Biochimie ( 1991 ) 73, 621-624 © Soci6t6 franc;aise de biochimie et biologie mol6culaire / Elsevier, Paris

621 Short communication

Dimethyl sulfoxide enhances lipid synthesis and secretion by long-term cultures of adult rat hepatocytes FM De La Vega*, T Mendoza-Figueroa** Department of Pharmacology and Toxicology, Centro de lnvestigaci6n y de Estudios Avanzados del IPN, AP i4-740, Mexico City 07000, DF Mexico (Received 20 September 1990; accepted 28 January 1991 )

Summary - - Dimethyl sulfoxide (DMSO) was tested for its effects on lipid metabolism of long-term cultures of adult rat hepatocytes. The addition of 1% DMSO to 3T3-hepatocyte cultures was not toxic to cells and in fact treated cultures maintained better their characteristic morphology for up to 14 days of exposure. DMSO treatment increased 2-3 fold the de novo synthesis of total lipids from [n4Clacetate. The analysis by thin layer chromatography of cellular and secreted lipids revealed that DMSO increased the levels of cellular triglycerides, phospholipides and free and sterified cholesterol at 7 days of exposure while at 14 days there was also a 23-fold increase in medium secreted lipids. Additionally, DMSO increased the activity of glycerol-phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, by > 50% at either 7 or 14 days of exposure. These results show that 1% DMSO not only is not detrimental to cultured hepatocytes but also enhances lipid synthesis and secretion, both hepatic-differentiated functions.

dimethyl sulfoxide / hepatocyte primary culture / lipid secretion / lipid synthesis Introduction

Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used to solubilize water insoluble compounds [1]. Earlier reports suggested that DMSO could be toxic to liver cells [2]~ precluding its use as a vehicle for pharmacological and toxicological testing. However, several researchers have reported that DMSO promotes the expression of differentiated functions in a variety of cultured malignant cell lines [3-7]. Further, I s o m e t al [8] have shown that the addition of 2% (v/v) DMSO to the culture medium of primary cultures of rat hepatocytes allows the maintenance of differentiated functions for extended periods. We have previously described that adult rat hepatocytes culture I on a feeder layer of lethally treated 3T3 cells survive for several weeks while maintaining their typical morphology and ultrastructure. Additionally, *Present address: Department of Genetics and Molecular Biology, Centro de Investigaci6n y de Estudios Avanzados del IPN, AP 14-740, M6xico DF 07000, M6xico

**Correspondence and reprints Abbreviations: DMSO, dimethyl sulfoxide; dpm, disintegrations per min; GPD, L-glycerol 3-phosphate dehydrogenase; HDL, high density lipoproteins; SD, standard deviation; TLC, thin layer chromatography; VLDL, very low density lipoproteins.

these cultures retain for long time various liver-specific functions, such as albumin secretion, basal and inducible levels of cytochrome P-450, and synthesis and secretion of lipids to the culture medium [9]. Since the liver plays a central role in the metabolism of lipids and lipoproteins [ 10], it is a useful approach to use cultured liver cells to study the effects of drugs and chemicals that might affect hepatic lipid metabolism [11]. However, many of these compounds are lipophilic and firstly they should be solubilized in an appropriate solvent. This vehicle should not be toxic to cultured cells, and further it should not inhibit hepatic lipid metabolism. Therefore, we studied the effect of long-term exposure of 3T3-hepatocyte cultures to low levels of DMSO on the synthesis and secretion to the culture medium of various lipid species. The results described here show that exposure for up to 14 days to 1% DMSO enhances the synthesis and secretion of different lipids by cultured hepatocytes. Materials and Methods Hepatocytes were isolated from male Wistar rats (180-200 g) as previously described [9]. Hepatocytes were inoculated (4.5 x 105/35-mm dish) on a feeder layer of 3T3 cells [12] that had been lethally treated with mitomycin C (a gift from BrystolMyers Co, Denver, CO) [131 and inoculated one day earlier at

t~22

M De La Vega, T Mendoza-FigueI~,a

4 x IC~r ~ cclls/35-mm dish 191, The cultures were n laintained in Eagle's mediunl modified by Duibecco and V6gt supplemented xvith 7% calf serum (HyC!one, Logan, UT). 5 lag/ml insulin, 0,1 uM d-biotin and 10 lajmi of hydrocortisone (Sigma Chemical Co, St Louis, MO) (complete medium). Treated cultures received 24 h after seeding 1% (v/v) DMSO (Sigma Chemical Co) dissolved in the culture medium. Cultures of ieth-,dly treated 3T3 cells were maintained with complete medium with or without DMSO to assess the contribution of the feeder layer to the observed effects. Culture medium was changed daily (I ml/35-mm dish). For transmission electron microscopy, cells were processed as described [141. To measure the incorporation of [14Clacetate into cellular and secreted lipids, 0.2 laCi/ml of [l,2-t4Clacetic acid (55 laCi/ mmoi, New England Nuclear, Boston, MA) were added to the cultures at the indicated culture times I9, 14]. After 24-h incubation, the medium was removed and the cells were washed, scraped an disrupted by sonicat~.on. Lipids from both culture media (secreted lipids) and cells (cellular lipids) were extracted as described by Cham and Knowles 1151 and analyzed by thin layer chromatography using a mobile phase of heptane:di-isopropyl ether:acetic acid t60:40:4) I161. The radioactive lipids that comigrated with purified standards of triglycerides, phospholipids, cholesterol and cholesterol esters, were determined by liquid scintillation. Cell extracts used to determine enzyme activities were prepared as described [17]. Malic enzyme (L-malate:NADP oxidoreductase [decarboxylatingl; EC I. 1.1.40) and L-glycerol 3-phosphate dehydro-

genase (EC I.!.1.8) activities were determined as described elsewhere [1 "~-201. Protein was determined by the method of Lowry et a, [211. Unless otherwise indicated, result~ are expressed as the mean + standard deviation (SD) of determinations from 3 or 4 culture dishes in a typical experiment; similar results were obtained in 3 independent experiments. Statistical comparison ~¢as perfo~xned with the unpaired Student's t-test.

Results and Discussion H e p a t o c y t e s m a i n t a i n e d for either 1 or 2 w e e k s in culture m e d i u m c o n t a i n i n g 1% D M S O better retained their characteristic p o l y h e d r i c a l m o r p h o l o g y and s h o w e d a d e c r e a s e d n u m b e r o f c y t o p l a s m i c lipid droplets with respect to untreated o n e s (fig 1a - d ) . This result is consistent with that o f I s o m e t a l [8] w h i c h h a v e reported the m a i n t e n a n c e o f the typical hepatocyte m o r p h o l o g y in long-term cultures b y the addition o f 2 % D M S O to culture m e d i u m . U n d e r the electron m i c r o s c o p e w e f o u n d only m i n o r alterations in the h e p a t o c ) t e ultrastructure, such as an increase in the m i c r o f i l a m e n t b u n d l e s through the c y t o p l a s m and especially u n d e r the p l a s m a t i c m e m b r a n e (data not shown).

Fig 1. Effect of D M S O on cell morphology. Hepatocytes were cultured in complete medium for 7 (a) or 14 days (c) or in complete medium supplemented with 1% DMSO for 7 (b) or 14 days (d). Phase contrast optics. Bar = 50 l,tm.

623

D M S O e n h a n c e s lipid s y n t h e s i s a n d s e c r e t i o n

To study the effect of DMSO exposure on de novo lipid synthesis and secretion by hepatocyte cultures, we measured the incorporation of ['4C]acetate into cellular, secreted and total (cellu'-" plus secreted) lipids. DMSO treatment increased 2-3 fold the [14C]acetate incorporation into total lipids fi'om 28.9 + 4.5 × 103 to 82.6 + 7.9 x 103 dpm/dish (mean + SD, n = 4, P < 0.001) and from 48.0 + 4.5 x 103 to 92.6 + 28.7 x 103dpm/dish (P < 0.05), after 7 and 14 days of expesure, respectively. To further investigate the particular lipid species increased by DMSO treatment, we analyzed the lipid extracts by TLC (fig 2). We found that after 7 days of exposure DMSO enhanced the total incorperation of [~4C]acetate in all the analyzed species, due mostly to an increase in cellular radiolabeled lipids (fig 2A-D). However, after 14 days of DMSO treatment the increase in [t~C]acetate incorporation into total lipids was due to an increase in both cellular and secreted lipids (fig 2 E-H). At this culture time, there was a 2-3-fold increase in the secretion of all lipid species (fig 2E-H). These results agree with the morphological observations (fig 1) showing a reduced number of cytoplasmic lipid droplets after 14 days of exposure to DMSO. Thus, these results are compatible with the hypothesis that DMSO enhances tissue-specific gene expression as evidenced by the accumulation of cell type characteristic products [3-8]. Here, there was an accumulation of radiolabeled lip~ds in the culture medium of DMSO treated cultures. The contribution of the feeder layers of these effects waq negligible, as studies with cultures of 3T3 cells revealed. The incorporation of the labeled precursor into lipids of 3T3 cells was 1-5% of that found in 3T3-hepatocyte cultures, except for phospho-

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more, the secretion of labeled lipids by 3T3 cells was not significant (data not shown), To determine if DMSO affects lipid synthesis pathways, we assayed the activities of malic enzyme and glycerol-phosphate-dehydrogenase (GDP), marker enzymes of fatty acid and glycerolipid synthesis, respectively [22]. DMSO treatment increased the activity of GPD from 26.7 + 14.3 to 40.8 + 4.7 (mean + SD, P < 0.05, n = 3) and from 26.3 + 3.3 to 67.5 + 10.6 (P < 0.05, n = 3) nmol N A D . mg protein-' min-' at 7 and 14 days of exposure, respectively. On the other hand, DMSO treatment did not significantly change the activity of malic enzyme, although there was a tendency to increase (data not shown). Even if the activity of these enzymes is an indirect measure of the lipid synthesis pathways [22], all the results combined strongly suggest that DMSO increases the synthesis of glycerolipids ( c f fig 2). We did not determine the secretion of the different lipoprotein classes by the hepatocytes; however, an increase in lipopretein export can be expected (VLDL or HDL; eg [23]). It remains to be investigated if D M S O affects selec-

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Fig 2. Effect of DM$O on [14C]acetate incorporation into hepatocyte cellular and secreted lipids. Hepatocytes were cultured for 7 (A-D) or 14 days (E-H) in complete medium unfilled bars) or complete medium supplemented with .'~ DMSO (filled bars). Then, cultures were incubated with 0.2 laCi/ml of ['4C]acetate for 24 h. The incorporation of r l4Clac, t~te ;ntn medium (secreted) and cellular lipids was measured after extraction of lipids and their separation by TLC. Total incorporation was the sum of radioactivity in medium and cells. Bars represent values of mean + SD (n = 4) in a typical experiment. Asterisks indicate significant difference with respect to control values (*P < 0.05; **P < 0.01).

tively the secretion of one or another lipoprotein species. In summary, we have shown that DMSO en|lances l;n;A

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cultures, both hepatic-differentiated functions. Our results are consistent with previous studies and using other criteria demonstrate that the addition of low amounts of DMSO to primary cultures of hepatocytes is not detrimental to this cell type [24], but favors the permanence of hepatic differentiated functions [8]. It should be noted that in the 3T3-hepatocyte cultures DMSO is not necessary to preserve hepatocyte differentiated functions, since hepatocytes can retain several liver-specific functions for long culture periods without DMSO addition [9]. Therefore, our results imply that the addition of 1% DMSO to the culture medium further enhances the expression of hepatic function. How DMSO promotes the expression of tissue-specific functions is still under discussion. Some authors suggest that DMSO could have a direct effect in gene expression [3-7, 25]. Furthermore, recent results of Isom et al [26] suggest that DMSO promotes the persistence of some liver-specific mRNAs in cultured hepatocytes. Since low concentrations of DMSO are not toxic to cultmed liver cells, its use is doubly advantageous.

624

M De La Vega, T Mendoza-Figueroa

D M S O is a suitable vehicle to deliver water-insoluble substances into cultured h e p a t o c y t e s for p h a r m a c o logical or toxocological testing, with the further benefit o f p r o m o t i n g differentiation m a i n t e n a n c e .

10 11 12

Acknowledgments We are indebted to A Hern,'indez and M de Lourdes L6pez for technical assistance and photographic artwork, and to W KuriHarcuch for making helpful suggestions during the developmerit of the research. FMV was supported by a studentship from the Consejo Naciona! de Ciencia y Tecnologia (CONACYT), Mexico, during part of this work. This research was partially supported by CONACYT grant P219CCOL882377.

References

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1 Mathew T, Karunanithy R, Yee MH. Natarajan PN (1980) Hepatotoxicity of dimethylformamide and dimethylsulfoxide at and above the levels used in some aflatoxin studies. Lab hn,est 42, 257-262 2 Rubin I. (1983) Toxicologic update of dimethyl sulfoxide. Ann NF Acad Sci 411, 6-9 3 Higgins PJ, Borenfretmd E (1980) Enhanced albumin production by malignantly transformed hepatocytes during in :itro exposure to dimeth2¢Isulfo×ide. Biochem B;ophys Acta 610, 174---180 4 Scher W, Waxman S (1983) Effects of dexamethasone and phorbol myristate acetate in mouse erythroleukemic cells by dimethyl sulfoxide, proteases, and other compounds. Ann NY Acad Sci 411, 1800190 5 Rifkind RA, Sheffery M, Profous-Jucheika HR, Reuben RC, Marks PA (1983) Induction of globin gene expression during erythroid cell differentiation. Ann NY Acad Sci 411,141-149 6 Higgins P (1983) Cell-cycle related events in the dimethyl sulfoxide-induced commitment of hepatic tumor cells to -..~1 a - ' l ~ t ~ l -

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335 7 Tapiero H, Zwingelstein G, Fourcade A, Portoukalian J (1983) The effect of dimethyl sulfoxide on the membrane dynamics and the phospholipid composition of 2 different cell lines. Ann IVY Acad Sci 411,383-388 8 Isom H, Secott T, Georgeoff I, Woodworth C, Mummaw J (1985) Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci USA 82, 32523256 9 Kuri-Harcuch W, Mendoza-Figueroa T (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41, 148-157

24

25 26

Levy RI (1988) Currently available lipid-lowering agents. Hosp Pratt 23 (suppl 1), 14-21 Forte TM (1984) Primary hepatocytes in monolayer culture: a model for studies on lipoprotein metabolism. Ann Rev Physio143, 403-410 Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17, 299 Macpherson I, Bryden A (1971) Mitomycin treated cells as feeders. Exp Cell Res 69, 240°241 Mendoza-Figueroa T, Hermindez A, L6pez ML, KuriHarcuch W (1988) Intracytoplasmic triglyceride accumulation produced by dexamethasone in adult rat hepatocytes cultivated on 3T3 cells. Toxicology 52, 273286 Cham BE, Knowles BR (1976) A solvent system for depilidation of plasma or serum without protein precipitation. J Lipid Res 17, 176-- 181 Kates M (1972) Techniques in lipidology. In: Laboratory Techniques in Biochemiso~y and Molecular Biology (Work TS, Work E, eds) American Elsevier, NY, 502 Kuri-Harcuch W, Green H (1977) Increasing activity of enzymes on pathway of triacylglicerol synthesis during adipose conversion of 3T3 cells. J Biol Chem 252, 2158-2 ! 60 Wise EM, Ball EG (1964) Malic enzyme and lipogenesis. Proc Natl Acad Sci USA 52, 1255-1263 Kosak LP, Jensen JT (1974) Genetic and developmental control of multiple forms of e-glycerol 3-phosphate dehydrogenase. J Biol Chem 249, 7775-7781 Kuri-Hacuch W, Wise LS, Green H (1978) Interruption of the adipose conve, sioia of 3T3 cells by biotin deficiency; differentiation without triglyceride accumulation. Cell 4, 53-59 Lowry OH, Rosenburg N J, Farr AL, Randal RJ (1951) Protein measurement with the folin reagent. J Biol Chem 193, 265-275 Newsholme EA, Start S (1981 ) Regulation in Metabolism. John Wiley and Sons, Chichester, 293 Davis RA, Engelhorn SC, Pangrun H, Weinstein DB, Steinberg D (i979) Very low density iipoprotein synthesis and secretion by cultured rat hepatocytes. J Biol Chem 254, 2010-2016 Santone KS, Kroschel DM, Powis G (1986) Enhanced formation of ethane and n-penthane by rat hepatocytes in the presence of dirnethyl sulfoxide. Biochem Pharmacol 35, 1287-1292 Kharasch N, Thyagarajan BS (1983) Structural basis for biological activities of dimethyl sulfoxide. Ann NY Acad Sci 41 l, 391--402 Isom H, Georgeoff I, Salditt-Georgieff M, Darnell Jr JE (1987) Persistence of liver-specific messeger RNA in cultured hepatocytes: different regulatory events for different genes. J Cell Biol 105, 2877-2885