Hindawi Publishing Corporation
Journal of Toxicology
Volume 2013, Article ID 545802, 11 pages
��eci�c Metabolic Fin�er�rint of a Dietary E��osureto
a Very LowDose of Endosulfan
Cécile Canlet, Marie Tremblay-Franco, Roselyne Gautier, Jérôme Molina, Benjamin Métais,
FlorenceBlas-YEstrada, and LaurenceGamet-Payrastre
INRA, TOXALIM (Research Centre in Food Toxicology), UMR 1331, INRA/INP/UPS, 31027 Toulouse, France
Correspondence should be addressed to Laurence Gamet-Payrastre; firstname.lastname@example.org
Received 13 July 2012; Accepted 20 November 2012
Academic Editor: M. Teresa Colomina Fosch
Copyright © 2013 Cécile Canlet et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Like other persistent organochlorine pesticides, endosulfan residues have been detected in foods including fruit, vegetables, and
�sh. e aim of our study was to assess the impact of a dietary exposure to low doses of endosulfan from foetal development
until adult age on metabolic homeostasis in mice and to identify biomarkers of exposure using an1H-NMR-based metabonomic
approach in various tissues and bio�uids. �e report in both genders an increase in plasma glucose as well as changes in levels of
factors involved in the regulation of liver oxidative stress, con�rming the prooxidant activities of this compound. �ome metabolic
changes were distinct in males and females. For example in plasma, a decrease in lipid LDL and choline content was only observed
in female. Lactate levels in males were signi�cantly increased. In conclusion, our results show that metabolic changes in liver could
be linked to the onset of pathologies like diabetes and insulin resistance. Moreover from our results it appears that the NMR-based
metabonomic approach could be useful for the characterization in plasma of a dietary exposure to low dose of pesticide in human.
Many epidemiological studies have shown that exposure to
pesticides is a risk factor for human health, as evidenced
by the positive correlation between professional exposure to
these compounds and an increase in the incidence of various
human diseases (reviewed in Merhi et al. ). e general
population is also exposed to pesticides mainly via food
intake. us many people have a lifelong exposure to low
doses of pesticides, the impact of which on human health is
not yet known.
Organochlorine (OC) pesticides are among the most
frequent contaminants found in the environmental com-
partments because they persist in the environment and
bioaccumulate in organisms, partly due to their lipophilic
properties . Endosulfan is a chlorinated cyclodiene pesti-
several European Union countries. Nevertheless, the general
population continues to be exposed: like other persistent
organochlorine pesticides, endosulfan residues have been
to humans through both accidental and intentional exposure
as a moderately hazardous (class II) pesticide ; however,
it is genotoxic  and is an endocrine disrupter displaying
xenooestrogenic activity . Endosulfan has been shown to
be toxic to the liver, kidney, nervous system, and repro-
ductive organs of laboratory animals [8–10]. Exposure to
this compound can modify the activity of some enzymes
involved in oxidative stress and xenobiotic metabolism, as
well as testosterone metabolism and clearance . Recently
Casabar et al.  showed that endosulfan was a strong
activator of the pregnane X receptor (PXR) and an inducer
of cytochrome P450 (CYP) 2B6 and CYP3A4, so it may
have an impact on the metabolism of their substrates. A
with endosulfan led to changes in glucose levels, histological
degenerative changes in the pancreas and endocrine distur-
bances. Endosulfan may also be a risk factor for children
whose parents have been exposed since the accumulation of
2 Journal of Toxicology
organochlorine compounds in fat tissue during the mother’s
. e transfer of OC pesticides from mother to foetus
was demonstrated via the detection of the pesticides in the
the newborn . Moreover, Pathak et al.  suggested that
levels of some organochlorine pesticides such as endosulfan
in the maternal �uid or tissue are associated with preterm
delivery and increased foetal oxidative stress. Pre- and post-
natal exposure to endosulfan is reported to affect biogenic
amines and amino acids in the prefrontal cortex . Whilst
the toxicological effects of endosulfan have been studied in
vitro and in vivo, the impact of long-term exposure to low
stage to adulthood has not yet been reported.
An NMR spectroscopy-based metabonomic approach
coupled with pattern recognition technology is an effec-
tive way of characterizing the biochemical response of an
organism to contaminant exposure and identifying potential
biomarkers of exposure [16, 17]. Metabonomics has proven
of many toxic compounds [18–20].
the impact of dietary exposure to a low dose of endosulfan
on metabolic homeostasis in mice exposed from foetal
development until adult age. We investigated biomarkers
of dietary exposure using an1H-NMR-based metabonomic
approach in various tissues and bio�uids.
2. Materials and Methods
2.1. Reagents. Endosulfan was purchased from Fluka (Riedel
de Haën, France) and was added as a component of rodent
nuggets at a dose of 30𝜇𝜇g per kg. is dose allowed the
Joint FAO/WHO Meeting on Pesticide Residues  that
was extrapolated for mice on the basis of mean body weight.
Information on the toxicity of endosulfan was obtained from
Agritox (AFSSA, 2005. http://www.dive.afssa.fr/agritox/) or
ExToxNet (ExToxNet: http://extoxnet.orst.edu/).
(1.5mg) was dissolved in 1ml methanol, and then 100𝜇𝜇L of
mice to ingest the equivalent of the Acceptable Daily Intake
(ADI 0.006mg/kg food/day)) de�ned for humans by the
2.2. Preparation of Pesticide-Enriched Feed. Endosulfan
methanol (�nal quantity of endosulfan 150𝜇𝜇g). e whole
mixture powder (vitaminic mixture 200, Scienti�c animal
Food Engineering (Safe), France). e vitamin powder
rotavapor for 30 minutes at 45∘C to evaporate the solvent
and then mixed for 50 minutes at room temperature with the
the rodent nuggets (5kg). e vitamin powder enriched
with endosulfan was then sent to the INRA animal feed
preparation unit (UPAE INRA Paris France) where vitamins
this solution (containing 150𝜇𝜇g endosulfan) was mixed with
with acetone (9mL) and dispersed on 10g of the vitamin
containing 150𝜇𝜇g endosulfan was then homogenized in a
resulting solution (containing 150𝜇𝜇g endosulfan) was mixed
(1%) and mineral complement (7%) were mixed with the
other components of the rodent nuggets. e presence of
endosulfan was quanti�ed by Euro�ns (Nantes, France).
e �nal quantity was around 30𝜇𝜇g/kg of food. Control
feed was analyzed for the presence of the endogenous
pesticide (endosulfan) and also for the presence of the most
common pesticides found in the environment (including
the organophosphorus pesticide organochloride, pyrethroid,
and PCB). Results showed that nuggets in control food do
not contain pesticides at the detectable level (0.01mg/kg).
feed was prepared as described above with a mixture of
methanol/acetone (1/1, V/V) but lacking pesticide. Control
were purchased from Charles River Laboratories, France.
Mean body weights were 20 and 24g for females and males,
respectively. Females (5 per lot) were fed from mating until
gestation and lactation with the control diet or the diet
enriched with pesticide. Weaned pups (from 6 to 8 per lot)
were then fed with the same diet as their respective parent
for an additional period of 11 weeks. Food consumption
was monitored and the body weight was measured. Animals
were sacri�ced by cervical dislocation, and the spleen, liver,
and kidneys were weighed. Bone marrow was extracted from
the femur of treated or nontreated males and females as
previously described [1, 22]. e liver was excised, weighed,
and frozen rapidly in liquid nitrogen. In addition, blood
samples were taken from the facial artery of each animal
and added to a glass vial containing heparin (Multivette,
2.4. Metabonomic Studies
2.4.1. Sample Preparation for1HNMR Spectroscopy. Plasma
samples (100𝜇𝜇L) were diluted with 500𝜇𝜇L deuterium oxide
sonicated in an ice bath for 1min. Samples were centrifuged
(D2O) before being placed in 5mm NMR tubes.
Bone marrow cells were mixed with 600𝜇𝜇L D2O then
transferred to NMR tubes.
For liver samples the organ extraction method used here,
which derives from the Folch procedure , was adapted
from the method described by Waters et al. . Samples of
for 10min at 4∘C at 5000g, and the supernatants were
liver tissue (∼100mg) were homogenized with a Polytron PT
resulting powders were reconstituted in 600𝜇𝜇L of deuterium
184.108.40.206H Nuclear Magnetic Resonance (NMR) Analyses. All
nance frequency using an inverse detection 5mm1H-13C-
2100 in 2mL acetonitrile/H2O (50/50, v/v) containing 0.1%
and the supernatants were removed and lyophilised. e
oxide (D2O). e reconstituted solutions were transferred to
15N cryoprobe attached to a CryoPlatform (the preampli�er
butylated hydroxytoluene (BHT) in an ice-water bath. e
homogenates were centrifuged for 10min at 4∘C at 5000g,
NMR spectrometer operating at 600.13MHz for1H reso-
Journal of Toxicology
e1HNMR spectra of plasma and bone marrow sam-
Gill (CPMG) spin-echo pulse sequence with presaturation
with a total spin-echo delay (2n𝜏𝜏) of 40ms to attenuate
width of 12ppm and an acquisition time of 2.28s. Prior
to Fourier transformation, an exponential line broadening
function of 0.3Hz was applied to the FID.
300K using the standard1H pulse sequence, accumulating
128 free induction decays into 32K data points with a
relaxation delay of 2s. A 12ppm spectral width was used.
e data were apodized with an exponential function using
a line broadening of 0.3Hz prior to Fourier transformation.
To con�rm the chemical structure of the metabolites of
1H-13C HSQC (Heteronuclear Single Quantum Coherence
broad signals from proteins and lipoproteins. A total of 128
transients were acquired in 32K data points using a spectral
e1HNMR spectra of liver extracts were acquired at
samples. For the1H-H COSY NMR experiment, a total of
32 transients were acquired in 4096 data points. A total of
256 increments were measured in F1 using a spectral width
of 10ppm and an acquisition time of 0.28s. e data were
Fourier transformation (FT).1H-13C HSQC NMR spectra
were collected in selected samples with
relaxation delay of 2.5s was used between pulses, with a
weighted using a sine-bell function in 𝑡𝑡1and 𝑡𝑡2prior to
refocusing delay equal to 1/41𝐽𝐽C−H(1.78ms). A total of 2048
180ppm in F1. e data were multiplied by a shied Qsine-
bell function prior to FT.
Spectral assignment was based on matching 1D and 2D
data to reference spectra in a home-made reference database,
as well as with other databases (http://www.bmrb.wisc.edu/
and http://www.hmdb.ca/), and reports in the literature.
1H detection. A
were acquired with spectral widths of 10ppm in F2 and
2.4.3. Data Reduction and Multivariate Statistical Analyses.
lytik) to integrate 0.04ppm wide regions corresponding to
the 𝛿𝛿 10.0–0.5ppm region. e 𝛿𝛿 5.1–4.5ppm region, which
was normalized to the total spectral area. Multidimensional
statistical analyses of NMR data were performed using
Simca-P11 soware (Umetrics, Umeå, Sweden). Principal
components analysis (PCA) was applied to the pareto-
scaled spectral data to reveal treatment-related patterns.
Projection to latent structure discriminant analysis (PLS-
DA) was also performed to improve the classi�cation of the
different groups of mice. Dummy variables, containing 0 and
1, were created to describe the class membership of each
observation. e number of components in the PLS models
and 𝑄𝑄2were used as measures for the robustness of a pattern
includes the water resonance, was excluded. To account
for differences in sample amounts, each integrated region
was chosen by cross-validation (7-fold). e parameters 𝑅𝑅2
recognition model. 𝑅𝑅2is the fraction of variance explained
by a component, and cross-validation of 𝑅𝑅2gives 𝑄𝑄2which
is test can determine whether the speci�c classi�cation of
individuals in the designated groups is signi�cantly better
than any other random classi�cation in two arbitrary groups
this study), while the 𝑋𝑋 matrix (NMR buckets) is kept
values calculated and compared
to the values of the “original” model. As a result of this
values of the “original” model (on the right
of biplot) and the “permuted” models are displayed versus
biplot. Valid models correspond to a negative 𝑄𝑄
tion (OSC) with the treatment as a correction factor. e
to the treatment (confounding factors such as physiological,
experimental, or instrumental variation).
e identi�cation of major metabolic perturbations
within the pattern recognition models was achieved by anal-
ysis of corresponding loading plots and variable importance
in the projection (VIP). VIP, a global indicator measuring
the in�uence of each variable on the PLS components, was
separation of experimental groups. An arbitrary threshold of
reveals the fraction of the total variation predicted by a
component; it is a measure of the difference between two (or
more) groups. Typically a robust model has 𝑅𝑅2> 50% and
In addition, the statistical signi�cance and validity of
the PLS-DA results were assessed using a permutation test.
. In this procedure, the 𝑌𝑌 matrix (group membership)
unchanged. For each “permuted” sample, a PLS-DA model
is randomly permuted a number of times (200 times in
is constructed, the 𝑅𝑅
the correlation between the “original” 𝑌𝑌 matrix and the
the regression line.
VIP >1.0 was chosen to select the variables.
Imported data were mean centered and Pareto scaled for
all PLS analyses.
Univariate statistical tests using 𝑅𝑅 soware were also
and liver samples. Each bucket was treated as an indepen-
dent variable. Statistically signi�cant changes between the
distributions of treated animals and the control group were
was considered signi�cant. e soware 𝑅𝑅 and the packages
endosulfan-treated to control mice for bone marrow, plasma
assessed using a parametric Student’s 𝑡𝑡-test as well as the
used in this work were downloaded freely from the website
nonparametric Kruskal-Wallis test. A value of 𝑃𝑃 ? 0.05
weight between endosulfan-treated and control groups, and
there was no difference in the weight of the liver, or brain
between the groups (data not shown). However a slight but
signi�cant decrease was observed in kidney in females.
4Journal of Toxicology
F 1: PLS-DA score plot based on the
exposed male (red circle, 𝑛𝑛
1HNMR spectra of
plasma samples from nonexposed males(green triangle, 𝑛𝑛 ? ?),
??), nonexposed females (blue
diamond, 𝑛𝑛 ? ?), and exposed female mice (black square, 𝑛𝑛 ? 1?)
(𝑄𝑄?? 0.?0, 𝑅𝑅?? ??.?%, and A ? 3 (Latent Variables �tted)).
To identify the metabolic �ngerprint characterizing
dietary exposure to endosulfan at low doses, high-resolution
1HNMR spectra were recorded from samples of bone mar-
row, plasma, and liver of control and endosulfan-treated
14-week-old mice. irty-one metabolites were identi�ed,
and Table 1 lists the compounds assigned based on their
1D and 2D spectra. From these results, PLS-DA analysis of
each tissue was carried out as described in the materials
and methods section; this highlighted the different effects of
pro�les of each gender are quite distinct, female and male
mice were considered independently in order to identify
the metabolic changes resulting from endosulfan treatment.
Intercept values of permutation test, showing the robustness
of the models, are presented in Table 2 for PLS-DA models
per sex. All metabolic changes are summarized in Table 3.
�.�. Meta�olic �ro�les of �las�a fro� Endosulfan-Treated
Mice. Pattern recognition of the
plasma of mice exposed to dietary endosulfan is shown in
Figure 1. e PLS-DA score plots clearly distinguish samples
of exposed mice from those of nonexposed mice along PLS-
DA component 1. PLS-DA models were built to model the
differences between endosulfan-exposed males separately to
those of females (Figures 2(a) and 2(c)). ese models were
and 𝑅𝑅?? 0.??? and 𝑄𝑄?? 0.83 for males). Permutation tests
in treated and untreated animals, in females and males,
respectively. Discriminant metabolites were summarized in
Table 3. Dietary exposure of female mice to endosulfan led
to a decrease in plasma LDL and/or VLDL. Biochemical
(data not shown). Dietary exposure of females to endosulfan
also led to a signi�cant decrease in choline levels and a
clear increase in glucose levels. e categories of metabolites
identi�ed in plasma samples from exposed male mice were
1HNMR spectra from
robustandpredictable(𝑅𝑅?? 0.?8?and𝑄𝑄?? 0.?5forfemales
showed that the model was not over�tted (Table 2). Loading
plots in Figures 2(b) and 2(d) show discriminant metabolites
levels which were also observed in both genders. Lactate
levels were increased in males exposed to endosulfan but no
�.�. Meta�olic �ro�les of �one Marro� fro� Endosulfan-
and methods section, separated all four groups of bone mar-
rowsamplesasshowninFigure3(𝑅𝑅?? 0.??5and𝑄𝑄?? 0.?4).
component 1, whereas animal gender appears separately
along PLS-DA component 2. To further clarify the impact of
treatment in the sample groups of each gender, other PLS-
male and female sample groups as shown in Figures 4(a) and
Bone marrow from endosulfan-exposed and nonexposed
mice appear as distinct metabolic groups along PLS-DA
4(c). For both gender groups the1HNMR pro�les derived
from endosulfan-treated mouse samples were distinguished
from those of the controls using PLS-DA analysis. ese
𝑄𝑄?? 0.?? for females and 𝑅𝑅?? 0.??5 and 𝑄𝑄?? 0.??
4(d) show discriminant metabolites in treated and untreated
animals in females and males, respectively. Discriminant
metabolites were summarized in Table 3. For both female
and male mice dietary exposure to endosulfan resulted in a
decrease in fatty acids in bone marrow, characterized by the
models were robust and predictable (𝑅𝑅?
for males). Permutation tests showed that the model was
not over�tted (Table 2). Loading plots in Figures 4(b) and
? 0.??? and
long chain lipid (CH?)𝑛𝑛signal and by an increase in valine
and glutamate/glutamine were increased; in contrast male
mice had increased levels of acetate, tyrosine, and inosine
upon exposure to endosulfan whereas the level of lactate and
ATP/ADP/AMP decreased. e level of choline compounds
and isoleucine. Gender differences were clearly observed:
in treated females the levels of lysine, alanine, succinate,
�.�. Meta�olic �ro�les of ��ueous �i�er E�tracts fro�
Endosulfan-Treated Mice. e PLS-DA score plots of liver
samples clearly separated exposed mice from nonexposed
mice along PLS-DA component 1 (Figure 5). Gender was
the distinction between male and female mice was not clear.
PLS-DA models were then built to model the differences
between exposed males and females separately (Figures 6(a)
0.?84 and 𝑄𝑄?? 0.??5 for females and 𝑅𝑅?? 0.??1 and 𝑄𝑄??
6(d) show discriminant metabolites in treated and untreated
animals, in females and males respectively. Discriminant
metabolites were summarized in Table 3. In males and
females, hepatic metabolic disturbances were characterized
by a net decrease-oxidized glutathione concomitantly with
an increase of taurine and betaine levels, suggesting a per-
turbation of oxidative status upon endosulfan exposure. In
females glucose levels were decreased whereas lactate levels
and 6(c)). ese models were robust and predictable (𝑅𝑅??
not over�tted (Table 2). Loading plots in Figures 6(b) and
Journal of Toxicology5
T 1:1H and13C resonance assignments with chemical shis, multiplicity, and 𝐽𝐽-couplings for signals identi�ed in plasma, bone marrow,
Acetate 1.92 (s, CH3)/25.9
and liver in mice.
𝛿𝛿Hppm (multiplicity, coupling constant, assignment)/𝛿𝛿CppmBiological matrice
Liver, bone marrow
1.48 (d, 𝐽𝐽 = 7.1Hz, CH3)/18.9; 3.78 (q, 𝐽𝐽 = 7.2Hz, CH)/53.1
3.27 (s, CH3)/56.1; 3.91 (s, CH2)/68.9;
2.54 (d, 𝐽𝐽 = 15Hz, 2CH)/48.7; 2.70 (d, 𝐽𝐽 = 15Hz, 2CH)/48.7
3.41 (m, CH)/72.3; 3.54 (m, CH)/73.9; 3.71 (m, CH)/75.4; 3.83 (m, CH)/74.2; 3.84 (m,
CH)/63.4; 4.64 (d, CH, 𝐽𝐽 = 8Hz)/98.6
2.16 (m, CH2)/29.1; 2.46 (m, CH2)/33.6; 3.78 (m, CH)/56.7
4.51 (m, CH2)/73.3; 6.15 (d, 𝐽𝐽 = 5.9Hz, CH)/89.5; 8.27 (s, CH, ring)/155.2; 8.58 (s, CH,
Liver, bone marrow
Liver, plasma, bone
Plasma, bone marrow
Liver, plasma, bone
Choline 3.22 (s, N(CH3)3)/56.9; 3.52 (m, NCH2)/70.0; 4.05 (m, CH2)/58.3
3.93 (s, CH2)/56.4; 3.03 (s, CH3)/39.9
CH)/63.3; 5.23 (d, CH, 𝐽𝐽 = 3.8Hz)/94.8
2.06 (m, CH2)/29.6; 2.36 (m, CH2)/36.1; 3.78 (m, CH)/56.7
3.25 (m, CH)/76.9; 3.41 (m, CH)/72.3; 3.48 (m, CH)/78.4; 3.73 (m, CH)/63.3; 3.90 (m,
Liver, bone marrow
Liver, plasma, bone
2.18 (m, CH2)/29.1; 2.55 (m, CH2)/34.2; 2.98 (m, CH)/41.7; 3.31 (m, CH)/41.6; 3.78 (m,
CH2)/46.2; 3.78 (m, CH)/56.7
Liver, bone marrow
Glycerophosphocholine 3.22 (s, N(CH3)3)/56.9; 3.68 (m, NCH2)/68.9; 4.33 (m, CH2)/62.1
Liver, plasma, bone
Liver, plasma, bone
Glycine3.56 (s, CH2)/44.3
4.28 (m, CH)/88.2; 4.44 (m, CH)/73.1; 6.11 (d, 𝐽𝐽 = 5.7Hz, CH ring)/90.9; 8.23 (s, CH
0.94 (d, 𝐽𝐽 = 6Hz, CH3)/23.6; 0.97 (d, 𝐽𝐽 = 6Hz, CH3)/24.7; 1.71 (m, CH)/27.3; 1.71 (m,
0.88 (m, CH3); 1.30 (m, (CH2)n); 1.40 (m, CH2); 5.30 (m, CH = CH)
0.87 (m, CH3)/14.7; 1.28 (m, CH2)/34.6;
2.12 (s, CH3); 2.18 (m, CH2); 2.64 (t, 𝐽𝐽 = 7.2Hz, CH2)
Glycogen3.66 (m, CH)/79.3; 3.98 (m, CH)/75.8; 5.41 (m, CH)/102.2;
0.93 (t, 𝐽𝐽 = 7.2Hz, CH3)/13.9; 0.99 (d, 𝐽𝐽 = 7.2Hz, CH3)/17.4
ring)/148.9; 8.32 (s, CH ring)/142.7
1.33 (d, 𝐽𝐽 = 7.2Hz, CH3)/22.9; 4.11 (q, 𝐽𝐽 = 7.2Hz, CH)/71.2
Liver, plasma, bone
Lipids (LDL, VLDL)
Plasma, bone marrow
Liver, plasma, bone
Lysine1.48 (m, CH2)/24.6; 1.72 (m, CH2)/29.1; 1.91 (m, CH2)/32.7; 3.01 (m, CH2)/42.1
7.60 (dd, 𝐽𝐽 = 7.9 and 5Hz, CH ring); 8.25 (m, CH ring); 8.58 (m, CH ring); 8.92 (s, CH
Phenylalanine7.43 (t, 𝐽𝐽 = 7Hz, CH ring); 7.38 (t, 𝐽𝐽 = 7Hz, CH ring); 7.32 (d, 𝐽𝐽 = 7Hz, CH ring)
3.22 (s, N(CH3)3)/56.9; 3.60 (m, CH2)/69.4; 4.18 (m, CH2)/60.7
3.26 (t, 𝐽𝐽 = 7.3Hz, CH2)/49.9; 3.43 (t, 𝐽𝐽 = 7.3Hz, CH2)/38.1
6.90 (m, CH ring); 7.18 (m, CH ring)
(d, 𝐽𝐽 = 8.10Hz, CH ring)/144.5;
Liver, plasma, bone
Liver, plasma, bone
Liver, plasma, bone
Liver, plasma, bone
4.14 (m, CH)/86.9; 4.38 (m, CH)/76.4; 5.88 (m, CH ring)/104.9; 5.90 (m, CH)/92.1; 7.88
0.99 (d, 𝐽𝐽 = 7Hz, CH3)/19.5; 1.05 (d, 𝐽𝐽 = 7Hz, CH3)/20.7; 2.28 (m, CH)/31.9
6 Journal of Toxicology
0.5 0.7 0.9
−0.1 0.3 0.1
0.5 0.7 0.9
F 2: PLS-DA (a) score plot and (b) loading plot based on the1HNMR spectra of plasma samples from nonexposed (blue diamond,
𝑅𝑅?? 9?.?%, and A ? 3).
𝑛𝑛 ? ?) and exposed female mice (black square, 𝑛𝑛 ? 1?) (𝑄𝑄?? 0.95, 𝑅𝑅?? 98.9%, and A ? ?). PLS-DA (c) score plot and (d) loading plot
T 2: Intercept values (𝑅𝑅
based on the1HNMR spectra of plasma samples from nonexposed (green triangle, 𝑛𝑛 ? ?) and exposed males (red circle, 𝑛𝑛 ? 9) (𝑄𝑄?? 0.83,
?) of permutation test for the PLS-DA models.
of a purely random model. 𝑅𝑅
intercept <0.05 indicate a robust model.
e intercept values (𝑅𝑅
were increased. By contrast in male mice dietary exposed
to endosulfan, no changes were observed in glucose level
whereas lactate level was decreased. In addition, in male
mice, 5 metabolites were found to distinguish from females:
alanine, glutamate/glutamine, and glycogen whose levels
decreased, and choline whose level increased.
?) represent the values of 𝑅𝑅
intercept <0.3 and 𝑄𝑄
in France in 2006. However, recent EFSA reports have
shown that residues of this compound are still found in
some fruits and vegetables in the European Union . In
addition, due to its capacity to bioaccumulate, endosulfan is
exposure to this contaminant commonly affects the central
nervous system [15, 27], immune , and reproductive
systems . ese effects have been at least partly linked
to an increase in oxidative stress [30–32]. Occupational
exposure to pesticides is oen linked to an increased risk
of developing certain pathologies in adults and children [1,
33]. e general population is also exposed to pesticides
mainly through the presence of contaminants in food, where
consumers are exposed to low doses of pesticides alone or in
mixtures throughout their life. Although consumer exposure
is different from occupational exposure in mode, quantity,
in�uence of such exposure on health is needed. Moreover,
since exposure of the general population to pesticides cannot
Journal of Toxicology7
T 3: Summary of the relative metabolite changes in plasma, bone marrow, and liver upon long term dietary exposure of mice to low
doses of endosulfan.
Major metabolitesDiscriminant NMR buckets
1.26; 1.30; 1.34; 1.38
0.90; 1.30; 1.34
3.18; 3.22; 4.02; 4.22; 4.30
3.26; 3.38; 3.42; 3.46; 3.50; 3.54;
3.82; 3.90; 5.22
1.66; 1.70; 1.74; 3.02
2.10; 2.14; 2.34; 2.38; 2.46
????;∗𝑃𝑃 ? ???? as compared with control.
2.18; 2.54; 2.58; 3.78
6.90; 7.18; 7.22
4.42; 4.38; 6.14; 8.26; 8.58
Comparisons among groups were performed by Kruskal-Wallis test. Changes are relative to control samples: ↘: decrease; ↗: increase;∗∗∗𝑃𝑃 ? ?????;∗∗𝑃𝑃 ?
0 0.20.1 0.3 0.4 0.5
F 3: PLS-DA score plot based on the?HNMR spectra of bone
𝑛𝑛 ? ?) and exposed female mice (black square, 𝑛𝑛 ? ?) (𝑄𝑄2? ???4,
marrow samples from nonexposed male (green triangle, 𝑛𝑛 ? ?),
exposedmale(redcircle,𝑛𝑛 ? ?),nonexposedfemale(bluediamond,
𝑅𝑅2? ????%, and A ? 4).
be quanti�ed, the development of approaches to enable
identi�cation of biological markers of exposure to these
compounds is of great interest for epidemiological studies.
In this study, we investigated the metabolic �ngerprint
of a dietary exposure to low doses of endosulfan using an
?HNMR-based metabonomic approach on bone marrow,
liver, and plasma in male and female mice. Our results
show that dietary exposure to endosulfan is characterized
by speci�c metabolic �ngerprints in plasma, liver, and bone
marrow of mice exposed to dietary endosulfan. e PLS-DA
models obtained in this study are valid and robust, as shown
LDL levels especially in females and biochemical changes to
the membrane composition of bone marrow cells (lipids and
choline/phosphocholine). e variations in taurine, betaine,
and oxidized glutathione in both genders could be linked to
a disturbance of oxidative status upon endosulfan exposure;
glucose, lactate variations could be useful as biomarkers for
liver function changes in both males and females. Some
metabolic changes appear to be gender-speci�c (e.g., plasma
lipids LDL and hepatic levels of lactate and glucose).
permutation test (Table 2). e main changes observed were
an increase in plasma glucose in both gender, a decrease in
4.1. Endosulfan-Induced Disturbances in Energy Metabolism.
Changes to the level of metabolites involved in energy
8Journal of Toxicology
−0.45 −0.35 −0.25 −0.15 −0.05 00.100.20 0.30
−0.7 −0.5 −0.3 −0.1
−0.30 −0.20 −0.1000.100.200.300.40
𝑅𝑅2? ??.?%, and A ? 4).
𝑛𝑛 ? ?) and exposed female mice (black square, 𝑛𝑛 ? ?) (𝑄𝑄2? 0.?2, 𝑅𝑅2? ??.6%, and A ? 2). PLS-DA (c) score plot and (d) loading plot based
on the1HNMR spectra of bone marrow samples from nonexposed (green triangle, 𝑛𝑛 ? ?) or exposed male (red circle, 𝑛𝑛 ? ?) (𝑄𝑄2? 0.??,
−0.32 −0.24 −0.16 −0.08
F 5: PLS-DA score plot based on the
aqueous liver extracts sampled from nonexposed male (green
female (blue diamond, 𝑛𝑛 ? 16) and exposed female mice (black
1HNMR spectra of
triangle, 𝑛𝑛 ? 14), exposed male (red circle, 𝑛𝑛 ? 1?), nonexposed
square, 𝑛𝑛 ? 16) (𝑄𝑄2? 0.6??, 𝑅𝑅2? ?3.3%, and A ? 3).
metabolism were observed upon dietary exposure to low
doses of endosulfan during the pre- and postnatal period.
increase in plasma glycaemia and a decrease in endogenous
hepatic glucose. e altered glucose levels could be related to
the induction of the glycolytic pathway since a concomitant
increase in liver lactate levels was also observed in females
upon exposure. In contrast, in male mice the increase in
plasma glucose levels appeared to be more associated with
a decrease in activity of the hepatic glycolytic pathway,
since lactate levels were decreased in the liver. Insulin
levels observed in exposed male and female mice. Indeed,
endosulfan has already been shown to affect glucose levels in
that are responsible for insulin secretion [13, 34]. Although
it cannot be con�rmed from the data presented here, our
showed that, even at low doses, endosulfan caused changes
to endogenous glucose content in the liver and plasma levels
that could be linked to the onset of pathologies such as
diabetes or insulin resistance. A physiological response to
such hyperglycaemia could increase the consumption of
glucose by muscle and fat tissue. is needs to be con�rmed
by further experiments. Our results are also in agreement
with Lee et al., who observed that serum concentrations of
O� pesticides are positively and signi�cantly correlated with
high fasting glucose in a population of nondiabetic human
Taurine levels in male mice were increased in liver upon
endosulfan exposure. Taurine is an important metabolite
involved in bile acid synthesis, osmoregulation, and intracel-
lularcalcium levels . Itis generally regarded asa sensitive
betaine levels were signi�cantly increased in the liver of
both genders. Betaine and taurine are important osmolytes
and appeared to be critical for proper liver function .
Osmolytes have been shown to affect protein stability against
a wide variety of adverse environmental conditions . e
increase of betaine or taurine could be linked to hepatic
Journal of Toxicology9
−0.8 −0.6−0.4 −00.20.2 0.4
F 6: PLS-DA (a) score plot and (b) loading plot based on the1HNMR spectra of aqueous liver extract samples from nonexposed (blue
circle, 𝑛𝑛 ? 15) (𝑄𝑄2? 0.93, 𝑅𝑅2? 99.1%, and A ? 3).
diamond, 𝑛𝑛 ? 1?) and exposed female mice (black square, 𝑛𝑛 ? 1?) (𝑄𝑄2? 0.9?5, 𝑅𝑅2? 98.4%, and A ? 2). PLS-DA (c) score plot and (d)
loading plot based on the1HNMR spectra of aqueous liver extract samples from nonexposed (green triangle, 𝑛𝑛 ? 14) or exposed male (red
could re�ect liver physiopathological change.
4.2. Endosulfan-Induced Changes in Choline Metabolism.
Choline is synthesized by the liver. It plays an important role
in the integrity and composition of cell membranes. Phos-
phocholine is the most abundant phospholipid in biological
membranes and, together with other lipids, forms the char-
acteristic bilayer structure of cells and regulates membrane
integrity. Our results suggest that dietary exposure to low
doses of endosulfan could lead to changes of haematopoietic
cell membrane composition. Changes to the biochemistry of
membrane lipids aer treatment with endosulfan have also
been observed by other authors [38, 39]. Changes to the
membrane composition of bone marrow cells could have a
major impact on the proliferative activity of haematopoietic
cells. e increased choline content may be also linked to
increased cell division since choline values have been shown
to be predictive of proliferative activity of glioma [40–42].
is point is currently under investigation in our lab.
At the plasma level, as choline is the main component
of VLDL, the decreased level of choline in females upon
exposure to endosulfan could be mediating the decrease of
this plasma lipoprotein. Biochemical analysis of plasma also
showed a signi�cant increase in triglyceride content that
result is in agreement with the clear decrease of plasmatic
glycerol levels in both genders since this metabolite is an
indirect indicator of TG hydrolysis.
Choline is also a precursor for the synthesis of acetyl-
choline whose synthesis depends on the capture of choline
in the blood. e level of circulating choline could have
an impact at the neuronal level by accelerating neuronal
transmission. is could be compared with the neurotoxicity
of endosulfan which has been described elsewhere .
Taken together, these results suggest that dietary expo-
sure to endosulfan even at low doses could interfere with a
number of physiological processes that regulate cell division,
lipid metabolism, and neuronal transmission.
4.3. Endosulfan-Induced Disturbance in Oxidative Stress
Metabolism. e levels of oxidized glutathione in the liver
were decreased in both males and females upon dietary
exposure to endosulfan, suggesting a cellular response linked
to oxidative stress, although it cannot be con�rmed from the
glutathione level have been observed between endosulfan
levels between the two groups could be due to an efficient
GSH generation in endosulfan-exposed animals through the
increase of betaine level (trimethylglycine). Indeed betaine is
a primary methyl donor for S-adenosylmethionine which is
known to regulate glutathione concentrations under condi-
tions of oxidative stress in the liver .
e decrease in oxidized glutathione levels in the liver
of male mice was associated with a concomitant decrease in
glutamine and glutamate in the liver upon endosulfan expo-
sure. Glutamate is a precursor for synthesis of glutathione
10Journal of Toxicology
and in this way forms a part of the key antioxidant system.
Glutamine and glutamate cycle back and forth, converting
from one form to the other during normal body metabolism.
Our results are in agreement with reports that endosulfan
induces oxidative stress, particularly in male mice [32, 38,
45], and studies are under investigation to con�rm this
As a fuel for rapidly dividing cells, glutamine contributes
towards a healthy immune system, especially in the rapid
production of white blood cells during an infection. e
increased level of glutamine in female mouse bone marrow
the immune system .
Our metabonomic approach has enabled the detection of
metabolic disturbances following dietary exposure to a low
associated with oxidative stress in the liver as well as changes
in hepatic glucose metabolism.
Given the increase in plasma glucose levels following
endosulfan exposure, we believe that metabonomics will
tools for assessing the real exposure to pesticides in epidemi-
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