# Differential Toxicological Interaction among Arsenic, Cadmium, Lead, and Mercury on MCF 7 Cell Line

Authors: Charles K. Klutse; Kelly Mack; Katherine Squibb; Ali B. Ishaque
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

Evaluation of joint toxic action of metal ion mixtures is one of the priority research areas due to the simultaneous occurrence of metals in the environment and the health risk they posed to humans and the environment as a mixture. Individual and composite mixture acute toxicities of arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb), which are among the top eight toxic chemicals, were characterized at varying concentrations.  MCF 7 cell lines were exposed to individual and composite mixtures containing the four metal ions in the proportion of their EPA's MCL for 24 hours, and the concentration-response data were generated spectrofluorometrically. Acute toxicities were estimated based on the uptake of fluorescence diacetate dye. Toxicological interactions among the four metals were profiled, based on computed interactive index. Results demonstrated that the toxicity of each of the metal ions was enhanced in the composite mixture, and the metals demonstrated differential interactions in a concentration dependent manner. Lead, the least toxic among the four metals, showed the highest enhancement (23-to 64-fold) in toxicity when in the mixture. Interaction among the four metals was largely additive although there was slight departures form additivity at the two extremes of the concentration range.


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## Body

## 1. Introduction

Mixtures of
arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) are commonly encountered
in food, water, and other parts of the environment as a result of human and
natural activities. Humans are therefore coexposed to these metals
simultaneously or sequentially through several exposure routes leading to
consequential health risk [1]. The four metals are among the top eight
contaminants in the site frequency count by Agency of Toxic Substances and
Disease Registry’s (ATSDR-) completed exposure pathway site count report [2] and EPA’s highest priority hazardous substances, and importantly, these metals are implicated
in the environmentally-induced cancers and hormonal disrupting activities [3]. It
is therefore imperative that toxicity studies of these metals in combination
are adequately pursued.Composite mixtures of metals
have been found to elicit toxicological interactions (antagonistic, additive,
and synergistic) [4–7]. Studies  have shown that on the basis of Dutch and US
water quality criteria, mixtures of commonly found metals in water do  not adequately protect aquatic lives  [8–10]. Ishaque et al. [11] showed
that composite mixture of As, Cd, Hg, and Pb demonstrated synergistic effects
on Vibrio fischeri. Bae et al., [12] found that interactive effects of
the metals were dependent on the concentration, and this differing interactive effect
was linked to biochemical response of the cells to the metal intoxication [13–16].  Combined effect of
mercury ions and dithiothreitol (DTT) increased cellular concentrations of GSH
in hepatoma cell cultures [17].Evidence is therefore mounting regarding the link between biochemical response and the interactive effect of chemical
mixtures. Although organisms are rarely exposed or
survive the exposure at the acute level, it is important to understand the
interactive effect of chemical mixtures at all levels in order to understand
the toxic effect of chemical mixtures in their totality. Different types of
mixture compositions have been used in studying the interactive effect of
chemical mixtures. In some studies compositions were based on the toxicity of
each individual component [12, 18]. In other studies the basis for the mixtures
included quality criteria, composition at Superfund sites, and average
composition in the environment [8–10]. Although these compositions have shed light on the interactive
effects of metal mixtures, none of them is conclusive because ratio of metals
in the natural environment is variable, and LD50 depends on the
organism, its state of health, and the exposure route.  To glean more information on the adverse effects of chemical
mixtures, it is equally important to investigate mixture composition based on
EPA’s maximum contamination levels (MCL) since this criterion is one of the
benchmarks for assessing water quality.In this study composite
mixture of As, Cd, Hg, and Pb with composition based on the ratio of the EPA’s
MCL of each metal was used in acute toxicity studies. This is to help
understand the interactive effect of these metals when they accumulate in the
environment at this ratio and at higher concentrations. It also offers an
alternative to modeling the impact of metal mixtures with composition based on
environmental pollution regulation.The aim of this research was to characterize the interactive effects
(additive, synergistic, and antagonistic) of As, Cd, Hg, and Pb in quaternary
mixtures using an MCF 7 cell line in
vitro system. The MCF 7 cell lines were chosen for the study due to the
ability of these cells to withstand harsh conditions which allowed the use of
higher exposure levels. This preliminary study aimed at  demonstrating
a proof of principle in the use of EPA’s MCL ratio for the study of metal
mixtures.


## 2. Materials and Methods

### 2.1. Cell Culture and Exposure

As (1 mg/mL in 2%
KOH), Cd (1 mg/mL in 0.5 N nitric acid), Pb (1 mg/mL in 2% nitric acid), and Hg (1 mg/mL
in 10% nitric acid), all atomic absorption standard solutions, were purchased
from Acros Organic (New Jersey).
Dimethyl sulfoxide (DMSO) and Fluorescence Diacetate Dye (FDA) were purchased
from Sigma-Aldrich Co (St. Louise ,
MO). MCF 7 cell lines, Trypsin-EDTA,
and Fetal Bovine Serum (FBS) were purchased from American Type Culture
Collection (ATTC) (Manassas ,
VA).  Minimum Essential Medium (MEM) alpha 1x,
Dulbecco’s Phosphate Buffered Saline (PBS), MEM without phenol, and penicillin-streptomycin
were purchased from GIBCO Invitrogen (Grand
Island , NY).  MCF 7 cells were grown in MEM alpha 1x
supplemented with 10% FBS and 1% penicillin streptomycin and incubated for 24 hours at37°C in a 5% CO2 incubator to allow the cells to
grow, and form a monolayer in the flask. Cells grown to 75%–85% confluence were
washed with PBS, trypsinized with 3 mL of 0.25% (v) trypsin-0.0.3% (v) EDTA,
diluted with fresh medium, and counted for experimental purposes.To determine the
individual and the combined toxicities of the metals, MCF 7 cells were seeded
in black sterile 96-well (1×104 cells/well) plates and
placed in a CO2 incubator for 24 hours for attachment. After the
incubation period, cells were exposed to serial dilutions of the individual and
the composite mixture prepared in MEM (without phenol) supplemented with 5%
penicillin-streptomycin. The highest concentration for the individual chemicals
was 80 mg/L. The mixture of the four metals was made by mixing As, Cd, Hg, and
Pb stock solutions in the ratio of their EPA MCL 10:5:2:15, respectively,
representing a starting concentration of 20, 10, 4, and 30 mg/L,
respectively.  The first row of each
plate was used as control (medium without cells), and the second row was used as
negative control (cells without metals). Treated cells were incubated for 24
hours.


### 2.2. Cell Viability Test by Fluorescence Diacetate (FDA) Dye-Spectrofluorometric Method

Treated cells
were taken out of the incubator, exposure media was removed, and cells were
washed with 100.0μL PBS. This procedure was done carefully to avoid detaching
cells from the bottom of the wells. Each of the wells was then treated with
100.0 μL of the diluted working FDA solution (10 μg/mL). The treated plates were
placed in the incubator for 45 minutes. This allowed the surviving cells to be
stained by the FDA giving them a fluorescent green color. Cell culture plates
were read with Fluoroskan Ascent FL 374 (ThermoLabsystems ,
Finland), and the
readings converted to percent survival by comparing each reading to the nonexposed
controls. Sigmoidal model logistic (3 parameters: (y = A/(1 + exp(B-Dx)), where y
= response, x = concentration, and A, B, and D are the 3 parameters) was used to
fit the data. Based on the data, the 3 parameters (A, B, and D) were estimated,
and an equation was generated for each curve fitted. Concentration addition was
used for the determination of toxicity of a mixture of chemicals expected to
exhibit a combined effect. II=(4,i=1∑(Ci/LCxi))=1, where II is the
interaction index, LCxi is
the concentration of the ith mixture
component that elicits x% effect when
applied singly, and Ci is the concentration of the
respective metal in the mixture when the mixture elicits the same effect. Each
(Ci/LCxi) is the concentration of a mixture component scaled
for its relative toxicity generally termed Toxic Unit (TU) of that component
[19].Using Statistix
appropriate statistical tools were applied to the results to determine  significant differences among the means of
the various toxicities, as well as to determine the existence and nature of
trends in the treatment level means. Regression analysis was done to determine
the correlation between the interaction index and the concentration of the
metals.


## 2.1. Cell Culture and Exposure

As (1 mg/mL in 2%
KOH), Cd (1 mg/mL in 0.5 N nitric acid), Pb (1 mg/mL in 2% nitric acid), and Hg (1 mg/mL
in 10% nitric acid), all atomic absorption standard solutions, were purchased
from Acros Organic (New Jersey).
Dimethyl sulfoxide (DMSO) and Fluorescence Diacetate Dye (FDA) were purchased
from Sigma-Aldrich Co (St. Louise ,
MO). MCF 7 cell lines, Trypsin-EDTA,
and Fetal Bovine Serum (FBS) were purchased from American Type Culture
Collection (ATTC) (Manassas ,
VA).  Minimum Essential Medium (MEM) alpha 1x,
Dulbecco’s Phosphate Buffered Saline (PBS), MEM without phenol, and penicillin-streptomycin
were purchased from GIBCO Invitrogen (Grand
Island , NY).  MCF 7 cells were grown in MEM alpha 1x
supplemented with 10% FBS and 1% penicillin streptomycin and incubated for 24 hours at37°C in a 5% CO2 incubator to allow the cells to
grow, and form a monolayer in the flask. Cells grown to 75%–85% confluence were
washed with PBS, trypsinized with 3 mL of 0.25% (v) trypsin-0.0.3% (v) EDTA,
diluted with fresh medium, and counted for experimental purposes.To determine the
individual and the combined toxicities of the metals, MCF 7 cells were seeded
in black sterile 96-well (1×104 cells/well) plates and
placed in a CO2 incubator for 24 hours for attachment. After the
incubation period, cells were exposed to serial dilutions of the individual and
the composite mixture prepared in MEM (without phenol) supplemented with 5%
penicillin-streptomycin. The highest concentration for the individual chemicals
was 80 mg/L. The mixture of the four metals was made by mixing As, Cd, Hg, and
Pb stock solutions in the ratio of their EPA MCL 10:5:2:15, respectively,
representing a starting concentration of 20, 10, 4, and 30 mg/L,
respectively.  The first row of each
plate was used as control (medium without cells), and the second row was used as
negative control (cells without metals). Treated cells were incubated for 24
hours.


## 2.2. Cell Viability Test by Fluorescence Diacetate (FDA) Dye-Spectrofluorometric Method

Treated cells
were taken out of the incubator, exposure media was removed, and cells were
washed with 100.0μL PBS. This procedure was done carefully to avoid detaching
cells from the bottom of the wells. Each of the wells was then treated with
100.0 μL of the diluted working FDA solution (10 μg/mL). The treated plates were
placed in the incubator for 45 minutes. This allowed the surviving cells to be
stained by the FDA giving them a fluorescent green color. Cell culture plates
were read with Fluoroskan Ascent FL 374 (ThermoLabsystems ,
Finland), and the
readings converted to percent survival by comparing each reading to the nonexposed
controls. Sigmoidal model logistic (3 parameters: (y = A/(1 + exp(B-Dx)), where y
= response, x = concentration, and A, B, and D are the 3 parameters) was used to
fit the data. Based on the data, the 3 parameters (A, B, and D) were estimated,
and an equation was generated for each curve fitted. Concentration addition was
used for the determination of toxicity of a mixture of chemicals expected to
exhibit a combined effect. II=(4,i=1∑(Ci/LCxi))=1, where II is the
interaction index, LCxi is
the concentration of the ith mixture
component that elicits x% effect when
applied singly, and Ci is the concentration of the
respective metal in the mixture when the mixture elicits the same effect. Each
(Ci/LCxi) is the concentration of a mixture component scaled
for its relative toxicity generally termed Toxic Unit (TU) of that component
[19].Using Statistix
appropriate statistical tools were applied to the results to determine  significant differences among the means of
the various toxicities, as well as to determine the existence and nature of
trends in the treatment level means. Regression analysis was done to determine
the correlation between the interaction index and the concentration of the
metals.


## 3. Results

Individual cytotoxicities of the four metals As, Cd, Hg, and Pb showed concentration-dependent
effects on MCF 7 cells (as shown Table1).  Hg was found to be the most toxic followed by
Cd, As, and Pb in descending order of toxicity. The mean LC50 for
Hg, Cd, As, and Pb were 0.565, 0.745,
1.65, and 24.5 mg/L, respectively. The order of toxicity of the four metals
remained the same at various LCs estimated. The coefficient of variation (CV) ranged from 1.97% to 37.29% indicating
good repeatability. There was higher variability in the mean lethal
concentrations at lower concentrations than at higher concentrations. Hg showed
the highest variability.Table 1
Individual toxicities (mean±SD) of the metals As, Cd, Hg, and Pb at various lethal concentrations
(LCs) with Hg being the most toxic and Pb the least toxic.
LCxConcentration
(mg/L)AsCdHgPbLC200.22±0.070.387±0.0030.11±0.0411±3LC300.78±0.020.52±0.050.30±0.0716±3LC401.22±0.080.64±0.060.4±0.120±2LC501.6±0.10.74±0.060.6±0.124±2LC602.1±0.20.85±0.070.7±0.128±1LC702.6±0.30.97±0.070.9±0.232±1LC803.3±0.41.12±0.081.1±0.237±1The concentrations
of each metal in the composite mixture eliciting LC20 to LC80 are shown in Table 2. The following ranking for toxicity of metals in the
mixture was obtained: Hg>Cd>As>Pb. The percent mortality of
cells increased with increasing concentration of the metal mixture. CVs ranged
from 7.24% to 13.36% indicating high repeatability. As expected, all the metals demonstrated
significantly (P=.0003) higher toxicity when they were in mixtures than when
they were administered individually (Tables 1 and 2).Table 2
Toxicities (mean±SD)
of the metals As, Cd, Hg, and Pb when present in a mixture at ratio of
10:5:2:15, respectively, at various lethal concentrations, with the four metals
showing higher toxicities than when used individually.
LCxConcentration
(mg/L)AsCdHgPbLC200.11±0.020.056±0.0080.022±0.0030.17±0.02LC300.31±.020.15±.010.061±0.0040.46±0.03LC400.47±0.040.23±0.020.093±0.0080.70±0.06LC500.62±.060.31±0.030.12±0.010.92±0.09LC600.77±0.080.38±0.040.15±0.021.1±0.1LC700.9±0.10.48±0.050.19±0.021.4±0.2LC801.1±0.10.57±0.060.23±0.031.7±0.2Toxic units (TU)
for various LCs were estimated by scaling the concentration of each mixture
component for its relative toxicity. Results are shown as a histogram in Figure1. The values for Cd increased with increasing concentration, while those for
As decreased with increaseing concentrations.  The values for Hg and Pb did not change over
the concentration range used in this study (Figure 1).  Pb had the lowest mean TU values (0.0353)
followed by Hg (0.218), Cd (0.381), and As (0.398).  From Figure 1, TU values for As and Cd showed
higher variability at lower concentration, and As appeared to contribute more to
the toxicity of the mixture (higher TU value) than all the other components of
the mixture. At higher concentrations, Cd became the highest contributor to the
toxicity of the mixture. All the TU values for each of the metals at all the
LCs were lower than one indicating synergistic effects.Figure 1
Toxic unit  (Mean±SD)  of TU of
the metals As, Cd, Hg, and Pb at various percentage cytolethalities. Cd shows
higher contribution to the toxicity at higher cytolethality percentages, whereas
Pb shows the least contribution among the four metals at all percentages.By using
randomized complete block analysis of variance, the mean values of the interaction
indices (II) calculated from TU values showed significant (P=.0036) differences
among the various LCs. At lower concentrations, the mean of the interaction
indices was less than one (LD20, II = 0.86) indicating a
synergistic effect among the four metals tested. The interaction at higher
concentrations is more than one (LD80, II = 1.13) indicating an
antagonistic effect among the four metals tested. Although the II values did
not differ significantly from unity, Tukey all pairwise comparison showed three
categories of II values, and also there was a general increase in the
interaction index with increase in concentration. The concentration-response
curves generated for the single metals and their composited mixtures are shown
in Figures 2(a) and 2(b). It could be seen that Pb showed significantly less
toxicity compared to the rest of the metals.(a) Concentration-percentage
mortality curves for the individual metals. The insert is an enlargement of the
curves in the concentration range of 0 to 5 mg/L (Pb showed low toxicity).  Each data point is the mean of four readings, and the errors bars represent standard deviation. (b) Concentration-percentage
mortality curve for the composite mixture of the metals. Concentration ratio of 1 contains each metal at its EPA MCL concentration.
(a)(b)


## 4. Discussion

The results of
this study clearly showed that the four metals (As, Cd, Hg, and Pb) were more
toxic when they were present in combination than when administered
individually.  At all percentage effects
tested, the concentrations estimated for individual toxicity were higher than
the concentrations for combined toxicity. The toxicities (LC50) of the four metals As, Cd, Hg, and Pb in mixture for 24-hour exposure
were 3, 3, 4, and 27 times higher than their individual toxicities, respectively
(Tables 1 and 2). The toxicity of  Pb (the least toxic metal among the four) was
enhanced  by several fold when in combination with the four metals. This indicates
that the cell detoxification process has been compromised by other components
of the mixture leading to increased toxicity of Pb. This agrees with the
observation made by Swiergosz-Kowalewska et al. [13]. They observed a negative
correlation between Cd level and GSH/GSSG ratio but positive correlation
between lead level and GSH/GSSG ratio and no significant effect of Pb level on
GSH. Also GSH-Cd complex has been reported to prevent intensive uptake of Cd
by cells [20, 21].It
has been suggested that either GSH or MT or both may play a major role during antagonistic
interactions among metals [12]. Antioxidative response (GSH and MT synthesis)
to metal intoxication in cells increases with increase in metal concentration [12–15]. The general increase in interaction index with increase in a concentration could be ascribed to the above observations. There was a positive
correlation (R2 = 0.946 at 95% confidence level) between interactive
indices and concentration of the metals. Results also showed differential interaction
among the four metals on MCF 7 cells in a concentration-dependent manner. At
lower concentration, the interaction index was less than unity indicating
synergistic interaction among the metals. As the concentration increased, the
interaction changed from synergistic to additive and finally antagonistic. Although
the interaction indices were not significantly different from unity, this
increase in interactive indices in concentration-dependent manner cannot be
ascribed to experimental variability alone due to the positive correlation
observed between the concentration and the interaction indices. Also, the trend
observed was partially consistent with results reported by Bae et al. [12] where antagonistic
effects were seen at higher concentrations and synergistic effects at lower
concentrations.  At higher concentration
of the metals, there is an increased level of detoxifying protein thereby
reducing the joint toxicity of the four metals [22, 23].The
mechanisms and the level of interaction of the four metals on MCF 7 cells need
to be fully established. To this end, further work is underway to profile the
level of GSH and MT in the cells with respect to the increasing concentrations
of the metals. Additional tests will also involve elimination of one of the four
metals at a time to identify the level of interaction.  Presently, studies being conducted on cells
with altered levels of GSH and/or MT to further explain the relevance of
detoxifying molecules in the interaction of the mixture of metals are nearing completion.In summary, a
combination of As, Cd, Hg, and Pb in the proportion of their MCLs showed three
types of interactions in MCF 7 cells in a concentration dependent-manner.  The combined toxicity changed from synergistic
to additive to antagonistic with increasing concentrations of the metal mixture.
These studies showed that multiple nonessential metals can exhibit
differential interactions in the environment and lead to complications in
predicting health risk to the environment.



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*Source: 789636-2009-05-05.xml*