Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes

0073-5655/82/0835-0842 $01.50/0 IN VITRO Vol. 18, No. 10, October 1982 © 1982 Tissue Culture Association, Inc.

P H E N O L S U L F O N P H T H A L E I N ~ P H E N O L R E D ; M E T A B O L I S M IN P R I M A R Y M O N O L A Y E R C U L T U R E S

O F A D U L T R A T H E P A T O C Y T E S

JAMES L. DRISCOLL, NANCY T. HAYNER, RHONDA WILLIAMS-HOLLAND, GERALDINE SPIES-KAROTKIN, PIERRE M. GALLETTI, AND HUGO O. JAUREGUI'

Department of Pathology, Rhode lsland Hospital (J. L. D., B. IV. H., G. S. K., H. 0. J.) and A rti[icial Organ Laboratory (N. T. H., P. M. G.), Division of Biology and Medicine,

Brown University, Providence, Rhode Island

(Received January 4, 1982; accepted July 12, 1q82}

SUMMARY

The sulfonic acid dye, phenolsulfonphthalein IPSP or phenol redk has been incor- porated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with/3-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20°-/o of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 1 0 0 , M there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cellsL Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye.

Key words: phenolsulfonphthalein; adult rat hepatocytes; glucuronidation.

INTRODUCTION

The sulfonic acid dye, phenolsulfonphthalein {PSP or phenol redl, is commonly included as a pH indicator in tissue culture media and salt solu- tions ~1~. Most commercially available formulations contain 1 to 20 mg/1 (2.8 to 56 ,M~ PSP, concentrations generally regarded as innocuous for cell growth and metabolism.

Liver cell cultures have been used widely for metabolic studies ~2-5t due to the development of the collagenase perfusion technique for effective hepatocyte separation. Hepatocytes were often cultured in media originally developed for other cell types 12-4t, although recently media have

'To whom requests for reprints should be addressed.

been formulated specifically for better maintenance of hepatocytes in vitro ~5-8). However, the commercially available media used for liver cell culture routinely contain PSP despite long- standing reports of hepatic metabolism of the dye in several species {9-11). Chromatographic studies have shown that PSP is partly conjugated with glucuronic acid by the liver i l lL The chemical structures of PSP moieties involved in this conjugation, as well as the ionized and nonionized forms, are shown in Fig. 1.

The following investigation was undertaken to examine: Ia) the major metabolic pathway of PSP in primary cultures of adult rat hepatocytes, tb~ the time course and degree of metabolism of different PSP concentrations in culture, and {c) the possible toxic effect of PSP on certain hepatocyte functions.

835

836 DRISCOLL ET AL.

MATERIALS AND METHODS

Hepatocyte isolation. Hepatocytes were isolated as previously described (12) from 150 to 200 g male Wistar rats. When the liver was di- gested sufficiently, it was removed from the ani- mal and placed in a beaker containing additional collagenase solution. The liver was minced, and the suspension was poured into a spinner flask that contained modified Waymouth's medium (MWM) (MB 752/1 GIBCO, Grand Island, NY), supplemented with 17 mM glucose, 50 gg/ml gentamicin sulfate (GIBCO Labora- wries, Grand Island, NY), 15 mM HEPES, no PSP, 10 #M insulin, and 1/~M dexamethasone. The MWM was previously equilibrated with 95% 02:5% CO2. The suspension was incubated at 37 ° C for 25 min with slow stirring in a sealed spinner flask. After incubating the cell suspension was filtered through a 253 ;~m nylon mesh and centrifuged at 50 xg for 2 rain at 4 ° C. The cell pellet was washed twice with MWM and centrifuged. All of the steps above were done under sterile conditions. Finally, the cell pellet was re- suspended in 25 ml MWM, and aliquots were taken for total cell count and uptake of trypan blue. This uptake was measured after 5-min incubations with 0.4% of the dye at 25 ° C. Only suspensions with a dye uptake of 15% or less were used to plate cultures. The mean total yield of

(YELLOW A) (RED B)

OH

GLUCURONYL TRANSFERASE

~ O _ R 2

0 i Ri

(COLORLESS C)

,.s<

OH

FIG. 1. Chemical structures of PSP moieties in media. The ratio of A to B is pH dependent whereas C is dependent on the metabolic competence of the hepatocyte. Specific metabolites are not designated and possible sulfate moieties are not listed.

dye-excluding parenchymal cells per pedusion was 1.16 _ 0.7 x 108 cells/g wet liver (1 to 5% nonparenchymal cells}.

Hepatocyte cultures. Hepatocytes were diluted to a concentration of 4 x l0 s dye-excluding cells/ml in cold MWM prepared as above with addition of 1% fetal bovine serum (FBS) (KC Biological, Inc., Lenexa, KS). This suspension was gently stirred and allowed to equilibrate to room temperature while 5 ml aliquots were dis- pensed to 60 mm Permanox culture dishes (Lux Scientific, Newbury Park, CA) precoated with Vitrogen bovine tendon collagen (Flow Labs, McLean, VA). All cultures were placed on wire mesh racks and incubated at 37 ° C in an at- mosphere of 93% air:7% CO2 and 98% relative humidity. Collagen coated dishes containing MWM with no cells served as a control. At 3 h the PSP-free medium was changed to fresh medium containing either 0, 28 .2~M (10mg/l), or 282 ~M (100 mg/l) PSP (GIBCO, Grand Island, NY). Cell cultures and controls were harvested at 24, 48, and 72 h, and the medium was utilized for the determination of PSP and its metabolite, lactate dehydrogenase (LDH) (E.C. 1.1.1.27), glucose, and lactic acid. The attached cells were used for total protein and cellular LDH determinations. All determinations were done in five replicate cultures and controls at each time. Modified Waymouth's medium (with respective PSP concentrations) was changed on remaining cultures and controls at 24 and 48 h. Experiments relating PSP metabolism to cell number were car- ried out in a similar fashion by plating hepatocytes at cell concentrations of 0.5, 1.0, and 2.0 x 106 dye-excluding cells/dish.

Three experiments also were conducted to com- pare the capacity of the hepatocytes to metabolize PSP when continuously exposed to the dye (as above) versus discontinuous exposure for selected 24 h intervals. The cultures were divided into two sets (with controls): (a)those receiving MWM with 28.2 or 282 ~M PSP at every media change, and (b) those exposed to PSP only on the first, second, or third day.

The effect of substrate concentration on the rate of PSP metabolism at various culture ages was studied by exposing hepatocyte cultures and control plates in triplicate to 56, 113, 252, 454, and 562 ~M PSP in MWM for 2 h intervals at 3, 24, 48, and 72 h after plating. To maximize the change in absorbance between controls and cultures, 2.5 ml medium with PSP rather than 5 ml was used per 60 mm culture dish. Prior to PSP

PSP METABOLISM IN HEPATOCYTES 837

exposure, all cultures and controls were main- tained in M W M without PSP and had a daily medium change. Total protein and percent LDH leakage were monitored at each time point. The rate of metabolism was expressed as nanomole PSP per milligram protein x 2 h.

Cell line cultures. Two nonliver human cell lines also were tested for PSP metabolism. New- born human foreskin cells, MA-184 (Microbio- logical Associates, Bethesda, MD), were used between Passages 26 and 30. Human epithelioid cervical carcinoma cells, HeLa, were obtained from American Type Culture Collection (ATCC CCL 2, Rocky(lie, MD). Cells were grown in M W M (with 7% FBS and without PSP, insulin, or dexamethasone) for 102 passages before use. Fibroblast cultures were seeded with 2 × 105 or 4 × l0 s dye-excluding cells/dish; HeLa cultures with 2.5 x l0 s or 5 x 105 dye-excluding cells/dish. These cultures (plus controls) were incubated under the same conditions as the hepatocyte cultures, and samples representing similar numbers of cells withdrawn at 24 h intervals.

Biochemical assays. Media from cell cultures and controls were collected at the indicated times and stored at -20 ° C until assayed. After cen- trifugation, an aliquot of the medium (1 ml of 28.2/~M PSP medium or 0.1 ml of the 282/JM PSP medium) was added to 2 ml 0.5 M glycine- NaOH buffer (pH 10), and the absorbance at 546 nm was determined. Phenolsulfonphthalein metabolism by cells was calculated from the decrease in absorbance in cell culture samples as compared to that in controls as follows:

% PSP metabolism =- 1 --

(Optical Density 546 nm

cell culture media) (Optical Density

546 nm control - - no cells - - culture media)

x 1 O0

To ascertain the nature of the PSP-conjugate in the cell culture medium, 24 h hepatocyte cultures in PSP supplemented medium (at both concentrations 28.2/~M and 282 ~M) were used. A 3 ml aliquot of the medium was adjusted to pH 5 with 0.01 ml of glacial acetic acid and mixed with 0.3 ml of 10 mg/ml fl-glucuronidase/sulfatase (E.C. 3.2.1.31) (Sigma Chemical Co., St. Louis, MO) solution in 0.1 M acetate buffer (pH 5). This solution was incubated at 37 ° C for 30 to 45 rain; then an aliquot was added to the glycine- NaOH buffer, and the absorbance at 546 nm was

noted. Compared to the control, the recovery of the free drug is calculated as:

(Optical density 546 nm of enzyme

incubated culture media) % Recovery = × 100

(Optical density 546 nm of control - - no cells - - culture media)

Lactate dehydrogenase was assayed separately in culture medium and in hepatocytes, as described previously, by the pyruvate to lactate reaction, monitoring the rate of absorbance decrease at 340 nm at 37 ° C (12). Percentage of LDH leakage was defined as follows:

Units LDH (in culture medium -- media control) × 100

Units LDH tln culture medium -4- attacbed cells) - - media control

Glucose was assayed in the culture media by the glucose oxidase method of Trinder (13t. Samples were stored at -20 ° C before measurements. Results are expressed as micromoles depleted from the medium per milligram of protein. Lactic acid and total protein measurements were made, as described previously (12).

Thin layer chromatography. Five to ten micro- liter aliquots of both cell culture and control medium at 24 h were chromatographed on Whatman K2 microcrystalline cellulose plates. The samples were adjusted to pH 5 before spotting, and the solvent system employed was a 10:1 mixture of ethanol:water. The dried plates were sprayed with glycine-NaOH buffer (pH 10) to visualize the free dye. The PSP-conjugate was visualized after spraying the plates with a 10 mg/ml p-glucuronidase/sulfatase solution (pH 4), and incubated 15 min at 37 ° C. After drying, these plates were also sprayed with glycine-NaOH buffer (pH 10).

RESULTS

Demonstration of PSP conjugate and recovery. For the study of PSP metabolism by adult rat hepatocytes in culture, samples of medium at 24 h and controls were chromatographed on micro- cellulose plates (Fig. 2). When sprayed with pH 10 buffer the chromatograph of cell culture media displayed two distinct spots whereas the control showed only one. The larger red spot and the solvent front have the same R t value for both the control and culture media. A slower migrating

838 DRISCOLL ET AL.

spot (R, approximately 0.6) for the culture medium was yellow and barely visible.

A second chromatograph of medium and control samples was developed in identical fashion, but before spraying with alkaline buffer the plate was sprayed with /J-glucuronidase-sulfatase reagent and then incubated at 37 ° C for 15 min. The spraying with pH 10 buffer followed and now two red spots were visible for the culture medium and only one for the control.

To quantitate the amount of PSP metabolite formed during culture, the concentration of unconjugated dye for the hepatocyte medium and controls was determined spectrophotometrically, based on the color intensity of the media measured at 546 nm. For the hepatocyte medium samples the absorption at 546 nm was always less than the control medium (without cells), and this difference is equated to the concentration of the metabolite. (See Materials and Methods, for the pertinent calculations.) However, after incubating the hepatocyte medium with the glucuronidase-sulfatase reagent and then repeat- ing the dye assay, the controls and hepatocyte incubated media now exhibited equivalent ab- sorptions and color intensity. Based on 48 deter-

o q b J

._1

X

1.0

0 . 9 .

0.8'

0.7-

0 . 6 .

0 , 5 -

0 . 4 -

0,5-

0 .2

0.1

= i

0 1 ~ I D I I

I I I I I I I I

T Tr xrr ~" I "wr

FIG. 2. Thin layer chromatography of PSP media incubated with either hepatoeyte cultures (II, IV) or controls (I, III, V, VI), where R t is the location of the dye moieties relative to the solvent front. I - - PSP standard sprayed with glycine buffer (pH 10) where the spot with Rf of 0.95 is red. II - - Hepatocyte media sprayed with pH 10 buffer with yellow and red spots with respective R f of 0.6 and 0.95. III - - Control media sprayed with pH 10 buffer with pattern Mentical to I. IV - - Hepatocyte media sprayed with/3-glucuronidase reagent followed by incubation at 37 ° C and then sprayed with pH 10 buffer. Red spots noted at Rf 0.6 and 0.95. V - - Control media treated identical to IV, with red spot at Rf 0.95. VI - - PSP standard treated identical to IV with red spot at Rf 0.95.

20-

m

._1 O I~D I 0 .

w

Q_

24 4 8 72

TIME (Hours)

FIG. 3. Phenolsulfonphthalein metabolism in adult ra t

hepatocyte cultures. Values presented are the mean and SE of 25 experiments; 5 replicates per experiment. Percent metabolism per 24 h period in cultures exposed to 28.2 ~M PSP is presented by clear bars and 282 t,M PSP by shaded bars.

minations done in glucuronidase-sulphatase treated PSP hepatocyte media, the recovery of PSP dye from these media (free and conjugated) was 98.4 _ 4.9% relative to the controls. (See Materials and Methods percent of recovery calculations.)

PSP metabolism. Cellular PSP uptake, conjugation, and excretion of PSP-conjugate into culture media all contribute to the decrease in absorbance at 546 nm, as compared to media incubated without cells. The fraction of PSP metabolized was higher at a dye concentration of 28.2 ~M than at 282 ~M, and decreased by approximately half for each successive day of culture (Fig. 3L It did not matter whether the cultures had been exposed to PSP continuously or only for the period in which the determination of PSP metabolism was made (data not shown). The relationship between PSP metabolism and the number of hepatocytes seeded per culture dish was investigated at hepatocyte concentrations ranging from 0.5 to 2 x 106 cells. Protein and dye metabolism were assayed at 24, 48, and 72 h for both 28.2 ~M and 282 ~M PSP. Dye metabolism was directly proportional to total protein (P <0.01) (data not shown).

The absolute amount of PSP metabolized over a 24 h period (expressed as nanomoles PSP per milligram protein) was higher at high dye concentrations (Table 1). However, this metabolic process cannot be related to glucose consumption, lactate production, or cell viability as indicated by LDH leakage. In control experiments conducted with a similar number of human fibroblasts and


TABLE 1

T HE RELATIONSHIP OF PSP METABOLISM TO OTHER HEPATIC F UNCTIONS

Metabolic Functions a

Percent i Time in Culture b PSP (pM) PSP-Conjugatinns c Glucose Depletion d Lactate Production e LDH Leakage Net Protein/Cultureg

3-24h 0 - - 9+-6 7 _+ 1 11 +-4 1.80_+0.13 28.2 14_+2 9 +_4 7+_ 1 11 +_ 3 1.80_+0.13

282.0 94+_ 10 8 +_4 7 _+ 1 11 +_4 1.75_+0.13

24-48 h 0 - - 15+_4 8+_1 4_+2 1.64_+0.09 28.2 10 _+ 1 17 +- 4 8 _+ 1 5 +_ 2 1.70 _+ 0.07

282.0 60 +_ 5 19 _+ 4 8 +_ 1 4 _+ 2 1.63 _+ 0.08

48-72 h 0 - - 16 +_ 8 10 +_ 1 2 +_ 1 1.69 _+ 0.09 28.2 8 +_ 1 17 +_ 6 10 +_ 1 2 +- 0 1.72 +_ 0.08

282.2 34_+2 20+_6 12+_1 2_+1 1.69+_0.10

a Values are the mean +- SD of 15 determinations (3 experiments, 5 replicate samples per experiment). b Data are not cumulative, but only for the 24 h period indicated. c Nanomole PSP-conjugate per milligram protein. d Micromole glucose depleted per milligram protein. e Micromoles lactate produced per milligram protein. f Percentage LDH leakage was calculated after procedure described in Materials and Methods. g Milligram of protein per culture dish of attached cells. These values are corrected for protein content of collagen

coated dishes with no cells.

HeLa cells there was no significant metabolism of the dye (<5%).

When hepatocytes were incubated with PSP for 2 h, the rate of PSP metabolism was lower at 24 than at 3 h, and showed no further decline at 48 and 72 h (Table 2). The rate of PSP metabolism for 3 h old cultures when plotted against PSP concentration in the incubation medium is shown in Fig. 4. More than one kinetic component is evi- dent from this figure.

D ISCUSSION

Metabolic studies of organic anionic dyes such as bromosulfophthalein (BSP)(14-18) , dibromo- sulfophthalein (DBSP) (19), and indocyanin green (20) have been extended recently to in vitro systems of hepatocytes, but to our knowledge there are no previous reports of PSP metabolism by hepatocytes in culture. Therefore, the effects of media formulations containing PSP as a dye indicator on hepatocytes in culture are unknown.

Phenolsulfonphthalein is known to be metabolized in species other than the rat. Apparently the variation in blood concentrations after adminis- tration of the dye in these species reflects different rates of clearance by the kidney and liver

(9,21,22). The amount of dye metabolized in vivo has

been shown to vary with species, strain, and method of detection. H o m a n and Guarino (23) found that perfused Wistar rat liver excreted approximately 35% of PSP into the bile as the glucuronide, whereas perfused Gunn rat liver excreted no detectable conjugate. Har t and Schanker (24) have shown glucuronidation of PSP in Sprague Dawley rats, and Despopoulos (25) found evidence of two PSP metabolites in the bile of isolated perfused Long-Evans rat livers. How- ever, the glucuronide was the only metabolite demonstrated in vivo (25L In our experiments, 98.4 -+ 4 .9% of the PSP metabolized seems to be altered by means of the glucuronidation pathway.

TABLE 2

EFFECT OF CULTURE AGE ON THE RATE OF PSP METABOLISM BY HEPATOCYTE CULTURES

Rate of Metabolism a

PSP (#M)

Culture Age (h) 56 113 256 452 565

3 1.5b-+ 0.2 2.4+_0.4 8.9+--0.7 10.6+_0.5 12.2+_0.8 24 1.0 +_ 0.4 1.1 + 0.3 2.3 +_ 0.4 5.4 +_ 0.4 5.3 +_ 0.6 48 0.9+_0.5 1.1 _+0.3 2.2+0.5 4.8+-0.7 5.0-+0.6 72 1.2 +- 0.6 1.4 +_ 0.7 2.2 _+ 0.8 4.7 _+ 0.7 5.1 + 0.8

Rate is expressed as nanomoles PSP per milligram protein (2 h). b Data shown is the mean +- SD of three experiments, three replicates each.

840 DRISCOLL ET AL.

15 t t l

z LLI I - o IO rY ft.

E Q.

u~ 5 Q.

(/I

0 E c.

0 I I I I I I I I 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0

PSP { ~ M )

FIG. 4. The effect of PSP concentration on the rate of PSP metabolism by 3 h hepatocyte cultures. Three hour cultures were incubated with the indicated concentrations of PSP diluted in medium for 2 h. Each point is the mean of three determinations from a representative experiment.

Specific PSP metabolism by our cultured adult hepatocytes was demonstrated by treatment of hepatocyte incubated media with/3-glucuronidase which caused an increase in absorbance to control levels, and by visualization of the metabolite by thin-layer chromatography. The use of cell-free controls precludes simple protein adsorption to collagen or FBS as the primary reason for an adsorbance decrease in hepatocyte incubated media samples. Although it is conceivable that some PSP from the medium could partition into the lipid phase of hepatocyte cell membrane mate- rial and account for a nonspecific decrease in optical density readings, control incubations with HeLa cells and fibroblast cultures did not support this possibility. Indeed, PSP supplemented media incubated with an appropriate number of cells tthe number of HeLa or fibroblasts used in these experiments corresponds to similar amounts of protein per dish, as in the hepatocyte incubations}, showed no significant decrease in PSP adsorbance versus cell-free control media.

Inasmuch as the levels of LDH in the tissue culture media did not increase substantially with time, and this leakage usually is associated with terminal events in the cell as previously reported by us (12}, we are confident that our cells were viable.

The decrease in uptake and glucuronidation of PSP in hepatocyte cultures parallels other known phenotypic changes in culture, such as decrease in cytochrome P-450 activity (7). We noted a half- life of approximately 24 h for PSP metabolism that is unaffected by conditions of continuous versus discontinuous exposure of the cultures to the dye. However, the quality of the isolated cell preparations influenced subsequent activity in culture. Cells with lower LDH leakage at the time of isolation were found to metabolize a larger percentage of the dye (data not shownL Lowered ATP levels and a gradual switch toward anaero- bic metabolism, as indicated by increased lactate production, with time may have contributed to the waning metabolic competence of the cells.

The kinetic data for the rate of PSP metabolism are similar to that previously reported for rat lung slices by Gardiner and Schanker (26) who showed saturable and nonsaturable components. Using their methodology, one may calculate an apparent Km of 50/~M and an apparent V max of 1.4 -+ 0.2 ~M/mg protein for 2 h incubation for the saturable component which occurred primarily at PSP concentrations less than 100 #M. At higher PSP concentrations, simple diffusion may account for the proposed nonsaturable component, although the limited solubility of PSP above 600 ~M precluded testing saturation kinetics above this level.

The use of PSP in adult hepatocyte cultures seemed to be harmless in terms of cell toxicity. Al- though glucuronidation rendered a portion of the compound colorless, enough remained to be use- ful as a pH indicator.

We have demonstrated that PSP may be used as a sensitive indicator of functional activity in hepatocyte culture. The metabolism of both PSP and bilirubin are dependent on glucuronyl transferase activity. Inasmuch as the use of PSP avoids the technical problems associated with bilirubin (such as light sensitivity and insolubility} in aqueous media, it can replace bilirubin in the study of this transferase. However, it should be cautioned that PSP should not be used in tissue culture media when adult rat hepatocyte cultures are employed to test compounds (xenobiotics, etc.) that are partially or wholly metabolized by glucuronidation.

Organic anions are transported into hepatocytes by plasma membrane carriers with high affinity for sulfobromophthalein, bilirubin, and indocyanin green {27-29}. This transport pro- ceeds with a half time of only a few minutes (30)


in spite of a high affinity of plasma albumin for these organic anions t31,32 ~. The organic dye uptake is not sodium dependent, although purified gluthathione transferases seem to have a lower binding affinity than a lbumin for these anions, they apparently play an important role in this uptake t33,34). Our research indicates saturation of PSP uptake similar to that observed for bil irubin and BSP. Consequently, future experiments will be designed to assess the role of a lbumin in the binding of PSP and to investigate the possible competition of BSP and indocyanin green for the uptake of PSP into adult rat hepatocytes in monolayer cultures.

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34. Levi, A. J.; Gatmaitan, Z.; Arias, I. M. Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin sulfobromophthalein and other anions. J. Clin. Invest. 48: 2156-2167; 1969.

We thank Florence McCaffrey and Frances Gallagher for typing the manu- script and the Clinical Biochemistry Laboratory for the preparation of the LDH assay reagent.

This study was supported in part by N I H Grants HL-11945-11 and 1 R01 AM 26520-01A1.

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