Gene expression fingerprints of largemouth bass (Micropterus salmoides) exposed to pulp and paper mill effluents

Gene expression fingerprints of largemouth bass (Micropterus salmoides) exposed to pulp and paper mill effluents

Mutation Research 552 (2004) 19–34 Gene expression fingerprints of largemouth bass (Micropterus salmoides) exposed to pulp and paper mill effluents N...

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Mutation Research 552 (2004) 19–34

Gene expression fingerprints of largemouth bass (Micropterus salmoides) exposed to pulp and paper mill effluents Nancy D. Denslow a,b,∗ , Jannet Kocerha a , Maria S. Sepúlveda c , Timothy Gross c,d , Stewart E. Holm e b

a Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, USA Department of Biotechnology Program, University of Florida, PO Box 100156 HC, Gainesville, FL 32610, USA c Department of Physiological Sciences, University of Florida, Gainesville, FL, USA d USGS-Center for Aquatic Resource Studies, Gainesville, FL 32653, USA e Georgia-Pacific Corp., Atlanta, GA 30303, USA

Received 25 February 2004; received in revised form 10 May 2004; accepted 12 May 2004 Available online 10 July 2004

Abstract Effluents from pulp and paper mills that historically have used elemental chlorine in the bleaching process have been implicated in inhibiting reproduction in fish. Compounds with estrogenic and androgenic binding affinities have been found in these effluents, suggesting that the impairment of reproduction is through an endocrine-related mode of action. To date, a great deal of attention has been paid to phytoestrogens and resin acids that are present in mill process streams as a result of pulping trees. Estrogen and estrogen mimics interact directly with the estrogen receptor and have near immediate effects on gene transcription by turning on the expression of a unique set of genes. Using differential display (DD) RT-PCR, we examined changes in gene expression induced by exposure to paper mill effluents. Largemouth bass were exposed to 0, 10, 20, 40, and 80% paper mill effluent concentrations in large flow-through tanks for varied periods of time including 7, 28 or 56 days. Plasma hormone levels in males and females and plasma vitellogenin (Vtg) in females decreased with dose and time. Measurements of changes in gene expression using DD RT-PCR suggest that the gene expression patterns of male fish do not change much with exposure, except for the induction of a few genes including CYP 1A, a protein that is induced through the action of the Ah receptor in response to dioxin and similar polyaromatic hydrocarbons. However, in the case of females, exposure to these effluents resulted in an up-regulation of CYP 1A that was accompanied by a generalized down-regulation of genes normally expressed during the reproductive season. These antiestrogenic changes are in agreement with previous studies in bass exposed to these effluents, and could result in decreased reproductive success in affected populations. © 2004 Elsevier B.V. All rights reserved. Keywords: Pulp mill effluents; Estrogen; Estrogen receptor; Estradiol; Endocrine disruptors; mRNA; Differential display PCR; Largemouth bass

1. Introduction ∗

Corresponding author. Tel.: +1 352 392 9665; fax: +1 352 392 4441. E-mail address: [email protected] (N.D. Denslow).

The literature supplies several examples of altered reproduction in fish following exposure to pulp mill effluent whose prevailing bleaching process used

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.06.001

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elemental chlorine. Several studies have indicated changes in a number of physiological and biochemical responses including reduced ovarian development, reduced egg size, decreased fecundity (number of eggs spawned), delayed sexual maturation, and reduced levels of circulating sex hormones compared to fish from reference sites [1–7]. We have observed similar alterations in reproduction in largemouth bass (Micropterus salmoides) exposed to effluents from a paper mill at Rice Creek in Central Florida [8,9]. Some studies have attributed this observation to an endocrine-mediated event possibly through the androgen receptor or through the estrogen receptor [10–16]. At the time of this study, the paper mill at Rice Creek was using elemental chlorine (C) to bleach pulp in the first stage of bleaching followed by an extraction stage with sodium hydroxide (E). The final bleaching stages were sodium hypochlorite (H) and chlorine dioxide (D). Since the results from this study were gathered, the mill has gone through an extensive number of upgrades, some of which were designed to improve the quality of mill effluent. In part, these upgrades included the use of chlorine dioxide instead of elemental chlorine and oxygen and hydrogen peroxide instead of sodium hypochlorite in the bleaching stages and several modifications that mitigate process stream losses. These improvements have greatly reduced or eliminated recalcitrant chemicals from the effluent. Research conducted in the last few years suggests that chlorinated compounds are not the only key players, since reproductive alterations are still observed when fish are exposed to unbleached mill effluents, black liquor, and mechanical pulping effluents [17,18]. Several reports have implicated ␤-sitosterol, a plant sterol, as a possible significant factor contributing to the reproductive effects observed in fish exposed to paper mill effluents. In goldfish (Carassius auratus), injection of ␤-sitosterol causes reductions in plasma circulating levels of sex steroids and decreases in gonadal testosterone and pregnenolone production under in vitro conditions [19]. These changes mimic effects seen in paper mill-exposed fish. ␤-Sitosterol can also induce estrogenic effects in fish: it can bind to the rainbow trout (Oncorynchus mykiss) estrogen receptor and promote expression of the vitellogenin (Vtg) gene in vitro and in vivo [10,20]. These reported observations raised the hypothesis that chemicals inher-

ent within paper mill effluents were behaving as estrogens and that endocrine disruption was due in part by disruption of the estrogen-receptor pathway. The broader concern in mammals, including humans, is that exposure to estrogen or estrogen mimics could lead to DNA damage and then cancer [21,22]. However, to date, we are unaware of any studies that suggest that exposure to paper mill effluents induces the formation of neoplasms in fish. Moreover, no neoplasms were observed in fish from this study or other research our group has conducted in the Rice Creek area. There are some recent papers that show oxidative stress resulting from such exposures [23,24], which could lead to DNA mutations caused by hyperactive metabolites. The present study did not examine these possibilities, but instead looked at changes in gene expression induced by the exposures. Differential display (DD) RT-PCR is a powerful method to chart out molecular fingerprints of gene induction and suppression in fish treated with environmental contaminants. The exposures can be done in vivo—normally short-term exposures are sufficient— thus allowing an integrative final outcome, while the endpoint measurements, made at the molecular level, are specific for the mechanisms of action. These experiments bridge the distance between in vitro assays that measure interactions with the receptor in a non-physiological setup, which may make them poor predictors of physiological outcomes [25], and in vivo assays that measure physiological outcomes but cannot pinpoint a mechanism of action [26]. The main focus of this study was to examine changes in reproductive function in largemouth bass exposed to mill effluents and to correlate these changes with changes in gene expression fingerprints at the molecular level. We measured gene expression profiles using DD RT-PCR [27–30]. This technique allows the comparison of expressed mRNAs in controls and treated samples to find those that are differentially expressed. 2. Materials and methods 2.1. Description of effluent and paper mill plant At the time of this study, the paper mill in Palatka, FL produced a 50/50 mix of bleached/unbleached

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market pulp. The mill released an estimated 32 million gallons of effluent/day and production averaged 1452 air-dried metric tons (ADMT)/pulp/day from a furnish that consisted of 80% softwood and 20% hardwoods. Bleaching sequences were C90D10 EOP HDP and CEHD for the softwoods and hardwoods, respectively, where CD = mixture of chlorine (C) and chlorine dioxide (D) in proportions designated by subscripts; EOP = extraction with alkali and the addition of elemental oxygen (O) and hydrogen peroxide (P); H = hypochlorite; and DP = 100% D substitution with the addition of P. As is common in the US, effluents received secondary treatment. This consisted of both anaerobic (500-acre basin) and aerobic (500-acre basin) biological degradation with a retention period of 40 days. Treated effluents are discharged into Rice Creek, a tributary that runs for about 5 km before its confluence with the St. Johns River. Because Rice Creek is small, effluents can account for a large portion of its total flow (yearly average effluent concentration is estimated to be around 60%, with a range of 50%–97%). However, by the time effluents reach the St. Johns River, concentrations usually are below 10% (Georgia-Pacific, Holm, personal communication). This high effluent concentration is inconsistent with other mills in the US. For example, roughly 70% of mills accounted for less than 1% flow contribution to their respective receiving waters at average flow conditions, and an additional 24% accounted for less than 10% [31]. 2.2. Animals, holding facility, and exposure conditions Adult largemouth bass (American Sport Fish, Montgomery, AL, USA) were exposed to 10, 20, 40, or 80% effluents for 7, 28, or 56 days. A control group of fish received only well water. Fish were acclimated to the experimental location for 1 week before the experiment started. In addition, previtellogenic largemouth bass females were euthanized in September and were used as controls for changes in gene expression patterns that normally occur in females during the reproductive season. Exposure to paper mill effluents began the last week in December of 1998, and ended either the last week of January (28-day group) or February (56-day group). Seven-day exposures were conducted in late January (22–29). Thus, bass from

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the 7- and 28-day exposures were sacrificed the same week. Concentrations were chosen to represent effluent concentrations likely to be encountered by free-ranging fish inhabiting the mill effluent receiving water, Rice Creek. Fish were placed in 1500 L round, plastic, flow-through tanks located adjacent to the effluent discharge. The effluent used for the study was taken directly from the effluent discharge. Water used for the control tanks and to dilute the effluent was obtained from a well located near the tank system. Analysis of the well water indicated that it was free of contaminants [32,33]. The tank set up has been previously described in detail by Sepúlveda et al. [8]. Exposure of fish to the pulp and paper mill effluents was evaluated through the analysis of total (free and conjugated) resin acid bile concentrations (isopimaric, dehydroabietic, and abietic acids), as described previously [8,34,35]. The average dissolved oxygen (DO), temperature, and pH for the tank system were 6.3 ± 0.14 mg/L, 17 ± 0.3◦ C, and 7.7 ± 0.04, respectively. The average flow-through rate was 15 L/min. During the course of the study, fish were fed ad libitum once a week with a commercial pellet (“Floating Fish Nuggets”, Zeigler, Gardners, PA, USA). In a separate experiment, male largemouth bass were injected with 2 mg/kg 17␤-estradiol (E2 ) in DMSO, or just DMSO for a control, following the procedures previously described [36]. Fish were sacrificed 48 h after injection, when Vtg mRNA is maximally synthesized. 2.3. Body measurements, organosomatic indices, and reproductive endpoints At the end of each exposure period, fish were weighed and their total length was recorded. Condition factor was calculated as K = weight/length3 × 100. Plasma Vtg and sex steroids [11-ketotestosterone (11-KT) and E2 ] were measured as previously described [8,34]. Fish were euthanized with a blow to the head, then gonads and livers were excised and weighed for the determination of gonadosomatic (GSI) and hepatosomatic (HSI) indices (100 × gonad or liver weight/(body weight − gonad or liver weight) as described previously [8,34]. Pieces of liver were flash frozen in liquid nitrogen for gene transcription

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analysis. A representative sample of gonad was also saved in 10% buffered formalin for histological examination after standard hematoxylin and eosin staining. Previous communications by Sepúlveda et al. [8,34] describe these results. 2.4. RNA preparation Total RNA was isolated from the frozen liver samples using the Qiagen RNeasy Mini Kit RNA extraction columns. Traces of RNase were removed by treating the eluted RNA with proteinase K and traces of DNA were removed by DNase treatment [28]. DNA removal was assessed by control reactions in which reverse transcriptase was omitted in the differential display experiment. The quantity of the isolated RNA was determined by measuring the absorbance at 260 nm, and the integrity (or quality) of the RNA was assessed by determining the ratio of absorbances at 260 nm and 280 nm, and by observing the relative intensities of the two rRNA bands resolved by electrophoresis on a 1.5% formaldehyde-agarose gel. The 28S band should be about twice the intensity of the 18S band when stained with 1% ethidium bromide. 2.5. Gene induction fingerprints using differential display RT-PCR (DD RT-PCR) DD RT-PCR reactions were performed with the RNAimage mRNA Differential Display system (GenHunter, Nashville, TN) using one-base anchored oligo-dT primers (Table 1, [28]). DNase-treated total RNA (0.2 ␮g) was transcribed using 0.2 ␮M of the Table 1 Anchor primers and arbitrary primers used for DD RT-PCR Anchor primers

Arbitrary primers

H-T11 -C

AP-1; AP-2; AP-3; AP-7; AP-21 AP-1; AP-2; AP-3; AP-7; AP-8; AP-9; AP-25; AP-26 AP-1; AP-2; AP-3; AP-23

H-T11-A

H-T11-G

AP-4; AP-5; AP-6; AP-4; AP-5; AP-6; AP-21; AP-23; AP-24; AP-4; AP-10; AP-21;

A total of 29 anchor primer–arbitrary primer combinations were used. Only typical DD RT-PCR results are shown in the figures. Anchor primers are abbreviated by their last nucleotide in the figures.

anchor primer, and 100 U MMLV reverse transcriptase as described by the manufacturer. PCR reactions (20 ␮L) were performed using the RNAimage protocol as previously described [28,29] using anchor and arbitrary primers that are supplied with the kit (Table 1). The final reaction mixture contained 1/10th volume of the reverse transcription reaction described above, 0.2 ␮M anchor and arbitrary primers, 2 ␮M dNTP, 2.5 ␮Ci-[33 P]-dATP (2000–4000 Ci/mmol) and 1 U AmpliTaq DNA polymerase in the buffer provided with the kit. PCR amplified products were separated by electrophoresis on 5% denaturing Long Ranger gels. The gels were then dried under vacuum at 80 ◦ C and exposed to Biomax MR X-ray film for 18–72 h. 2.6. Northern blot analysis of Vtg mRNA Total RNA was isolated from individual livers as described above. Total RNA (15 ␮g) was separated in each lane of a 1% agarose formaldehyde gel as previously described [36]. Following separation, the RNA was transferred to a nylon membrane overnight using horizontal capillary transfer and crosslinked to the membrane using UV light. The cDNA probe against Vtg was generated by random primer labeling with ␣-33 P-dATP using Ambion’s (Austin, TX) Strip EZ DNA kit and hybridization was performed as previously described [36]. The blot was then exposed to a phosphor screen at room temperature for 48–72 h and visualized using a Typhoon 8600 imaging system (Molecular Dynamics, Piscataway, NJ). 2.7. Cloning of Cyp1A RNA was isolated from liver of largemouth bass exposed to either 0% or 80% paper mill effluent. A total of 3 ␮g of RNA was reversed transcribed to cDNA with Superscript II enzyme (Invitrogen, Carlsbad, CA). The following forward and reverse primers were then designed using the Oligo Primer Analysis software program, version 5 [37] to amplify a 730 bp fragment of CYP1A: (forward) 5 GAAAAAGATTGTTGGGGAGCACTA3 and (reverse) 5 GCAGCGCTTGTGCTTCATTGTGAG3 . The following thermocycler conditions were used: hold at 95 ◦ C for 2 min; 45 cycles at 95 ◦ C for 30 s, 70 ◦ C for 90 s; then 72 ◦ C for 10 min; and finally hold at 4 ◦ C until the reactions were removed from the thermocycler.

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All PCR reactions were performed using 10× buffer with MgCl2 , 5 U Amplitaq, 2.5 mM dNTP’s (Applied Biosystems, Foster City, CA) and 10 ␮M of each primer per 20 ␮l reaction. The PCR products were observed on a 1% agarose gel stained by ethidium bromide. A band of the appropriate size was excised from the gel and purified (Qiagen, Valencia, CA) following the protocol provided by the manufacturer. The purified cDNA product was ligated into a PGEM-T easy vector (Promega, Madison, WI) and transformed into Escherichia coli DH5␣ cells (Invitrogen) as previously described [36]. A positive clone was purified and sent to the University of Florida DNA Sequencing Core Facility for sequence identification and the resulting sequence was verified using BLAST. The sequence for largemouth bass Cyp1 A has been deposited in Gen Bank, accession number AY619695. 2.8. Real-time PCR development The following primers for the CYP1A real-time PCR assay were designed using the Primer Express program (Applied Biosystems) based on the Cyp 1A DNA sequence: forward primer, 5 GCAGCGCCGCTTTCC3 and reverse primer, 5 CTGATGGCACTGAACTCAACAAGT3 . Specificity of the primers was checked by a dissociation curve using Sybr Green reagent (Applied Biosystems). Each reaction contained 10 pmol of oligos and 0.15 ␮g of reverse-transcribed RNA and was amplified using universal conditions as per instructions from Applied Biosystems. All unknown samples were extrapolated to a standard curve of the plasmid CYP1A constructed in 10 fold dilutions from 109 copies to 105 copies.

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would immediately be seen as a lowering of Vtg concentration in the plasma. In this time frame (January to February), males normally also increase steroidogenesis, with more of the final steroids being converted to 11-KT and less to 17␤-estradiol. Because the fish were sacrificed at different times in their cycles, each group is best compared to its 0% effluent control. The control fish used in this study progressed through the reproductive cycle as expected (Table 2) comparable to fish in other studies in which we have tracked plasma hormone and Vtg profiles for bass [8,9,34,38]. 3.1. Effect of exposure on females Exposure to 40% and 80% paper mill effluents reduced E2 levels in females from a normal level of ∼600 pg/mL at the end of January (28 days) to ∼450 pg/mL, and from a normal of 1300 pg/mL at the end of February to ∼700 pg/mL (Table 2). The effect was about equally pronounced for both the 40% and 80% fish. Exposure also had a pronounced negative effect on plasma Vtg (Table 2 and Fig. 1), decreasing in a dose dependent manner with exposure. Vtg is normally imported into developing oocytes during this time frame, so that the reduced concentrations observed in the plasma would be the sum of reduced synthesis and continued endocytosis into the developing eggs. 11-KT is not a significant hormone for

3. Results Fish were euthanized at different times of reproductive maturity. Seven-day and 28-day fish were harvested at the end of January, when plasma E2 and Vtg levels in females are still on a rise [38], whereas the 56-day fish were harvested at the end of February, closer to the time when plasma E2 and Vtg peak ([38], Table 2). During this time, Vtg is being actively transported into oocytes where it is packaged into egg yolk proteins to serve as a source of eventual nourishment for fry. Thus, in females a cessation of Vtg production

Fig. 1. Plasma Vtg measurements in female largemouth bass exposed to varying concentrations of paper mill effluents for 28 and 56 days. Asterisks indicate differences in relation to control group (analysis of variance, Dunnett’s multiple comparison test; α = 0.05). Standard error and sample size are as presented in Table 1 for the 40% and 80% effluent treatments. Number of females: 26, 31, and 34; and males: 24, 19, and 16 for 0%, 10%, and 20% effluent treatments, respectively.

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Table 2 Hormone (pg/mL) and vitellogenin (mg/mL) measurements of female and male largemouth bass exposed to three concentrations of pulp and paper mill effluents for 7, 28, or 56 days Parameters

0%

40%

80%

Females (7 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

656 ± 40 (7) 941 ± 86 (7) 1.2 ± 0.31 (7)

504 ± 107 (5) 610 ± 161 (5)∗ 1.0 ± 0.31 (5)

290 ± 12 (8) 451 ± 40 (8)∗ 0.39 ± 0.08 (8)∗

Females (28 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

570 ± 53 (26) 151 ± 7.8 (26) 1.3 ± 0.13 (26)

491 ± 38 (27) 265 ± 21 (28) 0.73 ± 0.09 (28)∗

408 ± 28 (29)∗ 214 ± 17 (29) 0.21 ± 0.04 (30)∗

Females (56 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

1298 ± 70 (21) 589 ± 40 (21) 3.0 ± 0.30 (23)

712 ± 35 (18)∗ 644 ± 29 (18) 1.1 ± 0.13 (18)∗

737 ± 33 (19)∗ 653 ± 21 (19) 0.35 ± 0.01 (19)∗

Males (7 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

923 ± 49 (7) 714 ± 72 (7) 0.23 ± 0.14 (6)

702 ± 91 (8)∗ 537 ± 61 (8) 0.06 ± 0.05 (8)

295 ± 8 (6)∗ 444 ± 38 (6)∗ ND (<0.001) (5)

Males (28 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

314 ± 22 (24) 653 ± 58 (24) 0.19 ± 0.06 (24)

358 ± 27 (21) 466 ± 38 (21)∗ 0.008 ± 0.004 (22)

369 ± 23 (20) 332 ± 29 (20)∗ 0.002 ± 0.002 (20)

Males (56 days) 17␤-Estradiol 11-Ketotestosterone Vitellogenin

407 ± 41 (20) 1052 ± 42 (20) 0.25 ± 0.07 (20)

657 ± 19 (21)∗ 750 ± 28 (21)∗ 0.18 ± 0.06 (21)

739 ± 15 (21)∗ 735 ± 39 (21)∗ 0.09 ± 0.05 (21)

Values presented are mean ± S.E. (sample size). Asterisks indicate differences in relation to control group (analysis of variance, Dunnett’s multiple comparison test; α = 0.05).

female fish and little if any change was observed in plasma levels, except for the 7-day exposure where significant decreases were seen. Exposure to 10% and 20% effluent did not have significant effects on any hormones or Vtg levels and are not represented in Table 2. To determine the effects of paper mill effluent on Vtg mRNA synthesis, we performed Northern blots on total RNA extracted from the livers of bass exposed to the different concentrations of effluents for 56 days (Fig. 2). Northern blots for duplicate fish at each effluent concentration are shown. The three bands observed (5.0 kb, 3.3 kb, and 1.7 kb) for Vtg mRNA are normally present even after high stringency washes [39]. While the results reported here are more qualitative than quantitative, there was an apparent dose-dependent decrease in hepatic Vtg mRNA levels, a pattern that was consistent with lowered plasma Vtg.

To analyze for other genes in females whose expression might be altered by exposure to the effluent, we performed DD RT-PCR with 29 different primer pairs (Table 1). Each group included hepatic RNA from three independent fish. In this analysis, we only consider bands that consistently appeared or disappeared in all three animals per treatment. As expected, the primer pairs amplified many different mRNAs from the total pool, most of which were not altered by the exposure to effluent (Fig. 3). Arrows in the figure point to bands that are either increased or decreased because of the treatment. Since DD RT-PCR is an amplification process, small differences in concentrations of the mRNAs might be masked. This method works best to distinguish large differences in transcript amounts. Notwithstanding these drawbacks, it is clear that several mRNAs were differentially regulated in 80% effluent treated fish compared to controls, with some

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Fig. 2. Northern blot analysis of total RNA extracted from the livers of male (M) and female (F) largemouth bass exposed to varying concentrations of paper mill effluents probeb for Vtg mRNA expression. Two female and male fish were randomly selected from each of the treatment groups for this analysis. Male samples for control and all treatments were blank. The figure shows only two representative samples; 0% and 80% for males. The arrows correspond to the sizes of the mRNA bands on the blot in kilobases.

genes up-regulated and others down-regulated, suggesting altered homeostasis of gene expression in the liver of females exposed to effluent. Because of the effects on Vtg mRNA (Fig. 2), we also compared gene expression patterns of control and 80% effluent treated females with pre-vitellogenic fish that had been sacrificed early in the fall (Fig. 4, “PF”, first three lanes), at a time when normal E2 values are low in female plasma. In addition, for a positive control, we included male fish that had been stimulated by E2 (Fig. 4, “E2M”, last three lanes). Regardless of treatment, there are several bands of mRNAs that are present in the livers of all animals. These are mRNAs that are constitutively expressed in liver tissue and are not affected by seasonality or treatment. In control female fish, the mRNAs for several genes are induced during the reproductive season, including genes for Vtg and zona radiata proteins (ZRPs), among others [36,40]. This set of genes are also induced in males that have been treated with E2 (last three lanes; [36]). Exposure of prime-reproductive females to 40% and 80% paper mill effluents, however, down-regulates this set of genes, making the treated females appear more like the pre-vitellogenic females.

3.2. Effect of exposure on males Exposure of male largemouth bass to 40% and 80% paper mill effluents reduced 11-KT (Table 2) from an average of ∼680 pg/mL for the 7-day and 28-day fish sampled at the end of January, to an average of ∼400 pg/mL, for the 80% treatment group. In the 40% treatment groups, only the 28-day exposure significantly reduced 11-KT levels. The exposures caused a decrease in E2 values, but only for the 7-day fish. Similarly, the 56-day exposure groups (40% and 80%) showed significant declines in 11-KT levels (from ∼1000 to 740 pg/mL) but interestingly this was accompanied by an increase in plasma E2 levels, suggesting that prolonged exposure changed hormone homeostasis in these fish. Control males used in this experiment had low values of plasma Vtg at the start of the experiment, probably due to a prior exposure to E2 in holding tanks. Although not statistically significant, Vtg concentrations in plasma of males appeared to decrease in a dose responsive manner, similar to the effects seen in females (Table 2). Males, however, did not have any Vtg mRNA (Fig. 2), suggesting that exposure to E2 occurred before the start of the experiment and was not due to the treatments [36].

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Fig. 3. Differential display of female largemouth bass livers exposed to control or 80% effluent for 56 days. Anchor primers HT11 -C and HT11 -G, and arbitrary primers AP-1, AP-21, and AP-10 were used in this analysis. Arrows point to amplified mRNA fragments that show the most evident differentially expressed genes in control and 80%-effluent treated fish.

To determine if the expression of any other hepatic genes was affected by exposure, we performed DD RT-PCR. There were only a few differences in gene expression patterns in the livers of males (Fig. 5). In fact, most of the primer pairs we used showed absolutely no difference between exposed fish (80%, 56 days) and controls, suggesting that few genes had altered expression in males (data not shown). Because of our initial hypothesis that males would be estrogenized by exposure to paper mill effluents, we compared gene expression profiles of effluent-exposed males to that of males injected with E2. None of the genes that are induced by E2 in males, however, were induced by the effluent exposure (Fig. 6). Of the seven gene expression differences observed among effluent-exposed and control bass, all were

up-regulated in the 80% effluent treatment after 56 days of exposure (Figs. 5 and 6). The scored bands were cut out from the gels, re-amplified by PCR, and cloned into plasmid vectors for sequence analysis. We were able to identify only one of the effluent induced genes, marked by name in Fig. 6 as CYP 1A. We used this fragment to clone a portion of the largemouth bass CYP 1A gene. The largemouth bass sequence that was obtained was highly similar (65% identity) to other fish CYP 1A sequences currently available in the databases (Fig. 7). Using this sequence, we designed a real-time PCR assay to measure CYP 1A expression (Fig. 8). Both female and male bass showed increased CYP1A expression after exposure to 40% and 80% effluents, paralleling in an inverse manner the effects seen with Vtg mRNA expression.

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Fig. 4. Differential display of female largemouth bass livers exposed to control (CF) or 80% effluent (80% F) for 56 days, compared to pre-vitellogenic females (PF) or males (E2M) treated with E2 . Anchor primers HT11 -A and arbitrary primers AP-23 and AP-25 were used in this analysis. Arrows on the right point to amplified mRNA fragments that are induced in males by E2 and that are induced naturally in females at the peak of their reproductive cycles. Arrows on left point to genes that are naturally down regulated during the reproductive cycle.

4. Discussion Exposure of largemouth bass to effluents released from the Rice Creek paper mill during 1999 resulted in reduced levels of circulating E2 in females and 11-KT in males, and in reduced levels of plasma Vtg in females. Exposure to these effluents, however, did not affect body weight, length, or condition factor for either sex (see [34] for a detailed discussion of these values). Females and males exposed to high effluent concentrations (40% and 80%) for at least 28 days had lower GSI values (overall declines of 22% and 35% for female and male bass, respec-

tively) whereas HSI were only increased in females [34]. Plasma Vtg and Vtg mRNA are both biomarkers of estrogenic action but they report differently on contaminant effects [41]. Plasma Vtg is normally present in the blood of females undergoing oogenesis. Because males do not produce eggs, Vtg can persist in the blood of E2- induced males for over a month after cessation of treatment [42]. Vtg mRNA, on the other hand, has a much shorter half-life compared to the protein and responds more quickly to hormonal changes. Indeed, we observed effects on the mRNA after only 7 days of exposure. In reproductively active females, Vtg is

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reproductive parameters and Vtg could be the timing of exposure [43]. Florida largemouth bass start vitellogenesis in late October and peak with this activity in March [38]. By the time our experiments started in late December, females had already allocated a considerable amount of Vtg into the developing oocytes. It is important to mention that in a subsequent study, female bass exposed to at least 10% effluent for 56 days produced fry that did not survive to swim-up stage, suggesting an impairment of egg quality [34]. 4.1. DD RT-PCR

Fig. 5. Differential display of male largemouth bass liver exposed to control (C) or 80% effluent for 56 days. Anchor primer HT11 -G, and arbitrary primers AP-1 and AP-2 were used in this analysis. Arrows point to amplified mRNA fragments that are differentially expressed in control and 80%-effluent treated fish.

deposited into eggs and its presence in the blood is transient, requiring a constant synthesis by the liver in order to maintain high concentrations in the blood. Thus, it was not surprising to see dramatic clearance of this protein in females that were exposed to the higher concentrations of effluents, even after only 7 days, once the fish apparently ceased to express the gene. Interestingly, fecundity, egg size and egg hatchability did not differ among the groups [8,34]. A possible explanation for the lack of association between

This method effectively compares the expression of mRNAs in control and treated samples on a one to one basis. There are three possible anchor primers and 80 possible arbitrary primers that can be used for a total of 240 primer pair combinations, to see all of the expressed genes in a tissue. In this set of experiments we used a total of 29 primer pairs to test the hypothesis that exposure to pulp mill effluents would induce genes normally under the control of E2 . Each primer pair set amplifies a different set of mRNAs, depending on the ability of the forward and reverse primers to bind to sequences within mRNAs, thus each primer pair combination reports on a different set of mRNAs. For some primer pairs there were no differences at all between control and treated fish suggesting that most of the genes are not affected by exposure. Other primer pairs showed differences because some of the genes amplified in these reactions were induced or repressed by the exposures. This method is open-ended and does not require prior knowledge of gene expression profiles. While it is possible to cut out the amplified segments and identify the genes they represent, and we did identify CYP1A, in this study we focused mainly on expression profile differences between controls and 80% effluent. 4.2. Effects of exposure on female largemouth bass In the case of females, the set of genes that is normally up-regulated by E2 during the reproductive process and that peak in the January to March time frame every year, was down-regulated by exposure to the effluents. Since endogenous E2 levels were down-regulated in these fish, it is likely that the down-regulation of this group of genes is due

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Fig. 6. Differential display of male largemouth bass livers exposed to control (C) or 80% effluent for 56 days or to E2 . Anchor primers HT11 -A and HT11 -C, and arbitrary primers AP-6 and AP-1 were used in this analysis. Arrows on the left point to amplified mRNA fragments that are differentially expressed in control and 80%-effluent treated fish and arrows on the right point to mRNAs that are induced by exposure to E2 . PDI, protein disulfide isomerase.

to low levels of endogenous E2 , rather than through a separate mechanism. We are currently investigating whether phytoestrogens, such as ␤-sitosterol that are frequently detected in paper and pulp mill effluents could be responsible for the down-regulation of steroidogenesis (Kocerha and Denslow, in preparation). The concomitant up-regulation of CYP 1A, however, could have played a role in the observed anti-estrogenic responses. Indeed, a similar pattern was observed in free-ranging largemouth bass females sampled downstream from this mill [44]. In this study, inverse relationships were observed between Vtg

and GSI and hepatic ethoxyresorufin-O-deethylase (EROD) activity, a measure of CYP 1A. English sole (Parophrys vetulus) exposed naturally to Puget Sound sediments contaminated with PCBs and PAHs, also showed significant correlations between chemical exposure, CYP 1A induction, and reduced concentrations of plasma E2 [45], and in vitro studies have shown a positive relationship between a compound’s antiestrogenicity and its ability to induce CYP1A proteins in fish [46]. These findings suggest alterations in the affinity of E2 for the estrogen receptor, probably due to Ah-receptor mediated changes in the phosphorylation state of the estrogen receptor [46]. Since

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N.D. Denslow et al. / Mutation Research 552 (2004) 19–34

LMB toadfish seabass killifish seabream scup trout Prim.cons.

LMB toadfish seabass killifish seabream scup trout Prim.cons.

LMB toadfish seabass killifish seabream scup trout Prim.cons.

LMB toadfish seabass killifish seabream scup trout Prim.cons.

LMB toadfish seabass killifish seabream scup trout Prim.cons.

250 260 270 280 290 300 | | | | | | ----------------------------------------KDNIPDITDSLIDHCEDRXL DFIPVLRFLPSTAMKKFLSINDRFDKFVKKIVSEHYATYNKDNIRDITDSLIDHCEDRKL DFIPVLQFLPSTTMKNFMDINARFNKFVQKIVSEHYTTYDKDNIRDITDSLIDHCEDRKL DFIPALQFLPNKSMKKFVNLNNRFNNFVQKIVSEHYSTFDKDNIRDITDSLIDHCEDRKL DFIPILQYLPSTSMKKFVSINDRFNAFVQKIVSEHYTTFDKDNIRDITDSLIDHCEDRKL DFXPIXQYLPSTXMKKFLNINDRFNTFVQKIVSEHYTTFDKDNIRDITDSLIDHCEDRKL DFIPILRYLPNRTMKRFMDINDRFNNFVQKIVSEHYESYDKDNIRDITDSLIDHCEDRKL **** ************* * DFIPILQ2LPST2MKKF33INDRFN2FVQKIVSEHYTT2DKDNIRDITDSLIDHCEDRKL 310 320 330 340 350 360 | | | | | | DENSNVQVSDDQIVGIVNDLFGAGFDTVSTALSWSVMYLVAYPEIQERLYQDLKDKVSVD DENCNVQVSDEKIVGIVNDLFGAGFDTVSTGLSWSVMYLVAYPEIQERLYQEIKDSVGTE DENANVQMSDEKIVGIVNDLFGAGFDTISTALSWSVMYLVAYPEIEERLYQELKENVGLD DENSNIQMSDEKIVGIVNDLFGAGFDTISTALSWAVMYLVAYPEVEERLYEEIKEKVGLD DENSNVQMSDEKVVGIVNDLFGAGFDTISTALSWSVMYLVAYPEIQERLYQEMKESVGLD DENSNVQMSDEKIVGIVNDLFGAGFDTISTALSWSVMYLVAYPEIQERLYQEMNETVGXD DENANIHVSDEKIVGIVNDLFGAGFDTISTALSWAVVYLVAYPEIQERLHQELKEKVGMI ***.*:::**:::**************:**.***:*:*******::***::::::.*. DENSNVQMSDEKIVGIVNDLFGAGFDTISTALSWSVMYLVAYPEIQERLYQELKEKVGLD 370 380 390 400 410 420 | | | | | | RSPRLSDRPNLPFLEAFILEMLRHSSFLPFTIPHCTTKDTSLNGYFIPKDTCVFINQWQI RMPLLSDRPSLPFLDAFILEIFRHSSFLPFTIPHCTTKNTSLNGYFIPKDTCVFINQWQI RTPLLCDRPNLPFLEAFILEIFRHSSFLPFTIPHCTSKDTSLNGYFIPKDTCVFINQWQI RTPVMSDRSNLPLLESFILELFRHSSYLPFTIPHCSTKDTSLNGYFIPKDTCVFVNQWQI RTPCLSDKPKLPFLEAFILEIFRHSSFLPFTIPHCSSKDTSLNGYFIPKDTCVFINQWQI RTPCLSDKPKLPFLEAFILETFRHSSFLPFTIPHCTSKDTSLNGYFIPKDTCVFINQWQI RTPRLSDKINLPLLEAFILEIFRHSSFLPFIIPHCTIKDTSLNGYFIPKDTCVFINQWQV * * :.*: .**:*::**** :****:*** ****: *:***************:****: RTP3LSDRPNLPFLEAFILEIFRHSSFLPFTIPHCT2KDTSLNGYFIPKDTCVFINQWQI 430 440 450 460 470 480 | | | | | | NHDPELWKDPSSFNPDRFLSADGTELNKLEGEKVMTFGLGKRRCIGEVFARNEVYLFLAI NHDPELWKDPFSFNPERFLSADGTELNRLEGEKVMLFGLGKRRCIGEVIARNEVFLFLAI NHDPELWKDPSSFNPDRFLSADGTELNKLEGEKVMVFGLGKRRCIGEVIARNGVFLFLAI NHDPELWKDPSMFIPDRFLSADGTEVNKQEGEKVLIFGLGRRRCIGEVIARNEVFLFLAI NHDPELWKDPSSFNPDRFLNTDGTELNKLEGEKMMVFGLGKRRCIGEVIARNEVFLFLAI NHDAELWKDPSSFNPDRFLNADGTEVNKLEGEKMMVFGMGKRRCIGEVIARSEVFLFLAI NHDPELWKEPSSFNPDRFLSADGTELNKLEGEKVLVFGMDKRRCIGEAIGHNEVYLFLAI ***.****:* * *:***.:****:*: ****:: **:.:******.:.:. *:***** NHDPELWKDPSSFNPDRFLSADGTELNKLEGEKVMVFGLGKRRCIGEVIARNEVFLFLAI 490 500 510 520 | | | | MVQKLHFSTIPGEPLDLTPEY---------------------IIQRLQFHMLPGEPLDMTPEYGLTMKHKRCQLRATMREKNEQIVQKLHFKTLPGEPLDMTPEYGLTMKHKRCHLRATMRASE--IIQKLHFYKLPGEPVDMTPEYGLTMKHKRCYLGVAMRAKDVQLVQNLRFHAKPGEPLDMTPEYGLTMKHKRCHLRAAMRSRNEELVQNLRFHSMPGEPLDMTPEYGLTMKHKRCQLRA--------LLQRLRFQEKPGHPLDMTPEYGLTMKHKRCQLKASMRPWGQEE ::*.*:* **.*:*:**** 2VQKL2FHTLPGEPLDMTPEYGLTMKHKRCQLRA2MRAKNE2E

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Fig. 8. Quantitative PCR analysis of CYP 1A expression in female (F) and male (M) fish exposed to varying concentrations of paper mill effluent for 56 days. For these analyses, we used four control and five exposed bass per dose. Statistical comparisons were done by Student’s T-test (α = 0.05).

CYP1A in fish does not participate in the catabolism of E2 , it is unlikely that increases in EROD activities are related to increases in the oxidative metabolism of this hormone [47]. Clearly, additional studies are needed for a better understanding on the involvement of CYP 1A on endocrine modulation and its potential effects on fish reproduction. 4.3. Effects of exposure on male LMB We had expected that treating male fish with high concentrations of effluent would result in activation of genes that are normally responsive to E2 . However, this hypothesis was not confirmed by our results. Instead, we found that genes that are normally turned on by E2 were not turned on in males. For most of the primer pairs that we used, gene expression patterns in male fish treated with effluent were very similar to 0% effluent controls and different from E2 -treated male

fish, suggesting that the Rice Creek effluents did not act as agonists through the estrogen receptor. Exposure of male fish to the paper mill effluents did, however, increase the differential expression of some genes even after only 7 days of exposure, suggesting the presence of bioactive compounds in these effluents. One of these genes is CYP 1A, a protein that is responsive to TCDD and similar polynuclear aromatic chemicals and has been reported to be induced in fish exposed to pulp and paper mill effluents [3]. The results with 56-day treated male fish were similar to the 7-day treatment group, suggesting that for monitoring events at the mRNA level, a short treatment study is sufficient. Other genes that are induced by the treatment remain to be identified. To verify the induction of CYP 1A, we cloned a fragment of the gene from bass and performed quantitative real-time PCR across all of the exposure concentrations at 56 days. From this more in depth analysis,

Fig. 7. Alignment of the amino acid sequence for the segment isolated for largemouth bass (LMB, Micropterus salmoides, accession number AY619695) CYP1A with other fish CYP1A’s, starting at amino acid position 280 for toadfish (Opsanus tau, accession number U14161), seabass (Dicentrarchus labrax, accession number AJ251913), killifish (Fundulus heteroclitus, accession number AF0268000), seabream (Sparus aurata, accession number AF005719), scup (Stenotomus chrysops, accession numbrt U14162), and rainbow trout (Oncorhynchus mykiss, accession number AF015660). There was a 65% identity in largemouth bass CYP 1A sequence compared to other species. The colored region shows the alignment to the LMB Cyp1A sequence, red residues are regions in which all sequences have the same amino acid, green residues represent sequences that have a conservative substitution, blue residues are highly variable regions. The bottom line of the alignment (prim cons) shows in bold and capital letters the sequence of the primary conserved regions of Cyp 1A, and the locations where more than one amino acid substitution has occurred.

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it is apparent that significant increase in the expression of this gene was only observed with 40% and 80% effluent. While these in-stream concentrations appear high, there are times during the year where effluent has been present in Rice Creek at these concentrations. There is abundant data suggesting that paper mill effluents have effects on fish [4,6] but the identities of the responsible compounds are not known. Dioxins and furans have been identified historically in paper mill effluents whose prevailing pulp bleaching process was elemental chlorine. However, the advent of chlorine dioxide as a replacement for elemental chlorine has basically eliminated TCDD in effluents and, as a consequence, fish body burdens have declined dramatically [48–50]. Nonetheless, other compounds may also have deleterious effects on fish reproduction. There is evidence for effects on mixed-function oxygenase induction at mills that do not use chlorine bleaching [7,24]. Other compounds such as fatty acids, resin acids and plant sterols [19] have also been implicated. Black liquor fractions have antiestrogenic activity when tested in mammalian recombinant receptor bioassays [51]. There is also excellent data showing that female mosquitofish (Gambusia affinis) become masculinized by paper mill effluents [52] and that aromatase activity is also targeted [53]. It is not surprising that such complex chemical mixtures as appear in paper mill effluents can at certain concentrations have diverse actions that lead to endocrine and reproductive anomalies. In summary, the effluents from the CEHD type paper mill at Rice Creek in 1999, before significant upgrades, reduced steroidogenesis in largemouth bass dropping concentrations of steroid hormones in both sexes and the levels of plasma Vtg and liver Vtg mRNA in females. A secondary effect was the induction of CYP 1A, possibly directly through the action of compounds on the Ah receptor. The role of this induction on the antiestrogenic effects observed deserves further studies. Other genes that are affected by these exposures still remain to be identified and are probably targeted by pathways other than the estrogen receptor signaling pathway. These results suggest that gene expression fingerprints can point to the final outcome of exposure and may help to elucidate the signaling pathways that are affected by complex mixtures of contaminants.

Acknowledgements This publication was made possible in part by grant number P42 ES 07375 from the National Institute of Environmental Health Sciences, NIEHS and in part by funding from Georgia-Pacific Corporation. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or Georgia-Pacific. The authors wish to acknowledge Christopher Bowman for supplying RNA samples from fish treated with E2 for controls. We also acknowledge technical help from Steve Li and Virginia Ritter.

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