Evaluation of genotoxicity of oral exposure
to tetravalent vanadium in vivo
Paola Villania, Eugenia Cordellia, Paola Leopardib, Ester Siniscalchib,
Enrico Veschettib, Anna Maria Fresegnaa, Riccardo Crebellib,∗
aSection of Toxicology and Biomedical Sciences, ENEA-CR Casaccia, Rome, Italy
bDepartment of Environment and Primary Prevention, Istituto Superiore di Sanit` a,
Viale Regina Elena, 299, 00161 Rome, Italy
Received 8 June 2006; received in revised form 10 July 2006; accepted 14 July 2006
Available online 22 February 2007
The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical
form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic
potential in vivo is not elucidated.
for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment,
micronuclei in both blood reticulocytes and bone marrow polychromatic erythocytes were determined; in addition, DNA lesions
detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined
by atomic absorption spectrometry.
Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight
increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups.
The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal
exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and
lack of significant genotoxic potential in vivo.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Vanadyl sulphate; In vivo; Genotoxicity; Comet; Micronucleus; Mouse
Vanadium is a trace element present in the natural
and occupational environment. It can be found in water,
rocks and soil at low concentrations as well as in fossil
fuels at relatively high concentrations. Vanadium exists
E-mail address: firstname.lastname@example.org (R. Crebelli).
+4, and +5, which modulate its toxicity (WHO, 1988).
Food is the main source of exposure to vanadium
for the general population, with an estimated daily
dietary intake of a few tens of micrograms (WHO,
though relatively high vanadium concentrations have
water from volcanic areas (WHO, 1988; Farias et al.,
2003). Another important source of vanadium exposure
0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
is from the use of dietary mineral supplements, which
can provide more than 10mgvanadium/day (Barceloux,
1999; EFSA, 2004).
Vanadium is poorly absorbed from the gastrointesti-
nal tract, thus ingested vanadium compounds are mainly
eliminated in feces. On the other hand, urine is the pre-
dominant route of elimination of absorbed vanadium.
The distribution of vanadium via blood circulation is
rapid: the highest concentrations of vanadium initially
sites are bone and muscles (Aragon and Altamirano-
Lozano, 2001; Ivancsits et al., 2002).
Several evidences indicate that vanadium is essen-
tial for different organisms, although the conclusive
demonstration of its essentiality for humans is still
lacking (Poggioli et al., 2001). Among the biological
actions of vanadium, an increase of glucose transport
and metabolism in different tissues and cell types has
been noted. This activity prompted its pharmacological
utilization as an alternative therapy in diabetic patients,
and as dietary supplement among bodybuilders. On the
other hand, depending on blood level, vanadium may
also cause several adverse effects in mammals, such
as haemopoietic changes, nephrotoxicity, reproductive
Domingo, 1996; Aragon and Altamirano-Lozano, 2001;
cinogenicity is concerned, no conclusion can be drawn
from the limited oral carcinogenicity studies performed,
while the positive findings from inhalation studies with
particles of vanadium pentoxide (IARC, 2003) are of
scarce relevance for evaluating potential risks from oral
Considerable attention has attracted in recent years
the assessment of the genotoxicity of vanadium com-
pounds. Adverse effects of pentavalent and tetravalent
vanadium compounds on chromosome integrity and
segregation have been observed in vitro. In particular,
pentavalent vanadium compounds have been reported
to induce micronuclei, DNA strand breaks, sister chro-
matid exchanges (SCE) and chromosomal aberrations
in different in vitro systems (Owusu-Yaw et al., 1990;
al., 1997; Ivancsits et al., 2002). Also vanadium tetrava-
lent compounds proved to be genotoxic in several in
vitro systems, inducing micronuclei and chromosomal
aberrations in human peripheral blood cells (Migliore
et al., 1993, 1995; Rodr` ıguez-Mercado et al., 2003), and
through the generation of free radicals (Shi et al., 1996;
itive findings were confirmed in some in vivo studies in
mice using acute intraperitoneal or intragastric adminis-
trations (Ciranni et al., 1995; Altamirano-Lozano et al.,
1996, 1999; Mailhes et al., 2003). However, the results
from these in vivo studies can be considered of lim-
ited use for hazard characterization of low dose repeated
oral exposures, as typically experienced by humans. To
fill this gap, in a previous study the genotoxic activity
of pentavalent vanadium was evaluated in mice treated
with a wide range of doses of sodium ortho-vanadate
in drinking water for 5 weeks (Leopardi et al., 2005). In
been applied to evaluate the genotoxic potential in vivo
of the tetravalent compound vanadyl sulphate. Effects
on chromosome integrity and segregation, visualized as
micronuclei, and primary DNA lesions detectable by
comet assay, have been assessed in somatic and germ
cells, and evaluated also with respect to tissue concen-
trations of the element at the end of the exposure period.
2. Materials and methods
s.r.l. (Udine, Italy), and acclimatized 1 week before treatment.
(22±2◦C, 55±15% relative humidity, on a 12h light/dark
cycle), with drinking water and laboratory rodent diet ad libi-
tum. Experiments were carried out in compliance with the
ethical provisions enforced by the European Union and autho-
rized by the National Committee of the Italian Ministry of
Health on the in vivo experimentation.
Vanadyl sulphate hydrate (VOSO4·5H2O), purity 99.99%,
MW 253, CAS 123334-20-3 was from Sigma–Aldrich (Milan,
Italy). Stock solutions of the chemical were prepared daily
by dissolving the blue crystalline powder in distilled water.
Their actual concentrations were determined by ICP-OES
spectrometry as described below. Tap water was used to dilute
VOSO4stock solutions and administered as such to control
2.3. Treatments and organ sampling
treatment group. In the first experiment, VOSO4was adminis-
tered in drinking water for 5 weeks at the concentrations of 10,
100, 500 and 1000mg/l. The top dose was selected as maxi-
mum tolerated dose on the basis of the results of a preliminary
range-finding experiment and of literature data (Ciranni et al.,
formed to check the effect of the low dose of vanadyl sulphate
on MnRETs observed in the first trial.
Consumption of drinking water, food intake, and body
weight gain were recorded twice a week during treatment
with VOSO4. For the analysis of micronuclei in reticulocytes
(RETs), blood samples (few microliters) were collected by
7, 14, 21, 28 and 35 days thereafter. At the end of treatment,
mice were sacrificed by cervical dislocation and liver, kidney,
spleen, testes, femours and tibias removed and weighted. In
cleus and comet assays; testis cells were analysed by comet
assay. Total vanadium concentration was determined in all
2.4. Isolation of cells
Whole blood obtained by tail venipuncture was used for
the cytogenetic analyses of reticulocytes. Bone marrow cells
were obtained by flushing both femours with phosphate buffer
saline (PBS). After centrifugation and resuspension in PBS,
the cell suspension was splitted and used for both micronu-
cleus and comet assays. Testes were minced in PBS, the
resulting cell suspension centrifuged and resuspended in cold
2.5. Optical emission spectrometric analysis
An aliquot (0.04–1.16g) of tissue was digested at 65◦C
with 0.3–1.7ml of fuming HNO3per mg of sample in 2ml
screw-cap polypropylene tubes. After digestion, 100–500?l
of rhodium (Rh) 1.00mg/l were added and the resulting solu-
tion was diluted to 5–25ml with demineralised water. Samples
were analysed by means of inductively coupled plasma emis-
sion spectrometry (ICP-OES) using a Perkin-Elmer Optima
4300 spectrometer and a Cetac ultrasonic nebulizer. Vana-
dium concentration was measured at 290.88, 292.40, 311.07
and 270.09nm and corrected using the internal standard (Rh)
signal detected at 343.49nm. Results were reported as micro-
grams of vanadium per grams of tissue (wet weight). Limit of
detection (LOD) and limit of quantitation (LOQ) of vanadium
in biological samples were 0.03 and 0.1?g/g (wet weight),
2.6. Mutagenicity tests
2.6.1. Micronucleus assay in bone marrow cells
Some drops of bone marrow cell suspension were spread
on slides; for each animal four slides were prepared. Air-
dried smears were then fixed in absolute methanol at room
temperature for 5min and stained with a 5% solution of
Giemsa in 0.01M phosphate buffer at pH 6.8 for 20min to
differentiate bone marrow polychromatic (PCE) from nor-
scored using a brightfield microscope. In the first experi-
ment the frequency of micronucleated PCEs was evaluated
analysing 2000cells/animal (1000 cells each of two scorers);
1000cells/animal were analyzed in the positive control group.
To assess bone marrow toxicity, the percentage of PCEs was
evaluated over 1000 total erythrocytes (PCEs+NCEs).
2.6.2. Micronucleus assay in blood reticulocytes
Five microlitres of whole blood were put on slides
coated with 10?l of a solution of 1mg/ml acrydine orange
(Sigma–Aldrich) in distilled water and examined under the
of micronucleated reticulocytes blind determined scoring
1000 reticulocytes for animal. The incidence of micronucle-
ated reticulocytes measured at t0served as control for each
treated group. To disclose possible toxic effects of treat-
ment on erythropoiesis, four cell types were distinguished
among the 1000 analysed cells according to Zijno et al.
2.6.3. Alkaline comet assay
The assay was performed essentially according to Singh et
electrophoresis, the slides were immersed in 0.3M sodium
acetate in ethanol for 30min. Microgels were then dehy-
drated in absolute ethanol for 2h and immersed for 5min
in 70% ethanol. Slides were air-dried at room temperature.
ethidium bromide (Sigma–Aldrich) and examined with an
Olympus fluorescent microscope. Slides were analyzed by a
computerized image analysis system (Delta Sistemi, Rome,
Italy). To evaluate the amount of DNA damage, computer
generated tail moment values were used. One hundred cells
2.7. Statistical analysis
Means of different experimental groups were compared by
two-tailed Student’s t-test. The effect of dose and duration
of treatment on the incidence of micronucleated reticulo-
cytes was evaluated by repeated-measures analysis of variance
(ANOVA). The limit of statistical significance was set at
3.1. General toxicity
Data on food and water consumption, and estimated
daily intake of tetravalent vanadium are summarized
in Table 1. Daily water consumption was significantly
possibly because of the effect of vanadyl sulphate on
the palatability of drinking water. This led to a less
than proportional increase in vanadium intake in the
high dose groups. Conversely, in the second experiment
an increased water consumption was observed in the
Food and water consumption, and vanadium intake along the 5 weeks of treatment with vanadyl sulphate
Dose Daily food
MeanS.E.Mean S.E.Mean S.E.MeanS.E.MeanS.E.
The number of mice/group is reported in Table 5.
aBased on mean group body weights, measured twice a week during treatment period.
bEstimated dose based on water consumption/body weight.
*p<0.01 vs. control group (Student t-test).
low dose group (2mg/l). No significant differences in
food consumption were observed among experimental
Data on body and organ weights are summarized
in Table 2. Treatment did not affect body weight gain
in the 5 weeks of exposure. In the first experiment
liver, spleen and kidney weights were significantly
increased in the low dose group (10mg/l); this differ-
ence was not confirmed in the second experiment. No
3.2. Vanadium distribution in body tissues
Data on spectrometric determinations of vanadium
concentrations in mouse tissues at the end of treat-
ment are shown in Table 3. In the first experiment a
dose-related, linear increase of vanadium content was
observed in bone tissues (R2=0.95 and R2=0.96 in
femours and in tibias, respectively). Vanadium concen-
trations in soft tissues were five- to ten-fold lower than
in bone on a wet weight basis. At the lowest dose tested
(10mg/l), vanadium content was below the LOQ in
Body weight gain and organ weights after 5 weeks of oral exposure to vanadyl sulphate
Dose Body weight gain (g) Organ weights (g)
MeanS.E. Spleen TestisKidney Liver
The number of mice/group is shown in Table 5.
*p<0.05 (Student t-test).
**p<0.01 (Student t-test).
Vanadium distribution (?gV/g wet tissue) in different organs after 5 weeks of oral exposure to vanadyl sulphate
FemursTibiasLiver Kidney SpleenTestis IntestineStomach
The number of mice/group is reported in Table 5. n.d., not detectable (<0.1?gV/g wet tissue).
all soft tissues. No large differences in vanadium con-
tent were observed among the soft tissues analysed. In
the second experiment, the spectrometric analysis was
extended to stomach and intestine: also in these site of
contact tissues vanadium content was below the LOQ
in the experimental conditions applied. Slightly higher
vanadium concentrations were measured in bone in the
data from the two experiments were compared by t-test,
no statistical significant differences at 0.05 level were
observed. The borderline signal measured in spleen and
testis at 2mg/l can be attributed to chemical contamina-
3.3. Mutagenicity tests
3.3.1. Micronucleus test in bone marrow cells
No increase in the incidence of micronucleated poly-
chromatic erythrocyte (MnPCEs) was observed in bone
marrow cells of mice treated with vanadyl sulphate
in a range of doses from 10 to 1000mg/l (Table 4).
The determination of the PCE/NCE ratio did not show
marrow toxicity in treated mice. A clear-cut increase of
MnPCEs was produced by the positive control methyl
methanesulfonate (MMS), with no evidence of toxic-
ity to bone marrow. The negative result obtained in the
main assay were confirmed in the second experiment,
designed for the assessment of low dose effects in retic-
ulocytes, where two low concentrations (2 and 10mg/l)
were tested (data not shown).
3.3.2. Micronucleus test in blood reticulocytes
The results of cytogenetic analyses of blood smears
are summarized in Table 5. The microscopic scor-
ing of reticulocyte types, characterized on the basis
of cytoplasmatic RNA content, did not point out any
treatment related toxic effect (data not shown). No dis-
tinct differences in the incidence of MnRETs were
observed among treated groups and sampling times.
Multiple comparisons of average values by Student t-
test highlighted a few statistically significant (p<0.05)
Results of micronucleus test in mouse bone marrow after 5 weeks of oral exposure to vanadyl sulphate
Dose Mice (n) ‰ frequency, mean (SE) Total MnPCEs/total PCEs analysed
(MnPCEs/2000 PCEs, individual data)
PCE (%), mean (SE)
10 4.6 (1.04)
5 60**(30)299/5,000(c)(47, 24, 46, 83, 99)(c)
a1/10 mouse died at the fourth week of treatment.bMMS, methyl methansulfonate.c1000 PCEs analysed per each animal.
*p<0.05 vs. negative control (Student t-test).
**p<0.0001 vs. negative control (Student t-test).
Incidence of micronucleated reticolocytes in peripheral blood of mice during 5 weeks of oral exposure to vanadyl sulphate
VOSO4·5H2O (mg/l)Mice (n)‰ frequency of micronucleated retycolocytes, mean (SE)
Week 0Week 1 Week 2Week 3 Week 4Week 5
10 2.4 (0.54)
a1/10 mouse died at the fourth week of treatment.
*Significantly (p<0.05) different from week 0 (Student t-test).
differences between MnRETs values measured at week
0 and along treatment in groups receiving the two low-
est concentrations of VOSO4(Table 5). However, the
analysis of data set by repeated-measures ANOVA did
and duration of treatment on the incidence of MnRETs
(p>0.05 for both). On the other hand, a highly signifi-
cant (p<0.001, Student t-test) increase in MnRETs (3.5
versus 21.7‰ in control and treated mice, respectively)
was induced by the positive control MMS, given by i.p.
24h before sacrifice.
3.3.3. Comet assay
The results of comet assays are summarized in
Table 6. No dose-related increase in mean tail moment
values was observed in bone marrow cells of treated
Results of comet assay in mouse bone marrow and testis cells after 5
weeks of oral exposure to vanadyl sulphate
DoseMice (n) Tail moment (μ), mean (SE)
Bone marrow cellsTestis cells
10 1.43 (0.34)
*Significantly (p<0.05) different from 0 (Student t-test).
experiment was not confirmed in the repeat experiment
with 2 and 10mg/l. No increase of mean tail moment
values was observed in testis cells. The positive con-
values of both bone marrow and testis cells, confirming
the sensitivity of the assay to in vivo induced genotoxic
The transition metal vanadium is present in trace lev-
els in all mammalian tissues, mainly in tetravalent form
(Bay et al., 1997). Although some therapeutic properties
have been attributed to vanadium (Domingo, 1996), the
depending on its circulating level and oxidation state
(Ciranni et al., 1995). Among toxic effects, particular
effects subsequent to DNA damage and chromosome
malsegregation, secondary to free radicals generation
(Shi et al., 1996; Sit et al., 1996).
In consideration of the potential toxicity of vanadium
and its ubiquitary occurrence in water and food items,
the assessment of the risk posed by the oral intake of
low doses of this metal is an important public health
sure to pentavalent vanadium (sodium ortho-vanadate)
(Leopardi et al., 2005): a wide range of doses were
given in drinking water for 5 weeks, the time required to
genotoxic effects in marrow and testis cells assessed by
micronucleus and comet assays. The same experimental
protocol was applied in this work for testing tetravalent
The results obtained did not show signs of overt
toxicity after ingestion of vanadyl sulphate: no signif-
icant effect on body weight gain during treatment and
organ weight at sacrifice was observed. Also the deter-
mination of the PCE/NCE ratio and the classification
of reticulocyte subtypes did not highlight toxic effects
toward haemopoietic cells. These results, together with
the low residual content of vanadium in soft tissues
at the end of treatment, suggest low general toxic-
ity and scarce bioavailability for orally administered
vanadyl sulphate, as previously observed for pentava-
of vanadium in mammals (Edel et al., 1984; Radike et
al., 2002; Mukherjee et al., 2004).
With respect to genotoxicity, repeated oral exposure
icant increases in DNA single strand breaks and other
lesions detectable by comet assay (e.g. abasic sites)
in bone marrow and testis cells, as well as micronu-
clei in bone marrow cells. These results are at variance
with those from a previous study (Ciranni et al., 1995),
where an increased incidence of micronuclei in bone
marrow was observed after the intragastric administra-
tion of a high dose of vanadyl sulphate (100mg/kg body
weight) as bolus. However, such discrepancy can be
composed bearing in mind the scarce bioavailability of
orally administered vanadium which suggest that, under
the repeated exposure regimen applied, internal expo-
sure levels may have been too low to elicit a detectable
The analysis of micronuclei in reticulocytes provided
less clear indications, showing a significant increase of
MnRETs in some low dose groups after a few weeks of
treatment. In principle the induction of micronuclei in
reticulocytes, but not in bone marrow, could indicate the
cell, e.g. peripheral blood reticulocytes deriving from
study, however, the lack of any relationship with dose
over a 500-fold dose range suggest that observed vari-
ation in the frequency of micronucleated reticulocytes
was confirmed by the analysis of data set by ANOVA,
incidence of MnRETs and administration of VOSO4.
Concerning the risk of heritable effects, previous
studies indicate that vanadium can be toxic to male
reproductive system, affecting sperm parameters and
inducing testicular damage (Domingo, 1996; Aragon
and Altamirano-Lozano, 2001; Aragon et al., 2005).
Indeed, several studies pointed out the detrimental
effects induced by pentavalent vanadium on male germ
still scanty. The results obtained herein did not highlight
any genotoxic effect in testis cells of treated animals,
suggesting that under the experimental applied the risk
of heritable effects may be absent or undetectable.
In conclusion, the overall experimental evidence pro-
vided by this study indicates that repeated oral exposure
kg body weight, may be devoid of significant genotoxic
effects both at somatic and germ cell levels. However, in
by vanadium on critical cell mechanisms, even in the
very low dose range (Sakurai, 1994; Barceloux, 1999),
it is suggested to maintain a cautelative approach in the
safety evaluation of vanadium compounds.
The authors are grateful to Dr. Andrea Zijno for
the statistical analysis of experimental data, to Mrs.
Celestina D’Ascoli for technical assistance, to Dr. Bar-
to Mr. Camillo Mancini and Mr. Agostino Eusepi for
assistance in animal treatment. This work was partially
supported by the Azienda Consorziale Servizi Etnei
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