In vitro and in vivo induction of heat shock (stress) protein (Hsp) gene expression by selected pesticides

ELSEVIER

Toxicology 112 (1996) 57-68

In vitro and in vivo induction of heat shock (stress) protein (Hsp) gene expression by selected pesticides D. Bagchi, G. Bhattacharya,

S.J. Stohs*

School of Pharmucy and Allied Health Professions, and the Cancer Center, Creighton University, 2500 Culiforniu Plaza, Omaha. NE 68178, USA Received I November 1995; accepted 6 March 1996

Abstract The chloroacetamide insecticide alachlor, polyhalogenated cyclic hydrocarbons endrin and chlordane and the organophosphate pesticides chlorpyrifos and fenthion induce oxidative tissue damaging effects including lipid peroxidation and nuclear DNA-single strand breaks. The mechanism involved in the induction of oxidative stress by these xenobiotics is unknown. No information is available regarding whether these pesticides can induce the expression of heat shock (stress) protein (Hsp) genes as a common protective mechanism against tissue damage. The pesticides were administered p.o. individually to female Sprague-Dawley rats in two 0.25 LD,, doses at 0 h and 21 h. The animals were killed at 24 h, and liver, brain, heart and lung tissues were removed to examine the induction of Hsps by Western and Northern blot analysis. In a separate series of experiments, cultured neuroactive PC-12 cells were treated 24 h with 50, 100 or 200 nM concentrations of these pesticides. Alachlor, endrin, chlorpyrifos and fenthion induced Hsp89a and Hsp89/3 in hepatic and brain tissues, as well as in cultured PC-12 cells. Chlordane induced some expression of Hsp89a but not Hsp89j in the hepatic and brain tissues of treated rats. Some expression of Hsp89/I was observed in lung tissues of endrin and alachlor treated animals. These findings were substantiated by Western blot analysis using Hsp90 antibody. Except chlordane all these pesticides induced enhanced synthesis of Hsp90 in cultured PC-12 cells. The results indicate striking tissue differences in the patterns of the Hsps induced by the pesticides which were used, and demonstrate that these pesticides can induce the expression of Hsp89a and Hsp89/3 genes in various target organs of rats. The results support the hypothesis that these genes may be mechanistically involved in protecting tissues against oxidative stress induced by structurally diverse pesticides. Keywords: Alachlor; Endrin; Chlordane; Chlorpyrifos; Fenthion; Sprague-Dawley rats; Liver; Brain; Lungs; PC-12 ceils

1. Introduction The structurally diverse chlorinated acetamide herbicides (CAH), polyhalogenated cyclic hydro*Corresponding author.

Heat shock (stress) proteins; Hsp89r; Hsp89b;

carbons (PCH) and organophosphate insecticides (OPS) are classified as pesticides or contaminants of pesticides, and environmental pollutants. CAH as alachlor, and PCH as endrin and chlordane, are halogenated and lipophilic (Murphy and Harvey. 1985; Kimbrough, 1985;

0300-483X/96/$1 5.00 8) 1996 Elsevier Ireland Ltd. All rights reserved PII 30300-483X(96)03350- I

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D. Bagchi et al. I Toxicology 112 (1996) 57-68

Murphy, 1986). OPS as chlorpyrifos or fenthion are organic triesters of phosphoric acid and phosphorothioic acid, and are also lipophilic and highly toxic (Murphy, 1986). Although alachlor, endrin, chlordane, chlorpyrifos and fenthion differ markedly in chemical structures and toxicological properties, all five xenobiotics induce oxidative stress and DNA damage in treated rats and cultured neuroactive PC-12 cells (Bagchi et al., 1995). These pesticides induced lipid peroxidation and DNA single strand breaks in brain and liver of treated rats, and produced enhanced chemiluminescence, lactate dehydrogenase leakage and DNA single strand breaks in cultured PC-12 cells (Bagchi et al., 1995). Neurotoxicity is a common characteristic of many of these compounds (Murphy, 1986; Shain et al., 1991; Davies, 1990; Rosenstock et al., 1990). Exposure of animals to these xenobiotics also elicits lipid mobilization, porphyria, hypothyroidism, hepatotoxicity, testicular atrophy, an increased liver to body weight ratio, and depletion of adipose tissues (Bach and Sela, 1984; Poland and Knutson, 1982; Shain et al., 1991; Stohs, 1995). A hallmark of OPS toxicity is the inhibition of acetylcholinesterase, neuronal degeneration, and changes in gait (Murphy, 1986). Another common biological property of these compounds is their ability to induce microsomal drug metabolizing enzymes (Viviani et al., 1978). Recent observations indicate that these xenobiotics induce the production of reactive oxygen species, with enhanced malondialdehyde production and glutathione depletion in the hepatic, brain and other tissues which may provide an explanation for the multiple types of toxic responses as well as the characteristic wasting syndrome (Metcalf et al., 1971; Morrison et al., 1992; Yamano and Morita, 1992; Julka et al., 1992; Akubue and Stohs, 1993; Naqvi and Hasan, 1992; Stohs, 1995). Ample evidence supports the hypothesis that reactive oxygen species mediate cell injury and lead to tumor promotion and carcinogenesis (Sun, 1990; Kehrer, 1993), and most of these xenobiotics have been implicated in tumor formation (Halliwell et al., 1992; Klaunig and Ruth, 1987; Williams and Numoto, 1984). Oxidative damage to proteins has become an import-

ant parameter under consideration to help explain oxygen free radical mediated tissue injury (Donati et al., 1990). Living organisms respond at the cellular level to unfavorable conditions such as heat or other stressful situations of many different origins by the rapid, vigorous, and transient acceleration in the rate of expression of a small number of specific heat shock/stress genes, resulting in the production of proteins commonly referred to as heat shock/stress proteins (Hsp) (Schlesinger et al., 1982). The Hsps are believed to assist cells to adapt or survive by a rapid but transient reprogramming of cellular metabolic activity to protect cells from further oxidative and thermal stress in responsive tissues (Harboe and Quayle, 1991; Pratt, 1993). Potential mechanisms for protection from oxygen free radicals by Hsp include prevention of protein degradation, inhibition of membrane lipid peroxidation or calcium intrusion, maintenance of ATP levels, and induction of classical scavengers such as superoxide dismutase or glutathione which itself plays a role in induction of Hsp (Freeman and Meredith, 1989). The present study was conducted to determine whether structurally diverse pesticides, which are known to induce oxidative stress, can initiate a common protective mechanism through the induction of the genes for heat shock proteins. The in vitro and in vivo induction of differential heat shock gene expression in cultured neuroactive PC-12 cells and in liver, brain, heart and lung by these pesticides was investigated. Oxidative stress inducible proteins by these pesticides were detected in cultured neuroactive PC-12 cells as well as in liver, brain, heart and lung by SDS-polyacrylamide gel electrophoresis. Western blot analysis was performed using antibody to Hsp90. The expression of pesticide-inducible genes was further monitored by Northern blot analysis using cDNA probes for 89cl kDa and 89B kDa Hsps. 2. Materials and methods Animals and treatment Female Sprague Dawley rats (Sasco Inc., Omaha, NE) weighing approximately 160-180 g were treated orally with alachlor (Monsanto, St.

2. I.

D. Bagchi et al. I To.rico1og.v 112 (1996)

Louis, MO), endrin, chlordane, chlorpyrifos and fenthion (Supelco, Bellefonte, PA) in two equal doses (0.25 LDso) of 2.25, 60, 300, 41 and 54 mg/kg body wt., respectively, in corn oil at 0 h and 21 h. Control animals received the vehicle. Analytical standard pesticides were purchased from Supelco (Bellefonte, PA). Analytical standard alachlor was a gift sample from Monsanto (St. Louis, MO). All animals were killed by decapitation at 24 h, and the liver, brain, lungs and heart were removed. 2.2. Neuroactive PC-12 cell cultures and pesticide treatment The PC-12 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained and grown in culture flasks in RPM1 1640 medium (GIBCO, Grand Island, NY) containing 10% heat-inactivated horse serum (Hyclone, Logan, UT) and 5% fetal bovine serum (Hyclone, Logan, UT). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO,. Trypsin solution was used to split cultures whenever they had grown to confluence. The number of cells was determined using a Coulter counter, and viability was checked by the Trypan blue exclusion method. The cultured cells (10’ cells) were treated individually with 50, 100 or 200 nM concentrations of alachlor, endrin, chlordane, chlorpyrifos or fenthion in two equally divided concentrations at 0 h and 21 h at 37°C. The incubation was continued for 24 h. The pesticide samples were dissolved in minimum volume of dimethyl sulfoxide (DMSO), and control samples were treated with identical volume of DMSO. 2.3. SDS-polyacrylumide gel electrophoresis and Ivestern blot anulysis Tissue homogenates of liver, brain, lungs and heart in 50 mM Tris chloride, pH 7.5, 1% nonidet P 40, 1 mM ethylene diaminetetraacetic acid tetrasodium salt (EDTA), 1 mM ethyleneglycolbis-(/&aminoethyl ether)N,N’-tetraacetic acid (EGTA) and 2m M phenylmethylsulfonyl fluoride (PMSF), and PC-12 cell lysates in 150 mM sodium chloride, 50 mM Tris chloride, pH 7.5. 1% nonidet P 40, 0.1% sodium deoxycholate,

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59

0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM EGTA and 2 mM PMSF, were individually boiled for 5 min in equal volumes of 2X SDS sample buffer (0.5 M Tris chloride, pH 6.8,4% SDS, 20% glycerol, 0.002% bromophenol blue and 10% 2-mercaptoethanol). SDS-polyacrylamide gel electrophoresis was performed according to the method described by Laemmli (1970). The boiled, denatured proteins (100 pg) were loaded on 7.5% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred from acrylamide gel onto an Immobilon PVDF nylon membrane (Millipore Corp., Bedford, MA) at 200 mA for 45 min at room temperature by using a semi-dry blot system. Polyclonal mouse antibody against Hsp90 was obtained from Affinity Bioreagents Inc., Neshanic Station, NJ. Hsp90 was detected by an alkaline phosphatase method using polyclonal rnouse antibody followed by biotinylated goat antimouse IgG (H +L) human IgG, and streptovidin-AP color development reagent (Bio-Rad, Hercules, CA) (Blake et al., 1984; Titus, 1991). 2.4. Northern blot analysis For Northern blot analysis, total RNA was extracted from the hepatic, brain, heart and lung tissues as well as from cultured PC-12 cells by the acid guanidium thiocyanate-phenol-chloroform method of Chomezymski and Sacchi (1987). RNA was quantitated spectrophotometrically at 260 nm. Integrity of RNA preparations and consistent sample loading were verified by electrophoresis in a 1% agarose gel containing ethidium bromide (0.1 pg/ml) and subsequent UV illumination. Twelve micrograms of total RNA from each sample was subjected to 1% agarose-2.2 M formaldehyde gel electrophoresis, and the samples were transferred to nylon membranes (Gene Screen Plus, NEN Research Products, Boston, MA) by capillary blot (Sambrook et al., 1989a). 20X SSC was prepared by dissolving 175.3 g of NaCl and 88.2 g of sodium Icitrate in 800 ml of water and adjusting the pH to 7.0 using a few drops of 10 N NaOH solution and adjusting the final volume to 1 liter with water (Sambrook et al., 1989b). 20X SSPE was prepared by dissolving 175.3 g of NaCl, 27.16 g of

60

D. Bagchi et al. I Toxicology I12 (1996)

NaH,PO, * H,O and 7.4 g of EDTA in 800 ml of water and adjusting the pH to 7.4 with 10 N NaOH and making up the final volume to 1 liter (Sambrook et al., 1989). Both 20X SSC and 20X SSPE were sterilized using an autoclave. The membranes were baked under vacuum at 80°C for 2 h, and prehybridized in a solution containing 50% formamide, 6X SSPE, 5X Denhardt’s reagent, 0.9% SDS, and 100 pg/ml denatured salmon testes DNA with a-[32P]-labeled Hsp cDNA probes at 42°C for 24 h. The Hsp89a probe was a 1337-bp pstI fragment of the human cDNA, and the Hsp89B probe was a 838-bp pstI fragment of the human cDNA (Stress Gene Biotechnologies Corp., Victoria, B.C., Canada). All the cDNA probes were labeled with c(-[~~P]dCTP (Amersham, Arlington Heights, IL) by using a random primed labeling kit (Boehringer Mannheim, Indianapolis, IN) with a specific activity of approximately 0.7 x lo9 dpm/pg. The membranes were washed twice with 2X SSC and 1% SDS at 60°C for 30 min and once with 0.1X SSC at room temperature for 30 min. Autoradiographs were obtained after exposure to Kodak X-Omat RP films (Sigma Chemical Co., St. Louis, MO) at -70°C. Each hybridization was repeated at least three times using different membranes. After hybridizing the blots with 32Plabelled Hsp89a and Hsp89/3 cDNA probes, the blots were stripped and rehybridized with a /Iactin cDNA probe (Oncor Inc., Gaithersburg, MD). The results from the rehybridization with the p-actin cDNA probe served as the control. The autoradiographs were quantitatively evaluated by determining the counts of radioactivity in the membranes using a Packard 1600CA TriCarb liquid scintillation analyzer (Meriden, CT). The results of radioactivity measurements were normalized relative to the data obtained for actin cDNA. Hybridization with /I-actin cDNA did not significantly increase the radioactivity counts, while both Hsp89cr and Hsp898 cDNA signifi-

57-66

cantly modulated the radioactivity with the membranes.

associated

2.5. Statistical methods Statistical comparisons were determined by using analysis of variance (ANOVA) with Scheffe’s S method as the post hoc test. Each value is the mean + S.D. of at least four experiments. A P < 0.05 was considered significant for all comparisons. 3. Results

3.1. SDS-polyacrylamide gel electrophoresis and Western blot analysis

Alachlor, endrin, chlordane, chlorpyrifos and fenthion induced overexpression of 90- and 60kDa proteins in hepatic, brain and lung tissues (Fig. lA-C, respectively). Enhanced expression of the 90-kDa protein was also observed in cultured PC-12 cells treated with pesticides (Fig. 1D). The 90-kDa protein was further recognized by Hsp90 polyclonal mouse antibody by Western blot analysis in all pesticide-treated hepatic tissues (Fig. 2A). Expression of Hsp90 was also observed in the pesticide-treated brain tissues, except in chlordane-treated animals, by Western blot analysis (Fig. 2A). The induction of Hsp90 in lung tissues was observed only in alachlor and endrin-treated animals (Fig. 2A). Expression of Hsp90 in pesticide-treated cultured PC12 cells agreed with the in vivo results in rats. Cultured neuroactive PC-12 cells were treated with 50,100 or 200 nM concentrations of alachlor, endrin, chlordane, chlorpyrifos and fenthion for 24 h. The 90-kDa protein was recognized by Hsp90 polyclonal mouse antibody by Western blot analysis in all pesticide-treated cultured PC-12 cells except in chlordane-treated PC-12 cells (Fig. 2B). Concentration dependent enhanced expression of Hsp90 was observed.

Fig. 1. SDS-polyacrylamide gel electrophoresis of the hepatic (A), brain (B), and lung (C) tissues from control and pesticide-treated rats, and cell lysates (D) from control and pesticide-treated cultured PC-12 cells. Lanes as follows: 1. control sample; 2, alachlor treated sample; 3, endrin-treated sample; 4, chlordane-treated sample; 5. chlorpyrifos-treated sample; 6, fenthion-treated sample.

61

D. Bugchi et (11.I Toxicology I I2 (1996) 57-68

123456

A

1

B

234

200 _) 200 +

C

123456

123456

D

200-+ 200 __)

116+

116+

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D. Bugchi rr ul. I Toxicology I I2 (1996)

62 1234567

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8

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Fig. 2. (A) Western blot nnalysis of protein samples derived from the organs of control and pesticide-treated rats. Lanes as follows: 1, control brain; 2, alachlor-treated brain; 3, end&-treated brain; 4, chlordane-treated brain; 5, chlorpyrifos-treated brain; 6, fenthion-treated brain; 7, control liver; 8, alachlor-treated liver; 9, endrin-treated liver; 10, chlordane-treated liver; 11, chlorpyrifos-treated liver, 12, fenthion-treated liver; 13, control lung; 14, alachlor-treated lung; 15, endrin-treated lung. (B) Western blot analysis of protein samples derived from control and pesticide-treated cultured PC-12 cells. Lanes as follows: 1, control DMSO (50 nM); 2, control DMSO (100 nM); 3, control DMSO (200 nM); 4, alachlor (50 nM); 5, alachlor (100 nM); 6, alachlor (200 nM); 7, endrin (50 nM); 8, endrin (100 nM); 9, endrin (200 nM); IO, chlordane (50 nM); 1I, chlordane (100 nM); 12, chlordane (200 nM); 13, chlorpyrifos (50 nM); 14, chlorpyrifos (100 nM); 15, chlorpyrifos (200 nM); 16, fenthion (50 nM); 17, fenthion (100 nM); 18, fenthion (200 nM).

3.2.Efects

of pesticides on hsp gene expression

The induction of the expression of Hsp89a and Hsp89/3 genes is shown in Fig. 3A and B, respectively. Alachlor, endrin, chlorpyrifos and fenthion dramatically induced expression of Hsp89a in both brain and hepatic tissues, and chlordane induced some expression of Hsp89a in both of these tissues (Fig. 3A). Endrin, alachlor, chlorpyrifos and fenthion induced overexpression of Hsp89jI in both hepatic and brain tissues. No induction of this Hsp was observed in the hepatic or brain tissues of rats treated with chlordane. Some expression of Hsp89B was noted in the lung tissues of alachlor and endrin treated rats (Fig. 3B). Endrin, alachlor, chlorpyrifos and fenthion induced overexpression of Hsp89a and Hsp898 in cultured PC-12 cells (Fig. 4A and B, respectively). Concentration-dependent overexpression of Hsp89~ and Hsp89/? was observed. However, no effect was observed following treatment with chlordane. The quantitation of Hsp gene expression re-

sults in rats and cell cultures as obtained from the counts of the radioactivity (32P counts/min) is shown in Tables 1 and 2, respectively. Table 1 demonstrates the radioactivity (3zP counts/min) of Hsp89a and Hsp89b mRNA expression in different tissues. Alachlor, endrin, chlorpyrifos and fenthion induced increases of 2.1-, 2.4, 3.4and 3.3-fold in brain Hsp89a, respectively, while in liver this same protein increased by 3.9-, 2.8-, 3.3-, and 2.7-fold, respectively. With respect to these same four pesticides in the brain, Hsp89fl increased 2.6-, 2.9-, 2.0-, and 2.2-fold, respectively, while in the liver increases of 3.0-, 3.4-, 2.7- and 2.6-fold occurred, respectively. Chlordane induced increases of 1.7- and 2.1-fold in the brain and liver Hsp89a, respectively. In the lung, alachlor and endrin induced increases in Hsp89B of 2.8- and 2.9-fold, respectively. Table 2 depicts the radioactivity (PJ2 counts/ min) associated with Hsp89ct and Hsp89/? mRNA expression in control and pesticide-treated cultured PC-12 cells. The cultured PC-12 cells were incubated with 50, 100 or 200 nM concentrations

D. Bagchi et al. I Toxico1og.v 112 (1996) 57-68

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-

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15

Fig. 3. Northern blot analysis of RNA derived from the organs of control and pesticide-treated rats. Twelve micrograms of total RNA was subjected to I % agarose-2.2 M formaldehyde gel electrophoresis and hybridized with Hsp 89~ cDNA probe (A) or Hsp89/f cDNA probe (B). Lanes as follows: 1, control brain; 2, alachlor-treated brain; 3, endrin-treated brain; 4, chlordane-treated brain; 5, chlorpyrifos-treated brain; 6, fenthion-treated brain; 7, control liver; 8, alachlor-treated liver; 9, endrin-treated liver; IO, chlordane-treated liver; 11, chlorpyrifos-treated liver; 12, fenthion-treated liver; 13, control lung; 14, alachlor-treated lung; 15, endrin-treated lung. The positions of the 28 S and I8 S ribosomal RNA bands after staining with ethidium bromide are indicated on the left.

A

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ia

2ase

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la

Fig. 4-Northern blot analysis of RNA derived from control and pesticide-treated cultured PC-12 cells. RNA was subjected to I % agarose-2.2M formaldehyde gel electrophoresis and hybridized with Hsp89r cDNA probe (A) or with Hsp89B cDNA probe (B). For experimental details, see Section 2, Materials and methods. Lanes as follows: I, control DMSO (50 nM); 2, control DMSO (IO0 nM); 3-control DMSO (200 nM); 4, alachlor (50 nM); 5-alachlor (100 nM); 6, alachlor (200 nM); 7 -endrin (50 nM); 8, endrin (100 nM); 9, endrin (200 nM); IO, chlordane (SO nM); II, chlordane (100 nM); 12, chlordane (200 nM); l3-chlorpyrifos (50 nM); 14, chlorpyrifos (100 nM); 15, chlorpyrifos (200 nM); 16, fenthion (50 nM): 17, fenthion (IO nM): 18, fenthion (200 nM).

D. Bugchi et (11.I Toxicology I I2 (1996) 57-68

64 Table 1 Radioactivity

(32P counts/min)

associated

with Hsp89r

and Hsp89p

Hsp89u

610 1288 1485 1050 2072 2006

expression

in different

tissues of rats

Hsp89P

Brain Control Alachlor Endrin Chlordane Chlorpyrifos Fenthion

mRNA

Liver rfr 80” k 104b _+ 122” f 94’ k 175d + 197d

570 2220 1604 1171 1896 1564

Brain * + k + f f

90” 143h 126 99d 158’ 133’

607 1576 1750 745 1216 1313

Liver + f k f f *

65” 96” 84’ 46“ 69’ 71’

550 t 1635 + 1861 + 703 + 1491 + 1428;

Lung 46” 117” 129h 81’ 133h.d 125d

542 1519 1577 650 614 691

+ 39” f 142” f 139h f 77”,’ + 83”.’ f64’

Female Sprague-Dawley rats were treated orally with 0.25 LD,, doses of alachlor, endrin, chlordane, chlorpyrifos or fenthion in corn oil at 0 and 21 h. Liver, brain and lung tissues were obtained at 24 h. RNAs were isolated and subjected to Northern blot Hsp89a and Hsp898 probes. Values are expressed as mean counts/min f S.D. of at least four analyses using 32P-labeled experiments. Values with non-identical superscripts in each column are significantly different (P < 0.05).

respectively, while at the same concentration the expression of Hsp89/? increased 5.7-, 5.6-, 6.1and 6.8-fold as compared to control values. At 200 nM concentrations of alachlor, endrin, chlorpyrifos and fenthion the expression of Hsp89c( increased by approximately 5.2-, 3.7-, 6.1- and 5.8-fold, respectively, while the expression of Hsp89P increased by approximately 5.7-, 5.1-, 6.0- and 5.6-fold as compared to the control values. Small (l.l- to 1.3-fold) increases in Hsp89c( and Hsp89P were observed following incubation with chlordane.

of the pesticides and the concentration-dependent effects of the pesticides were determined. At 50 nM concentrations of alachlor, endrin, chlorpyrifos and fenthion the expression of Hsp89cr increased by approximately 3.8-, 3.2-, 3.6-, and 4.1-fold, respectively, while the expression of Hsp89B increased approximately 4.0-, 3.0-, 4.9and 5.3-fold, respectively, as compared to control values. The expression of Hsp89cr increased 4.5, 4.6-, 5.1- and 5.2-fold following incubation of cultured PC-12 cells with 100 nM concentrations of alachlor, endrin, chlorpyrifos and fenthion,

Table 2 Radioactivity PC-12 cells

(32P counts/min)

Treatment

Control (DMSO) Alachlor Endrin Chlordane Chlorpyrifos Fenthion

associated

with Hsp89a: and Hsp89p

mRNA

in control

and pesticide-treated,

cultured

Hsp89B

Hsp89r 50 nM

100 nM

637 2427 2042 787 2287 2642

716 3219 3315 893 3626 371 I

f. 52” + 21 lb f 187’ f 64d f 218”,’ I223

expression

k + + k f

f

200 nM 73” 255” 308h 62’ 275h 307h

754 3957 2821 987 4573 4388

+ + f f f &

50 nM 66” 265” 257’ 71d 422h 414”

596 2392 1808 682 2938 3182

& + + t F +

34 181” 178’ 71d 232’ 274’

100 nM

200 nM

634 361 I 3544 756 3885 4312

787 & 55” 4456 + 32Xh 4030 f 413h 941 + 90’ 4744i411” 4418 f 409h

+ 39” + 263” f 295h + 84’ + 367h.d ; 323d

Cultured PC-12 (I x 10’) cells were incubated individually with either 50 nM, 100 nM or 200 nM of alachlor, endrin, chlordane, chlorpyrifos or fenthion, and total RNA was isolated after 24 h using the guanidium isothiocynate method. The RNAs were run on 1% agarose gels, transferred to nylon membranes, and Northern blot analyses were performed by using “P-labeled HSP89r and HSP89fi probes. Values are expressed as mean counts/min & S.D.of at least four experiments. Values with non-identical superscripts in each column are significantly different (P < 0.05).

D. Bagchi et al. I Tosico1og.v II? (1996) 57-68

4. Discussion The structurally diverse CAH, PCH and OPS have been previously reported to induce oxidative stress, enhance malondialdehyde production and lactate dehydrogenase leakage, and decrease glutathione peroxidase and superoxide dismutase activity (Akubue and Stohs, 1993; Julka et al., 1992; Naqvi and Hasan, 1992; Metcalf et al., 1971; Morrison et al., 1992; Stohs, 1995; Yamano and Morita, 1992), which may contribute to the production of toxicities by these pesticides. These pesticides also modify basic membrane mechanisms as permeability to nonelectrolytes and transport of cations mediated by ionophores (Antunes-Madeira and Madeira, 1979). The effectiveness of these xenobiotics as pesticides parallels their relative toxicities to mammals (Ames, 1992; Cantor et al., 1993; Stohs, 1995). Relatively low concentrations of these pesticides, ranging from 10v5 to 10e4M, have been reported to induce significant tissue damaging effects (Antunes-Madeira and Madeira, 1979; Botham, 1990). These lipophilic pesticides may accumulate preferentially in lipid domains of cells and organisms (Ames, 1992; Brooks, 1974; Domenech et al., 1977; Stohs, 1995), resulting in localization in or near membranes. Thus, a homogeneous distribution of these xenobiotics can not be assumed. Previous studies (Bagchi et al., 1995) have demonstrated that administration of endrin, chlordane, alachlor, chlorpyrifos and fenthion results in the in vitro and in vivo induction of hepatic and brain lipid peroxidation, production of chemiluminescence, increased DNA single strand breaks, and increased lactate dehydrogenase leakage, suggesting that the reactive oxygen species and/or free radicals may be involved in the toxic manifestations of these pesticides. The data also reflected the tissue specificity of the pesticides with respect to these responses. Donati et al. (1990) have demonstrated the ability of oxygen free radicals to induce heat shock proteins in vivo. Oxidative injuries by UV radiation, sodium arsenite or cadmium are well known inducers of heat shock (stress) proteins in cells

65

(Bannai et al., 1991). Studies involving the possible cross protection between heat shock and oxidative injury have demonstrated that heat shock (stress) proteins protect human monocytic cells not only from thermal but also from further oxidative injury as well as from tumor necrosis factor (TNF)-induced cell lysis (Donati et al., 1990). The present study indicates that different classes of pesticides induce the expression of Hsp genes in target organs. However, the degree of mRNA induction was dependent upon the pesticide employed, and varied from organ to organ. The production of the Hsp90 proteins in response to the pesticides may serve as a protective mechanism against further damage. The results from SDS-PAGE experiments have shown induction/overexpression of several protein bands in tissues of pesticide-treated rats and cultured PC12 cells. Western blot analysis has indicated the involvement of Hsp90 (Fig. 2A,B) which was further substantiated by Northern blot analysis (Figs. 3A,B, 4A,B). Hsp proteins are highly conserved in nature at the amino acid sequence level and act as physiological “house-keepers”. They play important roles in infection and autoimmunity (Harboe and Quayle, 1991), such as binding to other polypeptides and allowing them to either reach their correct intracellular destination in the cell, transporting other proteins across membranes, or preventing other proteins from misfolding (Pelham, 1984; Rothman, 1989). The uniqueness of Hsps include their ability to recognize “unstructured” protein regions present in other polypeptides (Pelham, 1984; Rothman, 1989). Eukaryotic: Hsps have been shown to tightly bind with the tumor promoter protein p53 (Clarke et al., 1989), and may therefore modulate tumor promotion. Studies on the mechanism of steroid receptor/ hormone action have uncovered a pivotal role for the Hsp90 or 90-kDa stress proteins in cell regulation (Pratt, 1993). Synthesis of Hsp90 increases approximately 3- to 5-fold after activation of the stress response, and phosphorylation of a‘t least 12 isoforms occurs. Newly synthesized pp60”” from Rous sarcoma virus-transformed cells rapidly associates with 90-kDa stress proteins

66

D. Bagchi rt al. I Toxicology

and other cellular proteins (Welch, 1990). In this complex state, src protein is unable to exhibit its normal tyrosine kinase activity (Welch, 1990), an enzyme which is involved in oncogenic activity. In the present studies, significant increases in both Hsp89a and Hsp89fl were observed in treated animals and cultured cells. Most dramatic induction of Hsp89a was observed in brain tissues from endrin and fenthion-treated rats, whereas alachlor and chlorpyrifos showed some expression of this Hsp in the brain. Little induction of Hsp89a and Hsp89fi was observed in brain tissues of chlordane-treated rats. In hepatic tissues, varying degrees of expression of Hsp89c( and Hsp89P were observed in animals treated with all of the pesticides. No induction of Hsp89a was observed in heart or lung of the pesticidetreated animals (data not shown). Furthermore, no induction of Hsp89P was observed in heart of pesticide-treated rats. In the lungs, significant expression of Hsp89/_3was noted only in endrin and alachlor treated rats. In the cultured PC-12 cells the greatest induction of Hsp89c( and Hsp898 was observed with the OPS pesticides chlorpyrifos and fenthion, No induction of Hsp 89~ and 89/3 was observed with chlordane. The differential responses of the tissues which were examined may reflect pharmacokinetic (toxicokinetic) differences of the pesticides. In summary, the results demonstrate that differential heat shock (stress) gene expression occurs in a number of organs as well as cultured cells following treatment with selected pesticides which are known to induce oxidative stress. The results indicate striking regional and cell type differences in the patterns of induction of the Hsp mRNAs by different classes of pesticides, suggesting that different organs and cell types respond differently to these pesticides. This study demonstrates that these pesticides can induce the expression of HSP89cr and HSP89P genes in various target organs in rats as well as cultured PC-12 cells, and the results support the hypothesis that expression of HSP89c( and HSP898 genes may be involved in modulating the oxidative stress and toxicity induced by these pesticides. Thus, expression of Hsp genes may constitute a common mechanism of protection

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against oxidative tissue damage induced by structurally diverse xenobiotics. Acknowledgments

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