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Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes
Life S iencea, Vol . 18, pp . 1139-1144, 1976 Printe in the II .S .A .
MAINTENANCE OF MICROSOMAL CYTOCHROMES b 5 AND
Pergamon Prass
P-450
IN PRIMARY CULTURES OF
PARENCHYMAL LIVER CELLS ON COLLAGEN MEMBRANES . George Michalopoulos, Gerald L . Sattler, and Henry C . Pitot Departments of Oncology and Pathology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wtswnstn,
53706 .
(Received in final form April 7, 1976) SUMMARY In previous reports from various laboratories, the levels of the microsamal cytochromes b S and P-450 In hepatocytes in primary culture have been found to bé very low and difficult to measure . The studies reported in this paper demonstrate that c~tochromes b and P-450 to hepatocytes cultured on floating collagen membranes f~r periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of 4 liver in vivo . Addition of high levels of hydrocortisone (10 M) to theculture medium for periods up to 10 days resulted In further increases In the levels of these cytochromes . Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained . Cytochrome P-450, In association with various mixed-function oxygenases present within hepatocellular mlcrosames, plays a key role to the metabolism of drugs, carcinogens and other xenobiotics (1) . While various systems for the culture of parenchyma) liver cells have recently been described (2-5), the intracellular levels of microsomal cytochrame P-450 present In such liver cell cultures appear to be very low in comparison to that seen in hepatocytes in vivo (6-9) . This circumstance has bean a serious limitation in the utilization of liver cell culture systems for the study of the mode of action and hepatospecific metabolism of various drugs and carcinogens . Recently we have reported a system for the culture of adult rat hepatocytes on floating collagen membranes (10) in which the morphology of the cell monolayers is quite similar to that of the liver _in vtvo . In this system the hepatocytes are capable of responding to hydrocortisone and dibutyryl cyclic AMP by rapid Increases in the levels of tyrosine aminotransferase for periods of more than three weeks in culture . In this report, the levels of mlcrosomal cytochromes b and P-450 in the presence and absence of hydrocortisone have been investiga5 ted as a function of the age of hepatocytes in culture on floattng collagen membranes . Materials and Methods Adult, Sprague-Dawley, male rats, weighing between 180-250 grams and maintained on 30$ protein diet, fed ad libitum were used as the source of cells for culture . The liver was perfused with collagenase (Sigma, Type I) 1139
1140
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
and the cells isolated according to the method of Berry and Friend (11) as modified by Bonney et al . (2) . The isolated cells were piated on previously prepared and washed collagen gels (10) . Four hours after inoculation of the cells the medium was changed and the gels were detached tram the periphery of the plates . Within the subsequent 4-5 days the cells aggregated to form a continuous monolayer and the floating gel reduced in size to 1/4-1/6 of Its original diameter (10) . The cell aggregation - end the formation of the monoIn layers requires the continuous presence of 10 M insulin in the medium . the complete absence of insulin the parenchymal hepatocytes degenerate within 3-4 days . The cells were maintained to L-15 medium (Gibco) supplemented with albumin (2006 mg/100 ml), fetal calf serum (5$), glucose (150 mg/100 ml) insulin (10 M), penicillin (100 1+9/ml), streptomycin (100 i+g/ml) with dally changes . For analysis of the cytochrome content the gels were cooled on ice and homogenized with a tight fitting glass homogenizes in 0 .5 M Tris-HC1, pH 7 .5 . The homogenates were spun at 12,000 x g for 10 min to precipitate the nuclei, mitochondria, lysosomes and insoluble collagen fragments . The sedimented pellets were assayed later for DNA and the supernatants were frozen at -70 ° C for the subsequent measurement of the cytochromes . The mlcrosomes were prepared by centrifugation of the thawed supernatant at 108,000 x g for 90 minutes . The sedimented microsomal pellets were resuspended by sonicatlon in 0 .5 M Tris-HC1, pH 7 .5, In a final volume of 2 .2 ml . Spectral assays were performed in a Cary spectrophotometer (Model 15), according to the method of Omura and Sato (12) after calibration with a crystal For the determination of cytochrome b the microsomal susof holmium oxide . pension in the sample cuvette was reduced with sodium dtt~htonite and the O .D . 4 ~q was measured . To measure cytochrome P-450, the microsamal suspensions 2 in 4 66th the reference and the sample cuvette were reduced with sodium dithionlte and the contents of the sample cuvette subsequently reacted with carbon monoxide . DNA was assayed by Burton's modification of the diphenylamine reaction Protein was assayed according to the method of Lowry (15) . Albumin, (13) " hydrocortisone hemisuccinate, sodium, and crystalline bovine insulin were purchased from the Sigma Chemical Company . Results Levels of microsomal cytochromes In hepatocytes as a function of their time to culture : In Table I may be seen the levels of mtcrosomal cytochromes expressed as plcomoles per mg mtcrosomal protein in the presence of insulin with and without hydrocortisone . Values for the levels of cytochrome b and P-450 In normal liver cells to vivo are presented for comparison . ItShould be noted that after only asingle day in culture the level of cytochrome P-450 has decreased to only a third that seen in normal liver in the presence of insulin alone but is maintained at much higher levels when hydrocortisone is also present in the medium . By the fifth day in culture scarcely more than 5$ of the original cytochrome P-450 specific activity of liver is still present regardless of the presence of hydrocortisone . From the 5th to 10th days of culture, cytochrome P-450 activity slowly decreases in the cells grown in the presence of insulin alone ; in contrast, in the cells grown in the presence of hydrocortisone the .001) . The changes to cytochrome Increases after 5 days in culture (p cytochrome b 5 follow the same pattern but are less dramatic and in the presence of hydrocortisone there is no significant change in the level of this cytochrome after the third day in culture .
Vol . 18, No . 10
114 1
Cytochromes in Liver Call Cultures
In Figure 1 the data are represented graphically as ptcomoies of cytochrome per microgram DNA ; the patterns of change of mlcrosomal cytochromes In Fig . 1 TABLE 1 Microsomal Cytochrome Levels + in Hepatocytes Cultured on Floating Collagen Membranes to the Presence of Insulin (I) : Hydrowrtisone (HC) Days in Culture 1 3 5 7 10 Normal liver
~Signlftcance of difference
Cytochrome b I I+HC S 465 362 248 183 155
* * 3 3 :
26 63 32 28 26
606 280 178 211 244
679 3 23
* * 3 3 3
54 58 57 60 43
P
0 .1 N .S . N .S . N .S . N .S .
Cytochrome P-450 I I+HC 157 * 14 65 : 4 65 3 8 393 9 33 3 5
365 * 61 110 : 22 54 * 9 803 1 123 3 7
Significance of difference P P P
0 .05 N .S . N .S . 0 .05 0 .001
480 3 24
+Values are the averages of 3-4 samples expressed as pieomoles eytochrame/mg mlcrosamal protein 3 standard error of the mean . These values are for the cytochrome P-450 complex and do not include cytochrome P-420 . Comparison of I and I+HC values by students t test .
N .S . ~ not significant .
FIG . 1 The levels of microsomal cytochrame b (A) and P-450 (B) of hepatocytes if~ medium containing insulin (10 - M) and fetal calf serum (5$) in the presence (--0----0--) or absence (-~ - -) of hydrocortisone (10 M) . Values are expressed as picomoles per Rg DNA 3 standard error . Each point represents the average value of 3-4 individual assays of mlcrosames from 40 x 106 cells . The 0 time points are taken from liver irnnedlately after the initiation of perfusion .
114 2
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
expressed on a per kg DNA basis) appear similar to those of Table I (data expressed as per milligram microsomal protein) . The levels of cytochrome P-450 in the cells grown in the presence of hydrocortisone are higher than in the calls grown in the absence of hydrocortisone, especially for the first 0 .001) days in culture . (p 0 .05) and 10th (p Effects of h drocortisone on the wavelen the of the absor taon s ectrum of the microsomal cytoçhrome P- 50 and P- 20 :
eaks
In Figure 2 the Soret spectra of the cytochrome P-450 complex and cytochrome P-420 from microsomes of normal liver and of the hepat$cytes cultured for 3 days in the presence and absence of hydrocortisone (10 M) are shown .
FIG . 2 Spectra of microsomal cytochromes in cellg cultured for 3 days with insulin (10 M) and fetal calf serum 5% In the presence (B) o~ absence (C) of hydrocortisone (10 M), compared to the same spectrum for normal liver microsomes (A) . For the spectra of microsomes from cultured cells, cell material from 60 culture plates 6 (60 rnn) was used (approxlmataly 75 x 10 cells) . Concentrations of microsomal protein 1n the cuvettes was approximately 2 .5 Wg/ml .
FIG . 3 A : Combined levels of cytochromes P-450 and P-420, expressed as nanomoles per milligram microsomal protein t standard error, as a function of the days in culture . The 0 time points as in Fig . l . B: Percentage of cytochrome P-420 to the sum of cytochromes P-450 and P-420 in relation to the days in culture . The amount of cell material used for the assay at each time point in Fig . 1 . The 0 time point as in Fig . l . Symbols as in Fig . l .
Vol . 18, No . 10
Cytochromes in Liver Call Cultures
114 3
The maximum of the spectral peak for mtcrosamal cytochrome P-450 for nor~al liver and from cells cultured in the continuous presence of insulin (10 M) and 5$ fetal calf serum are the same (449 .5 and 449 .4 rm respectlvely~ 6 In those cells cultured in the continuous presence of hydrocortisone (10 M) and fetal calf serum (5$) the absorption maximum of the cytochrome P-450 complex is located at 448 .0 rm . One might speculate that the appearance of cytochrome P-448 (P l - 450) might be a result of the induction of cytochrome P-448 by hydrocortisone in a manner analogous to its induction by polycycllc hydrocarbons . Levels of cytochrome P-420 : Cytochrome P-420 was always found in these cultured liver cells in substantial amounts as compared to the levels of cytochrome P-450, regardless of the method of preparation and treatment of microsomes . This Is In contrast to microsamal preparations from normal rat liver where insignificant amounts of cytochrome P-420 were found under the same conditions of preparation . In Ftg . 3A the combined amount of cytochrome P-450 and P-420 in the cultured cells is shown (in nanomoles per milligram microsomal protein) as a function of the days to culture . In Fig . 3B the percentage of cytochrome P-420 contributing to the sum of P-450 and P-420 is shown as a function of the days in culture . The relative amounts of P-420 fluctuatè between 20$ and 63$ of the total of P-450 and P-420 . Cytochrome P-420 might be present to increased amounts in cultured hepatocytes, due to alterations to cell metabolism resulting from the adaptation of the cells to the culture conditions ; e .g . cytochrome P-420 may be a result of the breakdown of cytochrome P-450 within the cells due to increased intracellular proteolyttc activity . High levels of P-420 have been reported in hepatamas by Mlyake et al . (14) . Discussion The development of systems for the primary culture of hepatocytes in recent years (2,3,4,5,10) has allowed the study and evaluation of parameters of liver metabolism in systans where the milieu of the hepatocytes can be well defined . In contrast to short-lived suspensions of isolated hepatocytes, the hepatocytes 1n primary culture survive for much longer periods of time . Furthermore, due to the virtual absence of cell replication in primary cultures, these cultured hepatocytes appear to exhibit metabolic patterns and control mechanisms similar to adult hepatocytes in vivo , unlike permanent liver cell lines where the cultured cells are separated from the _in vivo phenotype by a long series of mitotic events . In all the reports on the levels of microsomal cytochromes in hepatocytes in primary culture systems published to date (6-9), the levels of cytochrames fall rapidly within the first 24 hours In culture or are not readily measurable . Some studies (6,7,8) have demonstrated that the levels of cytochrame P-450 to cultured hepatocytes decrease to approximately 20$ of the value seen in normal liver within the first 24 hours to culture . No information was presented to these studies on the levels of the P-450 complex during subsequent days of culture . In the system reported herein the levels of microsomal cytochromes are about 70$ that of liver In vivo after 24 hours in culture and remain at readily measurable levels in cultured hepatocytes for at least 10 days . Previous studies (6,7) have reported no effect of drugs or hormones on the levels of cytochrome P-450 in cultured hepatocytes during the first 24 hours . In this report hydrocortisone reduces the initial decrease 1n the P-450 complex seen 1n cultured hepatocytes, Increases the levels of this cytochrome in cells that have been to culture for more than 5 days and causes
1144
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
a shift of the maximum of the absorption peak to 448 rm . These findings further support the evidence (10) that hepatocytes cultured on floating collagen membranes offer a relatively unique system for the study of hepatocyte function In vitro . Acknowledgement The authors wish to thank Dr . Charles Kasper and Dr . Jerry Ztmnerman for very helpful suggestions during the preparation of this report . This work was supported In part by a grant from the Natlonal Cancer Institute (CA-07175) : G .M . is a postdoctoral trainee In Biochemical Pathology of the Natlonal Institutes of General Medical Sciences . References 1. 2. 3.
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
Mtcrosomes and Drug Oxldations . Estrabrook, R . W ., and Gillette, J . R . and Letbman, K . C . editors, (1972) Drug Metabolism and Disposition 1, 1-482 . R . S . BONNEY, J . É . BECKER, P . R . WALKER, and V . R . POTTER . In vitro 399-413 (1974) . M . W . PARIZA, J . E . BECKER, J . D . YAGER, R . J . BONNEY, and V . R . POTTER . In "Differentlatfon and control of malignancy of tumor cells (ed . W . Nakahara, T . Sugimura and H . Sugano) University Park Press, Baltimore, Md . J . Cel l Biology 59 M . D . BISSEL, L . E . HAMMAKER, and U . A . HAYES . 722-734 (1973) . J . ALWEN and GALLHAI-HATCHARD, J . J . J . Cell Science 11 249-260 (1972) . M . D . BISSEL and P . S . GUZELIAN . in Gene Fxpresslon and Carcinogenesis in Cultures Liver (Academic Press) . pp . 119-136 (1975) . P . S . GUZELIAN and M . D . BISSEL . Fed . Proc ., Abstr . 1181 (1974) . D . M . BISSEL, P . S . GUZELIAN, L . E . HAMMAKER, and R . SCHMID . Fed . Proc ., Abstr . 123 (1974) . I . S . OWENS and D . W . NEBERT . Mol . Pharmacol . 1 1 94-104 (1975) . G . MICHALOPOULOS and H . C . PITOT . Exptl . Cel l Rés . 94 70-78 (1975) " N . M . BERRY and D . S . FRIEND . J . Cel l Blol . 43 506-520 (1969) . 2370-237 8 (1964) . T . OMURA and R . SATO . J . Blol . Chem . Biochem . J . 62 315- 323 (195 K . BURTON . J . Blol . Chem . 249 1980 - 1987 Y . MIYAKE, J . L . GAYLOR, and H . P . MORRIS . (19741 " 0 . H . LOWRY, N . J . ROSENBROUGH, A . L . FARR, and R . J . RANDALL . J . Biol . Chem . 19 3 265-275 (1951) .
2.
MAINTENANCE OF MICROSOMAL CYTOCHROMES b 5 AND
Pergamon Prass
P-450
IN PRIMARY CULTURES OF
PARENCHYMAL LIVER CELLS ON COLLAGEN MEMBRANES . George Michalopoulos, Gerald L . Sattler, and Henry C . Pitot Departments of Oncology and Pathology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wtswnstn,
53706 .
(Received in final form April 7, 1976) SUMMARY In previous reports from various laboratories, the levels of the microsamal cytochromes b S and P-450 In hepatocytes in primary culture have been found to bé very low and difficult to measure . The studies reported in this paper demonstrate that c~tochromes b and P-450 to hepatocytes cultured on floating collagen membranes f~r periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of 4 liver in vivo . Addition of high levels of hydrocortisone (10 M) to theculture medium for periods up to 10 days resulted In further increases In the levels of these cytochromes . Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained . Cytochrome P-450, In association with various mixed-function oxygenases present within hepatocellular mlcrosames, plays a key role to the metabolism of drugs, carcinogens and other xenobiotics (1) . While various systems for the culture of parenchyma) liver cells have recently been described (2-5), the intracellular levels of microsomal cytochrame P-450 present In such liver cell cultures appear to be very low in comparison to that seen in hepatocytes in vivo (6-9) . This circumstance has bean a serious limitation in the utilization of liver cell culture systems for the study of the mode of action and hepatospecific metabolism of various drugs and carcinogens . Recently we have reported a system for the culture of adult rat hepatocytes on floating collagen membranes (10) in which the morphology of the cell monolayers is quite similar to that of the liver _in vtvo . In this system the hepatocytes are capable of responding to hydrocortisone and dibutyryl cyclic AMP by rapid Increases in the levels of tyrosine aminotransferase for periods of more than three weeks in culture . In this report, the levels of mlcrosomal cytochromes b and P-450 in the presence and absence of hydrocortisone have been investiga5 ted as a function of the age of hepatocytes in culture on floattng collagen membranes . Materials and Methods Adult, Sprague-Dawley, male rats, weighing between 180-250 grams and maintained on 30$ protein diet, fed ad libitum were used as the source of cells for culture . The liver was perfused with collagenase (Sigma, Type I) 1139
1140
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
and the cells isolated according to the method of Berry and Friend (11) as modified by Bonney et al . (2) . The isolated cells were piated on previously prepared and washed collagen gels (10) . Four hours after inoculation of the cells the medium was changed and the gels were detached tram the periphery of the plates . Within the subsequent 4-5 days the cells aggregated to form a continuous monolayer and the floating gel reduced in size to 1/4-1/6 of Its original diameter (10) . The cell aggregation - end the formation of the monoIn layers requires the continuous presence of 10 M insulin in the medium . the complete absence of insulin the parenchymal hepatocytes degenerate within 3-4 days . The cells were maintained to L-15 medium (Gibco) supplemented with albumin (2006 mg/100 ml), fetal calf serum (5$), glucose (150 mg/100 ml) insulin (10 M), penicillin (100 1+9/ml), streptomycin (100 i+g/ml) with dally changes . For analysis of the cytochrome content the gels were cooled on ice and homogenized with a tight fitting glass homogenizes in 0 .5 M Tris-HC1, pH 7 .5 . The homogenates were spun at 12,000 x g for 10 min to precipitate the nuclei, mitochondria, lysosomes and insoluble collagen fragments . The sedimented pellets were assayed later for DNA and the supernatants were frozen at -70 ° C for the subsequent measurement of the cytochromes . The mlcrosomes were prepared by centrifugation of the thawed supernatant at 108,000 x g for 90 minutes . The sedimented microsomal pellets were resuspended by sonicatlon in 0 .5 M Tris-HC1, pH 7 .5, In a final volume of 2 .2 ml . Spectral assays were performed in a Cary spectrophotometer (Model 15), according to the method of Omura and Sato (12) after calibration with a crystal For the determination of cytochrome b the microsomal susof holmium oxide . pension in the sample cuvette was reduced with sodium dtt~htonite and the O .D . 4 ~q was measured . To measure cytochrome P-450, the microsamal suspensions 2 in 4 66th the reference and the sample cuvette were reduced with sodium dithionlte and the contents of the sample cuvette subsequently reacted with carbon monoxide . DNA was assayed by Burton's modification of the diphenylamine reaction Protein was assayed according to the method of Lowry (15) . Albumin, (13) " hydrocortisone hemisuccinate, sodium, and crystalline bovine insulin were purchased from the Sigma Chemical Company . Results Levels of microsomal cytochromes In hepatocytes as a function of their time to culture : In Table I may be seen the levels of mtcrosomal cytochromes expressed as plcomoles per mg mtcrosomal protein in the presence of insulin with and without hydrocortisone . Values for the levels of cytochrome b and P-450 In normal liver cells to vivo are presented for comparison . ItShould be noted that after only asingle day in culture the level of cytochrome P-450 has decreased to only a third that seen in normal liver in the presence of insulin alone but is maintained at much higher levels when hydrocortisone is also present in the medium . By the fifth day in culture scarcely more than 5$ of the original cytochrome P-450 specific activity of liver is still present regardless of the presence of hydrocortisone . From the 5th to 10th days of culture, cytochrome P-450 activity slowly decreases in the cells grown in the presence of insulin alone ; in contrast, in the cells grown in the presence of hydrocortisone the .001) . The changes to cytochrome Increases after 5 days in culture (p cytochrome b 5 follow the same pattern but are less dramatic and in the presence of hydrocortisone there is no significant change in the level of this cytochrome after the third day in culture .
Vol . 18, No . 10
114 1
Cytochromes in Liver Call Cultures
In Figure 1 the data are represented graphically as ptcomoies of cytochrome per microgram DNA ; the patterns of change of mlcrosomal cytochromes In Fig . 1 TABLE 1 Microsomal Cytochrome Levels + in Hepatocytes Cultured on Floating Collagen Membranes to the Presence of Insulin (I) : Hydrowrtisone (HC) Days in Culture 1 3 5 7 10 Normal liver
~Signlftcance of difference
Cytochrome b I I+HC S 465 362 248 183 155
* * 3 3 :
26 63 32 28 26
606 280 178 211 244
679 3 23
* * 3 3 3
54 58 57 60 43
P
0 .1 N .S . N .S . N .S . N .S .
Cytochrome P-450 I I+HC 157 * 14 65 : 4 65 3 8 393 9 33 3 5
365 * 61 110 : 22 54 * 9 803 1 123 3 7
Significance of difference P P P
0 .05 N .S . N .S . 0 .05 0 .001
480 3 24
+Values are the averages of 3-4 samples expressed as pieomoles eytochrame/mg mlcrosamal protein 3 standard error of the mean . These values are for the cytochrome P-450 complex and do not include cytochrome P-420 . Comparison of I and I+HC values by students t test .
N .S . ~ not significant .
FIG . 1 The levels of microsomal cytochrame b (A) and P-450 (B) of hepatocytes if~ medium containing insulin (10 - M) and fetal calf serum (5$) in the presence (--0----0--) or absence (-~ - -) of hydrocortisone (10 M) . Values are expressed as picomoles per Rg DNA 3 standard error . Each point represents the average value of 3-4 individual assays of mlcrosames from 40 x 106 cells . The 0 time points are taken from liver irnnedlately after the initiation of perfusion .
114 2
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
expressed on a per kg DNA basis) appear similar to those of Table I (data expressed as per milligram microsomal protein) . The levels of cytochrome P-450 in the cells grown in the presence of hydrocortisone are higher than in the calls grown in the absence of hydrocortisone, especially for the first 0 .001) days in culture . (p 0 .05) and 10th (p Effects of h drocortisone on the wavelen the of the absor taon s ectrum of the microsomal cytoçhrome P- 50 and P- 20 :
eaks
In Figure 2 the Soret spectra of the cytochrome P-450 complex and cytochrome P-420 from microsomes of normal liver and of the hepat$cytes cultured for 3 days in the presence and absence of hydrocortisone (10 M) are shown .
FIG . 2 Spectra of microsomal cytochromes in cellg cultured for 3 days with insulin (10 M) and fetal calf serum 5% In the presence (B) o~ absence (C) of hydrocortisone (10 M), compared to the same spectrum for normal liver microsomes (A) . For the spectra of microsomes from cultured cells, cell material from 60 culture plates 6 (60 rnn) was used (approxlmataly 75 x 10 cells) . Concentrations of microsomal protein 1n the cuvettes was approximately 2 .5 Wg/ml .
FIG . 3 A : Combined levels of cytochromes P-450 and P-420, expressed as nanomoles per milligram microsomal protein t standard error, as a function of the days in culture . The 0 time points as in Fig . l . B: Percentage of cytochrome P-420 to the sum of cytochromes P-450 and P-420 in relation to the days in culture . The amount of cell material used for the assay at each time point in Fig . 1 . The 0 time point as in Fig . l . Symbols as in Fig . l .
Vol . 18, No . 10
Cytochromes in Liver Call Cultures
114 3
The maximum of the spectral peak for mtcrosamal cytochrome P-450 for nor~al liver and from cells cultured in the continuous presence of insulin (10 M) and 5$ fetal calf serum are the same (449 .5 and 449 .4 rm respectlvely~ 6 In those cells cultured in the continuous presence of hydrocortisone (10 M) and fetal calf serum (5$) the absorption maximum of the cytochrome P-450 complex is located at 448 .0 rm . One might speculate that the appearance of cytochrome P-448 (P l - 450) might be a result of the induction of cytochrome P-448 by hydrocortisone in a manner analogous to its induction by polycycllc hydrocarbons . Levels of cytochrome P-420 : Cytochrome P-420 was always found in these cultured liver cells in substantial amounts as compared to the levels of cytochrome P-450, regardless of the method of preparation and treatment of microsomes . This Is In contrast to microsamal preparations from normal rat liver where insignificant amounts of cytochrome P-420 were found under the same conditions of preparation . In Ftg . 3A the combined amount of cytochrome P-450 and P-420 in the cultured cells is shown (in nanomoles per milligram microsomal protein) as a function of the days to culture . In Fig . 3B the percentage of cytochrome P-420 contributing to the sum of P-450 and P-420 is shown as a function of the days in culture . The relative amounts of P-420 fluctuatè between 20$ and 63$ of the total of P-450 and P-420 . Cytochrome P-420 might be present to increased amounts in cultured hepatocytes, due to alterations to cell metabolism resulting from the adaptation of the cells to the culture conditions ; e .g . cytochrome P-420 may be a result of the breakdown of cytochrome P-450 within the cells due to increased intracellular proteolyttc activity . High levels of P-420 have been reported in hepatamas by Mlyake et al . (14) . Discussion The development of systems for the primary culture of hepatocytes in recent years (2,3,4,5,10) has allowed the study and evaluation of parameters of liver metabolism in systans where the milieu of the hepatocytes can be well defined . In contrast to short-lived suspensions of isolated hepatocytes, the hepatocytes 1n primary culture survive for much longer periods of time . Furthermore, due to the virtual absence of cell replication in primary cultures, these cultured hepatocytes appear to exhibit metabolic patterns and control mechanisms similar to adult hepatocytes in vivo , unlike permanent liver cell lines where the cultured cells are separated from the _in vivo phenotype by a long series of mitotic events . In all the reports on the levels of microsomal cytochromes in hepatocytes in primary culture systems published to date (6-9), the levels of cytochrames fall rapidly within the first 24 hours In culture or are not readily measurable . Some studies (6,7,8) have demonstrated that the levels of cytochrame P-450 to cultured hepatocytes decrease to approximately 20$ of the value seen in normal liver within the first 24 hours to culture . No information was presented to these studies on the levels of the P-450 complex during subsequent days of culture . In the system reported herein the levels of microsomal cytochromes are about 70$ that of liver In vivo after 24 hours in culture and remain at readily measurable levels in cultured hepatocytes for at least 10 days . Previous studies (6,7) have reported no effect of drugs or hormones on the levels of cytochrome P-450 in cultured hepatocytes during the first 24 hours . In this report hydrocortisone reduces the initial decrease 1n the P-450 complex seen 1n cultured hepatocytes, Increases the levels of this cytochrome in cells that have been to culture for more than 5 days and causes
1144
Cytochromes in Liver Cell Cultures
Vol . 18, No . 10
a shift of the maximum of the absorption peak to 448 rm . These findings further support the evidence (10) that hepatocytes cultured on floating collagen membranes offer a relatively unique system for the study of hepatocyte function In vitro . Acknowledgement The authors wish to thank Dr . Charles Kasper and Dr . Jerry Ztmnerman for very helpful suggestions during the preparation of this report . This work was supported In part by a grant from the Natlonal Cancer Institute (CA-07175) : G .M . is a postdoctoral trainee In Biochemical Pathology of the Natlonal Institutes of General Medical Sciences . References 1. 2. 3.
5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
Mtcrosomes and Drug Oxldations . Estrabrook, R . W ., and Gillette, J . R . and Letbman, K . C . editors, (1972) Drug Metabolism and Disposition 1, 1-482 . R . S . BONNEY, J . É . BECKER, P . R . WALKER, and V . R . POTTER . In vitro 399-413 (1974) . M . W . PARIZA, J . E . BECKER, J . D . YAGER, R . J . BONNEY, and V . R . POTTER . In "Differentlatfon and control of malignancy of tumor cells (ed . W . Nakahara, T . Sugimura and H . Sugano) University Park Press, Baltimore, Md . J . Cel l Biology 59 M . D . BISSEL, L . E . HAMMAKER, and U . A . HAYES . 722-734 (1973) . J . ALWEN and GALLHAI-HATCHARD, J . J . J . Cell Science 11 249-260 (1972) . M . D . BISSEL and P . S . GUZELIAN . in Gene Fxpresslon and Carcinogenesis in Cultures Liver (Academic Press) . pp . 119-136 (1975) . P . S . GUZELIAN and M . D . BISSEL . Fed . Proc ., Abstr . 1181 (1974) . D . M . BISSEL, P . S . GUZELIAN, L . E . HAMMAKER, and R . SCHMID . Fed . Proc ., Abstr . 123 (1974) . I . S . OWENS and D . W . NEBERT . Mol . Pharmacol . 1 1 94-104 (1975) . G . MICHALOPOULOS and H . C . PITOT . Exptl . Cel l Rés . 94 70-78 (1975) " N . M . BERRY and D . S . FRIEND . J . Cel l Blol . 43 506-520 (1969) . 2370-237 8 (1964) . T . OMURA and R . SATO . J . Blol . Chem . Biochem . J . 62 315- 323 (195 K . BURTON . J . Blol . Chem . 249 1980 - 1987 Y . MIYAKE, J . L . GAYLOR, and H . P . MORRIS . (19741 " 0 . H . LOWRY, N . J . ROSENBROUGH, A . L . FARR, and R . J . RANDALL . J . Biol . Chem . 19 3 265-275 (1951) .
2.