Toxicity and microsomal enzyme induction effects of several polybrominated biphenyls of firemaster

F U N D A M E N T A L A N D APPLIED T O X I C O L O G Y 2"313-321 i1~82)

Toxicity and Microsomal Enzyme Induction Effects of Several Polybrominated Biphenyls of Firemaster' GHAZ! A. DANNAN, STUART D. SLEIGHT and STEVEN D. AUST2 Departments of Biochemittry and Pathology, Michigan State University, Ea~t Lansing, MI 48824

ABSTRACT

Toxicity and Microsomal Enzyme Induction Effects of Several Polybrominated Biphenyls of Firemaster. Dannan, G.A., Sleight, S.D, and Aust, S.D. (1982). Fundam. Appl. ToxicoL 2:313-321. Some toxicological and pharmacological effects of 2,4,5,2',5'-penta- (congener !). 2.3.4,2'.4',5'hexa- (congener 5), 2.4.5,3'.4',5'-hexa- (congener 6), 2,3,4,5,3',4',-hexa- (congener 7)° and 2,3,4,5,2',Y,4'-heptabromobiphenyl (congener 9) were evaluated in male rats given a single 90 mg/kg ip injection and killed seven days later. Only congener 7 depressed body weight gain, spleen and thymus weights, and caused severe histopathologicai changes in the thymus. Congener 7 caused the largest increase in liver weight and the most changes in liver pathology while congener I failed to enlarge this organ and caused the mildest ultrastructural changes. Liver microsomes v, ere isolated and evaluated for enzyme induction from =11treated rats except those administered congener 6. which was previously identified as a mixed-type enzyme inducer (Dannan et el., 1978b). All congeners increased the liver microsomal cytochrome P-450 content, but only congener 7 shifted the carbon monoxide difference spectrum absorption maximum to 448.0 nm. The microsomal ethyl isocyanide difference spectrum 455/430 nm ratio was increased the most by congener 7 (3 fold). All congeners increased c~tochrome P-450 reductase and microsomal epoxide hydrase activities by nearly !.5-3 fold. Congener 7 failed to induce aminopyrine-N-demethylase activity but the remaining congeners increased it by 2 fold. Congener 7 was the most effective inducer of benzola]pyrene hydroxylase and p-nitrophenol UDP-glucuronyl transferase. These results add to the suggestion that the presence of an ortho halogen on a polyhalogenated biphenyi does not completely abolish toxicity or 3-methylcholanthrenc-type microsomal enzyme induction. INTRODUCTION Firemaster is a complex mixture of brominated biphenyls (PBB) which contaminated the environment and people of Michigan (Carter, 1976) and probably other areas (DiCarlo. 1978). The mixture has twelve to fourteen major PBB congeners, ten of which have been structurally identifi(,d (Moore et al., 1980; Moore and Aust, 1978; Moore et el., 1978a; Dannan et el., 1982a). The mixture causes a mixed-type induction of liver microsomal drug metabolizing enzymes similar to the combined effects of phenobarbital (PB) and 3-methylcholanthzPa.¢t o| ~.h~ ¢¢,~¢=¢ch v, l t pzcscnfed at the ASPET Fall Mcct;ng. Rochctt¢¢. Mmne$otao Aufwtt 17-21. t980. Ab'.l. No. 14. =To ~hom correspondcnc¢ should be addre,~cd at the Department of Biechcmictry.

rene ¢MC) (Dent et al., 1976a.b). Firemaster also causes a number of toxic effects that are typically associated with similar Dolyhalogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCB} and 2,3.7,8-t etrachlorod,benzo-pdioxin (TCDD), except the dose required to cause the typ=cal toxic effects is quite higher than that needed of TCDD (Parkinson and Safe, 1981). In addition to liver enlargement and porphyria, neoplastic nodules were seen at high doses of Firemaster (Strik. 1978; Kimbrough eta/., 1978). The mixture also suppresses the immune response in mice and rats (Fraker, 1980; Fraker end Aust, 1978; Dannan et aL, 198213) and causes chicken edema and reduced egg hatchability (Polin et el., 1979). All of the PBB congeners, however, are not eqoally effective in producing these and other related biological effects, For example, a m o n o . o r t h o brominated congener (2,4,5.3'.4'pentabromobiphenyl) was found to be predominantly an MCtype inducer and relatively toxic since it was immunosuppressive and gave rise to somewhat unique h=stopathological and ultrastructural changes in the liver (Dannan et aL, 1982b} A mixed-type induction of liver microsomal er~zymeswas caused by another m o n o . o r t h o substituted congener, 2,4.5,3'.4'.5 'hexabromobiphenyl (congener 6) (Dannan e t a / . , 1978b), which was nearly as toxic te the immune system of inbred mice as Firemaster (Fraker and Aust, 1978). Three other PBB congeners, making up about 75% of Firemaster, with two halogens on corbons ortho to the biphenyl bridge, are strict PB-like inducers and cause a dramatic proliferation of the hepatic endoplasmic reticulum but no remarkable toxicities (Moore et el., 1978b, 1979; Besaw et el., 1978, Render et al., 1982). There are two reasons for characterizing the biolog;cal effects of the individual congeners of the mixture. First, it is estimated that nearly 90% of Michigan's residents~re conta minated with PBB which, due to their persistence, are expected to remain in their tissues for many years (WoHf et el., 1982). Since most human exposure occurred indirectly through contaminated meat and dairy products the possibility exists that the toxic PBB components may have been concentrated in the consumed food products. The composition of PBB extracted from the liver and milk of rats fed Firemaster was considerably different than that of the original mixture (Dannan et eL, 1978a). Therefore, studies with purified PBB congeners will help in predicting the toxicity and pharmacological effects related to human or environmental contaminations with PBB. Secondly, they will help develop the structureactivity relationships for toxicity and the type of microsomal enzyme induction.

Col~fril~ht 1~120So¢ltty of To=l¢ololcy Fund,,ment,,t

and Applied T o x i c o l o l w

(2) 11.12/82

313

D A N N A N . SLEIGHT A N D A U S T

We have examined four additional congeners of Firemaster for their toxicity, histopathological effects and type of hepatic microsomal drug metabolizing enzyme inductiort. These are 2.4.5,2'.5'-penta-. 2.3.4.2',4',5'-hexa-. 2,3,4,5,3",4'-hexa-, and 2.3,4.5,2",3".4"-hepta-bromobiphenyl. They will also be referred to as congeners 1, 5, 7, and 9, respectively, according to their order of elution during gas chromatography (Dannan et eL, 1992a). Some hepatic ultrastructural results from the administration of 2.4,5.3'.4",5'-hexabromobiphenyl (congener 6), previously identified as a mixed-type inducer (Dannan et aL, 1978b), are also presented. t

s 12

I0 I=

Rr

/

Br

Br

Br

D] Br Br

Br

B r ~ B r Br

/[5]

Jr

Br

Br er

FIG. I. Ca ~, ciwomatographic el~tlor~ prof~(es and ~trt=ctutes of Firema~ler FF-I, and purified c o n g e n e r / , cont;ener.S, conl~ener 7. and congener 9, Approxim,~¢ely 2, 0.2. 0.3, 0.6 and 0.8 ng of Firemaster FF-I and each or"the respective congeners was,iniected into an electron rapt ure detector (~Ni} equipped GC with a 6 ft. column packed with'3% OV-I. The iniector port. column, and detector were maintained at 280, 245, and 3.50°C, respectively; at tenuatlon, I0X Ib (amplmV). 314

METHODS Chemicals Polyethylene glycol (PEG. molecular weight approximately 400), PB and MC were purchased from Sigma Chemical Company, St. Louis, Missouri. All PBB congeners employed in this study were purified from Firemaster BP-6 (lot no. 6224-A) which was manufactured by Michigan Chemical Corporation. St. Louis, Michigan (Dannan eta/., 1982a). The GC elution profiles of Firemaster FF-1, which allegedly was involved in Michigan's contamination accident, and of purified congeners I, 5, 7, and 9 are shown in Fig, 1. Congener 6, whose GC elution profile is not presented but was studied previously (Oannan et al,. 1978b), was over 99% pure. All samples were also analyzed by GC/MS (Hewlett-Packard 5840A/5985) selective ion monitoring for brominated dibenzo-p-dioxins, napl~thalenes, and d;benzofurans. At nearly 1/ 1000 detection limit (2 pg PBB injected) none of the ions of concern could be identified. ~H-NMR spectral studies and assigned structures were i~reviously reported (Moore and Aust, 1978; Moore et eL, 197Ba, 1980; Dannan et el., 1982a). Animals Outbred male Sprague-Dawley rats (100 - 125 g) wece purchased from Spartan Research Animals, Inc., Haslett. Michigan and allowed an acclimation period of at least 48 hours before any treatments. Congeners 1, 5, and 9 were dissolved in PEG at a concentration of 30 mg/mL, while congeners 6 and 7 were dissolved at 22.5 m g / m L of the same vehicle (these two congeners are less soluble in PEG than the former): Each congener was administered into four rats one week before sacrifice as a single ip injection of 90 mg/kg body weight. PEG was also injected into four control rats at 3 mL/kg. All rats were given free access to feed and water except for the night before sacrifice when feed was removed. Pathological and ultrastructural evaluations One week after treatment, the rats were weighed and killed by asphyxiation with CO~. Liver, spleen and thymus weights were recorded for each rat. Tissue samples from the Hver, spleen, kidney, thyroid, thymus, trachea, lung, heart, adrenal, pancreas, and small intestine were fixed in 10% buffered forma|in, embedded in paraffin and sectioned at 5 ,~m. All tissue sections were stained with hematoxylin (Hi and eosin (El and the liver sections were also stained with oil red O. Prior to examination by electron microscopy the formalin-fixed liver tissues were postfixed in I% osmium and stained with uranyl acetate and lead citrate. Isolation o f microsomes Liver microsomes from each rat were isolated, washed, and stored as described before (Moore et el., 1978b).

Fundam. Appl. ToxlcoL (2)

Not'ember~December. 1982

T O X I C O L O G Y O F PRB C O N G E N E R S TABLE 1 B o d y a n d O r g a n W e i g h t s of R a t s A d m i n i s t e r e d

Average Daily Treatment

V a r i o u s PBB C o n g e n e r s ^

% of Body Weight

Body Weight Cain (g)

Liver

Spleen

Thymus,

Control

5.07:1 0.44

4.It 4" O.t4

O40.1"O.O5

0.J3 "LO.O3

2.4,5.2".5 % (Con~ener I}

.l.lt~O.3O

4.2o:~0l o

040"J. 0.05

0.44:t 0.04

2.3,4.2".4"05"( C o n g c p e r .~

.t.50~0.5o

531 "e057"

0.44 ~ 0 0 o

0.42 .:t: 0.03

2.3,4,5.3".4" (Con~4enet 7l

0.57 :t l.St n

O.5t~:t 0.-12"

0 3-1 ± 0.03l~

0.lo :t: 0.02n

2,3,4,5.2",3",4'.

4.20 :t 0.54

5.12.~" 0.2o"

0 48"L" 0 I0

0.3.t -t. 0 0 0

(Congener O) AEour r,~t~,rer tre.,rmont t'.'ere .~Jmim.,,lered a si~.lo q', iniectlon of 3 mL I'EGIk~ (Control)oteitherofthePBBcon~ener,,q oOm~tk~om, tseekbefole.,.~ri|we D.~t.t ,lfU ~'*~prc'.,.~,ed,1~nttoo~n~ SD. l*S=gnificantly difle~ent from m~,ar~t,t ¢o~trol u,,ivg ~t~klt,z~t'~,I-te,.t {p<0 05L E n z y m e assays Using the method of Omura and Sate (1964), cytochrome P-450 was assayed in 10% glycerol, while NADPH-cytochrome c reductase was assayed according to the method of Pederson et el. (1973L Aminopyrine demethylation was assayed as previously described (Moore et al., 1978b) and benzo[a]pyrene hydroxylation was assayed according to the method of Gielen et el. (1972). Epoxide hydrase was assayed by the method of Oesch et af. (1971 ), and the method of Lucim et at. (I 977) was used to assay the UDP-glycuronyltransferase activity with p-nitrop~enol. Ethyl isocyanide (EtNC) difference spectra were recorded as previously described (Dannan et aL, 1982b). RESULTS Congeners jr 5. 7, and 9 w e r e evaluated for their acute effects on the histologic and ultrastructural appearance of selected organs and for their induction effects on hepatic dr ug metabolizing enzymes. Congener 6, previously shown as a mixed-type (Dar,nan et aL, 1978b) is presently assessed only for it.~ effects on hepatic ultrastructure. Histological sections from the heart, spleen, kidney, stomach, small intestine, pancreas, testis, thyroid, and brain were aoparontty normal from all rats treated with any of the first four congeners. Congener 1 caused the least apparent histopathological changes in the liver, and liver weight was not significantly altered (Table 1 ). Rats pretreated with this congener had somewhat swollen hepatocytes and less apparent sinusoids, but like control beers, cytoplasmic vacuoles were not apparent (Fig. 2B). Although the three other congeners significantly increased liver weight. congener 7 had the most effect (60% increase). The most severe histopathological changes also resulted from this congener (Fig. 2D) w h i l e the least ~evaru effects were caused by congener 1 (Fig. 2g). Most treatments affected all portions of the liver Iobules. Congener 7 caused the most severe changes in the centralobular and tradznnal areas. Increased amounts of fat in liver of rats given any of the four treatments were confirmed by oil red O stain. Fat was especially prominent in the liver of rats given congener 7. Variable degrees of ultrastructu. ra! changes were also evident in electron micrographs of hepatocytes from rats administered all congeners including congener 6 (Figs. 3 and 4). All treatments caused proliferation

Fundsmentll =nd Applied Toxicology

(2) 11-12/82

of the smooth endoplasmic reticulum (SER) and increased the number of cytoplasmic vacuoles, but none caused any aporeciable changes in the mitochondria. The most pronounced and unique changes, t~owever, resulted from congener 7. In agreement with the histopathological findings in hepatocytes from rats treated with congener 7(Fig. 2D), the subcellular architecture of these hepatocytes was s;gnificantly altered (F;g. 3D). Mitochondria seemed to aggregate around the nucleus and were surrounded by the bulk of the proliferated SER, and laminated membranes seemed to form concentric arrays around one or more of the lipid vacuoles (Fig. 4A). Congener 1 seemed to cause the mildost cytoplasmic changes which were limited to a moderate increase in the SER and the n=~mbe,"of cytoplasmic vacuoles (Fig. 3BL Both congener 5 and congener 9 caused intermediate effects that were limited to proliferation of the SER and cytoplasmic vacuolation (Fsgs. 3C and 3E). Uitrastructural change~due to congener 6 were apparently similar to those caused .~w,,~.ngenor 7 but whorles of laminated membranes enclosing h~id vacuoles or mitochondria were. somewhat more apparent~Fig. 4B). Of all treatments, only congener 7 caused thymtc atrophy and decreased body weight gain. The average daily body weight gain of rats administered this congener was only 10% of control (Table 1). There was a 60% loss in thymic weight (Table 1 ) and a severe diminution in the number of thymocytec especially in the cortex (F,g. 5). The boundary between the cortex and medulla was indistinct. Macrophages could also be observed in thp remaining portion of the cortex. The hepatic microsomal drug metabolizing enzymes were also evaluated to characterize the type of induction caused by each congener (congener 6 was previously characterized as a mixed-type inducer) so that toxicity may be correlated with enzyme induction. Cytochrome c reductase activity and cytochrome P-450 content were invariably increased by all four congeners (Table 2). The reductase was increased by nearly 55% by congener ! and congener 7, whereas congener 5 and congener 9 doubled this enzyme activity. While all congeners induced cytochrome P-450 by 2-3 fold, congener 7 shifted the absorption maximum of the CO difference spectrum (Xmax) toward 448 am. Congener 1, had no effect on ,kmax, and 315

DANNAN. SLEIGHT AND AUST ....." "=,

, ~t . ,..,'~.''~,, .~

r b. ! ".

,•

"1 , ~ p . l"~ l j .

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iI.~ *

%.

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*, • . p

,} ~ ' + " . ~o'

,:,•'"

;-'. , t ~ . , ~ \

":'~

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"..:

.,- ; , . ~ . ,

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~. ",.,'.- • , ,'., ~," .." .~..'.~ .. A

B

D

C

E

FIC;. 2. Effects of vehicle and various PBB congeners on hepatic s t r u c t u r e 7 day~ at'ter treatment. Sections of liver from a control rat

administered .~n ip iniectionof P E C ~3 mL/kg} (A), and rat.streated with q0 mglkF. (ip)congener I (B),congener 5 (C). congener 7 (D), and con~ener 9 (E)• H and E ~;tain; m.lgnification, 160X•

congener 5 had an intermediate effect (,~max = 449.5 nm). These results seemed to be in full agreement with the 4 5 5 / 4 3 0 nm absorbance ratios of the EtNC difference spectra of dithionite reduced microsomes. Congener 7 tripled this ratio, while congener I changed it only slightly. Congeners 5 and 9 moderately increased the ratio by 79% and 50%. respectively. Aminopyrine-N-demethylase activity, normally induced by PB. was nearly doubled by all treatments except congener 7 which caused no change in this activity (Table 2). Epoxide hydrase, which is normally induced by PB but not by MC, was enhanced by all treatments inciiJding congener Z However, with the exception of congener 1, which increased it by nearly 72%. all the remaining treatments induced it by over 250%. 316

The extent of MC-likc induction was determined by assessing aryl hydrocarbon hydroxylase (AHH) activity using the fluorometric assay for benzo[aJpyrene hydroxylation (Gielen et aL. 1972)• Congener 7 was the most effective inducer of this activity (t6 fold) while congener 1 was almost noneffective (Table 2). Congeners 5 and 9 caused intermediate increases• Results of assays for microsomal p-nitrophenolUDP-glucuronyltransferase activity were similar with those for the AHH induction (Table 2).

DISCUSSION Three congeners used in this study. 2,4,5.2",5'-penta- (congener 1), 2.3,4,2',4",5'-hexa- (congener 5), and 2,3.4,5,2",3",4"hepta-bromobiphenyl (congener 9) have two bromines ortho to Fundam. App/. Toxlcoi. (2)

November/December. 19a2

TOXICOLOGY OF PBB CONGENERS

lq,,Y.~3. ,,..":.~.;",~.~;;af'~.;, ~-... '.* • : " " ~ ' ~ \~_~,:'r,"~',~ ." ;~

.t

" '~'.•"

,.r'~.~"

t~"

a

• * "...: "

rd

B

A

D

C

E

FIG. 3. Electron micro~raphs ot hepatocytes from a control rat administered (ipl 3 mL PEGIkg (A). and rats administered tip] o0 mglk 8 of congener 1 (5), congener J (C), congener 7 (D), or congener 9 (El. Uranyl acetaw-lead citrate st.tin, mai~nific,~tion, 4300X.

the biphenyl bridge, while the fourth compound, 2,3,4,5,3',4'hexabromobiphenyl (congener 7), is mono ortho substituted. Only the mono ortho substituted congener 7 significantly reduced body weight gain, the weights of the thymus and the spleen, and severely affected the structure of the thymus• The decline in body weight and the histological changes in the thymus due to congener 7 are typical of toxic PBB congeners such as 3,4,5,3".4",5"-hexabromobiphenyl (Render et al., 1982) and other toxic halogenated aromatic hydrocarbons such as TCDD (McConnell and Moore. 1979). Two other mono ortho substituted congeners in Firemaster. 2,4.5.3',4'.5"-hexabromobiphenyl (Dannan et aL, 1978b) and 2,4.5,3'4"-pentabromobiphenyl (Dannan et al., 1982b), have been found to cause an Fundzmentll =nd Applied Toxi¢ololcy

(21 11.12/82

MC-type enzyme induction and are considered toxic. Although both appeared less potent than 3.4.5,3".4',5".hexabromobiphenyl (Dannan et al•, 1978b. 1982b), a congener not yet identified in Firemaster. the pentabromobiphenyl was more immunosuppressive than the mixture itself (Dannan et a/., 1982b). The hepatic ultrastructural changes caused by congeners 6 and 7 in this study are very similar to those caused by another mono ortho substituted congener, 2.4.5.3'.4'-pentabromobiphenyl (Dannan et al., 1982b). All three treatments caused a marked disorganization in the normal pattern of the ER membranes and gave rise to concentric arrays of laminated membranes around one or more of the lipid vacuoles. TCDD has .'117

DANNAN. SLEIGHT AND AUST also been shown to cause similar changes including the formation of similar ultrastructural configurations which are refesred to as whorls or myelin figures (Norback and Allen, 1973: Jones and Butler, 1974; Gesiewicz et eL, 1980). A direct correlation seems to exist between the toxicity of a PBB congener and its ability to cause an MC-type induction of the hepatic microsomal drug metabolizing enzymes. Congener 1, for example, had little effect on the liver or the lymphoid organs and it had no apparent MC-like induction activity. Congener 7 was responsible for the most severe toxicity and it was the most potent MC-type inducer. A correlation can also be made between the partial MC-type induction effects of congener 6 (Dannan et al., 1978b) and the somewhat unique effects on liver ultrastructure, particularly the formation of whorled m e m b r a n e s (Fig. 4B). Similar correlations appear to exist between the toxicity and MC-typo induction potency of other PBB congeners and the polychtorinated biphenyl (PCB) analogues (Render et eL, 1982; Dannan eta/., 1982b; Parkinson and Safe, 1981; Yoshihara eta/., 1979; Yoshimura et a/., ! 979|. The question of whether an ortho halogenated biphenyl can resemble TCDD in being able to evoke similar toxicity and microsomal enzyme induction (MC-type) has been a controversial one. The original structure-activity relationship rules did not allow any ortho substituted congener to have any of these effects (Goldstein et eL, 1977; Gotdstein, 1979). Results

A

of several studies have indicated, however, that the mixedtype inducers among halogenated biphenyls are not that uncommon since a number of mono ortho substituted PCB or PBB congeners had. at variable degrees, the enzyme induction effects of both PB and MC (Dannan et el., 1978b. 1982b; Yoshihara et eL, 1979; Parkinson et al., 1979, 1980a,b; Robertson et al., 1980). These workers have recognized that a biphenyl with halogens at both pare carbons and at two or more m e t e carbons can cause an MC-type induction and a "l'CDD-like toxicity even if another halogen occupies one of the carbons ortho to the biphenyl bridge. In one study, the chlorinated analogueof congener 7 (2,3.4,5,3',4'-hexachlorobiphenyl) was found to be toxic and an MC-type inducer (Yoshihara et el., 1979). And the MC-type induction effects were confirmed by Parkinson et al. ~19B0a) who also reported a PB-type induction by the same compound. Our data indicate that 2.3,4,5,3',4'hexabromobiphenyl(congener 7) is quite toxic and an MC-type inducer, but is only a subtle or very weak PB-type inducer. Somewhat similar enzvme ~nduction effects by this brominated congener were reported by Robertson et al. (1981 ). Certain di-orlho substituted congeners have also been reported to evoke an induction response sim,lar to both PB and MC. The earlier findmgs of Stonard and Greig (1976) regarding mlxed-type induction by 2.3,4,2',3',4'. and 2,3,4,2',4',5'.hexa. chlorobiphenylhave more recently been confirmed by Pa¢kinson

B

FIC. 4. Electron microBraph~ of portions of hepatocytes from rats administered tip) 9D m~ of congener 7 (A) or congener 6 (B) per kg body weight a week earlier. Uranyl acetate-lead citrate stain, magnification, Z5 ISOX. 318

Fundam. Appl. ToxicoL 12)

November/December, 1~82

TOXICOLOGY Or PBB CONGENERS

A

B

FIG. 5. Sections of the thymus from rats 7 d,~ys ,'ffter injecting vehicle fA) ~3 mL PEC;fk~t m ¢on~,'ner 7 (B) (ao m~:lkf:). et al. (1980c, 1981). In this study the mixed-type induction

effects of congener 5 are in agreement with the similar results on the chlorinated analogue. 2.3,4,2".4",5"-hexachlorobiphenyl (Stonard and Greig, 1976; Parkinson et al., 1980c, 1981 ). The slight degree of MC-type induction by congener 9 is in agreement with that observed with the chlorinated analogue, 2.3,4,5,2".3",4"-heptachlorobiphenyl (Parkinson et al., 1981). However. the sample of congener 9 had nearly 3% of PBB contamination that can mainly be accounted for by congeners 5 and 6 (see Fig, 1), It seems that such contamination was responsible (at, least partly) for the slight degree of MC-type induction since at a dose similar to their concentration in congener 9 sample (nearly 3 mg/kg) congeners 5 and 6 atso resulted in a slight degree of MC-type induction (data not shown). We, therefore, conclude that congener 9. is predominantly a PB-type inducer with no or very slight MC-type induction effects. Including this study, nine of the twelve major congeners (each comprising nearly 0.5% or more o r Firemaster) have been examined for some pharmacological and toxicological effects in rats (Moore et a L 1978b, 1979; Dannan et aL, 1978b, 1982b; Besaw eta/., 1978; Akoso et a L 1980). Four of these congeners. 2.4,5,3",5'-penta-, 2,3,4,234",5'-hexa-. 2,4,5,3',4',5°-hexa-, and 2,3,4,5,3",4'-hexa-brominated biphenyl. totaling approximately 15% and 25% of Firemaster FF-1 and BP-6. respectively, have been classified as MC or mixed-type Fundamental and Applied Toxicology

[2) 11-12/82

inducers with variable toxic effects. The other five congeners comprising nearly 82% and 72% of FF-1 .and BP-6. respectively, are strict PB-type inducers and are apparently nontoxic. The three remaining congeners totaling less than 3% of either Firemaster mixture, with only partially resolved structures (Moore et al., 1980), are quite unlikely to cause toxicity or MC-like induction because they contain at least two bromines on carbons orH~o to the bridge carbons. A reconstituted mixture of nine purifiod congeners gave rise to ultrastructural changes and induction effects that are almost indistinguishable from those due to Firemaster BP-6 (accompanying manuscript). It therefore can be concluded that four of the congeners must account for most, if not all, of the toxic and MC-type induction effects of the crude Firemaster mixtures (FF- 1 or BP-6), A C K N O W L E D G E M E N TS

The authors wish tO recognize the excellent technical assistance of Debra Metcalf throughout this project, and would like to thank Esther Roege and Rick Jensen for performing the histopathological and ultrastructural studies. We would also like to acknowledge the secretarial assistance of Cathy M. Custer in the preparation of this manuscript. Supported by a grant from the Michigan Department of Agriculture. Michigan Agricultural Experiment Station Journal Article Number 10074. 319

DANNAN, SLEIGHT AND AUST TABLE 2 S o m e Hepatic Microsomal D r u g Metabolizing Parameters of Rat,, Administered Various PBB C o n g e n e r s ^ Cytochrome P-4,so s' (,kmax, nm)

NADPHCytochrome c Reducta~e c l/SO :~ 14

2.3.4.2",4".5"(Congener 3}

0.0.1 .= 0.08 (450.01 203,=0.47 1450.0) 3 00-t 0.2.2 (44o.0)

2,3.4,5.3".4'. {Copget~er 7} 2.3..1.5.2",3".4"{Congener 9)

Treatment Control Z.4.5,2'.5'-

(Congener I)

EtNC t4~5:.130 nm ratio}

Amlnopyrlne N-demelhylase c

Epoxldehydrasee

Benzola}pyrene Hydroxylase c

UDPglucuronyl Transferase c

056"/.0.03

1].2 .~" 0 5

131:t-l.0

1.3 ~ 0.3

48.7 :t" 7.0

204¢ 24 n

06o:1.0.03

23.4:1::2.80

22.t~33 n

2.3 ~ 0.4

47.2¢4.1

412~ 1as)

1.00= 0.00

21.7-I- J.4 I)

36.5.'I: 4.4 v

13.bTLo.q n

113.0± 5.3 u

2.7.1_'t'0,43 (448.0J

302.'~30 °

1.05.1"0.0~

11.o~20

34.4"r2.1 °

la.g'~ 1.8u

t57.7±7.0 D

2.78 ~ 0.24 (440.5)

30 ° ~' 2 ou

0.84 .1:0.07

20.8 "~ I b p

35.8 ::t'4.7 h

7 o ..'t: 1.7 n

77.5 "J: 0.7 t~

^Four rats per ~reat ment were administered a single ip itqection of 3 mL PEC/kg (Control} or either of the eBB congeners at q0 mglkg one week before s.wtifk e. D.lta are expressed as mean :I: SD. Itr~n'~oh,s/mR protein. Cnmole~,Jminmlg prolein. uSt,stist,cally differer, t from the mean of cc,ntro~ using Student's t-test (p
REFERENCES Akoso, 13.T., Sleight, 5.D. and Aust, S.D. (1980). Pathologic.d effects of purified pol)'bron'finated b;phenyt congeners in rats. J. thn. College Toxicol. In Press. Bes,tw, L.C., Moore, R.W., Dannan, C.A. and Aust, S.D. (IO78L Effects of 2.2",3.3".4,4".5,5"-ochl bromobiphen yl on m~crosom,fl drug metabolizing enzymes. 7he P/tarmacoL 20:251. Carter, L.J. (Io7o). MichJg.m's PBI3 incideol: Chemi¢.tl mix-up le.*ds to disaster. Sch'nce 1982:240-243. Dannan, G,A,, Moore, R.W. and Aust, S.O, ( }q78a). Studies on lhe microsom.d metabolism arid binding of polybromlnated biphenyls (PBI3s}. l'nviron, lleahh Pcr~pcct. 23:51-01. Dannan, G.A., Moore, R.W., Besaw, L.C. and Aust, S.D. (lq78b). 2,4,5.3',4',5'-Hexabromobiphenyl is both a 3-methylcholanthrene- ,sod a phenobarbit,ll-type inducer of microsom.d drug metabolizing en~,ymes. Biuchem. Biuph)s. Res. Comm. 85:450-458. Dannan, G.A., Mileskl, G.I. and Aust, S.D. I I qa2.a). Purification of polybrominated biphenyl congeners. J. ToxieoL Em iron. th'ahh 9"423-438 Dannan, G.A., Sleight, S.O., Fraker, P.J., Krehblel, J.D. and Aunt, S.D, (1082b). Liver microsom.d enzyme induction and toxicity studies with 2.4,5.3",4"-pentabromubiphenyl. ToxicoL AppL Pharmat eL 64:187-203. Dent, J.G., Netter, K.J. and Gibson, I.E. (I 076a). Effects of chronic administration of polybromir~,~ted biphenyls on parameters associated with hepatic drug metabolism. Re.~. Comnmn. £Ttem. Pathol. PharmaeoL 13:75-82. Dent, J.G., Netter, K.J. and Gibson, I.E. (lq7bb). The induction of hepatic microsomal metabolism in rats following acute administration of a mixture of polybrominated biphenyls. ToxicoL Appl. Pharmacol. 38:237-249. OiCar[o, r.l,,Seifter, J. and DiCarlo. V.l. (1978). Assessment of the hazards of polybrominated biphenyls. Environ. llealth Per~peet, 23:351-365. Fraker, P.J. (!98o). The antibody-mediated and delayed type hypersensitivity response of mice exposed to polybrominated biphenyls. Toxicol. AppL PharmacoL 53:1-7. l'raker, P.J. and Aust, S.D. (1978). The effect of polybrominated biphenyls on the immune response of Balbtc mice. In lnadverrant Modifications o f the Immune System (Asher, J.M., ed.), Office of Health Affairs, FDA, pp. 270-271. 32(}

Gasiewicz, T.A., Ho|scher, M.A. and Nee[, R.A. (19801, The effect of total p.lrenteral nutrition on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dio×in in tile rat. Toxicul. AppL Pharmacol. 54:469-488. Gielen, J,E., Goujon. F.M. and Nebert, D.W. (1o72). Ceneti:" rcgulotion of aryl hydrocarbon hydroxylase induction: II. Simple Mendelian expression in mouse tissues hl viva. J. Biol. Chcm. 247:1125-1137. Goldstein, J.A. (1079). The structure activity relationships of hJIogenated biphenyJs as enzyme inducers. In itcalth Effect.~ o f Halugenated Aromatic Itydrocarbons (W.J. Nicholson and J.A. Moore, eds.). Ann. N.Y. Acad. Sci., Vol. 320, pp. 164-]78. The New York Academy of Sciences, N e w York. Goldsteln, I.A., Hickman, P., Bergman, H., McKinney, J.D. and Walker, M.P. (1977). Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochromc P-450 or P-448. Chem. Biol. Inrerae. 17:bq-87. Jones, G. and Butler, W.H. (1974). A morphological study of the liver lesion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Pathol. 112:q3-q7. Kimbrough, R.D., Burse, V.W. and Liddle, J.A. (Iq78). Persistent liver lesions in rats after a single oral dose of polybromin,ded biphenyls (Firemaster FF-I) and concommitant P1313 tissue levels. Environ. lteahh Perspeet. 23:265-273. Lucier, G.W., Sonawane, B.R. and McDaniel, O.S. (1977). Glucuronidatlon and deglucuronidation reactions in hepatic and extrahepatic tissues during perinatal development. Drug. MetaboL Dispos. 5:27q,287. McConnell, E.E. and Moore, J.A, f1979). To,dropathology characteristics of the halogenated aromatics. In flcahh Effects o f Halogenated Aromatic Hydrocarbons (W.I. Nicholson and J.A. Moore, eds.), Ann. N.Y. Acad. Sci., Vol. 320, pp. 138-150. The New York Academy of Sciences, New York. Moore, R.W. and Aust, S.D. (1078). Purification and structural characteriration of polybrominated biphenyl congeners. Biochem. Biophys. Res. Commua. 84:936-q42. Moore, R,W., Dannan, G.A. and Aust, S.D, (1980). Structurefunction relationships for the pharmacological and toxicological effects and metaboli~;m of polybrominated biphenyl cong e n e r s . ' I n Molecular Basis o f Environmental Toxicity (R.S. Bhatnagar, ed.), Ch. 8, pp. 173-212. Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan. Fundam. AppL ToxlcoL t " 2 )

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TOXICOLOGY OF eBB CONGENERS Moore, R.IN., O'Connor, J.V. and Aust, S.D. (1978a). Identification of a major component of polybrorninated biphenyls as 2.2L3,4,4LS,,s'-heptabromobiphenyl. BulL Envlro~mn. Comam. ToxicoL 20:478-483. Moore, R.W., Sleight, S.D. and Aust, S.D. (IO78b). Induction of liver microsornal drug rnetaboli,'.ing en2yrnes by 2,2'.4,4",5,.5'hexabrornobiphenyl. ToxieoL AppL PharmacoL 44:300-321. Moore, R.YV., Sleight, S.D. and Aust, S.D. (1o791. Effects of 2.2"dtbromobiphenyl and 2.2',3,4.4',5,5'-heptabro.nobtphcnylon liver rnicrosomal drug metabolizing en;,ymes. ToxicoL AppL Pharmacol. 48:73-80. Norback, D.H. and Allen, J.R. (10731. Biological responses of the non-human primate, chicken, and rat to chlorinated dibentop-dioxin ingestion. Eat iron. lh'altls PerH~ecl. 5:233-240. Oesch, F., Jertna, D.M. and Daly, J. ( 1071 ). A radiomet ric assay for hepatic epoxide hydrase activity with [7)HI styrene oxide. Biochem. Biop/o'.*. Aria 227:085-001. Omura, T. and Sate, R. (10041. The carbon monoxide binding pigment of liver microsomes: I. Evidence fop its hernoprotein nature, d. BioL Chem. 230:2370-2378. Parkinson, A., Cockerline, R. and Safe, S. (] 070). Induction of both 3-rnethylcholanthrene-and phenobarbitone-type microsom.tl enzyme activity by a single polychlorinated biphcnyl isomer. Bioehem. PhatmaeuL 2o:25o-202. Parking.on, A., Cockerline, R. and Safe, S. (1080a). Polychlorinated btphenyl isomers and congeners as inducers of bat h 3-methylcholanthrene- and phenobarbitone-type rnierosomal en;,yrne activity. Chem. BioL hsteract. 20:277-289. Parkinson, A., Robertson, L., Safe, L. and Safe, S. tlOg0bL Polychlorinatud biphenyls as inducers of hepatic micro.,-ornal enzymes: Structure-activity rules. Chem. Bic,I. hfferact. 30:271-28,5. P..,rkin,,on, A., Rober stun, L.}V.and Safe, S. ( 1980c). Hep.tt ic rnigt osomal enzyme induclkm by 2,2'.3,3',4,4'- and 2,2',3".4,4',5 hexachlorobiphenyl. Life SoL 27:2333-2337. Parkinson, A., Robertson, L.W., Safe, I.. and Safe, S. (lO,glI Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Effects of di-orth, substitution. Chem. BinL Interact. 3.5:1-12. Parkinson, A. and Safe, S. (I081). Aryl hydrocarbon hydroxylase induction and its relationship to the toxicity of halogenated aromatic hydrocarbons. TuxiroL Era'iron. Chem. Rev. 4:1-40.

Fundamental and Applied Toxicology

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Pederson, T.C., Buege, J.A. and Au,~t, S.D. (IO731. Microsornal electron transport. The roJe of reduced nicotinarnide adenine dinucJeotide phosphate-cytoehrorne c reductase in liver micro:,ornal lipid peroxidation £ Biol. Clwm. 248.7134-714 I. Polin, D., Ringer, R.K. and Aust, S.D. {1o7ol. Effect of congeners of polybrorninated biphenyls on ha tchabilit y of chicken eggs. I. 2.2".4,4',5,5"-He,,abromobiphenyl v.~. eBB. Pier..Toe. t'.~TnL BaoL Meal 101:44-47. Render, J.A., Ausl, S.D. and Sleight, S.D. {1o82L Acute pat hological effects of 3,Y,4.4',5.5'-hexabromobiphenyl in rats: C'otnparison of its effects with Ftremaster BP.o and 2,2',4,4".5,5"hexabromobiphcnyl. 7bxirol. AppL Pharmact,I. 02:428-444. Robertson, L.W., Parkinson, A. and Safe, S. (1080). Induction of both cytochromes P-450 and P-448 by 2.3",4,4',5-pentabron~obipheny], a component of Firemaster. IlirJchem. BIol~h.r.~. Re.~. Commun. 02, l 'L~-132 Robert~on, L.W.. Parkin~on, A., Bandlera, S. and Safe, S. (1o81). Potent induction of rat liver micro.~omal, drug-metabolisms enzymes by 2.3,3',4,.t',$-hexabromobipheny].a component of Ftrenlaster. Chem. BinL IHh'raf ;. 35:13-24. Ston:.rd, M.D. and Greig, LB. ( 1O7o'1.Ddferent pat tern s of hi,pasSe micro,~.omal enzyme .,cttvity produced by adrziini.~tratit.n o{ pttre hexachlorobiphenyl isontt.r~ and hexachh~robt,rtzene. Cht'ltL ltioL htt~'ra~'l. 1S:30,~-370. Strik, J.J.T.W.A. (1078). Toxicity of eBBs v,'tth ~,pt,cialrt.feret,ee t,t porphrinogenic action and spectral h'ttt.r.'tvttoa with hepatic cytochrome P-45o. I't~vtron. lh'alth Pervwct. 23:1 o 7-175. Wolff, M.S., Anderson, H.A. and Selikoff, I.|. rlO82L t|unlao tissue bttrdt.ns of halogePat ed arorn3tlc chemwals m Mit hi,an. JA/IlA 247:2112-2116. Yoshihara, S., Kas~ano, K.. Yo,,hirnura, H.. Kuroki, H. and Masuda. Y. (1o79L Toxicological ass.essment of highly chlorin.'qed biphenyl congeners retained it. tht. Ytz.,.ho paSts'at.-. Chemotld;t'te 8:~31-538. Yoshirnura, It., Yoshihara, S., Oz-,wa, N. and Mlki, M. (lO7O) Possible correl;ttiorl between induction modes of hepatic enzymes by PCBs and their toxicity in rat~,. [n Ih,allh I.'/'J'eCl.~q f Ilalogc,tated Aromatic Ilvclrotarh,,tt.~ (W.I. Nicholson and J.A. Moore, eds.), Ann. N.Y. Acad. Sci., Vol. 320, pp. 17o-102. The New York Academy of Sciences, New York.

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