EXPERIMENTAL

CELL RESEARCH

194,157-160

(1991)

SHORT NOTE Mechanism of Maintenance of Liver-Specific Functions by DMSO in Cultured Rat Hepatocytes PIA VILLA,**’

PATRIZIA

ARIOLI,?

AND AMALIA

GuArrANIf’

*C.N.R. Center of Cytopharmacology, Department of Pharmacology, Via Vanvitelli 32, 20129 Milan, Italy; and fLaboratory of Experimental Liver Toxicology, Istituto di Ricer&e Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy

plemented media. It seems likely that under simple culture conditions requirements for specific regulation of the MM0 system or for preserving differentiation of hepatocytes are lacking. The loss of cytochrome P450dependent MM0 activities is usually temporarily prevented by coculture with nonparenchymal epithelial cells [5] or by supplementing the culture medium with a variety of compounds such as hormones [6], ligands [7], heme precursors [8], phenobarbital [9], and, more recently, DMSO [4]. The present study was undertaken to investigate the mechanism by which DMSO exerts its protective action on the cytochrome P450 system and differentiation in cultured rat hepatocytes.

The present study was undertaken to investigate the mechanism by which dimethylsulfoxide (DMSO) exerts its protective action on cytochrome P460-dependent activities and differentiation in cultured rat hepatocytes. Loss of cytochrome P450 is associated with a shortage of heme and reduced activity of &aminolaevulinic acid dehydratase: the addition of DMSO, which induces this enzyme in human hepatoma cells, is not able to affect it in hepatocytes in primary culture. DMSO is a strong scavenger of hydroxyl radicals and may destroy the reactive oxygen species formed under conventional culture conditions (i.e., 96% air and 5% CO,). In fact other powerful scavengers of oxygen radicals like dimethylthiourea, desferal, and catalase itself maintain higher levels of cytochrome P450 and higher activities of 7ethoxycoumarin O-deethylase during 3 days of culture. DMSO and the other scavengers are also able to retain features of the morphological and biochemical differentiation of hepatocytes such as the ability to induce tyrosine aminotransferase activity in response to glucocor0 1991 Academic Press, Inc. ticoids.

MATERIALS

INTRODUCTION

Dimethylsulfoxide (DMSO) is a dipolar, aprotic, organic solvent which is active in biological systems as a cryoprotectant and as a differentiating agent. Addition of l-2% (v/v) DMSO to the culture medium induces differentiation in tumor cells [l], induces heme synthesis in human hepatoma cells [2], and maintains differentiation [3] and microsomal monooxygenase (MMO) activities [4] in cultured rat hepatocytes. Rodent hepatocytes are one of the cell types which rapidly lose their highly differentiated functions such as microsomal MM0 activities when cultured with unsup1 To whom correspondence and reprint requests should he addressed at Laboratory of Experimental Liver Toxicology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.

AND

METHODS

Chemicals. The following compounds were used: collagenase type IV, insulin, hydtocortisone hemisuccinate, dexamethasone and catalase (from Sigma), DMSO and dimethylthiourea (DMTU) (from Janssen Chimica), and desferrioxamine (desferal) (from CibaGeigy). Other ordinary chemicals from various sources were of reagent grade. Liver cell culture. Hepatocytes prepared from fed male Crl:CD (SD)BR rats (211 k 13 g) by perfusing the liver with a collagenase solution were seeded in culture medium [4] supplemented with 5% v/v fetal calf serum (FCS). The medium, without FCS and with 7 X lo-’ M hydrocortisone hemisuccinate, was renewed 3,24, and 48 h after hepatocyte seeding. Hydrocortisone was omitted 24 and 48 h after plating when the cells were assayed for tyrosine aminotransferase (TAT) induction. Treatments of cell cultures. The scavengers of oxygen radicals were added to cell cultures at each one’s optimal concentration: 2% v/v (280 mM) DMSO, 25 mM DMTU, 1 mM desferal, 1500 U/ml catalase after 3 h of culture and every 24 h after the first treatment. Assays. (i) 7-Ethoxycoumarin deethylase (ECD) activity and cytochrome P450 content were assayed as described by Edwards et aL [lo] and Omura and Sato [ll], respectively; (ii) TAT activity was measured according to Granner and Tomkins [12]; (iii) d-aminolaevulinic acid dehydratase (ALA-DH) activity was assayed as described by Sassa [13]; (iv) catalase activity was measured according to Aebi 1141. Statistical significance of the results was analyzed by one-way analysis of variance.

157

0014~4827/91$3.00

Copyright Q 1991 by Academic Press, Inc. All rights of reproduction

in any form reserved.

158

SHORT

t=24h

1 -

DMSO

t=72h

250

:

CONTROL

NOTE

l

DMTU

i CATALASE

CONTROL

DMSO

DMTU

CATALASE

FIG. 1. Time-dependent effects of 280 m&f DMSO, 25 mM DMTU, and 1500 U/ml catalase on cytochrome P450 content in cultured rat hepatocytes. Cells were treated 3 h after seeding and every 24 h after the first treatment. The results are means + SE of three to four different hepatocyte preparations. RI Cyt. P450 content in fresh isolated hepatocytes. *P < 0.01 vs the corresponding control (Duncan’s test). OP c 0.05 and O”P < 0.01 vs catalase (Duncan’s test).

RESULTS

AND

DISCUSSION

As shown in Fig. 1 the cytochrome P450 level of rat hepatocytes declines to 57 and 36% of the initial value in fresh isolated cells after 24 and 72 h in culture, respectively. Addition of 2% (v/v) DMSO to a chemically defined medium with no FCS results in maintenance of a significantly higher content of cytochrome P450 (Fig. l), in accordance with previous results [ 151. Two possible factors have been considered to explain the mechanism by which DMSO exerts this protective effect. Loss of cytochrome P450-dependent MM0 activities in rodent-cultured hepatocytes has been partially associated with a shortage of heme [8] which may be related to the decrease of ALA-DH, the second enzyme in the heme biosynthetic pathway [ 161. DMSO has been described as inducing ALA-DH activity and heme synthesis in human hepatoma cells [2]. To verify this effect in our culture system ALA-DH activity was assessed during 3 days of culture in the presence and absence of DMSO. The basal enzyme activity decreased, as already described by Guzelian [16], but DMSO did not affect it (Table 1). Cytochrome P45O is also sensitive to microsomal lipid peroxidation [ 171 which occurs in hepatocytes both during the first day [l&19] and after 3 days of culture [20] under serum-free conditions. Lipid peroxidation could be a consequence of reactive oxygen species formed

under conventional culture conditions (i.e., 95% air and 5% CO,) which are hyperoxic for hepatocytes [18]. Hydrogen peroxide, the first reactive peroxide in the chain of peroxyradical formation, is formed in cultured hepatocytes [21]. DMSO is a strong scavenger of hydroxyl radicals [22] and this may contribute to its effect on the MM0 systern. In order to test the theory that oxygen radicals are involved, the effect of other scavengers of oxygen radicals was evaluated on the content of cytochrome P450 and on the deethylation of 7-ethoxycoumarin, a substrate oxidized by specific cytochrome P450 isoenzymes [lo]. TABLE 1 Effect of DMSO on &Aminolaevulinic (ALA-DH)

Treatment NONE NONE NONE DMSO”

Activity

in Cultured

Time in culture (hours) 0 24 12 72

Acid Dehydratase Rat Hepatocytes ALA-DH activity (nmol porphobilinogen/ h/mg protein) 8.98 5.38 4.62 4.46

a DMSO (280 mi%f) was added 3 h after hepatocyte every 24 h after the first treatment. * P < 0.01 vs fresh isolated cells (Dunnett’s test).

+ + + f

0.11 0.14* 0.32* 0.21* seeding and

SHORT

DMTU, like DMSO, is a powerful scavenger of hydroxyl radicals OH’ [23] and catalase is a scavenger of hydrogen peroxide. These compounds maintain a significantly higher content of cytochrome P450 after 24 and 72 h of culture (Fig. 1). After 24 h ECD activity too is significantly higher in the cells treated with the scavengers, DMSO and DMTU being more efficient than catalase (Fig. 2). Desferal, a specific iron chelating agent which is required in the reaction of Haber and Weiss [19], also maintained a higher activity (Fig. 2). After 72 h of culture ECD activity was kept higher by the treatment with DMTU; desferal, catalase, and DMSO even induced it above the level in the fresh isolated cells (Fig. 2). Only DMSO had this inducing effect on specific cytochrome P450 isoenzymes, probably because a much higher concentration of DMSO can be used without toxicity; a lower concentration (70 mM) had an effect comparable to the other scavengers [4]. Superoxide dismutase, which scavenges the superoxide radicals O,*- but produces H,O,, had no effect on MM0 activities in cultured rat hepatocytes (data not shown). Part of the effect of DMSO may be ascribed to its ability to maintain a higher activity of catalase. As previously described for cultured mouse hepatocytes [24] the activity of this antioxidant enzyme declines to about 68 and 19% of that found in fresh isolated cells after 1 and 3 days of culture, respectively. DMSO maintained the initial content of catalase (740 + 76 vs 781

159

NOTE

+- 28 pmol H,O,/min/mg protein in fresh hepatocytes) after 1 day; the content was significantly higher (P < 0.05) (250 -t 28) than in untreated cells (150 f 15 pmol H,O,/min/mg protein) after 3 days of culture. DMSO not only influenced cytochrome P450 and catalase activities but also improved the morphology and maintained the biochemical differentiation of cultured hepatocytes [3, 41. It has already been shown that DMSO maintains the basal TAT activity and its induction rate by glucocorticoids, which declines with time in culture [4]. These effects are shared by the other scavengers DMTU and desferal which, in fact, improved the cell morphology and conserved the initial TAT activity after 3 days of culture (Table 2). In the presence of dexamethasone the induction rate of the enzyme was maintained by treatment with all scavengers at a significantly higher level than in untreated cells (Table 2). Involvement of oxidative stress in the survival of cultured hepatocytes has been recently claimed in a study on the effect of phenobarbital, which also maintains functional and differentiated hepatocytes in culture for a long time [9] both by stabilizing the cell membranes and reducing the mitochondrial formation of active oxygen species [25]. These results appear to underline that the property of DMSO as a scavenger of oxygen radicals plays a role in the maintenance of liver-specific functions, such as higher cytochrome P450-dependent activities, higher

t= 24h

loo-

CONTROL

DMSO

DMT

MSFERAL

: L50-

,lALASE

CONTROL

0 MT U DESFERAL

CATAIASE

FIG. 2. Time-dependent effects of 280 mA4 DMSO, 25 mM DMTU, 1 mM desferal, and 1500 U/ml catalase on ECD activity in cultured rat hepatocytes. Cells were treated 3 h after seeding and every 24 h after the first treatment. The results are means k SE of four to five different hepatocyte preparations. b3 ECD activity in fresh isolated hepatocytes. *P < 0.01 vs the corresponding control (Duncan’s test); OP -z 0.01 vs desferal and catalase (Duncan’s test); AP < 0.01 vs DMTU, desferal and catalase (Duncan’s test); OP i 0.05 vs catalase (Duncan’s test).

160

SHORT

TABLE Effect

2.

2

of DMSO, DMTU, Desferal and Catalase on the Basal Activity and Induction of TAT in Hepatocytes

Treatment

Time in culture (hours)

None None DMSO” DMTU” Desferal” Catalase” Dexamethasoneb Dexamethasoneb + DMSO” Dexamethasoneb + DMTU” Dexamethasoneb + desferal” Dexamethasoneb + catalase’

TAT activity (mU/mg protein)

0 72 72 72 72 72 72

15.56 6.85 13.38 13.40 13.81 6.64 19.12

72

54.67 t- 1.78ott+

72

42.14 f 4.53O*tt

72

34.82 + 0.960vt

72

28.02 + l.OS”

* 0.82 T!Z0.49* _t 0.86°stt + o.90Qtt + 0.72°stt + 0.40* Z!Z1.24

a DMSO (280 mM), DMTU (25 n&f), desferal (1 mM), and catalase (1500 U/ml) were added 3 h after cell seeding and every 24 h after the first treatment. b Dexamethasone (10-r M) was added 48 h after cell seeding. The results are means ? SE of three to four different hepatocyte preparations. * P < 0.01 vs fresh isolated cells (Dunnett’s test). o P < 0.01 vs the corresponding control (Duncan’s test). t P c 0.05 and ttP < 0.01 vs the corresponding cells treated with catalase. A P < 0.01 vs the corresponding cells treated with DMTU and desferal (Duncan’s test).

of enzymes, and higher hormonal induction enzymes in rat hepatocytes in primary culture. content

This work was supported by the C.N.R. “Target nology and Bioinstrumentation.”

of

Project on Biotech-

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Received November 13, 1990 Revised version received January

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