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Effects of pentachlorophenol and tetrachlorohydroquinone on mitogen-activated protein kinase pathways in Jurkat T cells. (Articles).

When Jurkat human T cells were incubated with 20 [micro]M of pentachlorophenol (PCP) or its metabolite, tetrachlorohydroquinone (TCHQ), for 10 hr, flow cytometric analyses revealed marked increase in the number of apoptotic cells. DNA fragmentation was also observed in these cells. TCHQ was more potent than PCP in causing apoptosis. After incubation with 20 [micro]M TCHQ for 1 hr, all mitogen-activated protein kinases (MAPKs) examined [i.e., extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N[H.sub.2]-terminal kinase (JNK)] were phosphorylated, whereas no clear phosphorylation was induced by PCP. TCHQ-induced apoptosis was markedly suppressed by treatment with a p38 inhibitor (SB203580) and mildly (but significantly) suppressed by treatment with a MAPK/ERK kinase inhibitor (U0126). When cells were treated with both inhibitors at the same time, TCHQ-induced apoptosis disappeared almost completely. PCP-induced apoptosis was also suppressed by SB203580 and/or U0126. Nevertheless, treatment with LL-Z1640-2, which inhibits JNK phosphorylation, did not suppress the apoptosis caused by either TCHQ or PCP. Thus, p38 and ERK appear to be important signal transduction pathways leading to apoptosis in a human T-cell line exposed to a ubiquitous pollutant or its metabolite in the general and occupational environment. Key words: apoptosis, Jurkat cells, mitogen-activated protein kinases, pentachlorophenol, tetrachlorohydroquinone. Environ Health Perspect 110:139-143 (2002). [Online 10 January 2002] /abstract.html


Generally, chlorinated phenols have strong biological effects. Of all chlorinated phenols, pentachlorophenol (PCP) seems to have been produced and used in the largest quantities; annual production of PCP all over the world was estimated to be 25,000-90,000 tons at its peak (1). Because PCP is inexpensive and has potent biocidal effects against a broad spectrum of species, PCP and its salts have been used as algicides, bactericides, fungicides, herbicides, insecticides, and molluscides (2). Although its use has been reduced greatly and even banned in some countries, PCP is still used widely in the preservation of wood (1,2). Hence, PCP is ubiquitous in the environment. For instance, it has been detected in ambient air of rural mountain areas, and PCP levels of 5.7-7.8 ng/[m.sup.3] have been found in urban areas (2). PCP concentrations in groundwater can be 3-23 [micro]g/L in wood-treatment areas and concentrations in the milligrams per liter range can be found near industrial discharges. As a result, the general population is exposed to PCP through the ingestion of water (0.01-0.1 [micro]g/L) and food (up to 40 [micro]g/kg). People may be exposed to PCP in treated items such as textiles, leather, and paper products, and especially through inhalation of indoor air contaminated with PCP; PCP concentrations up to 25 [micro]g/[m.sup.3] have been found in rooms treated 1 to several years earlier (2). Workers handling PCP can be exposed to much higher doses of PCP (1). Thus, health effects of PCP among workers, as well as among the general population, are of great concern.

Acute exposures to high doses of PCP in accidental or suicidal intoxication are often fatal and are associated with extreme weakness, hyperpyrexia, and profuse sweating (2). These signs can be explained by the effects of PCP itself, that is, uncoupling of oxidative phosphorylation in mitochondria (3). In contrast, although effects of lower doses of PCP and tetrachlorohydroquinone (TCHQ), the major metabolite of PCP (4), on diverse biological systems (including the immune system) have been suggested (5-9), neither clinical pictures nor mechanisms of their effects have been clarified. We therefore examined effects of PCP and TCHQ on mitogen-activated protein kinases (MAPKs)--extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N[H.sub.2-] terminal kinase (JNK)--in a human T-cell line (Jurkat cells) because recent studies have shown that MAPKs play important roles in cellular responses to various harmful signals (10).

Materials and Methods

Cell culture and treatments. Jurkat human T cells (RIKEN Cell Bank, Tsukuba, Japan) were grown in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 [micro]g/mL streptomycin (GIBCO BRL, Life Technologies, Inc., Rockville, MD, USA) in a humidified atmosphere of 5% C[O.sub.2] and 95% air at 37 [degrees] C. Exponentially growing Jurkat cells were diluted at 3 x [10.sub.6] cells in 6-well culture plates or 1 x [10.sub.5] cells in 96-well culture plates and subsequently used for the experiments.

For each experiment, we prepared fresh solutions of PCP (Sigma Chemical Co., St. Louis, MO, USA) and TCHQ (Sigma Chemical Co.) with dimethyl sulfoxide (DMSO). Jurkat cells were incubated with serum-free medium containing 5-20 [micro]M PCP or TCHQ for 1 or 10 hr at 37 [degrees] C. Untreated control cells were incubated with serum-free medium containing the same concentration of DMSO (0.1-0.3%) and were treated identically to the cells exposed to PCP or TCHQ.

The MAPK/ERK kinase (MEK) inhibitor U0126 (Promega, Madison, WI, USA) and a specific inhibitor of p38, SB203580 (Calbiochem, La Jolla, CA, USA) were dissolved in DMSO. Cells were preincubated with serum-free medium containing either 0.1-0.2% DMSO, 50 [micro]M U0126, 50 [micro]M SB203580, or 50 [micro]M U0126 plus 50 [micro]M SB203580 for 30 min, and then incubated with 0.1% DMSO, 20 [micro]M PCP, or TCHQ for 10 hr. A radicicol-related macrocyclic non-aketide compound, LL-Z1640-2 (provided by Pharmaceutical Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan), which has been shown to inhibit the activation of JNK (11,12), was also dissolved in DMSO. Cells were preincubated with serum-free medium containing either 0.002% DMSO or 50, 100, or 200 ng/mL LL-Z1640-2 for 30 min and then incubated with 0.1% DMSO or 20 tam TCHQ for 10 hr.

WST-8 assay. We determined cell viability using the WST-8 assay. Cell Count Reagent SF (10 [micro]L; Nacalai Tesque, Kyoto, Japan), which consists of 5 mM WST-8 (4-[3-(2-methoxy-4-nitrophenyl)2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate sodium salt), 0.2 mM 1-methoxy-5-methylphenazinium methosulfate, and 150 mM NaCl, was added to each well of 96-well culture plates. After incubation for 1 hr at 37 [degrees] C, the absorbance of each well was measured at 450 nm with a reference wavelength at 655 nm.

Flow cytometric analysis. Jurkat cells (3 x [10.sup.6]) incubated with PCP or TCHQ for 10 hr were washed with phosphate-buffered saline (PBS) and fixed in 70% ethanol on ice for 30 min. The fixed cells were washed twice with PBS, incubated with 250 [micro]g/mL RNase at 37 [degrees] C for 1 hr, washed again twice, and then stained with 50 [micro]g/mL propidium iodide solution. The DNA content of cells (1 x [10.sup.4] for each sample) was determined with a Coulter EPICS XL (Coulter Electronics, Miami, FL, USA). Apoptotic cells were estimated by the percentage of cells in the sub-[G.sub.1] peak as described previously (13).

DNA fragmentation. We performed DNA fragmentation analysis using the method of Herrmann et al. (14) with slight modifications. After the incubation with PCP or TCHQ for 10 hr, Jurkat cells were washed with PBS and treated with lysis buffer (1% NP-40, 20 mM EDTA, 50 mM Tris-HCl, pH 7.5) for 30 rain on ice. After centrifugation at 1,600 g for 5 min, cell lysates were incubated with 1% SDS and 5 mg/mL RNase for 2 hr at 56 [degrees] C, and then with 2.5 mg/mL proteinase K for 4 hr at 37 [degrees] C. DNA was precipitated with 1/2 vol. 10 M ammonium acetate and 2.5 vol. ethanol, dissolved in loading buffer, separated by electrophoresis in 1.5% agarose gel containing 0.5 [micro]g/mL ethidium bromide, and visualized under UV light.

Western immunoblotting. After incubation with PCP or TCHQ for 1 hr, Jurkat cells were washed with PBS and lysed with SDS-polyacrylamide gel Laemmli sample buffer. Cell lysates were collected, sonicated, and boiled for 5 min. Aliquots equivalent to 4 x [10.sup.5] cells were subjected to SDS-PAGE on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia Biotech, Buckinghamshire, England). The membrane was blocked with 5% nonfat milk or 1% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 for 1 hr at room temperature. We used the following antibodies: phospho-p44/42 MAPK (Thr202/ Tyr204) antibody, phosphorylation state-independent p44/42 MAPK antibody, phospho-p38 MAPK (Thr180/Tyr182) antibody, phosphorylation state-independent p38 MAPK antibody, phospho-SAPK/JNK (Thr183/Tyr185) antibody, and phosphorylation state-independent SAPK/JNK antibody (Cell Signaling Technology, Inc., Beverly, MA, USA). The membrane was incubated overnight at 4 [degrees] C with the primary antibody diluted 1:1000 in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20. We detected protein using the Phototope-HRP Western blot detection kit (Cell Signaling Technology, Inc.). The bands on the developed film were quantified with NIH Image Version 1.61 (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis. Results were expressed as mean [+ or -] SEM. We determined statistical significance using one-way analysis of variance followed by the Bonferroni multiple comparison test. When two groups were compared, we used the Student t-test, p < 0.05 was considered as statistically significant.


The cell viability of Jurkat cells assayed with WST-8 conversion was 92.2 [+ or -] 0.9, 79.8 [+ or -] 1.7, and 74.9 [+ or -] 1.2%, respectively, for cells treated with 5, 10, and 20 [micro]M TCHQ for 10 hr and 99.8 [+ or -] 1.5, 97.5 [+ or -] 1.1, and 85.5 [+ or -] 0.5% (n = 4) of untreated control cells for those treated with 5, 10, and 20 [micro]M PCP for 10 hr, respectively. Thus, TCHQ treatment reduced cell viability more than PCP treatment at each concentration (p < 0.01).

When Jurkat cells were incubated with 20 [micro]M TCHQ for 10 hr, a marked increase in the number of apoptotic cells was found with flow cytometric analysis (Figure 1). Incubation with 20 [micro]M PCP for 10 hr also increased the number of apoptotic cells, but this increase was less significant than that with the same dose of TCHQ (p < 0.05; Figure 1). Consistent with the results of flow cytometric analysis, DNA fragmentation occurred in cells treated with 20 [micro]M TCHQ (Figure 2, lane 5) and PCP (Figure 2, lane 9). In contrast, incubation with TCHQ or PCP at concentrations higher than 20 [micro]M induced apoptosis less markedly (data not shown).


We determined the phosphorylation status of MAPKs after incubation with TCHQ or PCP for 1 hr, when cell viability of Jurkat cells was not changed as evaluated by the WST-8 assay (data not shown). After incubation with TCHQ for 1 hr, we found significant phosphorylation of ERK (Figure 3A), p38 (Figure 3B), and JNK (Figure 3C) at 20, 10, and 7.5 [micro]M TCHQ, respectively. In contrast, incubation with PCP did not induce clear phosphorylation of MAPKs (Figure 3). The levels of total (phosphorylation state-independent) ERK, p38, and JNK were not changed by the incubation with any concentration of TCHQ or PCP (Figure 3). In the time-course study, the levels of the phosphorylated form of ERK and p38 increased after 15 min and that of JNK increased after 1 hr in response to 20 [micro]M TCHQ exposure (data not shown).


Treatment with an MEK inhibitor, U0126 (50 [micro]M), suppressed the TCHQ-induced increase in the number of apoptotic cells mildly but significantly (p < 0.01; Figure 4). In contrast, treatment with a p38 inhibitor, SB203580 (50 [micro]M), protected cells more markedly from TCHQ-induced apoptosis (Figure 4). The difference in the magnitude of suppression of apoptosis between cells treated with U0126 and SB203580 was significant (p < 0.01; Figure 4B). When cells were treated with U0126 (50 [micro]M) and SB203580 (50 [micro]M) at the same time, TCHQ-induced apoptosis was abolished almost completely (Figure 5). Treatment with U0126, SB203580, or both compounds together also reduced the number of apoptotic cells in Jurkat cells treated with PCP (25.8 [+ or -] 0.7%, n = 6) to 21.4 [+ or -] 0.6% (p < 0.05, n = 3), 13.0 [+ or -] 0.8% (p < 0.001, n = 3), and 5.7 [+ or -] 0.3% (p < 0.001, n = 3), respectively. In contrast, treatment with > 50 ng/mL LL-Z1640-2, the concentration that suppressed TCHQ-induced phosphorylation of JNK (data not shown), did not reduce the number of apoptotic cells in cells treated with TCHQ (Figure 6). However, treatment with LL-Z1640-2 (200 ng/mL) alone did not induce apoptosis (Figure 6).



Both flow cytometric and DNA fragmentation analyses indicated apoptosis in Jurkat cells treated with 20 [micro]M PCP or 20 [micro]M TCHQ for 10 hr. The flow cytometry showed that apoptosis was more marked when cells were incubated with TCHQ than with PCP. Consistent with this, the WST-8 assay showed that treatment with TCHQ reduced cell viability more markedly than treatment with the same dose of PCP. Wang and colleagues (15,16) reported that TCHQ induced apoptosis in mouse fibroblasts and human bladder carcinoma cells, but not in human liver cells, whereas PCP induced no apoptosis in either human cells (16). These findings and ours suggest that exposure to PCP or TCHQ induces apoptosis depending on the cell type and that human lymphocytes are sensitive to both PCP and TCHQ, but more so to TCHQ. TCHQ has been detected in urine of humans occupationally exposed to PCP (17,18). The concentration of PCP found in blood from workers exposed to PCP (4,730 [micro]g/L) (19) was similar to the concentration we used in the present study (20 [micro]M PCP). Thus, lowered lymphocyte count seen in workers with high PCP concentrations in blood (9) might be due to apoptotic cell death of lymphocytes. However, it should be noted that we observed apoptosis in Jurkat cells that were incubated with PCP or TCHQ in the absence of serum, whereas more than 96% PCP may be bound to human plasma protein (20). Hence, it remains to be determined whether apoptosis is induced in peripheral lymphocytes following exposures to PCP or TCHQ in vivo as well.

MAPKs are a family of serine/threonine protein kinases that transmit extracellular signals into the nucleus (21). Three subfamilies of MAPKs have been identified: ERK, p38, and JNK, which is also known as stress-activated protein kinase or SAPK. MAPK cascades have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation, and apoptosis (10,21). Previously, we found that heavy metals that can affect the immune system, such as cadmium, inorganic mercury, and organotin compounds, activate MAPKs in CCRF-CEM human lymphoblastoid cells prior to the development of apoptosis (12,13,22). We therefore determined the phosphorylation status of ERK, p38, and JNK in Jurkat human T cells treated with PCP or TCHQ. As a result, we found that treatment with TCHQ induced marked phosphorylation of p38 as well as ERK and JNK in Jurkat cells without changing the total protein levels of each MAPK. In contrast to the treatment with TCHQ, treatment with PCP did not induce MAPK phosphorylation clearly at the same doses (5-20 [micro]M and incubation period (1 hr).

TCHQ has been found to induce DNA singlestrand breaks and increase the level of 8-hydroxy-2-deoxyguanosine, whereas these effects were not observed after exposures to PCP (23-25). Therefore, TCHQ and PCP may differ in their potentials to damage DNA, and that might account for the differences in MAPK phosphorylation and subsequent development of apoptosis.

MAPKs can be activated in response to other chlorinated compounds. For example, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) have been reported to activate ERK and JNK in various types of cells (26-32). Unpurified technical grade PCP is often contaminated with other chlorinated compounds including PCBs, TCDD, and o,p'-DDT (1,2). It is therefore possible that alterations of lymphocytes seen in workers or in the general population may be partly due to effects of these contaminants. However, our results do not appear to be affected by contaminants because we used purified PCP and TCHQ.

The responses to inhibitors of MAPKs observed in the present experiments are consistent with the involvement of MAPK phosphorylation in TCHQ or PCP-induced apoptosis. SB203580, a p38 inhibitor (33), suppressed apoptosis markedly, and U0126, a potent inhibitor of both activated and nonactivated forms of MEK1/2 (34), also suppressed apoptosis mildly but significantly. When cells were treated with both inhibitors at the same time, TCHQ-induced apoptosis disappeared almost completely. These observations indicate that p38 and ERK play a major role in TCHQ-induced apoptosis to a different extent and that the two pathways might work synergistically. Because PCP-induced apoptosis was also suppressed by SB203580 and U0126, inhibition of basally activated forms of p38 and ERK might be partially effective. In contrast, treatment with LL-Z1640-2, which suppresses JNK phosphorylation (11,12), did not protect cells from apoptosis. This indicates that although JNK phosphorylation was most marked among the MAPKs examined, JNK did not play a major role leading to the apoptosis.

PCP inhibits oxidative phosphorylation (3) and also induces morphologic alterations in the mitochondria (35). Therefore, to clarify the mechanisms of PCP- and TCHQ-induced apoptosis, mitochondrial functions including membrane potential and cytochrome c release remain to be determined.

Thus, the present results indicate that some effects of PCP and its metabolite may be prevented by modifying MAPK pathways. However, more experiments are necessary before such an application is considered because their in vivo effects have not been clarified.


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Bambang Wispriyono, Masato Matsuoka, and Hideki Igisu

Department of Environmental Toxicology, University of Occupational and Environmental Health, Kitakyushu, Japan

Address correspondence to M. Matsuoka, Department of Environmental Toxicology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Telephone: +81-93-691-7404. Fax: +81-93-692-4790. E-mail:

We thank K. Takehana and T. Kobayashi (Pharmaceutical Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan) for providing LL-Z1640-2.

This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Received 11 May 2001; accepted 9 August 2001.
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Author:Igisu, Hideki
Publication:Environmental Health Perspectives
Date:Feb 1, 2002
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