Xenobiotica the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies G. A. E. Van ‘t Klooster, F. M. A. Woutersen-Van Nijnanten, W. R. Klein, B. J. Blaauboer, J. Noordhoek & A. S. J. P. A. M. Van Miert To cite this article: G. A. E. Van ‘t Klooster, F. M. A. Woutersen-Van Nijnanten, W. R. Klein, B. J. Blaauboer, J. Noordhoek & A. S. J. P. A. M. Van Miert (1992) Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies, Xenobiotica, 22:5, 523-534 To link to this article: http://dx.doi.org/10.3109/00498259209053115

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Date: 25 September 2015, At: 01:11

XENOBIOTICA,

1992, VOL. 22,

NO.

5, 523-534

Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies

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G. A. E. VAN ’t KLOOSTER*, F. M. A. WOUTERSEN-VAN NIJNANTEN, W. R. KLEIN?, B. J. BLAAUBOERS, J. NOORDHOEKS and A. S. J. P. A. M. VAN M I E R T University of Utrecht, Department of Veterinary Basic Sciences, Division of Pharmacology, Pharmacy and Pharmacotherapy; Department of General and Large Animal Surgery; and 1Research Institute of Toxicology, Yalelaan 2, 3584 CM Utrecht, T h e Netherlands

Received 3 August 1991; accepted 22 January 1992

1. A procedure for the isolation and primary culture of hepatocytes from goat and cattle is described. Hepatocyte culture performance was monitored for 5 1 h by measuring viability, cytochrome P-450 maintenance, dealkylation of scoparone and ethylmorphine, and glucuronidation of phenol red. 2. Culture medium composition is discussed in relation to differences between splanchnic blood composition of ruminant and monogastric animal species. Main differences are in glucose and volatile fatty acid concentrations. Modified Williams’ E culture medium did not yield higher culture performance than non-modified Williams’ E. 3. Coating of culture dishes with either collagen or fibronectin did not improve culture performance. 4. Williams’ E, although developed for rodent cells, proves to be a suitable basal medium for ruminant hepatocytes. In this medium, culture quality is high for at least several days. 5. In cultured goat hepatocytes, biotransformation rate for scoparone amounted to

20 nmol/mg protein per h, for ethylmorphine 96 nmol/mg protein per h and for phenol red 2 nmollmg protein per h. Biotransformation activity in cow hepatocytes is approximately half that in goat hepatocytes.

Introduction Isolated hepatocytes have found a great number of applications in drug metabolism studies, including the evaluation of species differences. Several reports have been published on the use of suspensions of hepatocytes derived from agricultural species, including ruminants (Clark et al. 1976, Suolinna and Winberg 1985, Forsell et al. 1985, Shull et al. 1987, Aiello and Armentano 1987, Hoogenboom et al. 1989, Upreti et al. 1991). However, the longevity of hepatocytes in suspensions is too short to allow studies such as enzyme induction and the expression of many drug-induced toxicities (Chenery 1988, Blaauboer et al. 1990). Cultured hepatocytes are the only in vitro model suitable for long-term studies. This provides a system by which metabolites production, enzyme induction or inhibition and cytotoxicity may be studied. Nevertheless, even in hepatocytes in (primary) culture, retention of drug metabolism function and other differentiated *Address correspondence to: G. A. E. van ’t Klooster, University of Utrecht, Institute of Veterinary Pharmacology, Yalelaan 2, 3584 CM Utrecht, The Netherlands. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd

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5 24

properties of liver cells has often been possible for only a relatively short period of time (Chenery 1988, Blaauboer et al. 1990). T h e rapid decrease in culture of cytochrome P-450 content is of special importance because of the central role of the enzyme system in xenobiotic metabolism. Several papers indicate species differences in the loss of P-450 in hepatocyte cultures (Maslansky and Williams 1982, Mennes et al. 1988, 1991). Shull et al. (1986, 1987) describe a technique for isolation and use of suspensions and cultures of bovine hepatocytes. However, even in short-term cultures (8 h) these authors observed a rapidly decreasing drug-metabolizing capacity and a detachment of cells. They suggested a correlation with composition of the medium. Nutrient concentrations in the culture medium that are not in accordance with the in vivo environment of the cells appear to contribute to the loss of P-450 and differentiated cell functions (Paine and Hockin 1980, Lake and Paine 1982, Paine 1990). Based on this concept, most culture media have been designed for cells derived from monogastric animal species, particularly rodents. T h e rather exceptional gastrointestinal system of ruminant species results in striking differences in composition of portal blood (table 1). Therefore, an accordingly modified medium might be required for cultured ruminant hepatocytes. Furthermore, Shull et al. (1986,1987) suggested the use of coated culture dishes to improve ruminant hepatocyte culture performance. Several authors report improved maintenance of metabolic activity and viability of cultured hepatocytes if culture dishes are coated with constituents of the extracellular matrix (Rubin et al. 1979, Gjessing and Seglen 1980, Strom and Michalopoulos 1982, Jauregui et al. 1986). The aim of the present study was to investigate the effects of collagen or fibronectin coating of culture dishes combined with modified or regular Williams’ Medium E on the performance of cultured goat hepatocytes. Modifications of Williams’ Medium E included the addition of a mixture of volatile fatty acids and/or the reduction of glucose concentration, thus more closely approaching the in vivo environment of ruminant liver cells. Results obtained with three goats were compared to the performance of hepatocytes from a single cow. Total protein and P-450 maintenance, P-450-mediated metabolism of ethylmorphine and scoparone, glucuronidation of phenol red, and leakage of lactate dehydrogenase into the medium were used as criteria of hepatocyte function. Table 1 .

Peripheral plasma composition of monogastric and ruminant species and ruminant hepatocyte culture medium formulation (concentrations in mM). Culture media

Glucose Acetate Propionate (D)3-Hydroxy-butyrate Butyrates Valerates Lactate

Rat/man

Ruminants

Shull’

HGWE+VFA

4.0 03 002 0.07 002 0.02 1.5

2.0 1.2 0.02 (08)b 0.2 (0.8)b 0.01 (0.l)b 0.01 1.2

12.5 7.5 4.25 0.1 001

40

-

Plasma data from Lindsay and Leat (1975) and from Lomax and Baird (1983). ‘Data from Shull et ol. (1986). Portal concentrations.

1 .o 1.5 0.5 -

1 .o

Ruminant hepatocytes in biotransformation studies

525

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Methods and materials Chemicals Calcium-free modified Hanks’ balanced salt solution (HBSS), ethyleneglycol-bis(j-aminoethylether) N,N,N’,N’-tetraacetic acid (EGTA), bovine serum albumin (BSA, fraction V), glutamine, hydrocortisone-21 -hemisuccinate sodium, bovine pancreas insulin, gentamicin sulphate, limpet acetone powder (a crude extract with glucuronidase and sulphatase activity), calf skin collagen (CSC) and powdered Williams’ Medium E (WME), containing phenol red (10.7 mg/l medium) were all purchased from Sigma, St Louis, MO, USA. Half glucose Williams’ E (HGWE) was prepared by mixing 60% v/v W M E with 40% v/v glucose free Williams’ Medium E (Special, from Gibco, Breda, T h e Netherlands). Fibronectin (FBN), purified from human plasma, was kindly donated by the Central Laboratory for Blood Transfusion, Amsterdam, T h e Netherlands. Osmolarity was adjusted to that of W M E with 1.8g NaCI/l medium. Collagenase (type B, 0.565 Ujmg) and 8-glucuronidase (from Escherichia coli) were obtained from Boehringer Mannheim, Mannheim, Germany. Newborn calf serum (NCS) was from Flow Laboratories, Irvine, UK. Sodium propionate, sodium acetate, DL-lactic acid and sodium D-3-hydroxybutyrate were all from Fluka, Buchs, Switzerland. Scoparone (6,7-dimethoxycoumarin), purified according to Van Pelt et al. (1989), and scopoletin were obtained from Aldrich, Brussels, Belgium). Isoscopoletin was from C. Roth (Karlsruhe, Germany). Ethylmorphine and morphine were from OPG, Utrecht, T h e Netherlands. Norethylmorphine was kindly donated by Dr B. Lindstrom and Prof. A. Rane (National Board of Health and Welfare, Uppsala, Sweden). Surgery Three healthv castrated male dwarf eoats (aeed . . ~5 years. 45-50 ke) -. were derived from the laboratories’ flock. Preanaesthetic medication consisted of 0.4ml of a 1% w/v solution of acepromazine (VetranquiP, Sanofi, Maassluis, T h e Netherlands) and 0.2ml of a 0.5% solution of atropine sulphate. General anaesthesia was induced by injecting 10 mg/kg thiopental sodium (5% solution, NesdonaP, Rh8nePoulenc, France) and maintained with a mixture of nitrous oxide, oxygen and fluothane, inspired through an endotracheal tube. T h e abdomen was incised over 20 cm and the liver was carefully extricated from its connective tissues. Finally, the portal vein and accompanying vessels were cut and the liver was removed from the abdomen. The caudate lobe (approx. 30 g) was cut off. All blood was immediately washed out with ice-cold sterile saline (3G50ml/min) by inserting a 6 mm cannula, connected to an infusion system, in the central vein of the lobe. T h e flushed lobe was transported to the laboratory in ice-cold sterile phosphate buffered saline (PBS). In the present study, one 2-year-old Friesian Holstein cow weighing 430 kg was included. It was examined and found to be healthy and free of medication. The caudate process of the caudate lobe (64g) was removed essentially as described by Witzel et al. (1975), modified by Forsell et al. (1985) for hepatocyte isolation. Further handling was identical to goat caudate lobes. ~

I

Isolation of hepatocytes T h e procedure for hepatocyte isolation was based on Seglen’s method (Seglen 1976). In short, the liver lobe was placed in a perfusion funnel and retrograde perfusion was performed via a sterile 6 mm cannula in the central vein. This cannula was connected to the perfusion system which delivered perfusion buffers at a rate of 40ml/min. All bicarbonate buffered solutions were sterile, pH 7.4, 37°C and saturated with CO,/O, (95/5 v/v). The liver lobe was perfused with 500ml of HBSS containing 0 2 5 mM EGTA. Then the perfusion buffer was changed to 600ml of non-supplemented HBSS; 100ml of a 0.05% solution of collagenase in HBSS, supplemented with 0.25 mmol CaCI,, was recirculated through the lobe for 1720 min. Perfusion was stopped and the lobe was transferred to a laminar air cabinet. T h e liver capsule was torn off carefully and all non-perfused tissue was trimmed. Hepatocytes were dispersed in 100ml of a 2.5% solution of BSA in HBSS by gently shaking the forceps-handled liver lobe. The cell suspension was placed in a 37°C waterbath for lOmin under CO,/O, (95/5v/v) and carefully shaken each 2min. After sieving through a nylon mesh (0.125 mm), cells were washed by centrifugation (8Og, 5 min). T h e supernatant was replaced by Williams’ Medium E (WME). Cells were resuspended and washing procedure was repeated three times. The resulting cell suspension was diluted with the appropriate culture medium to a final concentration of lo6 viable hepatocytes per ml, as assessed by trypan blue exclusion. Hepatocyte cultures Hepatocytes were cultured at a density of approximately 4 x lo6 cells per 6 cm plastic Petri dish ( T C quality, Greiner, Alphen, T h e Netherlands) in 4ml WME or half glucose Williams’ E (HGWE, containing 1200 mg glucose/l). Media were supplemented with 5%v/v NCS, 1.67 mM glutamhe, 1opM hydrocortisone, 1 p~ insulin, gentamicin (50pg/ml), 1 mM CaCI, and 1 mM MgC1, (final concentrations). Four series of cultures were prepared: hepatocytes of series 1 and 2 were cultured in WME and those of series 3 and 4 in HGWE. Media of series 2 and 4 (HGWE+VFA in table 1)were supplemented with a

G . A. E. van’t Klooster et al.

526

mixture of volatile fatty acids (VFA), resulting in culture concentrations of 1.5 mM propionate, 1 mM acetate, 1 mM lactate and 0 5 mM D-3-hydroxybutyrate. In each series, cells were cultured in non-coated, collagen-coated and fibronectin-coated dishes. Cells were incubated in a humidified atmosphere of air (95%) and CO, (5%) at 37°C. Hepatocytes were allowed to attach for 3 h. Then media were replaced. Replacement media did not contain additional Ca2+ or Mg”.

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Rat hepatocytes Male Wistar rats, aged 12 weeks (390k25g body weight), were obtained from Charles River Wiga (Sulzfeld, Germany). Hepatocytes were isolated and cultured as described by Wortelboer et of.(1990) for comparative P-450 maintenance studies. Incubations and analysis At 25 h, culture medium was sampled for determination of phenol red (initially 30 PM) glucuronidation and replaced by serum free media. Phenol red glucuronidation was measured spectrophotometrically prior to and after b-glucuronidase treatment, as described by Driscoll et al. (1982). Replacement media contained either 0 2 mM scoparone or 2 5 mM ethyl-morphine. These compounds were used as P-450 marker substrates. Demethylation of scoparone yields scopoletin and isoscopoletin (Mennes et al. 1991), N-demethylation and 0-demethylation of ethylmorphine yields norethylmorphine and morphine, respectively (Nerland and Mannering 1978). After 2 h incubation, medium samples were taken. Samples were hydrolysed with limpet acetone powder at p H 4 5 . H.p.1.c. analysis for scoparaone and its metabolites was performed according to van Pelt et al. (1989). H.p.1.c. analysis of ethylmorphine was performed using a modification of the method of Ladona et at. (1989). Blank cultures were used for cytochrome P-450 and protein determination. Determinations were performed in freshly isolated hepatocytes (initial P-450,O h) and in cultured hepatocytes harvested at 3,27 and 5 1 h as previously described (Rutten et al. 1987). Lactate dehydrogenase (LDH) leakage was measured according to Bergmeyer (1965). Activity in the culture media was related to total LDH activity (activity in medium and in lysed cells).

Results Caudate lobes from 45-50 kg goats used in this study were 28-34 g. Each lobe yielded approx. 40 x lo6 cells/g (in total 12.5 g cells). Cell viability was 84--92%, as assessed by trypan blue exclusion. Average plating efficiency, expressed as protein content in attached hepatocytes after 3 h in culture relative to protein content in freshly isolated hepatocytes, was from 86% to 91%, reflecting initial viability. Cell yield from the bovine caudate lobe (64 g) was 14 g of 92%viable cells. Table 2 shows plating efficiencies and culture protein contents at 27 and 51 h for each medium (noncoated dishes). No significant differences in culture protein content were found for non-coated, CSC- and FBN-coated dishes. Hepatocytes attached rapidly (within 2 h) to culture dishes, irrespective of the dishes being coated with either CSC or FBN. With goats, the onset of cell stretching

Table 2.

Relative protein content’ (%) and plating efficienciesb (%) in non-coated dishes. Cow hepatocyte cultures

Goat hepatocyte cultures

WME WME VFA HGWE HGWE VFA

+ +

27 h culture time

51h culture time

Plating efficiencyb

27 h culture time

51h culture time

Plating efficiencyb

83k3 74+7 88k2 80+6

80+3 74+ 3 85f3 81 5 4

93+7 86+5 9713 8819

76 76 70 77

71 70 62 67

92 99 90 89

Data relative to protein content at 0 h. For goat (n= 3, means SD) protein in 4 ml freshly isolated cell suspension (0 h) ranged from 4.6 to 6.2 mg; in cow ( n= 1) 7.4 mg was found, indicating crowded cultures. bPlating efficiency (%) denotes the protein content at 3 h compared to 0 h. Abbreviations: W M E = Williams’ medium E, VFA= volatile fatty acids, HGWE = half glucose Williams’ medium E.

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Ruminant hepatocytes in biotransformation studies

Figure 1.

527

Ruminant hepatocytes in biotransformation studies.

Representative photographs from goat hepatocyte cultures at 12 h (left) and 27 h (right) in culture. Cells were cultured in Williams’ medium E in non-coated culture dishes. Magnification x 180.

was especially rapid in CSC- and FBN-coated dishes (within 2 and 3 h, respectively). In non-coated cultures, cell spreading was o.bserved after 6 h in culture. In all cultures, monolayers of cells were formed within 27 h (figure 1). Spreading of cow hepatocytes could be observed after 8 h. L D H leakage was lO-15% from 3 to 27h in goat hepatocyte cultures and decreased to 4-8% between 27 and 51 h (detailed results not shown). Coating tended to decrease the initial L D H leakage, especially if the culture medium contained VFA. In the latter cultures, L D H leakage was relatively high in VFA-supplemented cultures, irrespective of the basal medium being WME or HGWE. In cow hepatocytes initial L D H leakage was high (20-35%). Leakage between 27 and 51 h was still lO-15%. Relatively high L D H leakage was found in H G W E cultures. In contrast to goats, addition of VFA could prevent this higher leakage. Figure 2 shows the cytochrome P-450 content (per mg protein) from 0 to 51 h in non-supplemented and VFA-supplemented WME and HGWE cultures of goat hepatocytes. Overlapping graphs show the lack of effect of coating. After an initial decrease (10-20% within 3 h) in P-450 content, only a slow decrease in P-450 level was observed from 3 to 51 h. W M E and HGWE yielded identical results. Dish coating did not influence P-450 maintenance. T h e presence of VFA caused a larger initial P-450 loss (20% vs. lo%), but within 27 h the P-450 content was equal to not VFA-supplemented cultures (approx. 75% of 0 h P-450). P-450 maintenance in cultured cow hepatocytes (table 3) was nearly identical in WME, VFA-supplemented WME and VFA-supplemented HGWE, whereas HGWE cultures showed a lower performance. As with goats, coating was of no influence. Figure 3 shows comparative P-450 maintenance in goat, cow and rat hepatocyte cultures in W M E (non-coated dishes). T h e glucuronidation of phenol red, used as marker for conjugation, was visible from the loss of colour in the cultures after 22 h. In medium from goat hepatocyte

G. A. E. van’t Klooster et al.

528

100

G

80

.-m

‘‘ 4 ..

-

60

E

40

ap u C

00

0

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a

20 1 I

0 0

9

27

L

18

27

Culture time (hours1

Figure 2.

-

36

45

27

36

45

36

45

54

54

54

Effects of medium and attachment substratum on P-450 maintenance in goat hepatocyte cultures.

Results from non-coated, collagen (CSC)-coated and fibronectin (FBN)-coated dishes presented as overlapping graphs, are identical. Each point represents the mean SD of cultures in triplicate from three goats. P-450 content in freshly isolated hepatocytes served as 100% level. WME WME+VFA (volatile fatty acids), ---0---; HGWE (half (Williams’ medium E), -0-; HGWE+VFA, ....A..... glucose WME), --A--;

Table 3.

Effects of medium formulation and attachment substratum on total cytochrome P-450 maintenance in cow hepatocyte cultures. Total P-450 content (%)” WME

WME+VFA

HGWE

HGWEfVFA

Culture time 3 h Non-coated Collagen Fihronectin

90 89 87

94 101

88

91 89 100

91 93 97

Culture time 2 7 h Non-coated Collagen Fihronectin

67 64 58

63 71 71

57 57 50

61 65 63

Culture time 51 h Non-coated Collagen Fibronectin

38 32 33

37 35 40

23 28 24

35 35 36

‘Total P-450 maintenance (%) is expressed relative to total cytochrome P-450 content in freshly isolated hepatocytes. At 0 time hepatocytes contained 275.3 k 6 . 9 pmol/mg protein ( = 100%).Analyses were performed in triplicate. Abbreviations: W M E = Williams’ medium E, VFA =volatile fatty acids, HGWE = half glucose Williams’ medium E.

529

Ruminant hepatocytes in biotransformation studies 100

I

--a -

--

8o 60

ap

-

40

C

0

0

20

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a 0

0

3

0

18

27

Culture time (hour*)

Figure 3.

36

45

51

Comparative total cytochrome P-450 maintenance in cultures hepatocytes

Compared hepatocyte cultures were from castrated male Dwarf goat (--0--), Friesian Holstein and male Wistar rat (.... 0....). Hepatocytes were cultured in Williams' cow (--.. A-..) medium E on non-coated dishes. For goat (n = 3) and rat (n= 4), points represent means & SD. For cow (n= 1 ) maximal spreading in triplicate cultures is given.

cultures half of the phenol red was glucuronidated. The extent of glucuronidation did not differ significantly in the examined media or with coating (table 4). Similar results were found in cow hepatocytes, although total activity was only half of that in goat hepatocytes. In non-coated, non-supplemented W M E cultures the rate of formation of isoscopoletin, resulting from scoparone 6-O-demethylation, ranged from 15.2 to 22.8 nmol/mg protein per h; scopoletin formation from scoparone 7 - 0 demethylation ranged from 0.9 to 1.9 nmol/mg protein per h. Variation in rate of metabolite formation rate seems to result from inter-individual differences. In cow hepatoctyes metabolic rate was 6.8 and 0.5 nmol/mg protein per h for isoscopoletin and scopoletin formation, respectively. For ethylmorphine N-demethylation yielding norethylmorphine and 0-deethylation yielding morphine, metabolite formation rates in goat hepatocyte cultures (WME, non-coated) ranged from 51.5 to 121.5 and 6.0 to 13-5 nmol/mg protein per h, respectively. As with scoparone, in bovine hepatocytes ethylmorphine biotransformation was less than half that in goat cells: 2 4 6 nmol/mg protein per h for N-demethylation and 6.2 nmol/mg protein per h for 0-deethylation. T h e data above served as 100%levels in table 4, showing the effects of the culture conditions investigated on the metabolic activity in cultured goat hepatocytes. Results obtained with W M E and H G W E cultures were identical. In absence of VFA, coating with CSC or FBN did not yield significant differences in metabolic activity. Addition of VFA to W M E or H G W E tended to suppress biotransformation, especially in non-coated dishes. However, VFA appeared to be beneficial for bovine hepatocytes, especially if HGWE was the basal medium. For scoparone and ethylmorphine, compared to cultures in WME, total metabolic activity was only 57 f 10%in H G W E and 93 f8% in VFA-supplemented HGWE (means non-, CSCand FBN-coated cultures in triplicate fSD).

G . A. E . van't Klooster et al.

530 Table 4.

Effects of medium formulation and attachment substratum on metabolite formation rate in goat hepatocyte cultures.

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Rate of metabolite formation (%)" WME

WMEfVFA

HGWE

HGWE+VFA

Scopoletin Non-coated Collagen Fibronectin

100 103110 101+6

71 k 3b 81 & 10 89 16'

9 9 1 14 98f13 105f11

84f9 78f9C 83f6d

Isoscopoletin Nan-coated Collagen Fi bronectin

100 look8 96f8

71 +Sb 80f9 87 4'

89 f Sb 90+7 96f11

67+Sb 78 f 2'*' 79 +4dJ

Morphine Nan-coated Collagen Fi bronectin

100 9916 101 15

+

76+ lob 8319 ni 116

94f2 101 + 6 105f9

86&14 97113 90k16

Norethylmorphine Non-coated Collagen Fibronectin

100 98+6 106+11

71 + l o b 77+15 79+16

9212 98k7 101+3

75 & 14 83+15 80115

Phenol red glucuronide Non-coated Collagen Fibronectin

100 93&4 106+6

104+6 10412 102k2

74f lob 93 &4c 94 & 6'

+

+

78 n b 98 f 4e 100+5'

a Data ( n = 3, meansf SD) relative (%) to metabolic activity in goat hepatocytes cultured in WME in non-coated dishes. Significant vs. WME cultures in non-coated dishes. 'Significant vs. WME cultures in collagen-coated dishes. Significant vs. WME cultures in fibronectin-coated dishes. Significant vs. non-coated dishes, same medium. Abbreviations: W M E = Williams' medium E, VFA=volatile fatty acids, HGWE = half glucose Williams' Medium E. Statistics: p i 005, Student's t-test or ANOVA where appropriate.

Discussion The use of only one lobe for hepatocyte isolation, instead of the liver as a whole, limits the volume of buffers needed and allows an efficient perfusion because of the minimal cut surface and enclosing capsule. Wisniewski et al. (1987) investigated the uniformity of xenobiotic-metabolizing enzymes in microsomes from various liver lobes from cattle, sheep and goat. For a broad range of enzymic activities no significant differences are found. During EGTA perfusion, calcium and magnesium (and probably other cations as well) are thoroughly removed from the liver material. In many different biochemical processes, including cellular attachment and spreading, calcium and magnesium play an essential role (Gjessing and Seglen 1980, Ham 1981). Therefore, both elements were added to the culture medium during the attachment phase (0 to 3 h). To define a medium for ruminant hepatocytes, splanchnic blood composition in monogastric species, such as rat and man, was compared to that in ruminants.

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531

Glucose and volatile fatty acid (VFA) content provide the most obvious differences (table 1). Nearly the entire ruminant requirement of glucose is synthesized de novo in the liver (Young 1976, Wieghart et al. 1986). VFA, especially propionate, are the major gluconeogenic precursors in ruminants, which explains the relatively high VFA concentration in ruminant portal blood. Propionate concentration in peripheral blood remains low because of the extensive hepatic metabolism ( > 90%) of this compound at first pass. Butyrate is almost entirely metabolized to D-3hydroxy-butyrate, before entering the portal system (Lindsay and Leat 1975, Lomax and Baird 1983). Final concentrations of supplemented VFA in our media are still rather low, considering the extensive VFA metabolism in freshly isolated ruminant hepatocytes (Faulkner and Pollock 1986, Aiello and Armentano 1987, Aiello et al. 1989, Chow et al. 1990). Unfortunately, in pilot experiments (results not shown) doubling the VFA concentration decreased viability and metabolic activity, whereas propionate alone was not toxic at a concentration of 2 0 m ~ . Williams’ medium E (WME) (Williams and Gunn 1974) was used as basal medium in the present study, rather than Eagles’ minimal essential medium with Earle’s salts (EMEME), as proposed by Shull et al. (1986). EMEME lacks several amino acids and vitamins which are present in WME and the rather similar Waymouths’ MB 75211 basal medium (Waymouth 1959), which is often used in rat hepatocyte cultures. However, performance of goat hepatocytes cultured in nonsupplemented W M E was shown to be far superior to that in MB 752/1 (unpublished results). For both MB 752/1 and EMEME, the lack of several inorganic trace elements such as iron, zinc, copper and manganese, present in WME, might be involved in the relatively low culture performance observed. T h e absence of these elements is considered to be a major drawback for the general use of minimal essential medium in culturing non-transformed cells (Ham 1981). Several reports (Rubin et al. 1979, Gjessing and Seglen 1980, Strom and Michalopoulos 1982, Jauregui et al. 1986, Sawada et al. 1987) have been published on improvement of cellular attachment resulting in long-term survival and improved cell performance. I n the present study, coating with calf skin collagen or human plasma fibronectin did not significantly improve any of the parameters studied. In rat hepatocytes the production of attachment proteins has been shown to occur (Blaauboer and Paine 1979, Marceau et al. 1980). Apparently, a similar protein production in ruminant hepatocytes, or the content of attachment proteins in the supplemented serum (Yamada and Kennedy 1979), is sufficient for attachment. In oxidative biotransformation, the cytochrome P-450 enzyme system plays a key role. Therefore, culture P-450 content and its maintenance are major quality parameters in judging performance of cultures used in drug metabolism studies. An initial loss of P-450 is observed with hepatocyte cultures from rat and other species (Chenery 1988, Maslansky and Williams 1982, Mennes et al. 1988,1991, Paine 1990, Grant et al. 1985). T h e initial loss of P-450 in goat hepatocytes increases if VFA are present, but within 24 h P-450 content is identical to that in non-supplemented cultures (figure 2). T h e reason for this phenomenon, which was not observed in bovine hepatocytes, remains unclear. Especially with rodent cells, an ongoing loss of P-450 is found within the following 48 h to only 3 M O % of the P-450 content of freshly isolated hepatocytes (Paine 1990, Grant et al. 1985, Wortelboer et al. 1990). In hepatocytes from goat and cows (figure 3) and in cells derived from some other species, including man, P-450 is significantly better maintained than in rodent cells (Maslansky and Williams 1982, Mennes et al. 1988, Paine 1990).

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In rat hepatocytes some P-450 isozymes appear to degrade faster than others (Wortelboer et al. 1990, Steward et al. 1985). Therefore, in addition to total P-450 content, the maintenance of specific P-450 activities is a marker for culture performance. Metabolism of ethylmorphine and scoparone yielded metabolites each of which, in the rat, are formed by different P-450 isozymes (Nerland and Mannering 1978, Miiller-Enoch et al. 1984, 1985). Very little is known about the P450 isozymes in ruminant species, but an indication that isozyme patterns in goat and cow might be similar is found in the identical ratios for isoscopoletin :scopoletin, and morphine :norethylmorphine. T h e ratio of scoparone metabolites (10 to 12) in medium from goat and cow hepatocyte cultures is largely different from several other species (1 to 2 for rat, hamster and monkey (Mennes et al. 1991)). T h e relatively low activity in cow hepatocytes is in accordance with the overall low metabolic status of cattle in comparison to goats (van Miert 1986, 1989). Supplementing goat hepatocyte cultures with VFA tended to decrease biotransformation activity (table 4). Under these conditions, coating seemed somewhat beneficial. In contrast, VFA addition to bovine hepatocyte cultures could prevent low HGWE culture performance. Apparently, cow hepatocytes require higher medium glucose concentrations than goat hepatocytes that showed good performance in HGWE. Since VFA are used as glucose precursors in ruminants, the supplemented VFA seem to fill the need for glucose. We conclude that WME is a suitable medium for goat and cattle hepatocyte cultures. Neither addition of VFA, not coating culture dishes with either CSC or FBN, adds to the performance of cultured ruminant hepatocytes.

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