When administered acutely or subchronically, PBBs increase the activity of hepatic drug

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1 Environmentl Helth Perspectives Vol. 23, pp , 1978 Effects of Polybrominted Biphenyls on Kidney Function nd Activity of Renl Microsoml Enzymes by K. M. McCormck,* W. M. Kluwe,* V. L. Snger,t nd J. B. Hook* Polybrominted biphenyls (s) cuse heptic microsoml enzyme stimultion nd histopthologicl ltertions in severl orgns, including kidney. Concern bout effects of s on the helth of newborns hs incresed fter the discovery of s in milk of nursing mothers. Therefore, it ws of interest to investigte the effects of s on kidney function nd the ctivity of renl microsoml enzymes in dult nd immture nimls. Seven nd eleven dy old pups were treted with single IP injection of either penut oil or 150 mg/kg s (FireMster BP-6) in penut oil. Adult virgin rts were fed diet contining 0 or 100 ppm s for 30 or 90 dys. Tretment with s only retrded weight gin fter 90 dys exposure. Kidney-to-body weight rtio ws not ltered by s. Arylhydrocrbon hydroxylse ctivity ws incresed while epoxide hydrtse ctivity ws decresed (dults) or not ffected (immture rts) in kidney following tretment with s. Administrtion of s hd no effect on blood ure nitrogen, the clernce of inulin, p-minohippurte (PAH), or frctionl sodium excretion. Similrly, the in vitro ccumultion of PAH nd N-methylnicotinmide (NMN) by thin renl corticl slices nd mmonigenesis nd gluconeogenesis in renl corticl slices were not ffected by s. In conclusion, tretment with s resulted in modifiction of the ctivity of renl microsoml enzyme ctivities but hd no detectble effect on renl function. Introduction Polybrominted biphenyls (s) re fire retrdnts which contminted livestock nd poultry feed in Michign. The commercil product blended with feed ws FireMster BP-6, which is mixture of congeners; the mjor component is 2,- 2',4,4',5,5'-hexbromobiphenyl (1). Rts mintined on diet contining s hve enlrged livers s well s heptic nd renl histopthologicl ltertions (2-4). Renl lesions observed following tretment with 100 ppm octbromobiphenyl include petechil hemorrhge nd hyline degenertive cytoplsmic chnges (2, 3). Rt urinry protein is elevted fter subchronic dministrtion of s (5). These observtions suggest tht renl function my be impired by s. * Deprtment of Phrmcology, Michign Stte University, Est Lnsing, Michign t Deprtment of Pthology, Michign Stte University, Est Lnsing, Michign When dministered cutely or subchroniclly, s increse the ctivity of heptic drug metbolizing enzymes in rts (6, 7). Stimultion of heptic microsoml enzymes following s is similr to tht observed fter tretment with both phenobrbitl (PB) nd 3-methylcholnthrene (3MC) (6), two compounds which represent cytochrome P450 nd P-450 inducing gents, respectively (8). Currently, there is scrcity of informtion regrding s effects on the ctivity of extrheptic microsoml enzymes. Incresed ctivity of mixed function oxygenses my lter the metbolism of endogenous compounds nd xenobiotics. Concern bout potentil effects of s on mothers nd offspring hs been generted since high concentrtions of s hve been detected in humn milk (9). Young nimls my be prticulrly vulnerble to dmge cused by s. The kidney is morphologiclly nd functionlly immture t birth nd deficient in its cpcity to mintin homeostsis when chllenged with stressful stimuli. Therefore, it ws of interest to investigte the ef- April

2 fects of s on kidney function nd the ctivity of renl microsoml enzymes in dult nd immture nimls. Methods Adult, femle virgin nd lctting Sprgue- Dwley rts nd pups (10 nimls per litter) (Sprtn Frms, Hslett, Mich.) were mintined in cler polypropylene cges t 22 C with 12-hr light cycle (7 AM-7 PM) nd were llowed free ccess to food (Wyne Lb Blox) nd wter. Animls were cclimtized for t lest 3 dys prior to experimentl tretment. The 7- nd 11-dy old pups were treted with single IP injection of either penut oil (10 ml/kg) or 150 mg/kg s (FireMster BP-6, Michign Chemicl Corp., St. Louis, Michign) in penut oil, 10 m/kg. Following cclimtion, dult virgin rts were treted by substituting ground diet contining 0 or 100 ppm s (FireMster BP-6) for the Lb Blox. Rts were exposed to the diet for 30 or 90 dys. Enzyme ssys were performed 28 dys following injection of seven dy old pups nd fter 90 dys exposure of dult rts to the experimentl diet. Immeditely following scrifice, kidneys were excised, weighed nd then chopped into ice-cold 66mM Tris buffered to ph 7.4 with HCI. Renl postmitochondril superntnts were prepred by homogeniztion (Potter-Elvehjem homogenizer with Teflon pestle) in 3 volumes of 66mM Tris ph 7.4 followed by centrifugtion t 10,000g for 30 min. All ssys were performed on the dy of superntnt preprtion. Protein ws mesured (10) by use of bovine serum lbumin s stndrd. Enzyme ctivities mesured were epoxide hydrtse (EH) (11) nd rylhydrocrbon hydroxylse (AHH) (12, 13) Ȧnimls used for histologic exmintion hd been exposed to the experimentl diet for 90 dys. Pieces of kidney were cut nd fixed in 10% buffered formlin. After fixtion, these were embedded in prffin, sectioned t 5,um, nd stined with hemtoxylin nd eosin. Orgnic ion trnsport cpcity ws estimted in vitro 3 dys following injection of 11-dy-old pups nd fter 30 nd 90 dys exposure of dult rts to the experimentl diet. Following scrifice, kidneys were quickly removed, weighed, nd plced in icecold norml sline. Trnsport cpcity ws quntified s the bility of renl corticl slices to ccumulte representtive nion, p-minohippuric cid (PAH), nd ction, N-methylnicotinmide (NMN) (14, 15). Results were expressed s sliceto-medium (SIM) concentrtion rtio, where S is in units of mg/g wet tissue weight nd M is expressed in mg/ml medium. The bility of renl corticl slices 154 from rts treted with s for 90 dys to form mmoni nd glucose ws lso determined (16, 17). Net production of mmoni nd glucose ws expressed s gmole/mg wet tissue weight/hr. To determine renl function in vivo, nimls exposed to s for 30 or 90 dys were nesthetized with 50 mg/kg sodium pentobrbitl, IP, nd body temperture ws mintined t C by use of het lmps. A PE50 cnnul ws inserted into the bldder, nd urine ws collected under minerl oil in preweighed vils. The left femorl vein ws cnnulted for infusion. Both femorl rteries were cnnulted to monitor blood pressure nd to obtin blood smples. The infusion solution contined 1% inulin nd 0.6% PAH in norml sline. (3H)-Inulin (0.5,uCi/ml) nd (14C)-PAH (0.5,uCi/ml) were dded to the solution, which ws infused t mlmin using Hrvrd infusion pump. A minimum of 1.5 hr elpsed from the beginning of the infusion to initition of urine collection. The 30-min urine collections were tken with blood (400,ul) smpled t the middle of ech period. ('4C)-PAH, (3H)-inulin, nd sodium concentrtions were determined s previously described (18). Blood ure nitrogen (BUN) ws quntified (17) nd expressed s weight (mg) ure nitrogen per 100 ml of whole blood. Dt were nlyzed sttisticlly by rndomized complete block nlysis of vrince. Tretment differences were detected by the lest significnt difference test (19). The 0.05 level of probbility ws used s the criterion of significnce. Results Growth rte ws not ffected by single IP injection of 150 ppm (150 mg/kg) s or by 30 dys tretment with 100 ppm s; however, 90 dys dietry exposure to 100 ppm s retrded weight gin (Tble 1). The kidney to body weight rtio ws not ltered by tretment with s (Tble 1). Tble 1. Effect of s on body weight nd kidney to body weight rtio. Tretment Tretment, Kidney weight/ time, dys ppm Body weight, g body weight 30 Control ± ± ± Control ± ± ± 5.4b Acutec Control ± ± ± ± 0.08 Vlues re mens ± S.E.M. for four nimls. b Sttisticlly significnt difference from the respective control, p < c Seven-dy-old rts were given single tretment with s nd scrificed 28 dys lter. Environmentl Helth Perspectives

3 Administrtion of 150 ppm s to 7-dy-old rts incresed the ctivity of renl ryihydrocrbon hydroxylse (AHH) bove controls 28 dys lter (54% of control). Epoxide hydrtse (EH) ctivity ws not modified in kidneys of young rts by s. The ctivity of renl AHH in rts exposed to 100 ppm s for 90 dys ws incresed bove control (1116% of control), however, renl EH ctivity ws significntly decresed (14% of control) (Tble 2). Tble 2.?,ffect of s on renl AHH nd EH ctivities. EH, nmole AHH, fluorescence styreneglycol units/mg formed/ Tretment tretment, microsoml mg microsoml time, dys ppm protein-min protein-min 90 Control 2.94 ± ± ± 1.73b 0.02 ± 0.Olb Acutec Control 4.39 ± ± ± 8.22b 0.33 ± 0.03 Vlues re mens ± S.E.M. for three nimls. b Sttisticlly significnt difference from the respective control, p < c Seven-dy-old rts were given single tretment with s nd scrificed 28 dys lter. Degenertive histopthologicl ltertions were observed in kidney from rts treted with 100 ppm s for 90 dys (Fig. 1). Renl chnges included progressive obsolescence of glomeruli. Glomerulr tufts were shrunken. Bowmn's membrne, while not thickened, ws diminished in proportion to the shrinking tuft. A single focus of lymphocytes ws seen in one kidney. FIGURE 1. Renl tissue from rt fed diet contining 100 ppm s for 90 dys. There re shrunken glomeruli. Hemtoxylin nd eosin, 133 x. Tretment with s hd no effect on the in vitro ccumultion of PAH nd NMN by thin renl corticl slices (Tble 3) or mmonigenesis nd gluconeogenesis by renl slices (Tble 4). The clernce of inulin (glomerulr filtrtion rte, GFR), the clernce of PAH (effective renl plsm flow, ERPF) nd frctionl sodium excretion (%) were unffected by 30 or 90 dys exposure to diet contining 100 ppm s (Tble 5). Similrly, dministrtion of s for 30 or 90 dys hd no effect on blood ure nitrogen (BUN) (Tble 6). Tble 3. Effect of s on the ccumultion of PAH nd NMN. S/M rtio tretment, ppm PAH NMN 30 Dys Control ± ± ± ± Dys Control ± ± ± ± 0.17 Acuteb Control 8.29 ± ± ± ± 0.17 Vlues re mens + S.E.M. for t lest three nimls. btwo-week-old rts were given single tretment with s 72 hr prior to scrifice. Tble 4. Effect of feeding PPBs for 90 dys on mmonigenesis nd gluconeogenesis. Net production, tretment,,umole/mg wet tissue weight/hr" ppm Ammoni Glucose Control 3.39 ± ± ± ± 0.01 Vlues re mens + S.E.M. for four nimls. Discussion Polybrominted biphenyls stimulte heptic microsoml mixed function oxygenses in dult rts nd mice (6, 7, 20). The results of this investigtion indicte tht s lso lter the ctivity of renl microsoml enzymes in immture nd dult rts. Activity of AHH is incresed in kidney s in liver fter s; however, in contrst to heptic EH stimultion, renl EH ctivity is decresed (dults) or not ffected (immture rts) by tretment with s. Stimultion of AHH with concomitnt inhibition of EH in kidney my be of toxicologicl concern. Microsoml enzymes such s AHH cn ctlyze the formtion of very rective rene oxides from inert romtic compounds (21, 22). Arene oxides my be further metbolized to less toxic dihydrodiols by enzymes such s EH (22). Thus, since AHH ctivity is incresed while EH ctivity is April

4 Tble 5. Effect of feeding s on glomerulr filtrtion rte (GRF), effective renl plsm flow (ERPF), nd frctionl sodium excretion. Tretment GFR, ml/min ERPF, m/min Frctionl sodium excretion,% time, dys Control 100 ppm s Control 100 ppm s Control 100 ppm s ± ± ± ± ± ± ± ± 0.03 Vlues re mens ± S.E.M. for four nimls. Tble 6. Effect of feeding s on blood ure nitrogen. tretment, ppm Durtion, dys BUN, mg ure nitrogen/100 mllblood Control ± 1.92 Control ± ± 1.52 Vlues re mens ± S.E.M. for four nimls. decresed (dults) or not ffected (immture rts) in kidney following tretment with s, subsequent dministrtion of compound metbolized by these enzymes my result in incresed concentrtions of rene oxides nd possibly mplified nephrotoxicity. Weight gin ws only retrded following 90 dys exposure to 100 ppm s. Previous reports suggest tht this my be due to decresed food efficiency (5, 23). Kidney enlrgement ws not detected nd lthough glomerulr degenertive ltertions were observed following tretment with s for 90 dys, similr chnges were seen in controls but less frequently. Since more severe renl degenertive chnges were seen following tretment with 100 ppm octbromobiphenyl (2, 3), FireMster BP-6, which is mostly hexbromobiphenyl (/), my be less nephrotoxic. The pucity of prominent renl histopthologicl ltertions fter exposure to FireMster BP-6 correltes with our observtions regrding its lck of effect on renl function. Exposure to 100 ppm s for 30 or 90 dys hd no effect on BUN consistent with its lck of effect on GFR. Similrly, ERPF, filtrtion frction (GFR/ERPF) nd frctionl sodium excretion were unffected by s. Tretment with s hd no detectble effect on ny in i itro prmeters of renl function tested. Acute nd subchronic dministrtion of s to immture nd dult rts, respectively, modified the ctivity of renl microsoml enzymes. Altertions in enzyme ctivities were not correlted with chnges in renl function. This indictes tht s, t lest FireMster BP-6, my not be potent nephrotoxic gent. However, the chrcteristics of mixed function oxygense ltertion in kidney following tretment with s suggest tht this compound my predispose the kidney to toxicity produced by chemicls dministered subsequent to s. This investigtion ws supported in prt by the USPHS grnts ES00560 nd GM We wish to express our pprecition to C. Hermmnn nd L. Lepper for their excellent technicl ssistnce. We lso wish to thnk D. Hummel for preprtion of the mnuscript. REFERENCES 1. Sundstrom, G., Hutzinger, O., nd Sfe, S. Identifiction of 2,2',4,4',5,5'-hexbromobiphenyl s the mjor component of flme retrdnt FireMster BP-6. Chemosphere 5: 11 (1976). 2. Aftmosis, J. G., Culik, R., nd Lu, K. P. Toxicology of brominted biphenyls. I. Orl toxicity nd embryotoxicity. Toxicol. AppI. Phrmcol. 22: 316(1972). 3. Norris, J. M., et l. Toxicologicl nd environmentl fctors involved in the selection of decrbromodiphenyl oxide s fire retrdnt chemicl. J. Fire Flmmbility/Combustion Toxicol. 1: 42 (1974). 4. Corbett, T. H., et l. A toxicity of polybrominted biphenyls (FireMster BP-6) in rodents. Environ. Res. 10: 390(1975). 5. Sleight, S. D., nd Snger, V. L. Pthologic feture of polybrominted biphenyl toxicosis in the rt nd guine pig. J. Amer. Vet. Med. Assoc. 169: 1231 (1976). 6. Dent, J. G., Netter, K. J., nd Gibson, J. E. The induction of heptic microsoml metbolism in rts following cute dministrtion of mixture of polybrominted biphenyls. Toxicol. Appl. Phrmcol. 38: 237 (1976). 7. Dent, J. G., et l. Induction of heptic microsoml metbolism by mixture ofnpolybrominted biphenyls. J. Toxicol. Environ. Helth 3: 651 (1977). 8. Sldek, N. E., nd Mnnering, G. J. Induction of drug metbolism. 1. Differences in the mechnism by which polycyclic hydrocrbons nd phenobrbitl produce their inductive effects on microsoml N-demethylting systems. Mol. Phrmcol. 5:174 (1969). 9. DHEW Report. Finl report of the subcommittee on the helth effects of polychlorinted biphenyls nd polybrominted biphenyls. Deprtment of Helth, Eduction nd Welfre, Wshington, D. C., Lowry, 0. H., et l. Protein mesurement with the folin phenol regent. J. Biol. Chem. 193: 265 (1951). 11. Oesch, F., Jerin, D. M., nd Dley, J. A rdiometric ssy for heptic epoxide hydrtse ctivity with [7-3H] styrene oxide. Biochim. Biophys. Act 227: 685 (1971). 12. Nebert, D. W., nd Gelboin, H. V. Substrte-inducible microsoml ryl hydroxylse in mmmlin cell culture. I. Assy nd properties of induced enzyme. J. Biol. Chem. 243:6242(1968). 156 Environmentl Helth Perspectives

5 13. Oesch, F. Differentil control of rt microsoml "Aryl hydrocrbon" monoxygense nd epoxide hydrtse. J. Biol. Chem. 251:79(1976). 14. Cross, R. J., nd Tggrt, J. V. Renl tubulr trnsport: Accumultion of p-minohippurte by rbbit kidney slices. Amer. J. Physiol. 161: 181 (1950). 15. Smith, H. W., et l. The renl clernces of substituted hippuric cid derivtives nd other romtic cids in dog nd mn. J. Clin. Invest. 24: 388 (1945). 16. Roobol, A., nd Alleyne, G. A. 0. Control of renl cortex mmonigenesis nd its reltionship to renl cortex gluconeogenesis. Biochim. Biophys. Act 353: 83 (1974). 17. Kpln, A. Ure nitrogen nd urinry mmoni. In: Stndrd Methods in Clinicl Chemistry. S. Meites, Ed. Acdemic Press, New York, Pegg, D. G., et l. Effect of 2,3,7,8-tetrchlorodibenzo-pdioxin on renl function in the rt. J. Toxicol. Environ. Helth 2: 55 (1976). 19. Steel, R. G. D., nd Torrie, J. H. Principles nd Procedures ofsttistics. McGrw-Hill, New York, Frber, T. M., nd Bker, A. Microsoml enzyme induction by hexbromobiphenyl. Toxicol. Appl. Phrmcol. 29: 102 (1974). 21. Dley, J. W., Jerin, D. M., nd Witkop, B. Arene oxides nd the NIH shift: The metbolism, toxicity nd crcinogenicity of romtic compounds. Experienti 28: 1129 (1972). 22. Oesch, F. Mmmlin epoxide hydrses: Inducible enzymes ctlyzing the inctivtion of crcinogenic nd cytotoxic metbolites derived from romtic nd olefinic compounds. Xenobiotic3: 305 (1973). 23. Grthoff, L. H., et l. Biochemicl nd cytogenetic effects cused by ingestion of Arochlor 1254 or FireMster BP-6. J. Toxicol. Environ. Helth 3: 769 (1977). April