Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives

Cancer Letters, 4 (1978} 69--75 © Elsevier/North-Holland Scientific Publishers Ltd.

69

FURTHER IMPROVEMENTS IN THE HEPATOCYTE PRIMARY CULTURE DNA REPAIR TEST FOR CARCINOGENS: DETECTION OF CARCINOGENIC BIPHENYL DERIVATIVES

GARY M. WILLIAMS Naylor Dana Institute for Disease Prevention, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 (U.S.A.)

(Received 6 October 1977) (Revised version received 28 October 1977) (Accepted 22 November 1977)

SUMMARY

DNA repair in hepatocyte primary cultures was induced by simultaneous treatment with the carcinogen and [3H] thymidine for 18 h beginning immediately after attachment of cells. The unscheduled DNA synthesis elicited by carcinogens was determined by counting grains with an automatic grain counter. In 5 biphenyl derivatives, a correlation was found between carcinogenicity and the ability to induce DNA repair. Hence, the method offers promise as a means of screening chemical carcinogens.

INTRODUCTION

The measurement of DNA repair as unscheduled DNA synthesis (UDS) in cultured fibroblasts has been explored by Stich and associates as a screen for chemical carcinogens [9,11]. The potential of hepatocyte primary cell (HPC) cultures for such screening has been described [ 12,13]. HPC cultures provide 2 important advantages: the capability for procarcinogen activation and the virtual absence of replicative DNA synthesis to complicate the measurement of UDS. It has been demonstrated that a wide variety of procarcinogens will elicit autoradiographic UDS in HPC while noncarcinogenic analogs are inactive [14]. A preliminary report Michalopoulos et al. [5] also describes for hepatocyte preparations the stimulation by a variety of carcinogens of UDS measured by isolation of DNA. With these substantial indications of the responsiveness of such cultures to carcinogens, it becomes imperative to optimize and deliAbbreviations: HPC, hepatocyte primary cell; HU, hydroxyurea; TdR, thymidine; UDS, unscheduled DNA synthesis.

70 neate the sensitivity of the system and to validate its specificity for carcinogens or mutagens. The present report describes improvements in the protocol for the detection of autoradiographic UDS in HPC and the perfect correlation of the ability to elicit UDS and carcinogenicity in a series of biphenyl derivatives. In the development of this system, it was found that sensitivity was increased by simultaneous exposure to the carcinogen and [3H]thymidine (TdR) for utilization in repair synthesis [ 1 4 ] . This observation formed the basis for a modified protocol [10] reported in detail herewith in which simultaneous exposures are c o m m e n c e d at the end of the 1.5 h attachment interval [3] and continued overnight. This protocol permits the use of freshly isolated hepatocytes over a long interval for metabolic activation and the occurrence of DNA repair and allows convenient scheduling of procedures. The use of an automatic grain counter made measurement of UDS simpler and more precise. The measurement of repair synthesis b y acid-precipitable counts, although more rapid, is shown to be less sensitive than the measurement of autoradiographic UDS. MATERIALS AND METHODS HPC cultures Liver cells were obtained by dissociation procedures [15] that regularly yielded over 200 X 106 cells per 1 0 0 g b o d y wt. that were 79% viable. A 1.5 h attachment interval [3] resulted in a 42% attachment of hepatocytes over 90% viable, and produced primary cultures representing at least 30% of the liver [151. HPC culture test for UDS Simultaneous exposure to the test c o m p o u n d and [3H] T d R was commenced at the end of the 1.5 h attachment interval by washing the cultures with serumfree medium E [15] and feeding them serum-free medium containing the additions. Eighteen h later, the cultures were washed 3 times at 0.5 h intervals with medium containing I mM TdR. Following this, cells were processed and autoradiographs were prepared as previously described [ 13,14]. The amount of UDS was determined from nuclear and background grain counts made on a Count All Model 600 colony counter (Fisher Scientific Co, Springfield, New Jersey) with microse%pic attachment. Random counting was begun with the first cell visualized on the video monitor and continued with consecutive cells until a significant number of source and background grains were counted for a coefficient of variation of 0.05 [8]. Nuclear counts were obtained b y focusing the aperture over the nucleus, adjusting the focus and threshold until a white d o t indicating detection b y the instrument coincided with each grain, and then recording the digital count. Cytoplasmic counts of areas of the same size were made b y moving the aperture to 3 adjacent cytoplasmic locations. Net nuclear counts of UDS were calculated b y subtracting

71 the highest cytoplasmic background count from the nuclear count for each cell. A range o f doses was tested in each experiment, but only the result for the optimal dose are reported. Each dose was applied to triplicate coverslips and the data are the average + the standard deviation. DNA synthesis measurement 10 uCi/ml [3H] TdR was applied to cultures for 18 h. The cells were then detached into saline and the suspension adjusted to 10% trichloroacetic acid on ice. The acid-insoluble precipitate was sedimented by centrifugation at 3000 × g for 15 mins. The precipitate was collected and washed as previously described [14] and hydrolysed in 0.5 N perchloric acid. DNA was measured by the method of Burton [1] on the hydrolysate. Aliquots of the hydrolysed precipitates were counted in 10 ml of toluene-based scintillation fluid, and the supernatant in triton-containing scintillation fluid. Chemicals The sources were as follows: hydroxyurea (HU), Calbiochem, East Rutherford, N.J. : biphenyl, Eastman Kodak Co., Rochester, N.Y.; 2-aminobiphenyl and 4-aminobiphenyl, Aldrich Chemical Co., Metuchen, N.J.; 2',3-dimethyl-4aminobiphenyl (dimethylaminobiphenyl) and p-diaminobiphenyl (benzidine), Carcinogenesis Standard Reference Compound Bank, National Cancer Institute, Bethesda, Md. The 2-aminobiphenyl was found to be about 5% contaminated with 4-aminobiphenyl by combined gas chromatography-mass spectrometry and, therefore, was purified by silica gel column chromatography with elution by benzene-hexane.

RESULTS Repeat assays revealed that the noncarcinogen biphenyl and 2-aminobiphenyl did not elicit UDS, whereas the carcinogens, 4-aminobiphenyl, dimethylaminobiphenyl and p-diaminobiphenyl were positive (Table 1). 2-Aminobiphenyl is presumed to be a noncarcinogen because its derivative, 2-acetylaminobiphenyl, is noncarcinogenic [6], but it remains to be tested. The UDS produced by carcinogens could also be measured by liquid scintillation counting as increased acid-precipitable counts (Table 2), but the background was high. The background could be reduced 2/3 by HU (Table 2), an inhibitor of normal replicative DNA synthesis, but the increase produced by dimethylaminobiphenyl remained proportionately the same (Table 2). The finding of such high acid-precipitable counts in the presence of HU suggested the possibility of retention of radioactivity other than through incorporation into DNA. Therefore, some investigations were made on the characteristics of the background, In cultures exposed to [3H] TdR only, the autoradiographic background was lowest in areas of the emulsion where no cells were present and was slightly, but not significantly, higher over the cytoplasm than the nucleus (Table 3). The finding of comparable backgrounds over the cyto-

-_c + + + --

Biphenyl 2-aminobiphenyl 4-aminobiphenyl 2',3-dimethyl-4-amiiaobiphenyl p-diaminobiphenyl (benzidine) DMSO c o n t r o l

10 -3 10 -4 10 -4 10 -4 10 -4

0.2 2.0 71.8 54.1 27.8 0.8

+ 0.3 + 1.9 + 36.1 + 19.3 + 3.7 + 0.7

UDSb grains/nucleus

(M)

10 -2, 10 -3, 10 -3, 10 -3, 10 -3, --

HPC c u l t u r e t e s t

Toxic concentrationa

a S e p a r a t e e x p e r i m e n t s in m o s t o f w h i c h m o r e t h a n 1 d o s e was e x a m i n e d . b E x c e p t f o r b i p h e n y l , t h e results r e p o r t e d are f r o m t h e s a m e e x p e r i m e n t . c U n t e s t e d ; p r e s u m e d t o b e n o n c a r c i n o g e n i c b e c a u s e a c e t y l derivative is n o n c a r c i n o g e n i c .

Carcinogenicity

Compound

HPC/UDS TEST RESULTS FOR BIPHENYL DERIVATIVES

TABLE I

1 0 -s 1 0 -5 --

10 -4 10 -s 10 -4

Concentration (M)

0

0 5 11 3 0

6 5 11 3 17

No. of times positive

4

No. of times testeda

b0

73 TABLE 2 DNA SYNTHESIS IN THE PRESENCE OF BIPHENYLS Treatment

Experiment 1

None Biphenyl (10 -s M) Dimethylaminobiphenyl (10 -5 M) Hydroxyurea (10 -3 M) Hyd'roxyurea (10 -3 M) + Biphenyl (10 -s M) Hydroxyurea (10 -3 M) + Dimethylaminobiphenyl (10 -5 M)

ExPeriment 2

cpm/pg DNA a

Control %

cpm/pg DNA a

Control %

2100 2300 2900

100 110 138

1400 1100 2100

100 79 150

700 500

100 72

400 400

100 100

1000

143

600

150

a Each value represents 3 pooled flasks.

p l a s m a n d t h e n u c l e u s is c o n s i s t e n t w i t h b i n d i n g to m a c r o m o l e c u l e s o t h e r t h a n DNA. DISCUSSION A m o d i f i e d p r o t o c o l f o r d e t e c t i o n o f U D S in H P C c o m b i n e d t h e f o l l o w i n g a d v a n t a g e s : (1) t h e use o f fresh liver cells; (2) a l o n g d u r a t i o n o f e x p o s u r e to p e r m i t g r e a t e r a c t i v a t i o n a n d r e s u l t i n g repair; (3) c o n v e n i e n t scheduling o f p r o c e s s i n g ; a n d (4) s i m p l i f i e d grain c o u n t i n g using an a u t o m a t i c c o u n t e r . Using this p r o t o c o l , a c o r r e l a t i o n w i t h k n o w n c a r c i n o g e n i c i t y was o b t a i n e d in t h e b i p h e n y l derivatives, a s s u m i n g t h a t 2 - a m i n o b i p h e n y l is n o n c a r c i n o g e n i c . T h e U D S p r o d u c e d b y c a r c i n o g e n s in H P C c u l t u r e s c o u l d also be d e t e r m i n e d b y m e a s u r e m e n t o f acid p r e c i p i t a b l e r a d i o a c t i v i t y , w h i c h is a f a s t e r m e a n s o f TABLE 3 BACKGROUND IN CONTROL AUTORADIOGRAPHS Experiment

1 2 3

Background (grains/nucleus sized areas) a

Emulsion

Cytoplasm

Nucleus

2.2 -+ 1.5 1.1 -+ 1.3 1.5 -+ 1.4

12.3 -+ 4.2 4.4 +- 2.1 15.1 -+ 2.8

10.3 -+ 5.0 3.1 -* 1.7 12.8 ± 2.7

a Average of 3 slides per experiment.

74

testing agents, but several complications exist. Although the percentage of cells in DNA synthesis is only about 0.05% [4], a substantial background of replicatire DNA synthesis exists against which to detect UDS. Unfortunately, the acid-precipitable counts were only reduced 2/3 by HU, so the sensitivity was not greatly enhanced; i.e., carcinogen-induced incorporation was still only 140--150% of background. This background is probably due to binding of TdR to macromolecules other than DNA [7] since the crude acid-precipitated material contained protein, RNA and lipids in addition to DNA or to transfer of the label [2]. Thus, the measurement of UDS by autoradiography affords greater specificity (i.e., cells engaged in normal replicative DNA synthesis can be completely excluded on the basis of the amount of nuclear labelling) and sensitivity. Therefore, for screening purposes, autoradiography appears to be a satisfactory approach. Examination of untreated cultures clearly showed the autoradiographic background to be due to cellular retention of radioactivity that could not be eliminated even by washes with medium containing nonradioactive TdR. Nevertheless, the cytoplasmic background was easily quantified by the automatic counter and could be subtracted from the nuclear grains for calculation of the induced UDS. Also, since background was higher over the cytoplasm than over the nucleus, subtracting cytoplasmic counts from nuclear counts to calculate net nuclear grains actually produces a slight underestimation of the UDS. ACKNOWLEDGEMENTS

This study was supported by Contracts NO1-CP-55705 from the National Cancer Institute and No. 68-02-2483 from the Environmental Protection Agency. I thank C. Patrianakos and Dr. D. Hoffman for analysing and purifying the 2-aminobiphenyl. The excellent technical assistance of E. Bermudez and D. Scaramuzzino and the manuscript preparation by S. Ippolito and B. Meyer is gratefully acknowledged. REFERENCES 1 Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62, 315--323. 2 Bryant, B.J. (1966) The incorporation o f tritium from thyrnidine into proteins of the mouse. J. Cell Biol., 29, 29--36. 3 Laishes, B.A. and Williams, G.M. (1976} Conditions affecting primary cultures of functional adult rat hepatocytes. I. The effect of insulin. In Vitro, 1 2 , 5 2 1 - - 5 3 2 . 4 Laishes, B.A. and Williams, G.M. (1976) Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro, 1 2 , 8 2 1 - - 8 3 2 . 5 Michalopoulous, G., Satler, G.L., O'Connor, D. and Pitot, H.C. (1977) Unscheduled DNA synthesis induced in hepatocellular suspensions and primary cultures of hepatocytes by procarcinogens. Proc. Am. Assoc. Cancer Res., 18,246.

75 6 Miller, E.C., Sandin, R.B., Miller, J.A. and Rusch, H.P. (1956) The carcinogenicity of compounds related to 2-acetylaminofluorene. III. Aminobiphenyl and benzidine derivatives. Cancer Res., 1 6 , 5 2 5 - - 5 3 4 . 7 Morley, C.G.D. and KingdOn, H.S. (1972) Use of 3H-thymidine for measurement of DNA synthesis in rat liver - - a warning. Anal. Biochem., 4 5 , 2 9 8 - - 3 0 5 . 8 Rodgers, A.W. (1973) Techniques of Autoradiography, pp. 356--357. Elsevier, Amsterdam, London and New York. 9 San, R.H.C. and Stich, H.F. (1975) DNA repair synthesis of cultured human cells as a rapid bioassay for chemical carcinogens. Int. J. Cancer, 1 6 , 2 8 4 - - 2 9 1 . 10 San, R.H.C. and Williams, G.M. (1977) The identification of chemical carcinogens using rat liver primary cell cultures for the detection of DNA repair and cell mediated mutagenesis. Proc. Am. Assoc. Cancer Res., 1 8 , 1 6 3 . 11 Stich, H.F., Kieser, D., Laishes, B.A. and San, R.H.C. (1973) The use of DNA repair in the identification of carcinogens, precarcinogens and target tissue. Proc. Can. Cancer Conf., 10, 125--170. 12 Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures; A possible screen for chemical carcinogens. In : Natl. Cancer Inst. Fourth Annu. Collaborative Conf., p. 173. 13 Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures; A possible screen for chemical carcinogens. Cancer Letters, 1 , 2 3 1 - - 2 3 6 . 14 Williams, G.M. (1977) Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res., 37, 1845--1851. 15 Williams, G.M., Bermudez, E. and Scaramuzzino, D. (1977) Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro, in press.