Accepted Manuscript

Title: INTERACTIONS BETWEEN CADMIUM AND

DEKABROMINATED DIPHENYL ETHERS ON BLOOD
CELLS COUNT IN RATSMULTIPLE FACTORIAL
REGRESSION ANALYSIS
Author: Marijana Curcic Aleksandra Buha Sanja Stankovic
c Evica
Vesna Milovanovic Zorica Bulat Danijela ukic-Cosi
Antonijevic Slavica Vucinic Vesna Matovic Biljana
Antonijevic
PII:
DOI:
Reference:

S0300-483X(16)30051-8
http://dx.doi.org/doi:10.1016/j.tox.2016.05.011
TOX 51663

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Toxicology

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2-12-2015
3-3-2016
12-5-2016

Please cite this article as: Curcic, Marijana, Buha, Aleksandra, Stankovic,
c, Danijela, Antonijevic,
Sanja, Milovanovic, Vesna, Bulat, Zorica, ukic-Cosi
Evica, Vucinic, Slavica, Matovic, Vesna, Antonijevic, Biljana, INTERACTIONS
BETWEEN CADMIUM AND DEKABROMINATED DIPHENYL ETHERS ON
BLOOD CELLS COUNT IN RATSMULTIPLE FACTORIAL REGRESSION
ANALYSIS.Toxicology http://dx.doi.org/10.1016/j.tox.2016.05.011
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INTERACTIONS BETWEEN CADMIUM AND DEKABROMINATED DIPHENYL ETHERS ON

BLOOD CELLS COUNT IN RATS - MULTIPLE FACTORIAL REGRESSION ANALYSIS
Marijana Curcic1,*, Aleksandra Buha1, Sanja Stankovic2, Vesna Milovanovic3, Zorica
Bulat1, Danijela uki-osi1, Evica Antonijevi1, Slavica Vuini4, Vesna Matovi1,
Biljana Antonijevic1
1

University of Belgrade Faculty of Pharmacy, Department of Toxicology Akademik

Danilo Soldatovi, Vojvode Stepe 450, 11221 Belgrade, Serbia

2

Clinical centre of Serbia, Laboratory of Medical Biochemistry, Pasterova 2, 11000

Belgrade, Serbia
3

Ministry of Agriculture and environmental protection, Ruze Jovanovica 27, 11070

Belgrade, Serbia
5

National Poison Control Centre, Crnotravska 17, 11000 Belgrade, Serbia

Corresponding author: Marijana Curcic, Ph.D., Professor Assistant; University of

Belgrade Faculty of Pharmacy, Department of Toxicology Akademik Danilo
Soldatovi, Vojvode Stepe 450, Belgrade, Serbia
e-mail: makitox@pharmacy.bg.ac.rs; phone: +381 11 3951248; mobile: +381 63 643 803

Abstract: The objective of this study was to assess toxicity of Cd and BDE-209 mixture on
haematological parameters in subacutely exposed rats and to determine the presence and type
of interactions between these two chemicals using multiple factorial regression analysis.
Furthermore, for the assessment of interaction type, an isobologram based methodology was
applied and compared with multiple factorial regression analysis. Chemicals were given by
oral gavage to the male Wistar rats weighing 200- 240 g for 28 days. Animals were divided in
16 groups (8/group): control vehiculum group, three groups of rats were treated with 2.5, 7.5
or 15 mg Cd/kg/day. These doses were chosen on the bases of literature data and reflect
relatively high Cd environmental exposure, three groups of rats were treated with 1000, 2000
or 4000 mg BDE-209/kg/bw/day, doses proved to induce toxic effects in rats. Furthermore,
nine groups of animals were treated with different mixtures of Cd and BDE-209 containing
doses of Cd and BDE-209 stated above. Blood samples were taken at the end of experiment
and red blood cells, white blood cells and platelets counts were determined. For interaction

assessment multiple factorial regression analysis and fitted isobologram approach were used.
In this study, we focused on multiple factorial regression analysis as a method for interaction
assessment. We also investigated the interactions between Cd and BDE-209 by the derived
model for the description of the obtained fitted isobologram curves. Current study indicated
that co-exposure to Cd and BDE-209 can result in significant decrease in RBC count, increase
in WBC count and decrease in PLT count, when compared with controls. Multiple factorial
regression analysis used for the assessment of interactions type between Cd and BDE-209
indicated synergism for the effect on RBC count and no interactions i.e. additivity for the
effects on WBC and PLT counts. On the other hand, isobologram based approach showed
slight antagonism for the effects on RBC and WBC while no interactions were proved for the
joint effect on PLT count. These results confirm that the assessment of interactions between
chemicals in the mixture greatly depends on the concept or method used for this evaluation.

Keywords: cadmium; decabrominated diphenyl ether; interactions; multiple; factorial

regression

INTRODUCTION
Humans are exposed to complex mixtures of chemicals present in the environment,
therefore evaluation of mixture toxicity constitutes a critical research need in toxicology (EC,
2009; Kortenkamp et al., 2007; Spurgeon et al., 2010). Since metals and persistent organic
pollutants (POPs) are chemicals of great toxicological importance proved to pose threat to
human health (Buha et al., 2013, 2015; Curcic et al., 2015; Linares et al., 2015; Matovi et al.,
2015; Milovanovic et al., 2015), the toxicity of their mixtures should be evaluated. Several
studies on different mixtures of heavy metals and POPs have been already carried out (Buha
et al., 2013; Curcic et al., 2012, 2014; Xu et al., 2015; Zhang et al., 2012). Nevertheless in the
response to the growing concern about the potential toxicity of these important environmental
mixtures further experimental studies are essential.
Toxicological profile of cadmium (Cd) is well established (ATSDR, 2012) and recent
epidemiological evidence have shown that environmental exposure to this toxic metal
increases total mortality (Nawrot et al., 2010). Polybrominated diphenyl ethers are as flame
retardants with BDE-209 being the major congener of widely used decaBDE technical
mixture. After its release into environment through production processes or from commercial
mixture-containing products, and particularly from e-waste sites, these persistent chemicals
2

have potential for bioaccumulation and biomagnification through the food chain (Domingo et
al., 2008, 2012; Dorta et al., 2013; Hardy et al., 2008; Linares et al., 2015; Pereira et al.,
2015). The presence of both Cd and BDE-209 has been confirmed in various human tissues
(ATSDR, 2004 and ATSDR, 2012; EFSA, 2011, 2012; Fontain et al., 2008; Karlsson et al.,
2007; Roosens et al., 2010) thus raising the question whether their mixture can produce toxic
effects different from the effects of single chemicals.
The statistical and modeling approach used for the evaluation of interactions among
chemicals in the mixtures represents a great chalenge in chemical mixture toxicology
research. Different methods for the analyses of interactions have been proposed so far
(Gennings et al., 2005; Groten et al., 2001; Teuschler et al., 2002) indicating that different
methodologies can assess the risk differently. These findings point out that the development
of new and more advanced testing methodologies is crucial for the toxicology of mixtures.
The objective of this study was to assess toxicity of Cd and BDE-209 mixture on
haematological parameters in subacutely exposed rats and to determine the presence and type
of interactions between these two chemicals using multiple factorial regression analysis.
Furthermore, for the assessment of interaction type, an isobologram based methodology was
applied and compared with multiple factorial regression analysis.

MATERIALS AND METHODS

Chemicals
Decabrominated diphenyl ether (BDE-209, purity 98%) (Sigma-Aldrich, St. Louis,
MA, USA), cadmium-chloride (CdCl2xH2O, purity 99.99%) (Merck, Darmstadt, Germany)
and dimethyl sulfoxide (DMSO, 99.9%) (Sigma-Aldrich, St. Louis, MA, USA) were
purchased commercialy.
Experimental animals
Male Wistar rats were used in the experiment, body mass of 200-240 g. Animals were
kept in plastic cages, under controlled climate conditions, at the temperature of 20-24 C, air
humidity of 40-60% and dark:light cycles 12h:12h. Food and water were available ad libitum.
Treatment of experimental animals was in accordance with the guidance for the studies on
experimental animals No. 9667-1/2011 - Ethical Committee, Military Medical Academy,
Belgrade, Serbia.
Experimental protocol
After 14 days addaptation, animals (8 per group) were given orally, by gavage, single
oral dose Cd or BDE-209 or their mixtures in volume of 0.5 ml/kg, during 28 days. Design 24
3

was used in the experiment, as already described by Curcic et al. (2012). Control group was
treated with DMSO only. Three groups of rats were treated with 2.5, 7.5 or 15 mg Cd/kg/day.
These doses were chosen on the bases of literature data and reflect relatively high Cd
environmental exposure (ATSDR, 2012; Brzska et al., 2011; Buha et al., 2013; PiatMarcinkiewicz et al., 2001). Three groups of rats were treated with 1000, 2000 or 4000 mg
BDE-209/kg/bw/day, doses proved to induce toxic effects in rats (Curcic et al., 2015; van der
Ven et al., 2008). Furthermore, nine groups of animals were treated with different mixtures of
Cd and BDE-209 containing doses of Cd and BDE-209 stated above.
During the exposure, water and food intake were recorded weekly, while body weight,
clinical signs of toxicity and behaviour were monitored on a daily basis.
Blood samples were collected from the carotid artery and 6% K2EDTA was used as an
anticoagulant. Automatic commercial haematological analyser (Horiba ABS, PENTRA 80,
Paris, France) was used for blood cells counting: red blood cells (RBC), white blood cells
(WBC) and platelets (PLT).
Statistical data analysis
Statistically significant differences among data (p < 0.05) were tested using analysis of
variance (ANOVA) followed by post hoc Tukey test (Statistica 7.0 software).
Multiple factorial regression analysis was used to determine type of interactions
between Cd and BDE-209 (Bois et al., 1986; Gennings et al., 2005; Teuschler et al., 2002).
Independent variables (Cd and BDE-209 doses) were centered on the scale from -1 to +1 so
that the values of -1, -0.67, 0 and 1 corresponded to the doses of 0, 2.5, 7.5 and 15 mg Cd
/kg/day while the values of -1, -0.5, 0 and 1 corresponded to the doses of 0, 1000, 2000 and
4000 mg BDE-209 /kg/day.
Following equation of multiple factorial regression analysis was used:
Y = 0 + 1X1 + 2X2 + 12X1X2 + ...
where Y is dependent variable i.e. measured effect, X is independent variable corresponding
to centered dose of Cd or BDE-209, 0 is intercept, 1 and 2 are regression coefficient
evaluated by multiple factorial regression. Addend in equation 12X1X2 characterises the type
of interaction: if the value of 12 is positive, interaction is synergistic, when negative,
antagonism is assumed, while zero value implies additivity (Bois et al., 1986). Level of
statistical significance was set to be p < 0.05 (Statistica 7.0).
Software PROAST (RIVM, Bilthoven, Netherland) was used for the other approach
applied for the type of interactions assessment based on isobologram curve fitting. Fitted

isobologram curves were obtained on the basis of the values of RBC, WBC or PLT counts in
controls and in three groups treated with mixtures containing: lowest doses of both chemicals;
medium doses of both chemicals; highest doses of both chemicals. Derived models for the
description of isobologram curves can be linear or exponential. Linear model represents
additivity, while exponentional one suggests interactions; hence, deviation from the straight
line indicates synergism or antagonism. Benchmark doses (in software Critical Effect Dose CED) and lower confidence intervals of benchmark doses of 5% (CEDL5) were also derived
for the effects of mixtures on investigated haematological parameters (Slob, 2002).

RESULTS

Co-exposure to Cd and BDE-209 induced significant RBC decrease in four of totally

nine groups treated with mixtures when compared with controls. The same effect was
achieved with all doses of BDE-209, but only with the highest dose of Cd when given alone.
Significant elevation of WBC count when compared to the control group was obtained for all
the applied doses of BDE-209 given with the highest dose of Cd, for all the applied doses of
Cd given with the highest dose of BDE-209 and in the group treated with 7.5 mg Cd/kg
b.w./day and 2000 mg BDE-209/kg b.w./day. In the group treated with highest doses of both
substances significant difference was also obtained when compared with the group treated
with the highest dose of Cd only. Furthermore, the same effect was obtained for the mixture
of the highest dose of BDE-209 and the lowest dose of Cd when compared with the group
given the same BDE-209 dose only. Concerning the single chemicals effect on WBC count,
statistically significant increase was observed for the highest and medium doses of Cd and
BDE-209 when compared with controls. Application of mixtures did not induce significant
changes in PLT count if compared with controls. Generally, mixtures did not produce
significant changes in PLT levels if compared with PLT count obtained in groups given Cd or
BDE-209 only, i.e. statistical significant increase was observed just in one group when
compared with Cd groups and only in two groups if compared with BDE-209 groups.
Decabrominated diphenyl ether induced significant decrease in PLT count in all investigated
groups when compared with controls, while Cd did not cause any effect. All the statistical
differences were obtained by ANOVA followed by post hoc Tukey test (Table 1).

In order to define whether interactions between Cd and BDE-209 occur, as well as to

determine the type of interactions, two approaches were used: multiple factorial regression
and the methodology based on isobologram fitted curves.
Results of multiple factorial regression analysis are presented in Table 2. Significance
was observed only for RBC count with 12 0.713, indicating synergism between Cd and BDE209.

The obtained results of multiple factorial regression analysis are also presented in Figures 1, 2
and 3, as 3D surface plot (A) and 2D plot obtained by 3D surface rotation (B).

The type of interactions was also defined by the application of fitted isobologram
curves and contrary to multiple factorial regression analysis, the shape of isobologram
suggested slight antagonism between Cd and BDE-209 effect on RBC count (Figure 4A).
Slight antagonism was also observed for the effect on WBC count (Figure 4B), while no
interaction, i.e. additivity occurred for the effect on PLT count since linear model was
observed (Figure 4C).
Following CEDL5 values for mixtures were calculated: 0.353 (corresponding to 10.15
mg Cd/kg/day and 2706 mg BDE-209/kg/day) and 0.151 (corresponding to 8.63 mg
Cd/kg/day and 2302 mg BDE-209/kg/day), for the effects on RBC and WBC counts,
respectively. CEDL5 value for platelets was not calculated since a linear model (E1) was
obtained indicating no dose response relationship (Figure 4). Based on the calculated CEDL5
value for mixtures, the effect of mixtures on WBC count could be regarded as a critical one.

Data are fitted with PROAST software. CES (critical effect size) for this endpoint was set at 5% (horizontal
dotted line). Corresponding CED (critical effect dose) is represented by the vertical dotted line. L05 and L95
represent the lower and upper bound of 95% confidence level.

DISCUSSION
Toxicological evaluations of chemical mixtures, although recognised for many years
as a reason for human health and environmental concern, still represent great challenge for
scientific community (Kortenkamp et al. 2007). It is well known that even low level exposure
over decades to a complex cocktail of pollutants, especially persistent ones, can result in

toxicological risk, although exposure to single components does not induce adverse effects.
Furthermore, mixtures can induce toxicological outcomes that can differ from the effects of
single chemicals as a result of their interactions (Cassee et al. 1998; Kortenkamp et al. 2007).
In this study toxicity of Cd and BDE-209 mixture was investigated on hematopoietic
system as one of the most sensitive systems and therefore suitable to evaluate the hazardous
effects of chemicals (Hfer et al., 2004). Furthermore the effects of single chemicals on RBC,
WBC and PLT counts were assessed, giving the possibility to determine whether these
chemicals act in an additive, antagonistic or synergistic manner.
A wide variety of concepts and methods were proposed to study the presence and type
of interactions between components in mixtures, depending on the number of chemicals in the
mixture and on the extent to which the toxicity of mixture needs to be assessed. Hence,
making the right choice is of paramount importance for a successful mixture toxicity study
(Feron and Groten, 2002; Groten, 2000). In this study, we focused on multiple factorial
regression analysis as a method for interaction assessment proposed by Bois et al. (1986).
Multiple factorial regression analysis results in the equation that reflects response surface
model providing that all 16 groups used in our 24 design are covered and involved in
conclusion on interactions between chemicals. We also investigated the interactions between
Cd and BDE-209 by the derived model for the description of the obtained fitted isobologram
curves (Slob, 2002), although this approach considers only the effect of 4 groups used in our
24 design.
Current study indicated that co-exposure to Cd and BDE-209 can result in significant
decrease in RBC count when compared with controls. The same effect was observed for all
doses of BDE-209 and only for the highest dose of Cd given alone. Cadmium ability to
reduce RBC count has been already documented and was explained by suppression of
hematopoietic tissues, impaired erythropoesis and altered RBC membrane permeability
(Ashour and El-Shemi, 2014; El-Demerdash et al., 2004; Horiguchi et al., 2011). The
observed effect of BDE-209 on RBC could be at least partly explained by impaired RBC
membrane permeability caused by oxidative stress induction since our previous in vitro study
(Curcic et al., 2014) as well as in vivo study (Milovanovi et al., 2015) confirmed BDE-209
prooxidative activity. Co-exposure to Cd and BDE-209 induced increase of WBC count if
compared to controls and the same effect was obtained for both toxic agents when given
alone. Elevated number of WBC could be explained by inflammatory processes documented
for Cd and certain BDE congeners (Demensku et al., 2014; Krocova et al., 2000; Xu et al.,
2015). Mixture of Cd and BDE-209 as well as Cd given alone did not influence PLT count,
7

although BDE-209 treatment induced decrease of PLT count if compared with controls. Our
findings indicate that mixture of Cd and BDE-209 can alter immune response and can
produce toxic effect on haematological system.
Multiple factorial regression analysis used for the assessment of interactions type
between Cd and BDE-209 indicated synergism for the effect on RBC count and no
interactions i.e. additivity for the effects on WBC and PLT counts. On the other hand,
isobologram based approach showed slight antagonism for the effects on RBC and WBC
while no interactions were proved for the joint effect on PLT count. These results confirm that
the assessment of interactions between chemicals in the mixture greatly depends on the
concept or method used for this evaluation.
Analysis of presence and type of interactions between investigated chemicals by
multiple factorial regression analysis and isobologram based approach did not give coherent
results indicating that determining more reliable concepts for assessment of interactions
between chemicals remains daunting challenge in the toxicology of mixtures.

Acknowledgement
This work was partly supported by the Ministry of Education, Science and
Technological Development of Serbia (Project III 46009).
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11

Figure 1.The impact of different centered doses of Cd and/or BDE-209 on RBC count: 3D
response surface plot (A) and 2D plot obtained by rotation of 3D response surface plot (B)

Figure 2. The impact of different centered doses of Cd and/or BDE-209 on WBC count: 3D
response surface plot (A) and 2D plot obtained by rotation of 3D response surface plot (B)

12

Figure 3. The impact of different centered doses of Cd and/or BDE-209 on PLT count: 3D
response surface plot (A) and 2D plot obtained by rotation of 3D response surface plot (B)

Figure 4. Isobologram fitted curves for the effect of different centered doses of Cd and/or
BDE-209 on RBC (A), WBC (B) and PLT (C).

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Table 1. Blood cells counts determined in blood of rats orally exposed to different doses of
Cd and/or BDE-209 during 28 days.
Cd doses
(mg/kg/day)

BDE-209 doses
(mg/kg/day)

RBC (x 106/l)

WBC (x 103/l)

PLT (x 103/l)

7.9 1.6

11.6 4,4

952.2 192.0

2.5

6.1 1.5

18.3 1.0

750.1 122.9

7.5

6.2 1.4

18.8 2.9 a

753.2 313.1

15

5.1 0.6 a

20.2 3.1 a

609.5 137.9

1000

5.0 0.8 a

17.8 1.3

515.0 272.5 a

2000

5.1 1.4 a

20.1 3.0 a

581.5 282.3 a

4000

5.3 1.1 a

23.9 6.1 a

572.7 259.6 a

2.5

1000

6.0 1.2 a

15.7 1.4

797.0 32.3

7.5

1000

6.5 0.8

18.1 0.7

876.5 258.1

15

1000

5.9 0.8 a

22.0 4.3 a

634.5 299.6

2.5

2000

5.8 0.9

16.8 2.3

733.7 229.2

7.5

2000

6.1 1.9 a

19.9 4.7 a

984.2 351.7 b

15

2000

6.7 1.2

25.2 6.9 a

655.5 331.3

2.5

4000

6.5 1.4

17.8 4.0 a,b

1141.3 259.4 b,c

7.5

4000

6.7 1.5

20.7 2.9 a

725.0 251.1

15

4000

6.3 1.6

28.9 11.1 a,c

771.5 201.6

Values are presented as the means SD (n = 8). Statistically significant differences (ANOVA, Tukey test)
obtained for each parameter between groups are indicated by: a vs. control, b mixtures vs. corresponding BDE209 group, c mixtures vs. corresponding Cd group; p < 0.05.

Table 2. Blood cells counts determined by multiple factorial regression analyses.

Blood cells count (Y)

RBC (x 106/l)

= 6.149 + 0.043 X1 - 0.013 X2 + 0.713 X1 X2

WBC (x 103/l)

= 20.489 + 3.609 X1 + 3.247 X2 - 0.444 X1 X2

PLT (x 103/l)

= 756.5 21.1 X1 + 32.9 X2 + 47.9 X1 X2

1X1 +

2X2 +

12X1X2

X1 centered dose of Cd; X2 - centered dose of BDE-209; bolded coefficient indicates significant interaction
between Cd and BDE-209, p < 0.05.

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