Oxidative Stress and Bioindicators of Reproductive Function in Pulp and Paper Mill Effluent Exposed White Sucker

Ken D. Oakes*, Mark E. McMaster{dagger}, Andrea C. Pryce*, Kelly R. Munkittrick{ddagger}, Cam B. Portt§, L. Mark Hewitt{dagger}, Dan D. MacLean and Glen J. Van Der Kraak*,1

* Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; {dagger} National Water Research Institute, Environment Canada, Burlington, Ontario, Canada L7R 4A6; {ddagger} Department of Biology, University of New Brunswick, Saint John, New Brunswick, Canada E2L 4L5; § Cam Portt and Associates, 56 Waterloo Avenue, Guelph, Ontario, Canada N1H 3H5; and Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Received February 7, 2003; accepted April 11, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) collected from environments receiving pulp and paper mill effluent discharges in northern Ontario. Samples were collected over an eight-year period adjacent to three pulp and paper mills using a variety of processing and bleaching techniques. Fish collected downstream of pulp and paper mills within the Moose River basin exhibited elevated hepatic and gonadal 2-thiobarbituric acid reactive substances (TBARS), the presence of which is indicative of oxidative stress in these tissues. Within the Jackfish Bay system, exposure to pulp and paper mill effluent did not elevate hepatic or gonadal TBARS. Hepatic cytochrome P4501A activity (CYP1A) and fatty acyl-CoA oxidase (FAO) activities were frequently increased in livers of Moose River basin fish exposed to pulp and paper mill effluent, while lower activities of both enzymes were found within fish from the Jackfish Bay system. This suggests that oxidative stress may be related to CYP1A and FAO activities. Within the Moose River system, increases in measures of oxidative stress (TBARS, FAO) were generally coincident with decreased levels of 17 ß-estradiol; however, testosterone was often lower in Jackfish Bay system fish without any commensurate changes in oxidative stress. The suite of reproductive and oxidative stress parameters measured in this study varied between seasons and mills suggesting responses to effluent are dynamic and effects are complicated by different receiving environments. The relationship between gonad size, gonadal oxidative stress, and circulating plasma steroids remains unclear.

Key Words: oxidative stress; 2-thiobarbituric acid reactive substances; fatty acyl-CoA oxidase; hepatic cytochrome P4501A; pulp and paper mill effluent; plasma steroids, fish reproduction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased interest in environmental pollutant-induced oxidative stress, and knowledge of the interactions between free radicals, related oxidants, and cellular systems, has increased the profile of reactive oxygen species (ROS) in toxicology (Conway et al., 1989Go; Fatima et al., 2000Go; Livingstone, 2001Go). When ROS production overwhelms the endogenous protection afforded by specific degradative enzymes, antioxidant vitamins, and other radical scavengers, the resulting damage to cellular constituents is known as oxidative stress. Several studies have implicated ROS as the agents responsible for impaired reproductive function in organisms experiencing oxidative stress. In mammals, low concentrations of ROS impair ovarian development by negatively affecting steroid hormone biosynthesis, possibly via the oxidative modification of proteins, lipids, carbohydrates, and DNA (Behrman and Preston, 1989Go; Margolin et al., 1990Go; Schlezinger et al., 1999Go). Our earlier studies demonstrate that oxidative stress, as quantified by increases in 2-thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LPO), is associated with exposure to pulp and paper mill effluent at locations where reproductive impairment of fish in the receiving environment has been observed (Munkittrick et al., 1998Go, 2000Go; Oakes and Van Der Kraak, in press).

Much research has been devoted to identifying the effluent constituent(s) responsible for the altered steroidogenic biosynthetic capacities in exposed fish (McMaster et al., 1995Go, 1996Go). Of the effluent constituents examined, particular attention has been paid to phytoestrogens (MacLatchy and Van Der Kraak, 1995Go) and a variety of compounds promoting cytochrome P4501A (CYP1A) metabolism of endogenous substrates (Payne et al., 1987Go); however, these are unlikely to be solely responsible for the changes observed in circulating steroids (Munkittrick et al., 1994Go; Van Der Kraak, 1998Go). At this time, it is not known whether oxidative stress is a significant contributory mechanism to the reproductive dysfunction experienced by these fish.

The specific constituents within pulp and paper mill effluents that may promote reproductive dysfunction, ROS generation, and oxidative stress are not known. However, highly reactive ROS can be generated by known effluent ligands through increased fatty acyl-CoA oxidase (FAO) activity and by incomplete CYP1A substrate oxidation (Conway et al., 1989Go; Park et al., 1996Go; Schlezinger et al., 1999Go). Prolonged induction of these enzyme systems through continuous exposure to ligands in pulp and paper mill effluent may be responsible for generating significant oxidative stress, and may also contribute to reproductive impairments through cellular alterations in liver and gonadal tissue. While little is known regarding FAO responses to pulp and paper mill effluent exposure, increases in CYP1A activity, while not always present, are one of the most widely recognized biochemical responses associated with exposure to pulp and paper mill effluent (Munkittrick et al., 1994Go).

This study investigates oxidative stress and bioindicators of reproductive function in wild white sucker (Catostomus commersoni) populations exposed to effluent from three pulp and paper mills in northern Ontario. We have measured hepatic and gonadal TBARS as indicators of oxidative stress associated with lipid peroxidation, hepatic CYP1A, and FAO activity as possible contributors to reactive oxygen species generation, and gonad size and circulating sex steroids as indicators of reproductive condition. In order to investigate the factors contributing to oxidative stress and reproductive impacts, we evaluated the responses of fish collected from mills using different process types, different wood furnish, and different bleaching sequences over a number of years and seasons.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study sites.
White sucker were collected from preselected reference and pulp and paper mill effluent exposed locations over an 8-year period (Table 1Go). Two of the mills examined in this study (Smooth Rock Falls and Terrace Bay, ON) were bleached kraft mills discharging secondary-treated bleached kraft mill effluent (BKME). The third mill at Kapuskasing was a thermomechanical (TMP) mill, which did not employ secondary treatment at the time of sample collection (1994) but introduced this treatment shortly thereafter. Mill process modifications which may affect effluent composition are described for the Moose River system in Munkittrick et al.(2000)Go; however, little information is available for the Jackfish Bay system effluent composition beyond that described in Hewitt et al.(2000)Go.


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TABLE 1 Description of Mills, Effluent Discharges, and Sampling Occasions
 
White sucker from the Moose River system (Kapuskasing and Mattagami Rivers) were collected using overnight gill net sets (10 cm stretch mesh) immediately upstream and downstream of the pulp and paper mill effluent discharges unless indicated otherwise. Impoundments associated with each mill prevented migration between the upstream and downstream stations. Detailed limnological information for the Kapuskasing and Mattagami River sampling stations and a thorough description of both mills and the renovations they underwent during the course of the study are described elsewhere (Munkittrick et al., 2000Go).

Unlike the Moose River system, it was not possible to collect fish upstream and downstream of the effluent discharge within the Jackfish Bay system. Briefly, the Terrace Bay mill releases BKME into Blackbird Creek (48°49' N, 87°00' W) about 15 km upstream of the discharge of this tributary into Jackfish Bay on Lake Superior (Fig. 1Go). Fish residing in Jackfish Bay are exposed to the effluent discharge and are believed to spawn almost exclusively in Sawmill Creek, a clean water tributary of Jackfish Bay. In the fall, white sucker were captured using overnight gill net sets (10 cm stretch mesh) from Jackfish Bay near the effluent discharge (48°50' N, 86°58' W). This location receives no other industrial or municipal effluents and has no permanent residential development. In the spring, fish were collected in Sawmill Creek during the spawning migration using hoop nets placed across the width of the stream. The reference location, Mountain Bay on Lake Superior (48°56' N, 87°50' W), receives no effluent of any kind and is considered relatively free of anthropogenic influences. During the fall, white sucker were collected by gill net directly from Mountain Bay. In the spring, white sucker from Mountain Bay were captured during their spawning migration up a Mountain Bay tributary, Little Gravel River.



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FIG. 1. Map of Jackfish Bay system on north shore of Lake Superior. Study site enlargements inset.

 
After capture, fish were separated by size and initially sexed using external morphology with definitive sex and maturity determined after the animal was killed. Only sexually mature fish were used in the present study, although immature fish were also sampled to determine effects of maturity on oxidative stress endpoints, which were reported elsewhere (Oakes and Van Der Kraak, in press). For blood collection, fish were immobilized ventral side up in a foam block and bled via caudal puncture (5cc 21G11/2 syringe and needle) with immediate blood transfer to a 7 ml heparinized (100 U.S.P. units) Vacutainer® and placed on ice for up to 6 h. Plasma was collected after centrifugation at 3000 x g for 10 min, frozen in liquid nitrogen, and subsequently stored at -20°C until analysis. Fish were killed by a sharp blow to the head followed by severance of the spinal cord. These sampling methodologies received approval from our local animal care committees. Measurements of fork length, total weight, gonad and liver weight were taken and gonadosomatic indices (GSI = gonad weight/[total body weight – gonad weight]) and liver-somatic indices (LSI = liver weight/[total body weight – liver weight]) were calculated. Portions of liver and gonad were snap frozen in liquid nitrogen, followed by laboratory storage at -80°C prior to analysis. Care was taken to expedite the sampling process to minimize time from death of the fish to immersion of tissues in liquid nitrogen. Tissue remained frozen until analysis.

Biochemical Measures
TBARS assay.
The TBARS assay was used to quantify oxidative damage (lipid peroxidation) in fish liver and gonad samples as described previously (Oakes and Van Der Kraak, in press; Ohkawa et al., 1979Go). This method involves the reaction of malondialdehyde (MDA), a degradation product of lipid peroxidation, with 2-thiobarbituric acid (TBA) under conditions of high temperature and acidity to generate a fluorescent adduct that was measured spectrofluorometrically. TBARS were determined on 10% (w/v) whole tissue homogenates containing butylated hydroxytoluene (BHT) in the homogenizing buffer according to the assay optimization for white sucker tissue described in Oakes and Van Der Kraak (in press).

TOSC assay.
The total oxyradical scavenging capacity (TOSC) assay of Winston et al.(1998)Go, employing modifications described by MacLean et al. (in press), was used to assess changes in total antioxidant status of hepatic tissue in fish exposed to pulp and paper mill effluent. Briefly, the basis of the TOSC assay is the oxidation of {alpha}-keto-({gamma}-methiolbutyric acid) (KMBA) by 2,2'-azobis-amidinopropane dihydrochloride (ABAP) with the evolution of ethylene as a quantifiable end product. A second degree polynomial standard curve was established using a serial dilution (0–100 µM) of the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid). Trolox scavenges peroxyl radicals formed by the thermal homolysis of ABAP, thereby reducing the rate of ethylene formation. Inhibition of ethylene production by tissue samples, relative to that of the Trolox standard curve, was used to quantify total tissue scavenging capacity as Trolox equivalents. The assay was performed on the supernatant (105,000 g) from 20% (w/v) liver homogenates in STE buffer (0.25 M sucrose, 10 mM Tris-hydrochloride, and 1 mM ethylenediamine tetraacetate [EDTA]). Supernatant (10 µl) was diluted in 990 µl of STE buffer, of which 100 µl was added to each sample reaction vial. All sample reactions were analyzed in duplicate using 12.5 ml disposable septa-sealed culture vials. Reaction vials were prepared in advance with each vial containing 700 µl of 100 mM potassium phosphate buffer (pH 7.4) and 100 µl of 0.2 mM KMBA. A third reaction vial containing 100 µl of 20 µM stock solution of Trolox in lieu of cytosolic supernatant were run with each sample as an internal standard. The reaction vials were capped using rubber septa and 100 µl of 20 mM ABAP solution was injected into the vials at 3 min intervals. The ABAP injections were randomized among treatments. The vials were agitated on a vortex mixer for ~5 s, then placed immediately into a reciprocating waterbath shaker (~30 cycles/min) held at a constant 39°C. Preliminary experiments had established a 90 min incubation as the appropriate time required to surmount any lag-phase potentially caused by antioxidants present in tissue. After completion of the 90 min incubation period, a 3 ml gas sample was withdrawn from the headspace of the reaction vials and injected into a Varian CP-3380 gas chromatograph equipped with a 1.8 m x 3 mm Poropak Q stainless steel column, a 1 ml sample loop and a flame ionization detector (FID). The injector, column, and detector temperatures were 50°C, 60°C, and 340°C, respectively. High grade helium was used as the carrier gas at a flow rate of 22 ml/min with a typical run time of 2.3 min.

FAO activity.
The FAO activity assay quantifies production of the ROS H202 specifically generated by an enzyme unique to peroxisomal ß-oxidation, fatty acyl-CoA oxidase (EC 1.3.99.3). Production of H202, via the direct transfer of electrons to oxygen by fatty acyl-CoA oxidase, is quantified using lauroyl-CoA as the enzymatic substrate with concurrent measurement of the oxidation of 4-hydroxyphenylacetic acid to a flourescent product in a horseradish peroxidase coupled reaction (Poosch and Yamazaki, 1986Go).

FAO activity was assessed using crude peroxisomal fractions prepared by a modification of the techniques described by Small et al.(1985)Go and Moyes et al.(1990)Go. In brief, 10% (w/v) homogenates (10 passes using a teflon homogenizer at 1100 rpm) were prepared using 100 mg frozen liver pieces in homogenizing buffer containing 0.3 M mannitol, 10 mM HEPES, and 1 mM EGTA (pH 7.2). Iced homogenates were sonicated in two 10 s bursts at 35% maximal output using a Sonics and Materials Inc. Vibracell VC50. A 15 s interval between bursts allowed for cooling on ice. Homogenates were then centrifuged (4°C) for 5 min at 3000 x g to sediment unbroken tissue and heavy mitochondria. Supernatants were collected and centrifuged (4°C) for 20 min at 18,000 x g to make a peroxisome-enriched pellet which was resuspended in 1.0 ml of homogenizing buffer after vigorous vortexing. We found >90% of the FAO activity within the original tissue sample was contained within this peroxisomal fraction relative to other fractions.

Aliquots of each peroxisome enriched fraction (50 µl) were added to four 12 x 75 mm culture tubes containing 500 µl of assay cocktail comprised of 60 mM potassium phosphate buffer (pH 7.4) containing 4 U/ml horseradish peroxidase, 1 mM hydroxyphenylacetic acid, 20 µM flavin adenine dinucleotide (FAD), 0.2 mg/ml Triton X-100, 8.3 µM rotenone, and 1 mM sodium azide. Two culture tubes contained assay cocktail alone and two culture tubes received assay cocktail containing lauroyl CoA (100 µM).

Samples were added in indirect light (FAD is light sensitive) and incubated in the dark at 37°C for 30 min with the reaction terminated by the addition of 1.5 ml chilled 100 mM sodium carbonate buffer (pH 10.5) containing 2 mM potassium cyanide. H202 concentrations were determined relative to a standard curve of zero to 12.6 nmols H202 incubated with substrate-free assay cocktail. Fluorescence was measured on a Perkin Elmer Luminescence Spectrometer LS50 with excitation at 318 nm (slit width 3) and emission at 405 nm (slit width 3). The difference in fluorescence with and without lauroyl-CoA in assay cocktail was used to indicate fatty acyl-CoA oxidase activity. Protein concentrations were measured using the Bio-Rad protein-assay kit (Bio-Rad Laboratories). Fatty acyl-CoA oxidase activities were expressed both as nmols H202/g liver and nmols H202/min/mg protein.

Initial studies were conducted to ensure that, under assay conditions, H202 was exclusively produced by peroxisomal ß-oxidation and was not attributable to mitochondrial contamination. Mitochondria are recognized as a significant source of ROS including H202 (Carreras et al., 2002Go; Panaretakis et al., 2001Go). While cyanide is often used to eliminate contributions from mitochondrial contamination, our assay conditions preclude this as cyanide inhibits both horseradish peroxidase and mitochondrial activity (Poosch and Yamazaki, 1986Go). We therefore evaluated the ability of rotenone and sodium azide to specifically inhibit mitochondrial contributions to H202 production beyond those achieved using the detergent Triton X-100, which solubilizes the electron acceptors of the respiratory chain (Ballantyne, 1994Go; Moyes and Crockett, 1994Go). The addition of 8.3 µM rotenone and 1 mM sodium azide significantly reduced FAO activity in both reference and pulp mill effluent exposed fish populations without significantly altering the relative differences between the two sites suggesting the mitochondrial contribution had been reduced (Fig. 2Go).



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FIG. 2. Fatty acyl-CoA oxidase activity in female white sucker crude peroxisomal fractions with exposure to mitochondrial inhibitors Triton X-100 (0.02%), rotenone (8.3 µM), and sodium azide (1 mM). Reference sites (open bars) and effluent exposed sites (solid bars) represent the means (± SE) of 8 fish. Different lower case letters indicate significant differences (one-way ANOVA and Tukey’s HSD; p < 0.05).

 
CYP1A activity (ethoxyresorufin O-deethylase [EROD] assay).
CYP1A (EROD) activity was measured on S9 microsomal fractions with fluorescence determined using a 96-well microplate reader according to the established protocol described by Hodson et al.(1996)Go.

Sex steroids.
Plasma testosterone and 17 ß-estradiol were measured by radioimmunoassay after ethyl ether extraction using established protocols (McMaster et al., 1992Go).

Statistics.
TBARS, TOSC, and FAO activity were checked for homogeneity of variance and normality within year prior to analysis by two-way ANOVA for each year with site and sex as the main factors (SPSS software). Where more than two sites were sampled, differences between sites were detected by Tukey’s HSD. Differences were considered to be statistically significant when p < 0.05.

All data for CYP1A activity, LSI, GSI, and steroid hormone titres were evaluated for significance using two-tailed Student’s t-test. Student’s t-tests for all data were based on the summary statistics as means, SD, and sample sizes were available from several early data sets where raw data was unavailable. The SD and variance for each treatment were calculated, as was the pooled variance and the denominator for the t-statistic, with the resulting t-statistic evaluated against the tabulated critical value for {alpha} = 0.05. Where data was available for individual fish, correlation analysis among reproductive and oxidative stress endpoints was performed within river system and by sex among similar sized fish using Microsoft Excel.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TBARS
There were marked differences in white sucker hepatic TBARS responses with exposure to pulp and paper mill effluent between fish collected from the Moose River and Jackfish Bay systems (Table 2Go). Within the Moose River system in all years sampled, hepatic TBARS were increased with effluent exposure, and levels in females were always higher than in males. Within the Jackfish Bay system, hepatic TBARS did not change with effluent exposure. Hepatic TBARS in females from Jackfish Bay were higher than males in spring 1999 and fall 2000, but there was no difference in hepatic TBARS between the sexes in spring 1996 and 2001.


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TABLE 2 Hepatic TBARS (nmols TBARS/g Wet Tissue Weight) in White Sucker Collected Adjacent to Pulp and Paper Mills from the Moose River and Jackfish Bay Systems
 
Gonadal TBARS measured in fish from the Mattagami River in fall 1997 were significantly increased with exposure to pulp and paper mill effluent (Table 3Go). In 1998, only ovarian tissue was available from the Mattagami River, and TBARS did not change with effluent exposure. In contrast to results from the Mattagami River, white sucker from Jackfish Bay in fall of 2000 had decreases in TBARS with effluent exposure that were greater in female fish than in males.


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TABLE 3 Gonadal TBARS (nmols TBARS/g Wet Tissue Weight) in White Sucker Collected Adjacent to a Subset of the Pulp and Paper Mills from the Moose River and Jackfish Bay Systems Examined in this Study
 
TOSC
TOSC was significantly increased in female, but not male white sucker, with pulp and paper mill effluent exposure in the Mattagami River in the fall of 1997 (Table 4Go).


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TABLE 4 Hepatic TOSC (nmols Trolox Equivalents/g Wet Liver Weight) in White Sucker Collected Immediately Upstream and Downstream of a Pulp and Paper Mills on the Mattagami River in 1997
 
Hepatic FAO Activity
There were pronounced differences in apparent FAO response with effluent exposure between activities expressed as nmols H202/g liver and those protein normalized to nmols H202/min/mg protein (Table 5Go). FAO activities (expressed as nmols H202/g liver) in fish from the Kapuskasing River in 1994 had a significant response to effluent exposure but the response differed by sex, with increases in activity more pronounced in male than female fish. In the Mattagami River in 1995, exposed fish had higher FAO activities than reference fish and activities in females were higher than in males. In 1997 in the Mattagami River, FAO activities in females were higher with effluent exposure, whereas levels in males remained similar between reference and effluent exposed fish. There were no differences in FAO activities with either sex or exposure in the Mattagami River in 1998. When FAO activities were expressed as nmols H202/min/mg protein, there were no response differences between males and females in any year (Table 5Go). In 1994, 1995, and 1998, FAO activities were higher in exposed fish than reference fish, but there were no differences observed with effluent exposure in 1997. Hepatic FAO activities within the Moose River system were not significantly correlated with hepatic TBARS in female white sucker when FAO activity was expressed as nmols H202/g liver (r = 0.147; p = 0.245, n = 68) or when expressed as nmols H202/min/mg protein (r = 0.053; p = 0.674, n = 68). Similarly, male white sucker FAO activities were not significantly correlated with hepatic TBARS when FAO activity was expressed as nmols H202/g liver (r = 0.224; p = 0.079, n = 68) or when expressed as nmols H202/min/mg protein (r = 0.196; p = 0.125, n = 68).


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TABLE 5 Hepatic FAO Activity in Male and Female White Sucker Collected Upstream and Downstream of Pulp and Paper Mill Discharges on the Kapuskasing and Mattagami Rivers (Moose River System) in Northern Ontario
 
FAO activities (expressed as nmols H202/g liver) in white sucker from the Jackfish Bay system were higher in females than males in all years examined (Table 6Go). Fish collected in spring 1996 had significantly increased FAO activity with effluent exposure, but changes in FAO activity with effluent exposure were not apparent in fish collected after this time. When FAO activities were expressed as nmols H202/min/mg protein, levels in females were higher than males in the spring of 1996 and 2001. In spring 1999, there were no differences in FAO activity with sex or exposure. In fall 2000 there was a significant increase in FAO activity with effluent exposure, but the effect depended on sex with the response being much more pronounced in males than females. Hepatic FAO activities within the Jackfish Bay system were significantly correlated with hepatic TBARS in female white sucker when FAO activity was expressed as nmols H202/g liver (r = 0.298; p = 0.016, n = 64) but not when expressed as nmols H202/min/mg protein (r = 0.121; p = 0.337, n = 64). Similarly, male white sucker FAO activities were significantly correlated with hepatic TBARS when FAO activity was expressed as nmols H202/g liver (r = 0.391; p < 0.001, n = 64) but not when expressed as nmols H202/min/mg protein (r = 0.226; p = 0.071, n = 64).


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TABLE 6 Hepatic FAO Activity in White Sucker Collected during Their Spawning Migrations from a Reference Bay and a Bay Receiving Effluent Discharges from a Pulp Mill Near Terrace Bay, Ontario (Jackfish Bay System)
 
Liver- and Gonadosomatic Indices and CYP1A Activity
Within the Moose River system, female white sucker liver somatic indices (LSIs) were significantly increased with exposure to pulp and paper mill effluent only in fall 1994, while exposed males had significantly increased LSIs in 1994 and fall 1997, and significantly decreased LSIs in the fall of 1998 (Table 7Go). Female white sucker from the Jackfish Bay system had significantly decreased LSIs with exposure to pulp and paper mill effluent in spring 1996 and 2001, while male LSIs were significantly decreased in spring 1996 and significantly increased in fall 2000.


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TABLE 7 Liver-Somatic Indices (LSI) in White Sucker Collected Adjacent to Pulp and Paper Mills from the Moose River and Jackfish Bay Systems
 
In terms of gonadal development, within the Moose River basin, white sucker GSIs were significantly decreased with exposure to pulp and paper mill effluent only in females from spring 1995 and the fall of 1998 (Table 8Go). Male and female white sucker from the Jackfish Bay system had significantly lower GSI with exposure to pulp and paper mill effluent in spring 1996 and fall 2000.


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TABLE 8 Gonadosomatic Indices (GSI) in White Sucker Collected Adjacent to Pulp and Paper Mills from the Moose River and Jackfish Bay Systems
 
Within the Moose River Basin, white sucker CYP1A was significantly increased with pulp and paper mill effluent exposure in females from spring 1995, and both sexes from the fall 1997 and 1998 collections (Table 9Go). In the Jackfish Bay system white sucker, there was a significant decrease in female CYP1A activity in spring 1999, but significant increases in both sexes from fall 2000.


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TABLE 9 CYP1A Activity (Ethoxyresorufin O-Deethylase [EROD] Assay) in White Sucker Collected Adjacent to Pulp and Paper Mills from the Moose River and Jackfish Bay Systems
 
Plasma Steroids
Within the Moose River basin, only females from the fall of 1997 had significantly reduced circulating testosterone titres with effluent exposure (Fig. 3Go). In the Jackfish Bay system, male white sucker experienced a significant decrease in circulating testosterone with effluent exposure in spring collections from 1996, 1999, and 2001 and in the fall of 2000, while levels in females were only reduced in spring 1996 (Fig. 4Go).



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FIG. 3. Plasma testosterone (pg/ml) in white sucker collected in spring and fall from the Kapuskasing River and Mattagami River within the Moose River system. (a) Kapuskasing R. fall 1994 females, and (b) males; (c) Mattagami R. spring 1995 females, and (d) males; (e) Mattagami R. fall 1997 females, and (f) males; and (g) Mattagami R. fall 1998 females, and (h) males. Reference (upstream) sites are denoted by open bars and effluent exposed sites are denoted by solid bars. Each bar represents the mean (± SE) of 12 fish with the exception of Mattagami R. spring 1995, which represents 11 reference and 10 effluent exposed fish. Statistical differences were detected by Student’s t-test with the exception of testosterone in Kapuskasing R. Fall 1994 males, which was analyzed by Mann-Whitney U test due to unequal variance. Significant differences (p < 0.05) are indicated by different lower case letters.

 


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FIG. 4. Plasma testosterone (pg/ml) in white sucker collected in spring and fall from the Jackfish Bay system. (a) Spring 1996 females and (b) males; (c) spring 1999 females and (d) males; (e) fall 2000 females and (f) males; and (g) spring 2001 females and (h) males. Other details as in Figure 3Go.

 
Circulating 17 ß-estradiol in female white sucker exposed to pulp and paper mill effluent was reduced within the Moose River system in the fall of 1994 and 1997 as well as the spring of 1995 (Fig. 5Go). Within the Jackfish Bay system, effluent exposed females had significantly increased 17 ß-estradiol in the spring of 1996 and significantly decreased 17 ß-estradiol in the fall of 2000. Spring 1999 17 ß-estradiol information was unavailable.



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FIG. 5. Plasma 17 ß-estradiol (pg/ml) in female white sucker collected in spring and fall from the Moose River and Jackfish Bay systems. (a) Kapuskasing R. fall 1994; (b) Mattagami R. spring 1995; (c) Mattagami R. fall 1997; (d) Mattagami R. fall 1998; (e) Jackfish Bay spring 1996; (f) Jackfish Bay fall 2000; and (g) Jackfish Bay spring 2001. Each bar represents the mean (± SE) of 12 fish with the exception of Mattagami R. spring 1995 which represents 11 reference and 10 effluent exposed fish, and Jackfish Bay spring 1996 which represents 21 fish from each station. Other details as in Figure 3Go.

 
Hepatic TBARS were not significantly correlated with reduced 17 ß-estradiol or testosterone in female white sucker (r ≤ 0.190; p ≥ 0.370, n = 48) or testosterone in male white sucker (r ≤ 0.145; p ≥ 0.486, n = 48). Similarly, there were no correlations between gonadal TBARS and 17 ß-estradiol or testosterone in female white sucker (r ≤ 0.279; p 0.232, n = 36), while insufficient sample sizes precluded meaningful analysis in males. FAO activity (expressed both as nmols H202/g liver and H202/min/mg protein) was not significantly correlated to 17 ß-estradiol or testosterone in female white sucker (r ≤ 0.256; p ≥ 0.337, n = 48) or testosterone in male white sucker (r ≤ 0.438; p ≥ 0.089, n = 48).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To our knowledge, this is the first study to measure both oxidative stress and bioindicators of reproductive function in wild fish exposed to pulp and paper mill effluent. Fish caught downstream of effluent discharges in the Moose River basin had consistently higher levels of oxidative stress as evidenced by higher hepatic and gonadal TBARS relative to upstream reference fish. In contrast, there were no differences in hepatic TBARS with effluent exposure in fish from the Jackfish Bay system, and gonadal TBARS were reduced with exposure (although this was only examined at one point in time). Our findings of increased oxidative stress in effluent exposed white sucker from the Moose River system are consistent with the increased TBARS (in gill and kidney, but not liver tissue) found in freshwater catfish (Heteropneustes fossilis) exposed to pulp mill effluent (Fatima et al., 2000Go).

We measured TOSC to quantify the endogenous lipophilic and hydrophilic resistance of hepatic tissue to ROS and oxidative stress (Regoli et al., 1998Go; Winston et al., 1998Go). We initially predicted TOSC would be lower in fish exposed to pulp and paper mill effluent due to reductions in antioxidants by ROS generated through FAO and CYP1A activities. As increased TBARS indicated oxidative stress was occurring with effluent exposure, reductions in ROS scavenging capacity was anticipated. Paradoxically, we found the opposite relationship where increased TOSC suggested increased antioxidant capacities in hepatic tissues of female fish exposed to pulp and paper mill effluent. Previous analysis on these samples showed that while hepatic ascorbic acid was increased (Oakes and Van Der Kraak, in press), hepatic retinoids were markedly decreased with effluent exposure (Alsop et al., in press). The increase in hepatic TOSC in pulp and paper mill effluent exposed female white sucker, while probably not physiologically relevant, does appear to primarily reflect increased hepatic ascorbic acid content. As the TOSC assay did not reflect the marked declines in retinoids, it appears either retinoids are not acting as antioxidants under assay conditions, or the assay, at least as used in the present study, tends to emphasize the hydrophilic antioxidant status of white sucker tissue (MacLean and Oakes, unpublished data). We specifically evaluated TOSC on whole cytosol preparations, comparable to those used by other assays in this study, which could have reduced assay sensitivity due to differing protein concentrations between treatments. Protein can nonspecifically contribute to 20–30% of cytosolic TOSC (Regoli et al., 1998Go; Winston et al., 1998Go). However, the use of whole cytosol, which more closely approximates conditions in vivo, is reported to overcome TOSC contributions by proteins. Whole cytosol soluble antioxidants act as fast-reacting molecules which minimize the nonspecific reaction of proteins with radicals (Regoli et al., 1998Go).

FAO activity was measured to determine whether the oxidative stress we found could have resulted from the obligatory ROS production associated with peroxisomal ß-oxidation. Changes in FAO activity with effluent exposure were generally consistent with results from hepatic TBARS for fish from both the Moose River and Jackfish Bay systems, confirming the induction of this enzyme as a useful indicator of exposure (Mather-Mihaich and DiGiulio, 1991Go). While increases in fish FAO activity with exposure to pulp and paper mill effluent have been demonstrated, fish have far weaker peroxisomal responses to classical inducers than mammalian species and elevated FAO activity may not always generate significant oxidative stress (Mather-Mihaich and DiGiulio, 1991Go; Scarano et al., 1994Go).

There were significant differences in FAO responses depending on whether activity was expressed per gram tissue (nmols H202/g liver) or protein normalized (nmols H202/min/mg protein). While many enzyme activities linked to oxidative stress (CYP1A, FAO) are protein normalized, most oxidative stress parameters (TBARS, lipid hydroperoxides), including those in this article, are reported on a per gram tissue basis (Mather-Mihaich and DiGiulio, 1991Go; Oakes and Van Der Kraak, in press). To better evaluate their relationship to oxidative stress, antioxidant enzyme activities have also been reported on a per gram tissue basis (Palace et al., 1996Go). For comparison purposes, we and others report FAO activity both tissue and protein normalized with the caveat that nmols H202/g tissue is intended to primarily reflect ROS tissue concentrations, while nmols H202/min/mg protein is intended to primarily reflect changes in enzymatic activity (Scarano et al., 1994Go). However, FAO activity was significantly elevated with effluent exposure more frequently when expressed on a per gram tissue basis than when protein-normalized, most likely reflecting increased hepatic protein concentrations with pulp and paper mill effluent exposure rather than any changes in FAO activity. Higher hepatic protein content with effluent exposure may reflect increased peroxisomal protein (Yang et al., 1990Go) but may also reflect increases in CYP1A and other proteins of the aromatic hydrocarbon (Ah) receptor gene battery (Nebert and Gonzalez, 1987Go), which are known to be elevated with effluent exposure at these specific pulp and paper mills (Hewitt et al., 2000Go; Munkittrick et al., 1992bGo). Studies using the peroxisome proliferator gemfibrozil demonstrated similar responses to our study in that significant increases in rainbow trout hepatic FAO activity were observed only when expressed on a per gram basis and not when protein normalized (Scarano et al., 1994Go). Regardless of the source of the increased protein, protein normalized FAO activity may not reflect the oxidative stress burden experienced by the organism as accurately as the amount of H202 produced per gram of tissue, as confirmed by correlation analysis in the present study. Overall, FAO activity appears to be a plausible source of ROS contributing to the oxidative stress responses observed in fish exposed to pulp and paper mill effluent.

CYP1A activity has been widely used as an indicator of exposure to pulp and paper mill effluent, the efficacy of which is confirmed in this study. Significant increases in CYP1A activity have been correlated with hepatic TBARS in fish exposed to halogenated hydrocarbons (Palace et al., 1996Go), which are often constituents of pulp and paper mill effluents (Munkittrick et al., 1994Go). CYP1A enzymes, when bound to substrate such as halogenated hydrocarbons or other compounds that resist hydroxylation, produce ROS (Schlezinger et al., 1999Go), which may contribute to oxidative stress in effluent exposed fish. Hydrogen peroxide (H2O2) and superoxide anion may be produced by CYP1A enzymes when effluent constituents effectively uncouple CYP1A electron transfer; sufficient ROS may be produced to inactivate the enzyme itself, while also damaging adjacent biomolecules (Park et al., 1996Go; Schlezinger et al., 1999Go; Tindberg and Ingelman-Sundberg, 1989Go). While CYP1A activity is a plausible source of the oxidative stress experienced by these fish, it is unlikely to be the sole free-radical generating mechanism as TBARS responses were not always paralleled by CYP1A activity, and most ROS are localized both spatially and temporally to the site of generation. For example, elevated TBARS could be partially attributable to small amounts of extra-hepatic CYP1A activity, but it is unlikely this system could be responsible for the magnitude of some observed gonadal oxidative stress increases (Nebert and Gonzalez, 1987Go; Oakes and Van Der Kraak, in press) indicating other mechanisms of ROS production must be applicable in these tissues.

While CYP1A activity in the Moose River system tended to be elevated with pulp and paper mill effluent exposure, this was not always the case for fish from the Jackfish Bay system. As the majority of sampling periods in the Jackfish Bay system were spring collections, there is some evidence CYP1A in spawning fish may not be as readily inducible as other seasons, perhaps due to hormonal inhibition (Bezte et al., 1997Go). Our data appears to support this hypothesis, as only the fall collections from Jackfish Bay had increased CYP1A activity; however, other factors, such as depuration of inducing compounds, may also be responsible. In the course of their spring spawning migration, fish exposed to pulp and paper mill effluent in the Jackfish Bay system spend an unknown and presumably variable amount of time in clean water awaiting proper environmental cues to begin moving into spawning streams (Munkittrick et al., 1992aGo). While "staging" in clean water, the effluent exposed fish may depurate short-lived ROS-inducing compounds (Hewitt et al., 2000Go; Munkittrick et al., 1998Go) or products of ROS damage (or both) resulting in minimal CYP1A, FAO, and TBARS increases. Overall, we feel CYP1A activity is a potentially important source of ROS in pulp and paper mill effluent exposed fish. However, as only specific effluent substrates can generate ROS during CYP1A activity, generalized statements relating the relationship between CPY1A activity and ROS generation cannot reliably be made.

In the present study, LSI was sometimes increased within the Moose River system, but was also found unchanged or decreased. However, earlier studies in the Moose River basin, particularly within the Kapuskasing River, demonstrated LSIs consistently elevated with effluent exposure (data not shown). While increased LSI has historically been reported with effluent exposure at the Jackfish Bay mill (Munkittrick et al., 1992aGo), this index was unchanged as often as it was increased during the years of this study (Table 7Go). The relationship between LSI and oxidative stress was inconsistent in the populations examined in this study and was likely influenced by a number of variables including increased metabolic or CYP1A activity, peroxisomal protein production, or changes in nutritional status or lipid content (Oakes and Van Der Kraak, in press; Servos et al., 1992Go).

GSI was sometimes depressed with pulp and paper mill effluent exposure, but was never increased. Reductions in GSI are significant in that historically they appear to be correlated to reduced egg size, delayed maturity, and reduced levels of circulating steroids (Munkittrick et al., 1992aGo, 1998Go). However, within the scope of this study, there does not appear to be a consistent correlation between reduced GSI and reduced levels of circulating testosterone and 17 ß-estradiol (Table 10Go).


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TABLE 10 Summary of Statistically Significant Physical and Biochemical Changes in Hepatic TBARS, FAO (nmols H202/g Liver), CYP1A (EROD), LSI, GSI, Testosterone (T), and 17 ß-Estradiol (E2, Females Only)
 
Within the Moose River system, we found increased measures of oxidative stress (TBARS, FAO activity) were generally coincident with decreased levels of 17 ß-estradiol in fish exposed to pulp and paper mill effluent. However, testosterone concentrations within the Moose River system did not decrease in the same manner. Within the Jackfish Bay system, testosterone levels in male fish were always reduced with effluent exposure. While testosterone and 17 ß-estradiol in females did not change with effluent exposure in fall surveys, 17 ß-estradiol, as in earlier surveys (McMaster et al., 1996Go; Munkittrick et al., 1998Go) was significantly higher in our sole spring collection.

Depuration of short-lived oxidative stress products and inducing compounds are likely to have occurred within the spring Jackfish Bay system fish (Hewitt et al., 2000Go; Oakes and Van Der Kraak, unpublished data); however, reductions in circulating steroids from this specific pulp and paper mill effluent are known to persist after two weeks in clean water, a time frame potentially approximating the clean water depuration experienced during the spawning migration (Munkittrick et al., 1992bGo). Reductions in steroids might reasonably persist long after evidence of oxidative stress are no longer present in effluent exposed fish. While this study demonstrates some evidence of population level oxidative stress co-incident with reduced circulating steroids, there were few significant correlations between gonadal or hepatic oxidative stress and steroid titres within individual fish. This may be due, in part, to the many uncontrolled variables associated with field collections. Ongoing studies are examining the relationship between circulating steroids and oxidative stress with fish exposed to pulp and paper mill effluent under more controlled laboratory exposure conditions.

Throughout this study, there were marked differences in most measures of oxidative stress and indicators of reproductive function between fish collected from the Moose River and the Jackfish Bay systems. We believe these differences are largely attributable to the vastly dissimilar receiving environments, rather than a reflection of differing effluent toxicity. For example, effluent discharged by the mill at Kapuskasing accounts for a minimum of 0.39% of the total Kapuskasing River discharge during high water (May), and a maximum of 4.9% of discharge during low water conditions (March). Similarly, the mill at Smooth Rock Falls contributes a minimum 0.82% of total Mattagami River discharge during high water (May) to a maximum 4.1% of discharge during low water (March) (based on flow information in Munkittrick et al., 2000Go). While these are major river systems, they are not comparable to the dilution factor provided by Lake Superior (12,100 km3). While it is very difficult to compare the effects of toxicant introductions to lotic and lentic environments, we believe that dilution in the receiving environment, as well as potential depuration issues at Jackfish Bay, are largely responsible for the response discrepancies between the two river systems. Recent caging studies in Blackbird Creek (Fig. 1Go) confirm the effluent entering Jackfish Bay is capable of inducing oxidative stress and altering reproductive responses as observed in the Moose River system (Oakes et al., unpublished data), suggesting dilution in the Lake Superior receiving environment is at least partially responsible for the ameliorated responses.

While we conclude FAO and CYP1A activity are plausible sources of ROS generation, they are dominantly hepatic enzyme systems. Since there is little gonadal FAO or CYP1A activity, other mechanisms such as immune system responses or redox-active metal species must contribute to increases in gonadal, and perhaps hepatic, oxidative stress (Goksøyr and Husøy, 1998Go; Livingstone, 2001Go). Exposure to xenobiotics can greatly induce production of ROS from macrophages and neutrophils, enough to generate significant oxidative damage. Fatima et al.(2000)Go reported significant increases in extra-hepatic oxidative stress in tissues such as the kidney and gill which were attributed to phagocyte-activating constituents in pulp and paper mill effluent producing a burst of cytotoxic ROS. Other potential sources of increased free radical production include the decomposition of enzymatically and nonenzymatically formed peroxides, metal ion release (Fe2+, Cu2+) from storage sites or accumulated from the effluent, haem protein release, mitochondrial damage, or raised intracellular Ca2+ levels (Livingstone, 2001Go; Oakes and Van Der Kraak, in press).

The present study indicates that differing mill process types, furnish, and bleaching sequences may not significantly modify oxidative stress and reproductive responses in exposed fish. Over the 8 years of the study, there were no significant temporal trends in the endpoints we examined suggesting ROS generation is largely independent of process type or modifications. Our findings are supported by other studies demonstrating increased oxidative stress with effluent exposure at numerous pulp and paper mills using differing bleaching sequences (with and without chlorine), differing furnish, and differing effluent treatment strategies (Fatima et al., 2000Go; Mather-Mihaich and DiGiulio, 1991Go; Oakes and Van Der Kraak, in press). We have demonstrated that oxidative stress is present in effluent exposed fish from some aquatic systems with impaired bioindicators of reproduction, although there is currently insufficient evidence to link these responses. Different receiving environments, seasons, and sex collectively influence and complicate interpretation of oxidative stress endpoints. Due to the transitory nature of reactive oxygen species, we are currently investigating the onset and depuration of oxidative stress and reproductive impairments experienced by fish with pulp and paper mill effluent exposure under laboratory and field conditions.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the technical assistance of J. Ballantyne, D. Boyter, C. Chisholm, K. Daynes, J. Jardine, M. Maj, J. Rajotte, I. Smith, D. Stevens, and G. Tetreault. We thank the anonymous reviewers for their constructive comments, which greatly strengthened the article. This work was supported by Canadian Network of Toxicology Centers (CNTC), Department of Fisheries and Oceans (DFO), Environment Canada, and Natural Sciences and Engineering Research Council (NSERC) of Canada grants to G.V.D.K. K.D.O. received financial support from CNTC, DFO, NSERC, and the Ontario Graduate Scholarship program.


    NOTES
 
1 To whom correspondence should be addressed at Dept. of Zoology, University of Guelph, 50 Stone Rd. East, Guelph, ON, Canada N1G 2W1. Fax: (519) 767-1656. E-mail: gvanderk{at}uoguelph.ca. Back


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