TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

115,224-233

( 1992)

Exposure to Bleached Kraft Pulp Mill Effluent Disrupts the PituitaryGonadal Axis of White Sucker at Multiple Sites G. J. VAN DER KRAAK,* *Department Department

K. R. MUNKITTRICK,~

M. E. MCMASTER$’

C. B. PORTT,§ AND J. P. CHANC$

of Zoology. University of Guelph. Glrelph. Ontario, Canada NlG 2WI; IGreat Lakes Laboratory,fbr Fisheries and Aquatic Sciences. of Fisheries and Oceans. 867 Lakeshore Road. Burlington. Ontario, Canada L7R 4A6: *Department of Biolog}: University qf Waterloo. Waterloo. Ontario, Canada N2L 3Gl; §C. Portt & Associates, 56 Waterloo Avenue, Gtrelph, Ontario. Canada NlH 3H5: and IlDepartment qf Zoolo~p?: University ofAlberta, Edmonton. .4lberta, Canada T6G 2B9 Received

September

30. 199 I; accepted

April

7. I992

A231 87, suggestingthat BKME effects on ovarian function are selective and do not reflect a general impairment of ovarian function. BKME-exposed fish had plasmalevelsof testosterone DER KRAAK, G. J., MUNKITTRICK, K. R., MCMASTER, M. E., glucuronide proportionately lower than those of referencefish, PORTT, C. B., AND CHANG, J. P. (1992). Toxicol. Appl. Pharsuggestingthat there are site differencesin the peripheral memacol. 115,224-233. tabolism of steroids.Thesestudiesdemonstratethat BKME exOur recent studieshave demonstratedreproductive problems posure affects reproduction by acting at multiple sites in the in white sucker (Catostomus commersoni) exposedto bleached pituitary-gonadal axis. Q 1992 Academic press, hf. kraft pulp mill effluent (BKME) at Jackfish Bay on Lake Superior. These fish exhibit delayed sexual maturity, reducedgonadal size, reducedsecondarysexual characteristics, and circuOur previous studies demonstrated alterations in the relating steroid levelsdepressedrelative to thoseof referencepop- productive fitness of wild fish populations exposed to ulations. The present studieswere designedto evaluate sites in bleached kraft pulp mill effluent (BKME) at Jackfish Bay on the pituitary-gonadal axis of prespawningwhite suckeraffected Lake Superior. White sucker (Catostomus commersoni), by BKME exposure.At the time of entry to the spawningstream, plasmalevels of immunoreactive gonadotropin (GtH)-II (LH- which has been studied in the greatest detail, exhibit delayed type GtH) in male and female white sucker were 30- and 50- age to sexual maturation, decreased gonad size, and a refold lower, respectively, than the levels in fish from a reference duction of secondary sexual characteristics in a BKME-exposed population relative to reference populations (Mcsite. A single intraperitoneal injection of D-Arg6, Pro’N-Et sGnRH (sGnRH-A, 0.1 mg/kg) increasedplasmaGtH levelsin Master, 1991; McMaster d al., 1991, 1992; Munkittrick et male and female fish at both sites, although the magnitude of al., 1991). BKME-exposed female white sucker sampled the responsewas greatly reduced in BKME-exposed fish. Fish during vitellogenesis (period of ovarian growth) exhibited at the BKME site did not ovulate in responseto sGnRH-A, reduced levels of 17g-estradiol (Munkittrick et al., 1991). while 10 of 10 fish from the referencesite ovulated within 6 hr. which is the primary hormone responsible for regulation of Plasma 17a,20P-dihydroxy-4-pregnen-3-one (17,200-P) levels the synthesis of the yolk precursor protein, vitellogenin. were depressedin BKME-exposed fish and unlike fish at the These fish also exhibit decreasedlevels of testosteroneduring referencesite, failed to increasein responseto sGnRH-A. Tesboth vitellogenesis (Munkitttick et al.. 1991) where it functosterone levels in both sexesand 11-ketotestosteronelevels in tions as a precursor for 17P-estradiol. and at spawning maleswere elevated in fish from the referencesite but were not (McMaster et al., 1991) where this hormone functions in further increasedby GnRH treatment. In contrast, BKME-exposedfish exhibit a transitory increasein testosteronelevels in the feedback regulation of gonadotropin (GtH) secretion. responseto the GnRH analog. In vitro incubations of ovarian Male white sucker exposed to BKME also showed reductions follicles obtained from fish at the BKME site revealeddepressed in the circulating levels of testosterone and 1l-ketotestosterbasal secretion of testosterone and 17,200-P and reduced re- one (McMaster et al., 1991; Munkittrick et al., 199 1), which sponsiveness to the GtH analoghuman chorionic gonadotropin play a major role in testicular development and expression and to forskolin, a direct activator of adenylatecyclase.By comof secondary sexual characteristics. Both sexesof fish showed parison, ovarian follicles from fish collected at BKME and refreductions in the levels of 17a,20&dihydroxy-4-pregnen-3erencesitesproduced similar levels of prostaglandin E basally one (17,2Op-P) during the prespawning period (McMaster and in responseto a phorbol ester and calcium ionophore et al., 1991). 17,20&P functions in the induction of oocyte final maturation in females and has been associated with ’ Present Address: Department of Fisheries and Oceans, Burlington, Ontario, Canada. mobilization and hydration of spermatozoa in males. Exposure to BleachedKraft Pulp Mill Effluent Disrupts the Pituitary-Gonadal Axis of White Sucker at Multiple Sites. VAN

$5.00 Q 1992 by Academic Press, Inc. of reproduction in any form reserved.

004 1-008X/92

Copyright All rights

224

IMPACTS

OF BKME

ON REPRODUCTION

Although research on other species has not been as all encompassing, comparable reproductive impacts may occur in at least two other species found at Jackfish Bay, longnose sucker (Catostomus catostomus) (Munkittrick et al.. 1992a) and lake whitefish (Coregonus clupeaformis) (Munkittrick et al. 1992b). Fish from Jackfish Bay also exhibit increased hepatic mixed function oxygenase (MFO) activity as measured by catalytic activity against benzo[a]pyrene, diphenyloxazole, and ethoxyresorufin (McMaster et al., 1991; Munkittrick et al., 1991, 1992a,b). The basis of reproductive problems, and in particular reduced steroid hormone levels, in fish exposed to BKME are unknown. It has been hypothesized that the induction of MFO enzymes may accelerate the catabolism and excretion of sex steroids to such an extent that the fish cannot maintain physiological hormone levels (Okey, 1990). Previous studies on fish examining the impacts of BKME exposure and xenobiotics in general have focused almost exclusively on impacts associated with steroid metabolism rather than with steroid biosynthesis. The studies on steroid metabolism have not been consistent, as increased MFO activity has been shown to increase metabolism of androstenedione (Hansson and Rafter, 1983) and 17/3-[3H]estradiol (Forlin and Haux, 1985) while others have shown increased MFO activity to have no effect on testosterone (Lindstrom-Seppa and Oikari, 1989) or estradiol (Snowberger and Stegeman, 1987) metabolism. The possibility that reduced steroid levels in fish exposed to xenobiotics may occur secondary to hypothalamic, pituitary, and/or gonadal dysfunction has not been addressed. Recent extensive studies have shown that 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), which is a component of some BKMEs, affects reproductive function in rats by acting at both the pituitary and testis levels (Bookstaff et al., 1990a,b; Kleeman et al., 1990; Moore and Peterson, 1988; Moore et al., 1985, 1991). In the present studies, white sucker were collected during their spawning migration at a reference and a BKME-exposed site in order to evaluate the underlying basis of reproductive dysfunction associated with BKME exposure. Specifically, we determined (I) pituitary and gonadal integrity by measuring plasma GtH and reproductive steroid levels following an intraperitoneal injection of a superactive gonadotropinreleasing hormone (GnRH) analog, (2) functional competence of the ovary by measuring the irz vitro production of steroids and E series prostaglandins in response to hormone agonists and drugs, and (3) influences on the peripheral metabolism of steroids by determination of free and glucuronidated levels of testosterone in the plasma. METHODS Study site and fish collection. Jackfish Bay, located along the north shore of Lake Superior, receives BKME from a mill located in Terrace Bay. Ontario (Fig. I ). The mill produces 1200 air-dried metric tons of pulp per day and discharges approximately 120.000 m3 d-’ of effluent into the head-

IN FISH

225

waters of Blackbird Creek. The creek then carries the effluent approximately 15 km to Moberley Bay (48”5O’N. 86“SS’W). the western arm of Jackfish Bay, Lake Superior. Moberley Bay receives no other industrial or municipal effluents and has no permanent residential development. The effluent has received secondary treatment in an aerated lagoon since September 1989: prior to this time it passed through only a primary clarifier before discharge. At spawning time, the BKME-exposed white sucker migrate through the eastern arm of Jackfish Bay into Jackfish Lake. The white sucker spawn in Sawmill Creek: neither Sawmill Creek or Jackfish Lake receive pulp mill effluent. The amount of time fish spend in clean water (non BKME) prior to spawning is unknown and will depend on weather conditions during the spawning migration. For comparison, white sucker were collected from a spawning run at Mountain Bay (48O56’N. 87”5O’W) (Fig. I). These fish were collected from the Little Gravel River during the spawning migration. This reference site has been used previously to monitor impacts of BKME on fish populations (McMaster et al., 199 I; Munkittrick d al., 199 I; Smith el a/., 199 1) as it receives no industrial effluent or significant domestic effluent input. Prespawning fish were captured in overnight hoop net setsin both Sawmill Creek (BKME) and the Little Gravel River (reference) during mid May in 1990 and 199 I. In an attempt to get fish in similar reproductive states lish were taken on the first night in which large numbers of fish (in excess of 300) entered the spawning streams. Responsiveness to GnRH. The functional integrity of the pituitary-gonadal axis in the white sucker was evaluated by measuring the response to an intraperitoneal injection of a superactive GnRH analog. Male and female fish collected at BKME and reference sites received a single intraperitoneal injection of either salmon GnRH analog (D-Arg6, Pro’N-Et salmon GnRH; sGnRH-A at 0.1 mg/kg) or 0.9% saline. There were 10 fish per treatment group per sex. Fish weight ranged from 700 to I200 g. but did not differ between sites or treatments. Blood was collected from the caudal vessels using a heparinized syringe immediately prior to the injection (Time 0). and again 6 and 24 hr after injection. Blood was held on ice prior to the separation of the plasma by centrifugation; plasma was immediately frozen in liquid nitrogen. Females were assessedfor ovulation by applying gentle pressure to the abdomen at the 6-hr sample and following dissection at the 24-hr sample. Fish were held in mesh cages in the spawning streams between sampling times. Plasma GtH content was determined by radioimmunoassay (RIA) using the procedure described by Peter rt ul. (1984). This procedure, which is based on a GtH purified from common carp pituitaries for generation of the antisera and for assay standards, has been validated for measurement of GtH in the white sucker (Stacey et al., 1984). However, GtH values obtained using the current assay procedure are not directly comparable with the earlier studies by Stacey er al. (1984) owing to the use of a new GtH standard (common carp GtH II: see Van Der Kraak et al., 1992) which is approximately 0.5 times the immunological potency of carp GtH 378 used as the hormone standard in the earlier work. Plasma samples from males and females were assayed for testosterone and 17.20&P content by RIA using the methods described by Van Der Kraak and Chang ( 1990) and Van Der Kraak et al. (1989), respectively. Plasma samples from males were also analyzed for 1I-ketotestosterone content using the methods described by Wade and Van Der Kraak (1992). In vitro steroid and prostaglandin E production. In vitro incubation procedures used previously to evaluate the regulation of steroid production by goldfish periovulatory ovarian follicles (Van Der Kraak, 1990: Van Der Kraak and Chang, 1990) were used for studies conducted in 1990 to determine the steroidogenic responsiveness of white sucker ovarian follicles to human chorionic gonadotropin (hCG) and forskolin. Forskolin directly activates adenylate cyclase and thereby mimics GtH action by bypassing the GtH receptor. In brief. fish were killed by spinal transection and their ovaries were removed quickly and placed in Medium 199 (M 199, containing Hanks’ salts without bicarbonate: GIBCO, Burlington, Ontario) which was supplemented with 25 mM Hepes. 4.0 mM sodium bicarbonate, 0.01% streptomycin

226

VAN DER KRAAK

ET AL.

FIG. 1. Study site. Shaded areas represent land.

sulfate,and 0.1% bovine serum albumin (pH 7.2). Intact preovulatory follicles were then added in groups of 10 per well to a polystyrene tissue culture plate (Falcon 3047: Fisher Scientific Co., Toronto, Ontario). Immediately prior to the addition of test compounds, the incubation medium was replaced with Ml99 containing the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX; 0. I mM) together with graded dosages of hCG (0.1. 1.O. or IO W/ml) or forskolin (1 .Oor 10 PM); there were four replicate incubations per treatment. The final incubation volume was 1 ml. Follicles were incubated for 24 hr at 14-16°C. after which the incubation medium was collected and immediately frozen in liquid nitrogen. Aliquots of the media were assayed directly for testosterone and 17,20@-P content by RIA as described above. These experiments were replicated on four females from each site. Studies conducted in 1991 were expanded to include measurement of PGE production by ovarian follicles. Prostaglandins of the E series are produced in large quantities by ovarian follicles in the preovulatory period (Cetta and Goetz 1982; Kellner and Van Der Kraak, 1992) and are thought to function in coordination of ovulatory events. Ovarian follicles in groups of 10 were incubated with either M 199 alone, hCG (10 W/ml), or the combination of the phorbol ester phorbol l2-myristate 13-acetate (PMA: 400 nM) and calcium ionophore A23187 (1000 nM). Unlike the studies described for 1990, IBMX was not included in the follicle incubations as it is inhibitory to PGE production (Kellner and Van Der Kraak, 1992). Follicles were incubated for 24 hr at l4-16°C; there were four replicate incubations per treatment. Aliquots of the incubation media were assayed directly for PGE content using the RIA procedure described by Kellner and Van Der Kraak ( 1992). Testosterone production was determined for follicles incubated with

Medium 199 alone (controls) and with hCG. The experiment was replicated on ovarian follicles from four females at each site. Assessment of free and conjugated steroids. To evaluate the possible contribution of alterations in the peripheral clearance of steroids on circulating hormone levels.we determined the amounts of free and glucuronidated testosterone in plasma samples from male white sucker collected at the BKME and reference sites. Glucuronidation, the conjugation of glucuronic acid to steroid hormones. increases the water solubility of steroids and facilitates their excretion. Measurement of the levels of free and glucuronidated testosterone was based on a modification of the methods described by Van Der Kraak et al. (1989). Plasma samples (0.2 ml) were acidified by the addition of 0.6 ml of 0.6 M sodium acetate buffer (pH 4.5). The samples were then incubated with 0.2 ml of /%glucuronidase solution (5000 Sigma units/ml; Sigma, St. Louis, MO) or sodium acetate buffer alone for 24 hr at 37°C. Following extraction with diethyl ether, the testosterone content was determined as described above. The levels of testosterone-glucuronide were calculated as the difference of hormone content in samples treated with and without $-glucuronidase. Statistics. To determine the responsiveness to the GnRH challenge. differences in GtH and steroid levels between sites, treatment, and time were analyzed by three-way analysis of variance (ANOVA). Differences between sites at Time 0 were tested using one-way ANOVA. Fisher’s exact probability test was used to compare the number of ovulated fish between groups. Differences between sites in the production of steroids and PGE by ovarian follicles incubated in vitro were tested using one-way ANOVA. One-way

IMPACTS

OF BKME

ON REPRODUCTION

TABLE 1 Effects of Salmon GnRH Analog (0.1 mg/kg) on Ovulation Site BKME Reference

Treatment

Ovulation”

Saline sGnRH-A Saline sGnRH-A

o/10 O/IO l/IO lo/lo*

’ Number of fish ovulating/number of fish injected. * Significantly different from all other groups as determined by Fisher’s exact probability test (p < 0.01).

ANOVA was used to test for differences between sites in the levels of free and glucuronidated testosterone levels.

RESULTS Injection of sGnRH-A failed to induce ovulation over a 24-hr period in preovulatory fish collected at the BKME site. By comparison, all fish (10 of 10) from the reference site ovulated in response to sGnRH-A (Table 1). Ovulation was evident in all GnRH-injected fish from the reference site within 6 hr (Table 1). Ovulation was confirmed at necropsy (24-hr sample), and greater than 50% of oocytes were loose within the body cavity. Fish from the BKME site had markedly lower (p < 0.00 1) plasma GtH levels than fish from the reference site at Time 0 (Fig. 2). Although administration of sGnRH-A increased plasma GtH levels in fish at both sites, the peak levels in

Females

227

IN FISH

BKME-exposed fish treated with sGnRH-A did not reach basal levels in fish from the reference site. Plasma GtH levels in both males and females from the reference site were elevated (p < 0.001) 6 hr after treatment with sGnRH-A; this was transitory as GtH levels declined between the 6- and 24hr samples. By comparison, plasma GtH levels in BKMEexposed fish treated with sGnRH-A were elevated at the 6and 24-hr samples. At Time 0, circulating levels of testosterone and 17,20/3P were significantly lower in female white sucker from the BKME site (p < 0.00 1 and p = 0.012, respectively) (Fig. 3). Testosterone levels in reference females remained elevated at 6 hr relative to the BKME females (p < O.OOl), but these levels were unaffected by treatment with sGnRH-A (p = 0.80). In contrast, testosterone levels in sGnRH-A-injected BKME females were elevated at the 6-hr sampling time relative to the saline-injected BKME females (p = 0.015). Testosterone declined to very low levels at both sites between 6 and 24hr, irrespective of treatment with saline or sGnRHA (Fig. 3). 17,20&P levels were higher at all sampling times in reference females compared to BKME-exposed fish (a < 0.001) (Fig. 3). 17,206-P levels in females from the BKME site did not change in response to sGnRH-A administration (p = 0.17), whereas levels in fish from the reference site were elevated at 6- and 24-hr postinjection (p = 0.006) relative to saline-injected controls. All male fish were spermiating prior to sGnRH-A injection. At Time 0, males collected from the BKME site had significantly lower circulating levels of testosterone, 1 l-ketotestosterone, and 17,2Op-P than fish from the reference

Males

(ng/mL)

Gonadotropin

Gonadotropin

*

Rdsaline

+

RdO”RH

90 I

(q/ml) I

*

Rdsaline

4-

2d . . . .. .* _....... * 0

6

Tim::h)

.. 10

0 24

FIG. 2. Levels of GtH in plasma from female and male white sucker 0, 6, and 24 hr after an intraperitoneal injection of sGnRH-A (0.1 mg/kg) (asterisks) or saline (circles). Values represent the mean + SEM of measurements from 10 fish from a BKME (dotted line) and a reference (solid line) site.

228

VAN DER KRAAK

and 17,200-P responses to forskolin. Presentation of pooled data masks significant stimulatoty effects of hCG and forskolin on 17,200-P production by ovarian follicles from fish collected at the reference site. Production of 17,2Op-P by follicles from three of four fish used in these studies increased in a dose-dependent manner in response to hCG and forskolin. Ovarian follicles from the remaining fish produced very high levels of 17,206-P basally (2520 & 120) but did not change in response to either hCG or forskolin. Studies were expanded in 199 1 to include measurement of both testosterone and PGE production. As before, testosterone production by follicles from the BKME site was reduced basally (I-,= 0.003) and these follicles had diminished responsiveness to hCG (p = 0.0 12) (Fig. 6). HCG-stimulated testosterone production by follicles from the reference site was 4.7-fold higher than production by follicles from the

30- Testosterone (rig/ml) 25.

-8-

Rei saline

+++ FM GnRH 20.

BKME saline

15-

4

ET AL.

‘.

0 ’ 17,20 P (rig/ml) lo86-

Testosterone

15r ” 0

6

12

18

@g/ml)

24

Time (h)

FIG. 3. Levels of testosterone and 17,200-P in plasma of female white sucker 0. 6. and 24 hr after an intraperitoneal injection of sGnRH-A (0. I m&kg) (asterisks) or saline (circles). Values represent means f SEM of measurements on 10 fish from a BKME (dotted line) and a reference (solid line) site.

site (p = 0.001) (Fig. 4). Administration of sGnRH-A stimulated an elevation of testosterone levels in males from both sites by 6 hr (p = 0.014). By 24 hr, testosterone levels had declined in all groups to very low levels (Fig. 4). Levels of 1 I-ketotestosterone were unaffected by sGnRH-A treatment (Fig. 4) and declined with time in all treatment groups. Administration of sGnRH-A caused a transient increase in 17,2Op-P in males from the reference site. as the levels were higher than controls at both the 6- and 24-hr samples (p = 0.02). In contrast, sGnRH-A did not affect 17,20@-P levels in males from the BKME site at the 6-hr sample (p = 0.94) and these fish had significantly higher (p = 0.002) 17,20&P levels at the 24-hr sample (Fig. 4). Basal secretion of testosterone and 17,206-P were lower in ovarian follicles from white sucker collected at the BKMEexposed site (p = 0.001 and p = 0.004, respectively) (Fig. 5). Ovarian follicles from fish collected at the reference and BKME sites were responsive to hCG (a gonadotropin agonist) and forskolin (a direct activator of adenylate cyclase), although maximal steroid response in follicles from BKMEexposed fish was greatly reduced (Fig. 5). The maximal testosterone and 17,200-P responses for reference follicles to hCG were 2.7- and 16.9-fold higher than production by follicles from fish collected at the BKME site; comparable values, respectively, were 2.9- and 5.6-fold for the testosterone

,oo 1 l-KT (rig/ml) I

“k 60

+

Ref

+-

RefGnRH

.o

saline

BKME

saline

nl

” 17.20 7

P (rig/ml) I

6

12

18

24

Time (h) FIG. 4. Levels of testosterone, I 1-ketotestosterone, and 17.20/3-P in plasma of male white sucker 0.6. and 24 hr after an intraperitoneal injection of sGnRH-A (0.1 mg/kg) (asterisks) or saline (circles). Values represent means + SEM of measurements on 10 fish from a BKME (dotted line) and a reference (solid line) site.

IMPACTS 3ooo Testosterone I

OF BKME

ON REPRODUCTION

(pg/ml)

229

IN FISH

3500 ;BStoSterone (pglml) 3000

2500

t

T

2500

2000

2000 -

1500

1500 1000

1000 500

Control

10 NJ/ml hCG

0

1800

17,20 P (pg/ml)

1600 1400 1200 1000 800 600

Control

400 200 0

Ic

C:ontrol

0.1

1.0 hCG

2-Q

10

0.1 10 Forskolin

FIG, 5. 11,wifro production of testosterone and 17,20&P by white sucker ovarian follicles in response to graded dosages of hCG (0. I. I, and 10 IU/ ml) and forskolin (I and IO PM). Values are reported as the mean + SEM of pooled data based on quadruplicate measurements from four separate fish at the BKME (open bars) and reference (shaded bars) sites.

BKME site. In marked contrast, the PGE response did not differ in follicles obtained from fish at the BKME and reference sites basally (p = 0.16) or to the combined actions of PMA (a protein kinase C activator) and A23 187 (a calcium ionophore) (p = 0.37) (Fig. 6). White sucker from the BKME site had significantly lower levels of both free (nonconjugated) (p < 0.001) and glucuronidated (p = 0.002) testosterone, relative to fish from the reference site (Fig. 7). At the BKME site, testosterone glucuronide levels were below 500 pg/ml in all fish and were not detectable in 8 of 10 samples analyzed. At the reference site, testosterone glucuronide levels were approximately 50% of the free testosterone levels in the circulation.

PMA + A231 87

FIG. 6. In r’itro production of testosterone and PGE by white sucker ovarian follicles in response to either hCG ( IO IU/ml) (testosterone) or PMA (400 IIM) + A23187 (I000 nM) (PGE). Values are reported as the mean + SEM of pooled data based on quadruplicate measurements from four separate fish at the BKME (open bars) and reference (solid bars) sites.

reported for a population of white sucker in northern Alberta (Scott et al., 1984; Stacey et al., 1984), suggesting that the hormonal difference reported in this study is a consequence of exposure to BKME. Furthermore, at the BKME site, de-

Testosterone

lO---

g!

(ngiml)

m

BKME

m

Ref

6-

DISCUSSION The present study has shown that exposure to BKME was associated with significant reductions in circulating levels of GtH and sex steroids in prespawning white sucker collected from uncontaminated spawning sites. The hormone levels found in fish from the reference site were similar to the values

Free

Conjugated

FIG. 7. Free and glucuronidated testosterone levels in plasma from prespawning male white sucker. Values are reported as means + SEM for measurements from IO fish at the BKME site (open bars) and 6 fish at the reference site (shaded bars).

230

VAN

DER

KRAAK

creased steroid levels were found during four different stages of the reproductive cycle (McMaster et al., 199 1; Munkittrick et al., 1992b), in white sucker, longnose sucker, and lake whitefish (Munkittrick et al., 1991; 1992a,b), over a course of 4 years (McMaster et al., 1991; Munkittrick et al., 1991; present study), and relative to three reference sites (McMaster et al., 199 1; Munkittrick et al., 1991). The present studies also demonstrate for the first time that multiple sites within the pituitary-gonadal axis are affected by exposure to BKME. This includes diminished GtH secretion by the pituitary, depressed ovarian steroid biosynthetic capacity, and altered peripheral metabolism of steroids. The basis of reduced GtH secretion in white sucker exposed to BKME is unknown. The present studies demonstrate that BKME-exposed fish are responsive to a superactive GnRH analog, although the magnitude of the GtH response was greatly reduced relative to that of fish from the reference site (Fig. 2). The concentrations of GnRH were not evaluated in this study; however, the depressed GtH secretion in response to the sGnRH-A challenge suggests that a lesion exists at the level of the pituitary. Previous studies have shown that the neuroendocrine regulation of GtH secretion in teleosts involves a dual control, with GtH release stimulated by GnRH and inhibited by dopamine (see Peter et al., 1986). Although an increased dopamine inhibitory tone in BKMEexposed fish could contribute to reduced GtH secretion and depressed responsiveness to the GnRH analog, this seems to be an unlikely explanation given the prolonged increase in GtH secretion in BKME-exposed fish relative to that of fish from the reference site (Fig. 2). An important next step in defining the basis of GtH deficiency in these fish will be an evaluation of pituitary GtH content. In rats, TCDD exposure appears to increase the potency of androgens as feedback regulators of LH secretion (Bookstaff et al., 1990b). The possibility that BKME exposure affects fish in a similar manner remains to be investigated. White sucker exhibit a marked stress-induced depression of circulating sex steroid levels. Handling and sampling of saline-injected fish was associated with reduced levels of the C 19 steroids testosterone and 11-ketotestosterone (in males) whereas the C2 1 steroid 17,20/3-P shows little change over a 24-hr period. Similar stress-induced changes were reported for the marine snapper (Pagrzu auratzu) which shows a significant reduction in testosterone and 17/3-estradiol levels and a concomitant increase in 17,200-P levels when removed from the wild to confinement (Carragher and Pankhurst, 199 1). However, considerable species differences may exist, as several other teleost species including the coho salmon (Oncorhynchus kisutch) (Van Der Kraak et al., 1984) goldeye (Hiodon afosoides) (Pankhurst et al., 1986a), walleye (Stizostedion vitreum) (Pankhurst et al., 1986b), and goldfish (Carussizrs auratus) (unpublished) do not exhibit an acute reduction of circulating steroid levels in response to capture and routine handling procedures.

ET

AL.

The similarity of stress-induced changes in steroid biosynthesis in the white sucker and the marine snapper (Carragher and Pankhurst, 199 1) suggest that a common mechanism may be involved. Female fish showed no reductions in GtH or 17,2Op-P over time, making it unlikely that decreased GtH secretion is the primary cause of reduced steroid levels. It seems more likely that stress effects are mediated directly at the level of the ovary and testis. In support of this hypothesis, Carragher and Sumpter (1990) reported that cortisol, the primary stress hormone in teleosts, inhibits testosterone and 17/3-estradiol production by rainbow trout (Oncorhynchus mykiss) ovarian follicles incubated in vitro. Whether stress affects steroid production by acting at more than one site in the steroidogenic cascade is not known. A primary site of action appears to be at the level of 17/3-hydroxylase/ 17,20 lyase, leading to reduced testosterone secretion. A second lesion beyond testosterone formation may account for the more rapid disappearance of I l-ketotestosterone. Once again, species differences may exist, as acute and chronic stressors affected plasma testosterone levels to a greater degree than I I-ketotestosterone levels in mature male brown trout (Salvo tmtta) (Pickering et af., 1987). One of the questions we hoped to address by measuring the in vivo steroid response to sGnRH-A was whether reduced sex steroid levels in BKME-exposed fish occur secondary to altered pituitary function and reduced GtH secretion. However, interpretation of these data becomes complicated by the stress effects on steroid production. The inability of sGnRH-A to stimulate 17,20/?-P production in BKME-exposed fish suggests that factors other than GtH deficiency contribute to the low hormone titers. It was surprising that sGnRH-A did not increase testosterone levels in fish at the reference site but increased plasma testosterone levels at the 6-hr sample in BKME-exposed fish. These differences in the testosterone response between the two sites would not be predicted on the basis of the initial hormone levels or in terms of steroid biosynthetic capacity determined on the basis of in vitro testosterone production. One explanation for this difference is that stress effects on steroid biosynthesis are expressed at differential rates at the two sites. We are currently investigating this possibility. The studies assessing steroid production in vitro clearly demonstrate a reduced steroid biosynthetic capacity of ovarian follicles from BKME-exposed fish. As purified white sucker GtH is not available, we chose to determine the steroid response to a readily available GtH agonist (hCG) and to forskolin which activates adenylate cyclase. Adenylate cyclase activation increases the production of CAMP which is the intracellular mediator of GtH actions in teleosts (Kanamori and Nagahama, 1988; Van Der Kraak, 1990, 1992). While earlier studies have shown that teleosts exhibit considerable species differences in responsiveness to GtHs of mammalian origin (Bona-Gall0 and Licht, 198 1, 1983; Goetz, 1983), these studies demonstrate that hCG is a potent GtH agonist

IMPACTS

OF

BKME

ON

in the white sucker having comparable steroidogenic activity to forskolin. These findings demonstrate that the GtH receptor and adenylate cyclase, which represent key elements in the GtH signal transduction cascade, are functional in BKME-exposed fish, but whether the full capacity of these systems is intact or whether there are lesions downstream of cyclic AMP formation has not been evaluated. To evaluate whether reduced steroid production by follicles from BKME-exposed fish reflects a general impairment of ovarian function, we also determined the capacity of these follicles to produce PGE. Prostaglandins of the E series are the dominant prostaglandins secreted by ovarian follicles from several teleost species (Tan et al., 1987; Goetz et al., 1989; Kellner and Van Der Kraak, 1992) and are thought to function in the control of ovulation (Stacey and Goetz, 1982; Goetz, 1983). The production of PGE by goldfish ovarian follicles is stimulated by drugs which activate protein kinase C (PMA) and elevate intracellular calcium levels (ionophore A23 187); activators of CAMP formation inhibit PGE production (Kellner and Van Der Kraak, 1992). Basal and PMA + A23 187 stimulated production of PGE by white sucker ovarian follicles from BKME and reference sites did not differ, suggesting that the effects of BKME exposure are relatively specific to steroid production. Steroid production in fish, as in mammals, is regulated primarily by the rate at which the substrate cholesterol is mobilized to the mitochondrial enzyme responsible for pregnenolone formation (cytochrome P450 side chain cleavage). The activities of the steroidogenic enzymes which metabolize pregnenolone and its products are not generally rate limiting. For example, it has been shown in several species of fish that exogenous 25-hydroxycholesterol and pregnenolone are rapidly metabolized to other steroid products (Petrino et al., 1989; Van Der Kraak, 1992). Although the precise manner in which BKME exposure affects steroid production is unknown, considerable information is available from mammalian studies on the reproductive effects of chlorinated dibenzo-p-dioxins which may be bioactive components of BKME. In the male rat, TCDD causes an acute reduction of testosterone secretion by inhibiting the luteinizing hormone-induced mobilization of cholesterol to the side chain cleavage enzyme (Kleeman et al., 1990; Moore et al., 1991). Although decreases in the activities of the enzymes responsible for conversion of pregnenelone to testosterone have been reported following exposure to TCDD (e.g., Tofilon and Piper, 1982; Mebus et al., 1987) these changes are not thought to be of consequence to overall steroid production (Kleeman et al., 1990). Future studies should consider the extent to which these mechanisms contribute to steroid production in the white sucker. At least in the case of 17,200-P production, BKME exposure appears to act primarily at the enzyme level by decreasing the amount of 2Ophydroxysteroid dehydrogenase which mediates conversion of 17a-hydroxyprogesterone to 17,200-P. As production of

REPRODUCTION

IN

FISH

231

this enzyme is under the influence of GtH (Nagahama, 1987) these effects may relate directly to impaired GtH secretion. Increased levels of hepatic mixed function oxygenase (MFO) enzymes have been observed in several teleost species following exposure to BKME (McMaster et al., 199 1; Munkittrick et al., 199 1, 1992a,b). MFOs facilitate the excretion of xenobiotics by increasing their hydrophilicity through the addition of polar groups. MFOs also function in the synthesis and metabolism of endogenous compounds such as steroids, prostaglandins, and fatty acids (Rattner et al., 1989). It has been suggested that increased MFO activity may contribute to impacts on reproductive function through effects on the peripheral metabolism of sex steroids (see Okey, 1990). Given the stress-induced changes in the metabolism of steroids in the white sucker and the isolated study location, we felt that the traditional approach of following the disappearance of radiolabeled steroid was inappropriate to evaluate potential effects of BKME exposure on steroid metabolism. Consequently, we opted to determine the amounts of free and glucuronidated testosterone in plasma samples from male white sucker. These studies demonstrated a marked difference between sites, with fish from the BKME site having depressed levels of glucuronidated testosterone in the circulation. However, the underlying basis of this difference is unknown. It may reflect a reduction in the activity of the enzymes associated with glucuronidation or alternately enhanced activity of other steroid metabolizing enzymes such that the amount of substrate available for glucuronidation would be reduced. In support of the former mechanism, reduced amounts of the phase II MFO enzymes associated with steroid conjugation have been reported following exposure to BKME (Oikari et al., 1985). However, white sucker from Jackfish Bay have been shown to have levels of phase II enzymes comparable to reference fish in August samples (McMaster et al.. 1991; Smith et nf., 199 1). Irrespective of the mechanism involved, the significance of these differences in terms of effects on steroid clearance rates remains to be investigated. The present studies have demonstrated that BKME exposure affects the reproductive system by acting at multiple sites within the pituitary-gonadal axis. The extent of these disruptions and the magnitude of the hormone changes affected by BKME exposure were somewhat surprising in that the reproductive potential of spawning fish from BKME and reference sites were similar. There were no differences in the fertilization or survival of individual white sucker eggs or larvae, although there were significant decreases in egg size and larval size at 20 days posthatch (McMaster et al., 1992). White sucker from the BKME-exposed site are capable of spawning viable eggs and although these fish exhibit reduced spermatozoan motility there were no significant differences in milt volume, spermatocrit levels, or seminal plasma constituents.

232

VAN DER KRAAK

It is clear from the absence of ovulatory responses after GnRH injections, that the ability of fish from the BKME site to respond to ovulatory signals is depressed. It is unknown how white sucker from the BKME-exposed site are capable of successfully spawning with such aberrant GtH and steroid levels. Without question, the normal pattern of endocrine changes which accompany spawning are altered in BKME-exposed fish, even after their migration to clean water for spawning. Based on the apparent recovery of circulating levels of steroids in male white sucker during shortterm mill maintenance shutdowns (Munkittrick et al.. 1992a), these hormonal disruptions appear to be reversible. ACKNOWLEDGMENTS Funding for this study was provided by the Department of Fisheries and Oceans and the Research Advisory Council of the Ontario Ministry of the Environment (Grant 49463. The sGnRH-A was generously provided by Dr. J. Rivier, The Salk Institute, La Jolla, California.

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