Published by Oxford University Press 2005.

Effects of organochlorine insecticides on MAP kinase pathways in human HaCaT

keratinocytes : Key role of reactive oxygen species

Nathalie Ledirac*,‡,§, Sebastien Antherieu*,‡ , Anne Dupuy d’Uby*, Jean-Claude Caron† and

Roger Rahmani*

*Laboratoire de Toxicologie Cellulaire et Moléculaire, Centre de Recherche INRA, 400 route

des Chappes, 06903 Sophia-Antipolis, France.

†Galderma R&D, les Templiers, 2400 routes des Colles, 06410 Biot, France.

§Corresponding author. Tel. & Fax: +33 4 92 38 65 64

E-mail address: ledirac@antibes.inra.fr

Section : Environmental Toxicology

ToxSci Advance Access published May 25, 2005

Abstract : 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 MAPK cascades and more specifically to identify the mechanism

underlying OCs-induced ERK1/2 activation. OCs increased phosphorylated Raf, MEK1/2,

ERK1/2 and c-Jun in human HaCaT cells, but 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 PKC-Raf-MEK1/2 pathway.

We also showed that these insecticides induced the production of reactive oxygen species

(ROS). Pre-treatment with antioxidant molecule N-acetyl cysteine sharply decreased the level

of phospho-ERK1/2, 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 identifies a critical role of ROS in OCs-induced ERK activation probably by stabilizing

its phosphorylation.

Keywords: ERK1/2; Keratinocytes; Mitogen-activated protein kinases; Organochlorines;

Reactive oxygen species; Signal transduction

1 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,

heptachlor), chlorinated benzenes (such as hexachlorobenzene), and cyclohexanes (such as

lindane). Although these chemicals were widely used until the mid-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 human as an endocrine-

disrupting compound (Andersen et al., 2002; Scippo et al., 2004). The majority of OCs is also

considered Persistent Organic Pollutants (POPs). These POPs chemicals, including nine OCs

(aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex and

toxaphen), were targeted by the Stockholm Convention in May 2001, which aimed to

eliminate their productions and restrict or ban their uses in 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 leisure 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 therefore constitute an appropriate model to study 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 nucleus, they play a critical role in

controlling cell survival, proliferation, and differentiation (Chang and Karin, 2001). In

epithelial cells, and in particular in keratinocytes, deregulation of MAPK signaling pathways

can lead to hyperproliferation and altered differentiation, which in turn contribute to

photoaging, psoriasic epidermis and 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). ERK1/2

is mainly activated by mitogenic stimuli and 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.

However, even if a large number of studies have reported toxicological effects of OCs

in humans and animals, mainly by 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 the

inhibition of apoptosis (Okoumassoun et al., 2003). Also, heptachlor has been shown to

increase the amount of phosphorylated- ERK1/2 in human lymphocytes (Chuang and Chuang,

1998), first suggesting that heptachlor could be considered as a potent human mitogen.

However, more recent studies have demonstrated that heptachlor, like endosulfan, induces

apoptosis in those human lymphocytes (Kannan et al., 2000; Rought et al., 2000). In fact, in

contrast with the proliferating effects observed in animals, heptachlor provokes alterations of

cell cycle progression and induction of programmed cell death in human lymphocytes

(Chuang et al., 1999).

The aim of this study was to better understand the cellular events leading to OCs-

mediated toxicity and more particularly to investigate whether various compounds belonging

to the OCs family, such as dieldrin, endosulfan, heptachlor and lindane (figure 1), can activate

MAPKs and thus influence 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 that ROS production in MAPK signaling pathways were

investigated.

2 Materials and methods

2.1 Chemicals

Dulbeco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-

streptomycin, sodium pyruvate, Eagle’s non-essential amino-acids were from BioWhittaker

(Cambrex company, Walkersville, USA). Anti-phospho-p38, anti-phospho-c-Jun, anti-

phospho-MEK1/2, anti-phospho-Raf, 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).

2.2 Cell culture

HaCaT Cells (a generous gift from Prof. Fusenig, German Cancer Research Institute,

Heidelberg, Germany) were derived from a spontaneously immortalized human keratinocyte

but are non-tumorigenic epidermal cell line, and exhibit many of the morphological and

functional properties of normal human keratinocytes. HaCaT cells were cultured in DMEM

supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), sodium

pyruvate (1mM) and non-essential amino acids (0.1 mM). Cultures were incubated at 37°C in

a humidified atmosphere with 95% air and 5% CO2.

2.3 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

(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 µL of serum-free DMEM containing MTT

(0.5 mg/ml) were added to each well and incubated 2 h at 37°C. Finally, solutions were

removed, the water-insoluble formazan was dissolved in 100 µl DMSO, and absorbance was

measured at 550 nm.

2.4 ROS measurement

Intracellular ROS generation was assessed by using 2’, 7’-dichlorodihydrofluorescein

diacetate (H2DCFDA) from Molecular Probes (Eugene, Oregon, USA). HaCaT cells were

seeded in 12-well plates and grown to confluence. Then cells were treated for 90 min at 37°C

with OCs in the presence of 100 µM H2DCFDA (the stock solution was made in ethanol, so

that final concentration in the medium was 0.33%). After the incubation time, cells were

washed twice with cold PBS, and scraped in potassium buffer (10 mM pH7.4) / methanol

(v/v) completed with Triton X100 (0.1%). An aliquot of 100 µL was incubated in a black 96-

well plate and relative fluorescence intensity was determined by spectrofluorimetry (λex = 488

nm, λem = 520 nm).

2.5 Western blot analysis

HaCaT cells were plated in 6-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 µg/ml pepstatin

A and 10 µg/ml leupeptin). The protein concentration in each cell lysate was measured by

commercial method (BCA Protein Assay Kit), using BSA as the standard. Thirty micrograms

of total protein were resolved on 11% SDS-polyacrylamide gels electrophoresis and blotted

on PVDF membranes. Membranes were immunoblotted with anti-phospho-c-Jun (1:1000),

anti-phospho-p38 (1:2000) anti-phospho-Raf (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 4°C, or

anti-ERK2 (1:5000), anti-phospho-ERK1/2 (1:5000) antibodies for 1 h at room temperature.

Membranes were then reacted with horseradish peroxydase-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).

2.6 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.

2.7 Statistical analysis

The statistical differences between different treatment groups were determined by

Student's 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.

3 Results 3.1 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 ≈ 20-40 to 100 µM 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. However, it should be pointed out

that due to the poor solubility (≤ 200 µM) 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 exposure in complete

medium, and shows the absence of any relevant cytotoxicity at 25 µM, according to the

cytotoxicity tests used. We therefore used the subtoxic concentration 25 µM to study the early

events leading to toxic response.

3.2 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 exposure on

HaCaT cells. As shown in figure 3, OCs activate both ERK1/2 and c-Jun, but have no

induction effect on p38 MAPK phosphorylation, compared with that obtained with sorbitol-

induced osmotic stress (Figure 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 µM heptachlor in

human lymphocytes (Chuang and Chuang, 1998; Chuang et al., 1999), and clearly indicate

that OCs, endosulfan in particular, are able to strongly activate the ERK1/2 and JNK signaling

pathways, and therefore interfere with cellular functions.

3.3 Raf and MEK1/2 are required for OCs–induced ERK1/2 phosphorylation

Figure 4 shows the dose-dependent increase in ERK1/2 activation by the four OCs. Maximal

induction effect was observed for all the tested compounds at concentration of 25 µM, with a

strong effect of endosulfan as low as 10 µM. 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 clearly indicate that OCs of the cyclodiene group are more potent

activators. This difference 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 confirm 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 OCs-induced ERK1/2 activation, we analyzed

the effects of U0126, a specific inhibitor of MEK1/2, and geldanamycin, which destabilizes

Raf-1 and disrupts the Raf-MEK-ERK pathway (Schutle et al., 1996), on induced-ERK1/2

phosphorylation. 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.

3.4 Involvement of PKC in OCs-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 OCs-induced ERK1/2 activation, experiments were performed with calphostin C, a

highly specific inhibitor of PKC (Fig. 5B). Pre-treatment of HaCaT cells during 4 h with

calphostin C, entirely blocked OCs-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-Raf-MEK1/2 and PKC-

MEK1/2-dependent pathways (Schönwasser et al., 1998). This direct activation of MEK by

PKC, such as PKC-ζ, has already been demonstrated for insulin- and LPS-induced ERK1/2

phosphorylation (Monick et al., 2000; Sajan et al., 1999).

3.5 OCs- induced intracellular ROS generation

Many studies have shown that oxidative stresses induced JNK and p38 MAPK signaling

cascades, and to a lesser extent ERK1/2 pathway. Although OCs have been described to

increase ROS formation particularly in the liver, few studies have been conducted, which

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 cell-

permeable H2DCFDA to HDCF, which is subsequently oxidized by ROS to fluorescent DCF

(Myhre et al., 2003). Primaquine, a known pro-oxidant compound (Magwere et al., 1997),

was chosen as a positive control. Figure 6 illustrates DCF fluorescence in response to two

insecticides concentrations (25 and 50 µM), and shows dose-dependent increase in ROS

generation with dieldrin, endosulfan and heptachlor, without dose-dependence with lindane.

At 25 µM, all the tested compounds induce a significant increase in DCF fluorescence : 1.6

fold increase with lindane, 2 fold increase with dieldrin and endosulfan, and 2.5 with

heptachlor. Maximum effect is observed at 50 µM heptachlor (3.4 fold over DMSO control).

3.6 Effect of oxidative stress on ERK and JNK signaling pathways

To determine the role of OCs-induced ROS generation on both ERK and JNK activation, we

studied the effects of the antioxidant molecule N-acetyl cysteine (NAC). In order to determine

the optimal concentration of NAC that inhibits OCs-induced oxidative stress, HaCaT cells

were pre-treated for 24 h with various concentrations of NAC (1-10 mM). Figure 7 shows a

dose-dependent decrease of OCs-induced oxidative stress after NAC pre-treatment, and

demonstrates that oxidative stress induced by lindane and dieldrin was completely blocked

with 5 mM of NAC, whereas 10 mM were necessary to inhibit ROS production induced by

endosulfan and heptachlor.

Next we 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 OCs-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 since 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

OCs-induced ERK1/2 phosphorylation, but do not lead to the activation of the upstream

MAPKs. These data provide strong support of a crucial role of ROS in OCs-induced ERK1/2

phosphorylation, probably via a Raf-MEK-independent mechanism. Oxidative modifications

of other molecules including 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 OCs-induced c-Jun

phosphorylation, suggesting the possibility of a similar mechanism in OCs-induced JNK

activation.

4 Discussion The association between the levels of exposure to organochlorine pesticides and cancers is

still largely debated. 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 clearly been established yet, and

available data do not support the hypothesis that these chemicals increase the risk of breast

cancer. Nevertheless the toxicological effects of OCs have been extensively studied in

animals in which several studies have revealed that they provoke a vast range of disorders, but

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 µg/kg) and dieldrine (220 µg/kg), respectively (Mansour SA, 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 on 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 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 JA 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 and 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 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 strongly activated in HaCaT cells. The OCs-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 therefore demonstrate that these pesticides induce ERK1/2

phosphorylation via successive activations of PKC, Raf-1 and MEK1/2. These results are

consistent with a previous study that reported ERK1/2 activation by 50 µM 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 reactive oxygen species (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 can therefore have an impact on cell survival and cell death. A recent

study has reported that exposure to 10-100 µM 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 currently available on OCs-

induced oxidative stress in human keratinocytes, or on the impact of this oxidant injury on

cell signaling.

Under our experimental conditions, the four OCs tested enhance 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 OCs-induced ERK1/2 phosphorylation is in

part dependent 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; Peterson 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 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 ROS-induced MAPK phosphorylation

after OCs exposure, further experiments are under progress to investigate whether ERK and

JNK-directed phosphatases are inactivated. Nevertheless OCs-induced ERK activation is

completely blocked in the presence of U0126, indicating that inactivation of ERK

phosphatases would not be sufficient to drive by itself the increase in phosphorylated-

ERK1/2. Furthermore, the results presented here clearly demonstrate that OCs-induced ERK

activation require sequential activation of PKC, Raf and MEK1/2. Taken together, these

findings may contribute to better understand the mechanism underlying OCs-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 that of PKC. In addition the results presented here revealed that all the pesticides

tested stimulate ROS generation in HaCaT cells, and that this increase in ROS is partly

involved in ERK and JNK activation. However, MEK1/2 phosphorylation is not affected by

this oxidative stress. Thus we provide evidence that OCs-induced MAPK activation involves

both the classical MAPK signaling pathways, such as PKC and Raf-MEK1/2 activation

process for ERK1/2, and an additional ROS-dependent mechanism (figure 9) that should be

further investigated.

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Acknowledgements

This work was supported by a grant from the Région Provence-Alpes-Côte d’Azur and

Galderma R&D. English proof-reading by Philip Rousseau-Cunningham.

Footnotes

‡ These authors have equally contributed to the work.

FIGURE LEGENDS

Fig. 1. Chemical structure of the organochlorine insecticides selected.

Fig. 2. In vitro cytotoxicity of endosulfan and heptachlor. HaCaT cells were incubated for 24

h with different concentrations (0-200 µM) of heptachlor or (0-150 µM) of endosulfan, and

cytotoxicity was measured by MTT test. Each point is the mean ± S.D. of three independent

experiments replicated five times.

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 %

DMSO (C), 10 % FBS (S), 0.6 M sorbitol (So), 25 µM dieldrin (Die), 25 µM endosulfan

(End), 25 µM heptachlor (Hep), or 25 µM lindane (Lin), for 1 h. Equal amounts of total

protein lysates were separated by 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.

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 µM 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 ± S.D. of three separate samples. (*, p < 0.05;

**, p < 0.001; as determined by Student’s t test).

Fig. 5. Involvement of MEK, Raf, and PKC in OCs-induced ERK1/2 activation in HaCaT

cells (A) HaCaT cells were treated with 0.25 % DMSO (C) or insecticides (25 µM) for 1 h

after starving in serum-free media 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 µM U0126 for 30

min, 2 µM geldanamycin (GA) for 24 h, or 4 µM calphostin C (CC) for 4 h, followed by

stimulation with 0.25 % DMSO, or 25 µM 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

presence of DMSO. Values are the mean ± S.D. of three independent experiments. (*, p <

0.05; **, p < 0.001; as determined by Student’s t test).

Fig. 6. Intracellular generation of ROS after OCs exposure. HaCaT cells were treated with

0.25% DMSO (C), 200 µM primaquine (PQ), or 25 and 50 µM insecticides, in the presence of

100 µM H2DCFDA for 90 min at 37 °C. Cells were collected in phosphate / methanol buffer

as described in materials and methods, and analyzed by spectrofluorimetry. The results were

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 Student’s t

test).

Fig. 7. Effects of NAC on OCs-induced ROS generation. HaCaT cells were pre-incubated

with NAC (1-10 mM) for 24 h, and then treated with 25 µM insecticides or 0.25% DMSO

(C), in the presence of 100 µM of H2DCFDA for 90 min at 37°C. ROS generation was

quantified and analyzed as previously described. The results were 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 Student’s t test).

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 exposure with 10 mM N-acetyl

cysteine (NAC) or serum-free media. Then cells were stimulated with 0.25 % DMSO (C),

10% FBS (S) or 25 µM insecticides for 1 h. Phosphorylation of ERK1/2 and MEK1/2 (A), or

c-Jun (B), were 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 ± S.D. of three independent experiments. (*, p <

0.05; **, p < 0.001; as determined by Student’s t test).

Fig. 9. Summary of the proposed mechanism for MAPK activation by OCs. Exposure of

HaCaT cells to organochlorine pesticides 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 OCs-induced MAPK activation. Our study shows that OCs

induce MAPK activation via PKC and Raf-MEK activation processes, but also demonstrate

that the oxidative stress induced by these chemicals contributes in part to the increase in

ERK1/2 phosphorylation in a MEK and Raf independent manner.

Table 1

Comparative in vitro cytotoxicity of organochlorine insecticides in HaCaT cells.

Insecticides I. C. 50 (µM)

Serum + Serum −

Dieldrin > 200.0 > 200.0

Endosulfan 92.10 ± 2.59 39.60 ± 0.69

Heptachlor 109.6 ± 8.98 17.4 ± 0.57

Lindane > 200.0 > 200.0

In vitro cytotoxicity of insecticides was determined using MTT test. Cells were treated for 24

h with various concentrations of insecticides in presence or absence of serum in the medium.

Insecticides 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 ± S.E. from at least three independent experiments.

Figure 1

O

Cl

Cl

ClCl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

S

O

O

O

Cl

Cl

Cl

Cl

Cl

Cl

Dieldrin Endosulfan

Heptachlor Lindane

0

20

40

60

80

100

120

0 25 50 75 100 125 150 175 200

Concentration (µM)

Cel

l via

bilit

y (%

)Heptachlor

Endosulfan

Figure 2

Figure 3

C S Die End Hep Lin

P-ERK1/2

ERK2

C S Die End Hep Lin

P-c-Jun

c-Jun

C So Die End Hep Lin

P-p38

p38

*

*

**

*

*

0

5

10

15

20

25

30

Die End Hep Lin

Rel

ativ

e In

tens

ity (

Fol

d) 1 µM5 µM10 µM25 µM

Figure 4

P-ERK1/2

ERK2

DIE END HEP LIN

C 1 5 10 25 1 5 10 25 1 5 10 25 1 5 10 25

*

**

***

*****

** ** * **0

25

50

75

100

125

150

C Die End Hep Lin

Rel

ativ

e In

tens

ity

(% o

f con

trol

) CGACCU0126

**

Figure 5

P-MEK1/2

C Die End Hep Lin

P-Raf

MEK2

P-ERK1/2

P-ERK1/2

(B) C Die End Hep Lin C Die End Hep Lin

U 0126

ERK2

ERK2

GA CC

(A)

Figure 6

*

***

* **

****

0

100

200

300

400

500

600

700

C PQ Die End Hep Lin

Flu

ores

cenc

e (A

rbita

ry U

nits

) 25 µM

50 µM

Figure 7

***

** **

*

** **

***

0

100

200

300

400

500

0 1 5 10NAC (mM)

Flu

ores

cenc

e (A

rbita

ry U

nits

) CDieEndHepLin

****

**

0

50

100

150

200

C Die End Hep Lin S

Rel

ativ

e In

tens

ity

(% o

f con

trol

)

C

NAC

Figure 8

P-c-Jun

c-Jun

- + - + - + - + - + NAC

(B)

C Die End Hep Lin

P-MEK1/2

P-ERK1/2

C Die End Hep Lin S

- + - + - + - + - + - +

ERK2

NAC

(A)

Figure 9

Raf

MEK1/2

P-ERK1/2

PKC

Geldanamycin

U0126

NAC

Calphostin C

OCs?

JNK

ROS

ERK1/2 P-JNK

Ras

Cell Membrane