POLYCHLORINATED DIBENZOFURANS-POTENT INDUCERS OF RAT HEPATIC DRUG-METABOLIZING ENZYMES

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1 POLYCHLORINATED DIBENZOFURANS-POTENT INDUCERS OF RAT HEPATIC DRUG-METABOLIZING ENZYMES Sumie KAWANO and Kogo HIRAGA Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-kri, Tokyo 160, Japan Accepted November 29, 1977 Abstract-The effects of polychlorinated dibenzofurans (PCDFs), trace toxic con taminants of commercial polychlorinated biphenyl preparations ( PCBs), on the induction of hepatic drug-metabolizing enzymes were studied in the rat. PCDFs were about a thousand times more potent than PCBs (Kanechlor-500) as inducers of cytochrome P-450. Rats given 10 ƒêg/kg of PCDFs intraperitoneally for 3 days showed significantly increased hepatic cytochrome P-450 levels. At the highest dose tested, 1000 ƒêg/kg, a two-fold increase of cytochrome P-450 and a three-fold increase of p-nitroanisole demethylase activity were observed. PCDFs and 3-methylcholanthrene had quite similar effects on microsornal drug-metabolizing enzymes. Both drugs increased p-nitroanisole demethylase activity strikingly and aniline hydroxylase activity moderately, but produced little change in aminopyrine demethylase activity. Naphthoflavone, which is known to be a specific inhibitor of aryl hydrocarbon ƒ - hydro xylase induced by polycyclic aromatic hydrocarbons, inhibited at low concentrations p-nitroanisole demethylase activity of rats previously treated with both drugs. Further, both drugs increased the 455 nm to 430 nm peak ratios of ethyl isocyanide difference spectra. Following three daily doses of PCDFs (100 ƒêg/kg), cytochrome P-450 level and p-nitroanisole demethylase activity remained elevated for over 15 days, with a decrease to control levels after 30 days. Such indicates the slow excretion of PCDFs. Commercial polychlorinated biphenyl (PCB) preparations are contaminated with trace amounts of polychlorinated dibenzofurans (PCDFs) (1-4). Vos and Koeman (5) found a significant difference in toxicity, i.c., high mortality, chick edema-like lesions and liver necrosis, among three samples of commercial PCB preparations, despite the marked re semblance of gas chromatograms and mass spectra. Further, by means of column frac tionation, gas chromatography and mass spectrometric techniques, PCDFs were identified to be the compounds responsible for the differences in toxicity among the commercial PCB preparations (6). In 1968, an outbreak of poisoning occurred in northern and western Japan that involved at least 1000 people who had consumed rice bran oil highly contaminated with PCBs (Kanechlor-400), and such was termed "Kanemi Yusho" (7). However, recently, Naga yama et al. (3) and Nakagawa et al. (4) confirmed that the rice bran oil which was responsible this disease was contaminated with PCDFs to a significant level. These facts suggest that highly toxic compounds, such as PCDFs are easily produced as by-products in the process of PCB manufacture and under circumstances in the use of PCBs such as heating. As little work has been done concerning the toxicity of PCDFs (8-11), we examined the induction of hepatic drug-metabolizing enzymes by PCDFs in the rat and our results are reported herein.

2 Chemicals MATERIALS AND METHODS The disodium salt of glucose-6-phosphate, glucose-6-phosphate dehydrogenase (Baker's Yeast, Type V) and ethyl isocyanide were purchased from the Sigma Chemical Company. Ethyl isocyanide was distilled before use. NADP was from the Oriental Yeast Co., p nitroanisole and aniline were from Wako Pure Chemical Industries, and aminopyrine from the Sanko Seikagaku Kogyo Co. The polychlorinated dibenzofuran mixture (PCDFs) used was purchased from the Wako Pure Chemical Industries (Lot ) and its gas chromatogram profile showed only two peaks corresponding to tetra (12.00/,) and pentachlorodibenzofuran (88.0%). The polychlorinated biphenyl mixture (PCBs) used was Kanechlor-500 (KC-500) provided by the Kanegafuchi Industry Co. Animals and treatment Male Wistar-JCL rats weighing g were used. were given i.p. at doses of 1, 10, 100 and 1000 eg/kg daily for 3 days. PCDFs dissolved in olive oil PCBs, 10 mg/kg, phenobarbital (PB), 60 mg/kg, and 3-methylcholanthrene (3-MC), 20 mg/kg, were dissolved or suspended in olive oil, and administered by the same route for 3 days. All animals were sacrificed about 24 hr after the last injection. Preparation of microsomes Livers were removed, weighed and homogenized in three volumes of 1.15% KCl con taining 0.1 mm EDTA. The homogenates were centrifuged at 10,000 x g for 20 min. The supernatant was centrifuged at 105,000 x g for 1 hr. mixture and recentrifuged. and homogenized. Enzyme assays Pellets were suspended in KCl-EDTA Washed microsomes were resuspended in KCI-EDTA mixture For spectral study of hemoproteins, microsomes were stored at -80 C. The standard mixture (1.25 ml) containing 0.8 mm NADP, 4 mm glucose-6-phosphate, 5 mm nicotinamide, 5 mm MgC12, 0.2 U/ml of glucose-6-phosphate dehydrogenase, 56 mm sodium phosphate buffer (ph 7.4), 0.14 mm EDTA, the microsomal fraction derived from 50 mg of liver and a substrate (1.2 mm p-nitroanisole, 4 mm aminopyrine or 0.5 mm aniline) was previously incubated at 37'C for 5 min in the absence of substrate. The reaction was initiated by the addition of a substrate dissolved in acetone, and the tubes were shaken at 37'C for 10 min under air. The reaction was stopped by the addition of an equal volume of 100/ trichloroacetic acid and the mixture was centrifuged at 2,800 rpm for 15 min. O-Demethylation of p-nitroanisole was estimated by measuring the formation of p nitrophenol according to the method of Kato and Gillette (12). N-Demethylation of aminopyrine was measured by the formation of formaldehyde, using the method of Cochin and Axelrod (13). p-hydroxylation of aniline was determined by the formation of p aminophenol, as follows: 2.0 ml of trichloroacetic acid extract was mixed with 0.5 ml of 2 M Na2CO3 and 0.5 ml of I N NaOH containing 3 % phenol. After the mixture had stood at a room temperature of 25'C for 30 min, absorbance at 620 rim was determined.

3 Measurements of difference spectra Cytochrome b5 content was determined from the difference spectrum between dithionite reduced and oxidized microsomes, assuming that the molar extinction coefficient between 425 and 490 nm was 171 mm-1 cm-1 (14). Cytochrome P-450 content was determined by the method of Omura and Sato (15), from the CO difference spectrum of dithionite-reduced microsomes using an extinction coefficient of 91 mm-1 cm-i between 450 and 490 nm. The ethyl isocyanide difference spectrum at ph 7.4 was determined in a similar manner, except that instead of bubbling CO, ethyl isocyanide in a final concentration of 3.45 mm was added to the sample cuvette (16). The absorbance difference of 430 to 490 nm was used as an estimate of the 430 peak and difference in absorbance of 455 to 490 nm was used as an estimate of the 455 peak. Protein and phospholipid determination Protein was determined by the method of Lowry et al. (17), using bovine serum albumin as a standard. Phospholipids in the liver were extracted by the method of Folch et al. (18) and after ashed with perchloric acid, the resultant phosphor was determined by the method of Allen (19), with minor modifications. RESULTS Dose-response relationship of the induction of drug-metabolizing enzymes by PCDFs Table 1 shows changes in body weight and some hepatic components of rats adminis tered various doses of PCDFs for 3 days. These doses did not produce changes in body weight, TABLE 1. Effects of PCDF treatment on body weight and some hepatic components

4 Fin. 1. Log dose-response curves for induction of microsomal hemoproteins and drug-metabolizing enzymes by PCDFs. Treatment is described in the legend to Table 1. Control values of the lower (1 and 10 p g) and the hcigher (100 and 1000 fig) dose groups are as follows; cytochrome P-450, 0.87 and 1.02 nmoles/ mg of microsomal protein; cytochrome b 0.37 and 0.41 nmoles/mg of microsomal protein; p-nitroanisole demethylase activity, 13.9 and 13.6 nmoles of formed p-nitrophenol/mg of microsomal protein/10 min; aminopyrine demethylase acti vity, 90.7 and 94.6 nmoles of formed formaldehyde/mg of microsomal protein/10 min; aniline hydroxylase activity, 6.62 and 8.34 nmoles of formed p-aminophenol, nig of microsomal protein/10 min, respectively. Each point represents the mean for 3 (1 ig) or 5 animals. ; Cytochrome P-450;, cytochrome b,;, p-nitro anisole demethylase; A, aniline hydroxylase; 0, aminopyrine demethylase. but liver weight was increased remarkably at higher doses (100 and 1000 /tg/kg). Generally, no significant changes were observed in protein content, microsomal protein content and phospholipid content of the liver, except that at the highest dose, the former two showed a slight but significant decrease. The effects of PCDFs on hemoprotein levels and drug-metabolizing enzyme activities in microsomes are shown in Fig. 1. The administration of PCDFs produced dose-related increases in cytochrome P-450 level and p-nitroanisole demethylase activity in rat liver, but the latter increase exceeded by far that of the former. At the lowest dose tested, I Itg/ kg, there was a 27",o increase of demethylase activity, and maximal induction (about three fold) was produced by a dose of 1000 dig/kg. Induction of cytochrome P-450 was observed at 10 't-/'kg and the maximal induced level was only twice that of the control at a dose of 1000 lltg/kg. Cytochrome b5 level and aniline hydroxylase activity showed a distinct increase at the lowest dose, but the induction of these components does not seem to be related to the dose of PCDFs, as further augmentation of the dose produced no great increase. In contrast to these increases, aminopyrine demethylase activity decreased with increase in the dose, and at the highest (lose given, the activity was only 59", of the control.

5 These results show that PCDFs are very potent inducers of drug-metabolizing enzymes, but that the induced enzymes are distinct from noninduced enzymes for the substrate spe cificity, which rapidly metabolized p-nitroanisole, but slowly metabolized aminopyrine. Comparison of substrate specificity of drug-metabolizing enzymes induced by several typical inducel s Inducers of drug-metabolizing enzymes in the liver have been categorized into two groups (20, 21). One group of inducers, of which PB is a representative, enhances the metabolism of a large variety of substrates by liver cells: a second group represented by 3-MC stimulates the metabolism of only a few substrates. In Fig. 2 we have compared the catalytic activities of microsonics from rats treated with PCDFs with those treated with PB, 3-MC and PCBs. Four inducers all increased cytochrome P-450 content of the liver remarkably, but a striking difference was observed in the catalytic activities of induced enzymes. Microsomes from rats treated with PB and PCBs enhanced activities, metabolizing all three substrates, Fic;. 2. Comparative effects of PCDFs, PCBs, PB and 3-MC on the induction of microsomal hemoproteins and drug-metabolizing enzymes. Rats were given PCDFs (100 pg), PCBs (KC-500, 10 mg), PB (60 nig) or 3-MC (20 mg/kg/day) i.p. for 3 days. Each bar and vertical line represents mean and SD from 5 animals, respectively. Measurements were divided by the mean of the control values, and the mean and SD of each group were calculated from these values. Values are significantly different from respective control values, *P-"0.05,.,-*P-'0.01, "T " Control values are as follows: cytochrome P-450, 1.02 nmoles'nig of microsomal protein: cytochrome b5, 0.41 nmoles;'nig of microsomal protein: p-nitroanisole dernethylase, aniline hydroxylase and aminopyrine demethylase activities, 13.6, 8.34 and 94.6 nmoles of formed metabolite per nag of microsomal protein for 10 min, respectively.

6 p-nitroanisole, arninopyrine and aniline. On the other hand, microsomes from rats treated with PCDFs and 3-MC enhanced to a high degree the demethylation of p-nitroanisole and moderately the hydroxylation of aniline but did not increase the demethylation of amino pyrine. Inhibition of p-nitroanisole demethylase activity by a-naphthoflavone and metyrapone Nebert et al. (22) observed that hepatic hydroxylase activity induced by aromatic hydrocarbons differed from that in the basal enzymatic activity with respect to preferential inhibition of benzo(a)pyrene hydroxylation in vitro. a-naphthoflavone more effectively blocked the hydroxylase activity in induced mice by a factor of greater than 100, and con versely metyrapone was more effective by a factor of 10 in blocking the basal hydroxylase activity. Therefore, we examined the effects of specific inhibitors-a-naphthoflavone and metyrapone various drugs (Table 2). a-naphthoflavone on p-nitroanisole demethylase activity of microsomes from rats treated with inhibited p-nitroanisole demethylase activity by about 70 % at 10-4 M when the source of microsomes was 3-MC or PCDF-treated rats, although this concentration produced only % inhibition when microsomes of control, PB or PCB-treated rats were used. On the other hand, metyrapone at a lower concentration (10-4 M) inhibited the demethylase activity from the latter source by more than 20% and that from the former source by less than 10 x, but at a higher concentration of 10-3 M the activity was blocked by 50%, irrespective of the source of microsomes. Comparison of ethyl isocyanide difference spectra of microsomes from rats treated with several inducers Ethyl isocyanide binds to reduced cytochrome P-450 to give a difference spectrum with two maxima, one at 430 nm and the other around 455 nm. The distinct type of cytochrome P-450 that is produced after administration of polycyclic hydrocarbons shows an altered ethyl isocyanide difference spectrum with an increase in the maximum at 455 nm, and thus an altered ratio of the 455 nm and 430 nm peaks (21). The heights of the 455 nm and 430 nm TABLE 2. Effects of a-naphthoflavone and metyrapone in vitro on p-nitroanisole demethylase in liver of rats administered PCDFs, PCBs, P13 or 3-MC

7 TABLE 3. Microsomal ethyl isocyanide difference spectra in liver of rats administered PCDFs, PCBs, PB or 3-MC peaks and the ratios of the 455 nm and 430 nm peaks when microsomes from various sources were reacted with ethyl isocyanide, are summarized in Table 3. Microsomes from control, PB and PCB-treated rats showed similar 455 nm/430 nm peak ratios: 0.32, 0.51 and 0.40, respectively. 1-however, much different ratios were obtained with microsomes from PCDF and 3-MC-treated rats, i.e., 1.10 and 1.28, respectively. Time course of induction of microsomal drug-metabolizing enzymes following administration of PCDFs and PCBs Following three daily applications of PCDFs, cytochrome P-450 level and p-nitroanisole FIG. 3. Time course of induction of microsomal hemoproteins and drug-metabolizing enzymes in rat liver after administration of PCDFs and PCBs. After adminis tration of PCDFs (100!tg/kg), PCBs (10 mg,/kg) or solvent (olive oil) alone i.p. on day 0, 1 and 2, the rats were sacrificed on day 3, 8, 15 or 30. Each point represents the mean for 4 animals. Control;, PCDFs; /, PCBs.

8 demethylase activity in the liver rose rapidly, and maximum levels (2.2 and 2.8-fold increases, respectively) were attained by 8 days (Fig. 3). The elevations remained for 1 week and control levels were reverted to within 30 days. Aniline hydroxylase activity and cytochrome b5 level gradually increased for 2 weeks after administration of PCDFs, and reached maxi mum levels of two-fold on day 15. However, PCDFs had little effect on aminopyrine demethylase activity throughout the 30 day experimental period. Administration of PCBs elevated all components determined to a similar extent for 8 days. These levels remained for at least 1 week and control levels were reverted to within 30 days. enzymes. DISCUSSION In this report PCDFs were found to be potent inducers of microsomal drug-metabolizing Rats administered 100 /ig/kg of PCDFs for 3 days showed much the same effects as did animals given 20 mg/kg of 3-MC. Moreover, considering that 10 dig/kg of PCDFs and 10 mg/kg of KC-500 elevated hepatic cytochrome P-450 levels to a similar extent, PCDFs appeared to be a thousand times more potent inducers than PCBs. Thus far, polychlorinated dibenzodioxins (PCDDs) are considered to be extraordinarily potent inducers of microsomal drug-metabolizing enzymes (23). 2,3,7,8-Tetrachloro-dibenzodioxin (TCDD) is indeed 3 x 104 times more potent than 3-MC as an inducer of aryl hydrocarbon hydroxylase (24). PCDFs and PCDDs, structurally related compounds, are characterized by an ex ceedingly potent ability to induce drug-metabolizing enzymes. Commercial preparations of PCBs are contaminated with small amounts of PCDFs (1 to 20 ppm) (1-4). Such a trace contamination of PCDFs must not be neglected when we assess the toxicity of commercial PCB preparations. Rice bran oil which was respon sible for "Kanemi Yusho" proved to be contaminated with a significant amount of PCDFs. The concentration of PCDFs in rice bran oil was about 5 ppm, and as calculated per PCB in rice bran oil, amounted to 5,000 ppm (3). As judged from the potency of inducers in our present experiment, the effects of PCDFs on a person are not at all inferior to those of the major contaminant, KC-400. The administration metabolizing enzymes in the rat. of PCDFs and 3-MC had similar effects on microsomal drug Both drugs strikingly increased p-nitroanisole demethylase activity and moderately aniline hydroxylase activity, but produced little change in amino pyrine demethylase activity. a-naphthoflavone, which is known to be a specific inhibitor of aryl hydrocarbon hydroxylase induced by polycyclic aromatic hydrocarbons (25), in hibited at a low concentration p-nitroanisole demethylase activity in rats previously treated with PCDFs and 3-MC. Finally the ratios of 455 nm and 430 nm peak heights of ethyl isocyanide difference spectra strikingly increased when the rats were administered PCDFs and 3-MC. These characteristics all indicate that PCDFs are 3-MC type inducers of drug-metaboliz ing enzymes. Goldstein et al. (9) also observed that 2,3,7,8-tetrachlorodibenzofuran produced a remarkable change in the ethyl isocyanide difference spectra in chicks. Poland

9 and Glover (24) mentioned that the effects of TCDD on microsomal drug-metabolizing enzymes were similar to those produced by 3-MC, except for the greater potency and duration of action of TCDD. Alvares et al. (26) observed that microsomes from rats treated with Aroclor 1254, a commercial PCB mixture, had an absorption maximum of CO-difference spectrum at 448 rim, an increased 455 nm to 430 rim peak ratio of ethyl isocyanide difference spectrum, and an exceedingly increased benzopyrene hydroxylase activity; these characteristics are similar to those seen in 3-MC treated rats. However, unlike 3-MC, Aroclor 1254 treatment caused a significant enhancement of etliylmorphine demethylase. From these results they suggested that Aroclor 1254-induced cytochrome P-448 might be catalytically different from 3-MC induced P-448 or that the hemoprotein(s) induced by Aroclor 1254 might be a mixture of cytochrome P-448 and P-450. Our results with a commercial PCB mixture, KC-500, indicate rather a PB type induction. Little work has been done thus far on the effects of isomerically pure PCB compounds. Johnstone cat al. (27) and Fujita et al. (28) observed that several pure PCB compounds arranged from low to high chlorination elevated various enzymatic activities to a similar extent. Furthermore, Goldstein et al. (9) observed no change in ethyl isocyanide difference spectra of microsomes from chicks administered four pure hexachlorobiphenyl compounds, despite a marked increase of cytochrome P-450 content. These results seem to indicate a PB type induction. These data together with findings in the present experiment suggest several possibilities concerning the mode of action of commercial PCB preparations: (a) pure PCBs induce functionally different cytochrome(s) from PB type or 3-MC type cytochromes, (b) pure PCBs induce both types of cytochromes simultaneously, or (c) each isomer of PCBs induces different molecular species of cytochromes; (d) the possibility that 3-MC type cytochromes are induced by trace contamianants such as PCDFs and PCDDs cannot be ruled out. Following three daily administrations of PCDFs and PCBs, drug-metabolizing enzymes in rat liver maintained elevated levels for at least 2 weeks. These sustained inductions following the administration of PCDFs and PCBs probably resulted from the long biological half-life of these drugs. However, some differences were observed in the distribution in the body; PCDFs were exceedingly concentrated in the liver (29), while PCBs were dis tributed preferentially in adipose tissue (30, 31). Contrary to the action of these drugs, several reports indicate that the enzymes induced by 3-MC (24) and PB (21) rapidly decrease in activity to the control levels when the admin istration is terminated. 3-MC is rapidly metabolized, and 30% of 3-MC administered is excreted in the bile in I hr (32). The metabolism of PB is also very rapid (33). Acknonllcibcmcnt: Gratitude is due to.i. Nakagawa for gas chromatography of PCDF samples. I.L1"LI.LNCLS 1) ROACH, A.G. AND POMERANTZ, I.H.: The finding of chlorinated dibenzofurans in a Japanese

10 polychlorinated biphenyl sample. Bull. environ. Contam. Toxicol. 12, (1974) 2) BOWES, G.W., MULVIHILL, M.J., SIMONETT, B.R.T., BURLINGAME, A.L. AND RISEBROUGH, R.W.: Identification of chlorinated dibenzofurans in American polychlorinated biphenyls. Nature 256, (1975) 3) NAGAYAMA, J., KURATSUNE, M. AND MASUDA, Y.: Determination of chlorinated dibenzofurans in Kanechlors and "Yusho oil". Bull. environ. Contam. Toxicol. 15, 9-13 (1976) 4) NAKAGAWA, J., MORITA, M., AKIYAMA, K., HIGUCHI, Y. AND MIMURA, S.: Polychlorinated dibenzofurans in PCBs and Kanemi rice oil. Ann. Rep. Tokyo Metr. Res. Lab. Public Health 27-1, (1976) 5) Vos, J.G. AND KOEMAN, J.H.: Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues. Toxicol. appl. Pharmacol. 17, (1970) 6) Vos, J.G., KOEMAN, J.H., VAN DER MAAS, H.L., TEN NOEVER DE BRAUW, M.C. AND DE VOS, R.H.: Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenyls. Fd cosmet. Toxicol. 8, (1970) 7) TSUKAMOTO, H.: The chemical studies on detection of toxic compounds in the rice bran oils used by the patients of Yusho. Fukuoka Acta Medica 60, (1969) 8) MCKINNEY, J.D., CHAE, K., GUPTA, B.N., MOORE, J.A. AND GOLDSTEIN, J.A.: Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8-Tetrachlorodibenzofuran in chicks. I. Relationship of chemical parameters. Toxicol. appl. Pharmacol. 36, (1976) 9) GOLDSTEIN, J.A., MCKINNEY, J.D., LUCIER, G.W., HICKMAN, P., BERGMAN, H. AND MOORE, J.A.: Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8-tetra chlorodibenzofuran in chicks. II. Effects on drug metabolism and porphyrin ac cumulation. Toxicol. appl. Pharmacol. 36, (1976) 10) ARAKI, Y.: Comparison of enzyme-inducing activities of chlorinated dibenzofuran and chlorinated dibenzodioxin. Fukuoka Acta Medica 65, (1974) 11) NISHIZUMI, M., KURATSUNE, M. AND MASUDA, Y.: Comparison of hyperkeratosis induced by PCBs, PCDF and PCDD application. Firkuoka Acta Medica 66, (1975) 12) KATO, R. AND GILLETTE, J.R.: Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. exp. Ther. 150, (1965) 13) COCHIN, J. AND AXELROD, J.: Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. 125, (1959) 14) STRITTMATTER, P. AND VELICK, S.F.: The isolation and properties of microsomal cytochrome. J. biol. Chem. 221, (1956) 15) OMURA, T. AND SATO, R.: The carbon monoxide-binding pigment of liver microsomes.

11 activity relationship. Mol. Pharmacol. 9, (1973) 24) POLAND, A. AND GLOVER, E.: Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mol. Pharmacol. 10, (1974) 25) DIAMOND, L. AND GELBOIN, H.V.: Alpha-Naphthoflavone: An inhibitor of hydrocarbon cytotoxicity and microsomal hydroxylase. Science 166, (1969) 26) ALVARES, A.P., BICKERS, D.R. AND KAPPAS, A.: Polychlorinated biphenyls: A new type of inducer of cytochrome P-448 in the liver. Proc. natn. Acad. Sci. U.S.A. 70, (1973) 27) JOHNSTONE, G.J., ECOBICHON, D.J. AND HUTZINGER, O.: The influence of pure polychlorinated biphenyl compounds on hepatic function in the rat. Toxicol. appl. Pharmacol. 28, (1974) 28) FUJITA, S., TSUJI, H., KATO, K., SAEKI, S. AND TSUKAMOTO, H.: Effect of biphenyl chlorides on rat liver microsomes. Fukuoka Acta Medica 62, (1971) 29) MORITA, M., OISHI, S., MIMURA, S. AND HIRAGA, K.: Distribution and clearance of poly chlorinated dibenzofurans in the mouse. 31th Japan. Food Hyg. Soc. Meet., Tokyo, May (1976) (in Japanese) 30) BURSE, V.W., KIMBROUGH, R.D., VILLANUEVA, E.C., JENNINGS, R.W., LINDER, R.E. AND