Renal and hepatic microsomal enzyme stimulation and renal function following three months of dietary exposure to polybrominated biphenyls

TOXlCOLOGYANDAPPLIEDPHARMACOLOGY.

44,539-553(1978)

Renal and Hepatic Microsomal Renal Function Following Three to Polybrominated

Enzyme Stimulation and Months of Dietary Exposure Biphenyls

K. M. MCCORMACK, W. M. KLUWE, D. E. RICKERT,’ V. L. SANGER, AND J. B. HOOK* Departments of Pharmacology and Pathology, Michigan State University, East Lansing, Michigan 48824 Received

Juiy 28,1977;

accepted

October

19,1977

Renal and Hepatic Microsomal Enzyme Stimulation and Renal Function Following Three Months of Dietary Exposure to Polybrominated Biphenyls. MCCORMACK, K. M., KLUWE, W. M., RICKERT, D. E., SANGER, V. L., AND HOOK, J. B. (1978). Toxicol. Appl. Pharmacol. 44, 539-553. Polybrominated biphenyls (PBB) stimulate microsomal enzyme activity and produce a variety of toxic manifestations, including renal and hepatic histopathological changes. Therefore, it was of interest to determine the effect of chronic exposure to PBB on renal and hepatic microsomal enzyme stimulation and renal function. Adult Sprague-Dawley rats were fed diets containing 0 or 100 ppm of PBB for 3 months. Treatment with PBB retarded weight gain and increased the liver to body weight ratio but did not alter kidney to body weight ratio. Biphenyl-4. hydroxylase (BP-4-OH) and biphenyl-2-hydroxylase (BP-2-OH) activities were elevated in the kidney and liver following treatment with PBB. Exposure to PBB increased aryl hydrocarbon hydroxylase (AHH) activity in the kidney and liver. Epoxide hydratase (EH) activity was increased in the liver but decreased in the kidney following exposure to PBB. A three-month exposure to PBB had no effect on blood urea nitrogen, the clearance of inulin,p-aminohippurate (PAH), or fractional sodium excretion. Similarly, the in vitro accumulation of PAH and Nmethylnicotinamide (NMN) in thin renal cortical slices and ammoniagenesis and gluconeogenesis in renal cortical slices were not affected by PBB. In conclusion, chronic exposure to PBB resulted in significant alterations in renal and hepatic microsomal enzyme activities but had no detectable effect on renal function. These experiments suggest that alterations in microsomal enzyme activities following PBB do not lead to impairment of renal function; however, this compound may sensitize the kidney to toxicity produced by agents administered subsequent to PBB.

Polychlorinated biphenyls (PCB) are ubiquitous environmental contaminants that persist in the food chain (Bisebrough et al., 1968) and cause chloracne (Meigs et al., 1954) and a set of symptoms termed Yusho disease(Kuratsune et al., 1972) in humans. Of concern are the structurally analogous polybrominated biphenyls (PBB) which recently contaminated livestock and poultry feed in Michigan (Carter, 1976). The commercial product added to the feed was Firemaster BP-6, a mixture of PBB congeners (tetrabromo to octabromo) and the major component of which is 2,2’,4,4’5,5’-hexabromobiphenyl (Sundstrom et al., 1976). Although at present no ’ Present address: CIIT, P.O. Box 12137, Research Triangle Park, North Carolina 27709. *Author to whom correspondence should be sent to Department of Pharmacology, Michigan University, East Lansing, Michigan 48824. 539

State

0041-008X/78/0443-0539502.00/0 Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

540

McCORMACK

ET AL.

human health effects have been unequivocally attributed to PBB, dietary exposure to PBB results in a variety of toxic manifestations in animals.3 Cows that consumed feed containing PBB exhibited toxic signs including anorexia, decreased milk production, and increased frequency of urination (Jackson and Halbert, 1974). Interstitial nephritis and gross pathological changes including renal and hepatic degeneration, and hematomas and abcesses in the peritoneal and thoracic cavities were also observed in these animals (Jackson and Halbert, 1974). Rats fed 100 ppm of PBB have enlarged livers and hepatic lesions consisting of swelling, centrilobular cytoplasmic myelin bodies, and vacuolation (Aftmosmis et al., 1972; Norris et al., 1974; Corbett et al., 1975; Sleight and Sanger, 1976; Report, 19763). Some investigations have revealed that rats exposed to 100 ppm of PBB have kidney enlargement and renal lesions such as petechial hemorrhage and hyaline degenerative cytoplasmic changes (Aftmosmis et al., 1972; Norris et al., 1974; Corbett et aZ., 1975) as well as increased urinary protein (Sleight and Sanger, 1976). However, other investigators have not observed an increase in kidney weight (Sleight and Sanger, 1976; K. M. McCormack, S. Z. Cagen, D. E. Rickert, J. E. Gibson. and J. G. Dent, unpublished observations) or renal histopathological alterations (Sleight and Sanger, 1976). Polybrominated biphenyls and polychlorinated biphenyls represent a class of hepatic mixed function oxidase stimulators which exhibit characteristics of both phenobarbital (PB) and 3-methylcholanthrene (3MC) (Alvares et al., 1973; Stonard, 1975; Dent et al., 1977a,b), two agents which are distinct in their inducing effects (Sladek and Mannering, 1969; Lu et al., 1972). Polybrominated biphenyls also alter microsomal enzyme activities in rat kidney (K. M. McCormack, S. Z. Cagen, D. E. Rickert, J. E. Gibson, and J. G. Dent, unpublished observations). Significant concentrations of PBB were detected in kidney and liver following treatment with PBB (Rickert ef al., 1977). Since renal and hepatic histopathological and enzymatic alterations have been observed and drug elimination from plasma is enhanced (Cagen et al., 1977) following treatment with PBB, excretory function may be modified following dietary exposure to PBB. The effects of prolonged dietary exposure to PBB on the kidney and liver are not fully known. Available data are inadequate for assessment of renal and hepatic excretory function following dietary exposure to PBB. Thus, the purpose of this investigation was to determine renal function as well as mixed function oxidase activities, histopathological alterations, and concentrations of PBB in kidney and liver after 3 months of dietary exposure to PBB. METHODS Adult female Sprague-Dawley rats (Spartan Farms, Haslett, Michigan) were maintained in clear polypropylene cages at 22OC with a 12-hr light cycle (0700-1900 hr) and were allowed free access to food (Wayne Lab Blox) and water. After 3 days of acclimation, the experimental diet containing 0 or 100 ppm of PBB (Firemaster PB6, Michigan Chemical Co., St. Louis, Michigan) was substituted for the Lab Blox. Diet and tissues were prepared and analyzed for PBB content as previously described (Dent 3 Report. (1976). Final report of the subcommittee on the health effects of polychlorinated biphenyls polybrominated biphenyls. Department of Health, Education and Welfare, Washington, D.C.

and

PBB

AND

RENAL

FUNCTION

541

et al., 1977b). Enzyme assays, renal functional tests, tissue PBB quantification, and histologic examinations were performed after 3 months of exposure to the experimental diet. Animals used for enzyme assays were sacrificed by cervical dislocation and kidneys and livers were excised, weighed, and then chopped into ice=cold 66 mM Tris buffered to pH 7.4 with HCl (kidneys) or 1.15% KC1 (livers). Kidney postmitochondrial supernatants were prepared by homogenization (Potter-Elvehjem homogenizer with a Teflon pestle) in 3 vol of 66 mM Tris (pH 7.4) followed by centrifugation at 10,OOOg for 20 min. Liver microsomes were prepared (Netter, 1960) and resuspended (Dent et al., 1976). All assays were performed on the day of supernatant and microsomal preparation. Protein was measured (Lowry et al., 195 1) using bovine serum albumin as a standard. Enzyme activities measured were epoxide hydratase (EH) (Oesch et al., 1971) arylhydrocarbon hydroxylase (AHH) (Nebert and Gelboin, 1968; Oesch, 1976) and biphenyl-2-hydroxylase and biphenyl-4-hydroxylase (BP-2-OH and BP-4-OH) (Creaven et al., 1965). Animals used for histologic examination were sacrificed by cervical dislocation and pieces of liver and kidney were immediately cut and fixed in 10% buffered formalin. After fixation, these were embedded in paraffin, sectioned at 5 pm and stained with hematoxylin and eosin. Animals used to determine renal function in vitro were sacrificed by cervical dislocation and the kidneys were quickly removed, weighed, and placed into ice-cold normal saline. Organic ion transport capacity was estimated using renal cortical slices. Transport capacity was quantified as the ability of renal cortical slices to accumulate a representative anion, p-aminohippuric acid (PAH), and cation, N-methylnicotinamide (NMN) (Cross and Taggart, 1950; Smith et al., 1945). Results were expressed as the slice-to-medium (S/M) concentration ratio, where S equals milligrams per gram of wet tissue weight and A4 equals milligrams per milliliter of medium. The ability of renal cortical slices to form ammonia and glucose was also determined (Roobol and Alleyne, 1974; Kaplan, 1965). Net production of ammonia and glucose was expressed as micromoles per milligram of wet tissue weight per hour. To determine renal function in vim, animals were anesthetized with 50 mg/kg of sodium pentobarbital ip, and body temperature was maintained at 36 to 38°C using heat lamps. A PE50 cannula was inserted into the bladder and urine was collected under mineral oil in preweighed vials. The left femoral vein was cannulated for infusion. Both femoral arteries were cannulated to monitor blood pressure using a Statham transducer and a Beckman type RS dynograph and to obtain blood samples. The infusion solution contained 1% inulin and 0.6% PAH in normal saline. [3H]Inulin (0.5 &i/ml) and 114C]PAH (0.5 @i/ml) were added to the solution, which was infused at 0.019 ml/min using a Harvard infusion pump. A minimum of 1.5 hr elapsed from the beginning of the infusion to initiation of urine collection. A total of four 30-min urine samples was taken. Blood (400 ~1) was sampled at the middle of each urine collection. Following the initial clearance period, animals were infused with 1 : 4 rat plasma--saline solution (4% body weight) and three additional 30-min collections were taken. I 14CIPAH, 13H)inulin, and sodium concentrations were determined as previously described (Pegg et al., 1976). Mean systolic blood pressure was measured indirectly on unanesthetized animals using tail plethysmography. Blood urea nitrogen (BUN) was

542

McCORMACK

ET AL

determined (Kaplan, 1965) and expressed as milligrams of urea nitrogen per 100 ml of whole blood. Data were analyzed statistically by randomized complete block analysis of variance. Treatment differences were detected by the least significant difference test (Steel and Torrie, 1960). The 0.05 level of probability was used as the criterion of significance. RESULTS

Significant PBB concentrations were detected in the kidney and liver following 3 months of dietary exposure to PBB (Table I). Three months of dietary exposure to 100 ppm PBB; retarded weight gain and increased the liver weight to body weight ratio but did not alter the kidney to body weight ratio (Fig. 1). Renal postmitochondrial supernatant protein content was not affected by treatment with PBB; however, the administration of PBB significantly altered the relative activities TABLE TISSUE

PBB

I

CONCENTRATIONS TO RATS~

AFTER

PBB-FEEDING

Tissue Treatment

Control 100 ppm

Kidney NDb

of PBB

6.66 + 4.30’

Liver ND

369.00 + 55.05’

’ Values given are the means + SE for three animals. The units are micrograms per gram of wet tissue weight. b Not detectable. ’ A statistically significant difference from the respective control,p < 0.05.

of all renal enzymes investigated (Table 2). The activities of arylhydrocarbon hydroxylase ( 1116% of control value), biphenyl-2-hydroxylase (1100% of control value), and biphenyl-4-hydroxylase (550% of control value) were increased above controls while epoxide hydratase activity (14% of control value) was decreased below the control value. Microsomal protein was increased in livers of PBB-treated animals (132% of control value), as were the relative activities of all hepatic enzymes investigated (Table 2). AHH, EH, BP-2-OH, and BP-4-OH activities were 9242%, 214%, 23,538%, and 1598% of control values, respectively. Liver degenerative changes were observed following exposure to PBB. Hepatic cells were uniformly enlarged so that sinusoids appeared only as small, clear spaces with an occasional Kupffer cell visible. Many cells were vacuolated (Fig. 2). Focal necrosis was found occasionally, but midzonal necrosis was most apparent. Hepatic cells were disrupted and nuclei were absent in some cells and pycnotic in others. Many cells contained large myelin bodies. Some of these bodies were uniform in appearance, while others appeared to have a lighter, central mass surrounded by a wide, darker band (Fig. 3).

PBB

AND

RENAL

FUNCTION

543

8.0

SD

4.0

6 x 3.0 5 E 3 2.0 to

0.8

0.8

0.4

0.2

300

250

200

150

loo

50

FIG. 1. Effect of PBB on liver to body weight ratio, kidney to body weight ratio, and body weight. Rats were fed a diet containing 100 ppm of PBB for 3 months. Values given are the means ? SE for four animals. Asterisks indicate a statistically significant difference from the respective control,p < 0.05.

McCORMACK

ET AL

PBB

AND

RENAL

FUNCTION

545

Renal changes included progressive obsolescence of glomeruli. Glomerular tufts were shrunken and inactive or had been largely replaced by scar tissue. Bowman’s membrane, while not thickened, was shrinking proportionately to the shrinking tuft (Fig. 4). A single focus of lymphocytes was seen in one kidney. Exposure to PBB had no effect on the in vitro accumulation of PAH and NMN by thin renal cortical slices or ammoniagenesis and gluconeogenesis by renal slices (Table 3).

FIG. 2. Hepatic tissue from rat fed a diet containing Hematoxylin and eosin stain. x 100.

100 ppm PBB for 3 months.

There is vacuolation.

The clearance of inulin (glomerular filtration rate) and the clearance of PAH (effective renal plasma flow) were unaffected by treatment with 100 ppm of PBB (Table 4). Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were not different from controls before or after volume expansion (Table 4). Filtration fraction (GFR/ERPF) was unaffected by PBB. Urine flow rates (microliters per minute) were not different between control and PBB-treated animals. Fractional sodium excretion in control and PBB-exposed animals was not significantly different before or following

546

McCORMACK

ETAL.

TABLE

3

EFFECT OF PBB-FEEDING ON AMMONIAGENESIS, GLIJCONEOGENESIS,

AND THE ACCUMULATION AND NMN INRATS

OF

PAH

Net production @mol/mg of wet tissue wt/hr) Treatment

Ammonia

Control 100 ppm of PBB

Glucose

3.39 t 0.09 3.45 & 0.09

0.03 + 0.01 0.03 + 0.01

S/M Ratio Treatment Control 100 ppm of PBB

PAH

NMN

10.49 * 0.43 10.16 & 0.76

5.79 * 0.10 5.20 + 0.17

0 Values given are the means _+SE for four animals.

TABLE EFFECT

OF

4

PBB-FEEDING ON GLOMERULAR EFFECTIVERENALPLASMAFLOW

FILTRATION

GFR (ml/min) Periodb I

II III IV

2.46 2.86 2.77 2.40

& 0.16 + 0.5 1 + 0.59 If: 0.24

2.34 3.14 2.51 1.95

+ & + &

Control

0.21 0.43 0.17 0.27

10.87 10.95 9.61 10.39

+ k + *

“Values given are the means f SEforfour animals. b Rat plasma-saline (4?6 of body wt) was infused after Period

TABLE EFFECT

OF

Control 100 ppm of PBB

Unanesthetized 122.0 * 3.03 121.5 + 2.40

a Values given are the means units are millimeters of Hg.

100 ppm of PBB

0.63 1.94 1.54 ,1.77

8.77 10.67 9.05 10.66

I.

5

PBB-FEEDING ON BLOODPRESSUREINRATS~

Treatment

AND

ERPF (ml/min)

100 ppm of PBB

Control

(GFR)

RATE

(ERPF) INRATS~

+ SE for

MEAN

SYSTOLIC

Anesthetized 97.50 i 4.33 94.50 k 5.27 four

animals.

The

+ 0.97 _+ 0.78 + 1.73 k 1.95

PBB

FIG. 3. Hepatic bodies. Hematoxylin

AND

RENAL

tissue from rat fed a diet containing and oesin stain. x400.

541

FUNCTION

100 ppm of PBB for 3 months.

There

are inclusion

volume expansion (Fig. 5). Similarly, blood urea nitrogen (BUN) (Fig. 6) and mean systolic blood pressure of either unanesthetized or anesthetized animals (Table 5) were not affected by PBB. DISCUSSION

Previous studies have revealed that PBBs are potent stimulators of both renal and hepatic microsomal mixed function oxidases in rats (Dent et al., 1976, 1977a,b; K. M. McCormack, S. Z. Cagen, D. E. Rickert, J. E. Gibson, and J. G. Dent, unpublished observations). Stimulation produced by PBB is similar to both phenobarbital and 3methylcholanthrene, which represent two different classes of enzyme inducers (Lu et al., 1972; Sladek and Mannering, 1969). The results of this investigation are consistent with these observations in that 3 months of dietary exposure to PBB markedly altered microsomal drug-metabolizing capabilities in liver and kidney.

548

McCORMACK

ET AL.

Hepatic enzymes sensitive to treatment with the cytochrome P-450~inducing agent phenobarbital (EH and BP4-OH) and those hepatic enzymes sensitive to treatment with the cytochrome P,-450-inducing agent 3-methylcholanthrene (AHH and BP-2OH) were all stimulated above control values following dietary exposure to PBB. Hepatic EH activity was similar to that observed in liver of lactating rats fed 50 ppm of PBB for 28 days (Dent et al., 1977b). However, AHH activity was 10 times higher, BP-

FIG. 4. Renal tissue from rat fed a diet containing and fibrotic glomeruli. Hematoxylin and eosin stain.

x

100 ppm of PBB for 3 months. 100.

There are shrunken

4-OH activity was 2 times higher and BP-2-OH activity was several orders of magnitude higher than activities observed in lactating rat liver (Dent et al., 1977b). These results confirm the suggestion that PBB are potent modifiers of microsomal xenobiotic-metabolizing enzymes in the liver. Arylhydrocarbon hydroxylase, BP-2-OH, and BP-COH were also stimulated above control values in the kidney following treatment with PBB. Renal EH activity, however, was significantly decreased following the administration of PBB. Stimulation of AHH with concomitant inhibition of EH in the kidney may be of toxicological concern.

3. ! PBB

AND

RENAL

549

FUNCTION

- - oppmmm -

100ppmPBBs

2

b I\ \

2

1.5

1.0

0.

a5

1.0 TIME( hrs)

1.5

2.0

FIG. 5. Effect of PBB on fractional sodium excretion. Rats were fed a diet containing 100 ppm of PBB for 3 months. After control determination, animals were volume expanded with 1 :4 rat plasma-saline (4% body weight) and three additional determinations were made. Values given are the means *SE for four animals. CONTROL looppm

FIG. 6. Effect of PBB on blood urea nitrogen. months. Values are means &SE for four animals.

Rats were fed a diet containing

PBBS

100 ppm.

PBB for 3

550

McCORMACK

ET AL

Modification of mixed function oxidase activities can result in altered susceptibility to toxic xenobiotics administered secondarily (Brodie et al., 1971; Daly et al., 1972; Oesch, 1973). The expression of toxicity following treatment with a secondary toxic agent may be dependent on the relative activities of various drug-metabolizing enzymes. Microsomal enzymes such as AHH may catalyze the formation of reactive arene oxides from inert aromatic compounds, which may then bind to cell components and elicit a toxic response (Daly et al., 1972; Oesch, 1973). Electrophilic metabolites, however, may be further metabolized to less reactive compounds. Epoxide hydratase may transform arene oxides to fess toxic dihydrodiols (Oesch, 1973). Therefore, since AHH activity is increased while EH activity is decreased in the kidney following exposure to PBB, secondary treatment with an agent metabolized by these enzymes may result in enhanced nephrotoxicity. Polybrominated biphenyls fed at 100 ppm for a 3-month period caused extensive hepatic necrosis. Vacuolation was common and in many cells nuclei were absent or pycnotic. Myelin bodies and clumped cytoplasm were also frequently observed, as was fibrosis. The effect of PBB was most severe in the midzonal regions of the lobule in contrast to the preponderance of centrilobular degenerative changes observed following shorter periods of exposure to 100 ppm of PBB (Aftmosis et al., 1972; Norris et al., 1974; Sleight and Sanger, 1976). Liver to body weight ratio was increased after treatment with PBB, which is consistent with previous reports (Dent et al., 1976; Sleight and Sanger, 1976) and reflects the histopathological alterations present in the liver following exposure to PBB. Weight gain was retarded by exposure to PBB, which may be due to decreased food consumption efficiency4 or anorexia; however, food consumption was not monitored. Increased hepatic microsomal enzyme activities and impaired mitochondrial function in the liver following treatment with PBB3p4 may contribute to this effect. Kidney to body weight ratio was not altered by exposure to PBB. Although glomerular degenerative changes were observed following PBB, similar changes were seen in control animals but less frequently. The significance of this quantitative difference in histopathological alterations is not readily apparent. Blood urea nitrogen is a standard measurement that pertains principally to glomerular function, and as filtration diminishes, the BUN increases. A three-month exposure to 100 ppm of PBB had no effect on BUN, which is consistent with its lack of effect on GFR prior to or following volume expansion. Similarly, ERPF and the filtration fraction (GFR/ERPF) were unaffected by PBB. In addition, mean systemic blood pressure was not affected by treatment with PBB. Nephrotoxic agents often modify the ability of the kidney to concentrate the urine and also affect the volume of urine excreted (Berndt, 1976). Sodium transport might also be altered by a substance that influences renal function. In these experiments, urine flow rates and fractional sodium excretion in control and PBB-exposed animals did not differ before or after stressful volume expansion. ’ Garthoff, L. H., Friedman, L., Hurley, N., Farber, T. M., Peters, E. L., Locke, K. K., Green, S., Sobotha, T. J., Moreland, F., Keys, J., Scalera, J., Rothlein, J., Taylor, M. J., Story, G., Graham, C. H., Marks, E., Cerra, F., and Sporn, E. M. (1975). Biochemical and cytogenetic effects caused by ingestion of Arochlor 1254 or Firemaster BP-6. Testimony of Dr. A. C. Kolbye at the Polybrominated Biphenyl Conference, Michigan Department of Agriculture, Lansing, Michigan, May 29.

PBB

AND

RENAL

FUNCTION

551

The in vitro renal slice technique may be more sensitive than in vivo whole body methods for determination of nephrotoxic effects on renal transport processesbecause blood flow effects are eliminated (Berndt, 1976; Hirsch, 1976). Furthermore, this in vitro technique had been routinely employed to determine the effect of chemicals on renal organic acid (PAH) and organic base (NMN) transport as well as on renal gluconeogenesisand ammoniagenesis(Hirsch, 1976). Treatment with PBB had no significant effect on any of these in vitro parametersof renal function concordant with in vivo results. Three months of dietary exposure to PBB markedly altered microsomal mixed function oxidase activities and caused histopathological changes in both liver and kidneys. Liver was affected more severely than kidney following treatment with PBB. This may relate in part to tissue distribution, as the concentration of PBB in liver was more than 50 times that of kidney concentrations. Alterations in enzyme activities and structural integrity were not correlated to changes in renal function determined by a variety of specific tests. This indicates that PBB may not be a potent nephrotoxic agent. However, the characteristics of microsomal enzyme stimulation in the kidney following treatment with PBB suggeststhat this compound may sensitize the kidney to toxicity produced by agents administeredsubsequentto dietary exposure to PBB.

ACKNOWLEDGMENTS This investigationwas supportedin part by the National Institute of EnvironmentalHealth Sciences,Grant No. ESO0560.We wishto expressour appreciationto C. Herrmann,L. Lepper, and B. Schoepkefor their excellenttechnicalassistance. We alsowishto thank D. Hummelfor her helpin preparingthe manuscript. REFERENCES J. G., CIJLIK, R., AND Lu, K. P. (1972).Toxicology of brominatedbiphenyls:I, Oral toxicity andembryotoxicity. Toxicol. Appl. Pharmacol. 22, 3 16. ALVARES, A. P., BICKERS, D. R., AND KAPPAS, A. (1973). Polycblorinatedbiphenyls:A new type of inducerof cytochromeP-448 in the liver. Proc. Nat. Acad. Sci. USA 70, 1321-1325. BERNDT, W. 0. (1976). Renalfunction tests:What do they mean?A review of renal anatomy, biochemistry,and physiology.Environ. Health Perspec. 15, 55-71. AFTMOSIS,

BRODIE,

B. B., REID, W. D., CHO, A. K., SIPES, G., KRISHNA, G. AND GILLETTE, J. R. (1971).

Possiblemechanismof liver necrosiscausedby aromatic organic compounds.Proc. Nat. Acad. Sci. USA 68, 160-164. CAGEN, S. Z., PREACHE, M. M., AND GIBSON, J. E. (1977). Enhanceddisappearance of drugs from plasmafollowingpolybrominatedbiphenyls.Toxicol. Appl. Pharmacol. 40,3 17-325. CARTER, L. J. (1976).Michigan’sPBB incident:Chemicalmix-up leadsto disaster.Science 192, 240-243. CORBETT, T. H., BEAUDOIN, A. R., CORNELL, R. G., ANVER, M. R., SCHUMACHER, R., ENDRES, J., AND SZWAKOWSKA, M. (1975).A toxicity of polybrominatedbiphenyls(FiremasterBP-6) in rodents.Environ. Res. 10,390-396. CREAVEN, P. J., PARKE, D. V., AND WILLIAMS, R. T. (1965). A fluorimetric study of the hydroxylation of biphenyl in vitro by liver preparationsof various species.Biochem. J. 96, 879-885. CROSS, R. J., AND TAGGART, J. V. (1950).Renaltubular transport: Accumulationof p-aminohippurateby rabbit kidney slices.Amer. J. Physiol. 161, 181-190.

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