Inhalative exposure to vanadium pentoxide causes DNA damage in workers: results of a multiple end point study.

Abstract

Inhalative exposure to vanadium pentoxide (V(2)O(5)) causes lung cancer in rodents.
The aim of the study was to investigate the impact of V(2)O(5) on DNA stability in workers from a V(2)O(5) factory.
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 microg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
V(2)O(5) causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.

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Vanadium pentoxide (V2O5) is used for the
production of metal alloys, for the manufac-
turing of lithium batteries and high-pressure
lamps, and for the synthesis of chemicals
[International Agency for Research on Cancer
(IARC) 2006]. Its annual production world-
wide is in the range of 60,000 tons (IARC
2006; Woolery 1997). Occupational expo-
sure to the oxide occurs at production sites,
during processing and refining of vanadium
ores and sludges, during manufacturing of
vanadium-containing products, in the course
of combustion of vanadium-rich fuels, and by
handling of catalysts in the chemical industry
(Plunkett 1987). Environmental exposure to
the metal and its oxides occurs via inhalation
in the vicinity of metallurgical plants or
through consumption of contaminated foods
(Barceloux 1999; IARC 2006). Although
foods contain low concentrations, nutrition is
the major source of exposure for the general
population (Barceloux 1999).
The National Toxicology Program (NTP
2002) found an increase of lung adenomas
and carcinomas in mice after inhalative expo-
sure to V2O5; this was paralleled by an
increased incidence of hyperplasia in lung tis-
sue. In male rats, the number of tumors was
elevated (nonsignificantly), whereas in females
no increase was found (IARC 2006; NTP
2002; Ress et al. 2003). These findings led to
a reevaluation of the metal oxide (IARC
2006) and to its classification as a group 2B
(“possible human”) carcinogen. One of the
problems encountered in the risk assessment
of V2O5is the lack of human data. According
to IARC (2006), inhalative exposure to V2O5
in vanadium plants exists worldwide, and sev-
eral hundred workers may be affected; further-
more, workers of other industries may also be
exposed. The occupational exposure limit in
Austria for V2O5in air is 0.05 mg/m3
(Bundesminister für Wirtschaft und Arbeit
2003). The Senate Commission of the
German Research Foundation [Deutsche
Forschungsgemeinschaft (DFG)] decided to
suspend the maximum allowed concentration
of V2O5in workplace air because of its sus-
pected carcinogenicity (DFG 2006). In the
United States, the National Institute for
Occupational Safety and Health and the
American Conference of Governmental
Industrial Hygienists established an occupa-
tional exposure limit of 0.05 mg/m3air
(Occupational Safety and Health Adminis-
tration 2006). Measurements of air concentra-
tions in vanadium factories yielded values in
the range of 0.02–5 mg/m3(IARC 2006).
Results of in vitro and animal studies indi-
cate that the oxide causes formation of reactive
oxygen species (Ingram et al. 2003, 2007;
Wang et al. 2003; Zhang et al. 2001) and
aneugenic effects (Migliore et al. 1993; Ramirez
et al. 1997; Zhong et al. 1994) and interferes
with DNA synthesis and repair (IARC 2006).
Because DNA damage and aneugenic processes
are known to play a role in the onset of human
cancer (Duesberg et al. 2005; Pitot 1986), evi-
dence of genetic damage in exposed humans
would support the assumption of increased
cancer risks. At present, only one study on the
influence of occupational exposure to V2O5
on DNA stability has been published; in that
study, Ivancsits et al. (2002) investigated
DNA migration in leukocytes using the stan-
dard single-cell gel electrophoresis (SCGE)
assay. The authors observed no indication of
damage and found no elevation in the fre-
quencies of sister chromatid exchanges (SCEs)
or the concentration of 8-hydroxy-2´-
deoxyguanosine in leukocytes. Lener et al.
(1998) found no SCEs or chromosomal aber-
rations in blood cells of children living in the
vicinity of a vanadium production site.
Our goal in the present study was to com-
prehensively evaluate the impact of inhalative
V2O5exposure on genetic stability. We moni-
tored DNA migration in leukocytes of workers
and matched controls with the standard SCGE
assay, and we monitored oxidized bases using
lesion-specific enzymes (Collins et al. 1993).
Furthermore, we measured the sensitivity
toward bleomycin (BLEO) and the repair of
lesions induced by this cytostatic agent
(Rajaee-Behbahani et al. 2001; Schmezer et al.
2001; Wei et al. 2005). BLEO sensitivity was
initially monitored in chromosomal aberration
assays (Hsu et al. 1989; Szekely et al. 2003)
and later in SCGE experiments (Schmezer
et al. 2001).
Additionally, we conducted cytokinesis-
block micronucleus cytome (CBMN Cyt)
assays with lymphocytes. This test is widely
used for the detection of DNA damage in
Environmental Health Perspectives • VOLUME 116 |NUMBER 12 |December 2008
1689
Research
Address correspondence to S. Knasmüller, Institute
for Cancer Research, Borschkegasse 8a, 1090
Vienna, Austria. Telephone: 43-1-4277-65142. Fax:
43-1-4277-6519. E-mail: siegfried.knasmueller@
meduniwien.ac.at
This project was supported by the Austrian
Workers Compensation Board, Vienna, Austria.
The authors declare they have no competing
financial interests.
Received 3 March 2008; accepted 31 July 2008.
Inhalative Exposure to Vanadium Pentoxide Causes DNA Damage
in Workers: Results of a Multiple End Point Study
Veronika A. Ehrlich,1Armen K. Nersesyan,1Christine Hoelzl,1Franziska Ferk,1Julia Bichler,1Eva Valic,2
Andreas Schaffer,3Rolf Schulte-Hermann,1Michael Fenech,4Karl-Heinz Wagner,5and Siegfried Knasmüller1
1Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, Austria; 2Austrian Workers Compensation
Board, Vienna, Austria; 3Department of Medicine II, Medical University of Vienna, Austria; 4Commonwealth Scientific and Industrial
Research Organisation, Human Nutrition, Adelaide, Australia; 5Department of Nutritional Sciences, University of Vienna, Austria
BACKGROUND: Inhalative exposure to vanadium pentoxide (V2O5) causes lung cancer in rodents.
OBJECTIVE: The aim of the study was to investigate the impact of V2O5on DNA stability in workers
from a V2O5factory.
METHODS: We determined DNA strand breaks in leukocytes of 52 workers and controls using the
alkaline comet assay. We also investigated different parameters of chromosomal instability in
lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome
method.
RESULTS: Seven of eight biomarkers were increased in blood cells of the workers, and vanadium
plasma concentrations in plasma were 7-fold higher than in the controls (0.31 µg/L). We observed
no difference in DNA migration under standard conditions, but we found increased tail lengths
due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes
(formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA
migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions
was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and
nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis
rates were higher, but only the latter parameter reached statistical significance.
CONCLUSIONS: V2O5causes oxidation of DNA bases, affects DNA repair, and induces formation of
MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer
and other diseases that are related to DNA instability.
KEY WORDS: comet assay, cytokinesis-block micronucleus assay, DNA damage, occupational expo-
sure, vanadium pentoxide. Environ Health Perspect 116:1689–1693 (2008). doi:10.1289/ehp.11438
available via http://dx.doi.org/ [Online 31 July 2008]

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humans (Fenech 2007). Micronuclei (MNs),
which are formed as a consequence of chro-
mosome breakage and/or aneuploidy (Fenech
and Morley 1985), correlate with the inci-
dence of cancer in humans (Bonassi et al.
2007). Also, we evaluated the frequencies of
nucleoplasmic bridges (NPBs) and nuclear
buds (Nbuds) in lymphocytes. NPBs are
assumed to occur when centromeres of dicen-
tric chromosomes are pulled to the opposite
poles of the cell at anaphase and provide a
measure of chromosome rearrangements
(Fenech 2006). Therefore, NPBs give direct
evidence of genome damage resulting from
misrepaired DNA breaks, which cannot be
detected when MNs are scored as the only
end point. Nbuds form as a consequence of
gene amplification (Fenech 2006). Amplified
DNA is selectively localized at specific sites of
the nucleus and eliminated through recom-
binogenic events during the S-phase of mitosis
(Shimizu et al. 1998, 2000).
Other parameters included in the present
study were necrosis and apoptosis, and the
nuclear division indices (Fenech 2006). We
measured plasma concentrations of folate and
vitamins B6and B12in both groups, because
deficiencies of these micronutrients may
increase MN levels (Fenech et al. 1997).
Furthermore, we determined the plasma
vanadium levels of the participants.
Materials and Methods
Study groups. We used the SCGE assay to
study DNA migration in whole-blood leuko-
cytes from 52 vanadium production workers
exposed to V2O5by inhalation and from
52 nonexposed controls (jail wardens).
Additionally, we analyzed lymphocytes of
24 workers and 23 controls using CBMN Cyt
experiments. We collected data concerning
age, weight, height, and smoking status with a
questionnaire (Table 1).
Workers are exposed to vanadium dust
during the entire shift (8 hr) and are required
to wear protective masks and gloves. The
study was approved by the Ethics Committee
of the Medical University of Vienna. After
obtaining informed consent, we collected
blood (2 × 10 mL) in heparin and EDTA
tubes (Vacutainer-Systems, Becton Dickinson,
Plymouth, UK). We stored blood samples at
4°C and transported them to the Institute of
Cancer Research at the Medical University of
Vienna. Whole blood was centrifuged at
623 × g for 10 min at 14°C (Sigma Laboratory
Centrifuge 4K15; Sigma Chemical Co., St.
Louis, MO, USA) and plasma was collected,
aliquoted, and stored at –80°C. We con-
ducted experiments between October 2004
and May 2005.
Exposure assessment. We determined vana-
dium concentrations in plasma using graphite
furnace atomic absorption spectrometry, as
described by Ivancsits et al. (2002), with
Zeeman background correction at a tempera-
ture of 2,450°C. The calibration curves for
vanadium were in the range of 0–40 µg/L, and
the detection limit was 0.3 µg/µL (5.9 nmol/L).
Measurements of vitamins B6and B12
and folate. We determined pyridoxal phos-
phate (the active form of vitamin B6) in
plasma by HPLC using commercial assays
(Immundiagnostic AG, Bensheim, Germany)
as described by Majchrzak et al. (2006). We
measured vitamin B12and folate in plasma
using commercial radioimmunoassays (IUL
Instruments GmbH, Königswinter, Germany)
(Majchrzak et al. 2006).
Leukocyte isolation and BLEO treatment
for SCGE assays. We used the protocol of
Buschini et al. (2002) for leukocyte isolation:
EDTA-anticoagulated blood was maintained
in erythrocyte-lysis buffer (155 mM NH4Cl,
5 mM KHCO3, 0.005 mM Na2EDTA,
pH 7.4) at 37°C for 5 min; centrifuged (200 ×
g, 5 min at 4°C); washed twice in phosphate-
buffered saline (PBS; pH 7.4); and resus-
pended in RPMI 1640 (Sigma-Aldrich
Chemie Gmbh, Munich, Germany) without
serum (Buschini et al. 2002). We used a modi-
fied version of the protocol for BLEO sensitiv-
ity and DNA repair measurements (Schmezer
et al. 2001). Leukocytes were treated with
10 µg/mL BLEO sulfate (Nippon Kayaku Co.
Ltd., Tokyo, Japan) at 37°C for 30 min. To
terminate the treatment, cells were washed
twice in PBS (pH 7.4). To measure DNA
repair capacity, we incubated a second batch of
BLEO-treated cells for 15 min (37°C) before
lysis and electrophoresis. Cell viability was
monitored with trypan blue (Lindl and Bauer
1994). Viability of all samples was ≥ 90%.
Alkaline SCGE (comet) assay. We per-
formed the SCGE assay according to the
guidelines of Tice et al. (2000). Briefly, cell
pellets were mixed with 60 µL 0.5% low-
melting-point agarose (Invitrogen Life
Technologies Ltd., Paisley, Scotland), trans-
ferred to precoated (1.5% normal-melting-
point agarose) glass slides, and sealed with a
coverslip. The slides were placed on ice for
5 min to allow solidification of the agarose.
After removing the coverslip, we immersed the
slides in lysis solution (2.5 M NaCl, 100 mM
Na2EDTA, 10 mM Tris, 1% Triton X, 10%
dimethyl sulfoxide, pH 10.0) and kept them
at 4°C for ≥ 1 hr. To prevent DNA damage,
lysis and all subsequent steps were conducted
under red light. After lysis, we placed the
slides on a horizontal gel electrophoresis unit
(C.B.S. Scientific, Solana Beach, CA, USA)
and allowed the DNA to unwind at 4°C in
alkaline electrophoresis buffer (300 mM
NaOH, 1 mM Na2EDTA, pH ≥ 12.5) for
20 min. Electrophoresis was performed at 25 V
and 300 mA for 20 min; then the slides were
covered with neutralization buffer (0.4 M
Trizma base, pH 7.5, 4°C) for 2 × 8 min and
air dried. The coded slides were stained with
ethidium bromide (20 µg/mL) and evaluated
under a fluorescence microscope (Nikon
027012; Nikon, Tokyo, Japan). We used an
automated analysis system (Helma and Uhl
2000) to acquire images, compute the inte-
grated intensity profile for each cell, estimate
the comet cell components, and evaluate
derived parameters. For each experimental end
point we analyzed three cultures, and we meas-
ured 50 randomly captured cells from each
slide. To quantify DNA damage, tail lengths
and tail moments were determined.
Determination of oxidized purines and
pyrimidines. We used a modified version of
the protocol published by Collins et al. (1993)
to measure endogenous formation of oxidized
DNA bases. Formamidopyrimidine glycosy-
lase (FPG) and endonuclease III (ENDO III)
were provided by K. Angelis (Czech Academy
of Sciences, Prague, Czech Republic).
After lysis of the cells, the slides were
washed in enzyme buffer (0.1 M KCl, 40 mM
HEPES, 0.5 mM Na2EDTA, 0.2 mg/mL
bovine serum albumin, 4°C, pH 8) for
2 × 8 min. Subsequently, agarose-embedded
cells were covered with 50 µL enzyme buffer
or with enzyme solutions (1.0 µg/mL). Gels
were sealed with a coverslip and incubated at
37°C in the dark for 45 min (ENDO III) or
30 min (FPG). Subsequently, the slides were
transferred into alkaline buffer for unwinding,
and electrophoresis was performed.
CBMN Cyt experiments. We isolated
lymphocytes according to the protocol of
Fenech (2000). Briefly, Histopaque 1077
(Sigma) was overlaid with RPMI 1640–diluted
blood and centrifuged at 318 × g at 14°C for
30 min. Subsequently, the cell layer was
removed, resuspended in RPMI, washed twice,
and centrifuged (318 × g, 14°C, 10 min).
Ehrlich et al.
1690
VOLUME 116 |NUMBER 12 |December 2008 • Environmental Health Perspectives
Table 1. Distribution of selected characteristics in exposed subjects and controls.
Variable
Age (years)
Body mass index
Cigarettes/day (no.)
Vanadium (μg/L)
Folate (μg/L)
Vitamin B6(ng/mL)
Vitamin B12(ng/L)
Controls (n = 52)
38.0 (32.00–44.50)
27.0 (24.00–28.50)
5.0 (0.00–17.00)
0.3 (0.24–0.39)
4.7 (3.04–6.40)
18.2 (14.38–21.55)
665.0 (428.5–866.5)
Exposed (n = 52)
43.0 (38.50–49.50)
26.0 (25.00–27.50)
7.0 (0.00–21.00)
2.2 (1.54–3.89)
3.4 (2.54–4.205)
19.8 (14.06–36.94)
968.0 (662.5–1215.0)
p-Value
> 0.05
> 0.05
> 0.05
<< 0.0001
> 0.05
0.016
0.013
All study subjects were male Caucasians and nonvegetarians. Values shown are median (25th–75th percentiles).

Page 3

We performed the CBMN Cyt test using
the cytochalasin B technique described by
Fenech (2007). We determined MNs, Nbuds,
and NPBs, as well as apoptotic and necrotic
cells, in samples from 24 workers and 23 con-
trols using the cytome approach (Fenech
2007). Slides were evaluated by one of the
authors (V.A.E.) who was trained in the labo-
ratory of M.F. at Commonwealth Scientific
and Industrial Research Organisation (Human
Nutrition, Adelaide, South Australia), which
has developed the CBMN Cyt assay and was
centrally involved in the standardization of
scoring criteria via the International Collabora-
tive Project on Micronucleus Frequency in
Human Populations (Human Micronucleus
Project 2008). For each participant, we pre-
pared lymphocyte cultures in duplicate. Each
culture contained 106cells in 750 µL RPMI
1640 supplemented with 100 U/mL penicillin,
100 µg/mL streptomycin, 10% fetal bovine
serum (Sigma), 2.0 mmol/L L-glutamine, and
30 µg/mL phytohemagglutinin (Invitrogen,
Carlsbad, CA, USA). Cultures were incubated
in round-bottom tubes (Becton Dickinson) at
37°C in a humidified atmosphere containing
5% CO2. Forty-four hours after mitogen
stimulation, we added cytochalasin B (final
concentration, 4.0 µg/mL; Sigma-Aldrich, St.
Louis, MO, USA) to block cell division. The
total incubation time of the cultures was 72 hr.
Subsequently, the cells were harvested and used
to prepare slides, which were air dried, fixed,
and stained with Diff Quick (Dade Behring,
Deerfield, IL, USA). From each participant, we
scored 2,000 binucleated cells using a light
optical microscope (Microphot-FXA; Nikon)
(Fenech et al. 2003).
Statistical analysis. We performed statisti-
cal analyses using Statistica 5.0 software
(StatSoft Inc., Tulsa, OK, USA). The results
are presented as medians and 25th–75th per-
centiles. All p-values were two-tailed, and
we considered differences to be significant at
p ≤ 0.05. We used the Mann-Whitney U-test
for comparisons between exposed subjects
and controls. We used Spearman’s correlation
coefficients to test univariate relationships
between different variables.
Results
Demographics of the study populations. The
characteristics of participants are summarized
in Table 1. Age and body mass index did not
differ between the groups. All individuals
were males and nonvegetarians. The control
group included 11 smokers and the exposed
group included 12. The concentrations of
folate were similar in both groups, whereas
levels of vitamins B6and B12were higher
(8.1% and 31%, respectively) in the exposed
group, possibly due to intake of supplements.
Vanadium levels were 7-fold higher in the
plasma of the exposed group.
DNA migration in leukocytes. Table 2
summarizes the results of the SCGE experi-
ments. We found no difference of DNA
migration between exposed individuals and
control subjects when the assay was carried out
under standard conditions (which reflect for-
mation of single- and double-strand breaks),
but we observed increased tail lengths and tail
moments in all other end points in the work-
ers. Formation of oxidized purines (detected
with FPG) was elevated by 7%, and the forma-
tion of pyrimidines (by ENDO III treatment)
was enhanced by 33%. Furthermore, the sensi-
tivity of the cells toward BLEO-induced DNA
damage was higher (25%) in the exposed indi-
viduals. When we monitored DNA migration
after a repair phase (15 min incubation after
BLEO treatment), we observed a decrease of
tail lengths (50%) and tail moments (57%) in
the controls, whereas in the workers we
observed no reduction of tail lengths and a less
pronounced decrease of tail moments (30%).
Frequencies of end points measured with
the CBMN Cyt assay. Data on the frequencies
of MNs, NPBs, Nbuds, apoptosis, and necrosis
rates, as well as the division indices [nuclear
divison index (NDI) and nuclear divison cyto-
toxicity index (NDCI)] are summarized in
Table 3. The number of MNs was 2.5-fold
higher in the workers, and the numbers of
NPBs and Nbuds were substantially increased
(7-fold and 3-fold, respectively) .
To find out whether V2O5exposure and
smoking cause synergistic effects, we com-
pared the frequencies of MNs and Nbuds in
exposed smokers and nonsmokers to those in
the corresponding controls. In nonexposed
individuals, the levels (means ± SD) of MNs,
Nbuds, and NPBs were similar in smokers and
nonsmokers respectively (MNs, 1.77 ± 1.62
and 1.50 ± 1.86; Nbuds, 0.33 ± 0.39 and 0.36
± 0.32; NPBs, 0.46 ± 0.81 and 0.45 ± 0.61).
In workers, the MN rates were lower in smok-
ers (4.17 ± 2.95 and 8.08 ± 4.54), whereas the
frequencies of NPBs and Nbuds were more or
less identical (NPBs, 6.83 ± 3.04 and 7.33 ±
2.50; Nbuds, 3.58 ± 2.76 and 3.30 ± 1.41).
The frequencies of necrotic and apoptotic
cells were elevated in the workers by 55% and
50%, respectively; for the latter parameter the
difference was not significant. The last two
rows of Table 3 show the nuclear division
indices. The division rates were similar in both
groups, regardless of whether we considered the
number of viable cells only (NDI) or also dead
cells (NDCI). These results indicate that metal
exposure has no significant impact on the pro-
liferative capacity of viable or total lymphocytes,
respectively.
Discussion and Conclusions
In this article we present the results of the first
comprehensive study on the impact of occu-
pational exposure to airborne V2O5on DNA
stability. For seven of eight end points, we
found significant differences between exposed
workers and controls.
The most important observation is the
2.5-fold higher frequency of MNs in lympho-
cytes of the exposed individuals (Table 3),
because a prospective study showed that MNs
in peripheral blood lymphocytes are a valid
DNA damage in workers exposed to V2O5pentoxide
Environmental Health Perspectives • VOLUME 116 |NUMBER 12 |December 2008
1691
Table 2. DNA migration in leukocytes of workers and unexposed subjects using different end points of the
comet assay.
Test condition
Standard conditions
Measure
TL
TM
TL
TM
TL
TM
TL
TM
TL
TM
Controls (n = 52)
4.3 (4.07–4.64)
2.4 (2.27–2.79)
4.5 (4.21–4.80)
2.5 (2.34–2.62)
7.3 (6.71–8.17)
3.5 (3.24–3.89)
13.2 (11.93–16.58)
8.7 (7.55–11.72)
6.6 (6.02–7.20)
3.7 (3.41–4.20)
Exposed(n = 52)
4.1 (3.89–4.44)
2.3 (2.16–2.59)
4.8 (4.14–5.39)
2.7 (2.27–3.24)
9.7 (7.84–12.08)
3.8 (3.18–6.03)
16.5 (13.55–23.56)
12.2 (10.07–15.98)
17.8 (13.62–19.65)
8.3 (7.10–11.39)
Δ° (%)
–5
–4
+7
+8
+33
+9
+25
+40
+172
+124
p-Value
> 0.05
> 0.05
0.0236
0.0007
0.0019
0.0023
<< 0.0001
<< 0.0001
<< 0.0001
<< 0.0001
FPG
ENDO III
BLEO
BLEO + DNA repair
Abbreviations: Δ°, difference between exposed subjects and controls; TL, tail length (μm); TM, tail moment. Values shown
are median (25th–75th percentiles) of 150 measurements per person.
Table 3. Frequencies of micronucleated lymphocytes and total numbers of MNs, NPBs, Nbuds, and apoptotic
and necrotic cells per 2,000 binucleated cells in workers and unexposed subjects.
End point
Total no. of MNs
Micronucleated cells
NPBs
Nbuds
Necrotic cells (%)
Apoptotic cells (%)
NDI
NDCI
Controls (n = 23)
2.0 (1.00–4.00)
2.0 (1.00–4.00)
1.0 (0.00–2.00)
1.0 (0.00–1.00)
13.5 (10.7–16.5)
3.0 (1.1–10.7)
1.9 (1.81–1.94)
1.7 (1.64–1.76)
Exposed (n = 24)
5.0 (2.50–9.00)
5.0 (2.50–8.00)
7.0 (5.00–9.00)
3.0 (2.00–5.00)
20.9 (19.4–25.9)
4.5 (3.6–5.2)
1.9 (1.86–1.96)
1.7 (1.61–1.70)
Δ° (%)
+150
+150
+600
+200
+55
+50
p-Value
0.0132
0.0132
<< 0.0001
<< 0.0001
<< 0.0001
> 0.05
> 0.05
> 0.05
0
0
Δ°, difference between exposed subjects and controls. Values shown are median (25th–75th percentiles).

Page 4

biomarker for predicting an increased cancer
risk in humans (Bonassi et al. 2007).
Furthermore, a smaller study showed strong
predictability of the MN biomarker in lympho-
cytes for cardiovascular disease risk (Murgia
et al. 2007). Because the levels of folate and vit-
amins B6and B12were equal or higher in the
workers (Table 1), we can exclude that the
increased MN levels in this group are due to
deficiencies of these micronutrients.
V2O5and other vanadium compounds
induce MNs in bone marrow cells of rodents
(Leopardi et al. 2005; Sun 1996). Furthermore,
DNA-damaging properties of vanadium com-
pounds have also been found in human and
animal cells in vitro (Ivancsits et al. 2002;
Kleinsasser et al. 2003; Migliore et al. 1993;
Ramirez et al. 1997; Rojas et al. 1996; Roldan
and Altamirano 1990; Zhong et al. 1994).
In the present study, we observed an
increase of other end points of chromosomal
stability (NPBs and Nbuds) in the workers.
The induction of NPBs and Nbuds was even
more pronounced than the increase in MN
levels (Table 3), and it is notable that a recent
case–control study on smokers showed that
elevated NPB and Nbud frequencies are more
strongly associated with lung cancer risk than
are MN rates (El-Zein et al. 2006).
We found no synergistic effect between
smoking and V2O5exposure in the present
study. Bonassi et al. (2003) conducted a
meta-analysis concerning the impact of smok-
ing on MN frequencies among subjects in
occupational and environmental surveys.
They concluded that only nonexposed heavy
smokers exhibit a significant increase of MNs,
whereas the MN frequency was not influ-
enced by smoking among individuals exposed
to genotoxic agents. In the latter group, even
slightly reduced MN levels were found in
smokers compared with nonsmokers (Bonassi
et al. 2003).
The only marker that was not elevated in
the workers in the present study was DNA
migration monitored in SCGE experiments
under standard conditions, which reflect
endogenous DNA damage such as single- and
double-strand breaks and apurinic sites (Tice
et al. 2000). This observation is in agreement
with results of an earlier investigation (Ivancsits
et al. 2002).
In vitro studies have shown that the metal
oxide induces DNA migration in blood cells
under standard conditions, but the doses
required to cause effects were substantially
higher than the plasma concentrations in the
workers in the present study (Ivancsits et al.
2002; Kleinsasser et al. 2003; Rojas et al.
1996). Furthermore, results obtained in SCGE
experiments with rodents indicate that vana-
dium compounds cause DNA migration in
inner organs (Altamirano-Lozano et al. 1996,
1999; Leopardi et al. 2005; Villani et al. 2007).
It is notable that the basal levels for tail
lengths are quite low in the present study. Our
values are in a range similar to those found in
other laboratories (Grossi et al. 2008; Undeger
and Basaran 2005; Yoshida et al. 2006; Zhang
et al. 2008) and also within the historical range
of our laboratory (Bichler et al. 2007; Hoelzl
et al. 2008; Steinkellner et al. 2005). Because
we monitored the cell viability before elec-
trophoresis, we can exclude that cell damage
accounts for the low values. Furthermore, the
results obtained with tail moment in the pre-
sent study are essentially identical to those
from tail length measurements.
Endogenous formation of oxidized DNA
bases (shown by treatment with the repair
endonucleases FPG and ENDO III) is higher
in the exposed group. Earlier investigations
found that vanadium-treated cells generate
hydrogen peroxide and superoxide radicals
(Huang et al. 2000; Shi and Dalal 1992;
Ye et al. 1999; Zhang et al. 2001). These
vanadium-induced radicals were shown to
cause damage to macromolecules and lipid
peroxidation (Donaldson et al. 1985), and it is
conceivable that they account for the oxidative
damage that we observed in the present study.
It is noteworthy that a number of human
studies found increased sensitivity toward
DNA damage by BLEO in individuals who
are at increased risk for different types of can-
cer (Schmezer et al. 2001). In the present
study, we found a strong difference between
workers and controls after BLEO treatment
and a 15-min repair phase (Table 2).
Although the tail lengths decreased by 50% in
the controls, we saw no reduction in workers
after the repair phase, and the tail moments
were decreased to a higher extent in the con-
trols. This indicates that the metal oxide inter-
acts detrimentally with DNA repair processes.
Ivancsits et al. (2002) found severe inhibition
of BLEO-induced repair by V2O5in SCGE
experiments with lymphocytes in vitro,
whereas the repair of ultraviolet C–induced
lesions was less affected. These findings can be
taken as an indication that V2O5inhibits
pathways that are required for the repair of
BLEO-induced lesions (homologous recombi-
nation repair, nonhomologous end joining,
and base excision repair) (Schlade-Bartusiak
et al. 2002; Schmezer et al. 2001; Wei et al.
2005) and, to a lesser extent, nucleotide exci-
sion repair, which is required to repair ultra-
violet C–induced lesions (Wei et al. 2005).
As shown in Table 3, we found that the fre-
quencies of necrotic cells were higher in the
workers. Also, programmed cell death (apopto-
sis) was increased, but this effect did not reach
significance. Induction of apoptosis by vana-
dium compounds has also been observed in ear-
lier in vitro studies (Au et al. 2006; Huang et al.
2000; Lampronti et al. 2005; Ray et al. 2006;
Wang et al. 2003; Ye et al. 1999), and it has
been postulated that activation of mitogen-
activated protein kinases by reactive oxygen
species and/or by an oxidant-independent path-
way may play a role (Chien et al. 2006; Choi
etal. 2003; Huang etal. 2000; Luo etal. 2003).
Statistical analyses of the different end
points showed no correlations between forma-
tion of oxidized purines and pyrimidines and
MN induction (p = 0.3992 and 0.4679, respec-
tively), which indicates that different mecha-
nisms are involved. Although oxidative damage
of DNA bases caused by V2O5is probably due
to release of reactive oxygen species, the forma-
tion of MNs may be due to its aneugenic prop-
erties, which have been found in in vitro studies
(Galli et al. 1991; Roldan and Altamirano
1990; Zhong et al. 1994) and are apparently
caused by disturbances of microtubule assembly
(Ramirez et al. 1997).
We also failed to find correlations between
vanadium plasma levels and formation of oxi-
dized purines (p = 0.2340), pyrimidines (p =
0.2895), and MN frequencies (p = 0.1571).
Earlier studies with workers exposed to metals
other than vanadium indicate that polymor-
phisms in repair genes have a strong impact on
MN formation (Iarmarcovai et al. 2006;
Mateuca et al. 2005, 2008), and it is conceiv-
able that they also play a role in the case of the
effects caused by V2O5.
Overall, our results show that inhalative
exposure to V2O5increases the levels of oxi-
dized bases and of MN, NPB, and Nbud fre-
quencies in blood cells and affects their DNA
repair capacity. It is notable that vanadium lev-
els similar to or even higher than those found
in our study have been detected in workers of
other vanadium industries and in welders
(Altamirano-Lozano et al. 1999; Huang et al.
2000; Ivancsits et al. 2002; Shi and Dalal
1992; Villani et al. 2007). Because the afore-
mentioned parameters are causally related to
diseases including cancer, our findings strongly
suggest that more protective measures and
periodical monitoring of the workers are
required. Furthermore, the current exposure
levels should be reduced to avoid health risks
due to vanadium-induced DNA instability.
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