Elevated Ovarian Follicular Apoptosis and Heat Shock Protein-70 Expression in White Sucker Exposed to Bleached Kraft Pulp Mill Effluent

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

147, 391–398 (1997)

TO978283

Elevated Ovarian Follicular Apoptosis and Heat Shock Protein-70 Expression in White Sucker Exposed to Bleached Kraft Pulp Mill Effluent David M. Janz,*,1 Mark E. McMaster,† Kelly R. Munkittrick,† and Glen Van Der Kraak* *Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and †Environment Canada, National Water Research Institute, 867 Lakeshore Road, Burlington, Ontario, Canada L7R 4A6 Received June 3, 1997; accepted August 22, 1997

Elevated Ovarian Follicular Apoptosis and Heat Shock Protein-70 Expression in White Sucker Exposed to Bleached Kraft Pulp Mill Effluent. Janz, D. M., McMaster, M. E., Munkittrick, K. R., and Van Der Kraak, G. (1997). Toxicol. Appl. Pharmacol. 147, 391–398. Exposure of feral fish populations to bleached kraft pulp mill effluent (BKME) results in a variety of negative impacts on reproductive fitness including reduced ovarian development, reduced egg size, decreased fecundity with age, delayed sexual maturation, and alterations in reproductive endocrine homeostasis at multiple sites along the pituitary– gonadal axis. The present study provides evidence of elevated apoptotic DNA fragmentation and increased expression of the 70-kDa heat shock protein (HSP70) in ovarian follicular cells from prespawning white sucker (Catostomus commersoni) exposed to BKME. Apoptosis is the molecular mechanism responsible for ovarian follicular atresia which is involved in various stages of vertebrate ovarian development such as follicular recruitment, growth, differentiation, and regression. In mammals, induction of HSP70 is associated with inhibition of hormonesensitive steroidogenesis and mediation of luteal regression. The 3*-end labeling of isolated ovarian follicular cell DNA revealed a 10-fold increase in the extent of apoptosis in BKME-exposed white sucker in comparison to follicles collected from a nearby reference site. Western blotting for ovarian follicular HSP70 levels showed increased expression of this protein in fish exposed to BKME. The elevated ovarian cell apoptosis and increased HSP70 expression in BKME-exposed fish were associated with reduced ovary size, decreased plasma testosterone, and increased plasma 17b-estradiol concentrations, but not induction of hepatic ethoxyresorufin O-deethylase activity. It is not known whether increased ovarian HSP70 expression in BKME-exposed fish is related to elevated apoptosis or represents a general response to environmental stress. Since apoptosis is regulated by several hormonal factors and conserved gene products, these data suggest that certain components of BKME increase ovarian cell apoptosis in fish via stimulation of cell death signaling. However, it is unclear whether BKME stimulates ovarian cell apoptosis directly or if this response occurs secondarily as a result of altered reproductive endocrine homeostasis. © 1997 Academic Press

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Present address: Department of Zoology, Oklahoma State University, 430 Life Sciences West, Stillwater, OK 74078.

Several studies have demonstrated alterations in endocrine homeostasis and reproductive fitness in feral fish populations exposed to pulp mill effluents (McMaster et al., 1996; Sandstro¨m, 1996). The mechanisms responsible for effects on reproductive function have been studied most extensively in northern Ontario populations of white sucker (Catostomus commersoni). Female white sucker exposed to bleached kraft pulp mill effluent (BKME) at Jackfish Bay, Lake Superior, exhibit a number of organismal and biochemical responses, including reduced ovarian development, reduced egg size, decreased fecundity (number of eggs per female) with age, delayed sexual maturation, and alterations in reproductive endocrine homeostasis at multiple sites along the pituitary– gonadal axis (McMaster et al., 1991; Munkittrick et al., 1991; Van Der Kraak et al., 1992). These effects were consistent during different stages of ovarian development, different years, different locations, and following a number of changes in effluent treatment aimed at reducing environmental impacts of BKME (Munkittrick et al., 1994; McMaster et al., 1996). Although no differences in fertility or survival of individual white sucker eggs were apparent between BKME and reference sites (McMaster et al., 1992b), these studies nonetheless indicate a suite of disruptions in reproductive homeostasis. Important toxicological questions remain concerning the mechanistic linkages between alterations in reproductive endocrine homeostasis and responses observed at the organ and individual levels. Atresia is a degenerative process by which ovarian follicles lose their integrity and are eliminated prior to ovulation in all vertebrate classes (Byskov, 1978). In mammals and presumably other vertebrates, the molecular mechanism responsible for ovarian follicular atresia is apoptotic cell death (Tilly et al., 1991; Hughes and Gorospe, 1991). Apoptosis or programmed cell death is an evolutionarily conserved physiological process involved in tissue remodeling, differentiation, and degeneration in a variety of cell types (Steller, 1995). There is considerable information concerning the intracellular pathways and genes involved in ovarian cell death in mammals (Tilly, 1996; Tilly et al., 1997a,b; Tilly and Perez, 1997). In the vertebrate ovary, apoptotic cell death is controlled by several hormones

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and growth factors (Hsueh et al., 1994). For example, gonadotropins, 17b-estradiol, and epidermal growth factor act as follicle survival factors as shown by their ability to suppress apoptotic cell death in cultured ovarian follicles from rats (Tilly et al., 1992; Billig et al., 1993; Chun et al., 1994) and rainbow trout (Oncorhynchus mykiss) (Janz and Van Der Kraak, 1997). In contrast, gonadotropin-releasing hormone (GnRH), androgens, and certain cytokines stimulate apoptosis in cultured ovarian follicles from rats (Billig et al., 1993, 1994; Gorospe and Spangelo, 1993). Perturbation of the hormonal signals controlling apoptosis may adversely affect ovarian growth, development, and differentiation. Since ovarian follicular growth, development, and death (apoptosis) in teleost fish are controlled by hormones and growth factors (Janz and Van Der Kraak, 1997; Van Der Kraak et al., 1997) and reproductive endocrine homeostasis in fish is altered by BKME exposure, we hypothesize that BKME affects ovarian development in white sucker by stimulating apoptosis in ovarian follicular cells. In addition, nonhormonal factors such as heat shock proteins (HSPs) may also be important in teleost ovarian function since increased expression of HSP70 is associated with reduced steroidogenesis and luteal regression in rats (Khanna et al., 1994, 1995). Therefore, we also examined the expression of ovarian HSP70 in white sucker exposed to BKME due to its potential importance in various aspects of ovarian function and known upregulation following exposure to a variety of environmental stressors. METHODS Study sites. Jackfish Bay (48°509N, 86°589W) is located on the north shore of Lake Superior and receives effluent from a bleached kraft mill in Terrace Bay, Ontario, producing 1250 air-dried metric tons of pulp per day. This mill releases approximately 100,000 m3day21 of effluent following secondary treatment. There are no other industrial, municipal, or agricultural discharges to Jackfish Bay. The mill uses approximately 32 softwood (70% spruce, 20% pine, 10% fir) and 31 hardwood (poplar with a small amount of birch) as feedstock. The chlorine substitution level varies between 50 and 70%. The bleaching sequences are DCPE.DED for the softwood line and C/DEDED for the hardwood line (D, chlorine dioxide; C, chlorine; P, hydrogen peroxide; E, sodium hydroxide). Effluent is treated in a 10-d aerated stabilization basin before discharge into Blackbird Creek, which carries the effluent 15 km to Jackfish Bay. White sucker leave Jackfish Bay and travel through an uncontaminated lake (Jackfish Lake) before spawning in Sawmill Creek. The fish are not exposed to effluent at the time of collection during the prespawning period. Fish were also collected from a nearby reference site located at Little Gravel River, which flows into Mountain Bay (48°569N, 87°509W) and receives no industrial or significant domestic effluents. Both sites have been used previously to monitor impacts of BKME on fish populations, and more detailed information regarding these sites can be found elsewhere (McMaster et al., 1991; Munkittrick et al., 1991, 1994; Van Der Kraak et al., 1992). Fish collection and sampling. Prespawning white sucker were captured in overnight hoop net sets during mid May 1996. To ensure white sucker from each site were at similar reproductive stages, they were collected on the first night where greater than 300 fish entered the spawning streams and females were separated from males and sampled between 1030 and 1400 hr. Blood was collected using a syringe from the caudal artery and stored in 10-ml heparinized vacuum tubes on ice for 6 to 8 hr prior to centrifugation and collection

of plasma. The liver was excised, weighed, and immediately frozen in liquid nitrogen. The ovaries were removed and weighed, and approximately 100 follicles from the first 6 fish were frozen immediately in liquid nitrogen for determination of apoptotic DNA fragmentation and HSP70 levels. Follicles were also collected for determination of fecundity, and a gonadosomatic index (GSI) was calculated as 1003[ovary weight 4 (body weight 2 ovary weight)]. DNA isolation and analysis. Total genomic DNA from ovarian follicles was extracted and phenol/chloroform-purified using a method adapted from Gross-Bellard et al., (1973) for use in intact tissues (Tilly and Hsueh, 1993) and modified as previously described for fish (Janz and Van Der Kraak, 1997). DNA was isolated from a pool of eight ovarian follicles, quantitated by absorbance at 260 nm, and stored at 220°C. The yield of DNA from eight follicles was 7.7 6 0.4 mg (mean 6 SEM; n512). DNA purity ranged from 86 to 95%. Aliquots of DNA (2 mg) from each sample were 39-end labeled with [a32P]dideoxy-ATP ([32P]ddATP; 3000 Ci/mmol; Amersham, Oakville, ON) using terminal deoxynucleotidyl transferase (Tdt; 25 U/ml; Boehringer Mannheim, Laval, QC) as described previously (Tilly and Hsueh, 1993). Radiolabeled DNA was loaded onto 2% agarose gels (1 mg/lane) and fractionated by electrophoresis for 2.5 hr at 100 V (6.5 V/cm) using TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) as the running buffer. A 123-base-pair DNA ladder (Gibco, BRL, Burlington, ON) was also 3’-end labeled and run on each gel as a standard. Following electrophoresis, the gel was dried in a slab-gel drier for 2 hr without heat. The dried gel was sealed in plastic wrap and exposed to Fuji XR X-ray film for 2– 4 hr at 280°C. Portions of each lane corresponding to low (,15 kb)-molecular-weight DNA were cut from the dried gel with a scalpel and counted using liquid scintillation counting to quantify the extent of apoptotic DNA fragmentation (Tilly and Hsueh, 1993). Western blotting. Detection of HSP70 in ovarian follicles was performed by immunoblotting. Approximately 50 follicles from individual fish were thawed and placed in ice-cold homogenization buffer (50 mM Hepes, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 1% (v/v) Triton X-100, 10% (v/v) glycerol, pH 7.5) containing 0.1 mg/ml Pefabloc (Boehringer Mannheim), 20 mg/ml trypsin inhibitor, 4.3 mg/ml leupeptin, and 1% (v/v) aprotinin (Sigma). Follicles were homogenized on ice using a glass–teflon homogenizer and centrifuged for 20 min at 10,000g and 4°C, and the resulting supernatant was stored at 280°C. Protein was determined using a variation of the Lowry method (Bio-Rad, Mississauga, ON). Total follicular protein (40 mg/lane) was separated using SDS–PAGE with 10% acrylamide minigels (Bio-Rad) and transferred to Hybond-C nitrocellulose membranes (Amersham, Oakville, ON) overnight at 30 V and 4°C. Immunoblotting was performed using a polyclonal rabbit anti-rainbow trout HSP70 antibody (Forsyth et al., 1997) at 1:5000 dilution for 1 hr at room temperature. This was followed by a 30-min incubation with a goat anti-rabbit IgG-horseradish peroxidase conjugate diluted 1:5000. Immunoreactive bands were detected using the ECL system (Amersham). Protein was also prepared as described above from rainbow trout ovarian follicles and run on each gel as a control. The optical density of bands was determined using NIH Image software. Radioimmunoassay. Plasma concentrations of testosterone and 17b-estradiol were determined by radioimmunoassay (RIA) following ether extraction, as described previously (McMaster et al., 1992a). Descriptions of the antisera used to measure testosterone and 17b-estradiol are reported in Van Der Kraak and Chang (1990) and Van Der Kraak et al., (1990), respectively. Plasma samples were assayed in duplicate. Interassay coefficients of variation were ,15% for each hormone. Ethoxyresorufin O-deethylase (EROD) assay. Hepatic EROD activities were determined in 10,000g postmitochondrial supernatants from individual fish livers using a direct fluorometric assay (Burke and Mayer, 1974), optimized for use in fish (Munkittrick et al., 1993). The reactions were performed at 25°C and fluorescence was measured using a Millipore Cytofluor 2350 fluorometer with excitation and emission wavelengths of 530 and 590 nm, respectively. Protein concentrations were determined (Bradford, 1976) using bovine serum albumin as the standard.

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OVARIAN APOPTOSIS IN FISH EXPOSED TO PULP MILL EFFLUENT

TABLE 1 Gonadosomatic Index (Ovary to Body Weight Ratio), Fecundity (Number of Ovarian Follicles per Fish), and Hepatic Ethoxyresorufin O-Deethylase (EROD) Activity in Prespawning White Sucker Exposed to Bleached Kraft Pulp Mill Effluent or Collected from a Reference Site Parameter

Sample

n

BKME

n

Reference

Gonadosomatic index (%) Gonadosomatic index (%) Fecundity Fecundity Hepatic EROD activity (nmol/min/mg protein) Hepatic EROD activity (nmol/min/mg protein)

Subsample Sample Subsample Sample Subsample Sample

6 100 6 53 6 19

13.63 6 0.84* 12.91 6 0.19** 25256 6 2438 27590 6 1169 1.393 6 0.277 1.519 6 0.207***

6 104 6 51 6 39

17.58 6 1.08 14.42 6 0.18 30,654 6 3107 28,262 6 923 0.870 6 0.133 1.035 6 0.074

Note. Values are means 6 SEM. Significantly different from reference site using one-way ANOVA: *p , 0.05, **p , 0.001, ***p 5 0.05.

Statistics. Differences between reference and BKME-exposed white sucker were detected using one-way analysis of variance (ANOVA). Heteroscedasticity was detected using Bartlett’s test for homogeneity of variances, and such data were log-transformed prior to ANOVA. Biochemical analysis of apoptosis and determination of HSP70 levels were performed on six fish from each site, representing a subsample of the total number of female fish collected for analyses of ovary weight, fecundity, plasma sex steroids, and EROD activity. Differences in ovary weight between sites were detected using analysis of covariance (ANCOVA) with body weight as covariate. For comparison purposes, data were presented both from the subsample and from the complete sample of fish.

a 10-fold greater extent of internucleosomal DNA fragmentation in ovarian cells from BKME-exposed white sucker in comparison to the reference fish (p , 0.001; Fig. 2B). Western blotting for ovarian follicular HSP70 indicated expression of this protein in both reference and BKME-exposed white sucker (Fig. 3A). Densitometric evaluation of immunoreactive bands revealed a greater extent of HSP70 expression in fish exposed to BKME (p 5 0.034, Fig. 3B). The densitometry was performed on both immunoreactive bands present at 70 kDa.

RESULTS

DISCUSSION

The subsample of BKME-exposed female white sucker (n 5 6) collected for determination of apoptosis and HSP70 levels had a reduced GSI relative to the reference fish (p 5 0.016). Similar results were obtained with the total female fish sample (p , 0.001; n 5 100 –104; Table 1). Fecundity was reduced in BKME-exposed fish (82% of the reference fish) but this difference was not statistically significant (p 5 0.20; n 5 6; Table 1). The total fish sample also showed no difference in fecundity between BKME-exposed and reference fish. Plasma testosterone concentrations in female fish exposed to BKME were approximately 25% of those in the reference fish (p , 0.001; Fig. 1) whereas 17b-estradiol concentrations were increased approximately twofold in BKME-exposed fish (p , 0.001; Fig. 1). No difference in hepatic EROD activity was found between sites using the smaller sample size (p 5 0.12; n 5 6) but EROD activity was induced approximately 1.5-fold in the total sample of prespawning female fish from the exposed site (p 5 0.05; n 5 19 –39; Table 1). An autoradiogram of 39-end-labeled DNA isolated from ovarian follicles and resolved using agarose gel electrophoresis is shown in Fig. 2A. There was no evidence of low-molecularweight DNA fragmentation in fish from the reference site. In contrast, there was a large extent of internucleosomal DNA fragmentation in 39-end-labeled DNA from BKME-exposed fish as indicated by low-molecular-weight ‘‘DNA ladders’’ after autoradiography (Fig. 2A). Quantification of low-molecular-weight DNA (,15 kb) excised from agarose gels revealed

Exposure of wild fish populations to BKME results in a variety of negative impacts on reproductive fitness. The present study provides evidence of elevated apoptotic DNA fragmentation and increased HSP70 expression in ovarian follicles from fish exposed to BKME. The 39-end labeling of isolated ovarian follicular cell DNA revealed a 10-fold increase in the extent of apoptosis in white sucker exposed to BKME when compared to a nearby reference site. Since apoptosis is believed to have physiological importance in various aspects of vertebrate ovarian development (Hsueh et al., 1994), elevated ovarian apoptosis may have implications for a number of the reproductive responses observed in fish exposed to BKME. The elevated ovarian cell apoptosis and HSP70 levels were associated with reduced ovary size, decreased plasma testosterone, and increased plasma 17b-estradiol concentrations in BKME-exposed fish. The reduced ovary weight observed in the present study is a common response in fish populations exposed to some pulp mill effluents (McMaster et al., 1991; Munkittrick et al., 1991, 1994). A recent review of effects of pulp mill effluents on life history variables in fish from North America and Scandinavia found that four of six species had reduced gonad size, 8 of 10 populations exhibited delayed sexual maturation, and overall, 14 of 24 studies demonstrated a significant negative impact on gonadal development (Sandstro¨m, 1996). Fewer studies have examined fecundity, although McMaster et al., (1991) reported reduced fecundity as a function of age, as well as decreased

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granulosa and theca cells which surround the oocyte. Cellular homeostasis is dependent on a balance between cell proliferation, differentiation, and death, and rapidly proliferating cell populations normally have high rates of apoptotic cell death (King and Cidlowski, 1995). Since apoptosis is the physiological counterpart of mitosis, increased ovarian somatic cell apoptosis in BKME-exposed white sucker may limit follicular growth during vitellogenesis. The decreased plasma testosterone and increased 17b-estradiol concentrations seen in the present study were consistent with previous studies in prespawning white sucker exposed to BKME (reviewed in McMaster et al., 1996). In many teleost species, rising gonadotropin (GtH) levels during the prespawning period are associated with both increased steroidogenesis and downregulation of aromatase activity in the ovary, leading to increased levels of C21 (progestins) and C19 (androgens) steroids and decreased levels of C18 steroids (estrogens) (Na-

FIG. 1. Plasma testosterone (A) and 17b-estradiol (B) concentrations in prespawning female white sucker collected downstream of a bleached kraft pulp mill (BKME) or from a nearby reference site. Data are means 6 SEM of 20 –21 fish. *Significantly different from reference site using ANOVA (p , 0.001).

egg diameter and egg mass, in white sucker exposed to BKME at Jackfish Bay. Other studies have reported no impact of BKME on fecundity (Adams et al., 1992; Swanson et al., 1992) or increased fecundity associated with reduced follicle size (Munkittrick et al., 1992). These results and those of the present study indicate that although impaired ovarian development can occur in fish exposed to pulp mill effluents, it is unclear whether reductions in ovary weight are due to decreased fecundity, decreased follicle size, or both. Increased apoptotic cell death in ovarian follicles has potential significance with regard to decreases in both fecundity and egg size. Although fecundity was not significantly reduced in this study, decreased fecundity with age (McMaster et al., 1991) may be a result of elevated follicular atresia via apoptosis throughout the lifespan of BKME-exposed white sucker. During vitellogenesis, the period of extensive growth in fish ovarian follicles, the yolk precursor vitellogenin is actively taken up by follicles, necessitating extensive proliferation of

FIG. 2. Apoptotic DNA fragmentation in prespawning white sucker ovarian follicles collected downstream of a bleached kraft pulp mill at Jackfish Bay, Lake Superior (BKME), and at a nearby reference site (REF). (A) Autoradiogram illustrating representative DNA integrity in a pool of eight follicles from reference and BKME-exposed fish. (B) Quantitative estimation of low-molecular-weight (,15 kb) DNA fragments. Data are means 6 SEM of six fish. *Significantly different from reference site using ANOVA (p , 0.001).

OVARIAN APOPTOSIS IN FISH EXPOSED TO PULP MILL EFFLUENT

FIG. 3. Levels of HSP70 protein in ovarian follicles from prespawning white sucker collected downstream of a bleached kraft pulp mill (BKME) and from a nearby reference site. (A) Western blot. (B) Densitometric estimation of band density determined using NIH Image software. *Significantly different from reference site using ANOVA (p , 0.05).

gahama et al., 1993). The pattern of lower testosterone and higher 17b-estradiol levels is consistent with the decreased circulating levels of GtH demonstrated previously in BKMEexposed fish (Van Der Kraak et al., 1992). Induction of hepatic cytochrome P4501A1-associated monooxygenase activities such as EROD has been one of the most consistent responses of fish to BKME exposure (Munkittrick et al., 1994; Sandstro¨m, 1996). Induction of EROD activity is a highly sensitive and specific biochemical marker of exposure to chemicals that bind to the aryl hydrocarbon receptor (AhR) such as polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (Okey et al., 1994). In this study, hepatic EROD activity was not induced in the BKME-exposed fish which displayed elevated ovarian apoptosis and HSP70 expression, and the larger female sample of BKME-exposed white sucker exhibited less than two-fold induction of EROD activity compared to the reference site. This may reflect the reduced potential for induction of EROD activity in prespawning and spawning female fish due to the influence of gonadal sex steroids (Forlin and Andersson, 1984; McMaster et al., 1991). EROD activity in BKME-exposed male white sucker was approximately two-fold greater in com-

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parison to the reference fish (K. R. Munkittrick, unpublished data). Studies in previous years at Jackfish Bay have reported three- to five-fold induction of EROD activity in prespawning female and male white sucker (Munkittrick et al., 1991; McMaster et al., 1991). Although the increased ovarian cell apoptosis and HSP70 expression were not associated with elevated EROD activities in the present study, the BKME-exposed fish have migrated out of Jackfish Bay into uncontaminated water to spawn. It is unknown how long prior to spawning that fish leave Jackfish Bay, but attempts to capture fish in the week before spawning usually yield only immature fish. EROD activity in fish exposed to effluent in Jackfish Bay declines in less than 7 days to reference levels (Munkittrick et al., 1997). The specific chemical(s) responsible for induction of EROD activity in fish downstream of pulp and paper mills are currently unknown. Within bleached kraft mills, chemicals able to induce EROD activity exist in spent cooking liquors (black liquor) and effluents from the first bleaching and extraction stages (Schnell et al., 1993; Burnison et al., 1996; Hodson et al., 1997). In addition, PAHs such as retene can be formed in some secondary treatment systems (Hodson, 1996). Much less is known concerning the chemicals in BKME responsible for adverse reproductive responses in fish. Nevertheless, induction of EROD activity indicates activation of the AhR, and certain AhR agonists such as PAHs are well recognized for their ovotoxicity in laboratory animals and association with ovarian P4501A1 induction (Mattison and Thorgeirsson, 1979; Mattison et al., 1983). Recent studies have shown that 7,12-dimethylbenz[a]anthracene (DMBA) causes both germ and granulosa cell apoptosis in vitro in murine ovarian follicles (Perez et al., 1997; Tilly et al., 1997). The apoptosis in DMBA-exposed ovarian follicles was associated with the presence of the AhR exclusively in oocytes and granulosa cells, suggesting involvement of the AhR in mediating PAH ovotoxicity, possibly via altered expression of genes associated with apoptosis (Tilly et al., 1997). In the present study we did not measure ovarian EROD activity since our previous work has found nondetectable levels of this enzyme (using catalytic methods) in ovarian follicles from white sucker and rainbow trout exposed to BKME and 2,3,7,8-tetrachlorodibenzo-p-dioxin, respectively (K. R. Munkittrick, unpublished data). However, a recent study detected cytochrome P4501A1 mRNA in the ovary of chinook salmon (Oncorhynchus tshawytscha) using reverse-transcriptase-competitive polymerase chain reaction (RT-cPCR), indicating that further research into the potential role of ovarian P4501A1-associated monooxygenases in fish is needed. Nevertheless, we are unable at this time to establish any association between apoptosis and induction of cytochrome P4501A1dependent monooxygenase activities in the teleost ovary. Ovarian follicular cell HSP70 was expressed in both reference and BKME-exposed fish in this study, with elevated levels observed in the fish exposed to BKME compared to reference fish. HSP70 is one of the most highly conserved proteins characterized to date, and is expressed in both consti-

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tutive and inducible forms in vertebrates (Georgopoulos and Welch, 1993). The constitutive form of HSP70 functions in normal cellular processes such as various aspects of protein maturation and translocation. Expression of the inducible HSP70 form is increased in response to a large variety of environmental stressors (Georgopoulos and Welch, 1993). The antibody used in the present study recognizes both forms of teleost HSP70, seen as a doublet of immunoreactive bands at 70 kDa on Western blots. However, we are unable at this time to distinguish between constitutive and inducible HSP70 forms using this antibody. We also observed an immunoreactive band at approximately 50 kDa in both reference and BKME-exposed fish that occurred more intensely in ovarian cells from fish exposed to BKME. It is not known whether this band represents degradation of HSP70 in vivo in BKME-exposed white sucker or occurred as a result of the preparation of ovarian follicular protein. However, ovarian protein was prepared on ice using protease inhibitors and in a similar fashion between reference and BKME-exposed fish. Nevertheless, the results indicate an elevated expression of HSP70 expression in ovarian follicles of white sucker exposed to BKME. Although very little is known regarding the importance of HSP70 in teleost ovarian physiology, recent studies in the mammalian ovary indicate that increased HSP70 expression is associated with inhibition of hormone-sensitive steroidogenesis and mediation of luteal regression (Khanna et al., 1994, 1995). We do not know whether the increased expression of follicular cell HSP70 in white sucker is related to ovarian function (e.g., steroidogenic capacity, apoptosis) during the prespawning period or represents a general response to environmental stress caused by BKME exposure. Although a number of possibilities exist, at this time we do not know the precise mechanism responsible for elevated apoptotic cell death in ovarian follicles of white sucker exposed to BKME. Ovarian follicular apoptosis may be stimulated due to the direct actions of chronic exposure to certain components of BKME, such as PAHs. However, in the present study the low induction of hepatic EROD activity in the fish exposed to BKME indicates minimal exposure of these fish to AhR agonists and suggests that pathways other than those mediated by the AhR may be involved in elevated apoptotic cell death. BKME is a complex industrial effluent consisting of other compounds with the potential to stimulate apoptosis, such as trace metals, phytosterols, and resin acids. Further research is needed to investigate the capacity of individual BKME components to stimulate apoptotic cell death in teleost ovarian follicles. The elevated ovarian cell apoptosis in the present study suggests that the steroid biosynthetic capacity of ovarian follicles may be directly affected in fish exposed to BKME via reductions in the follicle cell population available for steroid production. Preliminary in situ examination of apoptosis in histological sections of BKME-exposed white sucker follicles using Tdt-mediated dUTP nick end labeling (Gavrieli et al., 1992) indicated that both granulosa and thecal cells were

undergoing apoptosis in the BKME-exposed white sucker (Janz and Van Der Kraak, unpublished data). The potential effect of elevated apoptosis on steroidogenesis may also be contributing to altered sex steroid hormone levels observed at other stages of ovarian development in white sucker exposed to BKME (McMaster et al., 1991, Munkittrick et al., 1991, 1994). In other studies, we have observed an elevation in ovarian cell apoptosis in vitellogenic white sucker exposed to BKME (Janz and Van Der Kraak, unpublished). The possibility that certain BKME components affect steroidogenesis as a direct consequence of elevated ovarian somatic cell apoptosis is supported by recent studies in the mammalian ovary showing increased apoptosis and expression of cell death regulatory gene products independent of altered endocrine homeostasis (Springer et al., 1996a; Perez et al., 1997; Tilly et al., 1997a,b). Alternatively, BKME may stimulate apoptosis indirectly via alterations in hormone levels. Ovarian follicular apoptosis is regulated by a variety of hormonal factors (Hsueh et al., 1994), and BKME is known to disrupt endocrine homeostasis at multiple sites along the pituitary– gonadal axis in fish (Van Der Kraak et al., 1992). The reduced testosterone and elevated 17b-estradiol levels in BKME-exposed fish are consistent with previous studies which also revealed a significant reduction in plasma GtH-II concentrations (Van Der Kraak et al., 1992). Since gonadotropins are important follicle survival factors controlling apoptosis in mammals (Chun et al., 1994; Tilly and Tilly, 1995) and act similarly in teleost fish (Janz and Van Der Kraak, 1997), it is possible that reductions in circulating GtH levels in BKME-exposed white sucker may be involved in the elevated degree of ovarian follicular apoptosis. It seems somewhat paradoxical that 17b-estradiol would be elevated in those fish displaying a higher extent of ovarian cell apoptosis as this hormone functions as a follicle survival factor (Billig et al., 1993; Janz and Van Der Kraak, 1997) and would be associated with follicular viability. However, the contribution of 17bestradiol as a survival factor in prespawning fish is not known but may be minimal since the plasma concentrations of 17b-estradiol during the prespawning period in white sucker (1–2 ng/ml) are relatively low in comparison to other stages of ovarian development. In addition, other alterations in endocrine homeostasis which reduce levels of follicle survival factors may be involved during the prespawning period as well as other stages of ovarian development. Further research is needed concerning the potential linkages between apoptotic cell death, steroidogenesis, stress protein induction, and ovarian development in fish exposed to environmental contaminants. ACKNOWLEDGMENTS We thank Dr. George Iwama, Department of Animal Science, University of British Columbia, Vancouver, BC, for supplying the HSP70 antibody. We also thank Cam Portt (C. Portt and Associates, Guelph, ON) for field sampling of

OVARIAN APOPTOSIS IN FISH EXPOSED TO PULP MILL EFFLUENT white sucker and M. Maj for statistical analysis. D. de Boer, University of Guelph, performed RIAs and D. Boyter, Environment Canada, measured EROD activities. This study was funded by the Natural Sciences and Engineering Research Council of Canada, the Canadian Network of Toxicology Centres, the Canadian Chlorine Coordinating Committee, and Environment Canada.

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Hughes, F. M., Fong, Y. Y., and Gorospe, W. C. (1994). Interleukin-6 stimulates apoptosis in FSH-stimulated rat granulosa cells in vitro: Development and utilization of an in vitro model. Endocrine 2, 997–1002. Hughes, F. M., and Gorospe, W. C. (1991). Biochemical identification of apoptosis (programmed cell death) in granulosa cells: Evidence for potential mechanism underlying follicular atresia. Endocrinology 129, 2415–2422.

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