2013

http://informahealthcare.com/txmISSN: 1537-6516 (print), 1537-6524 (electronic)

Toxicol Mech Methods, 2014; 24(1): 1–12! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.843110

REVIEW ARTICLE

Vanadium carcinogenic, immunotoxic and neurotoxic effects:a review of in vitro studies

Iwona Zwolak

Department of Cell Biology, Institute of Environmental Protection, John Paul II Catholic University of Lublin, Lublin, Poland

Abstract

Deleterious health effects induced by inorganic vanadium compounds are linked withcarcinogenic, immunotoxic and neurotoxic insults. The goal of this review is to provide asummary of mammalian cell culture studies (from the 1990s to most recent) looking into themode of the above-mentioned adverse actions of vanadium. Regarding the carcinogenicitypotential, the key cell-based studies have evidenced the ability of vanadium to inducegenotoxic lesions, cell morphological transformation and anti-apoptotic effects in a certain typeof cells. Two contradictory effects of vanadium on the immune functions of cells have beenobserved in cell culture studies. The first effect involves reduction of cell immune responsessuch as vanadium-dependent inhibition of cytokine-inducible functions, which may underliethe mechanism of vanadium-induced immunosuppression. The second one involves stimula-tion of immune activity, for example, a vanadium-mediated increase in cytokine production,which may contribute to vanadium-related inflammation. So far, an in vitro evaluation ofvanadium neurotoxicity has only been reported in few articles. These papers indicate probablecytotoxic mechanisms resulting from exposure of neurons and glial cells to vanadium.In summary, this literature review collects in vitro reports on adverse vanadium effects andthus provides vanadium researchers with a single, concise source of data.

Keywords

Carcinogenic, immunotoxic, neurotoxic,toxicity mechanism, vanadium

History

Received 5 July 2013Revised 22 August 2013Accepted 25 August 2013Published online 11 December 2013

Introduction

Vanadium: an overview of toxic effects

Vanadium is a transition metal, which occurs naturally in

various minerals (such as vanadinite or carnotite), phosphate

rocks, iron ores, crude oil and coal (Moskalyk & Alfantazi,

2003). It is released to the atmosphere as a result of natural

processes such as emissions from volcanoes and formation of

continental dust (ATSDR, 2009). However, anthropogenic

activities mostly contribute to the release of vanadium into the

atmosphere. In particular, these include residual oil burning

for generation of heat and power and metallurgical works,

for example, steel plants and vanadium pentoxide producing

plants (IARC, 2006).

Food is the main source of vanadium intake for the general

population. The element taken in via food as vanadyl or

vanadate is absorbed from the gastrointestinal tract and

transported in blood by albumin or transferrin to various

organs and tissues, including the liver, spleen, kidneys,

testicles and bones. Vanadium may also be accumulated in

lungs via inhalation of air containing vanadium pentoxide

(WHO, 2000; Mukherjee et al., 2004). The normal concen-

trations of vanadium are suggested to be around 1 nmol l�1

for blood and serum (Sabbioni et al., 1996). Vanadate

(containing pentavalent vanadium) is transformed to vanadyl

(containing tetravalent vanadium) intracellularly in the pres-

ence of reduced glutathione (GSH), flavoenzymes or

NAD(P)H oxidases. Vanadyl, in turn, can be again oxidized

to vanadate with oxygen or hydrogen peroxide. Vanadium in

the þ4 oxidation state is considered to be the predominant

form of vanadium in cells (Aureliano & Gandara, 2005;

Korbecki et al., 2012). Binding of vanadyl to intracellular

biomolecules such as glutathione, cysteine, ascorbic acid,

nucleotides or carbohydrates has been reported. The above

interactions may play a role in detoxification of excess

vanadium (Baran, 2003). Furthermore, being similar in its

structure and charge to a phosphate anion, vanadate is an

efficient inhibitor of phosphatases (and other enzymes, for

example, dynein ATPase) or can form complexes with ADP

(ADPV) or NAD (NADV), and as such promote a range

of effects (Tsiani & Fantus, 1997; Korbecki et al., 2012).

Finally, formation of decameric vanadate species (decavana-

date) in the acidic compartments of the cell (such as

lysosomes or endosomes) has been hypothesized (Aureliano,

2011). Once formed, decavanadate was reported to be stable

Address for correspondence: Iwona Zwolak, Department of Cell Biology,Institute of Environmental Protection, John Paul II Catholic Universityof Lublin, Krasnicka Ave. 102, 20-718 Lublin, Poland. Tel: +48814454626. Fax: +48 814454610. Email: iglina@kul.lublin.pl

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

enough to induce specific responses on animal cells, which

were found to be different from actions induced by lower

oligomeric vanadates or monomeric vanadate (Aureliano &

Gandara, 2005).

There is no evidence that vanadium is an essential element

in humans. Nevertheless, the element is well known for its

in vivo and in vitro actions on carbohydrate and lipid

metabolism as an insulin-mimicking agent (Thompson &

Orvig, 2004). These insulin-like effects are probably induced

by vanadate, which, acting as a phosphate analog, inhibits

protein tyrosine phosphatases (PTPs). In turn, the inhibition

of PTPs contributes to restoring the signaling pathway of

glucose uptake by a cell (Rehder, 2012). More recently, anti-

diabetic activity of vanadium has been supported by clinical

trials in which an organic vanadyl complex, bis(ethylmalto-

lato)oxovanadium (IV), exhibited a high therapeutic potential

(Thompson et al., 2009). Additionally, some studies con-

ducted on animal and cell culture models indicate vanadium

compounds as candidate drugs for the treatment of cancer

(reviewed by Bishayee et al., 2010; Rehder, 2012). The

anti-tumor activity of vanadium is often linked with ROS

generation (Aureliano, 2011).

However, harmful effects following exposure to inorganic

vanadium compounds both in animals and in people are

well-evidenced, as described hereafter. These often

examined by research scientists include vanadium-induced

carcinogenicity, immunotoxicity and neurotoxicity. Below,

in vivo data on these toxic actions have been shortly

summarized.

Vanadium in the form of V2O5 has been classified by the

International Agency for Research on Cancer as possibly

carcinogenic to humans (IARC, 2006). This classification

was largely based upon a discovery that V2O5 can induce lung

tumor in male and female B6C3F1 mice following exposure

via inhalation for a period of two years (NTP, 2002). More

recently, V2O5 has been proven to act as a lung tumor

promoter in mice and its tumor promoting activity was

observed in strains of mice with higher susceptibility to V2O5-

induced pulmonary inflammation (Rondini et al., 2010).

Assem & Levy (2009) discussed in vivo and in vitro data

regarding carcinogenic, genotoxic and respiratory effects of

inorganic vanadium compounds to address the mechanism of

V2O5-induced lung cancer in mice. They noted that vanadium

possibly via acting as a phosphate analogue and/or ROS

generation elicits genotoxic effects (such as aneuploidy and

DNA damage) and affects phosphatydylinositol-3 kinase

signaling pathways, which has downstream effects on cell

growth and proliferation (Assem & Levy, 2009). They also

proposed various in vivo and in vitro studies (such as based

on pulmonary or nasal cells) to be conducted so their

results could be helpful in more accurate classification and

risk assessment of V2O5 and other vanadium compounds

(Assem & Levy, 2009).

Exposure to vanadium compounds may result in immuno-

toxicity, which has been demonstrated in human and animal

studies. For example, vanadium air pollution was associated

with the higher incidence of bacterial and viral infections

in Czech children (IARC, 2006). Occupational exposure of

workers to V2O5 dust-induced inflammation-related condi-

tions in the respiratory tract such as rhinitis, bronchitis and

pneumonitis as well as asthma bronchiale (WHO, 2000).

Animal studies have demonstrated inflammatory responses in

lungs following V2O5 treatment as well (Rondini et al., 2010;

Yu et al., 2011). Various neurotoxic effects of vanadium

on the central nervous system of laboratory animals have

been reported. These are, for example, disruption of the

blood–brain barrier (Avila-Costa et al., 2005) and memory

deterioration along with morphological alterations in hippo-

campal neurons in mice exposed to V2O5 (Avila-Costa et al.,

2006). Furthermore, myelin deficits have been observed in the

brains of newborn rats exposed to vanadate through lactation

(Soazo & Garcia, 2007).

The induction of oxidative stress is often linked with the

toxic effects of vanadium. Indeed, vanadium can increase

the formation of ROS as a result of bioreduction of vanadate

to vanadyl, reaction of vanadyl with H2O2, or interaction of

vanadium with mitochondria (Capella et al., 2002; Aureliano

& Gandara, 2005). However, some studies suggest that the

role of oxidative stress in vanadium-induced cellular injury

may be cell-specific. For example, antioxidants protected

Ma104 cells or L02 cells from toxic effects of vanadate,

whereas they could not ameliorate vanadate-mediated damage

to HepG2 cells (Capella et al., 2002; Wang et al., 2010).

Additionally, as it was shown by in vivo studies, dissimilar

vanadate species (decameric and lower oligomeric vanadates

or monomeric vanadate) can induce different effects on

oxidative stress markers (Gandara et al., 2005; Soares et al.,

2007c). Hence, the presence of individual vanadate forms at

the experimental conditions may influence the final results

and conclusions.

Cell culture models have long been found to be extremely

useful in toxicological research studies. Importantly, there are

cell culture-based toxicity tests, which have been validated or

are under validation to be used as alternatives to animal

testing to predict human toxicity of new chemical substances

in the regulatory assessment process. For example, phototoxic

effects of chemicals are currently determined with the use of

the mouse fibroblast cell line Balb/c 3T3 (CCAC/CCPA).

In addition, regulatory assessment of acute mammalian

toxicity, genotoxicity or skin toxicity can be at least partly

performed using cell culture models (CCAC/CCPA). In basic

Abbreviations

Cdk 1 (cdc2), cyclin-dependent kinase 1

H2O2, hydrogen peroxide

IFN, interferon

IL, interleukin

NaVO3, sodium metavanadate

Na3VO4, sodium orthovanadate

NH4VO3, ammonium metavanadate

O��2 , superoxide anion�OH, hydroxyl radical

8-OHdG, 8-hydroxy-20-deoxyguanosine

PI3K, phosphatidylinositol-3 kinase

ROS, reactive oxygen species

V2O5, vanadium pentoxide

VOSO4, vanadyl sulfate

2 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

research studies, cell culture assays have proved to be useful

particularly in evaluating the mechanism underlying toxic

effects of different compounds; in vitro results often help

to complete animal toxicological data. This also applies to

investigations on toxic effects of vanadium. For instance,

in vitro data showed that mitochondria of animal cells

are particularly sensitive to vanadium when it is given as

decameric vanadate (Soares et al., 2007a,b). Other in vitro

results indicated lysosomes as potential cellular targets for

metavanadate (Zwolak, 2013).

This review describes the results from mammalian cell

culture studies (from the 1990s to most recent) pertaining to

the health effects of vanadium, namely potential carcinogenic

activity, immunotoxic effects and neurotoxicity. The reason

for choosing these health hazards was that they are well

documented in animal studies of vanadium (described above).

Moreover, some of them (mainly immunotoxic effects) have

also been observed in humans, and as such are of special

concern among toxicologists.

Vanadium-induced carcinogenesis: in vitro-testing

This section of the article is focused on in vitro studies

evaluating vanadium effects on carcinogenesis-related

mechanisms: genotoxic endpoints, cell transformation,

deregulation of cell cycle and apoptosis. The principles of

the cell-based assays used to measure the above-mentioned

effects as well as their potential to predict carcinogenic

activity of chemicals have been excellently reviewed by

Breheny et al. (2011).

Genotoxic activity of vanadium compounds

In the literature, there are reviews of in vivo and in vitro

genotoxicity studies of vanadium. For instance, the Agency

for Toxic Substances and Disease Registry (ATSDR, 2009)

reviewed data on the genotoxicity of vanadium compounds

and concluded that both clastogenic effects and DNA damage

have been observed in in vitro and in vivo studies. Another

review (Assem & Levy, 2009) reported that vanadium

compounds are non-mutagenic to bacteria, but produce

DNA damage, clastogenicity and aneugenicity in some

cells. In the present paper, several genotoxicological studies

of vanadium performed with rodent and human cell culture

models have been summarized.

Early papers reported that vanadate (NH4VO3, V2O5) and

vanadyl (VOSO4) did not induce mutagenicity measured by

the frequency of 6-thioguanine resistant mutants in the V79

Chinese hamster cell line (Galli et al., 1991; Zhong et al.,

1994). Similarly, vanadate (V2O5) was negative in a sister

chromatid exchange assay with V79 cells (Zhong et al., 1994)

and human lymphocytes (Roldan-Reyes et al., 1997).

However, conflicting results for vanadate (V2O5) genotoxicity

were reported in a micronucleus assay; vanadate-induced

micronuclei in V79 cells (Zhong et al., 1994) but no increases

in micronucleus formation were observed in Syrian hamster

embryo (SHE) cells (Gibson et al., 1997). Positive genotoxic

activity of vanadate (V2O5) was demonstrated in an alka-

line comet assay with human blood leukocytes (Rojas

et al., 1996). Since the DNA damage observed in this study

was completely repaired within a 90-min recovery time,

researchers suggested production of DNA single-strand

breaks and/or alkali-labile sites (Rojas et al., 1996). Other

authors reported increases in hyperdiploid nuclei in cultured

human lymphocytes exposed to vanadate (V2O5) using

fluorescence in situ hybridization with DNA probes for

chromosomes 1 and 7 (Ramırez et al., 1997).

Ivancsits et al. (2002) designed a study that evaluated the

effects of vanadate (Na3VO4) on three different human

cell culture models: whole blood leukocytes, isolated non-

stimulated lymphocytes and human diploid fibroblasts

(initiated from skin biopsy). In this study, Na3VO4 at 0.5

and 1 mM (which corresponded to vanadium concentrations

in the serum of V2O5-exposed workers) did not increase

DNA strand breaks in lymphocytes measured by the alkaline

comet assay. In contrast, the same vanadate doses added to

fibroblast cultures induced DNA strand breaks, which were

rapidly repaired, indicating that DNA single strand breaks

were mainly produced (Ivancsits et al., 2002).

Furthermore, inorganic V compounds of different valences

(þ3 and þ4) have been reported to induce genotoxic effects,

as described hereafter. Exposure of human lymphocytes

to vanadium(IV) tetraoxide (V2O4) increased the frequency of

sister chromatid exchanges (SCEs) and chromosomal aberra-

tions (CAs) (Rodrıguez-Mercado et al., 2003; Geyikoglu &

Turkez, 2008) as well as micronuclear aberrations (Geyikoglu

& Turkez, 2008). A study with a comet assay demonstrated

that the type of DNA damage induced by vanadyl (VOSO4)

can be cell-specific; in human lymphocytes vanadyl-induced

DNA single-strand breaks, and formation of DNA double-

strand breaks was observed in HeLa cells (Wozniak &

Blasiak, 2004). The same method of DNA damage detection

(comet assay) was used by Rodrıguez-Mercado et al. (2011)

to test three different vanadium oxidation states (V2O3, V2O4

and V2O5) in terms of their genotoxic effects in human

peripheral-blood leukocytes in vitro. In this study, V2O3- and

V2O5-induced DNA single-strand breaks and V2O4 produced

DNA double-strand breaks (Rodrıguez-Mercado et al., 2011).

A summary of cell-based studies regarding the genotoxic

effects of vanadium, including concentrations of vanadium

compounds used and duration of vanadium treatment is given

in Table 1.

The mode of vanadium-induced genotoxicity is suggested

to be related to the vanadium prooxidant activity (Shi et al.,

1996). Support for this suggestion comes from an early study

of Shi et al. (1996), who examined the mechanism of the

genotoxic effects of vanadyl (VOSO4) using a cell-free model.

Incubation of vanadyl with 20-deoxyguanosine or DNA in a

phosphate buffer solution caused formation of 8-OHdG or

DNA strand breaks, respectively. The addition of catalase

(H2O2 scavenger), formate (�OH scavenger) or metal chela-

tors significantly abolished the observed genotoxic effects,

indicating that ROS may be responsible for vanadyl-induced

adverse changes (Shi et al., 1996). Later investigations

with cell culture models demonstrated that vanadyl-induced

oxidative DNA damage in human lymphocytes and HeLa

cells (Wozniak & Blasiak, 2004). In this study, the presence

of spin traps was able to ameliorate vanadyl genetic toxicity,

which supported the role of ROS in these effects (Wozniak &

Blasiak, 2004). Furthermore, the inhibitory actions of vanad-

ate on microtubule assembly and, consequently, alterations

DOI: 10.3109/15376516.2013.843110 An in vitro evaluation of vanadium toxic effects 3

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

in formation of the spindle apparatus have also been

suggested as a mechanism underlying vanadium-mediated

genotoxicity (Ramırez et al., 1997). However, the mode

of vanadate interference with microtubule functions is still

unclear.

Summing up, the in vitro reports on vanadium genotoxicity

presented in this review show that vanadate and vanadyl are

genotoxic to cultured mammalian cells, although the results

are sometimes conflicting. It is probable that the discrepan-

cies between certain data stem from the complex biochem-

istry of vanadium. First, the contribution of individual

oligomeric vanadate species to vanadium-induced DNA

damage should be taken into account. It is well known that

decameric and lower oligomeric vanadate forms can exert

dissimilar biologic effects with regard to cellular targets or

induction of oxidative stress markers (Aureliano & Crans,

2009). Therefore, it cannot be excluded that once formed

in experimental conditions specific vanadate species may

have different potential for genetic toxicity. Second, vanadate

or vanadyl can bind to a variety of bioligands (GSH, ATP

or amino acids) and these interactions can influence the

biological activity of vanadium as well (Aureliano, 2011).

However, further experimental investigations that would

address the above issues are needed.

Cell transformation assays

In vitro cell transformation has been defined as induction

of certain phenotypic alterations in cultured cells that are

characteristic of tumorigenic cells. The process is induced

by carcinogens and it occurs in an analogous way as some

stages of in vivo carcinogenesis (Combes et al., 1999). In cell

transformation assays, mammalian cells are exposed to a

tested compound and the features of morphological trans-

formation are monitored. There are three main types of rodent

cell transformation assays, namely the SHE, Balb/c 3T3 cell

and C3H/10T1/2 cell assays. In the SHE cell assay, the

measured endpoint is the formation of transformed colonies.

In Balb/c 3T3 and C3H/10T1/2 cell assays, formation of

transformed cell foci in a monolayer indicates a carcinogenic

potential of the tested compound (Combes et al., 1999;

Breheny et al., 2011).

Studies that examined the carcinogenic activity of inor-

ganic vanadium compounds using in vitro cell transformation

assays were performed in the 1990s. Rivedal et al. (1990)

demonstrated that vanadate (Na3VO4)-induced morphological

transformation of SHE cells after five-day exposure. In

another study, Balb/c 3T3 cells were exposed to vanadate

(Na3VO4) for four weeks, which resulted in formation of

transformed foci in Balb/c 3T3 cell culture (Sheu et al., 1992).

Sabbioni et al. (1993) investigated vanadate (NH4VO3) and

vanadyl (VOSO4) in a Balb/c 3T3 cell transformation

assay and showed transforming activity for vanadate after

72-h exposure.

The carcinogenic potential of vanadate (NH4VO3)

was studied in a two-stage (initiator-promoter) cell trans-

formation system using C3H/10T1/2 cells (Parfett & Pilon,

1995). Unlike the standard cell transformation assays

described above, the two-stage transformation assay allows

detection of compounds that act as promoting agents inTab

le1

.T

he

gen

oto

xic

effe

cts

of

ino

rgan

icvan

adiu

mco

mp

ou

nd

sin

cell

-bas

edst

ud

ies.

Cel

lsV

anad

ium

com

po

un

dR

esu

lts

Ref

eren

ces

Ch

ines

eh

amst

erlu

ng

(V7

9)

cell

sV

OS

O4

(1–

7.5

mM

)N

H4V

O3

(0.5

–5

mM

)N

egat

ive

in6

-th

iog

uan

ine

resi

stan

ceas

say

Gal

liet

al.

(19

91

)C

hin

ese

ham

ster

lun

g(V

79

)ce

lls

V2O

5(1

–4mg

/ml)

for

24

hN

egat

ive

in6-t

hio

guan

ine

resi

stan

cean

din

sist

erch

rom

atid

exch

ange

assa

ys

Zhong

etal

.(1

994)

V2O

5(1

–3mg

/ml)

for

24

hIn

crea

sed

nu

mb

ero

fce

lls

wit

hk

inet

och

ore

-po

siti

ve

mic

ron

ucl

eiW

ho

leb

loo

dh

um

anle

uko

cyte

sV

2O

5(0

.3–

30

00mM

)fo

r2

hIn

du

ctio

no

fD

NA

stra

nd

bre

aks

atal

lco

nce

ntr

atio

ns

test

edR

oja

set

al.

(19

96

)H

um

anly

mp

ho

cyte

sV

2O

5(0

.3–

30

00mM

)fo

r2

4h

Ind

uct

ion

of

DN

Ast

ran

db

reak

so

nly

atth

eh

igh

est

con

cen

trat

ion

ste

sted

SH

Ece

lls

V2O

5(1

0–

25mg

/ml)

for

24

hN

egat

ive

inm

icro

nucl

eus

assa

yG

ibso

net

al.

(1997)

Hu

man

lym

ph

ocy

tes

V2O

5(0

.00

1–

0.1mM

)fo

r2

4h

Incr

ease

dfr

equ

ency

of

hy

per

dip

loid

nu

clei

Ram

ırez

etal

.(1

99

7)

Hu

man

lym

ph

ocy

tes

V2O

5(2

–6mg

/ml)

for

24

–7

2h

Neg

ativ

ein

sist

erch

rom

atid

exch

ange

assa

yR

old

an-R

eyes

etal

.(1

99

7)

Wh

ole

blo

od

hu

man

leu

ko

cyte

sIs

ola

ted

no

n-s

tim

ula

ted

hu

man

lym

ph

ocy

tes

0.5

–1

0mM

Na 3

VO

4fo

r2

2h

Ind

uct

ion

of

DN

Ast

ran

db

reak

so

nly

at5

and

10mM

Na 3

VO

4Iv

ancs

its

etal

.(2

00

2)

Dip

loid

hu

man

fib

rob

last

s(i

nit

iate

dfr

om

skin

bio

psy

)0

.5–

10mM

Na 3

VO

4fo

r2

2h

Ind

uct

ion

of

DN

Ast

ran

db

reak

sat

all

con

cen

trat

ion

ste

sted

Hu

man

lym

ph

ocy

tes

V2O

4(2

–1

6mg

/ml)

for

24

hIn

crea

sed

freq

uen

cyo

fC

As

Ro

drı

gu

ez-M

erca

do

etal

.(2

00

3)

V2O

4(2

–1

6mg

/ml)

for

48

hIn

crea

sed

freq

uen

cyo

fS

CE

sst

arti

ng

fro

mth

e4mg

/ml

trea

tmen

tH

um

anly

mp

ho

cyte

sV

OS

O4

(0.0

5–

1m

M)

for

1h

Ind

uct

ion

of

DN

Ast

ran

db

reak

sat

0.5

and

1m

MV

OS

O4

Wo

znia

k&

Bla

siak

(20

04

)H

um

ance

rvic

alca

nce

r(H

eLa)

cell

sV

OS

O4

(0.0

5–

1m

M)

for

1h

Ind

uct

ion

of

DN

Ast

ran

db

reak

sat

all

con

cen

trat

ion

ste

sted

Hu

man

lym

ph

ocy

tes

V2O

4(5

–2

0mg

/ml)

for

72

hIn

crea

sed

freq

uen

cyo

fS

CE

s,C

As

and

mic

ron

ucl

eiat

10

and

20mg

/ml

Gey

iko

glu

&T

urk

ez(2

00

8)

Wh

ole

blo

od

hu

man

leu

ko

cyte

sV

2O

3,

V2O

4,

V2O

5(1

–8mg

/ml)

for

2–

6h

V2O

3-

and

V2O

5-i

nd

uce

dD

NA

stra

nd

bre

aks

atal

lex

po

sure

tim

es(2

–6

h)

and

V2O

4-i

nd

uce

dD

NA

stra

nd

bre

aks

at4

and

6h

exp

osu

reR

od

rıg

uez

-Mer

cad

oet

al.

(20

11

)

4 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

carcinogenesis (Sakai, 2008). For the purpose of the assay,

C3H/10T1/2 cells were first exposed to a tumor initiator,

20-methylcholanthrene, and thereafter treated with the

tested compound, ammonium metavanadate. After a six-

week exposure to ammonium metavanadate, a significant

increase in the number of transformed foci was demonstrated,

indicating tumor promoting activity of this vanadium com-

pound (Parfett & Pilon, 1995).

Cell cycle and apoptosis analysis

The cell cycle is a set of events responsible for cell

duplication (Pucci et al., 2000). The key cell cycle processes

include DNA replication during S-phase and chromosome

segregation to the two new daughter cells during M-phase

(mitosis). Phases G1 and G2 precede S- and M-phases,

respectively. The transition through the successive cell cycle

phases (G1, S, G2 and M) is promoted by specific cyclin/Cdk

(cyclin-dependent kinase) complexes (Pucci et al., 2000).

When a cell encounters DNA damage, cell cycle progression

is arrested by the cell cycle checkpoint pathways. This gives

time for DNA repair. However, when the DNA damage is

severe, cell cycle checkpoint regulations direct the cell the

apoptotic pathway (Pucci et al., 2000).

Apoptosis (or programmed cell death) is energy-depen-

dent, tightly regulated self-destruction of cells. Specific

morphological and biochemical modifications that occur

during apoptotic cell death include cell shrinkage, plasma

membrane blebbing, chromatin condensation, altered expres-

sion of cell surface markers, activation of caspases and DNA

fragmentation (Elmore, 2007; Breheny et al., 2011). Cells

that are no longer needed are eliminated via apoptosis in a

range of normal physiological processes such as development,

immune reactions and aging (Elmore, 2007). Additionally,

apoptosis can be triggered by a variety of xenobiotics,

including metals, when the damage to cellular DNA cannot be

repaired. Elimination of aberrant cells via the apoptotic

pathway is crucial in preventing carcinogenesis (Rana, 2008).

Deregulation of cell cycle progression and/or apoptosis

may lead to cancer (Pucci et al., 2000). Suppression of

apoptosis is considered to play a key role in the development

of some cancers (Elmore, 2007). Interestingly, metals

classified as carcinogens, such as cadmium or hexavalent

chromium, are known to induce apoptosis (Rana, 2008).

Trying to reconcile the pro-apoptotic effects with the

carcinogenic potential of metals, Chen & Shi (2002) have

suggested that chronic exposure to metals may lead to an

imperfect apoptotic process during which an aberrant cell

can escape apoptotic cell death and thus turn into potentially

carcinogenic. Researchers who explore the potential carcino-

genic activity of vanadium compounds often examine

vanadium action on cell cycle checkpoints and the apoptosis

process, as described below.

Zhang et al. (2001) evaluated vanadate (NaVO3) effects

on cell cycle checkpoints in the human lung cell line A549.

The A549 cells represent a model of alveolar type II cells;

their usefulness for in vitro assessment of respiratory agent

toxicity has been discussed by Roggen et al. (2006). In the

study by Zhang et al. (2001), vanadate via generation of

H2O2 inhibited proliferation of A549 cells in G2/M phase.

Subsequent Western blot analysis showed that vanadate

caused a H2O2-dependent increase in the level of p21 and

Chk1 kinase, the latter leading to inhibition of Cdc25C

phosphatase, which probably resulted in inactivation of the

cyclin B/Cdk1 complex and inhibition of the transition from

the G2 to M-phase of the cell cycle (Zhang et al., 2001).

Further studies on the A549 cell line revealed that vanadate

upregulated cell cycle regulatory protein p21 (Cdk inhibitor)

via activation of mitogen-activated protein kinases (MAPKs),

p38 and ERK (extracellular signal-regulated kinase). ROS

(H2O2, O��2 and �OH) generated by vanadate in A549 cells

were found to be essential for stimulation of MAPKs

(Zhang et al., 2003). The ROS-dependent activation of

MAPKs has also been implicated in vanadate-induced

cell growth arrest of human normal liver L02 cells (Wang

et al., 2010).

The p53 protein is a transcription factor included in the

group of mammalian cell cycle checkpoint effector proteins

(Lukas et al., 2004). It is activated in response to DNA

damage either leading to transient cell cycle arrest (this allows

DNA repair and survival of a cell) or directing a cell to

apoptosis when the DNA damage cannot be repaired, which

prevents accumulation of aberrant cells with DNA mutations

(Amundson et al., 1998). The role of the p53 protein in

vanadate (NaVO3)-induced cell growth arrest has been

demonstrated in the JB6 Pþ mouse epidermal cell line

(Zhang et al., 2002b). This non-tumorigenic and anchorage-

dependent cell line is extensively used as a cell culture model

to study tumor promotion (Huang et al., 1998; Yasuda et al.,

2009). In JB6 Pþ cells, vanadate increased p53 activity, which

through activation of p21-induced cell cycle arrest at S-phase

(Zhang et al., 2002b). Growth inhibition caused by vanadate

may also proceed via a p21- and p53-independent mechanism,

as it was shown in p53-deficient mouse embryo fibroblasts,

in which vanadate treatment resulted in cell growth arrest at

G2/M phase (Zhang et al., 2002a).

Apoptosis induction due to vanadate (NaVO3) and vanadyl

(VOSO4) treatment has been shown in mouse epidermal JB6

Pþ cells (Ye et al., 1999a). A subsequent study demonstrated

that vanadate caused H2O2 generation and p53 activation,

which was required for vanadate-induced apoptosis of JB6 Pþ

cells (Huang et al., 2000). The authors suggested that

cells that are damaged by vanadate are eliminated via

apoptosis; however, damaged cells that escape apoptosis

may be responsible for vanadate-induced carcinogenesis

(Ye et al., 1999a). Studies performed with the human

epidermal cell line HaCaT demonstrated that vanadyl

(VOSO4) can induce apoptosis via a p53-independent

mechanism since HaCaT cells contain a transcriptionally

inactive p53 gene (Markopoulou et al., 2009).

The anti-apoptotic effects of vanadate in cell culture have

also been reported. For example, vanadate (NaVO3) inhibited

apoptosis induced by alkaloid K252a in human malignant

glioma T98G cells via restoration of the activity of anti-

apoptotic kinase Akt/PKB, possibly through a PI3K-depen-

dent pathway (Chin et al., 1999). Other in vitro studies have

reported that vanadate may transduce its mitogenic effects

on bone-derived cells by increasing the activity of MAPK-

dependent pathways (Laize et al., 2009). Furthermore, Tang

et al. (2007) addressed the role of cyclooxygenase-2 (COX-2)

DOI: 10.3109/15376516.2013.843110 An in vitro evaluation of vanadium toxic effects 5

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

in the anti-apoptotic effects of vanadate (V2O5). The COX-2

enzyme is suggested to contribute to the carcinogenesis

process (Rizzo, 2011). The study by Tang et al. (2007)

showed that V2O5 upregulated the expression of COX-2 in

human bronchial epithelial cells (Beas-2B cell line), yielding

an anti-apoptotic effect, which was suggested to be a potential

mechanism of vanadium-dependent carcinogenic transforma-

tion of human bronchial cells.

Collectively, the above studies indicate that the distinct

effects of vanadate on apoptosis may depend on the type

of exposed cells. Other factors which are also considered

to influence the anti- or pro-apoptotic effects of vanadate

include the use of different doses of vanadate (lower vanadate

doses were reported to inhibit apoptosis and higher concen-

trations of vanadate are pro-apoptotic) and the vanadate form

used in experiments (Chen & Shi, 2002).

Vanadium-induced immunotoxic effects:in vitro-testing

Immunotoxicity is defined as any adverse effect exerted on

the immune system resulting from exposure to xenobiotics,

including chemical pollutants and drugs. Immunotoxic

effects induced by these compounds can be divided into the

following manifestations: hypersensitivity (immune response

to an allergen leading to food allergy, allergic contact

dermatitis or respiratory hypersensitivity such as asthma),

immunosuppression (immune deficiency, reduced immune

response leading to higher susceptibility of organism to

infection or neoplasia), autoimmunity (immune response

against host’s own cells) and chronic inflammatory responses

(Luster et al., 2008; Rooney et al., 2012). Table 2 summarizes

the results of key cell-based studies (described below)

designed to evaluate vanadium-mediated immunotoxic

effects.

Immunosuppression-related effects of vanadium

An in vitro study that examined the mechanism of vanadate-

induced immunosuppression was performed on a mouse

macrophage-like cell line by Cohen et al. (1996). In this

study, vanadate (NH4VO3) treatment altered the immune

functions of macrophage-like cells causing such effects as

reduced IFNg binding to cells, decreased expression of

surface IFNg Class I receptors (IFNg binds to IFNg receptors

resulting in enhanced antibacterial activity of macrophages),

and lower density of the MHC I-A surface antigen (Cohen

et al., 1996). Another study demonstrated an inhibitory effect

of NaVO3 (used at 100 mM) on granulocyte O��2 production

and cytokine release (IFNg and IL-5) by phytohemagglutinin-

stimulated human peripheral blood mononuclear cells

(PBMCs) (Di Gioacchino et al., 2002). It should be added

however that in the same study a low concentration of NaVO3

(0.1mM) exerted the opposite effect on IL-5 release,

compared with 100 mM NaVO3, and stimulated its production.

The above observation indicates the dual effects of vanadate

on selected in vitro immune responses, which are dependent

on the dose of vanadate taken for examination (Di Gioacchino

et al., 2002).

A different mechanism of vanadium-mediated

immunosuppression, addressing the role of b-defensin (an

antimicrobial peptide, a mediator of innate immunity in

airway epithelium) has been examined by Klein-Patel et al.

(2006). In this report, pre-exposure of primary bovine tracheal

epithelial (BTE) cells to vanadate (V2O5) inhibited b-defensin

gene expression induced by stimuli given as bacterial

lipopolysacharide (LPS) or IL-1b (a pro-inflammatory

cytokine). The same results have been obtained in a human

lung cell line (A549), in which IL-1b-induced b-defensin

gene expression was inhibited by vanadate or vanadyl

(VOSO4) pretreatment (Klein-Patel et al., 2006).

Inflammation-related effects of vanadium

Inflammation is a protective response of an organism to

the biologically, chemically or physically mediated injury.

The process is initiated by inflammatory mediators, including

cytokines and chemokines released by injured tissue, which

attract leukocytes (mainly granulocytes) to the damaged site.

The attracted cells eliminate the agents that triggered

inflammation (for example, microbial pathogens) and

damaged host cells, which is essential to initiate wound

healing by platelets (Gomez-Mejiba et al., 2009; Fox et al.,

2010; Germolec et al., 2010). However, a persistent

inflammatory state can lead to excessive tissue injury and it

was shown to contribute to the development of many diseases,

such as allergy, asthma, atherosclerosis, autoimmune diseases

and cancer (Germolec et al., 2010). As mentioned earlier,

inflammatory responses have been observed in respiratory

of V2O5-exposed laboratory animals (Rondini et al., 2010;

Yu et al., 2011) and humans (WHO, 2000).

Several in vitro studies have been conducted on immune

and non-immune cells in order to elucidate various potential

mechanisms underlying vanadium-mediated inflammation.

For example, vanadate (NaVO3) was shown to induce

respiratory burst activity (production of ROS) of rat alveolar

macrophages via activating NADPH oxidase (a plasma

membrane enzyme essential for the production of ROS by

activated macrophages) (Grabowski et al., 1999). The authors

have implicated this effect in the initiation of inflammatory

responses in macrophages following vanadium exposure

(Grabowski et al., 1999). Furthermore, only vanadium rather

than other metal components of residual oil fly ash (ROFA)

has been demonstrated to induce secretion of IL-6 and IL-8

(both are pro-inflammatory cytokines) by human normal

bronchial epithelial cells (Carter et al., 1997). Other studies

have shown involvement of ROS in vanadate (V2O5)-

mediated IL-8 induction in human hepatoma cell line

Hep G2 (Dong et al., 1998) and human lung cancer cell

line NCI-H292 (Yu et al., 2011).

The allergic potential of vanadate (V2O5 and Na3VO4) was

studied on rat basophilic leukemia RBL-2H3 cells (Kitani

et al., 1998). Treatment of RBL-2H3 cells with either

vanadium compound (V2O5 and Na3VO4) in the presence of

H2O2-induced histamine release and leukotriene synthesis

(both are main inflammatory mediators of allergy). In the

same study, two different in vitro models, i.e. human

basophils and rat mast cells, also released histamine after

exposure to V2O5/H2O2 or Na3VO4/H2O2, indicating that the

stimulatory effect of vanadate on histamine release was

not specific only to RBL-2H3 cells. It was hypothesized that

6 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

Tab

le2

.A

sum

mar

yo

fce

ll-b

ased

stu

die

s,w

hic

hev

alu

ated

po

ten

tial

imm

un

oto

xic

effe

cts

of

ino

rgan

icvan

adiu

mco

mp

ou

nd

s.

Cel

lsV

anad

ium

trea

tmen

tR

esu

lts

Ref

eren

ces

Mo

use

myel

om

on

ocy

tic

mac

ro-

ph

age-

lik

e(W

EH

I-3

)ce

lls

NH

4V

O3

(10

0mM

)fo

r2

0h

Red

uce

db

ind

ing

of

IFNg

toW

EH

Ice

lls

Co

hen

etal

.(1

99

6)

Red

uce

dn

um

ber

of

acti

vel

yb

ind

ing

Cla

ssI

IFNg

rece

pto

rsIn

crea

sed

bin

din

gaf

fin

ity

of

Cla

ssI

IFNg

rece

pto

rsfo

rIF

Ng

Dec

reas

edb

ind

ing

affi

nit

yo

fC

lass

IIIF

Ng

rece

pto

rsD

ecre

ased

den

sity

of

MH

CI-

Asu

rfac

ean

tigen

sN

orm

alh

um

anb

ron

chia

lep

ith

elia

l(N

HB

E)

cell

sV

OS

O4

or

NaV

O3

(0.1

–0.7

5m

M)

for

2h

Incr

ease

dpro

duct

ion

of

IL-6

and

IL-8

Car

ter

etal

.(1

997)

Hu

man

hep

ato

ma

(Hep

G2

)ce

lls

V2O

5(1

–1

00mM

)fo

r1

8h

Incr

ease

dp

rod

uct

ion

of

TN

F-a

and

IL-8

Do

ng

etal

.(1

99

8)

Hu

man

bas

op

hil

s,R

BL

-2H

3ce

lls,

rat

mas

tce

lls

V2O

5(0

.3m

M)þ

H2O

2o

rN

a 3V

O4

(1m

M)þ

H2O

2fo

r6

0m

inIn

du

ctio

no

fh

ista

min

ere

leas

eK

itan

iet

al.

(19

98

)

Rat

bas

op

hil

icle

ukem

ia(R

BL

2H

3)

cell

sV

2O

5(0

.3m

M)þ

H2O

2o

rN

a 3V

O4

(1m

M)þ

H2O

2fo

r6

0m

inIn

du

ctio

no

fle

uko

trie

ne

syn

thes

is

Rat

alveo

lar

mac

rop

hag

esN

aVO

3(5

0–

10

00mM

)fo

r5

or

15

min

Ind

uct

ion

of

resp

irat

ory

bu

rst

acti

vit

yG

rab

ow

ski

etal

.(1

99

9)

GP

TE

cell

sV

OS

O4

(5mg

/cm

2)

for

8h

Incr

ease

dm

uci

nse

cret

ion

Jian

get

al.

(20

00

)H

um

anP

BM

Cs

NaV

O3

(10

0mM

)fo

r4

8h

Red

uce

dpro

life

rati

on

of

phyto

hem

agglu

tinin

-sti

mula

ted

cell

sD

iG

ioac

chin

oet

al.

(2002)

Red

uce

dp

rod

uct

ion

of

IFNg

and

IL-5

Red

uce

dp

rod

uct

ion

of

O�� 2

by

gra

nu

locy

tes

NaV

O3

(0.1mM

)fo

r4

8h

Incr

ease

dp

rod

uct

ion

of

TN

F-a

and

IL-5

Red

uce

dp

rod

uct

ion

of

O�� 2

by

gra

nu

locy

tes

BT

Ece

lls

V2O

5(0

.15

–2

0mg

/cm

2)

for

6h

and

then

wit

hli

po

po

lysa

cch

arid

e(L

PS

)fo

r1

8h

or

IL-1b

for

6h

Inh

ibit

ion

of

LP

S-

or

IL-1b-

ind

uce

dtr

ach

eal

anti

mic

rob

ial

pep

tid

e(T

AP

,b

ov

ine

b-d

efen

sin

)gen

eex

pre

ssio

nK

lein

-Pat

elet

al.

(2006)

Hu

man

lun

gca

rcin

om

a(A

54

9)

cell

sV

2O

5(0

.15

–2

.5mg

/cm

2)

or

VO

SO

4

(0.1

45

–2

.32mg

/cm

2)

for

6h

and

then

wit

hIL

-1b

for

6h

Inh

ibit

ion

of

IL-1b-

ind

uce

dh

um

anb-

def

ensi

n-2

gen

eex

pre

ssio

n

Hu

man

lun

gm

uco

epid

erm

oid

car-

cin

om

a(N

CI-

H2

92

)ce

lls

V2O

5(0

.2an

d1mg

/cm

2)

for

6o

r2

4h

Ind

uct

ion

of

mu

cin

(MU

C5

AC

)gen

eex

pre

ssio

nan

dm

uci

nse

cret

ion

Yu

etal

.(2

01

1)

V2O

5(1

mg/c

m2)

for

6h

Ind

uct

ion

of

IL-8

gen

eex

pre

ssio

n

DOI: 10.3109/15376516.2013.843110 An in vitro evaluation of vanadium toxic effects 7

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

vanadium can amplify the allergy in the presence of H2O2 at

the inflammation site (Kitani et al., 1998).

Vanadium and NF-iB protein

Nuclear factor-kappaB (NF-kB) protein family consists

of transcription factors that regulate the expression of genes

mediating inflammation, immune responses, cell proliferation

and survival (Oeckinghaus & Ghosh, 2009). The inactive

form of NF-kB associated with inhibitory IkB proteins resides

in the cytoplasm. Phosphorylation of IkB proteins mediated

by the IkB kinase complex leads to degradation of these

NF-kB inhibitors in proteosomes. Degradation of IkBs

releases the active form of NF-kB, which translocates to the

nucleus, where it controls the transcription of kB-dependent

genes. A large number of stimuli have been shown to activate

NF-kB, including bacterial and viral infections, cytokines

(TNFa, IL-1) and ROS (Oeckinghaus & Ghosh, 2009). Active

NF-kB regulates the transcription of about 200 genes

encoding such proteins as cytokines (IL-1, -2, -6 and -8),

chemokines, immunoregulatory proteins, cell adhesion mole-

cules, acute phase proteins and regulators of apoptosis

(Piotrowska et al., 2008).

As reviewed by Chen & Shi (2002), vanadate-induced

activation of NF-kB via IkBa degradation in the following

cell types: mouse macrophage RAW 264.7 cells, human

myeloid U937 cells and Jurkat E6.1 cells. Similarly, in

human airway epithelial cells, vanadyl (VOSO4)-induced

IkBa degradation and activation of NF-kB resulting in

kB-dependent transcription through an ROS-dependent

mechanism (Jaspers et al., 2000). Another study performed

on primary mouse T lymphocytes demonstrated that vanadate

(Na3VO4) might influence antigen-induced NF-kB activation

(Lee et al., 2001). The researchers found that although

preexposure to orthovanadate did not affect NF-kB activity

in T cells following 4-h induction with antigen, the same

vanadium compound reduced NF-kB activity in T cells after

30 min of antigen stimulation (Lee et al., 2001).

Although, as described above, vanadate and vanadyl

have been demonstrated to induce NF-kB activation, only

one in vitro study has been found in accessible literature,

which evidenced the involvement of the NF-kB pathway in

vanadium-induced immune responses. In that report, vana-

date-induced TNF-a production in mouse macrophage

RAW 264.7 cells via ROS generation and NF-kB activation

(Ye et al., 1999b).

Vanadium and mucin secretion

Airway mucus secretion and clearance plays an important role

in elimination of inhaled pollutants from the respiratory tract

thus minimizing contact of airway epithelial cells with toxic

compounds (Roggen et al., 2006). However, chronic mucus

hypersecretion can contribute to various diseases, including

asthma, chronic obstructive pulmonary disease and cystic

fibrosis (Rogers, 2007). A cause–effect relationship is

suggested between increased mucus secretion due to toxic

chemical exposure and respiratory immunotoxic effects

induced by such chemicals. This relationship has been

proposed to be useful in predicting the immunotoxic potential

of respiratory agents (Roggen et al., 2006). Two in vitro

studies have been found which addressed the role of vanadium

in mucus overproduction. Jiang et al. (2000) used a guinea

pig tracheal epithelial (GPTE) primary cell culture to study

mucin (a major protein component of mucus) secretion

induced by ROFA. In this report, vanadyl (VOSO4)-

dependent ROS generation was implicated in mediating

ROFA-induced mucin production by the examined cells

(Jiang et al., 2000). A recent study (Yu et al., 2011)

investigated the mechanism of vanadate (V2O5)-mediated

mucin production by human lung cancer cell line NCI-H292.

The researchers demonstrated that mucin secretion mediated

by V2O5 proceeded through activation of NF-kB and was

ROS independent (Yu et al., 2011).

Vanadium-induced neurotoxic effects:in vitro-testing

Only a few cell culture studies have been found in the

literature, which evaluated the mechanism underlying the

toxic effects of vanadium compounds on the central nervous

system. The mechanism of vanadate-induced neurodegenera-

tion has been investigated in rat primary cerebellar granule

progenitors (CGPs), which represent a model for study of

neuronal development (Luo et al., 2003) and in immortalized

rat dopaminergic N27 cells, which is a cell culture model

of Parkinson’s disease (Afeseh Ngwa et al., 2009). In both

culture models, vanadate-induced apoptotic cell death;

however, the mechanisms leading to apoptosis of the two

mentioned cell types were dissimilar. In CGPs, vanadate

(NaVO3) triggered apoptosis mainly through activation of the

Fas cell surface death receptor (initiates an extrinsic apoptosis

pathway) with subsequent caspase 8 and 3 activation, and this

apoptotic pathway was mainly dependent on c-Jun N-terminal

protein kinase activation; the involvement of ROS was

modest (Luo et al., 2003). In contrast, treatment of N27

cells with vanadate (V2O5)-induced ROS generation followed

by mitochondrial cytochrome c release into the cytoplasm and

caspase 9 and 3 activation leading to apoptosis (an intrinsic

apoptotic pathway) (Afeseh Ngwa et al., 2009). Two main

factors could influence the induction of different apoptotic

signaling pathways in vanadate-exposed cells, i.e. the type of

cells used for the examination and/or the presence of specific

oligomeric vanadates in the experimental conditions.

As mentioned in the introduction, the in vivo data

show deficits in myelin sheath formation in brains of

vanadate-exposed newborn rats (Soazo & Garcia, 2007).

The myelin sheath surrounds axons of neurons, and its role is

to accelerate a nerve impulse. It is formed by glial cells,

namely oligodendrocytes (in the central nervous system) or

Schwann cells (in the peripheral nervous system) (Harry

et al., 1998). The findings from a recent in vitro study

indicate that the adverse effects of vanadate on myelin

development during the post-natal period in rats can be

caused by vanadate-induced specific depletion of oligoden-

drocyte progenitors (OPCs) (Todorich et al., 2011). This was

evidenced by the higher sensitivity of primary rat OPCs to

vanadate (NaVO3) cytotoxicity in comparison to primary rat

astrocytes or mature oligodendrocytes. The exposure of OPCs

to NaVO3 resulted in ROS generation, which was suggested

to contribute to the vanadate-induced damage of these cells.

8 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

Interestingly, the cytotoxicity of metavanadate to an oligo-

dendrocyte progenitor culture was significantly exacerbated in

the presence of ferritin (an iron delivery protein) or TMH-

ferrocene (a lipophilic iron compound). The high iron

requirement during oligodendrocyte maturation was sug-

gested as the main reason for unique sensitivity of OPCs to

vanadium exposure (Todorich et al., 2011).

Concluding remarks

In this review, in vitro studies regarding the vanadium adverse

health effects have been summarized. On the basis of these

reports, some possible actions through which vanadium

compounds induce carcinogenic, immunotoxic and neurotoxic

effects can be proposed (Figure 1). The assessment of

vanadium-mediated genotoxicity, cell transformation, cell

cycle arrest and apoptosis in cell culture systems provides

additional data for our understanding of the potential

carcinogenic activity of vanadium compounds. The studies

of vanadium genotoxicity have firmly established that

vanadate and vanadyl have the ability to induce genotoxic

effects in cultured mammalian cells expressed as DNA strand

breaks, and chromosome and micronucleus aberrations. This

is in line with the results from cell transformation assays,

in which inorganic the vanadium compounds tested

proved positive. The evaluation of vanadium effects on cell

cycle- and apoptosis-related pathways supplements the

above observations. The cell cycle arrest and/or apoptotic

cell death, which play a protective role against genotoxic

stress, and accumulation of aberrant cells can be induced in

vanadyl or vanadate-exposed cells through the p53-dependent

pathway as well as the p53-independent mechanism. This

shows that mammalian cells damaged with vanadium can be

arrested in the cell cycle, which allows repairing DNA

damage, or they can be efficiently eliminated via apoptosis.

However, an opposite effect, i.e. suppression of apoptosis

following the vanadate treatment, has also been observed

in some cell lines. The reported mechanisms involved

vanadate-dependent upregulation of proteins linked to the

anti-apoptotic effects, namely phosphatidylinositol-3 kinase

or COX-2, which may result in aberrant growth of cells and

carcinogenesis process.

The data available in vitro have pointed out the potential

mechanisms by which inorganic vanadium compounds

mediate their immunotoxic effects. For example, vanadate-

stimulated respiratory burst (via activating NADPH oxidase)

of alveolar macrophages and induced production of pro-

inflammatory mediators such as IL-6 or -8 by airway

epithelial cells. These effects have been postulated to

contribute to vanadium-mediated inflammation in the

respiratory tract. Cell culture studies have also demonstrated

inhibitory effects of vanadyl or vanadate on some immune

responses, such as cytokine-inducible functions, which can

lead to decreased immunity against pathogens. The early

events leading to vanadium-induced inflammatory responses

have been suggested to involve ROS-dependent activation

of NF-kB protein. Indeed, NF-kB is a transcription factor,

which regulates the expression of various genes, including

those encoding pro-inflammatory mediators, and inorganic

vanadium compounds have induced the activity of NF-kB

in many cell lines. However, the direct linkage between

vanadium-dependent NF-kB activation and vanadium-

induced inflammatory responses remains to be investigated.

Our current knowledge of early signaling pathways leading

to vanadyl- or vanadate-mediated immunosuppression is

still very incomplete. In addition, it is not known why

inorganic vanadium compounds sometimes act as inflamma-

tory agents and sometimes induce immunosuppressive effects.

Presumably, the type of vanadium species, the vanadium

dose, the duration of vanadium exposure and the kind of cell

exposed to vanadium may influence vanadium-induced

immunotoxic effects.

The assessment of vanadium neurotoxicity in cell culture

systems is seldom reported in the literature. The available

data designate oxidative stress as a major factor involved

in vanadate-induced neurotoxicity with the exception of one

study, which pointed to ROS-independent activation of

MAPKs as the main mediators of the neurotoxic effects

of vanadate. Such discrepancies in vanadium neurotoxicity

studies may be expected due to various types of nervous

system-derived cells (different types of glial cells and

neurons) having diverse biological functions, which alto-

gether influences the mechanism of cellular response to

vanadium. Additional in vitro studies of vanadium focused

?

Neurotoxic effectsCarcinogenesis-related effects Immunotoxic effects

Antiapoptotic effects

ROS generation Induction of apoptosis inneurons and glial cells

In vitro exposure of mammalian cells to inorganic vanadium compounds

Induction of cyclooxygenase-2or phosphatidylinositol-3 kinase

Cell transformation

Genotoxic effects NF-κB activation ?

Synthesis ofproinflammatory cytokines

?

ROS generation

Figure 1. Main biological effects induced by inorganic vanadium compounds in mammalian cell culture models presented in this review. The linksbetween some vanadium actions can only be speculative (marked with?) as they still remain to be examined.

DOI: 10.3109/15376516.2013.843110 An in vitro evaluation of vanadium toxic effects 9

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

on neurospecific endpoints, for example, related with

neurotransmitters metabolism, could substantially contribute

to our understanding of neurotoxic effects of vanadium.

It is widely acknowledged that cell culture models are very

useful to study mechanistic toxicity of chemical substances.

Also, as described above, in vitro evaluation of vanadium

insults allows indicating the potential mode of vanadium

effects on specific mammalian cell types. However, since the

complex biochemistry of vanadium may influence the results

of experiments, thus more studies are needed to deal with this

issue. Finally, the ongoing development of in vitro techniques

and their application in vanadium research studies may help

in future expand our knowledge on adverse vanadium actions.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

References

Afeseh Ngwa H, Kanthasamy A, Anantharam V, et al. (2009). Vanadiuminduces dopaminergic neurotoxicity via protein kinase C deltadependent oxidative signaling mechanisms: relevance to etiopatho-genesis of Parkinson’s disease. Toxicol Appl Pharmacol 240:273–85.

Amundson SA, Myers TG, Fornace AJ. (1998). Roles for p53 in growtharrest and apoptosis: putting on the brakes after genotoxic stress.Oncogene 17:3287–99.

Assem FL, Levy LS. (2009). A review of current toxicological concernson vanadium pentoxide and other vanadium compounds: gaps inknowledge and directions for future research. J Toxicol EnvironHealth B 12:289–306.

ATSDR, Agency for Toxic Substances and Disease Registry, (2009).Toxicological profile for vanadium (Draft for Public Comment).Atlanta: U.S. Department of Health and Human Services, PublicHealth Service.

Aureliano M. (2011). Recent perspectives into biochemistry ofdecavanadate. World J Biol Chem 2:215–25.

Aureliano M, Crans DC. (2009). Decavanadate (V10O286�) and

oxovanadates: oxometalates with many biological activities. J InorgBiochem 103:536–46.

Aureliano M, Gandara RMC. (2005). Decavanadate effects in biologicalsystems. J Inorg Biochem 99:979–85.

Avila-Costa MR, Colın-Barenque L, Zepeda-Rodrıguez A, et al. (2005).Ependymal epithelium disruption after vanadium pentoxide inhal-ation, a mice experimental model. Neurosci Lett 381:21–5.

Avila-Costa MR, Fortoul TI, Nino-Cabrera G, et al. (2006).Hippocampal cell alterations induced by the inhalation of vanadiumpentoxide (V2O5) promote memory deterioration. NeuroToxicology27:1007–12.

Baran EJ. (2003). Model studies related to vanadium biochemistry:recent advances and perspectives. J Braz Chem Soc 14:878–88.

Bishayee A, Waghray A, Patel MA, et al. (2010). Vanadium in thedetection, prevention and treatment of cancer: the in vivo evidence.Cancer Lett 294:1–12.

Breheny D, Oke O, Faux SP. (2011). The use of in vitro systems to assesscancer mechanisms and the carcinogenic potential of chemicals.Altern Lab Anim 39:1–23.

Capella LS, Gefe MR, Silva EF, et al. (2002). Mechanisms of vanadate-induced cellular toxicity: role of cellular glutathione and NADPH.Arch Biochem Biophys 406:65–72.

Carter JD, Ghio AJ, Samet JM, et al. (1997). Cytokine production byhuman airway epithelial cells after exposure to an air pollutionparticle is metal-dependent. Toxicol Appl Pharmacol 146:180–8.

CCAC/CCPA. The Canadian Council on Animal Care. Available from:3rs.ccac.ca/en/testing-and-production/alternative-test-methods/tox-icity [last accessed May 2012].

Chen F, Shi X. (2002). Intracellular signal transduction of cells inresponse to carcinogenic metals. Crit Rev Oncol Hematol 42:105–21.

Chin LS, Murray SF, Harter DH, et al. (1999). Sodium vanadate inhibitsapoptosis in malignant glioma cells: a role for Akt/PKB. J Biomed Sci6:213–8.

Cohen MD, McManus TP, Yang Z, et al. (1996). Vanadium affectsmacrophage interferone-g-binding and -inducible responses. ToxicolAppl Pharmacol 138:110–20.

Combes R, Balls M, Curren R, et al. (1999). Cell transformation assaysas predictors of human carcinogenicity. Altern Lab Anim 27:745–67.

Di Gioacchino M, Sabbioni E, Di Giampaolo L, et al. (2002). In vitroeffects of vanadate on human immune functions. Ann Clin Lab Sci 32:148–54.

Dong W, Simeonova PP, Gallucci R, et al. (1998). Toxic metals stimulateinflammatory cytokines in hepatocytes through oxidative stressmechanisms. Toxicol Appl Pharmacol 151:359–66.

Elmore S. (2007). Apoptosis: a review of programmed cell death.Toxicol Pathol 35:495–516.

Fox L, Hegde S, Gumperz JE. (2010). Natural killer T cells: innatelymphocytes positioned as a bridge between acute and chronicinflammation. Microbes Infect 12:1125–33.

Galli A, Vellosi R, Fiorio R, et al. (1991). Genotoxicity of vanadiumcompounds in yeast and cultured mammalian cells. Teratog CarcinogMutagen 11:175–83.

Gandara RMC, Soares SS, Martins H, et al. (2005). Vanadate oligomers:in vivo effects in hepatic vanadium accumulation and stress markers.J Inorg Biochem 99:1238–44.

Germolec DR, Frawley RP, Evans E. (2010). Markers of inflammation.Methods Mol Biol 598:53–73.

Geyikoglu F, Turkez H. (2008). Boron compounds reduce vanadiumtetraoxide genotoxicity in human lymphocytes. Environ ToxicolPharmacol 26:342–7.

Gibson DP, Brauninger R, Shaffi HS, et al. (1997). Induction ofmicronuclei in Syrian hamster embryo cells: comparison to resultsin the SHE cell transformation assay for national toxicology programtest chemicals. Mutat Res 392:61–70.

Gomez-Mejiba SE, Zhai Z, Akram H, et al. (2009). Inhalation ofenvironmental stressors & chronic inflammation: autoimmunity andneurodegeneration. Mutat Res 674:62–72.

Grabowski GM, Paulauskis JD, Godleski JJ. (1999). Mediating phos-phorylation events in the vanadium-induced respiratory burst ofalveolar macrophages. Toxicol Appl Pharmacol 156:170–8.

Harry GJ, Billingsley M, Bruinink A, et al. (1998). In vitro techniquesfor the assessment of neurotoxicity. Environ Health Perspect 106:131–58.

Huang C, Ma WY, Young MR, et al. (1998). Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1transactivation and transformation in mouse JB6 cells. Proc NatlAcad Sci USA 95:156–61.

Huang C, Zhang Z, Ding M, et al. (2000). Vanadate induces p53transactivation through hydrogen peroxide and causes apoptosis.J Biol Chem 275:32516–22.

IARC, International Agency for Research on Cancer. (2006). IARCMonographs on the evaluation of carcinogenic risks to humans. Cobaltin hard metals and cobalt sulfate, gallium arsenide, indium phosphideand vanadium pentoxide. Vol. 86.

Ivancsits S, Pilger A, Diem E, et al. (2002). Vanadate inducesDNA strand breaks in cultured human fibroblasts at doses relevantto occupational exposure. Mutat Res 519:25–35.

Jaspers I, Samet JM, Erzurum S, et al. (2000). Vanadium-inducedkB-dependent transcription depends upon peroxide-induced activationof the p38 mitogen-activated protein kinase. Am J Respir Cell MolBiol 23:95–102.

Jiang N, Dreher KL, Dye JA, et al. (2000). Residual oil fly ash inducescytotoxicity and mucin secretion by guinea pig tracheal epithelial cellsvia an oxidant-mediated mechanism. Toxicol Appl Pharmacol 163:221–30.

Kitani S, Rajiva De Silva N, Morita Y, et al. (1998). Globalenvironmental pollutant substance vanadium activates mast cells andbasophils at the late phase in the presence of hydrogen peroxide.Environ Toxicol Pharmacol 6:1–12.

Klein-Patel ME, Diamond G, Boniotto M, et al. (2006). Inhibition ofb-defensin gene expression in airway epithelial cells by low doses ofresidual oil fly ash is mediated by vanadium. Toxicol Sci 92:115–25.

Korbecki J, Baranowska-Bosiacka I, Gutowska I, et al. (2012).Biochemical and medical importance of vanadium compounds. ActaBiochim Pol 59:1–6.

10 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

Laize V, Tiago DM, Aureliano M, et al. (2009). New insightsinto mineralogenic effects of vanadate. Cell Mol Life Sci 66:3831–6.

Lee K, Shen X, Konig R. (2001). Effects of cadmium and vanadiumions on antigen-induced signaling in CD4þ T cells. Toxicology 169:53–65.

Lukas J, Lukas C, Bartek J. (2004). Mammalian cell cycle checkpoints:signalling pathways and their organization in space and time. DNARepair 3:997–1007.

Luo J, Sun Y, Lin H, et al. (2003). Activation of JNK byvanadate induces a Fas-associated death domain (FADD)-dependentdeath of cerebellar granule progenitors in vitro. J Biol Chem 278:4542–51.

Luster MI, Simeonova PP, Germolec DR. (2008). Immunotoxicology.In ELS. Chichester: John Wiley & Sons Ltd, Available from: http://www.els.net [doi: 10.1002/9780470015902.a0000955.pub2].

Markopoulou S, Kontargiris E, Batsi C, et al. (2009). Vanadium-inducedapoptosis of HaCaT cells is mediated by c-fos and involves nuclearaccumulation of clusterin. FEBS J 276:3784–99.

Moskalyk RR, Alfantazi AM. (2003). Processing of vanadium: a review.Miner Eng 16:793–805.

Mukherjee B, Patra B, Mahapatra S, et al. (2004). Vanadium-an elementof atypical biological significance. Toxicol Lett 150:135–43.

NTP. (2002). NTP toxicology and carcinogenesis studies ofvanadium pentoxide (CAS No. 1314-62-1) in F344/N rats andB6C3F1 mice (inhalation). Natl Toxicol Program Tech Rep Ser 507:1–343.

Oeckinghaus A, Ghosh S. (2009). The NF-kB family of transcrip-tion factors and its regulation. Cold Spring Harb Perspect Biol 1:a000034.

Parfett CLJ, Pilon R. (1995). Oxidative stress-regulated gene expres-sion and promotion of morphological transformation induced inC3H/10T1/2 cells by ammonium metavanadate. Fd Chem Toxic 33:301–8.

Piotrowska A, Iz_ykowska I, Podhorska-Okołow M, et al. (2008). Budowabiałek z rodziny NF-kB i ich rola w procesie apoptozy. Postepy HigMed Dosw 62:64–74.

Pucci B, Kasten M, Giordano A. (2000). Cell cycle and apoptosis.Neoplasia 2:291–9.

Ramırez P, Eastmond DA, Laclette JP, et al. (1997). Disruption ofmicrotubule assembly and spindle formation as a mechanism for theinduction of aneuploid cells by sodium arsenite and vanadiumpentoxide. Mutat Res 386:291–8.

Rana SVS. (2008). Metals and apoptosis: recent developments. J TraceElem Med Biol 22:262–84.

Rehder D. (2012). The potentiality of vanadium in medicinal applica-tions. Future Med Chem 4:1823–37.

Rivedal E, Roseng LE, Sanner T. (1990). Vanadium compounds promotethe induction of morphological transformation of hamster embryocells with no effect on gap junctional cell communication. Cell BiolToxicol 6:303–14.

Rizzo MT. (2011). Cyclooxygenase-2 in oncogenesis. Clin Chim Acta412:671–87.

Rodrıguez-Mercado JJ, Mateos-Nava RA, Altamirano-Lozano MA.(2011). DNA damage induction in human cells exposed to vanadiumoxides in vitro. Toxicol In Vitro 25:1996–2002.

Rodrıguez-Mercado JJ, Roldan-Reyes E, Altamirano-Lozano M. (2003).Genotoxic effects of vanadium(IV) in human peripheral blood cells.Toxicol Lett 144:359–69.

Rogers DF. (2007). Physiology of airway mucus secretion and patho-physiology of hypersecretion. Respir Care 52:1134–46.

Roggen EL, Kristinsen Soni N, Verheyen GR. (2006).Respiratory immunotoxicity: an in vitro assessment. Toxicol InVitro 20:1249–64.

Rojas E, Valverde M, Herrera LA, et al. (1996). Genotoxicity ofvanadium pentoxide evaluate by the single cell gel electrophoresisassay in human lymphocytes. Mutat Res 359:77–84.

Roldan-Reyes E, Aguilar-Morales C, Frıas-Vaquez, S, et al. (1997).Induction of sister chromatid exchanges in human lymphocytes byvanadium pentoxide in combination with caffeine. Med Sci Res 25:501–4.

Rondini EA, Walters DM, Bauer AK. (2010). Vanadium pentoxideinduces pulmonary inflammation and tumor promotion in a strain-dependent manner. Part Fibre Toxicol 7:9.

Rooney AA, Luebke RW, Selgrade MK, et al. (2012). Immunotoxicologyand its application in risk assessment. EXS 101:251–87.

Sabbioni E, Kueera J, Pietra R, et al. (1996). A critical review on normalconcentrations of vanadium in human blood, serum, and urine.Sci Total Environ 188:49–58.

Sabbioni E, Pozzi G, Devos S, et al. (1993). The intensity ofvanadium(V)-induced cytotoxicity and morphological transformationin BALB/3T3 cells is dependent on glutathione-mediated bioreductionto vanadium(IV). Carcinogenesis 14:2565–8.

Sakai A. (2008). BALB/c 3T3 cell transformation assays for theassessment of chemical carcinogenicity. AATEX 14:367–73.

Sheu CW, Rodriguez I, Lee JK. (1992). Proliferation and morphologicaltransformation of BALB/3T3 cells by a prolonged treatment withsodium orthovanadate. Fd Chem Toxic 30:307–11.

Shi X, Jiang H, Mao Y, et al. (1996). Vanadium(IV)-mediated freeradical generation and related 20-deoxyguanosine hydroxylation andDNA damage. Toxicology 106:27–38.

Soares SS, Gutierrez-Merino C, Aureliano M. (2007a). Decavanadateinduces mitochondrial membrane depolarization and inhibits oxygenconsumption. J Inorg Biochem 101:789–96.

Soares SS, Gutierrez-Merino C, Aureliano M. (2007b). Mitochondria asa target for decavanadate toxicity in Sparus aurata heart. AquatToxicol 83:1–9.

Soares SS, Martins H, Duarte RO, et al. (2007c). Vanadium distribution,lipid peroxidation and oxidative stress markers upon decavanadatein vivo administration. J Inorg Biochem 101:80–8.

Soazo M, Garcia GB. (2007). Vanadium exposure through lactationproduces behavioral alterations and CNS myelin deficit in neonatalrats. Neurotoxicol Teratol 29:503–10.

Tang H, Sun Y, Xiu Q, et al. (2007). Cyclooxygenase-2 inductionrequires activation of nuclear factor of activated T-cells in Beas-2Bcells after vanadium exposure and plays an anti-apoptotic role. ArchBiochem Biophys 468:92–9.

Thompson KH, Orvig C. (2004). Vanadium compounds in the treatmentof diabetes. Met Ions Biol Syst 41:221–52.

Thompson KH, Lichter J, LeBel C, et al. (2009). Vanadium treat-ment of type 2 diabetes: a view to the future. J Inorg Biochem 103:554–8.

Todorich B, Olopade JO, Surguladze N, et al. (2011). The mechanismof vanadium-mediated developmental hypmyelination is related todestruction of oligodendrocyte progenitors through a relationship withferritin and iron. Neurotox Res 19:361–73.

Tsiani E, Fantus IG. (1997). Vanadium compounds biological actionsand potential as pharmacological agents. Trends Endocrinol Metab8:51–8.

Wang Q, Liu TT, Fu Y, et al. (2010). Vanadium compounds discriminatehepatoma and normal hepatic cells by differential regulation ofreactive oxygen species. J Biol Inorg Chem 15:1087–97.

WHO. (2000). Vanadium. In: WHO air quality guidelines for Europe.European Series. 2nd ed. World Health Organization, WHO RegionalPublications, Vol. 91.

Wozniak K, Blasiak J. (2004). Vanadyl sulfate can differentlydamage DNA in human lymphocytes and HeLa cells. Arch Toxicol78:7–15.

Yasuda M, Nishizawa T, Ohigashi H, et al. (2009). Linoleic acidmetabolite suppresses skin inflammation and tumor promotion inmice: possible roles of programmed cell death 4 induction.Carcinogenesis 30:1209–16.

Ye J, Ding M, Leonard SS, et al. (1999a). Vanadate induces apoptosis inepidermal JB6 Pþ cells via hydrogen peroxide-mediated reactions.Mol Cell Biochem 202:9–17.

Ye J, Ding M, Zhang X, et al. (1999b). Induction of TNFalpha inmacrophages by vanadate is dependent on activation of transcriptionfactor NF-kappaB and free radical reactions. Mol Cell Biochem 198:193–200.

Yu D, Walters DM, Zhu L, et al. (2011). Vanadium pentoxide (V2O5)induced mucin production by airway epithelium. Am J Physiol LungCell Mol Physiol 301:L31–9.

Zhang Z, Huang C, Li J, et al. (2001). Vanadate-induced cell growthregulation and the role of reactive oxygen species. Archiv BiochemBiophys 392:311–20.

Zhang Z, Chen F, Huang C, et al. (2002a). Vanadate induces G2/M phasearrest in p53-deficient mouse embryo fibroblasts. J Environ PatholToxicol Oncol 21:223–31.

DOI: 10.3109/15376516.2013.843110 An in vitro evaluation of vanadium toxic effects 11

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.

Zhang Z, Huang C, Li J, et al. (2002b). Vanadate-induced cell growtharrest is p53-dependent through activation of p21 in Cl41 cells. J InorgBiochem 89:142–8.

Zhang Z, Leonard SS, Huang C, et al. (2003). Role of reactiveoxygen species and MAPKs in vanadate-induced G2/M phase arrest.Free Radic Biol Med 34:1333–42.

Zhong BZ, Gu ZW, Wallace WE, et al. (1994). Genotoxicity of van-adium pentoxide in Chinese hamster V79 cells. Mutat Res 321:35–42.

Zwolak I. (2013). Comparison of five different in vitro assays forassessment of sodium metavanadate cytotoxicity in Chinese hamsterovary cells (CHO-K1 line). Toxicol Ind Health [Epub ahead of print].doi:10.1177/0748233713483199.

12 I. Zwolak Toxicol Mech Methods, 2014; 24(1): 1–12

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Uni

vers

ity o

f M

ontr

eal o

n 12

/02/

14Fo

r pe

rson

al u

se o

nly.