Chem.-Biol. Interactions, 45 (1983) 235-242 235 Elsevier Scientific Publishers Ireland Ltd.

S T A T U S O F R E D U C E D G L U T A T H I O N E I N P R I M A R Y C U L T U R E S O F R A T H E P A T O C Y T E S A N D T H E E F F E C T O N C O N J U G A T I O N O F B E N Z O [ a ] P Y R E N E - 7 , 8 - D I H Y D R O D I O L - 9 , 1 0 - O X I D E

HELEN MORRISON, VIVECA HAMMARSKIOLD and BENGT JERNSTROM*

Department of Forensic Medicine, Karolinska Instituter, S-104 01 Stockholm (Sweden)

(Received October 22nd, 1982) (Revision received December 17th, 1982) (Accepted December 20th, 1982)

SUMMARY

T h e i n t r a c e l l u l a r level of r educed g l u t a t h i o n e (GSH) and G S H con juga t ion h a v e been i nves t i ga t ed in p r i m a r y cell cu l tu re s of hepa tocy t e s i so la ted f r o m control ra ts , p h e n o b a r b i t o n e (PB) and 3 - m e t h y l c h o l a n t h r e n e (MC) t r e a t e d ra ts . T h e d a t a d e m o n s t r a t e t h a t in all cell cu l tu re s the G S H concen t r a t i ons show a t r i phas i c pa t t e rn : (i) w i th in I h of cu l tu re a n in i t ia l m a r k e d decrease to 50% of the levels found in f resh hepa tocy tes ; (ii) r ecovery of G S H concen- t r a t i o n s to above the levels observed in f resh cells. T h i s occurs a f t e r 6 h in cu l tu re wi th control cells and a f t e r 10-24 h w i th cells f r o m e i the r PB or MC t r e a t e d r a t s and was m o s t p r o m i n e n t in cells f r o m P B - t r e a t e d ra ts . (iii) A slow decl ine to be t ween 30 a n d 40 nmol G S H / m g pro te in f r o m 24 to 96 h in cu l ture .

Syn thes i s of G S H was s lower in cu l tu red cells f r o m PB t r e a t e d r a t s and th i s was conf i rmed by the r e s y n t h e s i s r a t e s w h e n d i e t h y l m a l e a t e (DEM) was used to deple te GSH. T h e f o r m a t i o n of G S H con juga tes wi th r acemic 7/~,8~- d ihydroxy-9a , 10 a - epoxy -7 ,8 ,9 ,10 - t e t r ahyd robenzo [a ]py rene (BPDE) was m e a s u r e d in control cells in suspens ion and a f t e r 3 and 24 h in cul ture . Desp i t e the decrease in G S H concen t r a t i ons obse rved be tween 1 and 4 h a f t e r cu l ture , the con juga t ion r a t e s were not decreased.

K e y w o r d s : G l u t a t h i o n e - - G l u t a t h i o n e c o n j u g a t i o n - - H e p a t o c y t e cu l tu res

*To whom correspondence should be addressed. Abbreviations: BP, benzo[a]pyrene; BPDE, racemic 7fl,8a-dihydroxy-9a,10a-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene; DEM, diethylmaleate; GSH, reduced glutathione; GSSG, oxidized glutathione; TGT, T-glutamyltranspeptidase; MC, 3-methylcholanthrene; PB, phenobarbitone.

0009-2797/83/$03.00 O 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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INTRODUCTION

Isolated hepatocytes in primary culture are useful for studying many aspects of drug metabolism and action which can not conveniently be examined in vivo. However, this application may be severely limited by changes in the levels of the drug metabolizing enzymes during culture. A number of studies have shown tha t concentrations of cytochrome P-450 and dependent monooxygenase enzyme activities are decreased by more than 60% during the first 24 h in culture [1-6]. Although the activity of monooxygenase enzymes plays a critical role in the activation of xenobiotics to the cytotoxic, mutagenic and carcinogenic forms [7] an important factor in the overall metabolism and detoxification of xenobiotics is the activity of the conjugating enzymes.

Conjugation with GSH plays an important role in detoxification of reactive species in isolated hepatocytes [8-10]. In rat hepatocytes the intracellular level of GSH is important in preventing intermediates of racemic trans.7,8- dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-dihydrodiol) and BPDE from reacting with the DNA. When the intracellular level of GSH is decreased to about 40% of the normal level, the DNA binding of both substrates increases substantial ly [11]. Moreover, a correlation between GSH levels and cellular damage has been suggested [12,13].

In this prel iminary report the stability of the intracellular GSH concen- trations over a 96 h culture period was investigated. The ability of cultured cells to synthesise GSH conjugates of BPDE was shown to be unaltered when compared to cells in suspension.

MATERIALS AND METHODS

( a ) Treatment of rats and preparation of cultured hepatocytes Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g) as

previously described [14]. The viability of the resulting cell suspension (20 × 10Scells/ml) was measured by trypan blue exclusion and NADH- penetration [12]. The cells were washed once with sterile Krebs-Hepes buffer and resuspended in prewarmed medium (1 x 10 s cells/5 ml medium). Aliquots (5-ml) of the cell suspension were dispensed onto collagen precoated tissue culture dishes. The cultures were incubated in a humidified atmosphere (5% CO~Jair) at 37°C. After allowing 4 h for the cells to attach to the collagen gels, unat tached cells were aspirated off and the medium renewed. Thereaf- ter the medium was changed every 24 h.

Cells were cultured from untreated rats, PB treated rats (1 mg/ml in the drinking water for 5 days before use) and from MC treated rats (50 mg/kg i.p. given 3 days before cell preparation).

(b) Preparation of medium and tissue culture dishes The medium used was based in Earle's balanced salt solution containing

10 ml 'BME Vitamin solution x l00 strength' (both from Gibco Biocult, Chemoferm AB, Stockholm) per litre medium.

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Amino acids were added at the same concentrations as found in RPMI 1640 [15] with the exceptions of cystine and methionine. Cystine was omitted from the medium [16] and the final concentration of methionine was increased from 0.1 to 0.25 raM. The medium also contained 100 ~M hydrocortisone.21- sodium succinate and 100 ~M 5-aminolaevulinic acid (Sigma Chemical Co., St. Louis, MO). After filtering the medium through a 0.22-~m sterile filter (Falcon, Labora AB, Stockholm) 5% foetal calf serum (Gibco biocult, Chemoferm AB, Stockholm) and 1 I.U. insulin (Zink-Pro- tamin-Insulin, Vi t rum AB, Stockholm) per litre of medium were added under sterile conditions.

The plastic tissue culture dishes (60 x 15 nm, from Falcon, Labora AB, Stockholm) were covered with collagen type HI (Sigma Chemical Co., St. Louis, MO). The collagen (0.5 mg/ml) was dissolved in 25% acetic acid and 2 ml dispensed onto each culture dish. The dishes were then left overnight at room temperature under UV light to dry, and were washed twice with 2 ml Krebs-Hepes buffer (pH 7.4) before use.

(c) Incubations with DEM To deplete intracellular GSH the culture plates and fresh cells in suspen-

sion (1 x 106 cells) were incubated for 30 minutes at 37°C with 0.01% DEM in DMSO [17]. The medium was then renewed and incubations continued to allow resynthesis of GSH. DMSO (20 ~l) was present in 3 ml medium and this concentration of DMSO had no effect on the cellular GSH content (data not shown).

( d) Analytical methods (i) Determination of GSH. For measurement of GSH in the cultured

hepatocytes the cells were suspended in 0 .1M Tris buffer (pH 7.4) by scraping with a ' rubber policeman'. Proteins were immediately precipitated from both cell and medium samples using 50 ~l/ml 70% perchloric acid. The GSH was derivatized with iodoacetic acid and Sanger's reagent as described by Reed and Beat ty [18]. The derivatives were analysed by HPLC using a ~Bondapak amine column (4x 100mm) and gradient elution [18]. Ap- propriate s tandards of both GSSG and GSH were analysed simultaneously and quantification was by the integrated peak areas.

(ii) Determination of GSH.BPDE conjugates. GSH-BPDE conjugates were analysed by a modification of the method of JernstrSm et al. [11]. Incubations containing 20 ~M [14C]BPDE (spec. act., 4306 dpm/nmol) were terminated after 30 min. The identity of the BPDE.GSH conjugate was previously confirmed by double isotope labelling and amino acid analysis [11].

(iii) Measurement of protein. Protein content of cell suspensions and cul- tures was determined by the method of Lowry et al. [19].

RESULTS

The intracellular levels of GSH measured in cells cultured from all groups of rats are illustrated in Fig. 1. The data show that within I h of culture and

238

80

60

~ 0 q

20

(a) control

-r } M C

0

60

z,c ~ °~o__ o 20

( c ) Phenobarbitone ' ' ' . . . . .

1 16 20 72 96 hours in culture

Fig. 1. Intracellular GSH. Each point is the mean of 4 determinations. At time zero the GSH in freshly isolated cells in suspension is represented.

in all three types of cells the GSH level init ially falls to approx. 50% of tha t in fresh cells. Resynthesis of GSH is fastest in control cell cultures and, within 4 h, the GSH levels approximate those measured in freshly isolated hepatocytes. In cell cultures from PB treated rats resynthesis of GSH occurs more slowly and the levels remain below 40 nmol/mg protein unti l 8 h af ter culture but, thereaf ter , rapid synthesis occurs and the level increases to 71 nmol GSH/mg protein at 10 h. Each culture type shows that, af ter this initial phase of deficiency, there is a marked elevation in GSH above the normal levels observed in fresh cell suspensions. In control cell cultures the

239

maximum GSH level (65nmol/mg protein) occurs after 6 h in culture, whereas with cell cultures from PB and MC treated rats the maximum occurs much later, at 24 h. This overshoot in GSH levels is most prominent in cultures from PB treated rats. There is a slow decline in GSH levels in all types of cell from 24 to 96 h in culture, but at 96 h the GSH level is still above 30 nmol/mg protein.

The initial decrease in GSH levels is not accompanied by a concomitant increase in either the intracellular level of oxidized glutathione (GSSG) or in the recovery of GSSG in the medium. The intracellular level of GSSG was measured at all t ime points and it varied between 5 and 10 nmol/mg protein throughout the culture period. Initially the medium did not contain significant concentrations of either GSH or GSSG but throughout the culture period (1-96 h) both GSH and GSSG were present in the culture medium.

At 24 h in culture there was no difference in the ability of cultured cells from control rats to synthesise GSH after depletion of intracellular GSH stores by DEM, compared with fresh cells in suspension (Fig. 2). Four hours after incubation with DEM the control cells had recovered the intracellular GSH to within physiological levels. Cultured cells from PB treated rats appeared to have a slower rate of synthesis of GSH than cells in suspension. It required 6 h incubation after the removal of DEM for the concentrations of GSH in cultured cells to recover compared with only 4 h incubation in cells in suspension.

The GSH conjugation of BPDE was measured in control cells in suspension and at 3 and 24 h after culture. These results are shown in Table I. Despite

50-

¢--

(3L cm30 E

t"

T

~D 10

(a) control

! i I

2 4 6

50- (b) Phenobarbitone

o

30 //~

incubation time, hours

Fig. 2. GSH levels i n f reshly isolated cells ( ~ - - © ) and 2 4 h cul tured cells ( ~ - - A ) af te r t r e a t m e n t wi th 0.01% DEM. Cells were exposed to DEM for 30 rain. The m e d i u m was then replaced (t ime = 0) and the cells incubated to allow recovery of GSH. Control resu l t s are the m e a n of 4 de te rmina t ions and the da ta f rom PB t rea ted ra t s are the m e a n of 2 de terminat ions .

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TABLE I

G L U T A T H I O N E C O N J U G A T I O N OF BPDE

Each resul t is the mean of 2 de terminat ions . Incubat ions contained 20 ~M [14C]BPDE and were carried out for 30 rain at 37°C.

nmol GSH conjugate/ml

F re sh cell suspens ions 9.7 F resh cell suspens ions a + DEM 6.0 3-h cul ture b 8.5 3-h cu l ture b + DEM 5.3 24-h cul ture b 9.4 24-h cul ture b + DEM 5.1

a 1 × 106 cells/ incubation. b 0.5-1 × 106 cells/ incubation.

the decrease in intracellular GSH concentrations observed in control cells between 1 and 4 h after culture, the conjugation system did not seem to be impaired. After 3 h in culture GSH conjugation of BPDE was decreased by only 12% compared with that of fresh cells. There was no difference between GSH conjugate synthesis by fresh cells and by cultured cells after 24h. Trea tment with DEM caused a 40% decrease in the synthesis of the GSH conjugate in each case.

DISCUSSION

Primary cultures of hepatocytes are being increasingly utilized as model systems for detection of drug genotoxicity [20-23]. It is important tha t in vivo activities of the detoxification reactions are maintained in culture, however, there have been few investigations into the activity of these reactions in cultured cells.

The results of the present study demonstrate tha t intracellular concen- trations of GSH can be maintained within physiological levels for up to 96 h in culture. However, Allen et al. [16] have reported decreased GSH concen- trat ions in hepatocytes 24 h after culture in medium containing no cystine and 0.2 mM methionine compared with medium containing 0.2 mM cystine and 0.2 mM methionine. They found that increasing the methionine concen- trat ion to 0 .5mM resulted in maintenance of GSH concentrations in the absence of cystine. In the present study maintenance of GSH was ac- complished at 0.25raM methionine in the absence of cystine. Reed and Beatty [18] found tha t methionine was a more efficient precursor of GSH, in hepatocyte suspensions than was cystine. Whether or not this also holds for hepatocytes in culture has still to be determined. Summer and Wiebel [24] have shown tha t continuous cell lines of mammal ian origin contain appreci- able amounts of GSH and express GSH transferase activities to varying extents. Decad et al. [25] have reported tha t the covalent binding of aflatoxin

241

B1 to cultured rat and mouse hepatocytes was increased 8-fold after DEM treatment. This demonstrates the influence of GSH availability in cultured cells on the toxicity of potential carcinogens. In the present study the concentration of GSH was found to fall to low levels between 1 and 4 h after culture. The reason for this is unclear but it would result in increased susceptibility to toxicity at this time and it is notable that there are a number of reports in the l i terature recently of genotoxicity studies carried out 1.5 h after culturing liver cells [26-28].

In the present study the decreased GSH concentrations in the early hours of culture cannot be explained by a concomitant rise in GSSG levels either intracellularly or in the medium. PB t rea tment of rats resulted in a decreased level and synthesis of GSH in the present experiments. There may be a rise in the activity of ~-glutamyltranspeptidase (~GT) shortly after culturing the cells. Edwards [29] has investigated the regulation of ~-GT in rat hepatocyte cultures and found a progressive rise in y-GT activity over several days in culture. However, he did not measure the enzyme activity before 24 h in culture.

Despite the decrease in intracellular GSH concentrations observed in cultured cells, the formation of BPDE,GSH conjugates in control cells after 3 h in culture was only 12% less than that in cells in suspension. Cultured cells from PB treated rats exhibited decreased levels and synthesis of GSH. Therefore any impairment in the GSH conjugation system is likely to be emphasized in these cells and the toxicity to xenobiotics may be potentiated.

ACKNOWLEDGEMENTS

This work was supported by NIH (Grant no. 1RC 1CA 26261-01) and by Swedish Cancer Society. The Cancer Research Program of the National Cancer Inst i tute (Bethesda, MD) is gratefully acknowledged for supplying the benzo[a]pyrene derivatives. The authors wish to thank Dr. J.R. Dawson for assistance with HPLC analyses of GSH and GSSG and Dr. J. Robertson for helpful comments on the manuscript.

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