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Toxicology and Applied Pharmacology 233 (2008) 92 – 99

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Roles of biomarkers in evaluating interactions among

mixtures of lead, cadmium and arsenic
Gensheng Wang a,⁎, Bruce A. Fowler b
a
Department of Experimental Radiation Oncology, U.T.M.D. Anderson Cancer Center, Houston, TX 77030, USA
b
Division of Toxicology and Environmental Medicine, ATSDR, Atlanta, GA 30333, USA
Available online 31 January 2008

Abstract

Human exposure to environmental chemicals is most correctly characterized as exposure to mixtures of these agents. The metals/metalloids,
lead (Pb), cadmium (Cd), and arsenic (As), are among the leading toxic agents detected in the environment. Exposure to these elements,
particularly at chronic low dose levels, is still a major public health concern. Concurrent exposure to Pb, Cd, or As may produce additive or
synergistic interactions or even new effects that are not seen in single component exposures. Evaluating these interactions on a mechanistic basis is
essential for risk assessment and management of metal/metalloid mixtures. This paper will review a number of individual studies that addressed
interactions of these metals/metalloids in both experimental and human exposure studies with particular emphasis on biomarkers. In general, co-
exposure to metal/metalloid mixtures produced more severe effects at both relatively high dose and low dose levels in a biomarker-specific
manner. These effects were found to be mediated by dose, duration of exposure and genetic factors. While traditional endpoints, such as
morphological changes and biochemical parameters for target organ toxicity, were effective measures for evaluating the toxicity of high dose
metal/metalloid mixtures, biomarkers for oxidative stress, altered heme biosynthesis parameters, and stress proteins showed clear responses in
evaluating toxicity of low dose metal/metalloid mixtures. Metallothionein, heat shock proteins, and glutathione are involved in regulating
interactive effects of metal/metalloid mixtures at low dose levels. These findings suggest that further studies on interactions of these metal/
metalloid mixtures utilizing biomarker endpoints are highly warranted.
© 2008 Elsevier Inc. All rights reserved.

Keywords: Lead; Cadmium; Arsenic; Mixtures; Biomarkers; Risk assessment

Introduction as leading constituents at superfund sites (Fay and Mumtaz,

With thousands of compounds in the environment and many
derived from anthropogenic sources, human exposure to chemi-
cals can be most correctly characterized as one of exposure to
mixtures (Centers for Disease Control and Prevention, 2005).
This is particularly true for metals that are not biodegradable
and are long-lived in the environment. Lead (Pb), cadmium
(Cd), and arsenic (As), as prime examples of these character-
istics, have been utilized in industry and other anthropogenic
sources for decades. Accumulation of these metals/metalloids in
ecosystems is a major source for human exposure and hence a
threat to human health. These elements have also been detected
⁎ Corresponding author. Unit 66, Department of Experimental Radiation
Oncology, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
TX 77063, USA. Fax: +1 713 794 5369.
E-mail address: genwang@mdanderson.org (G. Wang).
0041-008X/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2008.01.017
1996). Further, individuals have the potential to be simulta-
neously exposed to these metals in the workplace. Today high
dose exposure to these elements seldom happens in most
developed countries, but chronic low-dose co-exposures are still
a major public health concern.
Exposure to Pb, Cd, or As alone affects a number of major
target organ systems. (Please see Fowler et al., 2007; Nordberg
et al., 2007; Skerfving and Bergdahl, 2007 for reviews.) These
three metals/metalloids share several common mechanisms
underlying their toxicities, including production of oxidative
stress, reaction with sulfhydryl groups, and interference with
essential metals. In addition, stress proteins and antioxidant
enzymes have been proposed to provide common cellular
protective mechanisms against the element-induced toxicities
when they occur on an individual basis. Thus, investigation of
the interactive effects of Pb, Cd, and As mixtures in major target
organ systems provides a good model for predicting interactions
 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99
of chemical mixtures with components having common target
organs and mechanisms of action that may have applicability for
a wide variety of chemicals. Molecular biomarkers that identify
disturbances in cellular metabolic pathways that lead to cell
death or cancer are particularly valuable predictors.
In this paper individual studies addressing interactions of Pb,
Cd, and As in both experimental animal and human exposures
are reviewed. Comparisons of effects from relatively high and
low dose exposure levels are examined and common mechan-
isms of toxicity identified using biomarkers for Pb,Cd, and As
alone or as mixtures of these elements. Some of the molecular
factors which are known to mediate interactive effects of these
metals/metalloids and impacts on biomarker responses are also
discussed.
Interactions of Pb, Cd, and As in experimental systems
Elevated dose levels
Yanez et al. (1991) showed from rat experiments that the
acute toxicity of Cd and As is altered by concomitant Cd + As
treatment. The extrapolation of results from the single exposure
to the metals to predict those expected from co-exposure was
found to be misleading. Co-exposure to Cd (as cadmium
chloride, 2.6 mg/kg) and As (sodium arsenite, 10 mg/kg) via
one-time intraperitoneal injection resulted in more toxicity than
either metal alone; the 24 h LD50 values were noticeably dif-
ferent. Other observations, such as lower concentrations of Cd
in the liver of animals which were co-exposed to both Cd +
As compared to those exposed only to Cd, highlighted the
complexity of the sequelae and the usefulness of biomarkers in
understanding the underlying biological mechanisms.
Mahaffey, Fowler and co-workers reported the interactive
effects of Pb, Cd, and As on a number of rat organ systems
including the kidney at relatively high dose levels (Mahaffey
and Fowler, 1977; Fowler and Mahaffey, 1978; Mahaffey et
al., 1981). In these studies, rats were treated via diet for
10 weeks at doses of 200 ppm, 50 ppm, and 50 ppm with lead
acetate, cadmium chloride, and sodium arsenate, respectively.
The observed signs of renal toxicity, included swelling of
proximal tubule cells and pathognomonic lead intranuclear
inclusion bodies, was induced by Pb and Pb + As exposure.
Addition of Cd to the Pb treatment (e.g., Pb + Cd and Pb + Cd
+ As) significantly reduced these morphological effects.
Decreased Pb absorption/retention was proposed as respon-
sible for the reduction of Cd on Pb renal effects because the Pb
concentration in the kidney was reduced by 50–60% which
was associated with reduction of lead intranuclear inclusion
body formation. These inclusion bodies have been reported
(Goyer, 1971) to represent the main intracellular depot of Pb in
kidney tubule cells once formed. Alterations of kidney
morphology after Cd, or As, or Cd + As exposure were slight
and no increase in renal toxicity was observed for concomitant
exposure to Cd and As. On the other hand, animals in the Pb
+ Cd or the Pb + Cd + As treatment groups showed additive
increases in the urinary excretion of porphyrins (Fowler and
Mahaffey, 1978; Mahaffey et al., 1981) indicating that,
93
although measured tissue burdens of Pb were reduced, the
biologically available fraction of lead was actually increased.
The porphyrin biomarkers were hence of great value in
interpreting the potential toxic effects of combined exposure to
these toxic elements under these specified elevated dose levels.
Molecular mechanistic explanations for these findings were
provided in subsequent publications (Oskarsson and Fowler,
1985a,b). These later studies examined the bioavailability of
Pb within mitochondria and the formation of kidney lead
intranuclear inclusion bodies in mediating the bioavailability
of lead to heme pathway enzymes and development of the
characteristic lead-induced porphyrinuria pattern. Other in
vitro studies (Mistry et al., 1985, 1986; Goering and Fowler,
1984, 1985; Goering et al., 1986) examined the binding and
displacement of lead from kidney cytosolic lead-binding
proteins (PbBPs) first described by Oskarssson et al. (1982).
Donation of Zn to ALAD by these proteins as well as the Cd,
Zn binding protein metallothionein (MT) were reported by
Goering and coworkers (Goering and Fowler, 1984, 1985;
Goering et al., 1986, Goering and Fowler 1987a,b). Overall,
the results of these studies demonstrated the value of
metabolomic biomarkers (heme pathway enzymes and metallic
mixture-specific porphyrinuria patterns) in the interpretation of
analytical biomonitoring results and the molecular factors that
regulated these responses.
Genetic factors
Other factors such as genetic expression of the metal-binding
proteins (e.g. PbBPs and MT) noted above and arsenic methyl
transferases may also play an important role in mediating the
measured outcomes of concomitant exposures to metals, such as
cadmium and the metalloid arsenic.
Studies by Liu et al., 2000 concerning interactions of Cd and
As on nephrotoxicity examined combined exposures to these
elements on the mouse kidney. The animals were exposed to Cd
or As, or combined Cd + As for 4 months. Cd (as cadmium
chloride) was administered via feed at 100 ppm and As
(as sodium arsenite) via drinking at 22.5 ppm. These doses were
considered as maximum tolerated doses during 4–6 months of
exposure. As compared with single metals, combined exposure
to Cd + As significantly increased renal damage, evaluated by
monitoring urine N-acetyl-β-glucosaminidase, glucose, protein
excretion, blood urine nitrogen, and kidney morphology.
Elemental concentrations for both Cd and As in the kidney
tissue were comparable between single exposures and the com-
bined exposure. An examination of the interactive effect of
Cd and As on the kidney in metallothionein knockout mice
(MT-null) showed that both single Cd or As exposure and
combined Cd + As exposure produced more severe renal lesions
in MT-null mice than those observed in wild type mice though
Cd accumulation in MT-null mice was found to be significantly
less than that in wild type mice. Studies concerning the effects
of polymorphic forms of arsenic methyl transferase (AsMT) in
regulating the toxicity of As3+ in mice (Stýblo et al., 2002;
Aposhian et al., 2004; Li et al., 2005) highlighted the impor-
tance of polymorphisms in the metabolic pathway in mediating
94 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99
formation of toxic methylated arsenical intermediates and hence
the risk for arsenical toxicity.
Overall, the results of the above studies show that the genetic
expression of specific metal-binding proteins and methyl
transferase-mediated metabolic pathways will exert pronounced
effects on toxicity and measured biomarker endpoints and hence
affect the results of interactions among these elements.
Low dose exposure level studies
Lowest-observed-effect-level (LOEL) dose studies
In order to determine the interactive effects of Pb, Cd, and As
relative to single element exposures of these agents, lowest
observed effect level (LOEL) studies were conducted in rats
utilizing a two-way factorial design (Fowler et al., 2004;
Whittaker et al., 2004, 2005; Wang, 2005; Wang et al., 2002,
2003, 2004, 2005). For these studies, adult male Sprague–
Dawley rats were treated with lead acetate (25 ppm), cadmium
chloride (10 ppm), or sodium arsenite (5 ppm) alone or in
combinations via drinking water for 30, 90, and 180 days. The
dose levels selected were designated as LOELs based on a prior
series of drinking water exposure dose-response studies for Pb,
Cd, and As at 30, 90 and 180 days on an individual basis. The
interactive effects of Pb, Cd, and As on kidney, heme biosyn-
thesis pathway, oxidative stress, and stress proteins were then
examined. The results are described below.
Interactions and toxicokinetics. Toxicokinetic interactions
were previously observed in high dose metal/metalloid mixture
exposures (Mahaffey et al., 1981). Analysis of renal elemental
concentrations of Pb, Cd, and As showed an interactive effect on
the toxicokinetics among Pb, Cd, and As. As noted above, Cd
exposure markedly reduced renal lead concentrations, but
changes were also noted for a number of essential metals such
as Cu and Zn. In the LOEL dose studies of Pb, Cd and As,
described above, similar alterations in renal accumulation were
also observed. These interactions were metal/metalloid-and
time-specific. For example, As accumulation in tissue was
significantly reduced with co-exposure either to Pb or to Cd both
at 30- and 90-days. Concomitant Cd exposure reduced renal Pb
content, and concomitant As exposure reduced Cd accumulation
at 90 days relative to the groups receiving single element
exposures. Co-exposure to Cd significantly decreased renal Pb
concentrations at 180 days relative to groups not receiving Cd
(Wang, 2005). These findings were similar to those reported by
Mahaffey et al., 1981 at higher dose levels and 10 weeks of
exposure. As with the studies conducted by Mahaffey and
colleagues, interactive effects could be either alterations in the
processes of absorption, distribution, metabolism, or excretion.
These results were consistent with a study showing that sodium
arsenite significantly inhibited uptake of Cd by Chinese hamster
ovarian cells (Klug et al., 1988).
Interactive effects on biomarkers of nephrotoxicity and heme
biosynthesis. Increased kidney weight/body weight ratios,
increased urinary protein excretion, and abnormal non-specific
kidney morphology were observed in some combined exposure
groups at 180 days, but not in single exposures or at 30- and 90-
day time points. Combined exposure to Pb + As and Pb + Cd +
As for 30- or 90-days caused synergistic inhibition of blood
delta-aminolevulinic acid dedydratase (ALAD) as compared to
a single exposure either to Pb or As. When co-exposed to Pb,
Cd or As for 30 days, rats showed greater blood zinc proto-
porphyrin (ZPP) than those with single exposures. In general,
rats exposed to metals/mixtures for 90 days showed some
compensatory response compared to 30 and 180 days (Wang
et al., 2003), suggesting that duration of exposure is critical in
evaluating the interactions of metal mixtures particularly at low
dose levels.
Interactive effects on kidney oxidative damage. In the LOEL
Pb, Cd, As studies described above, when compared to controls,
protein carbonyls (PCs - a marker of protein oxidation) were
decreased after single exposure to these elements for 90 and
180 days). However, with co-exposure to these elements at the
same time points, the PC levels were increased (up to 20%)
relative to untreated controls. Induction of 8-hydroxy-2′-deoxy-
guanosine (8-OhdG, a marker of oxidative damage to DNA)
after a combined exposure to Cd + As for 180 days was poten-
tiated as compared with a single exposure to Cd or As. Expres-
sion of mRNA for antioxidant enzymes, Superoxide Dismutase
(SOD) and Glutathione Peroxidase (GPx), partially explained
the change of oxidative damage to proteins and DNA after
combined exposures (Wang et al., 2004).
Pb, Cd and As interactions and kidney stress protein responses.
The metal-specific stress protein, MT, and major classes of stress
proteins, such as heat shock proteins (Hsps) Hsp 32, Hsp 60, and
Hsp 70, were examined to explore the possible mechanisms
regulating the interactive effects of Pb, Cd, and As (Wang et al.,
2005). MT protein was expressed most in groups with Cd
treatment. The profile of MT expression location was similar
between single and combined treatments. Co-exposure to Pb + As
induced higher levels of MT protein than either Pb or As
exposure alone in the kidney tubule cells. Increased expression of
MT and Hsp 60 in the combined exposure groups at 90 days
could contribute to the compensatory effect at this time point.
Expression of mRNA for stress proteins was not always
consistent with their protein expression when exposed to metal/
metalloid mixtures at low doses.
An appreciation of the roles of molecular factors, which may
mediate biomarker responses, and ultimately cellular toxicity, is
particularly important for low dose exposure which is the most
commonly encountered exposure situation.
Interactions of Pb, Cd, and As in vitro
The interaction of Cd + As on the expression of stress
proteins was investigated both in rat and in human kidney cell
lines (Madden et al., 2002). Increased hsp 60, hsp 70, and hsp
90 were observed at a lower concentration (10 μM) for co-
exposure to Cd + As compared with a single exposure to Cd or
As (100 μM) in rat kidney cells. In human kidney cells,
induction of protein 70 occurred at a lower dose when there was
 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99
a combined exposure (1 μM) than when there was a single
exposure (10 μM). This suggested concurrent exposure to Cd +
As acted as a stronger stressor than exposure to either Cd or As
alone when given at same dose levels on an individual basis,
though this effect was metal-, cell line-and dose-specific. No
other toxic endpoints were examined in this study.
Another in vitro study (Bae et al., 2001) examined the
interactive effect of Pb, Cd, As and Cr on keratinocytes. The
interactions showed a trend of hormesis-additivity-synergism-
antagonism with increasing metal mixture concentrations. The
transition of the interaction types was due to the induction of
MT, i.e. MT levels followed the same trend.
Evaluation of biomarkers for metal mixtures
The traditional endpoints for renal injury, such as morphol-
ogy and biochemical parameters of renal functions, were effec-
tive in understanding the effects from chronic high dose metal
mixtures but were less sensitive and specific for delineating
biological changes at low dose level exposures. However,
oxidative stress and heme biosynthesis parameters are promis-
ing as endpoints in evaluating the toxicity of metal mixtures at
these lower levels.
Oxidative stress
Alteration of oxidative damage to macromolecules has been
shown to be sensitive both in high dose (Yáñez et al., 1991) and
low dose studies (Fowler et al., 2004), suggesting that oxidative
stress is a common mechanism for interactive effects of metal
mixtures. At low doses, the identification of oxidative damage
was more sensitive than traditionally used endpoints, such as
morphological changes. It is most likely that oxidative stress is a
sensitive endpoint for metal mixtures, because mitochondria,
the major intracellular source of reactive oxygen species (ROS),
are common targets for Pb, Cd, and As on an individual
exposure basis (Fowler and Mahaffey, 1978). It should be noted
that these metals also interrupt antioxidant enzymes in which
the active sites are sulfhydryl group or essential metals.
Analytical data on total tissue content of essential trace metals
(Mahaffey et al., 1981) showed that Cd exposure resulted in a
33% decrease in Fe in kidney, a 43% decrease in Fe in femur
and a 63% decrease in Fe in liver. Cd exposure also resulted in a
27% decrease in Zn in femur but a 20% increase in Zn content
of kidney. As exposure increased total renal content of Cu by
200% and increased liver content of Fe by 44%. These data
clearly indicate the exposure to the above toxic trace elements
also produce marked alterations of target tissue concentrations
of essential metals.
Heme biosynthesis parameters
Urinary porphyrin excretion patterns have been found to be
metal-specific and proposed to be a sensitive biological endpoint
in evaluating the toxicity of metal mixtures in high dose studies
(Fowler and Mahaffey, 1978). Blood ALAD and ZPP have also
been demonstrated to be sensitive for metal interactions at low
doses (Wang et al., 2003). Alterations of heme biosynthesis
indicators are not always reflective of injuries in all the major
95
target organ systems, but these endpoints still demonstrate the
overall biological activity of metal mixtures within the organism.
Regulation of interactions of metal mixtures in the kidney
Although the mechanisms by which these metals interact are
not fully elucidated, several molecules, including lead binding
proteins (PbBPs), MT, Glutathione (GSH) and Hsps, have
been suggested as important regulators of interactions in metal
mixture exposures:
Lead binding proteins (PbBPs)
Soluble cytosolic lead binding proteins in rat kidney and
brain (Oskarsson et al., 1982) and lead-binding proteins
associated with renal lead intranuclear inclusion bodies (Shelton
and Egle, 1982; Shelton et al., 1986) have been reported and
partially characterized. These molecules appear to play impor-
tant roles in mediating the intracellular bioavailability of Pb with
regard to ALAD (Goering and Fowler, 1984, 1985; Goering
et al., 1986) and the intranuclear movement of lead (Mistry et al.,
1985, 1986). Analogous PbBPs with similar dissociation
constants (Kds) for lead have also been reported in human
brain (Quintanilla-Vega et al., 1995) and kidney (Smith et al.,
1998). A brief review of the PbBPs has been previously pub-
lished (Fowler, 1998).
Metallothionein
(MT), a metal-specific stress protein, which is present in
many tissues as a number of isoforms, is critical in regulating
the nephrotoxic interaction of metal mixtures in the kidney. This
is supported by evidence from both high- and low-dose in vivo
studies and in vitro studies (Bae et al., 2001; Liu et al., 2000;
Wang et al., 2005). An in vivo study with MT-null mice directly
proved this. The effect of the sequence of exposure on inter-
action of Cd and As suggested a role for MT in toxicity
development of metal mixtures (Hochadel and Waalkes, 1997).
The induction of MT by one metal could impact the effects of
another metal through both toxicokinetics, such as transporta-
tion and accumulation, and toxicodynamics, such as sequestra-
tion of metals. The findings concerning the effects of
concomitant Cd exposure on Pb effects with regard to renal
injury and inhibition of blood ALAD could be due to altered
regulation of ALAD by the PbBPs and MT (Goering and
Fowler,1984, 1985,1987a,b; Goering et al., 1986).
Glutathione
GSH has been reported to play an important role in
development of metal mixture toxicity. The interaction of Cd +
As in the testis has been attributed to decreased (Diaz-Barriga
et al., 1990). Wang et al. (2005) found that kidney GSH levels
(measured as non-protein thiols) were variable at different time
points and were associated with an interactive effect of Pb + Cd +
As at low doses. GSH is a non-enzyme antioxidant, and its
regulation on interaction of metal mixtures could be through the
neutralization of oxidative radicals. GSH may also offer its
sulfhydryl group to protect enzymes or other active proteins
from insult by metal mixtures.
96 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99

Heat shock proteins (Hsps) conducted under occupational exposure conditions, a number of
Hsps, a group of universal stress proteins, which are primarily the more recent studies have reported results from environ-
localized in several cellular organelle systems, are commonly mental exposures in developing countries. Biomarkers have
altered under the situation of exposure to metal mixtures. The also proven highly useful in interpreting results and identifying
induction patterns of Hsps are potentially useful as biomarkers populations at special risk for toxicity.
of early cellular responses to exposure and mediator of toxicity.
The pattern of induction may delineate specific cellular re- Human occupational exposures to Pb, Cd, and As mixtures
sponses to a stressor and are hence of potential value as an early
response biomarker for the target organelle system. In addition, Garçon and co-workers (2004, 2007) assessed the effects of
once Hsps are induced, they provide protective effects to the combined chronic exposures to lead + cadmium in occupation-
organism or target cell population. Both in vitro low dose studies ally exposed workers on several biomarkers of renal toxicity.
(Madden et al., 2002) and in vivo studies (Wang et al., 2005) The results indicated that a number of oxidative stress markers
showed that Hsps were induced in a metal/metalloid, dose, and were altered by combined Pb + Cd exposures. Of these, urinary
time-specific manner. Examination of these proteins permits excretion of alpha-glutathione-S-transferase was a particularly
evaluation of the strength of the stressor and the ability of the useful biomarker of early changes in proxitubular structure and
organism to respond to the stressor. This is of particular function because it appeared to be predictive of subsequent
importance in evaluating risks associated with exposures to low clinical renal disease if exposures to these metals were not
dose metal mixtures. By combining other toxic endpoints, an reduced.
examination of Hsps can be used as either an endpoint or a Other studies in workers with combined exposures have
regulator in evaluating metal mixture toxicity. focused on genotoxic damage from these elements.
A summary overview table of the findings of interactions Studies by Wasowicz et al. (2001) demonstrated that essential
among Pb, Cd and As in the above experimental system studies trace elements (Cu, Se, Zn) were altered in workers with
is presented in Table 1. The results of these studies indicate that, combined exposures to Pb + Cd with statistically significant
overall, additive or more than additive interactions do occur decreases in the Se-dependent enzyme glutathione peroxidase in
among Pb, Cd, and As under combined exposure conditions. both erythrocytes and plasma, which was used as a biomarker of
oxidative stress in the combined Pb + Cd exposed group relative
Interactions among Pb, Cd and As in human exposure studies to controls. Statistically significant increases in the presence of
thiobarbituric acid reactive substances (TBARS) were also
In addition to studies in experimental systems, interactions reported in plasma of the Pb,exposed workers. Overall, results of
among Pb, Cd and As have been well-documented in human these studies indicated that combined exposures to Pb + Cd alter
exposure studies. While many of these studies have been the antioxidant capabilities of the blood. In agreement with these
findings, Palus et al. (2003) reported clastogenic and aneugenic
effects in peripheral lymphocytes; these effects were increased
Table 1 by a factor of two times that of controls. These genotoxic effects
Pb, Cd and As interaction studies in experimental systems were utilized as biomarkers of effect in these studies. Molecular
Reference Type of Elements Biomarker epidemiology studies by Hengstler et al. (2003) showed
experiment findings/endpoints combined exposures to Pb + Cd + Co produced DNA strand
Mahaffey and Fowler, 1977 In vivo Pb, Cd, As ALA, urinary breaks and suggested that these combined exposures produced
porphyrins oxidative stress coupled with an observed inhibition of repair of
Fowler and Mahaffey, 1978 In vivo Pb, Cd, As ALA, urinary 8-oxoguanine as the underlying mechanisms.
porphyrins
Mahaffey et al., 1981 In vivo Pb, Cd, As ALA, urinary
Human environmental exposures to Pb, Cd, and As mixtures
porphyrins
Liu et al., 2000 In vivo Cd, As Proteinuria
Fowler et al., 2004 In vivo Pb, Cd, As a ZPP, ALAD Roels et al. (1999) reported on the usefulness of specific
Wang et al., 2003 In vivo Pb, Cd, As a HSPs, MT, ZPP urinary biomarkers for assessing the nephrotoxic effects of Pb +
Yáñez et al., 1991 In vivo Cd, As Lipid peroxidation, Cd as well as inorganic Hg based upon studies in occupationally
GSH, MT
Mistry et al., 1985 In vitro Pb Chromatin binding,
exposed workers and the prospects for extrapolating these
PbBPs biomarkers to environmentally exposed populations. This report
Mistry et al., 1986 In vitro Pb, Cd, Ca, PbBPs, metal provides useful insights into the value of these biomarkers for
Fe, Zn displacement of assessing renal effects at elevated dose levels and suggests their
nuclear uptake value for addressing combined exposures to mixtures of these
Goering and Fowler, 1984 In vitro Pb ALAD
elements. Studies by de Burbure et al. (2003, 2006) evaluated the
Goering and Fowler, 1985 In vitro Pb ALAD
Goering et al., 1986 In vitro Pb ALAD utility of a suite of biomarkers for assessing the renal effects of
Goering and Fowler, 1987a In vivo Pb, Zn ALAD, MT Pb + Cd + As as well as inorganic Hg in children and adults
Goering and Fowler, 1987b In vivo Pb, Cd, Zn ALAD, MT with environmental exposures to these elements from living
Madden et al., 2002 In vitro Cd, As HSPs, MT around non-ferrous smelters. They reported minimal changes in
a
LOEL dose levels. the biomarkers where the smelters were in compliance with
 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99 97

environmental regulations and some modest alterations in
dopaminergic markers in children with these exposures indicat-
ing the need to consider the multi-organ systems (e.g. central
nervous system) in assessing the toxic effects of these elements.
Hong et al., (2004) and Nordberg et al. (2005) utilized a suite
of renal proteinuria biomarkers to study groups of persons in
China with combined exposures to Cd + As relative to controls
and to groups with primarily Cd or As exposures. They ob-
served that co-exposure to these elements produced more severe
renal effects than exposure to either element alone demonstrat-
ing the value of molecular biomarkers in delineating interactive
nephrotoxic effects between these two toxic elements under
environmental exposure conditions.
In Table 2 the findings are summarized for the human
exposure studies described above.
Future directions
The available studies provide evidence that the response of
an organism or cell to metal mixtures is not just a simple
additive response and more than additive responses are not
uncommon in both experimental system and human health
studies. Further investigations are definitely needed to provide
a more detailed picture before risk assessments can be applied
to metal mixture exposures. It is clear from the above studies
that molecular biomarkers are powerful tools for more finely
delineating the nature of interactive responses among metals/
metalloids and may provide useful insights into mechanisms
of action that are invaluable to modern risk assessment
practices.
effects of mixtures as NO(A)ELs for each chemical in a mixture
by providing mechanistic insights into ongoing processes that
will help to interpret the complicated nature of interactive
effects. Use of NO(A)ELs utilizing biomarkers will allow direct
testing to determine if the combination of two or more null
effects results in a positive effect following combined exposures
(e.g. potentiation). Here again the sensitivity and specificity of
molecular biomarkers are invaluable in detecting early or subtle
cellular responses to metal/metalloids at low dose exposures.
Additional metals or additional organ systems
As noted above, in addition to Pb, Cd, and As, other
metals such as mercury (Hg) and chromium (Cr) are also
ubiquitous in the environment. Additional interaction studies
will provide more realistic insights into human exposures to
these and other metal mixtures in the environment. With more
component chemicals in mixture studies, novel experimental
designs will be needed to efficiently evaluate interactions.
Interactive effects of metals on other organ systems, such as
nervous, cardiovascular, and reproductive systems, need more
investigation since those are also common target organs for
metals. Data from the available studies suggest that the
interaction of these metals could be variable in different
organs due to the varied ability to produce MT, GSH, and
Hsps. Measurement of molecular biomarkers for these
elements will greatly assist in the interpretation of data
from more complex metal exposure situations.
Epidemiologic studies and sensitive populations

Interactions of metal mixtures at no-observed-(adverse)-effect- Epidemiologic studies on metal mixtures are also clearly of
levels (NO(A)ELs) great value. A few available studies on the interactions of metals
in human populations only described the association between
Data from high dose or LOEL on interactions can be used for some adverse effects and metal concentrations in the same
extrapolation to NO(A)EL. However, data from high dose or tissues. Well-designed population studies could greatly con-
LOEL using biomarkers will assist investigations for interactive tribute to improved risk assessments and management of human
metal mixture exposures. Stress proteins and compensatory
responses play a role in the development of toxicity for metal
Table 2 mixtures. The capacity for stress responses in sensitive human
Studies on human exposures to Pb, Cd, and As mixtures populations, (e.g. children, pregnant women, and the elderly) is
Reference Exposure Site Element Biomarker/Endpoints usually regarded as being lower than that of a healthy adult in
deBurbure et al., 2003 Environmental Pb, Cd, Hg Proteinuria the general population. Some special animal models simulating
deBurbure et al., 2006 Environmental Cd, Pb, Serum prolactin, sensitive populations, for example, a gentamicin-induced model
Hg, As homovanillic acid of subclinical renal injury (Madden and Fowler, 2000) could be
Garcon et al., 2007 Occupational Pb, Cd ↑α Glutathione useful in addressing this issue.
transferase in urine,
ZPP, malondialdehyde
Garcon et al., 2004 Occupational Pb, Cd ↑α Glutathione Application of -omics techniques in metal mixtures
transferase in urine
Hengstler et al., 2003 Occupational Cd, Pb, Co DNA strandbreaks, There are two main public health concerns with mixtures:
8-OH DG 1) production of new toxicities not seen in individual components;
Hong et al., 2004 Environmental Cd, As Proteinuria
and 2) production of synergistic or potentiation effects (Conolly,
Nordberg et al., 2005 Environmental Cd, As Proteinuria, MT
Palus et al., 2003 Occupational Pb, Cd ↑Micronuceli, 2001). Evaluation of synergism for metal mixtures may use
DNA strandbreaks known toxic points from each component metal that are usually
Roels et al., 1999 Occupational Pb, Cd, Hg Proteinuria studied extensively. To identify a new toxic effect from metal
Wasowicz et al., 2001 Occupational Pb, Cd Decreased GPx, mixtures, combinations of toxicogenomics, proteomics, and
increased TBARS
metabolomics will be very helpful since these techniques can
98 G. Wang, B.A. Fowler / Toxicology and Applied Pharmacology 233 (2008) 92–99
examine many genes or proteins or sensitive and interrelated
metabolic pathways at the same time. Well designed experiments
employing these techniques are needed to elucidate the mechan-
isms underlying interactive effects of metal mixtures. In order for
“omic” biomarkers to reach their full potential, validation studies
which permit delineation of biomarkers of exposure from
biomarkers of effect or predictors of clinical disease outcomes
must be conducted. Such studies, while currently feasible, will be
expensive and time consuming.
Conflict of interest disclosure statement
The authors declare that they have no conflicts of interest.
Acknowledgment
Supported in part by USEPA STAR Grant #827161.
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