Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Polybrominated biphenyls: Inducers of hepatic microsomal enzymes and type a cytochrome P450 in the rat J. G. Babish & G. S. Stoewsand To cite this article: J. G. Babish & G. S. Stoewsand (1977) Polybrominated biphenyls: Inducers of hepatic microsomal enzymes and type a cytochrome P450 in the rat, Journal of Toxicology and Environmental Health, 3:4, 673-682, DOI: 10.1080/15287397709529601 To link to this article: http://dx.doi.org/10.1080/15287397709529601
Published online: 15 Oct 2009.
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Citing articles: 6 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Monash University Library]
Date: 11 November 2015, At: 19:14
POLYBROMINATED BIPHENYLS: INDUCERS OF HEPATIC MICROSOMAL ENZYMES AND TYPE A CYTOCHROME P 450 IN THE RAT J. G. Babish, G. S. Stoewsand
Downloaded by [Monash University Library] at 19:14 11 November 2015
Department of Food Science and Technology, Cornell University, Geneva, New York
A flame retardant used in the textile industry, known as FireMaster PB-6, consists of a mixture of polybrominated biphenyl isomers (PBBs) with the following reported percentage isomeric composition of various brominated biphenyis: tetrabromo, 2.0; pentabromo, 10.6; hexabromo, about 63; heptabromo, 13.2; and the remainder unidentified. Although these chemicals possess a low acute toxicity (the LD50 for rats is 21.5 g/kg), recent episodes of accidental ingestion indicate a possible high chronic toxicity. To evaluate the subchronic toxicity of PBB, rats were fed diets containing 0, 1, or 50 ppm of the PBB mixture. In the first experiment animals were killed every 5 days for 3 wk, while all animals in the second experiment were killed after 3 wk of consuming the experimental diets. The influence of PPBs was studied by determining weight gain, feed efficiency, relative liver weight (RLW), and the following hepatic parameters: microsomal enzyme activities (aryl hydrocarbon hydroxyiase, N- and O-demethylase), microsomal protein content, cytochrome P450 concentration (type a and type b), PBB residues, and total lipid. Relative liver weight, hepatic microsomal enzyme activities, microsomal protein, cytochrome P450, and total lipid content were increased at 50 ppm PBB. Maximal induction (~260% of control) of aryl hydrocarbon hydroxyiase occurred within 5 days of PBB administration, while N- and O-demethylase activities took twice as long to reach maximal levels (~300% of control). A blue shift of the Soret peak of cytochrome P450 from 450 to 448 nm was observed in the CO difference spectrum of microsomes from animals consuming 50 ppm PBB in their diets. Increases in both type a and type b subspecies of cytochrome P450 were observed at 1 and 50 ppm dietary PBB. However, the ratio a/b increased only at the 50 ppm level. From these results, it can be concluded that the toxicity of the brominated biphenyis in rats differs little from that of the chlorinated biphenyis of similar halogenation.
INTRODUCTION Polybrominated biphenyis (PBBs), manufactured under the trade name FireMaster PB-6 for use as a textile flame retardant since 1970, were We thank Mr. Walt Gutenmann and Dr. Don Lisk for their assistance, including PBB analysis, in this study. This investigation was supported in part by Hatch funds through the Director, New York State Agricultural Experiment Station. J. G. Babish's present address is Food and Drug Research Laboratories, Inc., Route 17C, Waverly, New York. Requests for reprints should be sent to G. S. Stoewsand, Department of Food Science and Technology, Cornell University, Geneva, New York 14456.
673 Journal of Toxicology and Environmental Health, 3:673-682,1977 Copyright © 1977 by Hemisphere Publishing Corporation
Downloaded by [Monash University Library] at 19:14 11 November 2015
674
J.G.BABISH AND G.S.STOEWSAND
estimated to have contaminated about 1 million chickens, with tissue residues in excess of 1 ppm and egg residues of approximately 0.3 ppm. Large losses of cattle and other livestock in Michigan were related to the consumption of PBB-contaminated feed (Hoeting, 1976). In such situations, the paucity of research concerning the chronic toxicity of PBBs led to the application of polychlorinated biphenyl (PCB) guidelines. However, work in our laboratories (Babish et al., 1975; Gutenmann and Lisk, 1975) suggested that although the effects of PCBs and PBBs may be qualitatively similar, quantitative differences may exist which make the potential toxicity of PBBs greater than that of PCBs. The purpose of this study was to determine whether low levels of PBBs consumed over a short period of time produce measurable changes in growth, microsomai enzyme activity, P 450 ligand binding characteristics, or hepatic iipid content. METHODS In the first experiment, individually caged weanling male SpragueDawley rats with initial body weights of 45-50 g were fed ad libitum the semipurified diet previously described (Babish and Stoewsand, 1975) for 3 wk. The animals were then divided into three groups with eight rats per group and placed on the experimental diets containing 0, 1, or 50 ppm of the PBB mixture. Addition of PBBs to the diets was effected by dissolving the PBBs in the corn oil required for formulation of the diet. All diets and distilled water were available ad libitum. Lighting in the animal quarters was set to provide 12 hr of light from 6 a.m. to 6 p.m. daily. Temperature and relative humidity were maintained at 23.5°C and 55%, respectively. To minimize circadian effects of microsomai enzyme activity, animals from each treatment were killed by decapitation between 6 and 8 a.m. following an 8 hr fast, 5, 10, 15, and 20 days after the introduction of PBBs into the diet. The livers were perfused with a cold 0.9% NaCI solution through the hepatic vein, weighed, sliced, and homogenized in four volumes of ice-cold 1.15% KG containing 20 mM Tris-HCI buffer, pH 7.4 (at 37°C), using a Potter-Elvehjem Teflon-glass homogenizer fitted to a mechanical drill. All microsomai enzyme analyses were performed on the 12,000 Xg supernatant (postmitochondrial fraction) obtained after two successive centrifugal steps: 2,000 Xg for 10 min and 12,000 Xg for 20 min. Centrifugation of the postmitochondrial supernatant at 105,000 Xg for 60 min produced a pellet, which was washed and resuspended in 40 mM Tris-HCI buffer, pH 7.4 (at 22°C), and recentrifuged at 105,000 Xg for 20 min. The washed microsomai pellet was resuspended in the 40 mM Tris-HCI buffer, pH 7.4 (at 22°C), and used for cytochrome P 4s0 quantitation. All solutions used in preparing both the postmitochondrial supernatant and 105,000 Xg fractions were maintained at 0°-4°C. Microsomai enzyme activities were determined within 4 hr of decapitation.
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675
The 2 ml incubation mixture used in the determination of microsomal enzyme activity contained 1.0 ml of the 12,000 X q supernatant, 0.15 M KCI, 20 m/W Tris-HCI buffer, pH 7.4 (at 37°C), 1.2 m/W NADP, 8.8 m/W isocitrate, 60 m/W MgCI2, 7.5 m/W semicarbazide HCI when assaying /V-demethylation, 0.18 unit of isocitrate dehydrogenase (Ls-isocitrate: NADP oxidoreductase, E.C. 1.1.1.42) and substrate in one of the following concentrations: aminopyrine, 7.6 m/W; benzo[o] pyrene, 100 /i/W; p-nitroanisole, 200 \iM. This mixture was incubated under ambient air in 30 ml beakers using a Dubnoff shaking incubator at 37°C. All observations were made during a time interval when reaction rates were linear. Reactions were initiated by addition of the substrate and terminated with 1 ml of a 20% trichloroacetic acid solution in the aminopyrine and p-nitroanisole assays and with 2 ml of ice-cold acetone in the benzo[o]pyrene assay. The /V-demethylation of aminopyrine was measured by determination of the formaldehyde formed using the colorimetric procedure of Nash (1953). Aryl hydrocarbon hydroxylase (AHH) assay was performed using benzofa]pyrene as the substrate and measuring the fluoresence of the 3-hydroxybenzo[o] pyrene produced, as previously described (Nebert and Gelboin, 1968). Microsomal p-nitroanisole Odemethylase activity was determined by measuring the product of the reaction, p-nitrophenol, as described by Kato and Gillette (1965). In the tables, all enzyme activities are expressed as nanomoles of product per milligram protein per hour. The quantity of cytochrome P4S0 in the 105,000 X^ pellet was determined by the method described by Schoene et al. (1972) using a Cary model 15 spectrophotometer. Under the conditions of the assay, the apparent molar extinction coefficient of the reduced P450-CO complex is 91 mAT'cm" 1 . Analytical procedures for the tissue residues of PBB have been described (Gutenmann and Lisk, 1975). Total hepatic lipid content was determined by the method of Folch et al. (1951). In the second experiment, animals (six males per group) and diets were treated exactly as described earlier with the exception of the spaced killing procedure. Instead, all rats consumed the experimental diets for 3 wk and were killed on the same day. Isolation of the 105,000 Xg pellet and n-octylamine binding studies were performed as described by Jefcoate and co-workers (1970). Type a cytochrome P 450 was calculated from the difference in absorption between 392 and 500 nm (minus 40% y4410 due to type b) using the molar extinction coefficient 65 m/W 1 c m " 1 . Type b cytochrome P 450 was calculated from the difference in absorption between 410 and 500 nm using the molar extinction coefficient 25 m/W"1 cm" 1 (Jefcoate et al., 1970). Ten microliters of /7-octylamine in methanol was added to 2 ml of microsomes (1-2 mg protein/ml) to make a final concentration of 1.0 m/W n-octylamine; TO /xl of methanol alone was
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J.G. BABISH AND G. S.STOEWSAND
added to the microsomal suspension in the reference cuvette. Total CO-binding hemoprotein in the microsomal suspensions was determined as described by Omura and Sato (1964) using 91 vnM'1 cm'1 for the molar extinction coefficient for the difference in absorption between the Soret maximum and 490 nm. Protein was determined in duplicate by the modification of the Lowry method described by Sutherland et al. (1949). Crystalline bovine serum albumin, fraction V, was used as the standard. The first experiment was statistically analyzed as a two-factor design, with time and level of PBB representing the factors. Experiment 2 was analyzed as a one-way completely randomized design, with level of PBB as the only factor. Tukey's multiple range and Isd tests were used to determine significant differences among treatments. All statistical analyses were performed on a desk-top calculator (Hewlett-Packard model 9820A) using formulas as described by Snedecor and Cochran (1972). RESULTS The effects of dietary administration of PBB on final body weight and relative liver weight (RLW) for both experiments are presented in Table 1. As can be seen from Table 1, no effect of PBB was observed on final body weight. Furthermore, no disparity existed in rate of growth or feed efficiency among the treatments. However, significant increases (/? 0.05) from the theoretical value of 1.0 in the controls; it decreased slightly but not significantly at the 1 ppm treatment, and dropped further (significantly with p < 0.01) in the animals consuming 50 ppm PBB in their diets. Binding of A7-octylamine with rat liver microsomes produced the spectra depicted in Fig. 2. Spectra from animals receiving 0 or 1 ppm PBB TABLE 4. Effect of Dietary Polybrominated Biphenyls on the Distribution of Type a and Type b Cytochrome Ptiaa
Dietary PPB concentration (ppm) 0 1
50
Type a 6 (nmol/mg microsomal protein)
Type b c (nmol/mg microsomal protein)
a/b
0.152 ± 0.0071 0.222 ± 0.0332 0.742 ± 0.0053
0.878 ± 0.033" 1.078 ± 0.073s 2.473 ±0.149 6
0.174 ± 0.0057 0.203 ± 0.0257 0.298 +0.017 8
0.954 ± 0.034' 0.919 ± 0.030' 0.821 ± 0.02610
"Values are means ± SEM of six observations; common numerical superscripts denote nonsignificant differences (p > 0.05). ^Species of CO-binding hemoprotein having iron in the high-spin state. Calculated from difference in absorption between 392 and 500 nm using the molar extinction coefficient 65 mAf~'cm~l. c Species of CO-binding hemoprotein representing the low-spin iron. Calculated from the difference in absorption between 410 and 500 nm using the molar extinction coefficient 25 nvWcrrT 1 . "Determined from CO difference spectrumusing the molar extinction coefficient 91 mM"' cm" 1 .
679
PBBs: MICROSOMAL ENZYMES AND P45O INDUCER
_429
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U
z < CQ
ex.
o
5 0 ppm DIETARY PBB 1 p p m DIETARY PBB ... CONTROL
FIGURE 2. Effect microsomes.
of dietary
polybrominated
biphenyls on n-octylamine binding in rat liver
exhibited the characteristic type II binding with minima at 410 nm and maxima at 432 nm. A shift in minimum and maximum absorption to 392 and 429 nm, respectively, was observed in rat liver microsomes obtained from animals consuming 50 ppm PBB in their diets. Hepatic PBB residue per moisture-free tissue and total lipid content after 2-3 wk of PBB consumption are presented in Table 5. Samples obtained early in the second week did not differ from those examined at the end of the third week with respect to PBB concentration or total lipid content. While a 70% increase in total hepatic lipid content relative to both the control and the 1 ppm PBB treatment was observed in the 50 ppm treatment, increasing the PBB concentration in the diet 50-fold produced a 125-fold increase in hepatic PBB residue.
680
J. G. BABISH AND G. S. STOEWSAND
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TABLE 5. Effect of Dietary Polybrominated Biphenyls on Hepatic Polybrominated Biphenyl Concentration and Total Hepatic Lipids" Dietary PBB (ppm)
PBB residue in liver6" (ppm dry weight)
% Hepatic lipids
0 1 50
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Polybrominated biphenyls: Inducers of hepatic microsomal enzymes and type a cytochrome P450 in the rat J. G. Babish & G. S. Stoewsand To cite this article: J. G. Babish & G. S. Stoewsand (1977) Polybrominated biphenyls: Inducers of hepatic microsomal enzymes and type a cytochrome P450 in the rat, Journal of Toxicology and Environmental Health, 3:4, 673-682, DOI: 10.1080/15287397709529601 To link to this article: http://dx.doi.org/10.1080/15287397709529601
Published online: 15 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 6 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [Monash University Library]
Date: 11 November 2015, At: 19:14
POLYBROMINATED BIPHENYLS: INDUCERS OF HEPATIC MICROSOMAL ENZYMES AND TYPE A CYTOCHROME P 450 IN THE RAT J. G. Babish, G. S. Stoewsand
Downloaded by [Monash University Library] at 19:14 11 November 2015
Department of Food Science and Technology, Cornell University, Geneva, New York
A flame retardant used in the textile industry, known as FireMaster PB-6, consists of a mixture of polybrominated biphenyl isomers (PBBs) with the following reported percentage isomeric composition of various brominated biphenyis: tetrabromo, 2.0; pentabromo, 10.6; hexabromo, about 63; heptabromo, 13.2; and the remainder unidentified. Although these chemicals possess a low acute toxicity (the LD50 for rats is 21.5 g/kg), recent episodes of accidental ingestion indicate a possible high chronic toxicity. To evaluate the subchronic toxicity of PBB, rats were fed diets containing 0, 1, or 50 ppm of the PBB mixture. In the first experiment animals were killed every 5 days for 3 wk, while all animals in the second experiment were killed after 3 wk of consuming the experimental diets. The influence of PPBs was studied by determining weight gain, feed efficiency, relative liver weight (RLW), and the following hepatic parameters: microsomal enzyme activities (aryl hydrocarbon hydroxyiase, N- and O-demethylase), microsomal protein content, cytochrome P450 concentration (type a and type b), PBB residues, and total lipid. Relative liver weight, hepatic microsomal enzyme activities, microsomal protein, cytochrome P450, and total lipid content were increased at 50 ppm PBB. Maximal induction (~260% of control) of aryl hydrocarbon hydroxyiase occurred within 5 days of PBB administration, while N- and O-demethylase activities took twice as long to reach maximal levels (~300% of control). A blue shift of the Soret peak of cytochrome P450 from 450 to 448 nm was observed in the CO difference spectrum of microsomes from animals consuming 50 ppm PBB in their diets. Increases in both type a and type b subspecies of cytochrome P450 were observed at 1 and 50 ppm dietary PBB. However, the ratio a/b increased only at the 50 ppm level. From these results, it can be concluded that the toxicity of the brominated biphenyis in rats differs little from that of the chlorinated biphenyis of similar halogenation.
INTRODUCTION Polybrominated biphenyis (PBBs), manufactured under the trade name FireMaster PB-6 for use as a textile flame retardant since 1970, were We thank Mr. Walt Gutenmann and Dr. Don Lisk for their assistance, including PBB analysis, in this study. This investigation was supported in part by Hatch funds through the Director, New York State Agricultural Experiment Station. J. G. Babish's present address is Food and Drug Research Laboratories, Inc., Route 17C, Waverly, New York. Requests for reprints should be sent to G. S. Stoewsand, Department of Food Science and Technology, Cornell University, Geneva, New York 14456.
673 Journal of Toxicology and Environmental Health, 3:673-682,1977 Copyright © 1977 by Hemisphere Publishing Corporation
Downloaded by [Monash University Library] at 19:14 11 November 2015
674
J.G.BABISH AND G.S.STOEWSAND
estimated to have contaminated about 1 million chickens, with tissue residues in excess of 1 ppm and egg residues of approximately 0.3 ppm. Large losses of cattle and other livestock in Michigan were related to the consumption of PBB-contaminated feed (Hoeting, 1976). In such situations, the paucity of research concerning the chronic toxicity of PBBs led to the application of polychlorinated biphenyl (PCB) guidelines. However, work in our laboratories (Babish et al., 1975; Gutenmann and Lisk, 1975) suggested that although the effects of PCBs and PBBs may be qualitatively similar, quantitative differences may exist which make the potential toxicity of PBBs greater than that of PCBs. The purpose of this study was to determine whether low levels of PBBs consumed over a short period of time produce measurable changes in growth, microsomai enzyme activity, P 450 ligand binding characteristics, or hepatic iipid content. METHODS In the first experiment, individually caged weanling male SpragueDawley rats with initial body weights of 45-50 g were fed ad libitum the semipurified diet previously described (Babish and Stoewsand, 1975) for 3 wk. The animals were then divided into three groups with eight rats per group and placed on the experimental diets containing 0, 1, or 50 ppm of the PBB mixture. Addition of PBBs to the diets was effected by dissolving the PBBs in the corn oil required for formulation of the diet. All diets and distilled water were available ad libitum. Lighting in the animal quarters was set to provide 12 hr of light from 6 a.m. to 6 p.m. daily. Temperature and relative humidity were maintained at 23.5°C and 55%, respectively. To minimize circadian effects of microsomai enzyme activity, animals from each treatment were killed by decapitation between 6 and 8 a.m. following an 8 hr fast, 5, 10, 15, and 20 days after the introduction of PBBs into the diet. The livers were perfused with a cold 0.9% NaCI solution through the hepatic vein, weighed, sliced, and homogenized in four volumes of ice-cold 1.15% KG containing 20 mM Tris-HCI buffer, pH 7.4 (at 37°C), using a Potter-Elvehjem Teflon-glass homogenizer fitted to a mechanical drill. All microsomai enzyme analyses were performed on the 12,000 Xg supernatant (postmitochondrial fraction) obtained after two successive centrifugal steps: 2,000 Xg for 10 min and 12,000 Xg for 20 min. Centrifugation of the postmitochondrial supernatant at 105,000 Xg for 60 min produced a pellet, which was washed and resuspended in 40 mM Tris-HCI buffer, pH 7.4 (at 22°C), and recentrifuged at 105,000 Xg for 20 min. The washed microsomai pellet was resuspended in the 40 mM Tris-HCI buffer, pH 7.4 (at 22°C), and used for cytochrome P 4s0 quantitation. All solutions used in preparing both the postmitochondrial supernatant and 105,000 Xg fractions were maintained at 0°-4°C. Microsomai enzyme activities were determined within 4 hr of decapitation.
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675
The 2 ml incubation mixture used in the determination of microsomal enzyme activity contained 1.0 ml of the 12,000 X q supernatant, 0.15 M KCI, 20 m/W Tris-HCI buffer, pH 7.4 (at 37°C), 1.2 m/W NADP, 8.8 m/W isocitrate, 60 m/W MgCI2, 7.5 m/W semicarbazide HCI when assaying /V-demethylation, 0.18 unit of isocitrate dehydrogenase (Ls-isocitrate: NADP oxidoreductase, E.C. 1.1.1.42) and substrate in one of the following concentrations: aminopyrine, 7.6 m/W; benzo[o] pyrene, 100 /i/W; p-nitroanisole, 200 \iM. This mixture was incubated under ambient air in 30 ml beakers using a Dubnoff shaking incubator at 37°C. All observations were made during a time interval when reaction rates were linear. Reactions were initiated by addition of the substrate and terminated with 1 ml of a 20% trichloroacetic acid solution in the aminopyrine and p-nitroanisole assays and with 2 ml of ice-cold acetone in the benzo[o]pyrene assay. The /V-demethylation of aminopyrine was measured by determination of the formaldehyde formed using the colorimetric procedure of Nash (1953). Aryl hydrocarbon hydroxylase (AHH) assay was performed using benzofa]pyrene as the substrate and measuring the fluoresence of the 3-hydroxybenzo[o] pyrene produced, as previously described (Nebert and Gelboin, 1968). Microsomal p-nitroanisole Odemethylase activity was determined by measuring the product of the reaction, p-nitrophenol, as described by Kato and Gillette (1965). In the tables, all enzyme activities are expressed as nanomoles of product per milligram protein per hour. The quantity of cytochrome P4S0 in the 105,000 X^ pellet was determined by the method described by Schoene et al. (1972) using a Cary model 15 spectrophotometer. Under the conditions of the assay, the apparent molar extinction coefficient of the reduced P450-CO complex is 91 mAT'cm" 1 . Analytical procedures for the tissue residues of PBB have been described (Gutenmann and Lisk, 1975). Total hepatic lipid content was determined by the method of Folch et al. (1951). In the second experiment, animals (six males per group) and diets were treated exactly as described earlier with the exception of the spaced killing procedure. Instead, all rats consumed the experimental diets for 3 wk and were killed on the same day. Isolation of the 105,000 Xg pellet and n-octylamine binding studies were performed as described by Jefcoate and co-workers (1970). Type a cytochrome P 450 was calculated from the difference in absorption between 392 and 500 nm (minus 40% y4410 due to type b) using the molar extinction coefficient 65 m/W 1 c m " 1 . Type b cytochrome P 450 was calculated from the difference in absorption between 410 and 500 nm using the molar extinction coefficient 25 m/W"1 cm" 1 (Jefcoate et al., 1970). Ten microliters of /7-octylamine in methanol was added to 2 ml of microsomes (1-2 mg protein/ml) to make a final concentration of 1.0 m/W n-octylamine; TO /xl of methanol alone was
Downloaded by [Monash University Library] at 19:14 11 November 2015
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J.G. BABISH AND G. S.STOEWSAND
added to the microsomal suspension in the reference cuvette. Total CO-binding hemoprotein in the microsomal suspensions was determined as described by Omura and Sato (1964) using 91 vnM'1 cm'1 for the molar extinction coefficient for the difference in absorption between the Soret maximum and 490 nm. Protein was determined in duplicate by the modification of the Lowry method described by Sutherland et al. (1949). Crystalline bovine serum albumin, fraction V, was used as the standard. The first experiment was statistically analyzed as a two-factor design, with time and level of PBB representing the factors. Experiment 2 was analyzed as a one-way completely randomized design, with level of PBB as the only factor. Tukey's multiple range and Isd tests were used to determine significant differences among treatments. All statistical analyses were performed on a desk-top calculator (Hewlett-Packard model 9820A) using formulas as described by Snedecor and Cochran (1972). RESULTS The effects of dietary administration of PBB on final body weight and relative liver weight (RLW) for both experiments are presented in Table 1. As can be seen from Table 1, no effect of PBB was observed on final body weight. Furthermore, no disparity existed in rate of growth or feed efficiency among the treatments. However, significant increases (/? 0.05) from the theoretical value of 1.0 in the controls; it decreased slightly but not significantly at the 1 ppm treatment, and dropped further (significantly with p < 0.01) in the animals consuming 50 ppm PBB in their diets. Binding of A7-octylamine with rat liver microsomes produced the spectra depicted in Fig. 2. Spectra from animals receiving 0 or 1 ppm PBB TABLE 4. Effect of Dietary Polybrominated Biphenyls on the Distribution of Type a and Type b Cytochrome Ptiaa
Dietary PPB concentration (ppm) 0 1
50
Type a 6 (nmol/mg microsomal protein)
Type b c (nmol/mg microsomal protein)
a/b
0.152 ± 0.0071 0.222 ± 0.0332 0.742 ± 0.0053
0.878 ± 0.033" 1.078 ± 0.073s 2.473 ±0.149 6
0.174 ± 0.0057 0.203 ± 0.0257 0.298 +0.017 8
0.954 ± 0.034' 0.919 ± 0.030' 0.821 ± 0.02610
"Values are means ± SEM of six observations; common numerical superscripts denote nonsignificant differences (p > 0.05). ^Species of CO-binding hemoprotein having iron in the high-spin state. Calculated from difference in absorption between 392 and 500 nm using the molar extinction coefficient 65 mAf~'cm~l. c Species of CO-binding hemoprotein representing the low-spin iron. Calculated from the difference in absorption between 410 and 500 nm using the molar extinction coefficient 25 nvWcrrT 1 . "Determined from CO difference spectrumusing the molar extinction coefficient 91 mM"' cm" 1 .
679
PBBs: MICROSOMAL ENZYMES AND P45O INDUCER
_429
Downloaded by [Monash University Library] at 19:14 11 November 2015
U
z < CQ
ex.
o
5 0 ppm DIETARY PBB 1 p p m DIETARY PBB ... CONTROL
FIGURE 2. Effect microsomes.
of dietary
polybrominated
biphenyls on n-octylamine binding in rat liver
exhibited the characteristic type II binding with minima at 410 nm and maxima at 432 nm. A shift in minimum and maximum absorption to 392 and 429 nm, respectively, was observed in rat liver microsomes obtained from animals consuming 50 ppm PBB in their diets. Hepatic PBB residue per moisture-free tissue and total lipid content after 2-3 wk of PBB consumption are presented in Table 5. Samples obtained early in the second week did not differ from those examined at the end of the third week with respect to PBB concentration or total lipid content. While a 70% increase in total hepatic lipid content relative to both the control and the 1 ppm PBB treatment was observed in the 50 ppm treatment, increasing the PBB concentration in the diet 50-fold produced a 125-fold increase in hepatic PBB residue.
680
J. G. BABISH AND G. S. STOEWSAND
Downloaded by [Monash University Library] at 19:14 11 November 2015
TABLE 5. Effect of Dietary Polybrominated Biphenyls on Hepatic Polybrominated Biphenyl Concentration and Total Hepatic Lipids" Dietary PBB (ppm)
PBB residue in liver6" (ppm dry weight)
% Hepatic lipids
0 1 50
