* 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
1 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{at}antibes.inra.fr.
Received March 7, 2005; accepted April 28, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: ERK1/2; keratinocytes; mitogen-activated protein kinases; organochlorines; reactive oxygen species; signal transduction.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (1 mM), and non-essential amino acids (0.1 mM). Cultures were incubated at 37°C 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 µL of serum-free DMEM containing MTT (0.5 mg/ml) was added to each well and incubated for 2 h at 37°C. Finally, solutions were removed, the water-insoluble formazan was dissolved in 100 µl 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 37°C with OCs in the presence of 100 µM 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 µl was incubated in a black 96-well plate, and relative fluorescence intensity was determined by spectrofluorimetry (ex = 488 nm,
em = 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 µg/ml pepstatin A, and 10 µg/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 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 with anti-ERK2 (1:5000), anti-phospho-ERK1/2 (1:5000) antibodies for 1 h at room temperature. Membranes were then reacted with horseradish peroxidaseconjugated 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 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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
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). Pre-treatment 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 PKCRafMEK1/2-dependent and PKCMEK1/2-dependent pathways (Schönwasser et al., 1998
). This direct activation of MEK by PKC, such as PKC-
, has already been demonstrated for insulin-induced and lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation (Monick et al., 2000
; Sajan et al., 1999
).
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 cell-permeable 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 control. Figure 6 illustrates DCF fluorescence in response to two insecticide 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: 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 µM heptachlor (3.4-fold over DMSO control).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 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 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 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 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 OCs-induced 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 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 in progress to investigate whether ERK and JNK-directed phosphatases are inactivated. Nevertheless OC-induced 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.
|
![]() |
NOTES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ayub, S., Verma, J., and Das, N. (2003). Effect of endosulfan and malathion on lipid peroxidation, nitrite and TNF- release by rat peritoneal macrophages. Int. Immunopharmacol. 3, 18191828.[CrossRef][ISI][Medline]
Bagchi, D., Bagchi, M., Tang, L., and Stohs, S. J. (1997). Comparative in vitro oxygen radical scavenging ability of zinc methionine and selected zinc salts and antioxidants. Toxicol. Lett. 91, 3137.[CrossRef][ISI][Medline]
Bayoumi, A. E., Perez-Pertejo, Y., Ordonez, C., Reguera, R. M., Balana-Fouce, R., and Ordonez, D. (2000). Changes in the glutathione-redox balance induced by the pesticides heptachlor, chlordane, and toxaphene in CHO-K1 cells. Bull. Environ. Contam. Toxicol. 65, 748755.[CrossRef][ISI][Medline]
Bedogni, B., O'Neill, M. S., Welford, S. M., Bouley, D. M., Giaccia, A. J., Denko, N. C., and Powell, M. B. (2004). Topical treatment with inhibitors of the phosphatidylinositol 3'-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways reduces melanoma development in severe combined immunodeficient mice. Cancer Res. 64, 25522560.
Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell. Biol. 106, 761771.[Abstract]
Chang, L., and Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature 410, 3740.[CrossRef][ISI][Medline]
Chuang, L. F., and Chuang, R. Y. (1998). Heptachlor and the mitogen-activated protein kinase module in human lymphocytes. Toxicology 128, 1723.[CrossRef][ISI][Medline]
Chuang, L. F., Rought, S. E., and Chuang, R. Y. (1999). Differential regulation of the major cyclin-dependent kinases, cdk2 and cdc2, during cell cycle progression in human lymphocytes exposed to heptachlor. In Vivo 13, 455461.[ISI][Medline]
Chung, J. H., Kang, S., Varani, J., Lin, J., Fisher, G. J., and Voorhees, J. J. (2000). Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J. Invest. Dermatol. 115, 177182.[CrossRef][ISI][Medline]
Delescluse, C., Ledirac, N., de Sousa, G., Pralavorio, M., Lesca, P., and Rahmani, R. (1998) Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: Binding capability to the Ah receptor. Toxicol. Lett. 9697, 3339.
Fautrel, A., Chesne, C., Guillouzo, A., de Sousa, G., Placidi, M., Rahmani, R., Braut, F., Pichon, J., Hoellinger, H., Vintezou, P., et al. (1991). A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology. Toxicol. In Vitro 5, 543547.[CrossRef][ISI]
Gupta, A., Rosenberger, S. F., and Bowden, G. T. (1999). Increased ROS levels contribute to elevated transcription factor and MAP kinase activities in malignantly progressed mouse keratinocyte cell lines. Carcinogenesis 20, 20632073.
Gutkind, J. S. (2000). Regulation of mitogen-activated protein kinase signaling networks by G proteincoupled receptors. Sci STKE 40, re1.
Kalantzi, O. I., Hewitt, R., Ford, K. J., Cooper, L., Alcock, R. E., Thomas, G. O., Moris, J. A., McMillan, T. J., and Martin, K. C. (2004). Low dose induction of micronuclei by lindane. Carcinogenesis 25, 613622.
Kannan, K., and Jain, S. K. (2003). Oxygen radical generation and endosulfan toxicity in Jurkat T-cells. Mol. Cell. Biochem. 247, 17.[CrossRef][ISI][Medline]
Kannan, K., Holcombe, R. F., Jain, S. K., Alvarez-Hernandez, X., Chervenak, R., Wolf, R. E., and Glass, J. (2000). Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line. Mol. Cell. Biochem. 205, 5366.[CrossRef][ISI][Medline]
Ledirac, N., Delescluse, C., de Sousa, G., Pralavorio, M., Lesca, P., Amichot, M., Berge, J. B., and Rahmani, R. (1997). Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor. Toxicol. Appl. Pharmacol. 144, 177182.[CrossRef][ISI][Medline]
Lee, K., and Esselman, W. J. (2002). Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways. Free Radic. Biol. Med. 33, 11211132.[CrossRef][ISI][Medline]
Lemaire, G., Terouanne, B., Mauvais, P., Michel, S., and Rahmani, R. (2004). Effect of organochlorine pesticides on human androgen receptor activation in vitro. Toxicol. Appl. Pharmacol. 196, 235246.[CrossRef][ISI][Medline]
Levinthal, D. J., and Defranco, D. B. (2005). Reversible oxidation of ERK-directed protein phosphatases drives oxidative toxicity in neurons. J. Biol. Chem. 280, 58755883.
Lonsway, J. A., Byers, M. E., Dowla, H. A., Panemangalore, M., and Antonious, G. F. (1997). Dermal and respiratory exposure of mixers/sprayers to acephate, methamidophos, and endosulfan during tobacco production. Bull. Environ. Contam. Toxcicol. 52, 179186.
Magwere, T., Naik, Y. S., and Hasler, J. A. (1997). Primaquine alters antioxidant enzyme profiles in rat liver and kidney. Free Radic. Res. 27, 173179.[ISI][Medline]
Martindale, J. L., and Holbrook, N. J. (2002). Cellular response to oxidative stress: signaling for suicide and survival. J. Cell. Physiol. 192, 115.[CrossRef][ISI][Medline]
Monick, M. M., Carter, A. B., Flaherty, D. M., Peterson, M. W., and Hunninghake, G. W. (2000). Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages. J. Immunol. 165, 46324639.
Monsour, S. A. (2004). Pesticide exposureEgyptian scene. Toxicology 198, 91115.[CrossRef][ISI][Medline]
Moser, G. J., and Smart, R. C. (1989). Hepatic tumor-promoting chlorinated hydrocarbons stimulate protein kinase C activity. Carcinogenesis 10, 851856.[Abstract]
Myhre, O., Andersen, J. M., Aarnes, H., and Fonnum, F. (2003). Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol. 65, 15751582.[CrossRef][ISI][Medline]
Okoumassoun, L. E., Averill-Bates, D., Marion, M., and Denizeau, F. (2003). Possible mechanisms underlying the mitogenic action of heptachlor in rat hepatocytes. Toxicol. Appl. Pharmacol. 193, 356369.[CrossRef][ISI][Medline]
Persson, C., Sjoblom, T., Groen, A., Kappert, K., Engstrom, U., Hellman, U., Heldin, C. H., den Hertog, J., and Ostman, A. (2004). Preferential oxidation of the second phosphatase domain of receptor-like PTP-alpha revealed by an antibody against oxidized protein tyrosine phosphatases. Proc. Natl. Acad. Sci. U.S.A. 101, 18861891.
Reed, A., Dzon, L., Loganathan, B. G., and Whalen, M. M. (2004). Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides. Hum. Exp. Toxicol. 23, 463471.[CrossRef][ISI][Medline]
Rought, S. E., Yau, P. M., Guo, X. W., Chuang, L. F., Doi, R. H., Chuang, R. Y. (2000). Modulation of CPP32 activity and induction of apoptosis in Human CEM x 174 lymphocytes by heptachlor, a chlorinated hydrocarbon insecticide. J. Biochem. Mol. Toxicol. 14, 4250.[CrossRef][ISI][Medline]
Saiyed, H., Dewan, A., Bhatnagar, V., Shenoy, U., Shenoy, R., Rajmohan, H., Patel, K., Kashyap, R., Kulkarni, P., Rajan, B., et al. (2003). Effect of endosulfan on male reproductive development. Environ. Health Perspect. 111, 19581962.[ISI][Medline]
Sajan, M. P., Standaert, M. L., Bandyopadhyay, G., Quon, M. J., Burke, T. R., Jr., and Farese, R. V. (1999). Protein kinase C-zeta and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes. J. Biol. Chem. 274, 3049530500.
Schönwasser, D. C., Marais, R. M., Marshall, C. J., and Parker, P. J. (1998). Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, atypical protein kinase C isotypes. Mol. Cell. Biol. 18, 790798.
Schulte, T. W., Blagosklonny, M. V., Romanova, L., Mushinski, J. F., Monia, B. P., Johnston, J. F., Nguyen, P., Trepel, J., and Neckers, L. M. (1996). Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway. Mol. Cell. Biol. 16, 58395845.[Abstract]
Scippo, M. L., Argiris, C., Van De Weerdt, C., Muller, M., Willemsen, P., Martial, J., and Maghuin-Rogister, G. (2004). Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors. Anal. Bioanal. Chem. 378, 664669.[CrossRef][ISI][Medline]
Steinmetz, R., Young, P. C., Caperell-Grant, A., Gize, F. A., Madhukar, B. V., Ben-Jonathan, N., Bigsby, R. M. (1996). Novel estrogenic action of the pesticide residue beta-hexachlorocyclohexane in human breast cancer cells. Cancer Res. 56, 54035409.[Abstract]
Takahashi, H., Ibe, M., Nakamura, S., Ishida-Yamamoto, A., Hasimoto, Y., and Iizuka, H. (2002). Extracellular regulated kinase and c-Jun N-terminal kinase are activated in psoriatic involved epidermis. J. Dermatol. Sci. 30, 9499.[CrossRef][ISI][Medline]
Zou, E., and Matsumura, F. (2003). Long-term exposure to beta-hexachlorocyclohexane (beta-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells. Biochem. Pharmacol. 66, 831840.[CrossRef][ISI][Medline]
|