a SPSS software. The chi-square
test was used to determine the significance of an increase in the frequency of tailed
cells. The shape factor and migration were analyzed using the Student’s t-test.

RESULTS
The viability and the percentage of cells with and without tails are presented in
Table I. Viability was greater than 70% in all organs except for liver that showed a
dose response decrease. Analysis of tailed cells with chi-square test showed a sig-
246 Altamirano-Lozano et al.
TABLE I. Percentage of Tailed Cells, Untailed Cells, and Cell Viability in Different Mice Organs
Treated i.p. With Vanadium Pentoxide
V2O5 dose (µg/gb.w.)
Control 5.75a 11.50b 23.00c
Liver Tailed cells (%) 65.7 57.3 77.8 53.5
Untailed cells (%) 34.3 42.7 22.2 46.5
Viability (% ± SD) 100 ± 3.5 94.2 ± 3.5 62.0 ± 4.2 44.0 ± 4.5
Lung Tailed cells (%) 76.9 55.4 64.5 30.9
Untailed cells (%) 23.1 44.6* 35.5 69.1*
Viability (% ± SD) 100 ± 1.0 100 ± 3.5 100 ± 2.4 75.6 ± 1.2
Spleen Tailed cells (%) 70.9 47.4 27.5 69.6
Untailed cells (%) 29.1 52.6* 72.5* 30.4
Viability (% ± SD) 100 ± 2.8 94.5 ± 1.4 89.6 ± 7.8 100 ± 3.5
Heart Tailed cells (%) 69.0 43.9 33.8 26.3
Untailed cells (%) 31.0 56.1** 66.2** 73.7*
Viability (% ± SD) 100 ± 2.4 100 ± 3.2 100 ± 1.8 100 ± 2.7
Kidney Tailed cells (%) 72.0 53.5 76.0 40.8
Untailed cells (%) 28.0 46.4* 24.0 59.2**
Viability (% ± SD) 100 ± 1.4 100 ± 3.5 91.7 ± 1.2 88.2 ± 7.8
Bone Marrow Tailed cells (%) 67.4 63.4 69.9 64.6
Untailed cells (%) 32.6 36.6 31.1 35.4
Viability (% ± SD) 100 ± 2.1 100 ± 1.2 100 ± 2.2 100 ± 1.8
a
¼ LD50 value.
b
½ LD50 value.
c
LD50 value.
*P < 0.05 with chi-squared test.
**P < 0.01 with chi-squared test.

nificant effect in some organs. Lung and kidney showed a significant decrease in the
number of tailed cells with 1/4LD50 and LD50 treatment, while in the spleen signifi-
cant effects were observed at 1/4 and 1/2 of LD50. In heart cells a significant dose-
response increase was found, while in bone marrow and liver no clear effects were
observed (Table I).
Figures 1A to 6A show the average nuclear DNA migration from liver, lung,
spleen, heart, kidney, and bone marrow cells of mice assayed with different i.p. va-
nadium pentoxide doses, respectively. A dose response were observed in all organs
except for the hematopoietic system.
When the DNA damage was evaluated according to the value of the nucleus
ratio and grouped into three categories, we observed a decrease in the percent of
cells with low damage in relation to the dose in all organs. However, the organs
sampled displayed different sensitivities to vanadium treatment was expressed as
cells with high DNA damage (Table II, Figs. 1B-6B).

DISCUSSION
DNA damage induced by vanadium pentoxide was evaluated according to three
different parameters (i.e., tailed cells vs. non-tailed cells, DNA migration length val-
ues, and the number of the cells with different nucleus/tail ratios). This approach al-
lowed us a better understanding of the response of each organ to vanadium treatment.
 Genotoxicity of V2O5 in Mice 247

Fig. 1. DNA migration and DNA damage in mice liver cells induced by V2O5. A: DNA migration
(mean ± S.E.M.). Student’s t-test was employed for statistical significance. B: DNA damage. Cells
were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
248 Altamirano-Lozano et al.

Fig. 2. DNA migration and DNA damage in mice kidney cells induced by V2O5. A: DNA migration
(mean ± S.E.M.). Student’s t-test was employed for statistical significance. B: DNA damage. Cells
were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
 Genotoxicity of V2O5 in Mice 249

Fig. 3. DNA migration and DNA damage in mice heart cells induced by V2O5. A: DNA migration
(mean ± S.E.M.). Student’s t-test was employed for statistical significance. B: DNA damage. Cells
were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
250 Altamirano-Lozano et al.

Fig. 4. DNA migration and DNA damage in mice spleen cells induced by V2O5. A: DNA migration
(mean ± S.E.M.). Student’s t-test was employed for statistical significance. B: DNA damage. Cells
were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
 Genotoxicity of V2O5 in Mice 251

Fig. 5. DNA migration and DNA damage in mice lung cells induced by V 2O5. A: DNA migration
(mean ± S.E.M.). Student’s t-test were employed for statistical significance. B: DNA damage. Cells
were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
252 Altamirano-Lozano et al.

Fig. 6. DNA migration and DNA damage in mice bone marrow cells induced by V2O5. A: DNA
migration (mean ± S.E.M.). Student’s t-test was employed for statistical significance. B: DNA damage.
Cells were assigned to different classes according to their degree of DNA damage using the formula:
measurement of the tail/measurement of the head. Low damage (≤ 1); medium damage (≤ 2); high
damage (> 2).
 Genotoxicity of V2O5 in Mice 253
TABLE II. Percentage of Cells With High DNA Danage in Different Mice Organs Treated i.p.
With Vanadium Pentoxide
V2O5 dose (µg/gb.w.)
Mice organ Control 5.75a 11.50b 23.00c
Liver 0 8 21 42
Lung 0 20 12 33
Spleen 0 9 0 22
Heart 11 19 38 39
Kidney 2 4 11 47
Bone Marrow 5 6 37 8
a
¼ LD50 value.
b
½ LD50 value.
c
LD50 value.

The results obtained in this study showed that vanadium pentoxide caused a
significant increase in the length of DNA migration, although it did not show dose-
dependent, relationship in most tissues sampled (Table I). With respect to DNA mi-
gration, the i.p. treatment of vanadium pentoxide produced DNA damage in liver,
kidney, heart, lung, spleen, and bone marrow. However, some organs were more
susceptible to this kind of damage than others. Liver, heart, and kidney showed the
highest response, and all of them have been reported previously as target organs for
the action of vanadium [1,25]. Although the lung has also been reported as a target
organ for vanadium, the low sensitivity that was observed in this organ could be due
to the administration route used in this study.
Bone marrow showed a non-dose related increase in DNA migration length, and
in the percentage of cells with more damage, but we did not observe differences in the
number of tailed cells and cell viability. It is important to note that the response in these
cells to vanadium treatment was lower than in the other organs. This possibly could be
due to an excessive proliferative activity, so diluting the cells with damage or that in
these cells the DNA repair systems are induced. This result could explain why Giri et
al. [17] and Altamirano et al. [9] reported that in vivo treatment of vanadium com-
pounds do not induce SCE or chromosomal aberrations in mouse bone marrow cells.
Our results showed that organs with a higher rate of proliferation were less sen-
sitive, indicating that the kind of damage induced by vanadium pentoxide could be
removed when cells divide. In contrast, organs with low rate of proliferation are more
sensitive to this compound. This is in agreement with our previous report, where we
found a dose response in non-dividing lymphocytes but no DNA damage in prolifer-
ating lymphocytes [6]. It is important to note that organs with a low rate of prolifera-
tion had the capacity to accumulate vanadium and that in these tissues DNA damage
persisted after 24 h [26].
It is worthy of emphasis that in the SCGE assay damage was still observed after
24 hr, are usually the kind of damage detected by this method is repaired very rap-
idly [6,19]. Persistence of damage could be due to the balance between the amount
of vanadium and the repair capacity of each organ.

ACKNOWLEDGMENTS
M.V. was the recipient of a fellowship from CONACyT and DGEP. We are grateful
to Raymond R. Tice for his encouragement and valuable discussion of the manuscript.
254 Altamirano-Lozano et al.

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