Arylamine N-acetyltransferase activities in cell lines of mouse, rat, hamster and man differing in their sensitivity to 1,6-dinitropyrene

Toxicology Letters, 54 (1990) 71-76

71

Elsevier

TOXLET 02435

Arylamine IV-acetyltransferase activities in cell lines of mouse, rat, hamster and man differing in their sensitivity to 1,6-dinitropyrene

AK. Srivastava and F.J. Wiebel GSF-Institute of Toxicology, Gesellschaft fiir Strahlen- und Umweltforschung. Neuherberg (F.R.G.)

(Received 30 March 1990) (Revision received 4 June 1990) (Accepted 6 June 1990) Key words: N-Acetyltransferase;

Mammalian cell lines; Nitroaromatic compounds

SUMMARY This study was aimed at monitoring N-acetyltransferase activities of continuous cell lines, which differ in their sensitivity to the toxic effects of nitroaromatic compounds. Transferase activities were measured toward the acetyl acceptors sulfamethazine and p-aminobenzoic acid in partially purified preparation of cytosols. Cell lines such as hamster V79, BHK, rat hepatoma H4IIEC3G- or fibroblast 208F, which are sensitive to 1,6-dinitropyrene (1,6-DNP), possess high transferase activities ranging from 120-270 nmol/ min x mg protein. In contrast, human lung cells NCI-H322, mouse and rat hepatoma cells BWlJ and H5, respectively, which are resistant to 1,6-DNP contain no or low transferase activity of less than 15 nmol/min x mg. There was no apparent correlation between 1,dDNP sensitivity and acetyltransferase levels in a few cell lines, e.g. rat hepatoma HTC, 2sFou and 5L, which express intermediate transferase activities ranging from 25-50 nmol/min x mg protein. The results suggest that acetylation is an essential step in activating 1,6-DNP to toxic products in mammalian cells.

INTRODUCTION

Recent observations have shown that mammalian cell lines derived from various species and tissues differ greatly in their susceptibility to the toxic effects of nitropyrenes, notably 1,6_dinitropyrene (1,6-DNP) [l-4]. Analysis of the metabolism of 1,6DNP in various cell lines suggested that 1,6-DNP-sensitive lines, but not 1,6-DNPinsensitive lines, formed appreciable amounts of acetylated products [5]. Since both

Address for correspondence: Dr. F.J. Wiebel, GSF-Institute

of Toxicology, D-8042 Neuherberg, F.R.G.

037%4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

72

sensitive and insensitive lines were capable of reducing 1,6-DNP to its amino derivatives, it appeared possible that the different susceptibility is attributable to the presence or lack of acetyltransferases. Acetylation is thought to be a necessary step in activating carcinogenic arylamines [6-S]. Acetylation has also been shown to be involved in activating nitroaromatic compounds in bacterial test systems [9]. The cytosolic enzyme capable of acetylating arylamines has been found in tissues of several mammalian species [ 1O-121. To date, very little is known about the expression of acetyltransferase(s) in cells in continuous culture. In the present study we establish the conditions for measuring arylamine N-acetyltransferase (NAT) activity in preparations of cultured cells and monitor NAT activities in a series of cell lines which are of interest because of their greatly differing sensitivity to 1,6-DNP. MATERIALS

AND METHODS

Materials

Ammonium sulfamate, acetyl coenzyme A (AcCoA), p-aminobenzoic acid (PABA), sulfamethazine (SMZ), sodium nitrite, N-naphthyl ethylene diaminodihydrochloride, DEAE cellulose and Sephadex G-100 were obtained from Sigma Miinchen, F.R:G. The sources of culture media, antibiotics, calf serum, trypsin and plastic tissue culture plates have been described previously [ 131. Cell culture conditions

The origin and sources of the cell lines are given in Table I. All cell lines except BWlJ, 2sFou, H41IEC3G- and HepG2, were grown on 100 mm dishes in Dulbecco’s Minimum Eagle’s Medium containing 10% fetal bovine serum, 100 units/ml penicillmand 100 pg/ml streptomycin at 37°C in a humified atmosphere enriched with 7% COz. BWlJ, 2sFou, H4IIEC3G- and HepG2 cells were maintained in Ham’s F 12:NCTC 135 (1: 1) medium with 5% fetal bovine serum. Enzyme assay

Cells were used for determination of in vitro NAT activity 3-5 days after plating when they approached confluency. Cells were washed twice with ice-cold Dulbecco’s phosphate-buffered solution (pH 7.4; 0.1 M) and collected by scraping. NAT was partially purified according to Weber [14] using minor modifications. Briefly, the procedure involves precipitation with saturated ammonium sulfate (45-655&), gel filtration on Sephadex G-100, and ion exchange chromatography on DEAE cellulose. The reaction mixture for assaying NAT activity towards SMZ contained 200 nmol AcCoA and 100 nmol SMZ, toward PABA 4 pmol AcCoA and 2 pmol PABA in 1 ml of phosphate buffer (pH 7.4; 0.1 M). Reactions were started by addition of 0.1 ml of enzyme preparations containing 50-150 pg protein, carried out at 37°C with shaking for 15 min and stopped by addition of 2 ml 5% TCA. After centrifugation

73

5 0, Q

300

250

E” x .G E >

200

150

E z ,x ‘5 .-

100

50-

%

/

0-e

G Z

0

10

20 Fraction

Fig. 1. DEAE cellulose chromatography mental procedure,

see Materials

30

40

number

of NAT activity

toward

SMZ in cytosol

of V79 cells. For experi-

and Methods.

at 3000 rpm for 15 min, supernatants were taken for determination of free SMZ and PABA, according to Weber and Cohen [15] and Meisler and Reinke [16], respectively. NAT activities are expressed as nmol substrate acetylated/min x mg protein. At 37°C the reactions were linear up to 200 lug/ml cellular protein and 15 min of incubation. Cellular protein was determined following the method of Lowry et al. [17] using bovine serum albumin as standard. RESULTS

AND DISCUSSION

General properties

Preliminary experiments indicated that cytosol of cultured mammalian cells contains some component(s), possibly ‘esterases’, which interfere with the measurement of NAT activities. In order to eliminate these component(s), we partially purified the transferase(s) by column chromatography. Figure 1 depicts the elution profile of NAT activity in a preparation of V79 cells. Peak NAT activity appeared in fractions 14-18 exhibiting a shoulder in fractions 19-22. Similar elution profiles were found for the other cell lines tested. The pattern of elution was identical for acetylase activities toward PABA and SMZ in all cell lines. Deacetylase activities toward cr-naphthyl acetate eluted after fraction 45 (data not shown) and were thus well separated from NAT-containing fractions. Expression of NAT in continuous cell lines

Table I shows the peak activities of SMZ and PABA acetylation in 16 cell lines derived from various tissues and species. The activities ranged from 6 to 270 nmol/ min x mg protein. NAT was detectable in all cell lines, except for certain variants

74 TABLE I NAT ACTIVITIES TOWARD SMZ OR PABA IN VARIOUS CELL LINES Cell line’

Sensitivity

to 1,6-DNP”

NAT activity (nmol/min x mg protein) SMZ PABA

SMZ/PABA

Human

[I] NCI-H322, lung tumor [2] HepG2, hepatoma

13.6 + 11.6 k

4.4b 2.1

14.7 + 12.4 +

5.7 2.9

0.9 + 0.4 0.9 + 0.4

226.8 k 17.6 co.5 co.5 co.5 41.2 + 20.1 148.5 + 29

1.2 f 0.1

Hamster

[3]V79, lung [4] V79/r-1,6-DNPld [S] V79/r- 1,6-DNP2d [6] V79/r- 1,6-DNP3d (71CHO, ovary [8] BHK, kidney

++++ + ++

270.5 + 6.4 co.5 co.5 co.5 56.9 f 23.3 177.9 & 27.8

1.4 f 1.1 1.2 + 0.4

Mouse

[9] BWIJ, hepatoma

_

6.0 +

3.9

7.7 +

3.3

11.4 * 43.7 f 33.1 * 43.5 f 121.9 k 25.6 & 123.4 +

4.3 20.2 14.4 7.4 19.1 5.8 16.2

8.8 f 22.4 + 23.9 f 36.6 f 135.9 f 30.4 f 116.9 &

3.7 11.9 5.9 23.3 13.7 13.9 24.8

0.8 + 0.2’

Rat

[lo] H5, hepatoma [I I] SL, hepatoma [12] SL/r-1,3-DNPI’ [ 13]2sFou, hepatoma [14] H4IIEC3G-, hepatoma [ 151HTC, hepatoma [16] 208F, fibroblast

+ f ++ +++ ++ ++

1.3 + 1.9 & 1.4 f 1.2 f 0.9 f 0.8 + 1.1 *

0.4 0.9 0.9 0.1 0.5 0.4 0.1

a From Roscher et al. [4]; number of plus signs denotes the severity of cytotoxic effects; + = very weak effects; - = no effect. b Mean f SD of 3 separate experiments. c Cell lines were obtained from the following sources: [I] Dr. H. Schuller, Dept. Pathobiol., COB. Vet. Med., Knoxville, TN, U.S.A.; [2] Dr. B. Knowles, Wistar Institute, Philadelphia, PA, U.S.A.; [3,7,8,15] Flow Laboratories, Meckenheim, F.R.G.; [9] Dr. G. Szpizer, Univ. Libre, Rhode St. Gezere, Belgium; [l&14] Dr. M. Weiss, Institut Pasteur, Paris, France; [16] Dr. J. Schmidt, Dept. Mol. Cell Pathol., GSF, Neuherberg, F.R.G. d Selected from V79 cells by their resistance to 1,6-DNP. e Selected from 5L cells by their resistance to 1,3-DNP. ‘Significantly (P< 0.05; Student’s t-test) different from ratios in [3,10,12,16].

of V79 cells. Since NAT activities of cultured cells were measurable only after partial purification, involving not yet accountable losses of enzyme protein, the specific NAT activities given in Table I have to be viewed as relative values. The ratio of NAT activities toward SMZ and PABA differed to some extent in

the various cell lines. In the majority of lines, rates of SMZ acetylation were somewhat higher than those of PABA acetylation. The different ratios of SMZ/PABA acetylation might be attributable to species or tissue specificity of a single NAT form which predominate in the test cell lines. They might also be due to the expression of two or more forms of NAT which vary in their relative amounts. Recently, the existence of at least 2 forms of NAT possessing distinct substrate specificities has been demonstrated in a number of tissues from rodents and man [ 18,191. A comparison of previous results on the cytotoxicity of 1,6-DNP in various cell lines [4] with the present findings shows that in 12 out of 16 cell lines the levels of NAT roughly correlate with the cytotoxicity of 1,6-DNP. V79, which exhibited the highest sensitivity toward 1,6-DNP, also contained the highest transferase activity. H4IIEC3G-, 208F, BHK and CHO cells, which are somewhat less sensitive, expressed lower NAT activities. The 1,6-DNP-insensitive lines NCI-H322 and BWlJ contained very little NAT activity. Furthermore, the 3 variants of V79, which had been selected by their resistance to the cytotoxicity of 1,6-DNP, failed to show detectable NAT activity. In a few cell lines, e.g. HTC, 2sFou or 5L, NAT activity was not associated with cytotoxicity. For example, NAT levels of 5L cells and their variants SL-r-1,3-DNPl, which are virtually insensitive to 1,6-DNP, were similar to those of HTC and 2sFou cells which are sensitive. The discrepant results might be due to the fact that 1,6-DNP has to undergo a series of reductions before it becomes a substrate for acetylases. The reductive steps are unlikely to be the same in such diverse cells types as those used in the present study. In fact, as shown earlier [5], the test cell lines differ considerably in their expression of reductases thought to be involved in reduction of nitroaromatic compounds. In addition, in vitro NAT activities do not necessarily predict the level of acetylated products in intact cells. Since the steady-state level of acetylated products is determined by the rates of acetylation and deacetylation, the latter reaction will be another important determinant for the toxicity of 1,6-DNP. Previous observations indicate that the level of esterases varied to a great extent in the cell lines tested [20]. In view of the multiplicity of reactions involved in the activation and inactivation of 1,6-DNP, and the semiquantitative nature of the specific NAT activities measured, it is not surprising that there is no strict correlation between NAT activities and 1,6-DNP toxicity in the test cell systems. However, it should be noted that a lack of NAT - or the presence of only very low activities - was invariably associated with high resistance of cells to the toxic effects of 1,6-DNP. In conclusion, the results suggest that acetylation is a necessary step in the metabolic activation of 1,6-DNP to toxic products in mammalian cells. ACKNOWLEDGEMENTS

We thank Mr. F. Kiefer for his excellent technical assistance. We also gratefully acknowledge Ms. K. Nougher’s expert secretarial help.

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