Hepatic P450 Enzyme Activity, Tissue Morphology and Histology of Mink (Mustela vison) Exposed to Polychlorinated Dibenzofurans

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1 Arh Environ Contm Toxiol (9) 57: DOI 1.17/s Hepti P45 Enzyme Ativity, Tissue Morphology nd Histology of Mink (Mustel vison) Exposed to Polyhlorinted Dienzofurns Jeremy N. Moore Æ John L. Newsted Æ Mrkus Heker Æ Mtthew J. Zwiernik Æ Sott D. Fitzgerld Æ Denise P. Ky Æ Xiowei Zhng Æ Eri B. Higley Æ Les L. Aylwrd Æ Kerrie J. Bekett Æ Roert A. Budinsky Æ Steven J. Bursin Æ John P. Giesy Reeived: 22 April 8 / Aepted: 8 Septemer 8 / Pulished online: 21 My 9 Ó The Author(s) 9. This rtile is pulished with open ess t Springerlink.om Astrt Dose- nd time-dependent effets of environmentlly relevnt onentrtions of 2,3,7,8-tetrhlorodienzop-dioxin equivlents (TEQ) of 2,3,7,8-tetrhlorodienzofurn J. N. Moore (&) M. J. Zwiernik X. Zhng E. B. Higley J. P. Giesy Deprtment of Zoology, Ntionl Food Sfety nd Toxiology Center, Mihign Stte University, Est Lnsing, MI 48824, USA e-mil: moorej42@msu.edu M. J. Zwiernik e-mil: zwiernik@msu.edu X. Zhng e-mil: howrd5325@yhoo.om E. B. Higley e-mil: higleyer@msu.edu J. N. Moore J. L. Newsted M. J. Zwiernik S. J. Bursin Deprtment of Animl Siene, Center for Integrtive Toxiology, Mihign Stte University, Est Lnsing, MI 48824, USA J. L. Newsted e-mil: jnewsted@entrix.om S. J. Bursin emil: ursin@msu.edu J. L. Newsted D. P. Ky ENTRIX, In., Okemos, MI 48823, USA D. P. Ky e-mil: dky@entrix.om M. Heker J. P. Giesy Deprtment of Biomedil Veterinry Sienes nd Toxiology Centre, University of Sskthewn, Ssktoon, SK, Cnd S7N 5B3 (TCDF), 2,3,4,7,8-penthlorodienzofurn (PeCDF), or mixture of these two ongeners on hepti P45 enzyme tivity nd tissue morphology, inluding jw histology, of M. Heker ENTRIX, In., Ssktoon, SK S7N 5B3, Cnd e-mil: mheker@entrix.om S. D. Fitzgerld Deprtment of Pthoiology nd Dignosti Investigtion, Dignosti Center for Popultion nd Animl Helth, Mihign Stte University, Est Lnsing, MI 48824, USA e-mil: Fitzgerld@dph.msu.edu L. L. Aylwrd Summit Toxiology, Flls Churh, VA 2244, USA e-mil: lylwrd@summittoxiology.om K. J. Bekett Stnte Consulting Servies, In., Topshm, ME 486, USA e-mil: kerrie.ekett@stnte.om R. A. Budinsky The Dow Chemil Compny, Midlnd, MI 48642, USA e-mil: rudinsky@dow.om J. P. Giesy Deprtment of Biology nd Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR Chin emil: jgiesy@ol.om J. P. Giesy Shool of Environment, Nnjing University, Nnjing 2193, Chin

2 Arh Environ Contm Toxiol (9) 57: dult rnh mink were determined under ontrolled onditions. Adult femle rnh mink were fed either TCDF (.98, 3.8, or 2 ng TEQ TCDF /kg w/dy) or PeCDF (.62, 2.2, or 9.5 ng TEQ PeCDF /kg w/dy), or mixture of TCDF nd PeCDF (4.1 ng TEQ TCDF /kg w/dy nd 2.8 ng TEQ PeCDF /kg w/dy, respetively) for 18 dys. Doses used in this study were pproximtely eight times greter thn those reported in prllel field study. Ativities of the ytohrome P45 1A enzymes, ethoxyresorufin O-deethylse (EROD) nd methoxyresorufin O-deethylse (MROD) were signifintly greter in livers of mink exposed to TCDF, PeCDF, nd mixture of the two ongeners; however, there were no signifint histologil or morphologil effets oserved. It ws determined tht EROD nd MROD tivity n e used s sensitive iomrkers of exposure to PeCDF nd TCDF in dult femle mink; however, under the onditions of this study, the response of EROD/MROD indution ourred t doses tht were less thn those required to use histologil or morphologil hnges. Reently, there hs een onern out the onentrtions of polyhlorinted dienzofurns (PCDFs), polyhlorinted dienzo-p-dioxins (PCDDs), nd polyhlorinted iphenyls (PCBs) in floodplin soil nd sediment from the Tittwssee River (Hilsherov et l. 3). The Tittwssee River flows into the Sginw River nd Sginw By, Mihign, USA s prt of the Lke Huron wtershed. Both field nd lortory-sed studies hve een onduted to ssess the potentil risks of these onentrtions of PCDD, PCDF, nd PCBs on terrestril nd quti orgnisms (Zwiernik et l. 8). The mink (Mustel vison) hs een utilized s sentinel speies for eologil risk ssessments t sites where ontminnts of onern re hemils tht n ind to the romti hydroron reeptor (AhR) suh s 2,3,7,8-tetrhlorodienzo-p-dioxin (TCDD) nd struturlly similr ompounds (Blnkenship et l. 8; Bsu et l. 7; Giesy et l. 1994; Tillitt et l. 1996). The mink is onsidered to e mong the more sensitive mmmls to TCDD nd relted ompounds (Bekett et l. 8; Hohstein et l. 1988, 1998). Mink hve reltively gret potentil for exposure to these persistent, ioumultive hemils (Bsu et l. 7). An eologil risk ssessment using previously estlished toxiity referene vlues (TRVs) derived primrily from studies of the effets of TCDD nd other AhR-tive ompounds on mink (Blnkenship et l. 8) nd onentrtions of TCDD equivlents (TEQs) in the diet nd tissues of mink inhiting the Tittwssee River hs een onduted (Zwiernik et l. 8). This study indited tht mink might e t risk of eing dversely ffeted y these ompounds, with hzrd quotients etween \1 nd 1 eing lulted. However, despite umulting reltively gret onentrtions of 2,3,4,7,8-penthlorodienzofurn (PeCDF) nd 2,3,7,8-tetrhlorodienzofurn (TCDF) in their livers, the ondition of individul mink from the more highly ontminted res of the Tittwssee River ws omprle or superior to tht of mink olleted in referene res nd the popultion ws roust (Zwiernik et l. 8). The inonsisteny etween the pprent helthy popultion nd the elevted hzrd quotient (HQ) estimtes might e due to severl ftors, inluding the following: (1) The World Helth Orgniztion (WHO) toxi equivlent ftor (TEF) vlues nd resulting TEQ onentrtions re onservtive nd might hve overestimted risk; (2) the toxiity referene vlues (TRVs) used to estimte the HQs might not hve een urte for mink due to lk of toxiologil informtion for the dominnt PCDF ongeners identified in mink t the site reltive to dt ville in the literture to derive TEFs; nd (3) uptke rtes, metolism, exretion, nd disposition of TCDF nd PeCDF might differ from TCDD or PCBs tht hve een studied in mink (Bekett et l. 8; Zwiernik et l. 8). A 18-dy dietry study ws onduted to (1) determine rtes of ssimiltion nd distriution of environmentlly relevnt doses of TCDF, PeCDF or omintion of the two ongeners in liver tissue of mink (Zwiernik et l. 8) nd (2) exmine the reltionship etween hemil exposure nd hepti ytohrome P451A enzyme tivities, potentil funtionl inditors of exposure to AhR gonists (Hhn 1998; Kwjiri nd Fujii-Kuriym 7; Whitlok 1999). Ethoxyresorufin O-deethylse (EROD) tivity is most diretly ssoited with the indution of hepti tivity of the ytohrome P451A1 enzymes, wheres methoxyresorufin O-deethylse (MROD) tivity is more ssoited with P451A2 enzymes. However, lthough oth enzymes n metolize either sustrte to some extent, the metolism of oth sustrtes provides vlule informtion s to P451A tivity in n orgnism reltive to its exposure to xenoiotis. The study ws onduted to lso (3) exmine reltionships etween EROD nd MROD tivity in liver to other morphologil nd histologil hnges in mink. This rtile presents the results of the effets of TCDF nd PeCDF on hepti EROD nd MROD tivities nd seleted morphologil nd histologil prmeters in mink. Mteril nd Methods Mink Husndry, Exposure, nd Neropsy Adult, femle, rnh mink were rndomly ssigned nd housed individully in wire-mesh reeder ges (61 m L 9 76 m W 9 46 m H) with wooden nest oxes

3 418 Arh Environ Contm Toxiol (9) 57: Tle 1 Dily dose nd onentrtions of TCDF nd/or PeCDF in the liver of mink (Mustel vison) Tretment Dily dose Liver onentrtion (ng TEQ/kg, ww) (ng TEQ/kg w/d) dy Men of 9 nd 18 dys Control TCDF \LOD \LOD \LOD PeCDF \LOD \LOD \LOD TCDF.98 NA 1.2 ± NA 2.3 ±.22 2 NA 7.1 ± 1.1 PeCDF.62 NA 52 ± NA 27 ± NA 16 ± 53 Mixture of TCDF 4.1 NA 1.4 ±.24 PeCDF 2.8 NA 36 ± 8 Note: Eh tretment group hd six mink nd the ontrol group hd eight mink. Control nimls were smpled t, 9, nd 18 dys; three treted nimls per single ongener dose group were smpled t 9 nd 18 dys. Two mink livers in the mixture group were nlyzed t 9 dys nd three mink livers were nlyzed t 18 dys. All onentrtions were onverted to 2,3,7,8-TCDD equivlents (TEQs) using TEFs of.3 for PeCDF nd.1 for TCDF (Vn den Berg et l. 6). NA indites tht smples were not olleted Liver onentrtions re presented s men ± 1SD LOD =.1 ng TEQ/kg, ww LOD =.1 ng TEQ/kg, ww (3 m L m W 9 25 m H) within n indoor fility t Mihign Stte University (MSU). A totl of 5 femle mink were distriuted mong 8 tretments with 6 individuls in eh of 7 furn-dosed groups (3 TCDF groups, 3 PeCDF groups, nd 1 TCDF plus PeCDF group) nd 8 femle mink in the ontrol group. Doses were expressed s TEQs (Tle 1) lulted y use of TEFs reported y Vn den Berg et l. (6). The test hemil for eh tretment ws dissolved in hexne to produe stok solution, nd liquots of the stok were then diluted ppropritely with 1 ml orn oil. The orn oil ontining the test hemil ws dded to the wter omponent of the mink diet nd mixed well in pddle mixer prior to the ddition of the other feed ingredients. After the ddition of ll of the dietry ingredients, the feed ws mixed for n dditionl 2 min. Eh morning for 18 dys, 25 g of feed ontining the furn ongener(s) were given to eh niml. This proedure ensured omplete ingestion of the ontminted feed, eliminting the need to mesure dily feed onsumption in order to estimte doses. After this feed ws onsumed, n dditionl 1 g of unontminted feed were given to eh niml. Wter ws provided d liitum. Full-spetrum lighting ontrolled y timer simulted the nturl light/drk yle for the Estern Stndrd Time Zone. Temperture ws mintined etween 13 C nd 28 C nd humidity rnged from 26% to 91%. Mink were oserved dily for signs of toxiity inluding derese in feed onsumption nd lethrgy. Individul ody msses (g) were mesured t the eginning of the study (Jnury 31, 6) nd every 3 dys therefter. Three nimls from the ontrol group were euthnized y sphyxition with ron dioxide t initition of the exposure ( dy) nd three nimls from eh of the eight tretment groups were euthnized t 9 nd 18 dys of exposure for susequent neropsy. Body mss (g) nd length (m), inluding nd exluding the til, were reorded for eh femle mink. Mink were exmined externlly nd internlly for overll ondition, nutritionl sttus, nd the presene of gross normlities. Livers were removed nd weighed. Susmples of the liver were frozen in liquid nitrogen for susequent mesurement of EROD nd MROD tivities. Approximtely 2. g of liver tissue ws pled in 1% formlin sline solution (1% formlin in.9% sodium hloride) for histologil exmintion. The remining liver ws pled in I-Chem Ò jrs (I-Chem, New Cstle, DE, USA) nd frozen t -2 C for susequent determintion of TCDF nd PeCDF onentrtions using high-resolution gs hromtogrphy/mss spetrometry (HR-GC/MS). In ddition, the spleen, kidneys, thymus, mesenteri lymph node, nd rin were removed nd preserved for susequent histologil exmintion. The hed ws pled in formlin sline solution for susequent histologil exmintion of mndiulr nd mxillry squmous epithelil ell prolifertion s desried y Bekett et l. (5). The lesion ws grded s mild, moderte, or severe sed on the numer nd size of foi of squmous ell prolifertion in the mxill nd mndile (Bekett et l. 5). The MSU Institutionl Animl Cre nd Use Committee pproved this study (AUF 12/5-165-). Chemils nd Regents Both PeCDF nd TCDF were otined from Austndrd In. (New Hven, CT, USA) nd dissolved in hexne to produe stok solution. Working solutions nd dilutions of PeCDF nd TCDF were prepred in pestiide residue nlysis-grde OmniSolv n-hexne (EM Siene, Lwrene, KS, USA). For iohemil nlyses, 7-ethoxyresorufin (7-ER) ws otined from Moleulr Proes (Eugene, OR, USA) nd 7-methoxyresorufin (7-MR) nd resorufin were otined from Sigm-Aldrih (St. Louis, MO, USA). All other iohemil regents, inluding NADPH, were otined from Sigm-Aldrih nd were regent grde or etter unless stted otherwise.

4 Arh Environ Contm Toxiol (9) 57: EROD nd MROD Quntifition Liver mirosomes were prepred y homogenizing.5 g of liver in Tris uffer (.5 M Tris nd 1.15% KCl, ph 7.5) nd entrifuged to otin the mirosoml frtion. The mirosoml pellet ws resuspended in mirosoml stiliztion uffer (2% glyerol,.1 M KH 2 PO 4, 1 mm EDTA, nd 1 mm dithiothreitol, ph 6.25) nd liquots were stored t -8 C. EROD nd MROD tivities were mesured using modifition of methods desried y Kennedy nd Jones (1994). The ssys were optimized nd onduted in 96-well pltes (Corning Costr Corp., Corning, NY, USA), in whih oth mirosoml ytohrome P45 tivity nd protein onentrtion were mesured simultneously using Fluorosn Asent miroplte fluorometer (Thermo Fisher Sientifi In., Wlthm, MA, USA). For EROD ssys, the rnge of the working resorufin stndrds ws 21 pmol/well. The retion mixture inluded 3. ll of mirosome preprtion in.5 M HEPES uffer (ph 7.8),.3 mm NADPH, nd 5 lm 7-ER per well. For MROD ssys, the working resorufin stndrd rnge ws 18 pmol/well. The retion mixture inluded 8 ll of mirosome preprtion in.5 M HEPES uffer (ph 7.8),.3 mm NADPH, nd 2.5 lm of 7-MR per well. Following the ddition of the sustrtes (7-ER or 7-MR), ll ssy pltes were preinuted for 1 min t 37 C prior to the ddition of NADPH to initite the retion. EROD nd MROD tivities were determined kinetilly y mesuring the formtion of resorufin every 2 min for 3 min. The retion ws terminted y dding 6 ll etonitrile (Burdik & Jkson, Muskegon, MI, USA) ontining.4 mm fluoresmine (Sigm-Aldrih, St. Louis, MO, USA) to eh well, followed y the determintion of protein onentrtions (Kennedy nd Jones 1994). EROD nd MROD tivities were determined from the liner rnge of the time urves for eh well nd the results were expressed s piomoles of sustrte onverted per minute per milligrm of protein (pmol/min/mg). Quntifition of PCDD, PCDF, nd TEQ To ensure tht o-ontminnts were not ftor in the study, the onentrtions of 17 individul 2,3,7,8-sustituted PCDF nd PCDD ongeners nd 12 individul PCB ongeners were mesured in the dietry items nd mink tissues s desried in Zwiernik et l. (8). Conentrtions of TEQ were lulted s the sum of the produts of the onentrtions of ongeners multiplied y their respetive TEF (Vn den Berg et l. 6). A surrogte vlue of one-hlf the method detetion limit (MDL) ws used for onentrtions less thn the MDL. Dt Anlysis All sttistil nlyses were performed with SAS (Version 9.1; SAS, Cry, NC, USA). Beuse of the nture of the prmeters, severl sttistil models were used for dt nlyses. The study ws designed for the pplition of oth fixed-effets models (test for differenes mong exposure groups) nd regression nlysis (orreltion of liver PeCDF nd TCDF onentrtions nd EROD nd MROD enzyme tivities). Prior to onduting sttistil omprisons, dt were tested for normlity using the Shpiro Wilkes test nd proility plots. If neessry, vlues were log-trnsformed to pproximte normlity. Differenes mong exposure groups were tested using one-wy ANOVA followed y Dunnett s test (PROC ANOVA). A sensitivity nlysis ws onduted to determine the is introdued y ssuming vlue of hlf the limit of quntifition (LOQ) for ensured dt sets. Results PCDF Conentrtions in Liver Conentrtions of TCDF nd PeCDF in livers of mink fed dily doses of TCDF, PeCDF, or mixture of the two ongeners did not differ etween 9 nd 18 dys; thus, single men onentrtion is presented (Tle 1). Conentrtions of TCDF in the liver vried mong doses, rnging from 3% greter thn the dily dose (.98 TEQ TCDF /kg ody weight (w)/dy) to 65% less thn the dily dose (2 TEQ TCDF /kg w/dy). Conentrtions of PeCDF in mink liver inresed signifintly with dose, with ioumultion ftors (BAFs) of 67 nd 179 for the two doses (.62 nd 9.5 ng TEQ PeCDF /kg w/dy, respetively). Conentrtions of TCDF nd PeCDF in livers of mink fed the TCDF/PeCDF mixture were similr to onentrtions in the livers of mink reeiving similr dose of the individul ongeners. Hepti BAFs sed on TEQ onentrtion were.34 for TCDF (4.1 ng TEQ TCDF /kg w/dy) nd 13 for PeCDF (2.8 ng TEQ PeCDF /kg w/dy). Gross Morphology nd Histology There were no tretment-relted hnges in gross morphology or histology. No externl lesions or normlities tht were ttriutle to tretment were oserved, nd the nutritionl sttus of ll mink, exept for one individul, ws lssified s good to very good. There were no signifint hnges in ody mss or liver mss over the ourse of the study (dt not presented). The most frequent histologil ltertion ws heptoellulr vuoltion tht ourred in ll groups nd, thus, ws not onsidered to e

5 42 Arh Environ Contm Toxiol (9) 57: tretment relted (Tle 2). There were few ses of ile dut hyperplsi nd miniml to mild minerliztion of renl medullry tuules tht ourred ross ll tretments. There ws numerilly greter inidene of ftty liver in mink fed only PeCDF ompred to the other groups (Tle 2). Periodontitis ws oserved in one mink from the ontrol group, ut this ws onsidered inidentl nd not tretment relted. Jw lesions lssified s mild were oserved t the termintion of the study in two mink from the 9.5-ng TEQ PeCDF /kg w/dy tretment group (Tle 2). One of these mink exhiited single yst onsisting of squmous epithelil ells (Fig. 1). However, the presene nd severity of this lesion ws not dose dependent nd, therefore, ws onsidered inidentl. EROD nd MROD Ativities Mink fed TCDF lone hd signifintly greter EROD nd MROD tivities in the liver ompred to ontrols. Beuse there were no signifint tretment y time intertions, enzyme tivities mesured fter 9 nd 18 dys of exposure were verged. Exposure to TCDF resulted in Tle 2 Inidene of gross nd histologil effets in femle mink exposed to either TCDF or PeCDF singly or s mixture through the diet for up to 18 dys TCDF (ng TEQ/kg w/dy) PeCDF (ng TEQ/kg w/dy) TCDF/PeCDF Pthologil end points Control Mixture Orl lesions Squmous epithelil osteoinvsion 1 Osetolsts nd one resorption 1 Periodontitis 1 Liver Heptoellulr voultion Periportl lymphoyti/plsmyti 1 Ftty liver Bile dut hyperplsi Kidney Medullry tuules or uroliths Infetion 1 1 Nephritis 1 Lymphoid ggregtes 2 Spleen Hemorrhge 1 Sr fissure 1 Note: Tretment onentrtions re estimted dily doses reported s TEQ vlues. Mmmlin TEF used were.3 for PeCDF nd.1 for TCDF (Vn den Berg et l. 6) Vlues given for eh end point represent the numer of findings (mink) ssoited with eh tretment group (n = 6 mink per tretment, n =8 ontrol) Mixture onsisted of 4.1 nd 2.8 ng TEQ/kg w/dy for TCDF nd PeCDF, respetively Fig. 1 A single yst onsisting of squmous epithelil ells within the mndiulr tissue t 49 () nd t 29 (). The rrow identifies the nest of ells proximl to the inisors

6 Arh Environ Contm Toxiol (9) 57: EROD (pmol/min/mg protein) MROD (pmol/min/mg protein) A B Control TCDF Dose (ng TEQ/kg w/d) Control TCDF Dose (ng TEQ/kg w/d) Fig. 2 Effets of TCDF on liver () EROD nd () MROD tivity in mink exposed for up to 18 dys vi the diet. Dt re presented s mens with error rs inditing stndrd devitions. Different letters indite signifint differenes (p \.5) etween dose groups. Control n = 8 nd eh TCDF dose n = 6 signifintly greter tivities of oth EROD nd MROD in mink t doses of 3.8 nd 2 ng TEQ TCDF /kg w/dy (Fig. 2). Additionlly, EROD nd MROD tivities in mink fed 2 ng TEQ TCDF /kg w/dy were signifintly greter thn tivities of those fed 3.8 ng TEQ TCDF /kg w/dy. Both EROD (Fig. 3) nd MROD (Fig. 3) tivities were positively orrelted with onentrtions of TCDF expressed s TEQ in the liver. Exposure to PeCDF resulted in sttistilly signifint greter EROD nd MROD tivities reltive to ontrols (Fig. 4). Beuse there were no sttistilly signifint differenes in either EROD or MROD enzyme tivities t 9 nd 18 dys nd there were no intertions etween tretment nd the time the vlues of eh of these enzyme tivities t the two times were verged. EROD tivities in ll PeCDF-dosed groups were signifintly greter thn ontrol tivity (Fig. 4). EROD tivity in the 9.5 ng TEQ PeCDF /kg w/dy group ws signifintly greter thn enzyme tivities in the.62 nd 2.3 ng TEQ PeCDF /kg w/ dy dose groups. MROD tivities were lso signifintly greter thn ontrol tivities t ll PeCDF doses, with tivities in livers of mink fed 2.2 or 9.5 ng TEQ PeCDF /kg w/dy eing signifintly greter thn tivities in livers of mink fed.62 ng TEQ PeCDF /kg w/dy. Both EROD EROD (pmol/min/mg protein) MROD (pmol/min/mg protein) A B nd MROD tivities were positively orrelted with onentrtions of PeCDF expressed s TEQ in the liver (Fig. 5 nd ). The EROD nd MROD tivities in livers of mink fed mixture of TCDF (4.1 ng TEQ TCDF /kg w/dy) nd PeCDF (2.8 ng TEQ PeCDF /kg w/dy) were signifintly greter thn tivities in livers of ontrol mink (Fig. 6). EROD tivity in the livers of mink fed the mixture of TCDF nd PeCDF were similr to the tivities in mink fed 3.8 ng TEQ TCDF /kg w/dy nd those fed 2.2 ng TEQ PeCDF /kg w/ dy (Fig. 6). MROD tivity in livers of mink fed the mixture ws signifintly greter thn enzyme tivity in livers of mink fed 3.8 ng TEQ TCDF /kg w/dy ut did not differ from tivity in livers of mink fed 2.2 ng TEQ PeCDF / kg w/dy PeCDF (Fig. 6). Disussion Control Dy 9 Dy 18 Control Dy 9 Dy 18 PCDF Conentrtions in Liver y = Ln(x) R 2 = Liver TCDF Con. (ng TEQ/kg, ww) y = 32.99Ln(x) R 2 = Liver TCDF Con. (ng TEQ/kg, ww) Fig. 3 Regression nlysis etween () EROD nd () MROD tivities nd mesured TCDF liver onentrtions (ng TEQ TCDF /kg, ww) of mink exposed vi the diet for 9 nd 18 dys. Models in the figures represent the est fit of the dt sed on riteri given in the Mterils nd Methods setion 2,3,4,7,8-Penthlorodienzofurn umulted in the liver of the mink to muh greter extent thn did TCDF when

7 422 Arh Environ Contm Toxiol (9) 57: EROD (pmol/min/mg protein) MROD (pmol/min/mg protein) A B Control PeCDFDose (ngteq/kg w/d) Control PeCDFDose (ngteq/kg w/d) Fig. 4 Effets of PeCDF on liver () EROD nd () MROD tivities in mink exposed for up to 18 dys vi the diet. Dt re provided s mens with error rs inditing stndrd devitions. Different letters indite signifint differenes (p \.5) etween dose groups. Control n = 8 nd eh PeCDF dose n = 6 dministered s single ongener or in omintion with TCDF (Tle 1). Hepti sequestrtion of PeCDF reltive to tht of PCDDs nd other PCDFs, inluding TCDF, is onsistent with wht hs een reported in other studies with mmmls (Brewster nd Birnum 1987, 1988; DeVito et l. 1997). These studies hve shown tht PeCDF umultes in the liver of rodents y inding to hepti CYP1A2 protein (Dilierto et l. 1999) nd, presumly, PeCDF ould e sequestered in livers of mink y the sme mehnism (Zwiernik et l. 8). The lesser onentrtions of TCDF umulted in livers of the mink suggest n effiient elimintion nd/or metolism of the ongener. The BAF of this ongener hs een reported to e inversely proportionl to dose (Zwiernik et l. 8), whih suggests induile metolism of TCDF. This is similr to wht hs een reported in rodents (Ti et l. 1993). The ft tht the presene of PeCDF redued the umultion of TCDF in the liver of the mink to n even greter extent strengthens the rgument tht indution of CYP1A1 redued umultion of TCDF (Zwiernik et l. 8). The whole-ody hlf-time for elimintion of PeCDF oserved for mink in this study ws estimted to e *8 dys, wheres the hlf-time for elimintion of TCDF ws less thn hlf dy in mink (Zwiernik et l. 8). The hlf-time for elimintion for TCDF nd, EROD (pmol/min/mg protein) MROD (pmol/min/mg protein) PeCDF in the mink re less thn those reported for rodents. The hlf-time of TCDF is *2 dys in mie (DeVito et l. 1997) nd the hlf-time of PeCDF in the rt is more thn 6 dys (Brewster nd Birnum 1987). Histology A Control Dy 9 Dy 18 y = Ln(x) R 2 = Liver PeCDF Con. (ng TEQ/kg, ww) B Control Dy 9 y = Ln(x) R 2 = Dy Liver PeCDF Con. (ng TEQ/kg, ww) Fig. 5 Regression nlysis etween () EROD nd () MROD tivities nd mesured PeCDF liver onentrtions (ng TEQ PeCDF / kg, ww) of mink exposed vi the diet for 9 nd 18 dys. Models in the figures represent the est fit of the dt sed on riteri given in the Mterils nd Methods setion In this study, TCDF nd PeCDF, dministered singly or in omintion, t environmentlly relevnt doses for 18 dys did not result in hnges in gross morphologil or histologil end points (Tle 2) tht hve een reported for other studies in whih mink were exposed to dioxin or dioxin-like ompounds (Hohstein et l. 1988, 1998; Render et l.,, 1). Reent studies (Bursin et l. 6, 6) suggest tht very sensitive inditor of exposure of mink to environmentlly relevnt onentrtions of TCDD-like ompounds is prolifertion of mndiulr nd mxillry squmous epitheli. Previous studies hve indited tht rnh mink exposed to 24. lg of 3,3,4,4,5-penthloroiphenyl (PCB 126)/kg feed or 2.4 lg TCDD/kg feed (2.4 lg TEQ/kg feed or *3 ng TEQ/kg w/dy) developed linil signs of mndiulr nd mxillry squmous epithelil hyperplsi tht, in

8 Arh Environ Contm Toxiol (9) 57: EROD (pmol/min/mg protein) MROD (pmol/min/mg protein) A B Control TCDF PeCDF Mixture Doses (ng TEQ/kg w/d) Control TCDF PeCDF Mixture Doses (ngteq/kg w/d) Fig. 6 Effets of mixture of TCDF (4.1 ng TEQ TCDF /kg w/dy) nd PeCDF (2.8 ng TEQ PeCDF /kg w/dy) on liver () EROD nd () MROD tivities in mink exposed for up to 18 dys vi the diet. Single TCDF nd PeCDF doses re 3.8 ng TEQ TCDF /kg w/dy nd 2.2 ng TEQ PeCDF /kg w/dy, respetively. Dt re presented s mens with error rs inditing stndrd devitions. Different letters indite signifint differenes (p \.5) etween tretments. Control n = 8, TCDF n = 6, PeCDF n = 6, nd mixture n = 5 severe ses, resulted in the loss of teeth (Render et l. ; 1). Mink fed diets ontining onentrtions s little s.24 lg PCB 126/kg feed (.24 lg TEQ/kg feed or 3 ng TEQ/kg w/dy) (Bekett et l. 8) exhiited the lesion (Bekett, personl ommunition), s did mink fed diet ontining fish ontining PCBs, PCDDs, nd PCDFs tht provided n estimted dily dose of 1 ng TEQ/ kg w/dy (Bursin et l. 6). In the present study, only one niml, whih hd een fed 9.5 ng TEQ PeCDF /kg w/ dy, hd single yst of squmous epithelil ells t 18 dys. The onentrtion of PeCDF in the liver of tht mink ws 1.3 ng TEQ/g, ww (wet weight). In those mink studies in whih jw lesion inidene nd liver TEQ onentrtions were ssessed, results indited tht histologil lesions were evident in nimls with hepti TEQ onentrtions rnging from 4 to 75 ng/kg, ww in the liver (Bursin et l. 6, 6). Wild mink with histologil evidene of prolifertion of mndiulr nd mxillry squmous epitheli hd n verge onentrtion of 61 ng TEQ/kg, ww (Bekett et l. 5). There re two possile explntions for the srity of the jw lesions in the present study. One possiility is tht the ge t whih exposure ws initited ws too lte nd/or the durtion of exposure ws not suffiient. In the studies with rnh mink in whih effets were oserved t onentrtions similr or less thn those tested in this study, exposure egn in utero nd ontinued until mink were *7 months old (Bursin et l. 6, 6). In those studies in whih exposure periods rnged from 3 to 6 dys (Render et l., 1), the mink were pproximtely 6 weeks old nd the dose ws *3-fold greter thn the dose in the present study (3 ng TEQ/kg w/dy vs. 9.5 ng TEQ/kg w/dy). A seond possiility is relted to the speifi PCB/PCDD/ PCDF ongeners ontriuting to the TEQs. In studies of rnh mink utilizing individul ongeners, TEQs were provided y either TCDD or PCB 126 (Render et l.,, 1). In those studies of mink fed diets ontining ontminted fish, the mjority of TEQs were ontriuted y ongeners other thn furns; for exmple, in study tht ssessed the effets of feeding diets ontining fish from the Houstoni River, PCB 126 nd TCDD ontriuted 61% of the totl TEQs nd TCDF nd PeCDF ontriuted 4% (Bursin et l. 6, 6). In similr study utilizing fish from the Sginw River, PCB 126 nd TCDD ontriuted 39% of the totl nd TCDF nd PeCDF ounted for 25% of the totl. It is possile tht TCDF nd PeCDF re less effetive thn PCB 126 nd TCDD in induing prolifertion of mndiulr nd mxillry squmous epitheli. Furthermore, it hs een determined tht the effets of PCDFs nnot e urtely predited from the use of TEQ-sed TRVs developed from studies of PCDDs nd PCBs (Blnkenship et l. 8). This suggests tht there re differenes in the sensitivity of mink to PCBs nd PCDFs tht re not ppropritely refleted y the urrently utilized TEQ pproh (Vn den Berg et l. 6). Enzyme Indution Bsl EROD tivities mesured in livers of mink during this study fell within rnge of ontrol tivities tht hve een reported in other studies with mink (Brunström et l. 1; Käkelä et l. 1; Mrtin et l. 7; Shipp et l. 1998; Smits et l. 1995). Vlues in this study were similr to those reported y Smits et l. (1995), Käkelä et l. (1) nd Mrtin et l. (7) ut were less thn those vlues reported y Brunström et l. (1). However, given the inonsistenies etween ll of these studies reltive to experimentl design, ge, nd sex of nimls, s well s potentil ontminnts ssoited with their feed, diret omprison mong these studies is not possile. Given tht the sl EROD tivities in our study re similr to those enzyme tivities mesured in other studies, it n e ssumed tht the ytohrome P451A1 system ws funtioning properly.

9 424 Arh Environ Contm Toxiol (9) 57: To our knowledge, there hve een no reports of MROD enzyme tivities in mink to dte. Bsl MROD tivity ws less thn tht reported for EROD, whih is in ordne with studies in other mmmls suh s rts or monkeys (Luet et l. 199; Suzuki et l. 1; Wever et l. 1994) ut opposite to reports on other speies suh s vrious mie strins, hmster, or humns (Hmm et l. 1998; Wever et l. 1994). The reltive differene etween EROD nd MROD tivities ws greter (pproximtely fivefold) when ompred to tht reported for rts or monkeys (\twofold to threefold). It hs een previously reported tht the speifiities of orthologous forms of P45 s re expressed differently mong mmmlin speies. In rts, CYP1A1 nd CYP1A2 seletively tlyze EROD nd MROD, respetively, wheres in humns, CYP1A2 hs similr tivities for oth EROD nd MROD. From the dt presented here it ppers tht mink re ssoited more losely with the rt or monkey regrding their sl EROD/MROD profiles. However, further eluidtion of the speifiities of different forms of P45 s for the different lkylresorufin O-delkylses (AROD) is neessry to e le to ssign mink to ertin mmmlin metolism type. There were no signifint differenes in enzyme tivity in mink reeiving dily doses of TCDF nd/or PeCDF etween 9 nd 18 dys. This suggests tht mximum indution of CYP1As in livers of mink s funtion of time in response to the exposure with TCDF nd PeCDF ours erlier thn the first smpling time point t 9 dy. The EROD tivity in mink dosed with the mixture of TCDF nd PeCDF ws similr to enzyme tivity in those mink dosed with either TCDF or PeCDF, wheres MROD tivity ws similr to tivity in those mink dosed with PeCDF. This suggests tht indution resulting from the omintion of the two furn ongeners might not hve een dditive nd perhps ws due primrily to the tion of only one of the ongeners. Bsed on liver onentrtion dt inditing greter onentrtion of PeCDF ompred to TCDF, it is possile tht enzyme indution in those nimls reeiving the mixture ws due primrily to PeCDF. Alterntively, TCDF might lso hve ontriuted to the inrese in enzyme tivities, ut due to metolism, its onentrtion in the liver ws less thn tht of PeCDF. Aknowledgments This reserh ws supported y n unrestrited grnt from ENTRIX In. to Steven J. Bursin, John P. Giesy, nd Mtthew J. Zwiernik. Professor Giesy ws supported y n t-lrge Chir Professorship t the Deprtment of Biology nd Chemistry nd Reserh Centre for Costl Pollution nd Conservtion, City University of Hong Kong nd y n Are of Exellene Grnt (AoE P- 4/4) from the Hong Kong University Grnts Committee. Speil thnks go to Angelo Npolitno, Jeff Greenlee, C.P. Npolitno, Dvid Hmmn, Ptrik Brdley, Mihel Krmer, Nozomi Iked, Molly Wiersem, nd Meliss Shotwell for the expertise nd support they provided during the study. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution Nonommeril Liense whih permits ny nonommeril use, distriution, nd reprodution in ny medium, provided the originl uthor(s) nd soure re redited. Referenes Bsu N, Sheuhmmer AM, Bursin SJ, Elliott J, Rouvinen-Wtt K, Chn HM (7) Mink s sentinel speies in environmentl helth. Environ Res 13: doi:1.116/j.envres Bekett KJ, Millsp SD, Blnkenship AL, Zwiernik MJ, Giesy JP, Bursin SJ (5) Squmous epithelil lesion of the mndiles nd mxille of wild mink (Mustel vison) nturlly exposed to polyhlorinted iphenyls. Environ Toxiol Chem 24: doi:1.1897/4-241r.1 Bekett KJ, Ymini B, Bursin SJ (8) The effets of 3,3,4,4 5- penthloroiphenyl (PCB 126) on mink (Mustel vison) reprodution nd kit survivility nd growth. Arh Environ Contm Toxiol 54: 129. doi:1.17/s Blnkenship AL, Ky DP, Zwiernik MJ et l (8) Toxiity referene vlues for mink exposed to 2,3,7,8-tetrhlorodienzop-dioxin. 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Environ Toxiol Chem 25: doi:1.1897/5-46r.1 Bursin SJ, Aulerih RJ, Ymini B, Tillitt DE (6) Dietry exposure of mink (Mustel vison) to fish from the Houstoni River, Berkshire County, Msshusetts, USA: effets on orgn weights nd histology nd hepti onentrtions of polyhlorinted iphenyls nd 2,3,7,8-tetrhlorodienzo-p-dioxin toxi equivlene. Environ Toxiol Chem 25: doi:1.1897/ 5-47R.1 Bursin SJ, Bekett KJ, Ymini B et l (6) Assessment of effets in mink used y onsumption of rp olleted from the Sginw River, Mihign, USA. Arh Environ Contm Toxiol 5: doi:1.17/s y DeVito M, Dilierto J, Ross DG, Menhe MG, Birnum L (1997) Dose-response reltionships for polyhlogented dioxins nd dienzofurns following suhroni tretment in mie I. CYP1A1 nd CYP1A2 enzyme tivity in liver, lung, nd skin. 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