Impact of hexavalent chromium on

mammalian cell bioenergetics: phenotypic

changes, molecular basis and potential
relevance to chromate-induced lung cancer

P. L. Abreu, L. M. R. Ferreira,
M. C. Alpoim & A. M. Urbano

BioMetals
An International Journal on the Role of
Metal Ions in Biology, Biochemistry and
Medicine

ISSN 0966-0844

Biometals
DOI 10.1007/s10534-014-9726-7

1 23
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1 23
 Author's personal copy
Biometals
DOI 10.1007/s10534-014-9726-7
Impact of hexavalent chromium on mammalian cell
bioenergetics: phenotypic changes, molecular basis
and potential relevance to chromate-induced lung cancer
P. L. Abreu • L. M. R. Ferreira • M. C. Alpoim
A. M. Urbano
Received: 29 October 2013 / Accepted: 6 March 2014
Ó Springer Science+Business Media New York 2014
Abstract Occupational exposure to hexavalent
chromium [Cr(VI)] has been firmly associated with
the development of several pathologies, notably lung
cancer. According to the current paradigm, the evo-
lution of normal cells to a neoplastic state is accom-
panied by extensive metabolic reprogramming,
namely at the level of energy-transducing processes.
Thus, a complete understanding of the molecular basis
of Cr(VI)-induced lung cancer must encompass the
elucidation of the impact of Cr(VI) on metabolism.
Research in this area is still in its infancy. Nonetheless,
Cr(VI)-induced metabolic phenotypes are beginning
to emerge. Specifically, it is now well documented that
Cr(VI) exposure inhibits respiration and negatively
P. L. Abreu  M. C. Alpoim  A. M. Urbano (&)
Unidade de Quı́mica-Fı́sica Molecular and Departamento
de Ciências da Vida (Bioquı́mica), Faculdade de Ciências
e Tecnologia, Universidade de Coimbra, Rua dos Estudos,
3001-401 Coimbra, Portugal
e-mail: amurbano@ci.uc.pt
L. M. R. Ferreira
Department of Molecular and Cellular Biology, Harvard
University, Cambridge, MA, USA
M. C. Alpoim  A. M. Urbano
Centro de Investigação em Meio Ambiente, Genética e
Oncobiologia (CIMAGO), Faculdade de Medicina,
Universidade de Coimbra, Coimbra, Portugal
M. C. Alpoim
Centro de Neurociências e Biologia Celular, Coimbra,
Portugal
•
affects the cellular energy status. Furthermore,
preliminary results suggest that it also upregulates
glucose uptake and lactic acid fermentation. From a
mechanistic point of view, there is evidence that
Cr(VI) exposure can interfere with energy transducing
pathways at different levels, namely gene expression,
intracellular protein levels and/or protein function.
Loss of thiol redox control likely plays a key role in
these processes. The transcriptional networks that
control energy transduction can likewise be affected.
Data also suggest that Cr(VI) exposure might com-
promise energy transducing processes through
changes in the intracellular pools of their substrates.
This article reviews, for the first time, the information
available on Cr(VI) impact on mammalian cell
bioenergetics. It aims to provide a framework for the
understanding of the role played by bioenergetics in
Cr(VI)-induced carcinogenesis and is also intended as
a guide for future research efforts in this area.
Keywords Hexavalent chromium  Chromate-
induced lung cancer  Energy metabolism 
Warburg effect  Oxidative stress
Abbreviations
BDH b-Hydroxybutyrate dehydrogenase
Cr(III) Trivalent chromium
Cr(IV) Tetravalent chromium
Cr(V) Pentavalent chromium
Cr(VI) Hexavalent chromium
DCFH Dichlorodihydrofluorescein
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DHR Dihydrorhodamine and refractory industries. Initially used as pigments,

EC Energy charge their utilization greatly intensified after the discovery
ETC Electron transport chain that their inclusion in alloys confers hardness and
FDG-PET 18-Fluorodeoxyglucose positron resistance to corrosion (Barceloux 1999). Occupa-
emission tomography tional exposure to these compounds is associated with
G6PDH Glucose-6-phosphate dehydrogenase a variety of adverse health effects to the skin and
GAPDH Glyceraldehyde-3-phosphate respiratory tract. Most notably, it has been firmly
dehydrogenase established that they are carcinogenic to humans, as
GPx Glutathione peroxidase encountered in certain industries (IARC 1990). In
GSH Reduced glutathione addition, widespread environmental Cr(VI) contami-
GSR Glutathione reductase nation, due mostly to industrial waste disposal, fossil
GST Glutathione S-transferase fuel combustion, steel production and, possibly,
KGDH a-Ketoglutarate dehydrogenase tobacco smoke (Urbano et al. 2008), may pose a
LDH Lactate dehydrogenase NHBE Normal carcinogenic risk to the general population.
human bronchial epithelial According to the prevailing paradigm, cancer
MCAD Medium-chain acyl-CoA dehydrogenase results from the sequential acquisition of mutations
MDH Malate dehydrogenase in genes whose protein products govern cell prolifer-
NADPH Nicotinamide adenine dinucleotide ation and death (Fearon and Vogelstein 1990; Hahn
phosphate and Weinberg 2002). Genomic instability (Hanahan
OCR Oxygen consumption rate and Weinberg 2011) and faulty DNA repair (Urbano
OXPHOS Oxidative phosphorylation et al. 2011) are driving forces of this process. Cr(VI)
PDH Pyruvate dehydrogenase itself interacts poorly with DNA and other biomole-
PDK2 Pyruvate dehydrogenase kinase isoform cules, but several of the species formed in the course of
2 its intracellular reduction are very reactive towards
3-PGK 3-Phosphoglycerate kinase them (Salnikow and Zhitkovich 2008). Among these
PGM Phosphoglucomutase reactive species are trivalent chromium [Cr(III)],
PK Pyruvate kinase which is the final product of Cr(VI) reduction, the
PPP Pentose phosphate pathway unstable pentavalent [Cr(V)] and tetravalent [Cr(IV)]
Prx Peroxiredoxin reduction intermediates, reductant-specific thiyl and
ROS Reactive oxygen species carbon-based radicals and, possibly, reactive oxygen
SCO2 Synthesis of cytochrome c oxidase 2 species (ROS) (‘‘Molecular basis’’ section). Alto-
SOD Superoxide dismutase gether, they induce a wide spectrum of DNA lesions
TCA Tricarboxylic acid (adducts, crosslinks, oxidized bases, abasic sites, gaps
TPI Triosephosphate isomerase and breaks) and other genetic defects (sister chromatid
Trx Thioredoxin exchanges, microsatellite instability, micronuclei and
Trx1 Cytosolic thioredoxin chromosomal aberrations) (Urbano et al. 2008). The
Trx2 Mitochondrial thioredoxin observation that TP53 is disrupted in lung cancers
TrxR Thioredoxin reductase from men with occupational exposure to Cr(VI)
TrxR1 Cytosolic thioredoxin reductase (chromate workers) (Harty et al. 1996; Kondo et al.
TrxR2 Mitochondrial thioredoxin reductase 1997) (‘‘Impact on potential transcriptional mediators
Cr(VI) effects’’ section) is of note, as p53, the protein
product of this gene, is a transcription factor tradi-
tionally seen as the guardian of the genome (Lane
1992). The detection of microsatellite instability in
The carcinogenicity of hexavalent chromium these cancers (Hirose et al. 2002) is in line with the
[Cr(VI)]: basic information reported repression of DNA repair genes (Ali et al.
2011), an effect that was also observed in cultured
Hexavalent chromium [(Cr(VI)] compounds (chro- cells exposed to this carcinogen (Rodrigues et al.
mates) are extensively used in chemical, metallurgical 2009; Permenter et al. 2011).

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Cr(VI) influence on the type of energy metabolism
adopted by mammalian cells (Table 1)
In the 1920s, seminal studies by Otto Warburg
revealed an unexpected metabolic peculiarity of
cancer cells: a strong reliance on lactic acid fermen-
tation for the provision of metabolic energy, even in
the presence of ample oxygen (Warburg 1930). The
strong reliance of cancer cells on ‘‘aerobic glycoly-
sis’’, a rather inefficient process of energy generation,
together with the high energetic demands of contin-
uous proliferation (Kilburn et al. 1969), translate into
an avid consumption of glucose and other glycolytic
substrates, a phenotype ingeniously exploited in the
imaging technique 18-fluorodeoxyglucose positron
emission tomography (FDG-PET) (Fletcher et al.
2008).
The shift to a more fermentative metabolism under
aerobic conditions, which came to be known as the
Warburg effect, is part of an extensive metabolic
reprogramming that incipient cancer cells undergo as
they progress into a fully transformed state (Ferreira
2010). Intriguingly, at least part of this process appears
vital to the progressive adaptation of these incipient
cancer cells to the selective pressures that they will
face from an increasingly hostile microenvironment.
Largely ignored for many decades, metabolic repro-
gramming is now gaining general acceptance as a
major requirement for neoplastic transformation
(Hanahan and Weinberg 2011).
In mammalian cells, lactate production and oxygen
consumption are parameters commonly used to assess
the relative contributions of, respectively, fermenta-
tion and respiration to overall ATP generation. To this
day, the effects of Cr(VI) on both fermentation and
respiration were investigated in three studies only
(Goncalves et al. 2011; Ferreira et al. 2011; Cerveira
et al. 2013) (Table 1). Significantly, exposure to
Cr(VI) stimulated lactate production and inhibited
respiration in all three studies, despite the use of
different exposure regimens and/or cell types (cell
lines derived from a pheochromocytoma of the rat
adrenal medulla (PC-12) and from normal human
bronchial epithelium (BEAS-2B)). Two of these
studies also assessed Cr(VI) effects on glucose uptake
(Goncalves et al. 2011; Ferreira et al. 2011), and both
reported upregulation. Taken together, these changes
point towards a shift to a more substrate-demanding
energy metabolism.
The impact of Cr(VI) on respiration was assessed in
eleven additional studies, using a wide variety of
systems: rat kidney, heart and liver mitochondria
(Molina-Jijon et al. 2011; Ryberg and Alexander 1990,
1984; Fernandes et al. 2002; Lazzarini et al. 1985;
Garcia-Nino et al. 2013), rat hepatocytes (Ryberg and
Alexander 1984), rat thymocytes (Lazzarini et al.
1985), human gingival fibroblasts (Messer and Lucas
1999; Messer et al. 2000), normal human foreskin
fibroblasts (Liu et al. 2010), Chinese hamster lung
fibroblasts (V79 cells) (Andreoli et al. 1991) and
human embryonic hepatocytes (L-02 cells) (Xiao et al.
2012a) (Table 1). Inhibition of respiration was, once
again, a consistent outcome. The strength of the
inhibition and the time of exposure required to
produce it were, however, dependent on the experi-
mental conditions adopted. For instance, in one of the
studies, the oxygen consumption rate (OCR) of
isolated mitochondria was reduced immediately after
Cr(VI) addition, whereas 30 min of incubation were
required to produce a similar effect in intact cells
(Ryberg and Alexander 1984).
Cr(VI) impact on the cellular energy status
of mammalian cells (Table 2)
If insufficiently compensated by fermentation, inhibi-
tion of respiration by Cr(VI) might unbalance the
nucleotide pools and, ultimately, the cellular energy
status. Assessment of the impact of Cr(VI) on these
two parameters is of utmost importance, as they can be
taken as indicators of the physiological status of the
cell. Specifically, high adenylate energy charges (EC)1
(Atkinson 1968) signal the slowdown of metabolism,
while low ECs indicate its upregulation (Berg et al.
2012). The results of the studies carried out over the
last three decades are summarized in Table 2.
Significantly, in all but one of thirteen reported
studies, Cr(VI) treatment had a negative effect on the
intracellular ATP levels and/or on the overall energy
charge. The strength of the effect depended on the
experimental conditions, namely on exposure regimen
(Cr(VI) concentration and duration of the insult) and
model system (baby hamster kidney fibroblasts (BHK
1
EC = ([ATP] ? ‘[ADP])/[ATP] ? [ADP] ? [AMP]).
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Table 1 Cr(VI) influence on the type of energy metabolism adopted by mammalian cells
Parameter Systema Exposure regimen Effect Study
analyzed
Cr(VI) dose/ Duration
concentrationb

Glucose uptake BEAS-2B cells 0.1 and 1 lM 1–6 weeks Up-regulation Ferreira et al. (2011)
PC-12 cells 1, 2 and 5 lM 6h Goncalves et al.
(2011)
Lactate BEAS-2B cells 1 lM 48 h Up-regulation Cerveira et al. (2013)
production 0.1 and 1 lM 1–6 weeks Ferreira et al. (2011)
PC-12 cells 1, 2 and 5 lM 6h Goncalves et al.
(2011)
Oxygen Rat kidney 15 mg/kg, subcutaneous 48 h mOCR down- Molina-Jijon et al.
consumption mitochondria injection regulation (2011)
Rat liver 24 and 48 h Garcia-Nino et al.
mitochondria (2013)
0.0050–1,000 lM 5 min pre-incubation Fernandes et al. (2002)
0.01–0.5 mM 10 min pre-incubation Lazzarini et al. (1985)
20–3,600 lM Added to the O2 electrode Ryberg and Alexander
chamber (1984)
Rat heart 10–50 lM 3 min pre-incubation Ryberg and Alexander
mitochondria (1990)
BEAS-2B cells 1 lM 48 h cOCR down- Cerveira et al. (2013)
0.1 and 1 lM 1–6 weeks regulation Ferreira et al. (2011)
L-02 cells 4, 8, 16 and 32 lM 24 h Xiao et al. (2012a, b)
PC-12 cells 1, 2 and 5 lM 6h Goncalves et al.
(2011)
Normal human 20–500 lM Added to the assay mixture Liu et al. (2010)
fibroblasts
from infant
foreskin
Human gingival 2.5 lM 72 h Messer et al. (2000)
fibroblasts 6.7 lM 24 h
0.25–135 lM 24 and 72 h Messer and Lucas
(1999)
V79 cells 3 lM Added to the assay mixture Andreoli et al. (1991)
and 24 h
Rat thymocytes 0.125–1 mM 10 min pre-incubation Lazzarini et al. (1985)
Rat hepatocytes 4.7–267 lM 15 min pre-incubation Ryberg and Alexander
(1984)

mOCR oxygen consumption rate of isolated mitochondria, cOCR oxygen consumption rate of intact cells
a
BEAS-2B, cell line derived from normal human bronchial epithelium; L-02, cell line derived from normal human embryonic liver; PC-12, cell
line derived from a pheochromocytoma of the rat adrenal medulla; V79, cell line derived from lung tissue of a normal Chinese hamster
b
Added as a K2Cr2O7 or Na2CrO4 aqueous solution

cells) (Bianchi et al. 1980; Debetto et al. 1982; Bianchi
et al. 1982), rat thymocytes (Lazzarini et al. 1985),
V79 cells (Andreoli et al. 1991), rat hepatocytes
(Afolaranmi et al. 2011), PC-12 cells (Goncalves et al.
2011), L-02 cells (Xiao et al. 2012a; Yuan et al. 2012),
human gingival fibroblasts (Messer and Lucas 1999),
BEAS-2B cells (Ferreira et al. 2011; Cerveira et al.
2013) and kidney mitochondria of Cr(VI)-exposed rats
(Molina-Jijon et al. 2011). In two of the above-
mentioned studies, the impact of Cr(VI) on the
guanylate pools was also investigated, yielding results
similar to those obtained for the corresponding
adenylate pools (Bianchi et al. 1982; Lazzarini et al.
1985).

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Table 2 Impact of Cr(VI) on the cellular energy status of mammalian cells

Parameter Systema Exposure regimen Effect Study
analyzed
Cr(VI) dose/ Duration
concentrationb

Intracellular BHK cells 100–2,000 lM 15 to 180 min Decrease of ATP Debetto et al. (1982)
nucleotide 15 to 120 min levels Bianchi et al. (1982)
levels
68 and 680 lM 2h Bianchi et al. (1980)
V79 cells 3, 100 and 1,000 lM Andreoli et al. (1991)
L-02 cells 8, 16 and 32 lM 12 and 36 h Increase of ATP Yuan et al. (2012)
levels
24 h Decrease of ATP
levels
4, 8, 16 and 32 lM Decrease of ATP Xiao et al. (2012a, b)
Human gingival 0.25–135 lM 24 and 72 h levels Messer and Lucas
fibroblasts (1999)
BEAS-2B cells 1 lM 48 h Cerveira et al. (2013)
0.1 and 1 lM 1 to 6 weeks Ferreira et al. (2011)
Rat thymocytes 125–1,000 lM 10 min pre- Lazzarini et al.
incubation (1985)
Rat hepatocytes 50–250 lM (as CrO3) 3h Afolaranmi et al.
(2011)
Rat kidney 15 mg/kg, subcutaneous 48 h Molina-Jijon et al.
mitochondria injection (2011)
Rat thymocytes 125–1,000 lM 10 min pre- Increase of ADP Lazzarini et al.
incubation levels (1985)
BHK cells 100–2,000 lM 15 to 120 min Bianchi et al. (1982)
68 and 680 lM 2h Bianchi et al. (1980)
Rat thymocytes 125–1,000 lM 10 min pre- Increase of AMP Lazzarini et al.
incubation levels (1985)
BHK cells 100–2,000 lM 15 to 120 min Bianchi et al. (1982)
68 and 680 lM 2h Bianchi et al. (1980)
Rat thymocytes 125–1,000 lM 10 min pre- Decrease of GTP Lazzarini et al.
incubation levels (1985)
BHK cells 100–2,000 lM 15–120 min Bianchi et al. (1982)
Rat thymocytes 125–1,000 lM 10 min pre- Increase of GDP Lazzarini et al.
incubation levels (1985)
BHK cells 100–2,000 lM 15–120 min Bianchi et al. (1982)
Rat thymocytes 125–1,000 lM 10 min pre- Increase of GMP Lazzarini et al.
incubation levels (1985)
BHK cells 100–2,000 lM 15–120 min Bianchi et al. (1982)
Energy charge BEAS-2B cells 0.1 and 1 lM 1–6 weeks Decrease of energy Ferreira et al. (2011)
PC-12 cells 1, 2 and 5 lM 6h charge Goncalves et al.
(2011)
V79 cells 3, 100 and 1,000 lM 2h Andreoli et al. (1991)
Rat thymocytes 125–1,000 lM 10 min pre- Lazzarini et al.
incubation (1985)
BHK cells 100–2,000 lM 15–120 min Bianchi et al. (1982)
a
BEAS-2B, cell line derived from normal human bronchial epithelium; BHK, cell line derived from the kidneys of Syrian hamsters; L-02, cell
line derived from normal human embryonic liver; PC-12, cell line derived from a pheochromocytoma of the rat adrenal medulla; V79, cell line
derived from lung tissue of a normal Chinese hamster
b
Added as a K2Cr2O7 or Na2CrO4 aqueous solution, unless otherwise specified

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Cr(VI)-induced changes in the activities of proteins
directly involved in energy transduction
The alterations in bioenergetic fluxes discussed in
‘‘Cr(VI) influence on the type of energy metabo-
lism adopted by mammalian cells’’ section may be
the result of changes in the activities of proteins
directly involved in the relevant pathways, changes
in substrate availability, or both. Concerning sub-
strate availability, it was suggested that Cr(VI)-
induced impairment of respiration might be due to
depletion of NADH and NAD-linked substrates
(e.g., glutamate and citrate) due to their oxidation
by Cr species (Ryberg and Alexander 1984, 1990).
The findings that intermediate Cr(V) was capable
of oxidizing an equimolar amount of NADH in a
few seconds (Ryberg and Alexander 1990) and that
the NADH pool was depleted in Cr(VI)-treated
BHK cells (Bianchi et al. 1982) and isolated rat
heart mitochondria (Ryberg and Alexander 1990)
gave some support to this hypothesis. The observed
inhibition of mitochondrial dehydrogenases
(‘‘Effects on specific enzyme activities’’ section)
might also contribute to the depletion of NADH
pool. It must, however, be borne in mind that,
in vivo, Cr(VI) partitions only moderately to the
mitochondria (Wiegand et al. 1986; Rossi et al.
1988).
The following sections will be devoted to the
known Cr(VI) effects on the total activities of proteins
involved in energy transduction. It should be men-
tioned that several of the studies discussed in this
section were carried out at a time when the different
isoforms of most proteins were still unknown, as were
their relative contributions to cancer. In addition, most
of these studies were not specifically addressing
Cr(VI) carcinogenicity. For instance, evidence that
Cr(III)-ATP complexes can behave as competitive
inhibitors for various ATP-dependent enzymes,
including several kinases involved in glycolysis, came
from kinetic studies that used Cr(III)-ATP complexes
as probes for the naturally occurring substrates of
these enzymes, i.e., Mg(II)-ATP complexes (‘‘Effects
on specific enzyme activities’’ section). Although
structurally similar (Pauls et al. 1986), Cr(III)-ATP
complexes are extremely inert, whereas their Mg(II)-
ATP analogs, which constitute the major form of
intracellular ATP (Lippard and Berg 1997), are very
labile.
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Biometals
Effects on specific enzyme activities (Table 3)
It had been known since the 1940s that Cr(III), the
final product of Cr(VI) intracellular reduction, can act
as an enzyme cofactor. Specifically, it was shown that
Cr(III), but not any of the plethora of other metal ions
tested, could impart some activity to partially purified
phosphoglucomutase (PGM) (Stickland 1949), a gly-
cogenolytic enzyme. In addition, at suboptimal Mg(II)
concentrations, maximal PGM activity could still be
achieved upon Cr(III) addition (Table 3).
The first systematic investigation into Cr(VI)
influence on enzymatic activity was carried out in
the 1960s (Koutras et al. 1964). In this study,
incubation of human erythrocytes with Cr(VI) inhib-
ited glutathione reductase (GSR), but had no effect on
the activities of any of the other sixteen enzymes
investigated, including all glycolytic enzymes, lactate
dehydrogenase (LDH) and three pentose phosphate
pathway (PPP) enzymes (glucose-6-phosphate dehy-
drogenase (G6PDH), 6-phosphogluconate dehydroge-
nase and transketolase). Inhibition of GSR might be
related to its chromate-reductase activity, as this
inhibition was accompanied by reduction of Cr(VI)
to Cr(III). The chromate-reductase activity of GSR
probably resides in the two adjacent cysteine residues
found in the active site of the enzyme, which are
known to form a disulfide bond upon oxidation
(Schulz et al. 1978). In line with the results of this
investigation with human erythrocytes, the activity of
the glycolytic enzyme glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) remained unchanged upon
exposure of BEAS-2B cells to Cr(VI) (Myers et al.
2011). On the contrary, three glycolytic kinases
[hexokinase, 3-phosphoglycerate kinase (3-PGK)
and pyruvate kinase (PK)] were found to be inhibited
by Cr(III)-ATP complexes (Janson and Cleland 1974a,
b). The activity of G6PDH was also significantly
inhibited when human erythrocytes (Ahmad et al.
2011) and human gingival fibroblasts (Messer and
Lucas 1999) were exposed to Cr(VI). Finally, LDH
activity was found to be stimulated upon Cr(VI)
exposure in male C57BL/6J mice bronchoalveolar
lavage fluid (Tajima et al. 2010) and in rat blood
plasma (Garcia-Nino et al. 2013).
In their 1990 study revealing inhibition of mito-
chondrial respiration by Cr(VI) (‘‘Cr(VI) influence on
the type of energy metabolism adopted by mammalian
cells’’ section), Ryberg and Alexander also determined
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Biometals
the impact of Cr(VI) on the activity of 10 mitochon-
drial enzymes, including five tricarboxylic acid (TCA)
cycle enzymes: citrate synthase, isocitrate dehydroge-
nase (NAD? and NADP? isoforms), a-ketoglutarate
dehydrogenase (KGDH), succinate dehydrogenase
and malate dehydrogenase (MDH) (Ryberg and Alex-
ander 1990). The only three enzymes affected were all
dehydrogenases [KGDH, pyruvate dehydrogenase
(PDH) and b-hydroxybutyrate dehydrogenase
(BDH)] and all were inhibited. A NAD?-dependent
effect could be ruled out, as a similar inhibition was
observed when an alternative electron acceptor was
used. Oxidative stress might account for the observed
effects, since the activities of the nicotinamide nucle-
otide-linked mitochondrial oxidations are under the
control of the thiol/disulfide pool (Skrede et al. 1965).
Moreover, KGDH was found to be inactivated by
S-glutathionylation in the presence of high levels of
hydrogen peroxide (Applegate et al. 2008). This type
of protein modification, which can be induced under
conditions of redox imbalance (Dalle-Donne et al.
2009), will be further discussed in ‘‘Molecular basis’’
section. GAPDH, which is regulated at its highly
reactive Cys152 residue (Colell et al. 2009), and
enolase 1 are also regulated by reversible S-oxidation/
thiolation reaction under conditions of electrophile-
induced oxidative stress (Ishii and Uchida 2004).
Aconitase is another TCA cycle enzyme found to be
irreversibly inhibited upon Cr(VI) exposure, both in
BEAS-2B cells and in bovine airways, possibly due to
oxidation of its [4Fe-4S]2? active center (Myers et al.
2010).
The effects of Cr(VI) on the five mitochondrial
complexes involved in oxidative phosphorylation
(OXPHOS) have been investigated in a variety of
model systems, both in vivo (in rats (Molina-Jijon
et al. 2011), ex vivo (in bovine airways (Myers et al.
2010), in isolated mitochondria (Fernandes et al. 2002;
Garcia-Nino et al. 2013) and in intact cells (Messer
et al. 2000; Xiao et al. 2012a, b; Myers et al. 2010;
Luciani et al. 1979). The results obtained depended on
the model system employed, but, altogether, they
suggest that complexes I, II and V are more sensitive
to Cr(VI) exposure than complexes III and IV.
Significantly, Cr(VI) had mostly an inhibitory action
on these complexes, which could account for the
inhibition of respiration upon Cr(VI) exposure
(‘‘Cr(VI) influence on the type of energy metabolism
adopted by mammalian cells’’ section). However,
much further work will be needed to explore these
possibilities.
Effects on intracellular protein levels and gene
expression (Table 4)
There are several reports of changes in intracellular
protein levels upon Cr(VI) exposure, as summarized in
Table 4. In a rat lung epithelial cell line, the intracel-
lular levels of over 30 proteins were found to be altered
upon Cr(VI) treatment (Lei et al. 2008). Upregulated
proteins included the glycolytic enzymes GAPDH,
3-PGK isoform 1, enolase and the M2 isoform of PK.
On the contrary, two enzymes involved in aerobic
respiration, NADH2 dehydrogenase and enoyl-coen-
zyme A hydratase, were downregulated. Increased
GAPDH protein levels upon Cr(VI) treatment were
also observed in BEAS-2B cells (Cerveira et al. 2013),
whereas those of the catalytic subunit (subunit b) of
the mitochondrial H?-ATP synthase (b-F1ATPase)
were decreased (Cerveira et al. 2013) and those of
aconitase remained unchanged (Myers et al. 2010). In
another study that used FFC (osteoblast) and U937
(monocyte) cells, two-dimensional gel electrophoresis
revealed time, concentration and cell type-dependent
changes in the intracellular levels of two glycolytic
enzymes, enolase 1 and triosephosphate isomerase
(TPI) isoform 1 (Raghunathan et al. 2010). In FCC
cells, enolase 1 was upregulated after the first week of
exposure, while the levels of the same enzyme were
significantly reduced in U937 cells by the end of a
3-week exposure. Levels of TPI were also found to be
downregulated in U937 cells after 3 weeks of expo-
sure. Finally, the levels of fructose-1,6-bisphosphatase
1, a gluconeogenic enzyme, and of MDH were
increased upon exposure of female nude mice to
Cr(VI) (Pan et al. 2012). This latter enzyme is a TCA
cycle enzyme with recognized importance in hepatic
gluconeogenesis.
Upregulation of some of the above-mentioned
proteins has been recognized as critical for malig-
nancy. Namely, increased levels of GAPDH protein
were associated with in vitro malignant transforma-
tion, being essential for anchorage-independent
growth and ATP synthesis of transformed cells (Yun
et al. 2011), as well as with tumor aggressiveness and
poor patient prognosis in several cancers (Colell et al.
2009). The isoform 1 of the glycolytic enzyme 3-PGK
is strongly expressed in over 70 % of pancreatic ductal
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 Table 3 Effects of Cr(VI) on the specific activities of proteins directly involved in bioenergetics
Protein Systema Exposure regimen Effect Study
b

123
Cr(VI) dose/concentration Duration

PGM Commercial preparation 5 lM–2 mM (as Cr2(SO4)3) Added to the assay Some activity in the absence of its Stickland (1949)
mixture natural co-factor
HK Commercial preparation (from Not specified Added to the assay Inhibition of activity Janson and Cleland
yeast) mixture (1974b)
Phosphoglucose Human erythrocytes 1–100 lg Cr(VI) per mL of 45 min No effect on activity Koutras et al. (1964)
isomerase blood
PFK-1
Aldolase
TPI
GAPDH BEAS-2B cells 12.5, 25 and 50 lM 90 min Myers et al. (2011)
3-PGK Commercial preparation (from Not specified Added to the assay Inhibition of activity Janson and Cleland
yeast) mixture (1974a, b)
2,3- Human erythrocytes 1–100 lg Cr(VI) per mL of 45 min No effect on activity Koutras et al. (1964)
Phosphoglycerate blood
mutase
Enolase
PK Commercial preparation (from Not specified Added to the assay Inhibition of activity Janson and Cleland
rabbit muscle) mixture (1974a, b)
LDH Male C57BL/6 J mice 1,900 and 2,400 lg/kg body 2 days Stimulation of activity Tajima et al. (2010)
bronchoalveolar lavage fluid weight (as CrO3, by
intratracheal instillation)
Author's personal copy

Rat blood plasma 15 mg/kg, sc (subcutaneous 24 and 48 h Garcia-Nino et al.

injection) (2013)
Human erythrocytes 1–100 lg Cr(VI) per mL of 45 min No effect on activity of LDHA Koutras et al. (1964)
blood
PDH Rat liver mitochondria 1–100 lM Added to the assay Inhibition of activity Ryberg and
mixture Alexander (1990)
G6PDH Human erythrocytes 0.1–5.0 mM 1h (Ahmad et al. 2011)
Human gingival fibroblasts 0.25–135 lM 24 and 72 h Messer and Lucas
(1999)
6PGD Human erythrocytes 1–100 lg Cr(VI) per mL of 45 min No effect on activity Koutras et al. (1964)
TK blood
CS Rat liver mitochondria 1–100 lM Added to the assay Ryberg and
mixture Alexander (1990)
Biometals
 Table 3 continued
Protein Systema Exposure regimen Effect Study
Biometals

b
Cr(VI) dose/concentration Duration

Aconitase BEAS-2B cells 25 and 50 lM 3 or 6 h Inhibition of activity Myers et al. (2010)

Bovine airways 0.62 mg/cm2 (as ZnCr) 3h
IDH2 Rat liver mitochondria 1–100 lM Added to the assay No effect on activity Ryberg and
IDH3 mixture Alexander (1990)
SDH
MDH
KGDH Inhibition of activity
BDH
Mitochondrial L-02 cells 4, 8, 16 and 32 lM 24 h Inhibition of activity Xiao et al. (2012a)
complex I 16 and 32 lM Xiao et al. (2012b)
Rat kidney mitochondria 15 mg/kg, sc 48 h Molina-Jijon et al.
(2011)
24 and 48 h Garcia-Nino et al.
(2013)
BEAS-2B mitochondria 25 lM 3h Myers et al. (2010)
Bovine airways 0.62 mg/cm2 (as ZnCr)
Rat liver mitochondria 0–100 lM Added to the assay Fernandes et al.
mixture (2002)
Human gingival fibroblasts 6.7 lM 24 h No effect on activity Messer et al. (2000)
2.5 lM 72 h
Author's personal copy

Mitochondrial L-02 cells 4, 8, 16 and 32 lM 24 h Inhibition of activity Xiao et al. (2012a)

complex II 16 and 32 lM Xiao et al. (2012b)
Rat kidney mitochondria 15 mg/kg, sc 48 h Molina-Jijon et al.
(2011)
BEAS-2B mitochondria 25 lM 3h Myers et al. (2010)
Bovine airways 0.62 mg/cm2 (as ZnCr)
Rat liver mitochondria 0–1,000 lM Added to the assay Fernandes et al.
mixture (2002)
Human gingival fibroblasts 6.7 lM 24 h No effect on activity Messer et al. (2000)
2.5 lM 72 h

123
 Table 3 continued
Protein Systema Exposure regimen Effect Study
b

123
Cr(VI) dose/concentration Duration

Mitochondrial Rat kidney mitochondria 15 mg/kg, sc 48 h Inhibition of activity Molina-Jijon et al.

complex III (2011)
BEAS-2B mitochondria 25 lM 3h No effect on activity Myers et al. (2010)
Rat liver mitochondria 0–1,000 lM Added to the assay Fernandes et al.
mixture (2002)
L-02 cells 4, 8, 16 and 32 lM 24 h (Xiao et al. 2012a)
16 and 32 lM Xiao et al. (2012b)
Mitochondrial Rat kidney mitochondria 15 mg/kg, sc 48 h Inhibition of activity Molina-Jijon et al.
complex IV (2011)
Rat liver mitochondria 0–1,000 lM Added to the assay No effect on activity Fernandes et al.
mixture (2002)
L-02 cells 4, 8, 16 and 32 lM 24 h Xiao et al. (2012a)
16 and 32 lM Xiao et al. (2012b)
Mitochondrial BHK cells 2,000–10,000 lM 1 to 5 h Inhibition of activity Luciani et al. (1979)
complex V (ATP L-02 cells 4 and 8 lM 24 h Stimulation of activity Xiao et al. (2012a)
synthase)
16 lM No effect on activity
32 lM
Rat kidney mitochondria 15 mg/kg, sc 48 h Inhibition of activity Molina-Jijon et al.
(2011)
Rat liver mitochondria 0–1,000 lM 5 min Stimulation of activity Fernandes et al.
Author's personal copy

(2002)
BDH b-hydroxybutyrate dehydrogenase, CS citrate synthase, GAPDH glyceraldehyde-3-phosphate dehydrogenase, G6PDH glucose-6-phosphate dehydrogenase, HK hexokinase,
IDH2 isocitrate dehydrogenase (NADP), IDH3 isocitrate dehydrogenase (NAD), KGDH a-ketoglutarate dehydrogenase, LDH lactate dehydrogenase, MDH malate
dehydrogenase, PDH pyruvate dehydrogenase, PFK-1 6-phosphofructo-1-kinase, 6PGD 6-phosphogluconate dehydrogenase, 3-PGK phosphoglycerate kinase, PGM
phosphoglucomutase, PK pyruvate kinase, SDH succinate dehydrogenase, TPI 1 triose phosphate isomerase 1, TK transketolase
a
BEAS-2B, cell line derived from normal human bronchial epithelium; BHK, cell line derived from the kidneys of Syrian hamsters; L-02, cell line derived from normal human
embryonic liver
b
Added as a K2Cr2O7 or Na2CrO4 aqueous solution, unless otherwise specified
Biometals
 Author's personal copy
Biometals
adenocarcinomas (Hwang et al. 2006). Enolase 1,
another glycolytic enzyme highly expressed in several
tumors, acts as a plasminogen receptor, eliciting an
integrated humoral and cellular response in cancer
cells (Amedei et al. 2013), and helps transformed cells
to escape the apoptotic cascade, allowing for survival
during limited glucose and oxygen availability
through modulation c-Myc levels (the gene that
encodes enolase 1 also encodes the c-Myc binding
protein (MBP-1)) (Sedoris et al. 2010).
An early investigation on the impact of Cr(VI) on
gene expression employed PEPCK, the gene encoding
the gluconeogenic enzyme phosphoenolpyruvate car-
boxykinase, as a model inducible gene (Hamilton et al.
1998). In this investigation, both the basal and inducible
expression of PEPCK increased in the liver of chick
embryos, whereas basal expression decreased in rat
hepatoma H4IIE cells. The influence of Cr(VI) exposure
on gene expression was confirmed in several subsequent
studies. In Cr(VI)-treated cultures of the A549 human
lung carcinoma cell line, for instance, out of the 2,400
genes analyzed in a high-density oligonucleotide array,
70 were found downregulated and 150 upregulated (Ye
and Shi 2001). Among the latter were three that encode
proteins directly involved in bioenergetics: pyruvate
dehydrogenase kinase isoform 2 (PDK2), medium-
chain acyl-CoA dehydrogenase (MCAD) and ATP
synthase subunit C. PDK2 represses PDH and, conse-
quently, decreases the influx of carbon into the TCA
cycle and attenuates OXPHOS (Berg et al. 2012).
MCAD catalyzes the first step of b-oxidation and ATP
synthase subunit C is the main transmembrane subunit
of the mitochondrial H?-ATP synthase (Berg et al.
2012). In BEAS-2B cells, expression of LDHA varied
inconsistently over a 12-passage exposure to Cr(VI).
This gene encodes lactate dehydrogenase A (LDHA),
the enzyme that catalyzes the conversion of pyruvate to
lactate in the last step of lactic acid fermentation.
Interestingly, LDHA expression was consistently upreg-
ulated in RenG2 cells, a cell strain derived from BEAS-
2B cells upon chronic Cr(VI) exposure, followed by
selection of colony-forming cells (Rodrigues et al.
2009), a well-established procedure to detect tumori-
genic cells (Salmon et al. 1978).
Molecular basis (Tables 5 and 6)
Cr(VI) is essentially viewed as a classic genotoxic
agent and most bioenergetic changes observed upon
Cr(VI) treatment are likely consequences of different
forms of gene disruption (e.g., mutations, amplifica-
tions, deletions, overexpression and epigenetic silenc-
ing). Unfortunately, information on genes affected by
Cr(VI) is still sparse and does not contemplate any
gene encoding proteins directly involved in energy
transduction. There is, though, limited data concerning
well-known transcriptional regulators of energy trans-
duction (‘‘Impact on potential transcriptional media-
tors Cr(VI) effects’’ section).
The mutagenicity of the Cr(III)-DNA adducts
produced upon Cr(VI) exposure is now well estab-
lished (Zhitkovich 2005), but oxidative damage to
DNA is still viewed by many as the critical step in
Cr(VI)-induced genotoxic effects. In fact, it is
believed that oxidative stress makes a substantial
contribution to most Cr(VI)-induced effects. The fact
that peroxidases and other ROS scavengers protect
cells from Cr(VI), as reviewed elsewhere (Myers
2012), gives some support to this hypothesis. Cr(III)-
protein interactions (‘‘Effects on specific enzyme
activities’’ section) are also potential mediators of
some of Cr(VI)-induced bioenergetic effects. By
altering the specific activities of target proteins, these
interactions may disrupt the cellular processes in
which these proteins participate.
Protein carbonylation is a widely-used marker for
oxidative stress (Dalle-Donne et al. 2003). Costa and
collaborators have demonstrated that, in vitro, Cr(VI)
produces some hydrogen peroxide-dependent carbon-
ylation of albumin (Costa et al. 2002), but this type of
protein modification has not been examined in mam-
malian systems exposed to Cr(VI). There is, though,
one study reporting protein carbonylation upon Cr(VI)
treatment in Saccharomyces cerevisiae (Sumner et al.
2005). Interestingly, among the most highly oxidized
proteins were three glycolytic enzymes: aldolase,
enolase and hexokinase (isoforms 1 and 2). Within this
context, it is worth mentioning S-glutathionylation,
another important protein modification that can be
induced under conditions of redox imbalance. This
post-translational modification, which is involved in
the regulation of several cellular processes, consists in
the addition of glutathione to cysteine residues,
preventing the irreversible oxidation of protein thiols
(Dalle-Donne et al. 2009). This and other forms of
oxidation of protein thiols are thought to be major
mechanisms by which oxidative stress impacts on
protein function (Biswas et al. 2006).
123
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Biometals

Table 4 Cr(VI)-induced modulation of gene expression and intracellular levels of proteins directly involved in bioenergetics
Protein Systema Exposure regimen Effect Study
Cr(VI) dose/ Duration
concentrationb

PEPCK 14-day Chick 50 lmol/kg 1–6 h Increased basal and inducible Hamilton
embryo mRNA levels et al. (1998)
H4IIE cells 2 lM 4h Decreased basal mRNA levels
PDK2 Increased mRNA levels Ye and Shi
MCAD A549 cells 300 lM 2h (2001)
ATP synthase Increased mRNA levels of
subunit C
LDHA BEAS-2B cells 1 lM 48 h Decreased mRNA levels of Cerveira
subunit b et al. (2013)
12 passages Increased or decreased mRNA Rodrigues
levels depending on the et al. (2009)
exposure duration
RenG2 subclonal Cell line established and Increased mRNA levels
cellsc propagated in the
presence of Cr(VI)
GAPDH Rat lung 10 lM 24 h Increased protein levels Lei et al.
epithelial cells (2008)
BEAS-2B cells 1 lM 48 h Cerveira
et al. (2013)
3-PGK1 Rat lung 10 lM 24 h Lei et al.
epithelial cells (2008)
Enolase FFC cells 0.5 lM (as 3 weeks Increased Enolase 1 protein Raghunathan
CrO3) levels et al. (2010)
U937 cells Decreased Enolase 1 protein
levels
PKM2 Rat lung 10 lM 24 h Increased protein levels Lei et al.
NADH2 epithelial cells Decreased protein levels (2008)
dehydrogenase
Enoyl-
coenzyme A
hydratase
TPI U937 cells 0.5 lM (as 3 weeks Decreased protein levels Raghunathan
CrO3) et al. (2010)
Aconitase BEAS-2B cells 25 and 50 lM 3 or 6 h Unchanged protein levels Myers et al.
(2010)
FBP 1 Female nude 70 mM 36 h Increased protein levels Pan et al.
MDH mice (ICR— (topical (2012)
Foxn/nu strain) application)
FBP 1 fructose-1,6-bisphosphatase 1, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TPI 1 triose phosphate isomerase 1,
LDHA lactate dehydrogenase A, MCAD medium-chain acyl-CoA dehydrogenase, MDH malate dehydrogenase, PDK2 pyruvate
dehydrogenase kinase isoform 2, PEPCK phosphoenolpyruvate carboxykinase, 3-PGK1 phosphoglycerate kinase 1, PKM2 pyruvate
kinase 2
a
A549, cell line derived from a human lung adenocarcinoma; BEAS-2B, cell line derived from normal human bronchial epithelium;
FFC, cell line derived from a rat immortalized neonatal calvaria; H4IIE, cell line derived from a rat hepatoma; U937, cell line derived
from a human histiocytic lymphoma
b
Added as a K2Cr2O7 or Na2CrO4 aqueous solution, unless otherwise specified
c
Cell line established from a clone that formed when BEAS-2B cells that had been exposed to 1 lM Cr(VI) for 12 passages were
cultured at clonal density

123
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Biometals
Among the targets of S-glutathionylation are sev-
eral glycolytic enzymes and other metabolic proteins,
leading to the proposal that this modification might
coordinate the modulation of cellular metabolism by
oxidative stress. It would, thus, be important to
investigate the potential involvement of this modifi-
cation in the inactivation of metabolic enzymes
observed upon Cr(VI) treatment (‘‘Effects on specific
enzyme activities’’ section). One of the enzymes that
were inactivated upon Cr(VI) exposure, KGDH, was
found to be also inactivated by S-glutathionylation
(Applegate et al. 2008), as already mentioned. How-
ever, it is not yet known whether Cr(VI) treatment
elicits the S-glutathyonylation of KGDH.
Cr(VI) can induce oxidative stress by a variety of
mechanisms, namely through the generation of reac-
tive species during the course of its intracellular
reduction (‘‘The carcinogenicity of hexavalent chro-
mium [Cr(VI)]: basic information’’ section). When
discussing this type of damage, a strong emphasis is
normally placed on ROS and there are, in fact,
numerous reports of elevated ROS levels upon Cr(VI)
exposure (Table 5) (Liu et al. 2010; Xiao et al. 2012a,
b; Lei et al. 2008; Tajima et al. 2010; Kim et al. 2003;
Quinteros et al. 2008; Wang et al. 2006; Patlolla et al.
2009; Gao et al. 2002; Kim and Yurkow 1996; Bagchi
et al. 2001; Fu et al. 2008; Xie et al. 2013). Many of
these reports were accompanied by suggestions that
these reactive species may mediate some of the
cellular responses induced by Cr(VI), namely p53-
dependent cell cycle arrest, apoptosis, activation of
NF-jB and expression of metabolic enzymes (Lei
et al. 2008; Ye and Shi 2001). However, it should not
be forgotten that Cr(V) and Cr(IV) are also strong
oxidants themselves. Namely, it has been shown that
these species are able to generate oxidative damage to
DNA (Slade et al. 2005), possibly through direct
hydrogen abstraction by highly reactive Cr(IV)- and
Cr(V)-peroxo intermediates (Casadevall et al. 1999).
Reports of Cr(V) detection upon Cr(VI) treatment are
also abundant (Borthiry et al. 2008; Liu and Shi 2001;
Liu et al. 1994, 1995; Liebross and Wetterhahn 1992;
Myers et al. 2010, 2000; Jannetto et al. 2001; Ueno
et al. 2001; Sugiyama et al. 1993; Jennette 1982). It
must also be stressed that the methods used for ROS
detection in the above-mentioned studies present some
limitations. Particular important in the context of these
studies is their lack of specificity, being unable to
discriminate between ROS and high valence Cr
species. These methods are based on the oxidation of
the fluorescent probes dihydrorhodamine (DHR) and
dichlorodihydrofluorescein (DCFH) and it is now
known that they can be oxidized by several one-
electron-oxidizing species, namely redox active met-
als in the presence of oxygen or H2O2 (Kalyanaraman
et al. 2012). Importantly, it was demonstrated that
Cr(V) is capable of oxidizing DCFH and DHR and
generate their oxidized products (dichlorofluorescein
(DCF) and rhodamine, respectively) faster than
hydrogen peroxide (Martin et al. 1998). Additionally,
some studies proved that the peroxide-induced reduc-
tion of DCFH is dependent on the intracellular levels
of reduced glutathione (GSH) (Tampo et al. 2003), and
there is evidence that these levels are altered in cells
exposed to Cr(VI) (‘‘Interference with the antioxidant
defense systems’’ section).
Concerning the mechanisms of ROS upregulation,
it has been proposed that Fenton-like reactions
between H2O2 and Cr reactive intermediate species
can generate the hydroxyl radical (Valko et al. 2006).
However, in spite of several studies showing the
participation of Cr(V) and Cr(IV) in these reactions, in
which Cr(V) is continuously recycled into Cr(VI),
further enhancing ROS formation, their occurrence
within the cell is still controversial (O’Brien et al.
2003). Elevated ROS levels can also result from
impairment of respiration, as conditions that compro-
mise electron flow along the electron transport chain
(ETC) strongly increase electron leakage from this
chain and, consequently, production of superoxide
anion and, secondarily, other ROS (e.g., hydrogen
peroxide and hydroxyl radical) (Wallace 2005). In line
with this hypothesis, ROS formation at the level of
complex I was reported in Cr(VI)-exposed L-02 cells
(Xiao et al. 2012b). In turn, overproduction of ROS
may further inhibit respiration through oxidative
damage to the ETC. ROS upregulation can also result
from interference with the cellular antioxidant sys-
tems, which is responsible for scavenging ROS. Data
supporting this hypothesis are discussed in ‘‘Interfer-
ence with the antioxidant defense systems’’ section.
Regardless of the mechanisms involved in its
production, the downstream implications of oxidative
stress must be considered in relation to Cr(VI) impact
on mammalian cell bioenergetics. It was already
mentioned that the activities of certain metabolic
enzymes with a recognized role in carcinogenesis are
under redox control (‘‘Effects on specific enzyme
123
 Table 5 Cr(VI)-induced redox imbalance and interference with the antioxidant defense system
Parameter Systema Exposure regimen Effect Study
analized

123
Cr(VI) dose/ Duration
concentrationb
Redox imbalance

Intracellular L-02 cells 4, 8, 16 and 32 lM 12, 24, 36 and Increase of ROS levels Xiao et al. (2012a)
ROS levels 48 h
16 and 32 lM 24 h Xiao et al. (2012b)
Rat lung epithelial cells 10 lM 1–3 h Lei et al. (2008)
Rat calvarial osteoblasts 10 lM 15–120 min Fu et al. (2008)
Swiss mice liver 25, 50 and 100 mg/kg 1–5 days Wang et al. (2006)
(orally given)
Rat kidney and liver 0–10 mg/kg, ip 5 days Patlolla et al. (2009)
(intraperitonal injection)
Rat anterior pituitary 10 lM 2–8 h Quinteros et al. (2008)
gland
Rat liver mitochondria 12.5, 25, 50 and Added to the Xie et al. (2013)
100 lMc assay mixture
A549 cells 12.5–800 lM 30 min to 6 h Kim et al. (2003)
DU145 cells 2 mM Not specified Gao et al. (2002)
K562 cells 12.5 and 25 lM 24 h Bagchi et al. (2001)
HPBM 24 or 48 h
H4IIE cells 0.1–1,000 lM 20 min Kim and Yurkow (1996)
Author's personal copy

Male C57BL/6 J mice 1,900 and 2,400 lg/kg 2 days Tajima et al. (2010)
lung tissue extracts body weight (as CrO3,
by intratracheal
instillation)
Normal human 0.5–200 lM Added to the Liu et al. (2010)
fibroblasts assay mixture
Biometals
 Table 5 continued
Parameter Systema Exposure regimen Effect Study
Biometals

analized
Cr(VI) dose/ Duration
concentrationb
Redox imbalance

Intracellular Lymphocytes from Occupational exposure Not specified Decrease of GSH levels Quievryn et al. (2001)
GSH levels stainless steel welders
Human erythrocytes 10 and 20 lM Not specified Dlugosz et al. (2012)
1–100 lg Cr(VI) per 45 min Koutras et al. (1964)
mL of blood
Rat thymocytes 1 or 2 lM 60 or 120 min Debetto and Luciani (1988)
Rat liver hepatocytes 50–250 lM (as CrO3) 3h Afolaranmi et al. (2011)
Rat liver homogenate 15 mg/kg, sc 24 and 48 h Garcia-Nino et al. (2013)
and mitochondria (subcutaneous
injection)
LL 24 cells 5–200 lM 2h Increase of GSH levels Dubrovskaya and Wetterhahn
(1998)
Rat anterior pituitary 10 lM 2–8 h Quinteros et al. (2008)
gland
Male C57BL/6 J mice 1,900 and 2,400 lg/kg 2 days Increase of GSH levels in Tajima et al. (2010)
body weight (as CrO3, bronchoalveolar lavage fluid and
by intratracheal decrease in lung lysates
instillation)
FFC cells 0.5 lM (as CrO3) 3 weeks Increase of GSH levels Raghunathan et al. (2010)
U937 cells
Author's personal copy

BEAS-2B cells 5 lM 24 h No effects on GSH levels Myers et al. (2008)

100 lM 30 min
400 lM 10 min
A549 cells 0.5, 1 and 2 lMc 3, 8 and 24 h No statistical effect on GSH levels Caglieri et al. (2008)

123
 Table 5 continued
Parameter Systema Exposure regimen Effect Study
analized

123
Cr(VI) dose/ Duration
concentrationb
Redox imbalance

SOD A549 cells 300 lM 2h Increased mRNA levels Ye and Shi (2001)
L-02 cells 16 and 32 lM 24 h No effect on protein levels Xiao et al. (2012b)
Rat liver homogenate 15 mg/kg, sc 24 and 48 h Inhibition of activity Garcia-Nino et al. (2013)
and mitochondria
Rat kidney 15 mg/kg, ip 48 h Fatima and Mahmood (2007)
15 mg/kg, sc Molina-Jijon et al. (2012, 2011)
Swiss mice liver 25, 50 and 100 mg/kg 1–5 days Wang et al. (2006)
Swiss mice kidney (orally given) No effect on activity
Human erythrocytes 10 and 20 lM Not specified Inhibition of activity Dlugosz et al. (2012)
0.1–5. 0 mM 1h Stimulation of activity Ahmad et al. (2011)
Rat kidney and liver 0–10 mg/kg, ip 5 days Patlolla et al. (2009)
Cat L-02 cells 16 and 32 lM 24 h No effect on protein levels Xiao et al. (2012b)
Human erythrocytes 0.1–5. 0 mM 1h Inhibition of activity Ahmad et al. (2011)
Rat kidney 15 mg/kg, sc 48 h Molina-Jijon et al. (2012, 2011)
15 mg/kg, ip Fatima and Mahmood (2007)
Rat liver homogenate 15 mg/kg, sc 24 and 48 h Garcia-Nino et al. (2013)
and mitochondria
Rat anterior pituitary 10 lM 2–8 h Quinteros et al. (2008)
Author's personal copy

gland
Swiss mice liver 25, 50 and 100 mg/kg, 1–5 days Wang et al. (2006)
Swiss mice kidney (orally given) No effect on activity
Rat kidney and liver 0–10 mg/kg, ip 5 days Stimulation of activity Patlolla et al. (2009)
GSR Human erythrocytes 0.1–5. 0 mM 1h Inhibition of activity Ahmad et al. (2011)
1–100 lg Cr(VI) per 45 min Koutras et al. (1964)
mL of blood
Rat kidney 15 mg/kg, sc 48 h Molina-Jijon et al. (2012, 2011)
Rat liver homogenate 15 mg/kg, sc 24 and 48 h Garcia-Nino et al. (2013)
and mitochondria
Rat hypothalamus 1.92 mM (in drinking 30 days Stimulation of activity Nudler et al. (2009)
water)
Biometals
 Table 5 continued
Parameter Systema Exposure regimen Effect Study
Biometals

analized
Cr(VI) dose/ Duration
concentrationb
Redox imbalance

GPx A549 cells 300 lM 2h Increased mRNA levels Ye and Shi (2001)
BEAS-2B cells 10 lM 4h Decreased mRNA levels Andrew et al. (2003)
Human blood 20 lM Not specified Stimulation of activity Dlugosz et al. (2012)
Human erythrocytes 0.1–5.0 mM 1h Inhibition of activity Ahmad et al. (2011)
Rat kidney 15 mg/kg, sc 48 h Molina-Jijon et al. (2012, 2011)
Rat liver homogenate 15 mg/kg, sc 24 and 48 h Garcia-Nino et al. (2013)
and mitochondria
Rat anterior pituitary 10 lM 2–8 h Quinteros et al. (2008)
gland
Trx L-02 cells 16 and 32 lM 24 h No effect on protein levels Xiao et al. (2012b)
Redox status of BEAS-2B cells 10–180 lM 10–180 min Augmented oxidation of Trx 1 and Myers et al. (2008)
thioredoxin 25 and 50 lM 3h 2 Myers and Myers (2009)
NHBE cells 1.5, 3 and 6 h Myers et al. (2011)
Redox status of BEAS-2B cells 3h Augmented oxidation of Prx-3 and Myers and Myers (2009)
peroxiredoxin NHBE cells 1.5, 3 and 6 h Prx-1 Myers et al. (2011)
TrxR Human erythrocytes 0.1–5.0 mM 1h Inhibition of activity Ahmad et al. (2011)
BEAS-2B cells 25 and 50 lM 0–3 h Myers and Myers (2009)
NHBE cells 25 and 50 lM 1.5, 3 and 6 h Myers et al. (2011)
Author's personal copy

Male C57BL/6 J mice 1,900 and 2,400 lg/kg 2 days Stimulation of activity Tajima et al. (2010)
lung lysates body weight (as CrO3,
by intratracheal
instillation)

123
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Biometals

A549, cell line derived from a human lung adenocarcinoma; BEAS-2B, cell line derived from normal human bronchial epithelium; DU145, cell line derived from a brain
metastasis of a human prostate cancer; FFC, cell line derived from a rat neonatal calvaria; H4IIE, cell line derived from a rat hepatoma; HPBM, normal human donor peripheral
blood mononuclear cells; K562, human chronic myelogenous leukemic cells; L-02, cell line derived from normal human embryonic liver; LL 24, cell line derived from normal
Cat catalase, GPx glutathione peroxidase, GSR glutathione reductase, GST glutathione S-transferase, Prx1 cytosolic peroxiredoxin, Prx3 mitochondrial peroxiredoxin, SOD
activities’’ section), as are the activities of several

Molina-Jijon et al. (2012, 2011)

transcriptional factors involved in metabolic control
(‘‘Impact on potential transcriptional mediators Cr(VI)

Garcia-Nino et al. (2013)

effects’’ section). Importantly, as the ETC is a major

Ahmad et al. (2011)

target of ROS, disruption of redox balance also
impacts on the mitochondrial membrane potential,
which in turn affects ROS production. This disruption
might also affect mitochondrial signaling (Goldenthal
Study

and Marin-Garcia 2004). Figure 1 summarizes some

of these aspects, which will be further discussed in the
sections that follow.

human lung; NHBE, Normal human bronchial epithelial cells; U937, cell line derived from human leukemic monocyte lymphoma cells
Interference with the antioxidant defense systems

The sophisticated antioxidant defense systems devel-

Stimulation of activity
Inhibition of activity

oped by aerobic cells to scavenge excessive ROS and

maintain redox balance comprise both enzymatic and
non-enzymatic components (Fig. 2). The antioxidant
enzymes superoxide dismutase (SOD), catalase and
Effect

different forms of peroxidases catalyze a network of

superoxide dismutase, Trx1 cytosolic thioredoxin, Trx2 mitochondrial thioredoxin, TrxR Trx reductase

reactions that convert ROS into innocuous molecules

(water, molecular oxygen). The efficacy of this
conversion depends upon the availability of GSH
and, ultimately, nicotinamide adenine dinucleotide
24 and 48 h

phosphate (NADPH). GSH supports also the activities

Duration

of the antioxidant enzymes involved in the mainte-

48 h
1h

nance of protein sulfhydryls in the reduced state

Added as a K2Cr2O7 or Na2CrO4 aqueous solution, unless otherwise specified

(Kregel and Zhang 2007) (Fig. 3). As can be appre-

ciated, NADPH is, once again, the ultimate electron
donor. Thus, in terms of redox homeostasis, catabo-
Exposure regimen

lism can be seen as a double-edged sword: it produces

concentrationb

15 mg/kg, sc
15 mg/kg, sc
Cr(VI) dose/

NADPH, but is also the major generator of endoge-

The Cr(VI) compound used was not specified by the authors
0.1–5.0 mM

nous ROS.
Cr(VI) exposure can interfere with the antioxidant
system at various levels, namely due to consumption
of antioxidant molecules in the intracellular reduction
of Cr(VI) and to disruption of the biological actions of
Rat liver homogenate
Human erythrocytes

antioxidant enzymes through interaction of these

and mitochondria

enzymes with species formed during Cr(VI) reduction.

Analysis of lymphocytes from stainless steel weld-
Rat kidney

ers revealed that chronic exposure to Cr(VI) signifi-

Systema

cantly depleted the cellular reservoirs of GSH

(Quievryn et al. 2001). This tripeptide is the most
prevalent free thiol and the major low molecular
Table 5 continued

Redox imbalance

weight recycling thiol-disulfide buffer in most living

cells (Schafer and Buettner 2001; Powis and Montfort
Parameter

2001; Meister and Anderson 1983). Abnormally high

analized

levels of GSH may be indicative of certain patholog-

GST

ical changes (Nair et al. 1991) and it was proposed that

b
a

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 Table 6 Potential transcriptional mediators of the bioenergetic reprogramming induced by Cr(VI)
Transcription factor Systema Exposure regimen Effect Study
Biometals

b
Cr(VI) dose/concentration Duration

c-Myc Male Wistar rat 4.8–24 mM (in drinking 60 days Increased c-Myc transcript Tsao et al. (2011)
water) and protein levels in
stomach and colon cells
BEAS-2B cells 1 lM 12 passages Increased or decreased Rodrigues et al. (2009)
c-Myc RNA levels
depending on the
exposure duration
10 lM 4h Decreased c-Myc RNA Andrew et al. (2003)
levels
RenG2 subclonal cellsc 1 lM Cell line established and Increased c-Myc mRNA Rodrigues et al. (2009)
propagated in the levels
presence of Cr(VI)
10T1/2 mouse embryo Not specified (as PbCrO4) Not specified Increased c-Myc RNA Landolph (1994)
fibroblasts levels
p53 L-02 cells 4, 8 and 16 lM 24 h Increased p53 protein levels Xiao et al. (2012a, b)
Male Wistar rat 4.8–24 mM (in drinking 60 days Decreased p53 transcript Tsao et al. (2011)
water) and protein levels in
stomach and colon cells
IMR90 cells 2, 5 and 10 lM 3h Unchanged p53 protein Reynolds and Zhitkovich
H460 cells levels (2007)
Isolated DNA 0.5–5 lM (as CrCl3) 30 min Formation of adducts and G Arakawa et al. (2006)
to T mutations in –NGG–
Author's personal copy

Mouse dermal fibroblasts 75 lM
Human lung fibroblasts 9 or 200 lM
Serum from former Occupational exposure
chromate workers (1.19–210.10 mg/m3
chromate dust in the work
area)
Lung cancer samples from Occupational exposure
chromate workers
sequences
2h Increased apoptosis in p53 Carlisle et al. (2000b)
wild-type mice
24 and 2 h, respectively Increased p53 protein levels Carlisle et al. (2000a)
Up to 23 years Increased levels of serum Hanaoka et al. (1997)
anti-p53 antibodies levels
8–38 years TP53 mutations in 20 % of Kondo et al. (1997)
the samples
Not specified GC to TA tranversions in Harty et al. (1996)
TP53 coding sequence

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 Table 6 continued
Transcription factor Systema Exposure regimen Effect Study
b

123
Cr(VI) dose/concentration Duration

HIF-1a BEAS-2B cells 1 lM 12 passages Increased or decreased HIF- Rodrigues et al. (2009)
1a RNA levels depending
on the exposure duration
5 lM 8 or 24 h Decreased Ni-induced HIF- Nemec and Barchowsky
1a protein levels (2009)
RenG2 subclonal cellsc 1 lM Cell line established and Increased HIF-1a mRNA Rodrigues et al. (2009)
propagated in the levels
presence of Cr(VI)
1HAEo cells 0–25 lM 1–24 h Increased HIF-1a protein Kaczmarek et al. (2007)
levels and activity
A549 cells 20 lM 4, 8 or 24 h Transient stabilization of Li et al. (2006)
HIF-1a protein
DU145 cells 0.625–100 lM 1–4 h Increased HIF-1a protein Gao et al. (2002)
levels
NF-jB Jurkat cells 2 lM 3h Increased NF-jB activity Shi et al. (1999)
0.02–5 lM Ye et al. (1995)
A549 cells 0–2.5 mM 35 min Decreased NF-jB activity Shumilla et al. (1998)
0, 1, 5 and 20 lMd 2h Shumilla et al. (1999)
a
1HAEo, cell line derived from normal human airway epithelium; A549, cell line derived from a human lung adenocarcinoma; BEAS-2B, cell line derived from normal human
bronchial epithelium; DU145, cell line derived from a brain metastasis of a human prostate cancer; H460, cell line derived from human large cell lung cancer; IMR90, cell line
derived from normal human lung; Jurkat, cell line derived from human leukemic T cells; L-02, cell line derived from normal human embryonic liver
Author's personal copy

b
Added as a K2Cr2O7 or Na2CrO4 aqueous solution, unless otherwise specified
c
Cell line established from a clone that formed when BEAS-2B cells that had been exposed to 1 lM Cr(VI) for 12 passages were cultured at clonal density
d
The Cr(VI) compound used was not specified
Biometals
 Author's personal copy
Biometals

Fig. 1 Potential
mechanisms of Cr(VI)-
induced loss of protein thiol
redox control and
downstream bioenergetic
implications. Cr(III)
trivalent chromium, Cr(IV)
tetravalent chromium, Cr(V)
pentavalent chromium,
Cr(VI) hexavalent
chromium, ETC electron
transport chain, GSH
reduced glutathione,
OXPHOS oxidative
phosphorylation, ROS
reactive oxygen species,
TCA tricarboxilic acid

its intracellular status may be a sensitive indicator of
the overall health of the cells and of their ability to
resist toxic challenge (Nair et al. 1991; Pani et al.
2010).
Decreased intracellular GSH levels upon Cr(VI)
exposure were also observed in human erythrocytes
(Koutras et al. 1964; Dlugosz et al. 2012), rat
thymocytes (Debetto and Luciani 1988), rat liver cells
(Afolaranmi et al. 2011), homogenate and mitochon-
dria (Garcia-Nino et al. 2013), and lung tissue from
male C57BL/6J mice (Tajima et al. 2010). Yet the
intracellular GSH pools of BEAS-2B cells remained
unaltered in two studies, even when much higher
Cr(VI) concentration were used (Myers et al. 2008;
Caglieri et al. 2008). The GSH pool also remained
unaltered in A549 cells (Caglieri et al. 2008). There
are also several reports of increased GSH levels
upon Cr(VI) treatment, namely in FCC and U937
cells (Raghunathan et al. 2009), normal human
lung fibroblasts (LL24 cells) (Dubrovskaya and

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Biometals

Fig. 2 ROS scavenging by

the mitochondrial
antioxidant defense system.
SOD superoxide dismutase,
Cat catalase, GPx
glutathione peroxidase, GSR
glutathione reductase, GSH
reduced glutathione, GSSG
glutathione disulfide, GRX2
glutaredoxin 2, Prx3
mitochondrial
peroxiredoxin, Trx2
mitocondrial thioredoxin,
TrxR2 mitochondrial
thioredoxin reductase

Fig. 3 Maintenance of protein sulfhydryls in the reduced state by mitochondrial thioredoxin (Trx2) and glutaredoxin (GRX2). GSR
glutathione reductase, GSH reduced glutathione, GSSG glutathione disulfide, TrxR2 mitochondrial thioredoxin reductase, Prot protein

Wetterhahn 1998), rat anterior pituitary gland cells
(Quinteros et al. 2008) and bronchoalveolar lavage
fluid from treated male C57BL/6 J mice (Tajima et al.
2010).
Two different groups, using different administra-
tion routes, reported a decrease of SOD and catalase
activities in the kidneys of rats exposed to Cr(VI)
(Fatima and Mahmood 2007; Molina-Jijon et al. 2011;
2012). Again in the kidneys of Cr(VI)-exposed rats,
another group observed a stimulation of these two
activities (Patlolla et al. 2009), but a fourth group
observed no significant alterations (Wang et al. 2006).
There are also reports of both decreased (Wang et al.
2006; Garcia-Nino et al. 2013) and increased SOD and
catalase activities (Patlolla et al. 2009) in the livers of
Cr(VI)-exposed rats or mice.
In Cr(VI)-exposed erythrocytes, the activities of
catalase, glutathione peroxidase (GPx) and GSR were
found to be inhibited, whereas that of SOD was either
stimulated (Koutras et al. 1964; Ahmad et al. 2011) or
inhibited (Dlugosz et al. 2012). Pre-incubation with
ascorbate restored GSR activity (Koutras et al. 1964).
Thus, redox reactions known to occur between this
enzyme and Cr(VI) (Myers et al. 2008) might explain

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the inhibition. Inhibition of GSR activity was also
observed in rat kidney (Molina-Jijon et al. 2011,
2012), whereas the opposite effect was reported in rat
hypothalamus (Nudler et al. 2009). On the other hand,
GPx inhibition was confirmed in rat kidney (Molina-
Jijon et al. 2011, 2012; Ahmad et al. 2011) and rat
anterior pituitary gland (Quinteros et al. 2008), but this
enzyme was found stimulated in total human blood
(Dlugosz et al. 2012). Other Cr(VI) effects include
overexpression of SOD and GPx in A549 cells
exposed to a high Cr(VI) concentration (300 lM)
(Ye and Shi 2001). The opposite effect on GPx
expression was observed with a different cell line
(BEAS-2B) and a lower Cr(VI) concentration
(10 lM) (Andrew et al. 2003). In L-02 cells, no
alteration of SOD, catalase and Trx levels was
observed (Xiao et al. 2012b).
Cr(VI) also interferes with the thioredoxin system,
which comprises the cytosolic (Trx1) and mitochon-
drial (Trx2) forms of Trx, the reductases responsible
for maintaining these two small redox proteins in their
reduced states (TrxR1 and TrxR2, respectively) and
NADPH (Arner and Holmgren 2000). This system has
a major role in the maintenance of the intracellular
thiol-disulfide redox balance and, ultimately, in pro-
moting cell survival (Lu and Holmgren 2013). In
BEAS-2B cells, Cr(VI) caused a dose- and time-
dependent oxidation of both Trx1 and Trx2, as well as
inhibition of TrxR1 and TrxR2 activities (Myers et al.
2008, 2011; Myers and Myers 2009). Similar results
were observed in normal human bronchial epithelial
(NHBE) cells (Myers et al. 2011). A diminution in
thioredoxin reductase (TrxR) activity was also
observed in human erythrocytes (Ahmad et al.
2011), whereas the opposite effect was observed in
male C57BL/6J murine lung lysates (Tajima et al.
2010). These latter studies did not discriminate the two
isoforms of TrxR.
Oxidation of Trx, which causes its inactivation, is
expected to impact the function of Trx-dependent
proteins. Included in this group are the peroxiredoxins
(Prx), whose function is of the utmost importance in
the context of the antioxidant defense system, as they
are one of the major catalysts of hydrogen peroxide
reduction (Fig. 2) (Winterbourn 2008). A dose- and
time-dependent oxidation of Prx1 (cytosolic) and Prx3
(mitochondrial) was reported in both BEAS-2B and
NHBE cells treated with Cr(VI) (Myers and Myers
2009; Myers et al. 2011). The accumulation of
oxidized Prxs has been recently proposed as a
biomarker of oxidative stress (Poynton and Hampton
2014).
Finally, a recent study reported an increase in the
activity of glutathione S-transferase (GST) in Cr(VI)-
exposed human erythrocytes (Ahmad et al. 2011).
GSTs are a family of enzymes that catalyze the
nucleophilic addition of glutathione to electrophilic
acceptors, including toxic products, and it was
proposed that they could play a rate-limiting role in
the reduction of Cr(VI) to Cr(III) (DeFlora and
DeFlora 1989). The results obtained with human
erythrocytes contrast with those obtained in the
kidneys of rats intraperitoneally exposed to Cr(VI)
(Molina-Jijon et al. 2011, 2012).
Impact on potential transcriptional mediators
of Cr(VI) effects
In 1997, a study from Dang’s group (Shim et al. 1997)
challenged the traditional perception of central carbon
metabolism as a homeostatic, self-regulating network
of reactions essentially independent of signaling
pathways (Thompson 2011). This study revealed the
transactivation of a metabolic gene, LDHA, by Myc, a
transcription factor until then only associated with cell
cycle regulation and apoptosis (Zornig and Evan
1996). Later findings showed that Myc also activates
glycolytic and glutaminolytic genes and stimulates
mitochondrial biogenesis (Dang 2012). Altogether,
these findings firmly established Myc as a critical
regulator of central carbon metabolism. Of note, MYC
expression is deregulated in up to 70 % of human
cancers (Dang 2012).
It has recently been suggested that one of the roles
played by Myc may be amplification of gene expres-
sion programs already in place (Lin et al. 2012; Nie
et al. 2012). In the context of this review, it is
remarkable that, in mice engineered to specifically
overexpress MYC in hepatocytes, this overexpression
alone led to increased rates of lactic acid fermentation
and liver cancer initiation. Moreover, the increased
fermentative metabolism required continuous MYC
expression (Hu et al. 2011).
It is now known that the expression of genes related
to central carbon metabolism is also regulated by other
orchestrators of carcinogenesis, such as p53, HIF and
NF-jB (Levine and Puzio-Kuter 2010). Importantly,
the activity of these transcription factors has been
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shown to be modulated by the cellular redox status
(Kabe et al. 2005; Pouyssegur and Mechta-Grigoriou
2006; Liu et al. 2008), which is well known to be
perturbed by Cr(VI) insults (‘‘Molecular basis’’ and
‘‘Interference with the antioxidant defense systems’’
sections). This section summarizes the known effects
of Cr(VI) on the activity of the above-mentioned
transcription factors (Table 6).
Myc Studies on the impact of Cr(VI) exposure on
MYC expression are sparse and the results strongly
dependent on the model system employed. MYC
overexpression upon Cr(VI) exposure was observed
in 10T1/2 mouse embryo fibroblasts (Landolph 1994)
and in RenG2 cells (Rodrigues et al. 2009). In BEAS-
2B cells, there are both reports of downregulation
(Andrew et al. 2003) and random variation over a 12
passage exposure (Rodrigues et al. 2009). Finally,
in vivo experiments found that Cr(VI) treatment
increases MYC expression in stomach and colon in
Wistar rats (Tsao et al. 2011).
p53 p53 activation is best known for triggering cell
cycle arrest and/or apoptosis downstream of DNA
damage (Levine 1997; Vogelstein and Kinzler 2004)
and in response to other cellular stresses, including
hypoxia (Hammond and Giaccia 2005) and acidosis
(Williams et al. 1999). From a metabolic point of view,
p53 is central in controlling the balance between
fermentative and respiratory fluxes (Levine and Puzio-
Kuter 2010). It acts at different levels to repress
glycolysis, namely by preventing allosteric activation
of the glycolytic enzyme 6-phosphofructo-1-kinase
(Bensaad et al. 2006) and through inhibition of AKT
signaling (Feng and Levine 2010). AKT is a protein
kinase that acts downstream of growth factors to
stimulate cell growth. This stimulation is achieved by
fostering protein synthesis (Feng and Levine 2010) and
by augmenting glucose uptake and levels of glycolytic
enzymes (Elstrom et al. 2004). Concomitantly, p53
increases flux through OXPHOS by augmenting levels
of Synthesis of Cytochrome c Oxidase 2 (SCO2)
(Matoba et al. 2006) and through upregulation of
glutaminase 2. SCO2 is a chaperone essential for
assembly of complex IV of the ETC and glutaminase 2
catalyzes the conversion of glutamine into glutamate,
augmenting flux through the TCA cycle and,
ultimately, through OXPHOS (Hu et al. 2010). Thus,
inactivating mutations in TP53, as commonly found in
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tumors (Hollstein et al. 1991), have the potential to
shift cellular metabolism towards lactic acid
fermentation. Consistent with this hypothesis,
overexpression of the oncogene AKT sufficed to
trigger a Warburg effect in cancer cells (Elstrom
et al. 2004). Recent studies found that p53 also hampers
the PPP, which is normally upregulated in cancers to
meet the increased biosynthetic demands for reducing
power and ribose phosphate (Jones and Thompson
2009; Ramos-Montoya et al. 2006). Finally, loss of p53
function during carcinogenesis may preclude apoptosis
in response to hypoxia and acidosis, usually present
in the tumor microenvironment (Pouyssegur et al.
2006), thus allowing for the survival of incipiently
transformed cells.
TP53 is the gene most consistently mutated in
cancer (Hollstein et al. 1991). In the case of lung
cancer, the global mutation rate reaches 60–70 %
(Mitsuuchi and Testa 2002). As most of these muta-
tions are stabilizing mutations, mutant p53 protein
accumulates, triggering the production of anti-p53
polyclonal antibodies to levels detectable in the serum
of lung cancer patients (Lubin et al. 1995). High levels
of pantropic p53 proteins were also detected in the
serum of chromate-exposed workers, most predomi-
nantly in those with a past history of lung cancer
(Hanaoka et al. 1997; Khoury and Bourdon 2010).
However, there is some indication that the rate of
TP53 mutations in chromate lung cancers may be
significantly lower (20 %) than that reported for
common lung cancers (i.e., cigarette smoke related
lung cancers) (Kondo et al. 1997). According to an
epidemiological study, chromate-induced mutations
in TP53 are mostly G:C to T:A transversions in the
coding sequence (Harty et al. 1996), as is the case for
TP53 mutations in common lung cancers (Hollstein
et al. 1991). Cr(III)-DNA and Cr(III)-histidine-DNA
adducts that form upon the intracellular reduction of
Cr(VI) are likely pre-mutagenic, as it was shown, in
in vitro studies, that they occur preferentially in
regions of the TP53 gene often mutated in lung cancer
and do induce mainly G:C to T:A transversions
(Arakawa et al. 2006).
In mice, administration of chromate in the drinking
water caused a visible diminution of p53 transcript and
protein levels in isolated stomach and colon cells
(Tsao et al. 2011). In the same cells, MYC was found to
be upregulated, also at the transcript and protein
levels, constituting, together with p53 loss, a
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molecular signature of gastrointestinal cancer initia-
tion (Tsao et al. 2011). Interestingly, it was reported
that p53-deficient mice are more sensitive to Cr(VI)
oxidative damage than wild-type mice, exhibiting
more extensive lipid peroxidation and DNA damage in
the liver and brain (Bagchi et al. 2001).
Several in vitro studies have focused on the impact
of Cr(VI) on p53 activity, as reviewed elsewhere
(Urbano et al. 2012), which has been related with
redox control (Myers 2012). Patierno and colleagues
reported an up to sixfold increase in p53 protein levels
in human lung fibroblasts upon Cr(VI) treatment
(Carlisle et al. 2000a). These observations are consis-
tent with previous findings by the same group using
the same cells that Cr(VI) treatment elicited DNA
damage repair and cell cycle arrest at the S phase (Xu
et al. 1996), two cellular responses long known to be
caused by p53 upregulation (Levine and Oren 2009).
Later, these investigators reported that Cr(VI) induced
p53-dependent apoptosis in mouse dermal fibroblasts
(Carlisle et al. 2000b). More recently, it was reported
that treatment of human hepatocytes with Cr(VI) led to
p53-mediated cell cycle arrest, OXPHOS impairment
and ATP depletion (Xiao et al. 2012a). By contrast,
another group observed that p53 was not essential for
the induction of cell death in human lung epithelial
cells (H460) and primary human lung fibroblasts
(IMR90) (Reynolds and Zhitkovich 2007).
It is of note that chronic exposure of human foreskin
fibroblasts to Cr(VI) and several rounds of selection of
surviving cells led to the establishment of cell clones
more resistant to Cr(VI) exposure than their untreated
counterparts (Pritchard et al. 2005; Nickens et al.
2012). Curiously, this phenomenon appeared to be
p53-independent, as the surviving cells upregulated
p53 upon Cr(VI) exposure to the same extent of the
parental cells (Pritchard et al. 2005). Work from our
laboratory also provided indirect evidence of such
resistance (Costa et al. 2010). Altogether, these
observations lend support to a model where Cr(VI)-
induced carcinogenesis is initiated by a population of
cells in the respiratory tract that resist Cr(VI) cytotoxic
insults and accumulate genetic mutations that confer
them resistance to apoptosis and other cancer hall-
marks (Carlisle et al. 2000a; Nickens et al. 2012).
The mechanism through which Cr(VI) modulates
p53 activity has not been fully characterized. An
attractive possibility would be that Cr(VI)-induced
DNA damage is fully responsible by changes in p53
expression and activity. However, further studies
strongly suggest that Cr(VI) also impacts p53 function
at the protein level, by altering p53 phosphorylation
status (Wang and Shi 2001). Finally, oxidation of
cysteine residues in p53 differentially affects transac-
tivation of p53-target genes (Liu et al. 2008), poten-
tially establishing a link between Cr(VI)-induced thiol
redox changes and p53 function.
HIF-1a HIF-1a is upregulated in a wide array of
human cancers (Semenza 2003, 2010). Its protein
product, HIF-1a, combines with HIF-1b to form HIF-
1, a transcription factor involved in cellular oxygen
sensing (Semenza 2003, 2010). Under normoxic
conditions, HIF-1a is targeted for degradation by
dedicated prolyl hydroxylases (Bruick and McKnight
2001), preventing HIF-1 subunit assembly and traffic
into the nucleus. This hydroxylation reaction is slowed
down under hypoxic conditions, such as those found in
solid tumors (Vaupel and Harrison 2004). This is due
to both substrate (oxygen) limitation (Jaakkola et al.
2001) and prolyl hydroxylase inhibition by increased
ROS levels (Klimova and Chandel 2008). Some
studies have suggested that, under oxidative stress
conditions resulting from exposure to strong oxidants
such as Cr(VI), HIF-1a is stabilized by p38-mediated
phosphorylation (Gao et al. 2002; Myers 2012).
Once in the nucleus, HIF-1 orchestrates gene
expression to drive metabolic adaptation to hypoxia
(Semenza 2010). Genes involved in glucose uptake,
glycolysis and lactate production are activated, while
augmented expression of pyruvate dehydrogenase
kinase isoform 1 (PDK1) leads to repression of PDH
and, consequently, attenuates the TCA cycle and
OXPHOS (Semenza 2009). Respiration is further
downregulated through HIF-1-induced expression of
BNIP3, which triggers mitochondrial destruction via
autophagy (Zhang et al. 2008). Curiously, p53
deficiency (Ravi et al. 2000) and Myc upregulation
(Shim et al. 1997) also augment HIF-1 activity.
Recently, it was found that Myc, when present in high
levels, cooperates with HIF-1 to produce a Warburg
phenotype in a lymphoma cell line (Kim et al. 2007),
suggesting yet another mechanism to produce a
Warburg effect besides LDHA (Shim et al. 1997)
and AKT (Elstrom et al. 2004) constitutive activity.
Increased ROS production upon Cr(VI) exposure
(‘‘Molecular basis’’ section) suggests that HIF-1
activity might be a target in Cr(VI)-induced
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carcinogenesis. However, experiments testing directly
the impact of Cr(VI) on HIF-1 have failed to produce
consistent results. There is evidence indicating stabil-
ization of HIF-1a protein by Cr(VI) in DU145 human
prostate carcinoma cells (Gao et al. 2002), as well as in
the human lung cell lines 1HAEo- and A549 (Kacz-
marek et al. 2007). It has been proposed that this
stabilization may be achieved via inhibition of the
prolyl hydroxylases whose activity triggers HIF-1a
degradation (Kaczmarek et al. 2007). There is also a
report of increased HIF-1a expression in the malig-
nant RenG2 cell strain, whereas in the parental BEAS-
2B cell line this expression varied randomly over a 12
passage exposure (Rodrigues et al. 2009). Yet, other
experiments did not find any permanent effect of
Cr(VI) treatment on HIF-1a levels or HIF-1a-depen-
dent transcription (Li et al. 2006). There is also one
report of Cr(VI)-induced inhibition of HIF-1 stabil-
ization and binding to DNA (Nemec and Barchowsky
2009).
NF-jB Several studies in the literature have focused
on the effect of Cr(VI) on the NF-jB pathway, as
reviewed elsewhere (Urbano et al. 2012). This
transcription factor participates in various biological
processes, including cell migration and tissue
patterning during development (Bushdid et al. 1998).
It has also been shown to promote inflammation-
associated cancer via cell-autonomous (Cooks et al.
2013) and non-cell-autonomous (Yamamoto et al.
2013) mechanisms. Importantly for the purposes of the
present review, augmented NF-jB activity in p53-
deficient mouse embryonic fibroblasts has been
associated with increased glucose uptake and
glycolytic rates (Kawauchi et al. 2008). Along the
same line, Ras-induced transformation and increased
aerobic glycolysis in a p53 deficiency background
were visibly deterred upon NF-jB depletion. Hence,
NF-jB unchecked activity may participate in the
acquisition of cancer properties during tumorigenesis.
There is evidence from different research groups
that Cr(VI) impacts on NF-jB signaling, but the nature
of this impact has been very dependent on the
experimental system used. Specifically, results by
Hamilton and co-workers showed that the effects of
Cr(VI) and other heavy metals on the levels of NF-jB
activity are very cell type-dependent (Hamilton et al.
1998), while work from other groups collectively
showed that lower doses of Cr(VI) enhance NF-kB
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activity, while higher doses do not (Ye et al. 1995;
Chen et al. 2000, 2002; Wang et al. 2004). Shi and
colleagues showed that Cr(VI) induces NF-jB activity
via oxidative stress in Jurkat cells, a human T cell
leukemia cell line (Shi et al. 1999; Ye et al. 1995).
Further experiments revealed that Cr(VI) treatment
induces significantly more cell death in Jurkat cells
with constitutively blocked NF-jB signaling than in
wild-type cells (Chen et al. 2002). Consistently,
ectopic expression of anti-apoptotic genes partly
rescued NF-jB signaling-deficient Jurkat cells from
Cr(VI)-induced cell death, suggesting that NF-jB
exerts a protective role against Cr(VI) cytotoxicity
(Chen et al. 2002). Pointing in a different direction, the
Barchowski group reported that Cr(VI) inhibits NF-
jB in human lung carcinoma cells (Shumilla et al.
1998, 1999).
NF-jB activity is controlled by, among others,
redox factors. Intracellular ROS lead to phosphoryla-
tion of the NF-jB inhibitory complex IKK, conse-
quently releasing sequestration of NF-jB in the
cytoplasm and allowing it to traffic to the nucleus
and activate transcription of target genes. On the other
hand, an oxidizing environment in the nucleus, mainly
due to the upregulation of nitric oxide downstream of
NF-jB activation, leads to nitrosylation of Cys62 of
the p50 subunit of NF-jB, significantly reducing its
DNA binding affinity (Kabe et al. 2005). Cr(VI) could
therefore affect NF-jB activity by a thiol redox-based
mechanism.
Epigenetic modifications
There are several reports on Cr(VI)-induced epige-
netic modifications that should also be considered
when studying the mechanisms of Cr(VI) carcinoge-
nicity (Arita and Costa 2009). Cr(VI) treatment has
been shown to induce abnormal methylation of the
bacterial xanthine-guanine phosphoribosyl transferase
(gpt) reporter gene in the transgenic Chinese hamster
G12 lung cell line (Klein et al. 2002) and to alter
histone methylation in the A549 cell line, an effect that
was correlated with silencing of the DNA repair gene
hMLH1 (Sun et al. 2009). Epigenetic modifications in
lung cancers from chromate workers include aberrant
DNA methylation and gene silencing of the tumor
suppressor gene p16Ink4a (Kondo et al. 2006) and,
possibly, repression of hMLH1 (Takahashi et al.
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2005). Interestingly, some of these effects are in line
with the observation of microsatellite instability in the
above-mentioned cancers (Hirose et al. 2002). In the
context of the present review, the observed influence
of histone acetylation levels on the expression of genes
directly related to energy metabolism (Wellen and
Thompson 2012) is particularly meaningful. It is, thus,
feasible that this and other forms of epigenetic
deregulation might contribute to the metabolic shift
observed in tumors (Levine and Puzio-Kuter 2010).
Remarkably, the reverse connection also holds true,
i.e., deregulated metabolism can produce epigenetic
aberrations, contributing to its further deregulation
(Luo and Kuo 2009; Gerhauser 2012; Wellen et al.
2009; Wellen and Thompson 2012; Katada et al.
2012). For instance, global levels of histone acetyla-
tion depend on nuclear levels of acetyl CoA. a-
Ketoglutarate, a TCA cycle intermediate, can also
modulate gene expression via modifications of the
epigenome (Katada et al. 2012). To sum up, there is
some evidence linking Cr(VI) with epigenetic changes
potentially conducive to metabolic perturbation and
carcinogenesis, but no definitive studies have been
conducted yet.
Closing remarks
The first part of this article reviewed the literature
addressing the impact of Cr(VI) on the bioenergetic
phenotype of mammalian cells. Altogether, the results
obtained in fourteen independent studies provided
significant evidence that Cr(VI) inhibits respiration.
The opposite effect was found in terms of lactate
production, but experimental data supporting this
latter effect is more limited. Importantly, in all but one
of the twelve studies conducted so far, the cellular
energy status was negatively affected by Cr(VI)
exposure, probably reflecting a shift to a more
inefficient, fermentative metabolism, as observed in
most cancer cells. More studies are, nonetheless,
required to establish whether these changes in three
critical bioenergetic indicators are general responses
to Cr(VI) exposure.
As mentioned before, several of the species formed
in the course of the intracellular reduction of Cr(VI)
are very reactive towards various biomolecules, such
as DNA and proteins. This extreme chemical versa-
tility makes the elucidation of the molecular
mechanisms underlying chromate effects particularly
complex. As a consequence, the molecular basis of the
phenotypic changes observed upon Cr(VI) exposure
remains essentially elusive.
The bioenergetic changes described can result
from alterations in substrate availability, as well as
from direct perturbations to components of the
energy transduction pathways and/or to the tran-
scription and signaling networks that control these
pathways. Cr(VI) is believed to act as a classic
genotoxic agent, but current knowledge on Cr(VI)
effects on target genes is sparse. In particular, it
remains to be determined whether Cr(VI) disrupts
genes encoding proteins directly involved in energy
transduction processes. It would be interesting to
assess, for instance, whether the genes encoding the
isocitrate dehydrogenase isoforms IDH1 and IDH2
are affected, as these were found to be mutated in
a large variety of tumors (Reitman and Yan 2010).
There are several reports of Cr(VI)-induced
changes in the expression of genes and intracellular
levels of proteins directly involved in bioenergetics,
but their number is still too reduced to establish
definitive trends. The same applies to the studies
conducted thus far on the interaction of Cr reactive
species with enzymes and other proteins involved in
energy transduction, which produced results heavily
dependent on the model system. It was, nonetheless,
interesting to observe the upregulation of GAPDH and
the concomitant downregulation of b-F1ATPase, as
well as the inhibition of the mitochondrial respiratory
complexes, as these changes are in line with a shift to a
more fermentative metabolism.
The reported changes on the intracellular levels of
Cr(IV), Cr(V) and ROS, together with interference
with the antioxidant machinery, might disrupt the
intracellular redox balance, with important implica-
tions on the levels and specific activities of a vast array
of proteins, including those of redox-sensitive meta-
bolic enzymes and transcription factors, such as Myc,
p53, HIF-1 and NF-jB. Oxidation of Trx and Prx
might represent an initiating event, as suggested by
recent studies (Myers 2012). The modulation of the
above-mentioned transcription factors by Cr(VI)
needs to be further clarified, as evidence is rapidly
accumulating that they are active partners in the
metabolic rewiring that encompasses the Warburg
effect (Levine and Puzio-Kuter 2010). In particular,
future research efforts focusing on their involvement
123
 Author's personal copy
in Cr(VI) toxicity from a metabolic perspective should
be undertaken.
An increasing number of genome-wide microarrays
of different cell types exposed to Cr(VI) and other
heavy metals is becoming available (Gavin et al. 2007;
Permenter et al. 2011; Sun et al. 2011). Comparative
analysis of such large amounts of data on global gene
expression could be complemented with multidimen-
sional screenings assessing several metabolic param-
eters. Combining gene expression and metabolic
profiling (Ramanathan et al. 2005) data holds the
potential to unveil new mechanistic links between
transcription and metabolism-related genes affected
upon Cr(VI) treatment.
Finally, as in any other area of biological research,
it is crucial that future investigations are conducted in
biologically meaningful models. In particular, results
obtained using Cr(VI) concentrations that are overtly
cytotoxic should be interpreted with great care.
Acknowledgments Investigations from the authors’
laboratories described here were supported by Centro de
Investigação em Meio Ambiente, Genética e Oncobiologia
(CIMAGO), Portugal (Grants 26/07, to AMU, and 16/06, to
MCA) and by Fundação para a Ciência e a Tecnologia, Portugal
(Grant PEst-OE/QUI/UI0070/2011, to Unidade de Quı́mica
Fı́sica Molecular). The authors apologize to all colleagues
whose relevant work could not be mentioned and/or cited owing
to space limitations.
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