TOXICOLOGICAL SCIENCES 86(2), 444452 (2005)

doi:10.1093/toxsci/kfi192
Advance Access publication May 11, 2005

Effects of Organochlorine Insecticides on MAP Kinase Pathways in

Human HaCaT Keratinocytes: Key Role of Reactive Oxygen Species
Nathalie Ledirac,*,1,2 Sebastien Antherieu,*,2 Anne Dupuy dUby,* Jean-Claude Caron, and Roger Rahmani*
*Laboratoire de Toxicologie Cellulaire et Moleculaire, Centre de Recherche INRA, 400 route des Chappes, 06903 Sophia-Antipolis, France;
Galderma R&D, les Templiers, 2400 routes des Colles, 06410 Biot, France
Received March 7, 2005; accepted April 28, 2005

INTRODUCTION

Organochlorine pesticides (OCs) are organic compounds

that persist in the environment, bioaccumulate through the food
chain, and pose a risk of causing adverse effects to human
health and the environment. These pesticides, characterized by
their cyclic structure, number of chlorine atoms, and low
volatility, can be divided into four groups: dichlorodiphenylethanes (such as DDT), cyclodienes (such as dieldrin, endosulfan, and heptachlor), chlorinated benzenes (such as
hexachlorobenzene), and cyclohexanes (such as lindane).
Although these chemicals were widely used until the mid1
To whom correspondence should be addressed at Laboratoire de Toxicologie Cellulaire et Moleculaire, Centre de Recherche INRA, 400 Route des
Chappes, 05903 Sophia-Antipolis, France. Tel and Fax: 33 4 92 38 65 64.
E-mail: ledirac@antibes.inra.fr.
2
These authors contributed equally to the work.

1970s, most of them are now banned from use in developed

countries. However, they are still being produced and used in
other countries. Furthermore, one of these insecticides, endosulfan, is still in widespread use throughout the world despite
its known adverse effect on humans as an endocrine-disrupting
compound (Andersen et al., 2002; Scippo et al., 2004). Most
OCs are also considered persistent organic pollutants (POPs),
a category of chemicals that includes nine OCs (aldrin,
chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex, and toxaphen), targeted by the Stockholm
Convention in May 2001, which aimed to eliminate their
production and restrict or ban their use throughout the world
(http://www.pops.int/).
Many human epidemiologic and animal studies have shown
that exposure to OCs is positively correlated with endocrine
disruption (Lemaire et al., 2004), reproductive and immune
dysfunctions (Ayub et al., 2003; Reed et al., 2004; Saiyed
et al., 2003), and cancers (e.g., breast cancer [Kalantzi et al.,
2004; Zou and Matsumura; 2003]). Human exposure occurs
mainly by ingestion (from eating contaminated foods), inhalation, absorption through skin, and often during pest control
operations both at home and in resort areas. Therefore, to
investigate the toxicological effects of OCs after cutaneous
exposure, we used the spontaneously immortalized human
keratinocyte cell line, HaCaT (Boukamp et al., 1988). This
well-characterized cell line is still able to proliferate and
differentiate, and it has been shown to be a good model in
toxicology (Delescluse et al., 1998; Ledirac et al., 1997).
Moreover, these cells show good predictive results in comparison with keratinocyte and skin models, and they therefore
constitute an appropriate model for studying the molecular
mechanisms regulating keratinocyte growth and differentiation.
The mitogen-activated protein kinases (MAPKs) are serine/
threonine kinases that transduce signals from the plasma
membrane to the cell nucleus. They play a critical role in
controlling cell survival, proliferation, and differentiation
(Chang and Karin, 2001). In epithelial cells, in particular
keratinocytes, deregulation of MAPK signaling pathways can
lead to hyperproliferation and altered differentiation, which
in turn contribute to photoaging, psoriatic epidermis, and

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Organochlorine pesticides (OCs) are reported as potential

carcinogens in humans. The aim of this study was to investigate
the effects of four OCs (dieldrin, endosulfan, heptachlor, and
lindane) on mitogen-activated protein kinase (MAPK) cascades
and more specifically to identify the mechanism underlying OCinduced ERK1/2 activation. Organochlorine pesticides increased
phosphorylated Raf, MEK1/2, ERK1/2, and c-Jun in human
HaCaT cells, but they had no effect on p38 MAPK activation.
Moreover, blockade of Raf, MEK1/2, or PKC activation with
geldanamycin, U0126, or calphostin C inhibited ERK1/2 phosphorylation, demonstrating a PKCRafMEK1/2 pathway. We
also showed that these insecticides induced the production of
reactive oxygen species (ROS). Pre-treatment with the antioxidant
molecule N-acetyl cysteine sharply decreased the level of phosphoERK1/2 and had no effect on Raf and MEK1/2 activation,
suggesting a Raf-independent mechanism. This study indicates
that OCs strongly activate the ERK1/2 pathway, and it identifies
a critical role of ROS in OC-induced ERK activation, probably by
stabilizing its phosphorylation.
Key Words: ERK1/2; keratinocytes; mitogen-activated protein
kinases; organochlorines; reactive oxygen species; signal transduction.

ROS-DEPENDENT PHOSPHORYLATION OF MAPKS BY OCs

FIG. 1. Chemical structure of the organochlorine insecticides (OCs)

selected.

important cellular processes. In another respect, OCs such as

dieldrin and more recently endosulfan, have been reported to
induce reactive oxygen species (ROS) production in human
liver. Therefore, the induction of ROS production by these
molecules and the involvement of ROS production in MAPK
signaling pathways were also investigated.
MATERIALS AND METHODS
Chemicals. Dulbecos modified Eagles medium (DMEM), fetal bovine
serum (FBS), penicillin-streptomycin, sodium pyruvate, Eagles non-essential
amino-acids were from BioWhittaker (Cambrex company, Walkersville, MD).
Anti-phospho-p38, anti-phospho-c-Jun, anti-phospho-MEK1/2, anti-phosphoRaf, anti-c-Jun, anti-p38, anti-MEK2, and MEK1/2 inhibitor U0126 were
purchased from Cell Signaling (Beverly, MA). Anti-ERK2 was from Santa
Cruz (Santa Cruz, CA). Anti-phospho ERK1/2 and N-acetyl cysteine (NAC)
were from Sigma-Aldrich (St. Louis, MO). Raf inhibitor geldanamycin and
PKC inhibitor calphostin C were purchased from Alexis Biochemicals.
Organochlorine pesticides were from ChemService (West Chester, PA).
Cell culture. HaCaT cells (a generous gift from Prof. N. E. Fusenig,
German Cancer Research Institute, Heidelberg, Germany) were derived from
a spontaneously immortalized human keratinocyte but are a non-tumorigenic
epidermal cell line that exhibits many of the morphological and functional
properties of normal human keratinocytes. HaCaT cells were cultured in
Dulbeccos minimum essential medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 lg/ml), sodium
pyruvate (1 mM), and non-essential amino acids (0.1 mM). Cultures were
incubated at 37C in a humidified atmosphere with 95% air and 5% CO2.
Cell viability. The cytotoxicity of OCs was evaluated after 24 h of exposure
by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay, according to the procedure of Fautrel et al. (1991). Briefly,
HaCaT cells were seeded in 96-well plates and grown to confluence. Then cells
were treated with various concentrations of the tested pesticides for 24 h. The
next day, medium was removed and 100 lL of serum-free DMEM containing
MTT (0.5 mg/ml) was added to each well and incubated for 2 h at 37C. Finally,
solutions were removed, the water-insoluble formazan was dissolved in 100 ll
dimethyl sulfoxide (DMSO), and absorbance was measured at 550 nm.
ROS measurement. Intracellular ROS generation was assessed by using
2#,7#-dichlorodihydrofluorescein diacetate (H2DCFDA) from Molecular
Probes (Eugene, OR). HaCaT cells were seeded in 12-well plates and grown
to confluence. Then cells were treated for 90 min at 37C with OCs in the
presence of 100 lM H2DCFDA (the stock solution was made in ethanol, so that
the final concentration in the medium was 0.33%). After the incubation time,
cells were washed twice with cold phosphate buffered saline (PBS), and
scraped in potassium buffer (10 mM pH7.4) / methanol (v/v) completed with
Triton X-100 (0.1%). An aliquot of 100 ll was incubated in a black 96-well
plate, and relative fluorescence intensity was determined by spectrofluorimetry
(kex 488 nm, kem 520 nm).
Western blot analysis. HaCaT cells were plated in six-well plates and
grown to confluence. The confluent cells were serum starved for 24 h to
establish quiescence (except for p38 MAPK), and then stimulated with OCs for
the indicated periods and at the indicated concentrations. After treatments, cells
were scraped and resuspended in buffer A containing the protease inhibitor
cocktail (25 mM HEPES [pH7.5], 5 mM MgCl2, 5 mM EDTA, 5 mM DTT,
2 mM PMSF, 10 lg/ml pepstatin A, and 10 lg/ml leupeptin). The protein
concentration in each cell lysate was measured by a commercial method (BCA
Protein Assay Kit), using bovine serum albumin (BSA) as the standard. Thirty
micrograms of total protein were resolved by 11% sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis and blotted on polyvinyliden
difluoride (PVDF) membranes. Membranes were immunoblotted with antiphospho-c-Jun (1:1000), anti-phospho-p38 (1:2000), anti-phospho-Raf

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malignant transformation in human cancers, including those of

the cutaneous epithelium (Bedogni et al., 2004; Chung et al.,
2000; Takahashi et al., 2002). Mammals express at least four
MAPK subfamilies, extracellular signal-related kinases (ERK),
Jun amino-terminal kinases (JNK), p38 MAPK, and ERK5
(Gutkind, 2000). The ERK1/2 group is activated mainly by
mitogenic stimuli, and it has been linked to cell survival,
whereas the JNK and p38 MAPK pathways are primarily
activated by stress stimuli and are linked to the induction of
differentiation and apoptosis.
Although a large number of studies have reported toxicological effects of OCs in humans and animals, mainly by OCs
interfering with endocrine processes, the effects of pesticides
on MAPK cascades are poorly documented, and their underlying mechanisms remain unclear. Indeed, heptachlor,
a well-known liver tumor promoter in animals, triggers proliferation in rat hepatocytes both by the induction of ERK
phosphorylation and by the inhibition of apoptosis (Okoumassoun
et al., 2003). Heptachlor has also been shown to increase the
amount of phosphorylated ERK1/2 in human lymphocytes
(Chuang and Chuang, 1998), a finding that led to the suggestion
that heptachlor could be considered a potent human mitogen.
However, more recent studies have demonstrated that heptachlor, like endosulfan, induces apoptosis in human lymphocytes (Kannan et al., 2000; Rought et al., 2000). In fact, in
contrast with the proliferating effects observed in animals, in
human lymphocytes heptachlor provokes alterations of cell
cycle progression and induction of programmed cell death
(Chuang et al., 1999).
The aim of this study was to gain a better understanding of
the cellular events leading to OC-mediated toxicity and more
particularly to investigate whether various compounds belonging to the OCs family, such as dieldrin, endosulfan, heptachlor,
and lindane (Fig. 1), can activate MAPKs and thus influence

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(1:1000), anti-phospho-MEK1/2 (1:1000), anti-c-Jun (1:1000), anti-p38

(1:1000), or anti-MEK2 (1:1000) antibodies overnight at 4C, or with antiERK2 (1:5000), anti-phospho-ERK1/2 (1:5000) antibodies for 1 h at room
temperature. Membranes were then reacted with horseradish peroxidase
conjugated secondary antibodies (anti-mouse or anti-rabbit immunoglobulin G)
for 1 h at room temperature. After washing, blots were reacted using an ECL
detection kit (Amersham Biosciences, Piscataway, NJ).
Western blot densitometric quantitation. Films were scanned and quantitated with the Chemi Genus Bio Imaging System (SynGene, Sunnyvale, CA).
The amount of phosphorylated protein detected was quantified, and values from
multiple experiments were averaged and graphed. The Y-axis was presented as
arbitrary units. Error bars in each of the figures represent the standard deviation
of the mean.
Statistical analysis. The statistical differences between different treatment
groups were determined by Students t-test, and probability levels were noted as
*( p < 0.05) and **( p < 0.001). Data are expressed as means standard
deviations (SD) for at least three independent determinations for each
experimental point.

Cytotoxicity
Cytotoxicity studies of four OCs were performed by using
two different endpoints the MTT reduction and neutral red
(data not shown) tests. As shown in Table 1, significant
differences were observed between the tested compounds.
Indeed, endosulfan and heptachlor exerted a cytotoxic effect
with an IC50 ranging from
2040 to 100 lM, depending on
the absence or presence of serum in the medium, whereas dieldrin and lindane did not lead to cellular cell death, regardless of
concentration and culture conditions. It should be pointed out,
however, that because of poor solubility ( 200 lM) of the two
latter molecules in the culture medium, their IC50 values could
not be determined precisely. Nevertheless these experiments
allowed us to optimize experimental concentrations for the
induction studies. Figure 2 illustrates the dose-dependent
toxicity of the two most toxic insecticides after 24 h of

OCs Induce ERK1/2 and JNK but Not p38

MAPK Activation
To determine the effects of OCs on MAPK cascades, we
studied the ability of these molecules to activate the phosphorylation of ERK1/2, c-Jun, and p38 MAPK after 1 h of exposure
on HaCaT cells. As shown in Figure 3, OCs activate both
ERK1/2 and c-Jun, but they have no induction effect on p38
MAPK phosphorylation, compared with that obtained with
sorbitol-induced osmotic stress (Fig. 3, line So). Endosulfan
and heptachlor induce a strong activation of ERK1/2, above
that produced by FBS, whereas c-Jun is more strongly activated
by dieldrin and endosulfan, and to a lesser extent by heptachlor
and lindane. As expected, no effect was obtained on the
inactive forms of ERK, c-Jun, and p38 MAPK. These results
are in agreement with those previously obtained for ERK1/2
activation by 50 lM heptachlor in human lymphocytes
(Chuang and Chuang, 1998; Chuang et al., 1999), and they
clearly indicate that OCsendosulfan in particularare able
to strongly activate the ERK1/2 and JNK signaling pathways,
and therefore interfere with cellular functions.
Raf and MEK1/2 Are Required for OC-Induced ERK1/2
Phosphorylation
Figure 4 shows the dose-dependent increase in ERK1/2
activation by the four OCs. The maximal induction effect was

TABLE 1
Comparative In Vitro Cytotoxicity of Organochlorine Insecticides
in HaCaT Cells
IC

50

(lM)

Insecticides

Serum

Serum 

Dieldrin
Endosulfan
Heptachlor
Lindane

>200.0
92.10 2.59
109.6 8.98
>200.0

>200.0
39.60 0.69
17.4 0.57
>200.0

In vitro cytotoxicity of insecticides was determined using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Cells were treated for 24 h with various concentrations of insecticides in the presence or
absence of serum in the medium. Insecticide solutions were prepared in
DMSO so that the final concentration in the medium was 0.25%. IC50 was the
concentration that decreased viability to 50%. Values are expressed as mean
SE from at least three independent experiments.

FIG. 2. In vitro cytotoxicity of endosulfan and heptachlor. HaCaT cells

were incubated for 24 h with different concentrations (0200 lM) of heptachlor
or (0150 lM) endosulfan, and cytotoxicity was measured by the MTT
test. Each point is the mean SD of three independent experiments replicated
five times.

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RESULTS

exposure in complete medium, and it shows the absence of

any relevant cytotoxicity at 25 lM, according to the cytotoxicity tests used. We therefore used the subtoxic concentration
25 lM to study the early events leading to the toxic response.

ROS-DEPENDENT PHOSPHORYLATION OF MAPKS BY OCs

447

FIG. 3. Activation of ERK1/2, c-Jun but not p38 MAPK by OCs in HaCaT
cells. HaCaT cells were serum-starved for 24 h (not for p38 MAPK analysis)
and then treated with 0.25% dimethyl sulfoxide (DMSO; C), 10% fetal bovine
serum (FBS; S), 0.6 M sorbitol (So), 25 lM dieldrin (Die), 25 lM endosulfan
(End), 25 lM heptachlor (Hep), or 25 lM lindane (Lin), for 1 h. Equal amounts
of total protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylation levels of
ERK1/2, c-Jun, and p38 MAPK were determined by immunoblotting using the
appropriate phosphospecific antibodies and developed with ECL reagent, as
described in Materials and Methods. The results are representative of three
independent experiments.

observed for all tested compounds at concentration of 25 lM,

with a strong effect of endosulfan at levels as low as 10 lM. The
phosphorylation of both MEK1/2 and Raf was also induced by
OCs, with a lesser effect of lindane, a less potent activator than
other OCs (Fig. 5A). These results suggest the involvement of
Raf and MEK1/2 in ERK1/2 activation by OCs, and they
clearly indicate that OCs of the cyclodiene group are the more
potent activators, a difference that may be explained by their
different chemical structures. Experiments with DDT, a dichlorodiphenylethane, have produced similar results to those we
obtained with cyclodienes (data not shown), and they have
confirmed the existence of a relationship between the chemical
structure (polyclyclic structures by comparison with that of
lindane) and the level of ERK1/2-induced phosphorylation.
To further confirm the upstream mechanism of OC-induced
ERK1/2 activation, we analyzed the effects on induced-ERK1/
2 phosphorylation of U0126, a specific inhibitor of MEK1/2,
and geldanamycin, which destabilizes Raf-1 and disrupts the
Raf-MEK-ERK pathway (Schulte et al., 1996). As shown in
Figure 5B, U0126 completely blocked ERK1/2 activation.

Surprisingly, although geldanamycin completely blocked any

activation of Raf phosphorylation by OCs (data not shown),
ERK1/2 was still weakly activated by dieldrin, endosulfan, and
heptachlor. These results suggest that OCs activate ERK1/2
through distinct pathways including the Raf-MEK signaling
cascade and a Raf-independent mechanism.
Involvement of PKC in OC-Induced ERK1/2
Phosphorylation
Protein kinase C (PKC) is involved in many cellular
responses and in particular in Raf-1 stimulation. In addition,
OCs were previously described as stimulating PKC activity
both in vitro and in vivo (Bagchi et al., 1997; Moser and Smart,
1989). Therefore, to examine the role of PKC in OC-induced
ERK1/2 activation, experiments were performed with calphostin C, a highly specific inhibitor of PKC (Fig. 5B). Pretreatment of HaCaT cells for 4 h with calphostin C entirely
blocked OC-induced ERK1/2 phosphorylation, suggesting
a key role of PKC. We also noted that the amounts of
phosphorylated Raf-1 were similar to those obtained by
treatment with geldanamycin (data not shown), suggesting
a direct PKC-MEK1/2 mechanism for the residual ERK1/2
activation observed with geldanamycin. Taken together, these
data clearly indicate that OCs can activate ERK1/2 via PKC
RafMEK1/2-dependent and PKCMEK1/2-dependent pathways (Schonwasser et al., 1998). This direct activation of MEK
by PKC, such as PKC-f, has already been demonstrated for
insulin-induced and lipopolysaccharide (LPS)-induced ERK1/2
phosphorylation (Monick et al., 2000; Sajan et al., 1999).

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FIG. 4. Dose-dependent increase in ERK1/2 activation by OCs in HaCaT

cells. HaCaT cells were serum-starved for 24 h and then treated with 0.25%
DMSO (C), or various concentrations ranging from 1 to 25 lM of dieldrin
(Die), endosulfan (End), heptachlor (Hep), or lindane (Lin) for 1 h. Both
phospho-ERK1/2 and total ERK2 were determined by immunoblotting. Levels
of phospho-ERK1/2 were determined by densitometric scanning of autoradiograms. The results are expressed as fold increases in band densities above those
found for the control, and values are the mean SD of three separate samples.
(*p < 0.05; **p < 0.001; as determined by Students t-test).

448

LEDIRAC ET AL.

control. Figure 6 illustrates DCF fluorescence in response to

two insecticide concentrations (25 and 50 lM), and shows
dose-dependent increase in ROS generation with dieldrin,
endosulfan, and heptachlor, without dose-dependence with
lindane. At 25 lM, all the tested compounds induce a significant increase in DCF fluorescence: a 1.6-fold increase with
lindane, a 2-fold increase with dieldrin and endosulfan, and
a 2.5-fold increase with heptachlor. Maximum effect is
observed at 50 lM heptachlor (3.4-fold over DMSO control).
Effect of Oxidative Stress on ERK and JNK Signaling
Pathways

OC-Induced Intracellular ROS Generation

Many studies have shown that oxidative stresses induced
JNK and p38 MAPK signaling cascades, and to a lesser extent
the ERK1/2 pathway. Although OCs have been described to
increase ROS formation, particularly in the liver, few studies
have been conducted that involved other human tissue or cell
types (Kannan and Jain, 2003). To determine whether OCs
treatments are associated with changes in intracellular ROS
levels in HaCaT cells, we measured oxidation of (H2DCF)
diacetate (H2DCFDA). Intracellular esterases convert cellpermeable H2DCFDA to HDCF, which is subsequently oxidized by ROS to fluorescent dichlorodihyro-fluorescein (DCF)
(Myhre et al., 2003). Primaquine, a known pro-oxidant
compound (Magwere et al., 1997), was chosen as a positive

FIG. 6. Intracellular generation of reactive oxygen species (ROS) after

OCs exposure. HaCaT cells were treated with 0.25% DMSO (C), 200 lM
primaquine (PQ), or 25 and 50 lM insecticides in the presence of 100 lM
H2DCFDA for 90 min at 37C. Cells were collected in phosphate/methanol
buffer as described in Materials and Methods, and analyzed by spectrofluorimetry. The results are expressed as means SD of at least three independent
experiments. Asterisks indicate significant differences from control (*p < 0.05;
**p < 0.001; as determined by Students t-test).

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FIG. 5. Involvement of MEK, Raf, and PKC in OC-induced ERK1/2

activation in HaCaT cells. A: HaCaT cells were treated with 0.25% DMSO (C)
or insecticides (25 lM) for 1 h after starving in serum-free medium for 24 h.
Analysis of both Raf-1 and MEK1/2 phosphorylation was performed by
immunoblotting using anti-phospho-Raf and anti-phospho-MEK1/2 antibodies.
The results are representative of three independent experiments. B: HaCaT
cells, serum-starved for 24 h, were preincubated (or not) with 10 lM U0126 for
30 min, 2 lM geldanamycin (GA) for 24 h, or 4 lM calphostin C (CC) for 4 h,
followed by stimulation with 0.25% DMSO, or 25 lM insecticides for 1 h.
Phosphorylation of ERK1/2 was analyzed by immunoblotting. Densitometric
data of phospho-ERK1/2 levels are plotted as percentage of maximal induction
caused by Die, End, Hep, Lin, respectively or observed in the presence of
DMSO. Values are the mean SD of three independent experiments. (*p < 0.05;
**p < 0.001; as determined by Students t-test).

To determine the role of OC-induced ROS generation on

both ERK and JNK activation, we studied the effects of the
antioxidant molecule N-acetyl cysteine (NAC). To determine
the optimal concentration of NAC that inhibits OC-induced
oxidative stress, HaCaT cells were pre-treated for 24 h with
various concentrations of NAC (110 mM). Figure 7 shows
a dose-dependent decrease of OC-induced oxidative stress after
NAC pre-treatment, and it demonstrates that the oxidative
stress induced by lindane and dieldrin was completely blocked
with 5 mM of NAC, whereas 10 mM of NAC was necessary to
inhibit ROS production induced by endosulfan and heptachlor.
We next examined the effects of NAC on ERK1/2 activation
by OCs. HaCaT cells were pre-treated for 24 h with 10 mM
NAC, and then stimulated for 1 h with the four insecticides. As
indicated in Figure 8A, NAC pre-treatment markedly decreases
the OC-induced ERK1/2 activation, whereas the corresponding
level of phospho-MEK1/2 remains unchanged. Moreover, NAC
pre-treatment has no effect on serum-induced ERK1/2 activation, as expected, because this activation is ROS-independent
and has no significant effect on basal ERK phosphorylation.
These results indicate clearly that ROS contribute to increase
the level of OC-induced ERK1/2 phosphorylation, but they do
not lead to activation of the upstream MAPKs. These data

ROS-DEPENDENT PHOSPHORYLATION OF MAPKS BY OCs

449

provide strong support of a crucial role for ROS in OC-induced

ERK1/2 phosphorylation, probably via a RafMEK-independent mechanism. Oxidative modifications of other molecules,
among them, ERK-specific phosphatases, may also contribute
to an increase in ERK1/2 phosphorylation level (Levinthal and
Defranco, 2005). Results presented in Figure 8B indicate that
pre-treatment with NAC partially prevents OC-induced c-Jun
phosphorylation, suggesting the possibility of a similar mechanism in OC-induced JNK activation.
DISCUSSION

The association between the levels of exposure to organochlorine pesticides and cancers is still a matter for significant
debate. Indeed, in addition to their known estrogenic characteristics, OCs have been especially implicated as risk factors for
breast cancers. Although OCs such as lindane and DDT have
been shown to increase cell proliferation of MCF-7 cells
(Steinmetz et al., 1996; Zou and Matsumura, 2003), no
mechanism has been clearly established yet, and available data
do not support the hypothesis that these chemicals increase the
risk of breast cancer. Although the toxicological effects of OCs
have been studied extensively in animals, and several studies
have revealed that they provoke a vast range of disorders, they
remain poorly documented in humans. Dietary contribution is
probably the most significant route of human exposure.
Maximum levels were found in vegetables, specifically in
Egypt; plants like potato tubers or strawberries could contain high
levels of lindane residues (850 lg/kg) and dieldrine (220 lg/kg),
respectively (Monsour, 2004). Although dietary intake has
decreased since 1970, tissue bioaccumulation, which is a common feature of insecticides, should not be ruled out. This could
lead to unexpectedly high intracellular OC concentrations at
hepatic and epidermal sites. Furthermore, during the application period, OCs may be found in surface water and reservoirs.
The general population can also be exposed to OCs during pest

FIG. 8. Effect of oxidative stress on ERK1/2 and JNK signaling pathways

in HaCaT cells. HaCaT cells were serum-starved for 24 h, followed by 24 h of
exposure with 10 mM N-acetyl cysteine (NAC) or serum-free medium. Then
cells were stimulated with 0.25% DMSO (C), 10% fetal bovine serum (FBS; S),
or 25 lM insecticides for 1 h. Phosphorylation of ERK1/2 and MEK1/2 (A), or
c-Jun (B) was analyzed by immunoblotting. The results shown are representative of three independent experiments. Densitometric data of phospho-ERK1/
2 levels are plotted as percentage of maximal induction caused by Die, End,
Hep, Lin, respectively or observed in presence of DMSO. Values are the mean
SD of three independent experiments. (*p < 0.05; **p < 0.001; as determined
by Students t-test).

control operations, both at home and in recreational areas.

Finally, the highest dermal and inhalation exposures documented were reported in farm workers involved in the spraying
of these insecticides. The mean dermal exposures to mixers and
sprayers of endosulfan via a tractor-mounted boom sprayer
and highboy were 16.18 and 8.06 mg/kg/day, respectively
(Lonsway et al., 1997). The authors concluded that not using
protective measures when spaying endosulfan could lead to
poisoning. The present study was therefore designed to assess
the toxicological effects of OCs in humans after acute dermal
exposure by using the HaCaT cell line, the most widely studied
keratinocyte cell line and to try to better understand the effects
of OCs on a population chronically exposed by dermal contact.
Because MAPKs are key enzymes in signal transduction, we
determined whether OCs had an inducing effect on MAPK
cascades, and we provided data supporting the existence of an
additional ROS-dependent mechanism in MAPK activation by
OCs. In this report we demonstrate that multiple members of
the MAPK family are stimulated by OCs and that ERK1/2 is

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FIG. 7. Effects of NAC on OC-induced ROS generation. HaCaT cells were

pre-incubated with NAC (110 mM) for 24 h, and then treated with 25 lM
insecticides or 0.25% DMSO (C), in the presence of 100 lM of H2DCFDA for
90 min at 37C. Reactive oxygen species generation was quantified and
analyzed as previously described. The results are expressed as means SD of at
least three independent experiments. Asterisks indicate significant differences
from control (*p < 0.05; **p < 0.001; as determined by Students t-test).

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results clearly indicate that ROS-dependent events induced by

OCs contribute to both ERK and JNK activation.
Therefore, to identify the cellular events leading to ROSinduced MAPK phosphorylation after OCs exposure, further
experiments are in progress to investigate whether ERK and
JNK-directed phosphatases are inactivated. Nevertheless OCinduced ERK activation is completely blocked in the presence
of U0126, indicating that inactivation of ERK phosphatases
would not be sufficient by itself to drive the increase in
phosphorylated-ERK1/2. Furthermore, the results presented
here demonstrate that OC-induced ERK activation requires
sequential activation of PKC, Raf, and MEK1/2. Taken
together, these findings may contribute to better understanding
of the mechanism underlying OC-induced alterations suspected to play a role in carcinogenesis.
In summary, we have shown that OCs activate ERK1/2 and
JNK cascades, but have no effect on p38 MAPK. This
activation of ERK1/2 by OCs results in Raf and MEK1/2
activation, as well as activation of PKC. The results presented
here also reveal that all the pesticides tested stimulate ROS
generation in HaCaT cells, and that this increase in ROS is
involved to some degree in ERK and JNK activation. However,
MEK1/2 phosphorylation is not affected by this oxidative
stress. Thus we provide evidence that OC-induced MAPK
activation involves both the classical MAPK signaling pathways, such as PKC and RafMEK1/2 activation process for
ERK1/2, and an additional ROS-dependent mechanism (Figure 9)
that should be further investigated.

FIG. 9. Summary of the proposed mechanism for mitogen-activated

protein kinase (MAPK) activation by OCs. Exposure of HaCaT cells to OCs
results in the activation of ERK1/2 and JNK signaling pathways. In addition,
these molecules induce the generation of intracellular ROS, which appears to be
critical for the OC-induced MAPK activation. Our study shows that OCs induce
MAPK activation via PKC and Raf-MEK activation processes, but it also
demonstrates that the oxidative stress induced by these chemicals contributes in
part to the increase in ERK1/2 phosphorylation in a MEK-independent and Rafindependent manner.

Downloaded from http://toxsci.oxfordjournals.org/ by guest on January 9, 2016

strongly activated in HaCaT cells. Organochlorine-induced

ERK1/2 activation is significantly reduced or completely
blocked by inhibitors of MEK1/2 (U0126), Raf-1 (geldanamycin), and PKC (calphostin C). In addition, our data indicate
that OCs induce Raf and MEK1/2 phosphorylation, and our
findings thus demonstrate that these pesticides induce ERK1/2
phosphorylation via successive activations of PKC, Raf-1, and
MEK1/2. Our results are consistent with a previous study that
reported ERK1/2 activation by 50 lM heptachlor in human
lymphocytes (Chuang and Chuang, 1998); they also clearly
demonstrate that insecticides of the organochlorine family
induce the same MAPK activation profile in HaCaT cells,
namely ERK and JNK, but not p38 MAPK activation.
Furthermore OCs have been shown to induce oxidative stress in
certain mammalian species (Bayoumi et al., 2000), and the ROS
generation has been described to interfere with various signaling
pathways, including MAPKs. Indeed all MAPK cascades are
known to be activated in response to oxidant injury (Gupta et al.,
1999; Martindale and Holbrook, 2002), and they can therefore
have an impact on cell survival and cell death. A recent study has
reported that exposure to 10100 lM endosulfan levels induced
apoptosis in human T cells via a bcl-2-independent mechanism
and suggested that endosulfan-induced apoptosis may be linked
to excessive ROS production (Kannan et al., 2000). However, the
mechanisms that mediate MAPK activation by oxidants and the
variability of mammalian sensitivity to oxidant injury remain to
be understood. Moreover, no data are available on OC-induced
oxidative stress in human keratinocytes, or on the impact of this
oxidant injury on cell signaling.
Under our experimental conditions, the four OC tested
enhanced the production of ROS in a dose-dependent manner.
When cells were pre-treated with increasing concentrations of
the antioxidant agent NAC, ROS induction was reduced, and
the phosphorylation of both ERK1/2 and c-Jun was partially
decreased. Interestingly, MEK and Raf phosphorylation was
not affected by NAC pre-treatment, suggesting that OCsinduced ERK1/2 phosphorylation is dependent in part on
a ROS-dependent mechanism downstream of the MEK1/2
level. Recent studies have demonstrated the role of oxidative
stress in the inactivation of phosphatase activity, and have
precisely described the mechanism of ROS regulation (Lee and
Esselman, 2002; Persson et al., 2004). Indeed, protein tyrosine
phosphatases (PTPs) were shown to be reversibly oxidized and
inactivated after H2O2 treatment in various cellular systems,
upon the oxidation of cysteine thiols. Moreover, inactivation of
phosphatase activity by H2O2 has been demonstrated to
contribute to MAPK activation, which could explain the
ROS-dependent increase of ERK and JNK phosphorylation
by OCs. More recently, a study on phosphatase inhibition
during oxidative stress in murine neuronal cells has shown that
glutamate-induced oxidative stress specifically inhibited the
phosphatase activity regulating ERK1/2 (PP2A, MKPs),
whereas other phosphatases, such as that regulating JNK, were
not affected (Levinthal and Defranco, 2005). In contrast, our

ROS-DEPENDENT PHOSPHORYLATION OF MAPKS BY OCs

ACKNOWLEDGMENTS
This work was supported by a grant from the Region Provence-Alpes-Cote
dAzur and Galderma R&D. English proofreading was done by Philip
Rousseau-Cunningham.

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