Biochemical and physiological sequelae to perinatal exposure to polybrominated biphenyls: A multigeneration study in rats

TOXICOLOGY

AND APPLIED

PHARMACOLOGY

59, 300-313

Biochemical and Physiological Polybrominated Biphenyls: K. M. MCCORMACK, The Center

jkw Environmental Michigan

Receirvd

(1981)

Sequelae to Perinatal Exposure to A Multigeneration Study in Rats

L. F. LEPPER,

D.

M.

WILSON,

Toxicology and Department State University, Etrst Lansing.

October

2, 1980; accepted

AND

of‘ Pharmacology Michigan 48824

January

J. B.

HOOK

and Toxicology.

26, 1981

Biochemical and Physiological Sequelae to Perinatal Exposure to Polybrominated Biphenyls: A Multigeneration Study in Rats. MCCORMACK, K. M., LEPPER, L. F.. WILSON, D. M.. AND HOOK, J. B. (1981). Toxicol. Appl. Pharmacol. 59, 300-313. Polybrominated biphenyls (PBBs) cross the placenta and have been detected in milk. Since biologically stable PBBs are transferred readily from mother to offspring, addition of PBBs to the food supply of one generation may result in exposure of subsequent generations. Therefore. we identified physiological and biochemical sequelae to perinatal exposure to PBBs. Rats were fed diets containing 0. 10, or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-10 and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the F, generation (F,-10 and F,100). F,-100 littermates were bred to produce F,-100 animals. Experiments were conducted in offspring from each generation at 28 days of age. Results from this investigation demonstrate that survival can be reduced and growth and development retarded by perinatal exposure to PBBs. PBBs transferred from one generation to the next produced liver enlargement, hepatic histopathologic alterations, hepatic and renal microsomal enzyme stimulation, a reduction in the duration of anesthesia produced by pentobarbital or a large dose of progesterone, and a decrease in the concentration of vitamin A in liver. Therefore, the health hazard associated with exposure to PBBs may not be limited to a single generation.

Polybrominated biphenyls (PBBs) are fire retardants which were added accidentally to livestock and poultry feed in Michigan (Dunckel, 1975). Brilliant and co-workers (1978) estimated that by 1976 over 85% of Michigan’s residents had detectable body burdens of PBBs. Although no human health effects have been attributed definitively to PBBs, a variety of alterations have been produced in other mammalian species. Body weight gain has been retarded and components of the immune and endocrine systems modified in several species following exposure to PBBs (DiCarlo er al., 1978; Kimbrough et al., 1978). Morphological and functional changes have been detected in livers and kidneys from animals 0041-0008X/81/080300-14$02.00/0 Copyright All

rights

k of

1981

by

reproduction

Academic in any

300 Pres. form

Inc. reserved.

fed PBBs (Sleight and Sanger, 1976; Moorhead et al., 1977,1978). Microsomal enzyme activity has been increased in liver and extrahepatic tissues, including kidney, by PBBs (Dent et al., 1976a,b; McCormack et al., 1978). PBBs were termed “mixed” inducers because they exhibited hepatic enzyme-stimulating characteristics of both phenobarbital and 3-methylcholanthrene (3-MC) (Dent et al., 1976a,b). Hepatic microsomal enzyme stimulation following PBBs may result from combined effects of individual congeners which are either phenobarbital-like (2,4,5,2’4’,5’-hexaand 2,3,4,5,2’.4’,5’-heptabromobiphenyl) or 3MC-like inducers, and congeners which produce both types of enzyme induction

EFFECT OF PBBs ON SUBSEQUENT

(2,4,5,3’,4’,5’-hexaand 2,4,5,3’,4’-pentabromobiphenyl) (Moore et al., 1978a, 1979; Dannan et al., 1978a; Robertson et al., 1980). Pharmacokinetic properties of individual PBB congeners depend on the position and degree of bromination. However, most PBBs are lipophilic, biologically stable, and accumulate in tissues, particularly those with a high fat content (Matthews et al., 1977, 1978). Tuey and Matthews (1980) estimated that the body burden halflife of 2,4,5,2’,4’,5’-hexabromobiphenyl in humans was 6.5 years. It was predicted that less than 10% of a single oral dose of 2,4,5,2’,4’,5’-hexabromobiphenyl would ever be excreted by healthy, normally fed male rats (Matthews et al., 1977). PBBs were detected in rat tissues even 300 days following treatment termination, confirming the remarkable stability and slow elimination of certain PBB congeners (McCormack et al., 1980). Retention of PBBs in tissues was correlated with hepatic and renal microsomal enzyme stimulation throughout the IO-month residual phase. Transplacental movement of PBBs has been reported in several species and PBBs have been detected in the milk of nursing mothers (Fries and Marrow, 1975; Brilliant er al., 1978). The most effective transfer of PBBs from mother to offspring occurs via milk (Rickert et ul., 1978). Exposure to PBBs transplacentally or by ingestion of contaminated milk has produced liver enlargement and increased activity of hepatic microsomal enzymes in rat pups (Dent et al., 1978; Moore et al., 1978b). Since biologically stable PBBs are transferred readily from mother to offspring, addition of PBBs to the food supply of one generation may result in exposure of subsequent generations. Therefore, it was of interest to identify physiological and biochemical sequelae to perinatal exposure to PBBs. There were three specific objectives of this investigation: (1) to further determine effects of perinatal exposure to PBBs on survival,

GENERATIONS

301

morphology, growth, and development; (2) to further determine effects of PBBs on metabolism of xenobiotic and endogenous compounds; and (3) to determine the persistence of effects of PBBs administration to a single generation on subsequent generations. METHODS Sprague-Dawley rats (at least eight per treatment group) were obtained on Day 2 (inseminated on Day 0) of pregnancy (Spartan Farms, Haslett, Mich.). They were housed in clear polypropylene cages at 22°C with a 12-hr light cycle (0700-1900 hr) and were allowed free access to food (Wayne Lab Blox, Anderson Mills, Maumee, Ohio) and water. On Day 8 of pregnancy, rats were fed diets containing 0, 10, or 100 ppm PBBs (Firemaster BP-6, Velsicol Chemical Co., St. Louis, Mich.) which were the diets fed until 28 days after parturition. These animals were designated as the F, generation. The diets were prepared as described by Dent and co-workers (1978). The two main congeners, 2,4,5,2’,4’,5’-hexabromobiphenyl and 2,3.4,5,2’,4’,5’-heptabromobiphenyl, account for approximately 56 and 27% of the Firemaster BP-6 mixture by weight, respectively (Moore er al., 1980). However, Firemaster BP-6 may contain 30 or more components including the following congeners: 2,2’-di-, 2,4,5,4’S’-penta-, 2,4,5,3’,4’-penta-. 2,3,4,2’,4’,5’-hexa-, 2,4,5,3’,4’,5’-hexa-, 2,3,6,2’,4’,5’hexa- 2,3,4,5.2’,3’,4’,5’-octa-, and 2,3,4,5.6,2’,3’,4’,5’nonabromobiphenyl (Moore et al., 1980). The treatment regimen was chosen to approximate maximal human exposure. The highest reported concentrations of PBBs detected in human milk were between 90 and 100 ppm (Cordle et ul., 1978). However, the median concentration of PBBs in milk from Michigan women may be much lower (less than 0.1 ppm) (Brilliant et al., 1978). Litters were normalized to 10 pups (5 males and 5 females) at birth. Offspring of F, rats were termed the F, generation and were further identified as F,-10 or F,-100 animals to signify the dose of PBBs to which the dams were exposed. Pups in the F, generation also were allowed free access to diets and were weaned onto Lab Blox containing no PBBs at 28 days postpartum. All rats were fed diets free of PBBs for the remainder of the investigation. After allowing lo-12 weeks for maturation, F, littermates were bred to produce the next generation (F,-10 and F,-100). Similarly, F,-100 littermates were fed control diet and bred at 14-16 weeks of age to produce Fn100 animals. Control values were obtained by pooling data from F,-0, F,-0, and F,-0 rats. Experiments were conducted in offspring at 28 days of age. The rate of postnatal development was assessed

302

MCCORMACK

in control, F,-10, F,-100. and F,-100 animals by monitoring daily the following parameters: pinna detachment, incisor eruption, fur development, and opening of the external auditory ducts, eyes, and vagina. Animals (at least three females per treatment group) used for histologic examinations were killed by cervical dislocation. For light microscopy, pieces of liver from control, F,-10, F,-100, and F,-100 rats and kidney from control and F,-100 rats were fixed in lo%, buffered formalin. After fixation samples were embedded in paraffin. sectioned at 5 /*m, and stained with hematoxylin and eosin. For electron microscopy, pieces of liver from control. F,-100 and F,-100 rats were fixed in Karnovsky’s fixative ( 1965) for approximately 48 hr. Tissues were then washed twice in 0.1 M cacodylate buffer and rewashed in water. Samples were postfixed in 1%’ osmium tetroxide in 0.1 M phosphate buffer, dehydrated in ethanol, and embedded in a mixture of Araldite, Epon. and Spurrs epoxy resins. Ultrathin sections were cut. stained sequentially with 2% uranyl acetate and 0.2% lead citrate, and viewed with a Philips 300 electron microscope. Animals (at least four per sex and treatment) used for determination oforgan weight-to-body weight ratios were killed by decapitation. The following organs were excised, blotted on gauze, and then weighed: liver, kidneys, heart. lungs. testes, ovaries, thyroid glands. and thymus. Animals (females from all treatment groups) used for microsomal enzyme assays were killed by cervical dislocation. Livers and kidneys were excised, weighed, and then chopped into 3 vol of ice-cold 1.15% KC1 (livers) or 66 mM Tris-HCI, pH 7.4 (kidneys). Postmitochondrial supernatants were prepared by homogenization (Potter-Elvehjem homogenizer with a Teflon pestle) followed by centrifugation at 10,OOOg for 20 min. All assays were performed on the day of supernatant preparation. Protein was measured by the method of Lowry ef rrl. (1951) using bovine serum albumin as a standard. Arylhydrocarbon hydroxylase (AHH) activity was determined fluorimetrically by the method of Nebert and Gelboin ( 1968) as modified by Oesch (1976). Epoxide hydratase (EH) activity was determined by the method of Oesch rf al. (1971). The concentration of vitamin A was quantified in livers from control, F,-10, F,-100, F,-100, and F,-100 females. Hepatic vitamin A concentrations were measured fluorimetrically by the method of Hansen and Warwick (1978). Duration of anesthesia following a single ip injection of 50 mgkg sodium pentobarbital. or 150 mgikg progesterone, was determined in females and males. respectively. Sleeping time was recorded as the interval from time of injection until the righting reflex was regained. Concentrations of PBBs in liver, kidney, lung.

ET AL thyroid. testis, ovary, and fat were determined using the following procedure. Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Powdered tissues were extracted four times with 30-m] portions of hexane. Pooled extracts were reduced in volume to l-2 ml which was placed on a Florisil (60- 100 mesh) column (500 x 200 mm). PBBs were eluted with 200 ml hexane which was evaporated to dryness under N, and then brought up to the desired volume with hexane. Quantitation was by gas-liquid chromatography on a Varian Model 2100 gas chromatograph (Varian. Palo Alto, Calif.) with a Sc’H electron capture detector using a 1.7-m v 2-mm-i.d. column packed with 1°C OV-I. Carrier gas (N,) flow and column temperature were 30 mlimin and 226°C. respectively. Gas chromatograms were examined for all peaks: however, only the height of the major peak (2,4.5.2’.4’,5’-hexabromobiphenyl) was calculated. Concentration of PBBs was expressed as micrograms of PBBs (2.4,5.2’,4’,5’-hexabromobiphenyl) per gram wet tissue weight. Data were analyzed statistically by analysis of variance. completely random design. 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 Prenatal exposure to PBBs did not affect length of gestation, litter size. incidence of gross external anomalies, or pup body weight at birth. However, the survival rateto-weaning was reduced in F,- 100 pups (87% of control value, Fig. 1). The postnatal physical maturation rate of F,-10 and F,100 animals did not differ from controls (Table 1). Although no effect was observed during the first week postpartum, development was retarded subsequently in F,-100 pups (Table 1). Fur development (secondary coat) and opening of the external auditory ducts, eyes, and vagina were delayed in F,100 animals. Body weight gain of F,-100 pups was decreased at 14 days of age and remained decreased at least through 84 days of age (80% of control value at 28 days of age: Fig. 2). The liver weight-to-body weight ratio was increased at weaning in F,-100 rats, their offspring (F,-100 animals) and F,-10 rats (165, 128, and 126% of control value,

EFFECT

0

F-10

5-10

OF PBBs

F-100

t-100

ON SUBSEQUENT

tion, focal necrosis, pyknotic nuclei, swelling, and intracytoplasmic myelin inclusion bodies. The severity of lesions in livers from F,-10 and F,-100 rats was comparable and much less prominent than in livers from F,100 animals. Electron microscopic changes in livers from F,-100 rats included cytoplasmic vacuolation, proliferation of smooth endoplasmic reticulum, myelin inclusion bodies, and altered mitochondria (Fig. 3). Histopathologic alterations were not detected in livers from F,,-100 animals or in kidneys. Activity of AHH was increased in livers from F,-10, F,-10. F,-100. and F,-100 animals at 28 days of age (approximately 400. 130. 1400, and 600% of control value. respectively: Fig. 4). Activity of EH was increased in livers from F,-10, F,-100, and F,-100 animals at 28 days of age (approximately 125, 250, and 130% of control value. respectively; Fig. 4). Activity of AHH was increased in kidneys from F,-10, F,-100, and F,-100 rats at 28 days of age (approximately 200, 700, and 32.5% of control value, respectively: Fig. 5). Activity of renal EH was not affected by perinatal exposure to PBBs (Fig. 5). F,,-100 animals did not exhibit a difference from controls in any response quantified in this investigation. Duration of anesthesia produced by 50

e-100

PBBs kwm) FIG. I. Effect of perinatal exposure to PBBs on neonatal survival until weaning. Rats were fed 0, IO. or 100 ppm PBBs from Day 8 of pregnancy until 18 days postpartum at which time all offspring (F,-IO and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the F1 generation (F,-IO and F,-100). F,-100 littermates were bred to produce F:,-100 animals. Values are means + SE for at least seven litters. An asterisk indicates a statistically significant difference from the control value. p i 0.05.

respectively; Fig. 2). Kidney, heart, lung, testis, ovary, thyroid, and thymus weightto-body weight ratios were not affected by PBBs (thymus weights were not recorded in F,-100 or F,-100 animals). Liver enlargement was associated with histopathologic alterations. Light microscopic changes in livers from F,-100, F,100, and F,-10 animals included vacuola-

TABLE POSTNATAL

DEWI.OPMENT

OF RATS

303

GENERATIONS

I

~OLI.OWING

PERINATAI

EXIWSURF.

7-o PBBs” .~

Treatment Parameter Pinna detachment Incisor eruption Fur development External auditory duct opening Eye opening Vaginal opening

Control 2.7 r 0.1 8.1 c 0.2 9.1 ‘t 0.1 II.6

t 0.2 I 0.2 37.2 + 1.3 14.7

F,-IO

F,-100

Fy 100

2.7 r 0.1 8.2 t 0.1 9.3 t 0.2

2.9 2 0.1 8.6 -c 0.3 10.5 t 0.‘”

2.8 -t 0.1 8.4 i 0.2 9.7 z 0.3

1 I .s t 0.3 14.9 + 0.4 39.4 + I.7

12.4 2 0.3” 15.4 -t 0.2” 40.6 2 1.8”

11.8 -’ 0.2 14.7 + 0.4 38.7 k 1.4

” Values are means in days +- SEM for at least five litters. I’ Significantly different from the control value. ~1 < 0.05.

304

MCCORMACK

mg/kg (ip) pentobarbital was reduced in F,-10, F,-10, F,-100, and F,-100 rats at 28 days of age (8, 80, 1, and 12% of control value, respectively; Fig. 6). The concentration of vitamin A was reduced in liver from F,-10, F,-100, and F,-100 animals at 28 days of age (60,43, and 72% of control value, respectively: Fig. 7). Duration of anesthesia produced by 150 mg/kg (ip) progesterone was reduced in F,-10, F,-100, and F,-100 animals at 28 days of age (25, 4, and 21% of control value, respectively; Fig. 8). Approximately onethird of control, F,-10, and F,-100 rats died from this dose of progesterone. Dose and generation-related effects were correlated with tissue concentrations of PBBs (2,4,5,2’,4’,5’-hexabromobiphenyl: Table 2). The concentrations of PBBs in tissues from F,-100 animals were approximately 5-30 times higher than in tissues from F,-100 or F,-10 animals and approximately 50- 1000 times higher than in tissues from F3-100 or F?-10 animals. The gas chromatographic profile of tissue from animals in all treatment groups had peaks corresponding to at least seven different PBB congeners (Fig. 9). Peaks 1,2,3,4,5,6, 7, 8, and 9 in the gas chromatographic profile of Firemaster BP-6 correspond to 2,4,5,2’,5’-penta-, 2,4,5,3’,4’-penta-, 2,3,6, 2’,4’,5’-hexa-, 2,4,5,2’,4’.5’-hexa-, 2,3,4,2’, 4’,5’,-hexa-, 2,4,5,3’,4’,5’-hexa-, unknown hepta-, 2,3,4,5,2’,4’,5’-hepta-, and 2,3,4,2’,3’,4’,5’-heptabromobiphenyl, respectively (Moore el ~11.. 1980). Peak 3 in the Firemaster BP-6 standard was not detected in tissues from any animals. The height of peaks 1 and 8 relative to 4 appeared to be lower in liver from F,-10 and F,-100 rats than the Firemaster BP-6 standard. The height of peaks 1, 2, and 5 relative to 4 appeared to be higher in liver from F,-10 and F,-100 rats than in liver from F,-10 and F,100 rats, respectively. The height of peaks 6, 7, 8, and 9 relative to peak 4 appeared to be lower in liver from F,-10 and F,-100 rats than in liver from F,-10 and F,-100 rats,

ET AL

0

t-10

s-10

c-100

F,-loo

F,-loo

Pans (pm)

FIG. 2. Effect of perinatal exposure to PBBs on body weight and the liver weight-to-body weight ratio. Rats were fed 0, 10, or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring tF,-10 and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the Fz generation (F,-10 and F,-100). F,-100 littermates were bred to produce F,-100 animals. Values are means 2 SE for at least seven litters. An asterisk indicates a statistically significant difference from the control value, p < 0.05.

respectively. 6, 7, 8, and to be lower in liver from

The height of all peaks (1,2,5, 9) relative to peak 4 appeared in liver from F:,-100 rats than F2-100 or F,-10 animals. DISCUSSION

Although PBBs cross the placenta, the most effective transfer of PBBs from mother to offspring occurs via milk (Rickert et al., 1978). At 14 days postpartum, whole carcass concentrations of PBBs were approximately 25 times higher in rat pups born to and nursed by dams fed 50 ppm PBBs from Day 8 of gestation than in pups born to PBBs-exposed dams (50 ppm) but nursed by control dams (Rickert et al., 1978). In this

EFFECT

FIG.

tensive smooth

3. Electron cytoplasmic endoplasmic

OF PBBs

ON

SUBSEQUENT

305

GENERATIONS

photomicrograph of liver from a F,-100 rat killed at 28 days of age. There is exvacuolation, myelin bodies, disruption of mitochondria, and proliferation of reticulum. Uranyl acetate and lead citrate stain. x8100.

inv estigation, a latent period of 1 to 2 weeks exi sted before growth, development, and SW ,vival were reduced postnatally in F,-100 Put 3s. This delay may have been related to

the time required for to concentrations in produce such effects. have contributed to

PBBs to accumul late tissues sufficient to Several factors nnay retarded growth ;and

306

MCCORMACK

development in F,-100 animals. For instance, pups may have had an aversion to milk containing high concentrations PBBs and consequently, reduced milk intake. Reduced body weight gain in adult rats exposed to PBBs in diet also has been attributed, at least in part, to decreased food efficiency (i.e., ratio of weight gain/food consumed) (Sleight and Sanger, 1976; Garthoff ef al., 1977). The liver weight-to-body weight ratio was increased and hepatic histopathologic alterations were produced in F,-10, F1-100, and F,-100 animals. Liver enlargement and pathologic changes were dose dependent and related directly to hepatic concentrations of PBBs. Increased liver weights

zi Y

Arylhydrocorbon Hydroxylare

* 1

t tivdrakxe

r

PBBr fppm)

Epoxide Hydratase

FIG. 5. Effect of perinatal exposure to PBBs on the activity of arylhydrocarbon hydroxylase and epoxide hydratase in kidney. Rats were fed 0, 10. or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-10 and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the F, generation (F,-10 and F,-100). F,-100 littermates were bred to produce F,,-100 animals. Values are means t SE for at least six female rats. An asterisk indicates a statistically significant difference from the control value, p < 0.05.

*

d gOE 5 8.0 zL;.: 6.0 tb -0 4.0 ;E 2.0 0.0

Arylhydrocarbon Hydroxyl.s. 6.0

*

400

10.0

E t

ET AL.

0

4-10

s-10

t-100

yoo

FjlOO

PBBs (ppm)

FIG. 4. Effect of perinatal exposure to PBBs on the activity of arylhydrocarbon hydroxylase and epoxide hydratase in liver. Rats were fed 0, 10, or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-10 and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the F2 generation (F,-10 and F,-100). F,-100 littermates were bred to produce F:,-100 animals. Values are means 2 SE for at least six female rats. An asterisk indicates a statistically significant difference from the control value. 11 < 0.05.

produced by PBBs may have been due to increased synthesis of protein and lipid. Hepatic microsomal protein has been increased following treatment with PBBs (Dent et al., 1976a,b, 1978). All liver lipid fractions including total fat, phospholipid, neutral lipid, and cholesterol also were increased in rats fed PBBs (Garthoff et cl/.. 1977). Increased hepatic cholesterol produced by PBBs may have been due to increased activity of P-hydroxy-fl-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) which is rate-limiting in cholesterol biosynthesis. Although activity of HMG-Co-A reductase has not been quantified following exposure to PBBs it has been

EFFECT

0

R-10

$40 PBBr

rpa

OF PBBs

poo

ON

SUBSEQUENT

Fj-loo

(Ppm)

FIG. 6. Effect of perinatal exposure to PBBs on the duration of anesthesia produced by pentobarbital, 50 mgikg ip. Rats were fed 0, 10. or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-10 and F,-100) were weaned onto control diet, allowed to mature sexually. and bred with littermates to produce the F, generation (Fz-10 and F,-100). F,-100 littermates were bred to produce F,-100 animals. Values are means ? SE for at least six female rats. An asterisk indicates a statistically significant difference from the control value, p < 0.05.

increased by treatment with phenobarbital or polychlorinated biphenyls (PCBs) (Gibbons and Mitropoulos, 1973; Kato and Yoshida, 1980). Compared to livers from control animals, livers from F,-10. F,-100, and F,-100 rats had cytoplasmic vacuolation. focal necrosis, pyknotic nuclei, swelling, and intracytoplasmic myelin inclusion bodies. Electron microscopic changes in livers from F,-100 rats included proliferation of smooth endoplasmic reticulum. presence of intracytoplasmic myelin inclusion bodies. and altered mitochondria. Proliferation of smooth endoplasmic reticulum in liver was correlated with increased activity of hepatic microsomal enzymes following perinatal exposure to PBBs. Activity of AHH was increased in liver from F,-10, F,-10, F,-100, and F,-100 but not F,-100 rats. Similarly, EH activity was increased in liver from F,-10, F,-100, and F,-100 animals. Hepatic EH activity has been increased by 2,4,5.2’,4’,5’-hexaor 2,3,4.5,2’,4’,5’-heptabromobiphenyl; however, these congeners had little effect on AHH activity (Moore et al., 1978a, 1980).

307

GENERATIONS

Activity of AHH in liver has been increased by 2,4,5,3’,4’,5’-hexaand 2,4,5,3’,4’-pentabromobiphenyl (Dannan et al., 1978a; Robertson et al., 1980) which were detected in liver, at dose- and generationdependent concentrations, from all rats exposed to PBBs. These congeners produce both phenobarbital-like and 3-MC-like enzyme induction and thus, may have contributed to the increase in activity of both EH and AHH in liver. Perinatal exposure to PBBs had no detectable effect on the kidney weight-to-body weight ratio or renal morphology. Since kidney enlargement and renal degenerative changes have been observed in dairy cows and mink fed PBBs (Firemaster FF-1 or BP-6) (Jackson and Halbert, 1974; Moorhead et al., 1978; Aulerich and Ringer, 1979) and in rats fed octabromobiphenyl (Norris et al., 1974). the nephrotoxicity of PBBs may be dependent on species and chemical form of PBBs administered. Nevertheless, the absence of renal histopathologic alterations in F,-100 animals is consistent with a previous report of a lack of effect of PBBs (Firemaster BP-6) on renal

Q

z

75

$

50

2

25

ii =

0

0

F-10 l+a

FpJ

Efloo

PBBs (Ppm) FIG. 7. Effect of perinatal exposure to PBBs on the concentration of vitamin A in liver. Rats were fed 0. 10, or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the Fz generation (F?-10 and F,-100). F,-100 littermates were bred to produce F,-100 animals. Values are means ? SE for at least eight female rats. An asterisk indicates a statistically significant difference from the control value, p < 0.05.

308

MCCORMACK

PBBs(pPm) FIG. 8. Effect of perinatal exposure to PBBs on the duration of anesthesia produced by 150 mg/kg progesterone, ip. Rats were fed 0. 10. or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-10 and F,-100) were weaned onto control diet. allowed to mature sexually. and bred with littermates to produce the F, generation (F,-10 and F,-100). FZ-100 littermates were bred to produce F,-100 animals. Values are means ? SE for at least eight male rats. An asterisk indicates a statistically significant difference from the control value, p < 0.05.

function in rats (McCormack rt al., 1978). Perinatal exposure to PBBs also had no effect on the activity of renal EH. However, activity of AHH was increased in kidneys from F,-IO, F1-100, and F,-100 rats. Increased microsomal MFO activity in Lyitro was correlated with a reduced duration of anesthesia following pentobarbital in FL-lo, F,-10, F,-100, and F,-100 rats. Many lipophilic xenobiotics including therapeutic agents and environmental chemicals are substrates for microsomal MFOs (Conney, 1967). Alterations in microsomal MFO activity produced by PBBs may affect the metabolism and consequently, the tox-

ET AL.

icity of certain xenobiotics administered subsequently. Pretreatment of mice with PBBs increased the toxicity of bromobenzene (Roes et al., 1977) which is metabolized to the putative hepatotoxicant bromobenzene-3,4-epoxide (Zampaglione et cl/. , 1973). Kluwe and co-workers (1978) reported that prior exposure to PBBs also increased the toxicity of other compounds activated metabolically including chloroform and carbon tetrachloride (Suarez or trl., 1972). Results from this investigation indicate that such toxic chemical interactions following PBBs may not be limited to a single generation. Endogenous compounds such as fatsoluble vitamins, fatty acids, and steroid hormones also are substrates for the MFO system (Conney, 1967). In this investigation. the concentration of vitamin A was reduced in livers from F,-10, F,-100. and F,-100 rats. Hepatic concentrations of vitamin A and body weight gain also have been reduced in rats by PCBs (Cecil et II/.. 1973: Innami et crl., 1976). Rats fed PCBs and large amounts of vitamin A gained more weight than those fed PCBs and only standard amounts of vitamin A (Innami rt r~l., 1976). Whether the reduction in hepatic vitamin A in F,-100 rats was sufficient to account for their retarded growth and development has not been determined. Similarly, little is known about effects of PBBs on vision, susceptibility to carcinomas produced by other chemicals administered subsequently, or reproductive

TABLE TISSUE Treatment Control F,-10

F,-10 F,-100 F,-100 F,- 100 ‘* Values b Sample

Liver 4.1 17.7 t 0.4 f 410.2 f 21.8 k 0.4 2

OF PBBs

CONCENTRATIONS

Kidney

3.6 0.1 40.6 3.2 0.1


FOLLOWING

Lung 1 0.6 0.2 11.2 1.3 0.4

co.1 5.1 2 0.9 t 32.4 t 8.7 t 0.6 k

are means in pg 2.4,5.2’,4’.5’-hexabromobiphenyyg not available.

2 PERINATAL


70

Testis

Thyroid

0.8 0.1 2.0 1.0 0.1

EXPOSURE

co. I 8.2 -t 5.3 0.2 f; 0.1 -1, I.8 i Cl.1 0.3 -t 0.1

PBBs”

ovary co. I 24.0 r 1.6 3.0 c 0.5 -0 lb.5 -+_ 2.6 0.6 c 0. I

-c SE for at least three

animals

Fat co. 161.7 f 6.7 f 1693.: r 159.5 + 4.9 -r at 28 days

I 24.2 I.Y 250.4 16.9 0.8

of age.

EFFECT

RETENTION

TIME

OF PBBs

ON

SUBSEQUENT

(mid

9. Gas chromatographic profiles and numbering system for the PBB congeners in Firemaster BP-6 and in liver from rats exposed to PBBs. Rats were fed 0, 10, or 100 ppm PBBs from Day 8 of pregnancy until 28 days postpartum at which time all offspring (F,-IO and F,-100) were weaned onto control diet, allowed to mature sexually, and bred with littermates to produce the F,-generation (F,-IO and F,-100). I;,- 100 littermates were bred to produce FS- 100 animals. Peak 3 in the gas chromatographic profile from the Firemaster BP-6 mixture was not detected in tissue samples. FIG.

capacity all of which involve functions of vitamin A. Since steroid hormones are endogenous substrates for microsomal enzymes, reproductive function also may be affected by alterations in steroid metabolism produced

GENERATIONS

309

by PBBs. The finding of a delay in vaginal opening in F,-100 rats confirms a previous report of delayed vaginal opening in rats exposed to PBBs (Harris er al., 1978). Retarded sexual maturation also was suggested by a reduction in the ventral prostrate weight-to-body weight ratio in pubescent male rats treated perinatally with PBBs (Johnston et al., 1980). Rats exposed to PBBs had lengthened estrous cycles (Johnston et fil., 1980). Rhesus monkeys had lengthened menstrual cycles after consuming PBBs, and this effect was associated with flattened and lengthened serum progesterone peaks (Lambrecht et a/. , 1978). Rhesus monkeys exposed to PBBs in the diet had hypoactive seminiferous tubules (Allen et al., 1978). Young bulls fed high doses of PBBs had testicular atrophy and reduced spermatogenesis (Jackson and Halbert, 1974). Responses to steroid hormones administered exogenously were reduced in rats pretreated with PBBs suggesting that metabolism of steroids was accelerated by PBBs (McCormack et cl/., 1979). Recently, several investigators have demonstrated that the irl 1’irr.o microsomal metabolism of progesterone, testosterone and estrogens was altered markedly in rats exposed to PBBs perinatally (Arneric er al., 1980; Newton et a/., 1980; Bonhaus et al.. 1981). The duration of anesthesia produced by a very large dose of progesterone reflects its rate of metabolism in \sil,o (Conney et ctl., 1966). The reduction in progesterone sleeping time in F,-10, F,-100, and FS-100 rats in our study indicates that alterations in metabolism of steroid hormones produced by PBBs may not be limited to a single generation. However, litter size and gestational length were not affected by PBBs in this investigation. Therefore, rats were able to compensate for alterations in steroid hormone metabolism to the extent that implantation and maintenance of pregnancy were possible. Mechanism(s) by which PBBs produce endocrine related alterations have not been demonstrated.

310

MCCORMACK

Physiological and biochemical alterations detected in weanlings were correlated with tissue concentrations of PBBs. At 28 days of age, the concentrations of 2,4,5,2’,4’,5’hexabromobiphenyl in tissues from F,-100 animals were approximately 5-30 times higher than in tissues from F,-100 or F,10 animals and approximately 50- 1000 times higher than in tissues from F3-100 or F,-10 animals. These results demonstrate that PBBs persisted in body fat animals in the F, and F, generations from weaning until pregnancy. During pregnancy and lactation, animals transferred most of their body burden of PBBs to offspring. This finding is consistent with a report by Vodicnik and Lech (1980) that mice transferred almost their entire body burden of 2,4,5,2’,4’,5’-hexachlorobiphenyl to nursing offspring. Of the first eight peaks in the gas chromatographic profile of Firemaster BP-6, all except peak 3 (2,3,6,2’,4’,5’-hexabromobiphenyl) were detected in liver from rats in the F,, F, and F3 generations. 2,3,6,2’,4’,5’Hexabromobiphenyl has been shown to be metabolized ifz \lirro (Dannan et crl., 1978b). Since 2,3,6,2’,4’,5’-hexabromobiphenyl has been transferred via milk (Dannan et al., 1978b), the absence of this congener from tissues in this investigation may have been due to its metabolism. The concentration of several congeners relative to the 2,4,5,2’,4’,.5’-hexabromobiphenyl concentration was altered in tissues compared to the Firemaster BP-6 standard. Differences in the pharmacokinetic properties of individual PBB congeners also were reflected by changes in the relative concentrations of certain congeners from one generation to the next. For example, the concentration of 2,4,5,3’,4’,5’-hexabromobiphenyl (peak 6) relative to 2.4,5,2’,4’,5’-hexabromobiphenyl (peak 4) was higher in liver of animals in the F, generation than in the Firemaster BP-6 standard: however, the relative concentration of 2,4,5,3’,4’,5’hexabromobiphenyl in liver decreased with

ET AL.

each subsequent generation. 2,4,5,3’,4’,5’Hexabromobiphenyl is the most toxic of the PBB congeners that have been tested (Moore et rrl., 1980). Therefore, the toxicity of the mixture of PBBs transferred from mother to offspring may decrease with each generation. Support for this hypothesis includes the finding of biochemical changes in F.,-10 but not F,-100 animals. Although these animals had similar hepatic concentrations of 2,4.5,2’,4’,5’-hexabromobiphenyl, the relative concentrations of other PBB congeners. including 2,4,5.3’,4’,5’-hexabromobiphenyl, appeared to be lower in liver from F,-100 than F,-10 animals. Results from this investigation demonstrate that survival can be reduced and growth and development retarded by perinatal exposure to PBBs. PBBs transferred from one generation to the next produced alterations in liver morphology and in the ability to metabolize endogenous and xenobiotic compounds. Therefore, the health hazard associated with exposure to PBBs may not be limited to a single generation. ACKNOWLEDGMENTS This investigation was supported in part by the National Institute of Environmental Health Sciences Grant ES00560 and a grant from the Michigan Department of Agriculture. We wish to express our appreciation to Dr. V. L. Sanger (Department of Pathology) and Dr. K. K. Baker (Center for Electron Optics) for assistance with histopathology, Dr. W. E. Braselton. Jr. (Department of Pharmacology and Toxicology) for assistance with PBBs quantification and D. K. Hummel and M. A. Pinto for help in manuscript preparation. Michigan State Agricultural Experimental Station Journal Article No. 9683.

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TUEY,