pathologic alterations in adult rainbow trout ... · aquatic toxicology 50 (2000) 287–299...

Aquatic Toxicology 50 (2000) 287–299

Pathologic alterations in adult rainbow trout, Oncorhynchusmykiss, exposed to dietary

2,3,7,8-tetrachlorodibenzo-p-dioxin

Gail L. Walter a,1, Paul D. Jones c,d,*, John P. Giesy b,c,d

a Department of Pathology, College of Veterinary Medicine, Michigan State Uni6ersity, East Lansing, MI 48824, USAb Department of Zoology, Michigan State Uni6ersity, East Lansing, MI 48824, USA

c Institute of En6ironmental Toxicology, Michigan State Uni6ersity, East Lansing, MI 48824, USAd Food Safety and Toxicology Building, National Food Safety and Toxicology Center, Michigan State Uni6ersity, East Lansing,

MI 48824, USA

Received 29 June 1999; received in revised form 31 January 2000; accepted 2 February 2000

Abstract

Adult female rainbow trout (Oncorhynchus mykiss) fed [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impreg-nated diet at 0, 1.8, 18 or 90 ng/kg food for up to 320 days were evaluated by clinical pathology, gross pathology andhistopathology procedures. Gross pathological changes were limited to a slight increase in the incidence of lesions ofthe caudal fins in the treated groups. Mixed mononuclear inflammatory infiltrates were present in multiple organs offish from control and treatment groups, but did not appear to be treatment related. Lesions associated with exposureto TCDD were observed histologically in liver and spleen after 100 and 250+ days of exposure. The livers ofTCDD-treated fish contained less hepatocellular glycogen, more mitotic figures, greater anisokaryosis, anisocytosis,nuclear chromatin clumping and margination. Prominent nucleoli were directly proportional to TCDD dose.Hepatocellular changes in fish exposed to TCDD also included single cell necrosis and clear cytoplasmic vacuolesconsistent with lipid. Some fish from all TCD exposed groups had lower lymphoid density compared to controls atall time intervals. Fish exposed to the highest TCDD dose had decreased peripheral leukocyte counts after 50 and 100days. The lowest observable adverse effect level (LOAEL) for these effects was 5.69 ngTCDD/kg in diet and 0.90 ngTCDD/kg liver. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Dioxin; Fish; Accumulation; Pathology; Histopathology; Toxicity

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1. Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) isthe by-product of industrial processes and py-rolytic reactions (Harrad and Jones, 1992; Rappeand Kjeller, 1994; Brzuzy and Hites, 1996).

* Corresponding author. Tel.: +1-517-4326333; fax: +1-527-4322310.

1 Present address, 325 Grandview Ave., Kalamazoo, MI49001, USA.

0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S 0166 -445X(00 )00095 -3

G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299288

TCDD has been shown to accumulate into thetissues of fish (Schmieder et al., 1995; Niimi, 1996;Delorme et al., 1998; Johnson et al., 1998; Tietgeet al., 1998). Some fish species, particularlysalmonids, are sensitive to the effects of TCDD(Walker and Peterson, 1994; Peterson et al., 1993;Newsted et al., 1995). While a number of chemicalsstructurally similar to TCDD can cause adverseeffects in fish through the same mode of action(Hanberg et al., 1990; Walker and Peterson, 1991;Newsted et al., 1995), TCDD is the most potent ofthe class of polyhalogenated, diaromatic hydrocar-bons (PHDHs; Giesy and Kannan, 1998). Thetoxicity of complex mixtures of PHDH is oftenexpressed as TCDD equivalents (TEQs; Safe,1987). TCDD equivalency factors have beenderived for fish (Van den Berg et al., 1998) so thatthe toxic potency of complex mixtures of Ah-recep-tor (Ah-R) active compounds to fish can be calcu-lated. However, to be able to interpret the potentialeffects of these complex mixtures on fish a dose-re-sponse relationship including a threshold for effectis needed. Previously, the toxicity of TCDD to fishhas been investigated in short-term studies atexposure concentrations greater than those foundin the environment. Furthermore, the vector ofexposure was generally via ‘intra peritoneal’ injec-tion, single gavage dose or from aqueous mediacontaining solvent carriers (Spitsbergen et al.,1991). Alternatively eggs have been exposed toconcentrations of TCDD in water above the com-pounds water solubility or TCDD was injected intoeggs (Walker and Peterson, 1991). While thesestudies established that TCDD is extremely toxic tofish, no reference doses were available.

The research presented here was conducted as adefinitive study of the long-term effects of smallconcentrations of TCDD in the diet of rainbowtrout. The study was designed to investigate theeffects of concentrations of TCDD in the diet thatwere similar to those currently in the environmentor that once occurred in the environment. Further-more, the greatest concentration of TCDD used inthe current study was less than the total concentra-tion of TEQs often measured in fish from the NorthAmerican Great Lakes (Jones et al., 1993; Giesy etal., 1999). The vector and rate of exposure wereselected to allow an estimation of ecological effects

of TCDD on a salmonid fish at environmentallyrelevant concentrations. In addition, the patholog-ical responses are catalogued such that they can beused as a functional measure of TCDD exposureunder field conditions. This paper presents only thepathological findings, detailed descriptions of theaccumulation disposition and effects on reproduc-tion and survival can be found elsewhere (Giesy etal., 2000; Jones et al., 2000).

2. Materials and methods

Adult (age class II; 350 g), female rainbow trout(Oncorhynchus mykiss) of the spring-spawningShasta strain were collected from the rearing pondsat the Stoney Creek Trout farm (Grant, MI). Fishwere sorted by sex and held at the Michigan StateUniversity Aquaculture facility until exposure be-gan in March of 1991. Fish were acclimated for 60days in the exposure tanks before the commence-ment of exposure.

Tritium labeled TCDD was synthesized andpurified at the Pesticide Research Center, MichiganState University. The radiochemical purity (\99.9%) and specific activity were confirmed by gaschromatography-mass spectrometry (GC-MS) andliquid scintillation counting. Concentrations ofTCDD in food and selected fish tissues wereconfirmed by GC-MS and ELISA (Enzyme linkedimmunosorbent assay). Food was spiked with both3H-labeled TCDD and non-labeled TCDD suchthat, while the TCDD dose varied, the TCDDspecific activities (DPM/pg TCDD; DPM=disin-tegrations per minute) were also varied so that theradiometric dose remained constant. In this wayfish were exposed to the same dose of radiation overthe course of the study (Table 1). Control fish werenot exposed to 3H.

Fish were exposed in 1700 l, flow-throughtanks. The flow rate was 71.5 l/h resulting inapproximately two turnovers of the water perday. Temperature was maintained at 12°C andphotoperiod was adjusted weekly to match ambi-ent outside conditions. Tanks were situated in anegative pressure facility with three levels of con-tainment for water and one for air. Control fishwere held in the same facility with the same

G.L. Walter et al. / Aquatic Toxicology 50 (2000) 287–299 289

containment but in an adjacent room to preventTCDD carry over between tanks.

Fish were fed Silver Cup Fish Feed (MurrayElevators, Murray, UT) with or without tritium-labeled TCDD for up to 320 days. Stock solutionsof TCDD were prepared in acetone and appliedto the food. The acetone was allowed to evapo-rate. TCDD-spiked food containing 5.4 54 or 270ng TCDD/kg was fed every third day while nospiked food was fed on the other 2 days. Thisresulted in average daily dietary concentrations of0, 1.8, 18–90 ng TCDD/kg moist weight of food(Table 1). The food contained a background con-centration of 24 ng/kg TCDD-EQ as determinedby the H4IIE bioassay (Sanderson et al., 1996) ofwhich less than 0.2 ng/kg was 2,3,7,8-TCDD. Theother compounds that contributed to the Ah-Ractivity were unknown, but could have been poly-chlorinated biphenyls (PCBs), polycyclic aromatichydrocarbons or short-lived ‘natural’ inducers.The quantity of food fed was adjusted throughoutthe experiment to maintain a constant ration of1.5% of body weight per day.

The experiment was initiated with 35 females ineach of the four exposure groups (three TCDDexposure concentrations and one control). Two tofour fish from each of the three treatment andcontrol groups, were evaluated for clinical pathol-ogy alterations and gross lesions after 50, 100,150, or 200 days of exposure. Additional fish werecollected and evaluated at various time points atthe time of spawning from day 255 to 320, anddata from these fish have been grouped togetheras a 250+ -day treatment group.

Fish were anesthetized by submersion in MS-222 (tricaine methane sulfonate). Blood was col-lected by venipuncture of the caudal vein andplaced on ice. Samples were collected in EDTA(ethylene diamine tetraacetic acid) for completeblood counts and without anticoagulant forserum chemistry evaluation. Clinical pathologytests were performed on the same day as bloodcollection. A 1:200 dilution in modified Dacie’ssolution was used to determine leukocyte countsby previously described methods (Blaxhall andDaisley, 1973; Campbell, 1988b). Erythrocytecounts and hemoglobin determinations were doneby voltage impedance (Counter ZBI) by previ-ously described methods (Campbell, 1988a). Leu-cocytes were classified by microscopic evaluationof Wright’s stained blood smears. Serum fromtwo fish was typically pooled to provide adequatesample volume for analysis. Serum chemistryevaluation included sodium, potassium, total car-bon dioxide, anion gap, iron, albumen, alkalinephosphatase, amylase, total bilirubin, urea nitro-gen, calcium, cholesterol, creatine kinase, crea-tinine, gamma glutamyl transferase, glucose,magnesium, phosphorus, sorbitol dehydrogenase,aspartate aminotranserase, alanine aminotrans-ferase, total protein and osmolality. Tests wereperformed on a tandem access analyzer (AbbottSpectrum) using reagents supplied by the manu-facturer. Tests were not modified from standardmethodologies used for mammals. Enzyme analy-sis was performed at 37°C. Osmolality was deter-mined by freezing point depression.

Table 1Concentrations of 2,3,7,8-TCDD, TCDD-EQs and radiometric doses in food

Dose group Control ng TCDD/kg diet

901.8 18

5.4B0.2 27054TCDD in food (ng/kg)Naa 541Specific activity (DPM/pg TCDD) 57.0 10.5

9491026974NaRadiometric dose (DPM/g food)24 28.9Bioassay (ng TCDD/kg food)b 46.9 154

TCDD only dose 0 22.94.9 129.8

a Na, Not applicable.b Background concentrations of dioxin like activity were measured using the H4IIE bioassay. The presence of this activity is

discussed in Section 2.


Fig. 1. Accumulation of TCDD in the livers of rainbow troutexposed to TCDD for up to 200 days. Values are mean of eachtreatment group; solid=1.8 ng TCDD/kg; dashed=18 ngTCDD/kg; dotted 90 ng TCDD/kg. Error bars indicate onestandard error of the mean. As the control group was notexposed to 3H-TCDD no values are available.

but one fish and samples of ovary were collectedfrom most fish. Following routine processing andparaffin embedding, 6-mm sections were preparedand stained with hematoxylin and eosin. PeriodicAcid Schiff (PAS) staining was also performed onsome sections. Incidence and severity of lesionswere enumerated and the values for treatmentgroups were compared to controls at each timeinterval.

3. Results

There were no statistically significant differ-ences in growth among treatment groups after 200days of exposure. TCDD was rapidly accumu-lated in the livers of TCDD-treated fish andreached a steady state after 50 days of exposure(Fig. 1 and Table 2). There was an apparent butnon-significant decrease in liver TCDD concentra-tion in all TCDD-treatment groups at 150 days.However liver concentrations increased again be-tween 150 and 200 days to reach levels equivalentto those observed at 100 days of exposure. Thisdecrease may represent a mobilization and trans-fer of lipid and associated TCDD to the develop-ing gonads. Relatively uniform increases in theovary/somatic index were noted over the entireexposure period (results not shown) so alterationsin TCDD distribution would indicate a specificphase of egg development. Details about tissueaccumulation and distribution of TCDD in thisexperiment are provided elsewhere (Giesy et al.,2000; Jones et al., 2000).

Anaesthetized fish were killed by concussionfollowed by cervical spinal cord transection. Liverand ovaries were removed and weighed, grosslesions were noted and samples for histopatholog-ical evaluation were taken within 5 min of deathand placed in 10% neutral buffered formalin.Samples of liver, midsection kidney, hematopoi-etic tissue, gill, and stomach were collected fromeach fish. Samples of spleen were obtained in all

Table 2Concentrations of 2,3,7,8-TCDD (pg/g wet weight) accumulated in rainbow trout livers during the 200-day feeding perioda

Interval (days) ng TCDD/kg dietControl

1.8 9018

1.65 (0.57)0.255 (0.06)Nab50 –0.312 (0.051)100 2.875 (0.72)Na 12.92 (2.75)

150 Na 0.218 (0.083) 1.717 (0.56) 9.933 (0.528)Na200 – 2.85 (0.639) 16.242 (2.21)

a Values are mean (S.D.).b Na, not applicable.


3.1. Gross pathology

The only consistent gross external lesion wasnecrosis of the caudal and occasionally anal fins.The incidence of fin lesions was slightly greater intreated groups than in the controls at the sametime intervals (Table 3) but this response was notdose dependent. Lesions were characterized histo-pathologically as epithelial erosion with mild sub-jacent fibrosis and occasional necrosis. The degreeof severity of lesions was not dose dependentsuggesting that these lesions were probably due toconfinement in the tanks.

3.2. Clinical pathology

No TCDD-related alterations in any serumchemistry parameters or in erythrocyte parame-ters were observed at any exposure time (resultsnot shown). The total number of leukocytes wasdecreased in the 90-ng TCDD/kg treatment dosegroup fish when compared to controls for samplesalthough there was not a clear dose-response rela-tionship across dose groups.

3.3. Histopathology

Inflammation was frequently present and ofteninvolved the peritoneal serosal or capsular sur-faces of the stomach, spleen and liver, the submu-cosa, muscularis and subserosa of the stomach,the ovary, and the parenchyma of the liver. Infl-ammatory infiltrates generally consisted of mixedmononuclear cells (lymphocytes, plasma cells,macrophages). Occasionally fibroblasts and neo-vascularization were increased on the stromal sur-

faces. These changes were not dose-related andoccurred with similar frequency among all treat-ment groups. Specific etiologic agent(s) were notidentified. Other organ-specific changes are ad-dressed individually.

3.4. Li6er

There were dose-related hepatocellular changesin all TCDD treated groups but not in untreatedfish (Table 4 and Fig. 2). The livers of control fishwere characterized by uniform small hepatocyteswith small, oval monomorphic nuclei, which hadevenly dispersed finely granular chromatin andrare mitotic figures. In the livers of fish exposed to18 or 90 ng TCDD/kg exposed fish, there wereincreased mitotic figures and nuclear changes, in-cluding variably enlarged nuclei (anisokaryosis),prominent nucleoli, marginated and/or coarselyclumped chromatin and vesiculation (Fig. 3). An-isocytosis due to increased hepatocellular size wasalso observed in 18 and 90 ng/kg treatmentgroups when compared to controls. Cells withyellow–brown, slightly refractile cytoplasmic pig-ment, suggestive of bile, were noted in the 18- and90-ng/kg TCDD treatment groups at 200 and250+ days (data not shown). Individual cell ne-crosis was occasionally observed in the livers offish exposed to 18 or 90 ng TCDD/kg (Table 4).Dose-dependent decreases in hepatocellular glyco-gen were also observed in all TCDD-treatedgroups. Hepatocytes of control fish containedmoderate to abundant glycogen in irregular, fibril-lar intracytoplasmic vacuoles which were stronglyPAS positive (Fig. 3). Hepatocellular vacuoliza-tion was less intense and cytoplasm was moredensely eosinophilic in TCDD-exposed fish (Fig.3). There was little PAS stain affinity in TCDD-treated fish. Sharply-outlined, PAS negative, clearvacuoles, consistent with lipid, were present insome fish in the 18- and 90-ng TCDD/kg treat-ment groups but were not seen in untreated fish.There were periportal, mixed mononuclear infl-ammatory infiltrates and capsulitis/peritonitis infish from control and treatment groups; therefore,these were not considered to be treatment related(Fig. 4). Thus the lowest observable adverse effectlevel (LOAEL) for these effects was 5.69 ng

Table 3Incidence (lesions/number of fish examined) of fin lesions(gross) in rainbow trout exposed to TCDD

Exposure time (days) ng TCDD/kg dietControl

18 901.8

0/4 2/450 1/3 0/4100 0/4 4/4 2/33/4

2/42/4 3/3150 2/40/2200 1/31/6 0/4


Table 4Incidence of liver lesions (lesions/number of fish examined) in rainbow trout exposed to TCDD

Exposure time ControlParameter ng TCDD/kg diet(days)

1.8 18 90

Glycogen decreased 0/3100 2/4 1/4 3/40/4 1/4150 4/4 2/3

200 1/4 2/4 0/4 2/3250+ 0/2 0/6 2/4 4/5

0/3 1/4Lipid-like vacuoles 1/4100 2/40/4150 2/4 3/4 2/30/4 0/4200 0/4 0/3

250+ 0/2 0/6 0/4 1/5

Increased mitotic figures 100 0/3 0/4 2/4 (1.5/10 hpf)a 3/4 (5/10 hpf)a

0/4 0/4150 3/4 (2/10 hpf)a 2/3 (3/10 hpf)a

1/4 (1/10 hpf)a 0/4 3/4 (1.7/10 hpf)a200 2/3 (5/10 hpf)a

1/4 (2/10 hpf)a 0/4250 0/5

Nuclear or cellular change 100 0/3 0/4 1/4 4/4150 0/4 0/4 4/4 3/3

0/4 3/4200 1/4 2/30/2 3/6 2/4250+ 4/5

0/3 0/4Individual cell necrosis 0/4100 1/4150 0/4 0/4 0/4 0/3200 0/4 0/4 0/4 1/3

0/2 0/6 2/4250+ 0/5

0/3 1/4Peritonitis liver 0/4100 1/41/4 0/4150 0/4 0/3

200 1/4 0/4 1/4 0/3250+ 0/2 0/6 0/4 0/5

1/3 1/4Inflammatory infiltrates 0/4100 1/41/4 1/4 1/4 2/31503/4 1/4200 1/4 2/30/2 0/6 2/4 1/5250+

100 3/3Inflammation/peritonitis 2/4 1/4 4/4abdominal, not liver

4/4 1/4150 1/4 0/33/4 0/4 2/4200 1/31/2 3/6 1/4250+ 2/5

a Mean number of mitotic figures per number of high power fields (hpf).

TCDD/kg in diet (geometric mean of 1.8 and 18 ngTCDD/kg) and 0.90 ng TCDD/kg liver (geometricmean of 0.285 and 2.85 ng TCDD/kg) based onmeasured liver concentrations at 200 days.

3.5. Spleen

There was a decrease in lymphoid density in allfish exposed to TCDD-treatments at all time

points. Occasionally the lymphoid tissue of un-treated fish was scant but was never as little as theTCDD-treated fish (Fig. 5). This lesser densitywas usually associated with an apparent increasein stromal and vascular structures and oftenvascular and/or sinus congestion. Occasional fociof individual cell necrosis and hemosiderosis werealso observed in the 18- and 90-ng TCDD/kgtreatment groups. Some fish in the TCDD treat-


ment groups were also characterized by multiplefoci with clusters of small endothelial-lined vascu-lar structures, which contained small amounts ofintraluminal eosinophilic fibrillar material or ery-throcytes (Fig. 6). These foci were often adjacentto large or moderately-sized blood vessels.

Inflammation occurred with similar frequencyin treatment and control groups and was notconsidered to be treatment related. Etiologicagent(s) were not identified. The inflammationwas characterized by the infiltration of mononu-clear cells into the splenic capsule with neovascu-larization and fibrous adhesions in some fish.Extension of this reaction through the capsuleinto the subcapsular parenchyma was uncommon.

3.6. Stomach

No dose-related toxicological effects on thestomach were observed. The most frequently ob-served gastric lesions included chronic inflamma-tion of the serosa with lymphocyte, mononuclearcell, and occasional plasma cell infiltration. Thisoften involved the neural, perineural and perivas-cular tissues with variable amounts of capsularthickening due to fibrovascular proliferation. In

Fig. 3. (A, B) Liver from an untreated fish (panel A) from afish fed 90 ng TCDD/kg food for 100 days (Panel B). There isabundant cytoplasmic glycogen, which appears as an emptyspace within the hepatocytes (straight arrow Panel A). Nucleiare uniform in size with fine granular chromatin (curvedarrow, panel A). There is decreased hepatocellular glycogen,slight variation in nuclear size and chromatin, and increasednumbers of mitotic figures (curved arrows, Panel B). Scale baron both panels represents 25 mm.

Fig. 2. Occurrence of histopathological lesions in the livers oftrout exposed to TCDD for up to 250+ days.

many samples, this reaction extended to themesentery, and in a few cases extended into themuscularis. This inflammation was observed in


untreated as well as all TCDD-treated groups.Infectious agents were not identified and the etiol-ogy could not be determined. Multifocal mineral-ization, usually involving the submucosa and

Fig. 5. (A, B) Spleen from an unexposed fish (Panel A) after100 days and from a fish fed 90 ng TCDD/kg food for 150days (Panel B). In the untreated fish there is a mixed popula-tion of lymphoid cells (arrow, Panel A), stromal cells (openarrowhead), erythrocytes (solid arrowhead), and occasionalmelanomacrophages (curved arrow, Panel A). In the exposedfish there are increased numbers of melanomacrophages(curved arrows, Panel B), stromal cells (open arrowhead), anderythrocytes (solid arrowhead). Lymphoid cells (arrow, PanelB) are decreased in number. Scale bar on panel A is 50 mm.Scale bar on panel B is 25 mm.

Fig. 4. (A, B) Liver from a fish fed 90 ng TCDD/kg food for50 days. There is prominent hepatocellular anisocytosis andanisokaryosis. Note variation in nuclear chromatin patternsand sharply outline vacuoles consistent with lipid (open arrow-head, Panel B). Infiltrates of lymphocytes (small arrow, PanelB) are present in the portal areas. Bile duct (curved arrow,Panel B), artery (large arrow), vein (solid arrowhead, Panel B).Scale bar on panel A is 25 mm. Scale bar on panel B is 50 mm.


occasionally the subserosa and/or muscularis, of-ten between inner and outer muscle layers, wasnoted in all treatment groups. In most cases,granulomatous inflammatory infiltrates were asso-ciated with the mineralized foci. Similar foci of

inflammation also occurred without evidence ofmineralization. Less common lesions includedlymphocytic or lymphohistocytic infiltration ofthe cardiac glands with loss or effacement ofglandular epithelium. Vacuolization, clefting andindividual cell degeneration of the submucosalbasilar zone was noted in many stomach speci-mens but was considered to be an artifact ofsample preparation.

3.7. Peritoneum

Peritonitis occurred in all fish from control andTCDD-treated groups with similar frequency andwas defined as the presence of inflammatory cellinfiltrates or fibrous to fibrovascular adhesions onthe serosal surfaces of one or more abdominalorgans or in the mesentery. Inflammatory cells inall cases were primarily macrophages with fewernumbers of lymphocytes and plasma cells, withvariable numbers of fibroblasts and fibrocytes.Etiologic agents were not identified.

3.8. Gill

Gill lesions were infrequent, mild in severityand occurred randomly between groups. Lesionsmost frequently noted were focal chronic granulo-mas, occasional blunting and fusion of secondarylamellae or lymphocytic infiltrates. Infectiousagents were not identified.

3.9. Hematopoietic tissue

Variation in numbers of melanomacrophageswas observed but could not be associated withany dose group or time interval. Despite consis-tent differences between total numbers of erythro-cytes or leukocytes in fish from the control and90-ng TCDD/kg dose group fish, histopathologi-cal alterations in hematopoietic tissue were rarelyobserved. Plasma cell infiltrates with adjacent fociof hemosiderosis were present in one control fishat 250+ exposure, and non-suppurative neuritis/ganglioneuritis was present in one fish fed 18 ngTCDD/kg in the diet for 150 days.

Fig. 6. (A, B) Spleen from a fish fed 90 ng TCDD/kg food for250 days. There are multiple foci of endothelial-lined vascularstructures (curved arrows, Panel A). Lymphoid density isdecreased. Erythrocytes (straight arrow, Panel B) andmelanomacrophages (open arrowheads, Panel B) are present.Scale bar on panel A is 50 mm. Scale bar on Panel B is 25 mm.


3.10. Kidney

Renal lesions were infrequent. Variations in thenumber of melanomacrophages were noted butwere not treatment or dose dependent. Intenselyeosinophilic droplets were observed in the renaltubular epithelium of a majority of the fish andwere not unique to any particular treatmentgroup.

3.11. O6ary

Ovaries generally contained eggs in varyingstages of development (primary and secondaryoocytes). The proportion of primary to secondaryoocytes varied among fish but was not associatedwith any dose group, time interval or other histo-pathological alteration. Occasionally, degenera-tion of oocytes was observed and, in some cases,was associated with mononuclear inflammation.In some, but not all cases, peritonitis and ovarianinflammation were both present. One fish fromthe 90-ng TCDD/kg treatment group (150 daysexposure) contained both ovarian and testiculartissue. This fish had ambiguous gonads on grossexamination.

3.12. Other tissues

Thymic and thyroid tissues were not obtainedfrom all fish; therefore definitive conclusions can-not be made. However, no apparent treatment-re-lated alterations were observed. Thyroid folliclesvaried in both size and height of follicular epithe-lium. Lymphocytic neuritis and perineuritis wasnoted in pancreatic tissue of one fish exposed to18 ng TCDD/kg after 100 days exposure. Ade-nomatous hyperplasia with multifocal interstitialmineralized concretions of the Corpuscle of Stan-nius was noted in one fish exposed to 1.8 ngTCDD/kg at 200 days exposure. Cardiac lesionswere limited to mixed mononuclear inflammatoryinfiltrates on the serosal (pericardial) surface ofone control fish at 150 days and within the adja-cent pericardial adipose tissue of one 18-ngTCDD/kg treatment group fish after 200 daysexposure. Histologic alterations were not iden-tified in interrenal, chromaffin, or ocular tissues.

4. Discussion

Liver vacuolization has been reported to bevariable in salmonids with glycogen deposits caus-ing variable distention of hepatocellular cyto-plasm (Yasutake and Wales, 1983). Two patternsof hepatocellular vacuolization were noted in thepresent study. Indistinct fibrillar vacuolizationwith varying prominence was associated with PASpositive stain reaction and was considered to beconsistent with glycogen. This vacuolization pat-tern was most consistently observed in untreatedfish and fish fed 1.8 ng TCDD/kg. Clear, sharply-outlined hepatocellular vacuolization was ob-served in the livers of some fish fed 18 and 90 ngTCDD/kg. The localization, appearance and lackof affinity for PAS suggest that the material waslipid. Although these patterns of hepatocellularvacuolization were not exclusive to specific treat-ment or control groups the trend suggests that, inthis study, vacuolar patterns were altered by ad-ministration of TCDD. Other treatment-relatedhepatic alterations were consistently found andincluded a variation in nuclear size and chromatinpattern, mild variation in hepatocyte size and/orincreased numbers of mitotic figures. These resultsare consistent with two effects associated withTCDD toxicity in mammals, wasting syndrome(Rozman, 1984) and fatty liver.

The hepatocellular effects of TCDD exposureon fish have been documented in previous studies(Fisk et al., 1997; Tietge et al., 1998). However,unlike our study, previous studies exposed eggs,fry or juvenile fish to waterborne TCDD or in-traperitoneal (i.p.) injection. Sinusoidal dilatation,hepatocellular swelling and necrosis, nuclear chro-matin margination, decreased glycogen and cyto-plasmic inclusions were observed followingexposure of eggs, yolk sac fry and juvenile rain-bow or lake trout to varying concentrations ofTCDD in water for 48–96 h (Helder, 1981; Spits-bergen et al., 1991). Juvenile rainbow trout ex-posed to a single i.p. injection of TCDD in dosesfrom 0.1 to 125 mg/kg (Spitsbergen et al., 1988)exhibited hepatic lesions characterized by bileduct hyperplasia and the presence of intracyto-plasmic eosinophilic inclusions. The incidence andseverity of both of these lesions varied among


strains. The absence of bile duct hyperplasia andhepatocellular eosinophilic inclusions in this studymay have been due to a number of factors, in-cluding strain of trout, lesser doses, age of fish atthe time of study, route of administration orpossible repair of lesions before the first histo-pathologic evaluation at day 100. Six weeks fol-lowing a single i.p. injection of either 0.06 or 3.06mg TCDD/kg, livers of juvenile rainbow troutcontained less glycogen compared to unexposedfish (van der Weiden et al., 1992). Livers of theexposed fish also exhibited hepatocellularswelling, hydropic degeneration, vacuolization,single cell necrosis and occasional focal necrosiswith mononuclear inflammation. Twelve weeksafter the single i.p. injection liver glycogen contenthad increased, and there was an increased numberof mitotic figures in the 3.06-mg/kg dose group(van der Weiden et al., 1992). In the currentstudy, mild mononuclear inflammatory infiltrateswere present in the liver to a similar degree in alltreatment groups. A number of fish exposed to 18and 90 ng TCDD/kg had livers which containedcells with yellow–brown slightly refractile gran-ules in the cytoplasm. These granules were sugges-tive of bile, although staining techniques to ruleout iron and hemosiderin were not conducted.Dietary exposure of fingerling rainbow trout to494 ng/kg TCDD for 91 days did not result in anydetectable gross lesions, although histologic exam-ination was not performed (Kleeman et al., 1986).This exposure was of shorter duration than ourstudy, but to a greater TCDD concentration thaneven the greatest TCDD concentration in ourstudy.

In the present study, multifocal gastric mineral-ization with associated granulomatous inflamma-tion was observed in some fish from every group,including controls. The stomach alterations ob-served are most consistent with mineralized fociof inflammation defined as visceral granulomas(Ferguson, 1988; Roberts, 1989) and were notconsidered to be related to TCDD exposure.Dose-related gastric lesions noted in previousstudies of trout include cardiac gland necrosis(Helder, 1981), degeneration of enterocytes (Spits-bergen et al., 1991) and atrophy or hyperplasia ofserous gastric glands which varied between hatch-ery strains (Spitsbergen et al., 1988).

Lymphoid involution of the thymus and spleenand hypocellularity of the hematopoietic tissuehave been reported following i.p. injection of ju-venile rainbow trout with 10 mg TCDD/kg (Spits-bergen et al., 1988). Thymic tissue was collectedand examined microscopically from a number ofthe fish in the present study. The amount oflymphoid tissue varied among fish but this wasattributed, in most cases, to sampling variation.Due to the size and location, piscine thymic tissueis a difficult organ to consistently sample andevaluate, especially in adults. Thymic tissues weredifficult to identify and age-related thymic involu-tion had most likely already occurred in these fish.

Increases in numbers of melanomacrophages,lymphoid depletion, and increased splenic ery-throcytes have previously been reported followinga single i.p. injection of 0.27–2.93 mg TCDD/kgin juvenile mirror carp (Cyrinus carpio ; van derWeiden et al., 1993). Melanomacrophages areconsidered to be repositories of end products ofcell breakdown, such as phospholipids, erythro-cytes, particulate matter and antigens (Ferguson,1988). In the spleens of TCDD-exposed fish, therewas evidence of increased degeneration oflymphocytes, and possibly erythrocytes whichwould lead to an increased need for functionalmacrophages to phagocytoze cellular debris. Thiscould account for both increased numbers ofmelanomacrophages and macrophages with yel-low–brown intracytoplasmic globular material,interpreted as hemosiderin.

The presence of multiple foci of endothelial-lined vascular structures were present in a fewTCDD-exposed fish at the 250+ time period.The significance of this observation is unknown,although it is possible that these structures mightrepresent vascular or lymphatic channels, whichformed or opened secondary to congestion orimpaired circulation.

In the present study, decreases in spleniclymphoid density observed in TCDD-treated fishwere accompanied by prominent splenic stroma,sinuses, vasculature and blood content. However,this may have been due to the presence of fewerlymphoid cells. The somewhat variable nature ofthe lymphoid depletion and lack of histopatho-logic changes in hematopoietic tissue observed in


this study, as compared to more severe lesionsobserved in previous studies, may be due to differ-ences in strain and age of fish, dose and route ofadministration of TCDD, duration of study andsampling times. Unlike mammalian bone marrow,hematopoietic tissue of fish has no bony spiculesor adipocytes with which to judge cellularity bycomparing the proportion of cells to fat. This lackof stromal support also causes fish hematopoietictissue to collapse when sectioned, which can maskmild to moderate changes in cellularity. Subtlechanges may be more easily distinguished in plas-tic-embedded thin sections (Spitsbergen et al.,1988), than in paraffin-embedded specimens asdone in this study.

Acknowledgements

This research was supported by a grant fromthe Biology Panel of the Exploratory ResearchProgram on the US-EPA (R814850). The assis-tance of R. Crawford, L. Williams, D. Tillitt, J.Newsted, K. Henkel, and W. Lawrence aregreatly appreciated. Dr S. Sleight of the Depart-ment of Pathology reviewed all of the histopathol-ogy conclusions.

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