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Effects of lactation and nursing on tissue concentrations of polybrominated biphenyls and on microsomal enzyme activity in mammary gland and liver in maternal rats
ENVIRONMENTAL
27, 1 lo-
RESEARCH
117 (1982)
Effects of Lactation and Nursing on Tissue Concentrations of Polybrominated Biphenyls and on Microsomal Enzyme Activity in Mammary Gland and Liver in Maternal Rats’ K. M. MCCORMACK
Received
AND J. B. HOOK
February
10. 1981
Redistribution of polybrominated biphenyls (PBBs) during the recovery period following lactation resulted in concentrations of PBBs that were higher in mammary gland and lower in liver and fat than at the end of lactation. Hepatic arylhydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH) activities were higher in PBB-treated rats during lactation than in the recovery period. However, transition from lactating to dry state had no effect on activity of mammary gland AHH in rats treated with PBBs. Microsomal enzyme activity and concentrations of PBBs in mammary gland and liver were reduced by nursing a second litter. Although redistribution and excretion of PBBs resulted in alterations in microsomal enzyme activity, all rats exposed to 100 ppm PBBs had microsomal enzyme activity in liver and mammary gland that was higher than that in controls. Persistent stimulation of mammary gland and hepatic microsomal enyzmes while mothers are exposed to PBBs may affect not only mothers and offspring suckling but subsequent progeny as well.
INTRODUCTION
Polybrominated biphenyls (PBBs) are fire retardants which were added accidentally to the food supply at Michigan (Dunckel, 1975). Brilliant and co-workers (1978) estimated that by 1976 over 85% of Michigan’s residents had detectable body burdens of PBBs. Pharmacokinetic properties of individual PBB congeners depend on the position and degree of bromination (Matthews et al., 1978). However, most PBBs are lipophilic and biologically stable, and accumulate in tissues, particularly those with a high fat content such as mammary gland. Matthews and co-workers (1977) predicted that less than 10% of a single oral dose of 2,4,5,2’,4’,5’-hexabromobiphenyl, the major congener in the mixture of PBBs added to livestock feed (Sundstrom et al., 1976), would ever be excreted by healthy, normally fed male rats. Body burdens of females are reduced by pregnancy and lactation because PBBs cross the placenta and are excreted in milk (Fries and Marrow, 1975; Brilliant et al., 1978); the most effective transfer of PBBs from mother to offspring occurs via milk (Rickert et al., 1978). The highest reported concentrations of PBBs detected in human milk were between 90 and 110 ppm on a fat basis (Cordle et al., 1978). However, the median concentration of PBBs in milk from Michigan women may be less than 0.1 ppm on a fat basis (Brilliant et al., 1978). During the dry period following lactation, PBBs in maternal I Michigan
State Agricultural
Experiment
Station 110
0013-935V8210101
lo-08$02.00/O
Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
Journal
Article
No.
9838.
EFFECTS
OF
PBBs ON
MATERNAL
RATS
111
tissues are redistributed (Fries et al., 1978). Fries and co-workers (1978) suggested that PBB residues in mammary gland fat and body fat equilibrated in cows during the dry period, resulting in higher concentrations of PBBs in nonlactating than in lactating mammary glands. Although few human health effects have been attributed to PBBs, a variety of morphological and biochemical alterations have been produced in other species (DiCarlo ef al., 1978; Kimbrough et al., 1978). Microsomal mixed function oxidase (MFO) activity was increased in liver and extrahepatic tissues, including mammary gland, by PBBs (Dent et al., 1977; McCormack et al., 1979). Modifications in the concentrations of PBBs in maternal tissues during the dry period following lactation or after nursing subsequent offspring may result in altered microsomal enzyme activity. Alterations in MFO activity in the mammary gland may affect not only the susceptibility of the mammary gland to deleterious effects of certain xenobiotics but also the quantity and chemical form of compounds transmitted via milk to suckling offspring. Therefore, it was of interest to determine if changes in tissue concentrations of PBBs subsequent to nursing offspring or during the recovery period following lactation were correlated with alterations in the activity of microsomal enzymes in mammary gland and liver. MATERIALS
AND METHODS
Sprague-Dawley rats were obtained on Day 2 (inseminated on Day 0) of pregnancy (Spartan Farms, Haslett, Mich.). Animals were maintained in clear polypropylene cases 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. Rats were fed diets containing 0, 10, or 100 ppm PBBs (Firemaster BP-6 Velsicol Chemical Co., St. Louis, MO.) from Day 8 of pregnancy until 28 days postpartum when pups were weaned. The composition of Firemaster BP-6 has been reported by Moore et czf.(1980). The diets were prepared as described by Dent et al. (1978). Litters were culled to ten pups each at birth. Effects of PBBs on the offspring are included in another report (McCormack et al., 1981). Six treatment groups of maternal rats were included in this investigation (0, 10, 100, O/O, 100/O, and loo/-). Each group contained at least four animals and was designated by the concentration of PBBs in diets consumed during the first or second pregnancy and lactation (Table 1). Three groups of rats were used in experiments when their first and only litter was weaned. These rats were designated as 0, 10, or 100 animals according to the concentration of PBBs in their diet. Animals in the other three groups were switched from a diet containing 100 ppm PBBs to a diet free of PBBs when their first litter was weaned (100/O and lOO/rats) or were never exposed to PBBs (O/O rats). Rats in the O/O or 100/O groups were rebred with untreated male Sprague- Dawley rats 6-10 weeks after the weaning of their first litter. Experiments were conducted in O/O and 100/O rats when their second litter was weaned. Rats in the lOO/- treatment group were not rebred. Experiments were conducted in lOO/- rats 14 weeks after their first and only litter was weaned. Animals were killed by cervical dislocation. Liver and mammary gland (ingui-
112
MC
CORMACK
AND
TABLE TREATMENT
GROUPS
Concentration Treatment group designation 0
10 100 o/o loo/o loo/-
First
litter
1
OF MATERNAL
of PBBs
in diet (ppm)
Second
litter
HOOK
RATS
EXPOSED
TO PBBs
Total exposure to PBBs in diet (weeks)
Recovery period (weeks)
0 10 100
0 0
0 100 100
0 0 b
12-16 12-16 14
0
U Rats were used in experiments when their first and only litter was weaned (28 days postpartum). b Rats were placed on diet free of PBBs when their first and only litter was weaned: however, they were allowed a 14-week recovery period before being killed.
nal) were excised, weighed, and then chopped into 3 vol of ice-cold 1.15% KC1 (livers) or 66 mM Tris-HCl, pH 7.4 (mammary glands). Liver was homogenized with a Potter-Elvehjem homogenizer and Teflon pestle. Mammary gland was homogenized with a Brinkmann Polytron (Brinkmann Instruments, Westbury, N.Y.). Postmitochondrial supernatants were prepared by centrifugation of homogenates at 10,OOOg for 20 min. All assays were performed on the day of supernatant preparation. Protein was measured by the method of Lowry et al. (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 hydrolase (EH) activity was determined by the method of Oesch et al. (1971). Concentrations of PBBs in liver, mammary gland (inguinal), and fat (perirenal) were determined using the following procedure: Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Each powdered tissue was extracted four times with 30-ml portions of hexane. The pooled hexane extracts were reduced in volume to l-2 ml which was placed on a Florisil(60100 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 Sc3H electron capture detector using a column, 1.7 m x 2 mm i.d., packed with 1% OV-1. Carrier gas (NJ flow and column temperature were 30 mYmin and 236”C, respectively. Concentrations of PBBs were determined from the height of the major peak (2,4,5,2’,4’,5’-hexabromobiphenyl) in the gas chromatograms. Concentrations of PBBs were expressed as micrograms of PBBs (2,4,5,2’,4’,5’-hexabromobiphenyl) per gram wet tissue. 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.
EFFECTS
OF
PBBs ON
MATERNAL
113
RATS
RESULTS
Treatment- and tissue-related differences in the distribution of PBBs were observed in maternal rats exposed to PBBs and killed when their litters were weaned (Table 2). Of the tissues examined, concentrations of PBBs were generally highest in fat and lowest in liver. Concentrations of PBBs in fat and liver were highest in rats exposed to 100 ppm PBBs and killed when their first litter was weaned (animals in the 100 treatment group). The concentration of PBBs in fat from nonlactating animals that were allowed a 1Cweek recovery period (loo/-) was 50% of that in fat from animals in the 100 treatment group. However, the concentration of PBBs in mammary gland from nonlactating animals that underwent a recovery period (100/-J was five times higher than that in these lactating rats (100). These results in rats confirm a previous report in cows (Fries ef al., 1978) that PBB residues in mammary gland fat and body fat equilibrate during the dry period following lactation. Rats in the 10 and 100/O treatment groups had similar tissue concentrations of PBBs. Concentrations of PBBs in fat, mammary gland, and liver from rats that nursed a second litter (10010) were 17, 8, and 22% of the respective tissue concentrations in rats that were treated similarly but not rebred (lOO/-). The body weight was reduced and the liver weight-to-body weight ratio was increased only in rats in the 100 treatment group (86 and 117% of 0 group values, respectively). However, activity of AHH was increased in liver from animals in the 10, 100/O, lOO/-, and 100 treatment groups (approximately 150, 200, 1200, and 2200% of 0 group value, respectively). Activity of EH also was increased in liver from animals in the 10, 100/O, lOO/-, and 100 treatment groups (approximately 150, 150, 200, and 300% of 0 group value, respectively; Fig. I). Activity of AHH was increased in mammary gland from animals in the 100/O, loo/-, and 100 treatment groups (approximately 150, 250, and 200% of 0 group value, respectively; Fig. 2). Activity of EH in mammary gland was not affected by exposure to PBBs. A statistical difference was not detected between 0 and O/O animals in any parameter quantified in this investigation. DISCUSSION
Concentrations of PBBs in maternal tissues were reduced markedly by nursing a second litter. These findings are consistent with those of Vodicnik and Lech TABLE TISSUE Treatment
0 010 10 100/o
” Values maternal
OF PBBs
CONCENTRATIONS Liver
RATS
TO PBBs”
EXPOSED
Mammary
1 I 0.5 1.3
loo/-
12.1
100
53.5 2 5.9
are means in fig animals at weaning.
2
IN MATERNAL
k 4.6
2,4,5.2’.4’,5’-hexabromobiphenyYg
co. co. 14.8 2 19.1 2 225.2 k 43.1 c
Fat
I I 3.6 4.7 21.6 10.3 wet
co. I 0.3 90.4 k 23.2 76.8 t 18.2 459.4 t 61.8 812.5 t 92.4 tissue
+
SE
for
at least
three
114
MC
CORMACK
AND
HOOK
Epoxide Hydrolare
0
o/o
10
10010
loo/-
100
TREATMENT
FIG. 1. Effect of PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydrolase in liver. Treatments are described in the text. Values are means i SE for at least four maternal rats at weaning. Asterisk indicates a statistically significant difference from the control values (0 or 010. P i 0.05).
(1980), who reported that mice transferred a large fraction of their body burden of 2,4,5,2’,4’,5’-hexachlorobiphenyl to suckling offspring. However, quantitative predictions regarding excretion of accumulated PBBs in human milk are not yet possible. The increase in concentration of PBBs in mammary gland that occurred during the recovery period in lOO/- rats did not result in an activity of mammary gland AHH higher than that in animals in the 100 treatment group. However, the reduction in concentration of PBBs in mammary gland from rats that nursed a second litter (100/O) was correlated with an activity of mammary gland AHH lower than that in nonlactating rats (loo/-). Activity of AHH and EH in liver reflected hepatic concentrations of PBBs which were highest in rats in the 100 treatment group. The reduction in concentration of PBBs in liver from 100/O rats was correlated with an activity of hepatic AHH and EH lower than that in 100 or lOO/animals. Results from this investigation demonstrate that microsomal MFO activity and
EFFECTS
OF
PBBs
ON
010
10
MATERNAL
RATS
115
ipoxide iydrolase
0
100/o
lOO/-
100
TREATMENT
FIG. 2. Effect of PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydrolase in mammary gland. Treatments are described in the text. Values are means r+ SE for at least four maternal rats at weaning. Asterisk indicates a statistically significant difference from the control values (0 or O/O),P < 0.05.
concentrations of PBBs in mammary gland and liver are reduced by nursing a second litter. Although microsomal MFO activity was reduced in tissues from 10010 rats relative to 100 or lOO/- rats, MFO activity in mammary gland and liver from 100/O rats remained increased above control (0 or O/O activity. A variety of lipophilic compounds 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 biological action of certain compounds administered subsequently. Pretreatment of mice with PBBs increased the toxicity of bromobenzene (Roes et al., 1977), which is metabolized to the putative hepatotoxicant bromobenzene-3,4epoxide (Zampaglione et al., 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 et al., 1972). Therefore, persistent stimulation of mammary gland and hepatic microsomal
116
MC
CORMACK
AND
HOOK
MFOs while mothers are exposed to PBBs may affect not only mothers offspring suckling but subsequent progeny as well.
and
ACKNOWLEDGMENTS This investigation was supported in part by National Institute of Environmental Health Sciences Grant ESOOS60 and a grant from the Michigan Department of Agriculture. We wish to express our appreciation to Dr. W. E. Braselton, Jr., for assistance with PBB quantification, L. F. Lepper. M. Steger. and D. M. Wilson for technical assistance. and D. K. Hummel for help in manuscript preparation.
REFERENCES Brilliant, L. B., Van Amburg. G., Isbister, J.. Humphrey. H., Wilcox, K., Eyster. J., Bloomer, A. W.. and Price, H. (1978). Breast-milk monitoring to measure Michigan’s contamination with polybrominated biphenyls. Lo~crt 2, 643-646. Conney, A. H. (1967). Pharmacological implications of microsomal enzyme induction. P/rcrr/r~ccco/. Rcl,. 19, 317-366. Cordle, F.. Corneliussen, P., Jelinek, L.. Hackley. B., Lehman, R., McLaughlin, J., Rhoden. R., and Shapiro, R. (1978). Human exposure to polychlorinated biphenyls and polybrominated biphenyls. E/~viro/r H~nltJt Pcrspcct. 24, 157- 172. Dent, J. G.. Cagen, S. Z., McCormack, K. M., Rickert, D. E., and Gibson, J. E. (1977). Liver and mammary arylhydrocarbon hydroxylase and epoxide hydratase in lactating rats fed polybrominated biphenyls. Lij)) Sci. 20, 2075-2080. Dent, J. G., McCormack, K. M.. Rickert, D. E.. Cagen, S. 2.. Melrose, P., and Gibson, J. E. (1978). Mixed function oxidase activities in lactating rats and their offspring following dietary exposure to polybrominated biphenyls. To.rico/. Appl. Phorm~oJ. 46, 727- 735. DiCarlo, F. J., Seifter, J., and DeCarlo, V. J. (1978). Assessment of the hazards of polybrominated biphenyls. Enr?ro,r. Hrrrlfh Prrsprct. 23. 351-365. Dunckel. A. E. (1975). An updating of the polybrominated biphenyl disaster in Michigan. J. Artier. Vet. Med. Assrx~. 167. 838-841. Fries, G. E., and Marrow, G. S. (1975). Excretion of polybrominated biphenyls into milk of cows. J. Dairy SC,;. 58, 947-951. Fries, G. F.. Marrow, G. S.. and Cook, R. M. (1978). Distribution and kinetics of PBB residues in cattle. Et/r,il-otr. Hcwlth Persp~ct. 23, 43-50. Kimbrough, R., Buckley. J., Fishbein, L., Flamm. G., Kasza. L., Marcus, W.. Shibko. S., and Teske, R. (1978). Animal toxicology. Etr~~iror~. Hertlth Pcrspect. 24, 173- 184. Kluwe, W. M., McCormack, K. M.. and Hook, J. B. (1978). Potentiation of hepatic and renal toxicity of various compounds by prior exposure to polybrominated biphenyls. Envirm. Health Prxyxct. 23, 241-246. Lowry, 0. H., Rosebrough, N. J., Farr. A. L.. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. .I. Biol. Chr~rr. 193, 265-275. Matthews, H. B., Kato, S., Morales. N. M.. and Tuey, D. B. (1977). Distribution and excretion of 2.4,5.2’,4’,5’-hexabromobiphenyl. the major component of Firemaster BP-6. J. To.uic~>/. E!triron. HcwltJt. 3, 599-605. Matthews, H., Fries, G., Gardner, A., Garhoff, L., Goldstein, J., Ku, Y., and Moore, J. (1978). Metabolism and biochemical toxicity of PCBs and PBBs. En~irorz. Health Prrspct. 24, 147- 155. McCormack, K. M., Melrose, P., Rickert, D. E., Dent, J. G., Gibson, J. E., and Hook, J. B. (1979). Concomitant dietary exposure to polychlorinated biphenyls and polybrominated biphenyls: Tissue distribution and arylhydrocarbon hydroxylase activity in lactating rats. Tc~.rico/. Appl. Phur/rluco/. 47, 95- 104. McCormack, K. M.. Lepper, L. F., Wilson, D. M., and Hook, J. B. (1981). Biochemical and physiological sequelae to perinatal exposure to polybrominated biphenyls: A multigeneration study in rats. To.uic~ol. Appl. Phortturcol. 59. 300-3 13. Moore, R. W., Dannan, G. A.. and Aust, S. D. (1980). Structure-function relationships for the pharmacological and toxicological effects and metabolism of polybrominated biphenyl congeners. Irr
EFFECTS
OF
PBBs ON
MATERNAL
RATS
117
“Molecular Basis of Environmental Toxicity” (R. S. Bhatnagar, Ed.), pp. 173-212. Ann Arbor Science Pub., Ann Arbor, Mich. Nebert, D. W., and Gelboin, H. V. ( 1968). Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. Biol. Che/tr. 243, 6242-6249. Oesch, F. (1976). Differential control of rat microsomal “aryl hydrocarbon” monooxygenase and epoxide hydratase. J. Biol. c‘he/tt. 251, 79-87. Oesch, F., Jerina. D. M., and Daly, J. (1971). A radiometric assay for hepatic epoxide hydrase activity with [7-3H]styrene oxide. Bid7i~u. Bioplzy.s. Aucr 227, 685-691. Rickert, D. E., Dent, J. G.. Cagen, S. Z., McCormack, K. M., Melrose, P., and Gibson, J. E. (1978). Distribution of polybrominated biphenyls after dietary exposure in pregnant and lactating rats and their offspring. f%r*if~
27, 1 lo-
RESEARCH
117 (1982)
Effects of Lactation and Nursing on Tissue Concentrations of Polybrominated Biphenyls and on Microsomal Enzyme Activity in Mammary Gland and Liver in Maternal Rats’ K. M. MCCORMACK
Received
AND J. B. HOOK
February
10. 1981
Redistribution of polybrominated biphenyls (PBBs) during the recovery period following lactation resulted in concentrations of PBBs that were higher in mammary gland and lower in liver and fat than at the end of lactation. Hepatic arylhydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH) activities were higher in PBB-treated rats during lactation than in the recovery period. However, transition from lactating to dry state had no effect on activity of mammary gland AHH in rats treated with PBBs. Microsomal enzyme activity and concentrations of PBBs in mammary gland and liver were reduced by nursing a second litter. Although redistribution and excretion of PBBs resulted in alterations in microsomal enzyme activity, all rats exposed to 100 ppm PBBs had microsomal enzyme activity in liver and mammary gland that was higher than that in controls. Persistent stimulation of mammary gland and hepatic microsomal enyzmes while mothers are exposed to PBBs may affect not only mothers and offspring suckling but subsequent progeny as well.
INTRODUCTION
Polybrominated biphenyls (PBBs) are fire retardants which were added accidentally to the food supply at Michigan (Dunckel, 1975). Brilliant and co-workers (1978) estimated that by 1976 over 85% of Michigan’s residents had detectable body burdens of PBBs. Pharmacokinetic properties of individual PBB congeners depend on the position and degree of bromination (Matthews et al., 1978). However, most PBBs are lipophilic and biologically stable, and accumulate in tissues, particularly those with a high fat content such as mammary gland. Matthews and co-workers (1977) predicted that less than 10% of a single oral dose of 2,4,5,2’,4’,5’-hexabromobiphenyl, the major congener in the mixture of PBBs added to livestock feed (Sundstrom et al., 1976), would ever be excreted by healthy, normally fed male rats. Body burdens of females are reduced by pregnancy and lactation because PBBs cross the placenta and are excreted in milk (Fries and Marrow, 1975; Brilliant et al., 1978); the most effective transfer of PBBs from mother to offspring occurs via milk (Rickert et al., 1978). The highest reported concentrations of PBBs detected in human milk were between 90 and 110 ppm on a fat basis (Cordle et al., 1978). However, the median concentration of PBBs in milk from Michigan women may be less than 0.1 ppm on a fat basis (Brilliant et al., 1978). During the dry period following lactation, PBBs in maternal I Michigan
State Agricultural
Experiment
Station 110
0013-935V8210101
lo-08$02.00/O
Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
Journal
Article
No.
9838.
EFFECTS
OF
PBBs ON
MATERNAL
RATS
111
tissues are redistributed (Fries et al., 1978). Fries and co-workers (1978) suggested that PBB residues in mammary gland fat and body fat equilibrated in cows during the dry period, resulting in higher concentrations of PBBs in nonlactating than in lactating mammary glands. Although few human health effects have been attributed to PBBs, a variety of morphological and biochemical alterations have been produced in other species (DiCarlo ef al., 1978; Kimbrough et al., 1978). Microsomal mixed function oxidase (MFO) activity was increased in liver and extrahepatic tissues, including mammary gland, by PBBs (Dent et al., 1977; McCormack et al., 1979). Modifications in the concentrations of PBBs in maternal tissues during the dry period following lactation or after nursing subsequent offspring may result in altered microsomal enzyme activity. Alterations in MFO activity in the mammary gland may affect not only the susceptibility of the mammary gland to deleterious effects of certain xenobiotics but also the quantity and chemical form of compounds transmitted via milk to suckling offspring. Therefore, it was of interest to determine if changes in tissue concentrations of PBBs subsequent to nursing offspring or during the recovery period following lactation were correlated with alterations in the activity of microsomal enzymes in mammary gland and liver. MATERIALS
AND METHODS
Sprague-Dawley rats were obtained on Day 2 (inseminated on Day 0) of pregnancy (Spartan Farms, Haslett, Mich.). Animals were maintained in clear polypropylene cases 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. Rats were fed diets containing 0, 10, or 100 ppm PBBs (Firemaster BP-6 Velsicol Chemical Co., St. Louis, MO.) from Day 8 of pregnancy until 28 days postpartum when pups were weaned. The composition of Firemaster BP-6 has been reported by Moore et czf.(1980). The diets were prepared as described by Dent et al. (1978). Litters were culled to ten pups each at birth. Effects of PBBs on the offspring are included in another report (McCormack et al., 1981). Six treatment groups of maternal rats were included in this investigation (0, 10, 100, O/O, 100/O, and loo/-). Each group contained at least four animals and was designated by the concentration of PBBs in diets consumed during the first or second pregnancy and lactation (Table 1). Three groups of rats were used in experiments when their first and only litter was weaned. These rats were designated as 0, 10, or 100 animals according to the concentration of PBBs in their diet. Animals in the other three groups were switched from a diet containing 100 ppm PBBs to a diet free of PBBs when their first litter was weaned (100/O and lOO/rats) or were never exposed to PBBs (O/O rats). Rats in the O/O or 100/O groups were rebred with untreated male Sprague- Dawley rats 6-10 weeks after the weaning of their first litter. Experiments were conducted in O/O and 100/O rats when their second litter was weaned. Rats in the lOO/- treatment group were not rebred. Experiments were conducted in lOO/- rats 14 weeks after their first and only litter was weaned. Animals were killed by cervical dislocation. Liver and mammary gland (ingui-
112
MC
CORMACK
AND
TABLE TREATMENT
GROUPS
Concentration Treatment group designation 0
10 100 o/o loo/o loo/-
First
litter
1
OF MATERNAL
of PBBs
in diet (ppm)
Second
litter
HOOK
RATS
EXPOSED
TO PBBs
Total exposure to PBBs in diet (weeks)
Recovery period (weeks)
0 10 100
0 0
0 100 100
0 0 b
12-16 12-16 14
0
U Rats were used in experiments when their first and only litter was weaned (28 days postpartum). b Rats were placed on diet free of PBBs when their first and only litter was weaned: however, they were allowed a 14-week recovery period before being killed.
nal) were excised, weighed, and then chopped into 3 vol of ice-cold 1.15% KC1 (livers) or 66 mM Tris-HCl, pH 7.4 (mammary glands). Liver was homogenized with a Potter-Elvehjem homogenizer and Teflon pestle. Mammary gland was homogenized with a Brinkmann Polytron (Brinkmann Instruments, Westbury, N.Y.). Postmitochondrial supernatants were prepared by centrifugation of homogenates at 10,OOOg for 20 min. All assays were performed on the day of supernatant preparation. Protein was measured by the method of Lowry et al. (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 hydrolase (EH) activity was determined by the method of Oesch et al. (1971). Concentrations of PBBs in liver, mammary gland (inguinal), and fat (perirenal) were determined using the following procedure: Samples were weighed and ground with sufficient anhydrous sodium sulfate to render them completely dry and pulverized. Each powdered tissue was extracted four times with 30-ml portions of hexane. The pooled hexane extracts were reduced in volume to l-2 ml which was placed on a Florisil(60100 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 Sc3H electron capture detector using a column, 1.7 m x 2 mm i.d., packed with 1% OV-1. Carrier gas (NJ flow and column temperature were 30 mYmin and 236”C, respectively. Concentrations of PBBs were determined from the height of the major peak (2,4,5,2’,4’,5’-hexabromobiphenyl) in the gas chromatograms. Concentrations of PBBs were expressed as micrograms of PBBs (2,4,5,2’,4’,5’-hexabromobiphenyl) per gram wet tissue. 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.
EFFECTS
OF
PBBs ON
MATERNAL
113
RATS
RESULTS
Treatment- and tissue-related differences in the distribution of PBBs were observed in maternal rats exposed to PBBs and killed when their litters were weaned (Table 2). Of the tissues examined, concentrations of PBBs were generally highest in fat and lowest in liver. Concentrations of PBBs in fat and liver were highest in rats exposed to 100 ppm PBBs and killed when their first litter was weaned (animals in the 100 treatment group). The concentration of PBBs in fat from nonlactating animals that were allowed a 1Cweek recovery period (loo/-) was 50% of that in fat from animals in the 100 treatment group. However, the concentration of PBBs in mammary gland from nonlactating animals that underwent a recovery period (100/-J was five times higher than that in these lactating rats (100). These results in rats confirm a previous report in cows (Fries ef al., 1978) that PBB residues in mammary gland fat and body fat equilibrate during the dry period following lactation. Rats in the 10 and 100/O treatment groups had similar tissue concentrations of PBBs. Concentrations of PBBs in fat, mammary gland, and liver from rats that nursed a second litter (10010) were 17, 8, and 22% of the respective tissue concentrations in rats that were treated similarly but not rebred (lOO/-). The body weight was reduced and the liver weight-to-body weight ratio was increased only in rats in the 100 treatment group (86 and 117% of 0 group values, respectively). However, activity of AHH was increased in liver from animals in the 10, 100/O, lOO/-, and 100 treatment groups (approximately 150, 200, 1200, and 2200% of 0 group value, respectively). Activity of EH also was increased in liver from animals in the 10, 100/O, lOO/-, and 100 treatment groups (approximately 150, 150, 200, and 300% of 0 group value, respectively; Fig. I). Activity of AHH was increased in mammary gland from animals in the 100/O, loo/-, and 100 treatment groups (approximately 150, 250, and 200% of 0 group value, respectively; Fig. 2). Activity of EH in mammary gland was not affected by exposure to PBBs. A statistical difference was not detected between 0 and O/O animals in any parameter quantified in this investigation. DISCUSSION
Concentrations of PBBs in maternal tissues were reduced markedly by nursing a second litter. These findings are consistent with those of Vodicnik and Lech TABLE TISSUE Treatment
0 010 10 100/o
” Values maternal
OF PBBs
CONCENTRATIONS Liver
RATS
TO PBBs”
EXPOSED
Mammary
1 I 0.5 1.3
loo/-
12.1
100
53.5 2 5.9
are means in fig animals at weaning.
2
IN MATERNAL
k 4.6
2,4,5.2’.4’,5’-hexabromobiphenyYg
co. co. 14.8 2 19.1 2 225.2 k 43.1 c
Fat
I I 3.6 4.7 21.6 10.3 wet
co. I 0.3 90.4 k 23.2 76.8 t 18.2 459.4 t 61.8 812.5 t 92.4 tissue
+
SE
for
at least
three
114
MC
CORMACK
AND
HOOK
Epoxide Hydrolare
0
o/o
10
10010
loo/-
100
TREATMENT
FIG. 1. Effect of PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydrolase in liver. Treatments are described in the text. Values are means i SE for at least four maternal rats at weaning. Asterisk indicates a statistically significant difference from the control values (0 or 010. P i 0.05).
(1980), who reported that mice transferred a large fraction of their body burden of 2,4,5,2’,4’,5’-hexachlorobiphenyl to suckling offspring. However, quantitative predictions regarding excretion of accumulated PBBs in human milk are not yet possible. The increase in concentration of PBBs in mammary gland that occurred during the recovery period in lOO/- rats did not result in an activity of mammary gland AHH higher than that in animals in the 100 treatment group. However, the reduction in concentration of PBBs in mammary gland from rats that nursed a second litter (100/O) was correlated with an activity of mammary gland AHH lower than that in nonlactating rats (loo/-). Activity of AHH and EH in liver reflected hepatic concentrations of PBBs which were highest in rats in the 100 treatment group. The reduction in concentration of PBBs in liver from 100/O rats was correlated with an activity of hepatic AHH and EH lower than that in 100 or lOO/animals. Results from this investigation demonstrate that microsomal MFO activity and
EFFECTS
OF
PBBs
ON
010
10
MATERNAL
RATS
115
ipoxide iydrolase
0
100/o
lOO/-
100
TREATMENT
FIG. 2. Effect of PBBs on activity of arylhydrocarbon hydroxylase and epoxide hydrolase in mammary gland. Treatments are described in the text. Values are means r+ SE for at least four maternal rats at weaning. Asterisk indicates a statistically significant difference from the control values (0 or O/O),P < 0.05.
concentrations of PBBs in mammary gland and liver are reduced by nursing a second litter. Although microsomal MFO activity was reduced in tissues from 10010 rats relative to 100 or lOO/- rats, MFO activity in mammary gland and liver from 100/O rats remained increased above control (0 or O/O activity. A variety of lipophilic compounds 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 biological action of certain compounds administered subsequently. Pretreatment of mice with PBBs increased the toxicity of bromobenzene (Roes et al., 1977), which is metabolized to the putative hepatotoxicant bromobenzene-3,4epoxide (Zampaglione et al., 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 et al., 1972). Therefore, persistent stimulation of mammary gland and hepatic microsomal
116
MC
CORMACK
AND
HOOK
MFOs while mothers are exposed to PBBs may affect not only mothers offspring suckling but subsequent progeny as well.
and
ACKNOWLEDGMENTS This investigation was supported in part by National Institute of Environmental Health Sciences Grant ESOOS60 and a grant from the Michigan Department of Agriculture. We wish to express our appreciation to Dr. W. E. Braselton, Jr., for assistance with PBB quantification, L. F. Lepper. M. Steger. and D. M. Wilson for technical assistance. and D. K. Hummel for help in manuscript preparation.
REFERENCES Brilliant, L. B., Van Amburg. G., Isbister, J.. Humphrey. H., Wilcox, K., Eyster. J., Bloomer, A. W.. and Price, H. (1978). Breast-milk monitoring to measure Michigan’s contamination with polybrominated biphenyls. Lo~crt 2, 643-646. Conney, A. H. (1967). Pharmacological implications of microsomal enzyme induction. P/rcrr/r~ccco/. Rcl,. 19, 317-366. Cordle, F.. Corneliussen, P., Jelinek, L.. Hackley. B., Lehman, R., McLaughlin, J., Rhoden. R., and Shapiro, R. (1978). Human exposure to polychlorinated biphenyls and polybrominated biphenyls. E/~viro/r H~nltJt Pcrspcct. 24, 157- 172. Dent, J. G.. Cagen, S. Z., McCormack, K. M., Rickert, D. E., and Gibson, J. E. (1977). Liver and mammary arylhydrocarbon hydroxylase and epoxide hydratase in lactating rats fed polybrominated biphenyls. Lij)) Sci. 20, 2075-2080. Dent, J. G., McCormack, K. M.. Rickert, D. E.. Cagen, S. 2.. Melrose, P., and Gibson, J. E. (1978). Mixed function oxidase activities in lactating rats and their offspring following dietary exposure to polybrominated biphenyls. To.rico/. Appl. Phorm~oJ. 46, 727- 735. DiCarlo, F. J., Seifter, J., and DeCarlo, V. J. (1978). Assessment of the hazards of polybrominated biphenyls. Enr?ro,r. Hrrrlfh Prrsprct. 23. 351-365. Dunckel. A. E. (1975). An updating of the polybrominated biphenyl disaster in Michigan. J. Artier. Vet. Med. Assrx~. 167. 838-841. Fries, G. E., and Marrow, G. S. (1975). Excretion of polybrominated biphenyls into milk of cows. J. Dairy SC,;. 58, 947-951. Fries, G. F.. Marrow, G. S.. and Cook, R. M. (1978). Distribution and kinetics of PBB residues in cattle. Et/r,il-otr. Hcwlth Persp~ct. 23, 43-50. Kimbrough, R., Buckley. J., Fishbein, L., Flamm. G., Kasza. L., Marcus, W.. Shibko. S., and Teske, R. (1978). Animal toxicology. Etr~~iror~. Hertlth Pcrspect. 24, 173- 184. Kluwe, W. M., McCormack, K. M.. and Hook, J. B. (1978). Potentiation of hepatic and renal toxicity of various compounds by prior exposure to polybrominated biphenyls. Envirm. Health Prxyxct. 23, 241-246. Lowry, 0. H., Rosebrough, N. J., Farr. A. L.. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. .I. Biol. Chr~rr. 193, 265-275. Matthews, H. B., Kato, S., Morales. N. M.. and Tuey, D. B. (1977). Distribution and excretion of 2.4,5.2’,4’,5’-hexabromobiphenyl. the major component of Firemaster BP-6. J. To.uic~>/. E!triron. HcwltJt. 3, 599-605. Matthews, H., Fries, G., Gardner, A., Garhoff, L., Goldstein, J., Ku, Y., and Moore, J. (1978). Metabolism and biochemical toxicity of PCBs and PBBs. En~irorz. Health Prrspct. 24, 147- 155. McCormack, K. M., Melrose, P., Rickert, D. E., Dent, J. G., Gibson, J. E., and Hook, J. B. (1979). Concomitant dietary exposure to polychlorinated biphenyls and polybrominated biphenyls: Tissue distribution and arylhydrocarbon hydroxylase activity in lactating rats. Tc~.rico/. Appl. Phur/rluco/. 47, 95- 104. McCormack, K. M.. Lepper, L. F., Wilson, D. M., and Hook, J. B. (1981). Biochemical and physiological sequelae to perinatal exposure to polybrominated biphenyls: A multigeneration study in rats. To.uic~ol. Appl. Phortturcol. 59. 300-3 13. Moore, R. W., Dannan, G. A.. and Aust, S. D. (1980). Structure-function relationships for the pharmacological and toxicological effects and metabolism of polybrominated biphenyl congeners. Irr
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OF
PBBs ON
MATERNAL
RATS
117
“Molecular Basis of Environmental Toxicity” (R. S. Bhatnagar, Ed.), pp. 173-212. Ann Arbor Science Pub., Ann Arbor, Mich. Nebert, D. W., and Gelboin, H. V. ( 1968). Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. Biol. Che/tr. 243, 6242-6249. Oesch, F. (1976). Differential control of rat microsomal “aryl hydrocarbon” monooxygenase and epoxide hydratase. J. Biol. c‘he/tt. 251, 79-87. Oesch, F., Jerina. D. M., and Daly, J. (1971). A radiometric assay for hepatic epoxide hydrase activity with [7-3H]styrene oxide. Bid7i~u. Bioplzy.s. Aucr 227, 685-691. Rickert, D. E., Dent, J. G.. Cagen, S. Z., McCormack, K. M., Melrose, P., and Gibson, J. E. (1978). Distribution of polybrominated biphenyls after dietary exposure in pregnant and lactating rats and their offspring. f%r*if~