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Spectral changes in liver microsomal cytochrome P-450 from benzanthracene-treated normal and partially hepatectomized rats
Chem.-Bid. Interactions, 8 (1974) 253-259
0 Elsevier Scientific Publishing Company, Amsterdam-Printed in The Netherhtnds
253
SPECTRAL CHANGES IN LIVER MICROSOMAL CYTOCHROME P-450 FROM BENZANTHIUCENE- TREATED NORMAL AND PARTIALLY HEPATECTOMIZED RATS*
P. W. F. FISCHER” ANDT. SPENCER” Department of Blodemistry, Queen’s Uniwsity, iCingsion,Ont. (Canah) (Received July 39th. 1973) (Revision received November 2&t, 1973) (Accepted January 3rd, 1974)
SUMMARY
The cytochrome P-450 component of the mixed function oxidase in rat liver microsomes is modified to a species with a reduced CO complex spectral peak at 448 nm when the animal is pretreated with ~n~nthra~ne (BA). The P-450 content of the microsomes is increased when a rat is treated with BA and in regenerating liver BA has a greater effeot on the cytochrome level. When protein synthesis is blocked by the administration of a~tinomycin D or puromycin there is a decrease in the P-450 content of the microsomes, but BA can still cause the conversion to the modified form in young female rats. The change also occurs in vitro when microsomes from young female rats are treated with BA; this change does not occur in adult female fats. It is proposed that the ability of BA to catalyze the change in the cytochrome structure depends upon the estrogen level of the animal.
JNTRODUCTION
Liver microsomal mixed function oxidases’ convert many endogenous and foreign compounds into more polar products2-s. It has been proposed that substrates bind to cytochrome P-450. This substrate-cytochrome complex is reduced by NADPH and interacts with atmospheric oxygen to form hydroxylated substrate, water and m-oxidized cytochrome P-450 (ref. 6). It has previously been shown7t8 that aryl * A portion of these results was presented at the 15th Annual Meeting of the Canadian Federation of Biologica Societies in Quebec, P.Q., June, 1972. 1 Present address: The Ranting and Rest Department of Medical Research, University of Toronto, Toronto, Ont, (Canada). 2 To whom reprint requests may be sent. Abbreviation: BA, 1,2-be~anthracaRe.
P. W. F. FISHER, T. SPENCER
254
hydrocarbon hydroxylase, which metabolizes a wide range of polycyclic hydrocarbons, can be induced lo-fold by BA in regenerating rat liver and 4-fold in normal liver. In contrast estrogen hydroxylase, which catalyzes the conversion of estradiol to 2hydroxyestradiol, is induced 2- to 5-fold in regenerating liver but is not inducible in normal liver. Following polycyclic hydrocarbon pretreatment, it has been shown that there is an increase in the cytochrome P-450 content of the microsomes as well as a shift in the absorption peak of the Soret spectrum from 450 nm to 448 nmg. This suggests that a new species of cytochromeis formed, referred toas P-448 or PI-450 (refs. 9,lO). It has so far been thought that cytochrome P-448 is formed through de rtoro protein synthesis independent of cytochrome P-450 formation”. The results presented here, however, suggest that its formation can be due to a conversion of preexisting cytochrome P-450 to P-448 in the presence of a polycyclic hydrocarbon. MATERIALSAND METHODS
Female Wistar rats (Bio Breeding Laboratories of Canada Ltd., Ottawa, Ont., Canada) were used for all studies. They were given free access to food (Rockland Laboratory mouse-rat diet) and water. Partial hepatectomy was performed following the procedure of HIGGINS AND ANDERSON’~ under ether anesthesia. All operations were cnrried out between 10.00 and 12.00 h. BA was injected intraperitoncally at a dose of 100 mg/kg in corn oil. Control animals received corn oil. Actinotnycin D and puromycin were injected as previously described7*8113. The animals were sacrificed and the livers perfused with 50 mM sodium citrate in physiological saline (pH 7.2). Microsomes were prepared by the methad of REMMER ct ~1.“’ and suspended in I50 tnM KCI, 50 mM Tris-HCI buffer pH 7.4 to give a final concentration of 2 mg protein/ml. The cytochromc P-450 present in the freshly prepared microsomes was reduced with a few crystals of sodium dithionite and carbon monoxide bubbled through the reduced suspension for 15 set at a rate which caused a minimum of foaming. The pH of the solution remained at 7.4. A difference spectrum TABLE I ARYL
HYDROCARBON
AND
ESTROGEN
HYDROXYLASE
ACTIVITIES
IN RAT LIVER
MICROSOMC9
AFTER
VARIOUS
TREATMENTS
Activities are expressed as pmoles product/30 min/mg protein f standard error of the mean. Hepatecromy was carried out at 11.OOh on day I. On day 2 the rats received actinomycin D (2OO,ug/kg) at 9.00. 11.00, 14.00 h or puromycin (5 nlg/kg) at 9.00, 11.00, 12.30, 14.00, 15.00. 16.00 h. BA (100 mg’kg) was given at t I .OO h. All animals were sacrificed at 11.00 h on day 3. Antil~iotic
Normd Corn oil
Aryl hydrocarbon hydroxylase Estrogen hydroxvlase
Actinomycin Puromycin Actinomycin Puromycin
185 k 25 D 275 5 33 79% 9 937 + 117 D 739 & 43 703 f 43
Hepatectornized BA
1064 753 1049 989 703 931
_ir 85‘ * 39 f 45 & 69 f 52 + 61
Corn oil
BA
16; rf .lb 280 * 70 172It 4 692 & 114 1182 f 76 652 & 37
2323 1210 1006 2100 1022 739
f f f f * f
I62 92 8 180 70 60
SPECTRALCHANGESIN BA-TKEATEDRATS
255
with reduced m~~rosomes in the reference cuvette was plotted between 500 and 400 nm in a Unicam SP 800 s~trophotometer. Generally labelled rH]BA was specially prepared by catalytic exchange with tritium gas (International Chemical and Nuclear Corp., Irvine, Calif., U.S.A.) and was purified by silica gel column chromatography using a benzene: ethanol (9 : 1) solvent system. The distribution of BA in liver microsomes was measured in normal and 24-h ~st-he~t~to~~ rats. They were injected with 100 mg [3H]BA (75 /Xi) per kg body weight and were sacrificed 24 h later. The uptake of radioactivity into the microsomes was measured after solubilization of the microsomal pellet in ‘Protosol’ (New England Nuclear Corp., Montreal, P.Q., Canada). Aryl hydrocar~n and estrogen hydroxylase activities were assayed on the same preparation as previously described’**. Activities are the mean values of duplicate assays on at least 3 animals. RESULTS AND DISCUSSION
Fig. 1 shows the cytochrome difference spectra of microsomes from normal and hepat~tomized 4-week-old female rats which had been treated with corn oil or BA. It can be seen that 24 h after hepatectomy there is no increase in the cytochrome P-450 content of the microsomes for the corn oil-treated rats. However, treatment of the rats with BA not only increases the P-450 content of the microsomes but also causes the position of the peak to move from 450 to 448 nm. This spectral shift has
I 430
1
440
450
460 nm
470
460
nm
Fig. 1. Difference spectra of CO-treated reduced microsomes against reduced microsomes; (a) microsomes from normal or partially hepatectomized rats (pretreated with 0. I ml corn oil); (b) microsomes from normai rats pretreated with BA fItI mg BA/kg;) (cl microsomes from partially hepatectomized rats pretreated with BA. All rats were 4.week-aid females; protein concentration is 2 mg per ml. Fig. 2. Difference spectra of CO-treated reduced microsomes against reduced microsomes; 4 preparations from partially hepat~tomi~d, 4-week-old female rats are treated with r’a) actinomycin D and BA, [bJ puromycin and BA, (cJ corn oil, (d) BA. The injection schedule is given in ref. 13; protein concentration is 2 mg per ml.
P. W. F. FISHER, T. SPENCER
-02 t. 30 440
450
460 nm
470
460
Fig. 3. Difference spectra of CO-treated reduced microsomes from 4-week-old female rats incubated in vitro with (a) methanol; (b) 20 nmoles benzpyrene added to 2 ml microsomal suspension (protein concentration is 2 mg/ml). Adult (lo-week-old) female rats show the same curve as (a) in the absence or presence of benzpyrene.
been found by other workers 9*10. The P-448 content is higher in the hepatectomized rats. These spectra were obtained from at least 3 animals and the microsomal preparations had the hydroxylase activities shown in Table I. The values are similar to those previously reported. We have shown7** that, following actinomycin D or puromycin treatment, the level of aryl hydrocarbon hydroxylase increases whereas there is no increase in the estrogen hydroxylase activity in BA-treated rats. We followed this injection procedure7+**r3 and the cytochrome spectra obtained for 4-week-old female rats showed a decre;lse in cytochromc P-450 after 24-h treatment with antibiotics (Fig. 2). The corresponding hydroxylase activities are shown in Table 1. This increase in aryl hydrocarbon hydroxylase activity in liver microsomes from actinomycin D and BAtreated rats without an increase in cytochrome P-450 content has also been reported by ALVARES et al. * 5. In these experiments there is a loss of cytochrome from the microsomes clue to its degradation without concomitant synthesis in the presence of the antibiotics. Values ranging from 24 to 50 h have been reported for the half life of cytochrome P-450 (refs. l&17) and the present data are within this range. However, the change in the position of the peak from 450 to 488 nm still occurs in the antibiotic and BA-treated animals. This implies that the change of P-450 to P-448 may be independent of de noeo protein synthesis or that any new cytochrome synthesized during the antibiotic treatment is the P-448 species. In a parallel experiment where rats received antibiotics but no BA, a similar decrease in the height of the cytochrome peak was observed but the absorption peak remained at 450 nm. To examine the possibility that P-448 could arise by the BA catalyzed conversion from pre-existing P-450, BA or benzpyrene was incubated with a microsomal preparation and the cytochrome spectrum measured. 2 ml of microsomes (2 mg protein/ml) from a young female rat were incubated with 20 nmoles of hydrocarbon in
SPECTRAL CONGAS
BA-T~A~
RATS
257
10 ~1 of methanol. (This is the amount of [3H]BA found in the microsomal fraction.) This mixture was shaken at room temperature by inverting the tube several times. The microsomes were reduced and the CO difference spectrum measured. The results in Fig. 3 show that for i~ature female rats there was a shit in the cytochrome peak from 450 to 448 mm but this peak did not occur with microsomes from adult female rats and did not occur with the solvent alone. The spectral change occurred in the absence of added coenzyme and was completed within the manipulation time. Incubation of microsomes with benzopyrene for 30 min before reduction with dithionite did not change the spectrum, so it is most unlikely that me~bolism of the hydrocarbon is involved in the structural changes. The same spectral shift was found when the microsomes were incubated with BA instead of benzpyrene. When 2 ml of microsomes (2 mg protein/ml) from adult female rats were incubated with 100 nmoles of BA or benzpyrene then a slight shift of peak from 450 nm to a lower wavelength was observed. The same spectrum was obtained whether benzpyrene was present or absent from the reference cell. These results show that it is possible to convert cytochrome P-450 to the P-448 species in vitro without concurrent protein synthesis in young female rats, whereas the higher level of estrogens in the adult female rat inhibits this conversion. The presence of BA in the liver microsomes was measured by the uptake of ~dioactive BA into normal and 24-h post-hepate~tomiz~ animals with sacrifice 24 h after BA injection. At this time the content of BA in the microsomal fraction of both operated and normal animals was the same (0.7 rug BA/mg microsomal protein). This is in contrast to the greater uptake of radioactivity into 700 g nuclear fraction of hepatectomized animals compared to the radioactive uptake into the normal rat liver nuclear fraction (0.4 and 0.15 pg BAfmg protein, respectively). These results suggest that the BA is present in microsomes from normal and regenerated liver, and may be able to convert the cytochrome P-450 to the modified P-443 species without protein synthesis. It is important to note that this spectral change was only seen in young female rats and did not occur, except partially at very high hydrocarbon concentration, in microsomal preparations from adult female rats. Other workersls*lg have reported that there is no in vitro effect of polycyclic hydrocarbons on cytochrome P-450. However, these workers have always used adult male rats. (Unpublished experiments in this laboratory have also found that this in vifro shift does not occur in male rats.) Thus it would appear that this change is dependent upon the sex hormone balance of the animal. The data in Fig. 2 show that in the antibiotic-treated partially hepatectomized rats there is a decrease in the cytochrome content in the presence of antibiotics but that the presence of BA still induces the change from P-450 to P-448. We have pr-eviously shown ‘~3that in animals receiving this dose of antibiotic and BA, estrogen hydroxylase induction is inhibited, whereas the level of aryl hydrocarbon hydroxylase increases to approximately half the value found when rats are treated with BA alone. It is suggested that P-450 is the normal form of the cytochrome and is the active species in the metabolism of estradiol. In the presence of the polycyelic hydrocarbon P-448 is formed which is more active than cytochrome P-450 for aryl hydrocarbon
258
P. W. F. FISHER,T. SPENCXR
hydroxylase activity. In the presence of the antibiotic there is no cytochrome P-450 synthesis and therefore no increase in estrogen hydroxylase activity, however BA still binds to the existing P-450 converting it to P-448 which causes the observed increase in aryl hydrocarbon hydroxylase activity. The extent of binding of BA to cytochrome P-450 depends upon the level of sex hormones in the liver. In the young female rat, with very low estrogen levels, BA binds to existing P-450 to convert it to P-448 whereas in older animals this change cannot take place with preformed P-450. This is probably due to the stabilization of the P-450 by the presence of sex hormones, so that the conversion of P-450 to P-448 can only occur during or soon after synthesis of the cytochrome. This proposal reconciles the present data with that of BIDDLEMAN AND MANNERING", MANNERING'~ AND GNOSSPELIUS et a/.lg. The model proposed here also explains the in vitro inhibition of aryl hydrocarbon hydroxylase by estradiol and the estrogen hydroxylase inhibition by benzpyrene 20. Both activities are inhibited to different extents when measurements are carried out on microsomal preparations from young or old, male or female rats. This shows the different effects of estrogen and androgens on the microsomal hydroxylase activities. Furthermore, the effects of BA differ in these 4 groups of rats. For example, BA-treated young female rats yield liver microsomes which are not inhibited by estradiol in the aryl hydrocarbon hydroxylase assay, whereas microsomes from control animals are inhibited in the same assay conditions. However, in the estrogen hydroxylase assay there is only a small difference in the extent of inhibition by benzpyrene between control and BAtreated rats. These observations would be expected if the in viva and in vitro effects of polycyclic hydrocarbons are the same as proposed in this model. ACKNOWLEDGEMENTS
This work has been supported by the Medical Research Council of Canada and we thank Mr. R. KOOLEN for competent technical assistance. REFERENCES 1
2 3 4 5 6
7 8
H. S.MASON, Mechanisms of oxygen metabolism, A&n. Enzymol., 19 (1957) 79-233. A. H. CONNEY, Pharmacological implications of microsomal enzyme induction, Phartttacol. Rev., 15 (1967) 317-366. L. G. HART AND J. R. Fours, Effects of acute and chronic DDT administration on hepatic drug metabolism in the rat, Proc. Sot. Expff. Biol. Med., I14 (1963) 388-392. H. V. GELBOIN, Carcinogens, enzyme induction and gene action, Advun. Cancer Res., 10 (1967) l-81. P. H. JELLINCKAND J. Woo, Changes in oestrogen metabolism induced in rat liver microsomes by subcutaneous pellets of oestrone and testosterone, J. Ettdocrinof.. 39 (1967) 99-104. R. W. ESTABROOK,A. G. HILDEBRANDT,J. BARON, K. J. NETTERAND K. LEIBMAN,A new spectral intermediate associated with cytochrome P-450 function in liver cells, Biochem. Biophp. Res. Cornmutt., 42 (1971) 132-139. T. SPENCERAND P. W. F. FISCHER,The induction of microsomal hydroxylases in regenerating rat liver, Chetn.-Biol. Interactiotts, 4 (1971/72) 41-47. P. W. F. FISCHERAND T. SPENCER,The induction of aryl hydrocarbon and estrogen hydroxylases in the rough and smooth endoplasmic reticulum in regenerating rat liver, (Ihem.-Biol. fttteractions, 5 (1972) 27-36.
SPECTRAL CHANGE8 IN BA-TREATED RATS
259
A. P. ALVARES,G. SCHILLING,W. LEVINAND R. KUNTZMAN,Studies on the induction of CObinding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene, Biochem. Biophys. Res. Commun., 29 (1967) 521-526. 10 N. E. SLADEKAND G. J. MANERING, Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats, Biochem. Biophys. Res. Commw~., 24 (1966) 9
668-673.
11 K. BXDLEMAN AND G. J. MANNERING, Induction of drug metabolism. V. Independent formation of cytochromes P-450 and PI-450 in rats treated with phenobarbital and 3-methylcholanthrene simultaneously, Mol. Pharmacol., 6 (1970) 697-701. 12 G. M. HIGGINSAND P. M. ANDERSON, Restoration of the liver in the white rat following partial surgical removal, Arch. Pathof., 12 (1931) 186-202. 13 M. WATANABE,V. R. Porrza AND H. P. MORRIS,Benzpyrene hydroxyla~ activity and its induction by methy~cholanthrene in Morris hepatomas, in host livers, in adult livers and in rat liver during development, Cancer Res., 30 (1970) 263-273. 14 H. REMMER,J. B. SCHENKMAN, R. W. ESTABROOK, H. SASANE,J. R. GILLETTE,D. Y. COOPER, S. NARASIMHULU AND 0. ROSENTHAL,Drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol., 2 (1966) 187-190. 15 A. P. ALVARES,G. SCHILLING,W. LEVINAND R. KUIUTZMAN, Alteration of the microsomal hemoprotein by 3-methylcholant~ene: effects of ethionine and actinomycin D, J. Pharm. Exptf. Therap., 163 (1968) 417-424. 16 L. ERNSTERAND S. ORRENIUS,Substrate-induced synthesis of the hydroxylating enzyme system of liver microsomes, Federufion Proc., 24 (1965) 1190-l 199. 17 1. M. ARIAS, D. DOYLE AND R. T. SCHIMKE,Studies on the synthesis and degradation of the endoplasmic reticulum of rat liver, J. Biol. Chem. 244 (1969) 3303-3315. 18 G. J. MANNERING, Properties of cytochrome P-450 as affected by environmental factors: Qualitative changesdue to administration of polycyclic hydrocarbons, Metffbol~sin, 20 (1971) 228-245. 19 Y. GNOSSFELIUS,H. THOR AND S. ORRENIUS, A comparative study on the effects of phenobarbital and 3,4-benzpyrene on the hydroxylating system of rat liver microsomes, Chem.-Biol. interacrions. 1 (1969/70) 125-137. 20 T. SPENCER,A comparison of aryl hydrocarbon and estrogen hydroxylase of rat liver, Enzymologia, 43 ( 1972) 301-3 10.
0 Elsevier Scientific Publishing Company, Amsterdam-Printed in The Netherhtnds
253
SPECTRAL CHANGES IN LIVER MICROSOMAL CYTOCHROME P-450 FROM BENZANTHIUCENE- TREATED NORMAL AND PARTIALLY HEPATECTOMIZED RATS*
P. W. F. FISCHER” ANDT. SPENCER” Department of Blodemistry, Queen’s Uniwsity, iCingsion,Ont. (Canah) (Received July 39th. 1973) (Revision received November 2&t, 1973) (Accepted January 3rd, 1974)
SUMMARY
The cytochrome P-450 component of the mixed function oxidase in rat liver microsomes is modified to a species with a reduced CO complex spectral peak at 448 nm when the animal is pretreated with ~n~nthra~ne (BA). The P-450 content of the microsomes is increased when a rat is treated with BA and in regenerating liver BA has a greater effeot on the cytochrome level. When protein synthesis is blocked by the administration of a~tinomycin D or puromycin there is a decrease in the P-450 content of the microsomes, but BA can still cause the conversion to the modified form in young female rats. The change also occurs in vitro when microsomes from young female rats are treated with BA; this change does not occur in adult female fats. It is proposed that the ability of BA to catalyze the change in the cytochrome structure depends upon the estrogen level of the animal.
JNTRODUCTION
Liver microsomal mixed function oxidases’ convert many endogenous and foreign compounds into more polar products2-s. It has been proposed that substrates bind to cytochrome P-450. This substrate-cytochrome complex is reduced by NADPH and interacts with atmospheric oxygen to form hydroxylated substrate, water and m-oxidized cytochrome P-450 (ref. 6). It has previously been shown7t8 that aryl * A portion of these results was presented at the 15th Annual Meeting of the Canadian Federation of Biologica Societies in Quebec, P.Q., June, 1972. 1 Present address: The Ranting and Rest Department of Medical Research, University of Toronto, Toronto, Ont, (Canada). 2 To whom reprint requests may be sent. Abbreviation: BA, 1,2-be~anthracaRe.
P. W. F. FISHER, T. SPENCER
254
hydrocarbon hydroxylase, which metabolizes a wide range of polycyclic hydrocarbons, can be induced lo-fold by BA in regenerating rat liver and 4-fold in normal liver. In contrast estrogen hydroxylase, which catalyzes the conversion of estradiol to 2hydroxyestradiol, is induced 2- to 5-fold in regenerating liver but is not inducible in normal liver. Following polycyclic hydrocarbon pretreatment, it has been shown that there is an increase in the cytochrome P-450 content of the microsomes as well as a shift in the absorption peak of the Soret spectrum from 450 nm to 448 nmg. This suggests that a new species of cytochromeis formed, referred toas P-448 or PI-450 (refs. 9,lO). It has so far been thought that cytochrome P-448 is formed through de rtoro protein synthesis independent of cytochrome P-450 formation”. The results presented here, however, suggest that its formation can be due to a conversion of preexisting cytochrome P-450 to P-448 in the presence of a polycyclic hydrocarbon. MATERIALSAND METHODS
Female Wistar rats (Bio Breeding Laboratories of Canada Ltd., Ottawa, Ont., Canada) were used for all studies. They were given free access to food (Rockland Laboratory mouse-rat diet) and water. Partial hepatectomy was performed following the procedure of HIGGINS AND ANDERSON’~ under ether anesthesia. All operations were cnrried out between 10.00 and 12.00 h. BA was injected intraperitoncally at a dose of 100 mg/kg in corn oil. Control animals received corn oil. Actinotnycin D and puromycin were injected as previously described7*8113. The animals were sacrificed and the livers perfused with 50 mM sodium citrate in physiological saline (pH 7.2). Microsomes were prepared by the methad of REMMER ct ~1.“’ and suspended in I50 tnM KCI, 50 mM Tris-HCI buffer pH 7.4 to give a final concentration of 2 mg protein/ml. The cytochromc P-450 present in the freshly prepared microsomes was reduced with a few crystals of sodium dithionite and carbon monoxide bubbled through the reduced suspension for 15 set at a rate which caused a minimum of foaming. The pH of the solution remained at 7.4. A difference spectrum TABLE I ARYL
HYDROCARBON
AND
ESTROGEN
HYDROXYLASE
ACTIVITIES
IN RAT LIVER
MICROSOMC9
AFTER
VARIOUS
TREATMENTS
Activities are expressed as pmoles product/30 min/mg protein f standard error of the mean. Hepatecromy was carried out at 11.OOh on day I. On day 2 the rats received actinomycin D (2OO,ug/kg) at 9.00. 11.00, 14.00 h or puromycin (5 nlg/kg) at 9.00, 11.00, 12.30, 14.00, 15.00. 16.00 h. BA (100 mg’kg) was given at t I .OO h. All animals were sacrificed at 11.00 h on day 3. Antil~iotic
Normd Corn oil
Aryl hydrocarbon hydroxylase Estrogen hydroxvlase
Actinomycin Puromycin Actinomycin Puromycin
185 k 25 D 275 5 33 79% 9 937 + 117 D 739 & 43 703 f 43
Hepatectornized BA
1064 753 1049 989 703 931
_ir 85‘ * 39 f 45 & 69 f 52 + 61
Corn oil
BA
16; rf .lb 280 * 70 172It 4 692 & 114 1182 f 76 652 & 37
2323 1210 1006 2100 1022 739
f f f f * f
I62 92 8 180 70 60
SPECTRALCHANGESIN BA-TKEATEDRATS
255
with reduced m~~rosomes in the reference cuvette was plotted between 500 and 400 nm in a Unicam SP 800 s~trophotometer. Generally labelled rH]BA was specially prepared by catalytic exchange with tritium gas (International Chemical and Nuclear Corp., Irvine, Calif., U.S.A.) and was purified by silica gel column chromatography using a benzene: ethanol (9 : 1) solvent system. The distribution of BA in liver microsomes was measured in normal and 24-h ~st-he~t~to~~ rats. They were injected with 100 mg [3H]BA (75 /Xi) per kg body weight and were sacrificed 24 h later. The uptake of radioactivity into the microsomes was measured after solubilization of the microsomal pellet in ‘Protosol’ (New England Nuclear Corp., Montreal, P.Q., Canada). Aryl hydrocar~n and estrogen hydroxylase activities were assayed on the same preparation as previously described’**. Activities are the mean values of duplicate assays on at least 3 animals. RESULTS AND DISCUSSION
Fig. 1 shows the cytochrome difference spectra of microsomes from normal and hepat~tomized 4-week-old female rats which had been treated with corn oil or BA. It can be seen that 24 h after hepatectomy there is no increase in the cytochrome P-450 content of the microsomes for the corn oil-treated rats. However, treatment of the rats with BA not only increases the P-450 content of the microsomes but also causes the position of the peak to move from 450 to 448 nm. This spectral shift has
I 430
1
440
450
460 nm
470
460
nm
Fig. 1. Difference spectra of CO-treated reduced microsomes against reduced microsomes; (a) microsomes from normal or partially hepatectomized rats (pretreated with 0. I ml corn oil); (b) microsomes from normai rats pretreated with BA fItI mg BA/kg;) (cl microsomes from partially hepatectomized rats pretreated with BA. All rats were 4.week-aid females; protein concentration is 2 mg per ml. Fig. 2. Difference spectra of CO-treated reduced microsomes against reduced microsomes; 4 preparations from partially hepat~tomi~d, 4-week-old female rats are treated with r’a) actinomycin D and BA, [bJ puromycin and BA, (cJ corn oil, (d) BA. The injection schedule is given in ref. 13; protein concentration is 2 mg per ml.
P. W. F. FISHER, T. SPENCER
-02 t. 30 440
450
460 nm
470
460
Fig. 3. Difference spectra of CO-treated reduced microsomes from 4-week-old female rats incubated in vitro with (a) methanol; (b) 20 nmoles benzpyrene added to 2 ml microsomal suspension (protein concentration is 2 mg/ml). Adult (lo-week-old) female rats show the same curve as (a) in the absence or presence of benzpyrene.
been found by other workers 9*10. The P-448 content is higher in the hepatectomized rats. These spectra were obtained from at least 3 animals and the microsomal preparations had the hydroxylase activities shown in Table I. The values are similar to those previously reported. We have shown7** that, following actinomycin D or puromycin treatment, the level of aryl hydrocarbon hydroxylase increases whereas there is no increase in the estrogen hydroxylase activity in BA-treated rats. We followed this injection procedure7+**r3 and the cytochrome spectra obtained for 4-week-old female rats showed a decre;lse in cytochromc P-450 after 24-h treatment with antibiotics (Fig. 2). The corresponding hydroxylase activities are shown in Table 1. This increase in aryl hydrocarbon hydroxylase activity in liver microsomes from actinomycin D and BAtreated rats without an increase in cytochrome P-450 content has also been reported by ALVARES et al. * 5. In these experiments there is a loss of cytochrome from the microsomes clue to its degradation without concomitant synthesis in the presence of the antibiotics. Values ranging from 24 to 50 h have been reported for the half life of cytochrome P-450 (refs. l&17) and the present data are within this range. However, the change in the position of the peak from 450 to 488 nm still occurs in the antibiotic and BA-treated animals. This implies that the change of P-450 to P-448 may be independent of de noeo protein synthesis or that any new cytochrome synthesized during the antibiotic treatment is the P-448 species. In a parallel experiment where rats received antibiotics but no BA, a similar decrease in the height of the cytochrome peak was observed but the absorption peak remained at 450 nm. To examine the possibility that P-448 could arise by the BA catalyzed conversion from pre-existing P-450, BA or benzpyrene was incubated with a microsomal preparation and the cytochrome spectrum measured. 2 ml of microsomes (2 mg protein/ml) from a young female rat were incubated with 20 nmoles of hydrocarbon in
SPECTRAL CONGAS
BA-T~A~
RATS
257
10 ~1 of methanol. (This is the amount of [3H]BA found in the microsomal fraction.) This mixture was shaken at room temperature by inverting the tube several times. The microsomes were reduced and the CO difference spectrum measured. The results in Fig. 3 show that for i~ature female rats there was a shit in the cytochrome peak from 450 to 448 mm but this peak did not occur with microsomes from adult female rats and did not occur with the solvent alone. The spectral change occurred in the absence of added coenzyme and was completed within the manipulation time. Incubation of microsomes with benzopyrene for 30 min before reduction with dithionite did not change the spectrum, so it is most unlikely that me~bolism of the hydrocarbon is involved in the structural changes. The same spectral shift was found when the microsomes were incubated with BA instead of benzpyrene. When 2 ml of microsomes (2 mg protein/ml) from adult female rats were incubated with 100 nmoles of BA or benzpyrene then a slight shift of peak from 450 nm to a lower wavelength was observed. The same spectrum was obtained whether benzpyrene was present or absent from the reference cell. These results show that it is possible to convert cytochrome P-450 to the P-448 species in vitro without concurrent protein synthesis in young female rats, whereas the higher level of estrogens in the adult female rat inhibits this conversion. The presence of BA in the liver microsomes was measured by the uptake of ~dioactive BA into normal and 24-h post-hepate~tomiz~ animals with sacrifice 24 h after BA injection. At this time the content of BA in the microsomal fraction of both operated and normal animals was the same (0.7 rug BA/mg microsomal protein). This is in contrast to the greater uptake of radioactivity into 700 g nuclear fraction of hepatectomized animals compared to the radioactive uptake into the normal rat liver nuclear fraction (0.4 and 0.15 pg BAfmg protein, respectively). These results suggest that the BA is present in microsomes from normal and regenerated liver, and may be able to convert the cytochrome P-450 to the modified P-443 species without protein synthesis. It is important to note that this spectral change was only seen in young female rats and did not occur, except partially at very high hydrocarbon concentration, in microsomal preparations from adult female rats. Other workersls*lg have reported that there is no in vitro effect of polycyclic hydrocarbons on cytochrome P-450. However, these workers have always used adult male rats. (Unpublished experiments in this laboratory have also found that this in vifro shift does not occur in male rats.) Thus it would appear that this change is dependent upon the sex hormone balance of the animal. The data in Fig. 2 show that in the antibiotic-treated partially hepatectomized rats there is a decrease in the cytochrome content in the presence of antibiotics but that the presence of BA still induces the change from P-450 to P-448. We have pr-eviously shown ‘~3that in animals receiving this dose of antibiotic and BA, estrogen hydroxylase induction is inhibited, whereas the level of aryl hydrocarbon hydroxylase increases to approximately half the value found when rats are treated with BA alone. It is suggested that P-450 is the normal form of the cytochrome and is the active species in the metabolism of estradiol. In the presence of the polycyelic hydrocarbon P-448 is formed which is more active than cytochrome P-450 for aryl hydrocarbon
258
P. W. F. FISHER,T. SPENCXR
hydroxylase activity. In the presence of the antibiotic there is no cytochrome P-450 synthesis and therefore no increase in estrogen hydroxylase activity, however BA still binds to the existing P-450 converting it to P-448 which causes the observed increase in aryl hydrocarbon hydroxylase activity. The extent of binding of BA to cytochrome P-450 depends upon the level of sex hormones in the liver. In the young female rat, with very low estrogen levels, BA binds to existing P-450 to convert it to P-448 whereas in older animals this change cannot take place with preformed P-450. This is probably due to the stabilization of the P-450 by the presence of sex hormones, so that the conversion of P-450 to P-448 can only occur during or soon after synthesis of the cytochrome. This proposal reconciles the present data with that of BIDDLEMAN AND MANNERING", MANNERING'~ AND GNOSSPELIUS et a/.lg. The model proposed here also explains the in vitro inhibition of aryl hydrocarbon hydroxylase by estradiol and the estrogen hydroxylase inhibition by benzpyrene 20. Both activities are inhibited to different extents when measurements are carried out on microsomal preparations from young or old, male or female rats. This shows the different effects of estrogen and androgens on the microsomal hydroxylase activities. Furthermore, the effects of BA differ in these 4 groups of rats. For example, BA-treated young female rats yield liver microsomes which are not inhibited by estradiol in the aryl hydrocarbon hydroxylase assay, whereas microsomes from control animals are inhibited in the same assay conditions. However, in the estrogen hydroxylase assay there is only a small difference in the extent of inhibition by benzpyrene between control and BAtreated rats. These observations would be expected if the in viva and in vitro effects of polycyclic hydrocarbons are the same as proposed in this model. ACKNOWLEDGEMENTS
This work has been supported by the Medical Research Council of Canada and we thank Mr. R. KOOLEN for competent technical assistance. REFERENCES 1
2 3 4 5 6
7 8
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