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DNA repair test in testing of coded carcinogens and noncarcinogens
Mutation Research, 97 (1982) 359-370 Elsevier Biomedical Press
359
Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens G.M. W i l l i a m s *, M . F . L a s p i a *+ a n d V.C. D u n k e l ** * Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, N Y 10595; and ** U.S. Food and Drug Administration, Bureau of Foods, Washington, DC 20204 (U.S.A.)
(Received 22 December 1981) (Revision received 16 March 1982) (Accepted 25 March 1982)
Summary The hepatocyte primary culture/DNA repair test was evaluated for its reliability using a series of coded samples. Among the 30 chemicals tested, 15 were general reference compounds and 15 were chemicals that had been tested for carcinogenicity in the U.S. National Cancer Institute Bioassay Program. The latter group were from the same lot that had been used for the in vivo testing and had also been tested for mutagenicity in the Ames test. From the group of 15 reference compounds, 5 were positive for DNA repair and all 5 were carcinogens. Of the 10 samples scored as negative, 4 were noncarcinogens and 6 were carcinogens. Among the 6 carcinogens were 3 compounds whose carcinogenicity probably does not involve the production of DNA damage. From the 15 coded chemicals that were tested for carcinogenicity by the NCI in long-term animal studies, 7 were scored as positive. 5 of these were judged carcinogenic in the in vivo bioassays and the other 2, which were also mutagenic in Salmonella, showed some indication of carcinogenicity. Of the 8 compounds that were scored as negative, 5 were noncarcinogenic. Among the 3 carcinogens that were not detected, there was at least one whose carcinogenicity probably does not involve DNA damage. Thus, the results of this study indicate that positive results in the hepatocyte primary culture/DNA repair test are highly specific for carcinogens and that the test is also highly sensitive in the detection of DNA-damaging genotoxic carcinogens.
÷ Current address: Department of Microbiology, New York Medical College, Valhalla, NY 10595. Abbreviation: HPC, hepatocyte primary culture. 0165-1161/82/0000-0000/$02.75 © Elsevier Biomedical Press
360
The hepatocyte primary culture ( H P C ) / D N A repair test (Williams, 1976, 1977, 1978) measures damage to DNA as repair synthesis in freshly isolated nonreplicating hepatocytes. This screening test for carcinogens combines intact cell metabolism in the cell typewith the broadest capability for xenobiotic biotransformation (Weisburger and Williams, 1982) together with a reliable biochemical end-point for genotoxicity (Dunkel and Williams, 1981). The autoradiographic light nuclear labeling of unscheduled DNA synthesis measured in the H P C / D N A repair test can be visually distinguished unequivocally from the dense nuclear labeling of replicative DNA synthesis seen only rarely in HPCs. This unscheduled DNA synthesis has been shown to represent true repair synthesis in parental DNA (McQueen and Williams, 1981), as in other hepatocyte systems (Andrae and Schwarz, 1981; Yager and Miller, 1978). Importantly, autoradiographic measurement of DNA repair permits the use of small samples of cells and allows determination of the fraction of cells which respond to a genotoxin. The H P C / D N A repair test has been shown to respond to carcinogens representing a wide variety of structural classes that require metabolic activation and to be negative for structurally related non carcinogens (Williams, 1980a, 1980b, 1981). It has also detected types of carcinogens that represented problems in bacterial mutagenicity assays (Williams and Laspia, 1979; Williams et al., 1980). The test has a defined protocol (Williams, 1980a) and has been duplicated in other laboratories (Bradlaw et al., 1981; Probst et al., 1981). An important aspect in the confirmation of the reliability of any screening test is its performance with samples of unknown identity. The H P C / D N A repair test reliably detected four coded samples of complex environmental materials (cigarette smoke condensate, roofing tar emission, diesel exhaust and coke oven emission) submitted by the U.S. Environmental Protection Agency (Ved Brat et al., 1982). We now describe the results of tests with coded compounds provided by the U.S. National Cancer Institute. These consisted of selected reference compounds and compounds that had been tested in long-term animal bioassays by the U.S. National Cancer Institute.
Materials and methods
Preparation of hepatocyte primary cultures HPCs are prepared from adult male F344 rats by the procedures of Williams et al. (1977). Conditions for the use of hepatocytes from mice, hamsters and rabbits have also been reported (Maslansky and Williams, 1981; McQueen et al., 1981, 1982). Perfusion is performed with a Bio-Fiber peristaltic pump (BioRad Labs, Richmond, CA). The tubing connected to the pump contains a bubble trap to prevent obstruction of small radicals of the portal vein during perfusion. The complete circulation system is cleaned with hot water and is sterilized by circulation of 70% ethanol followed by sterile water prior to perfusion. Animals are anesthetized with Nembutal Sodium Solution (Abbott Laboratories, North Chicago, IL) given at 50
361 mg/kg body weight i.p. A ventral midline incision is made from the xiphisternum to the pubic bone and the liver exposed. A 21-gauge butterfly needle is inserted in the portal vein and clamped in place with a serrafine forceps (Arista Surgical, New York, NY). Perfusion is then commenced with 200 ml of sterile Solution I at 37°C. This solution consists of 0.5 mM ethylene glycol-bis-(fl-aminoethyl ether) N-N'-tetracetic acid (EGTA) (Sigma, St. Louis, MO) in Ca 2÷ , Mg 2+ free Hanks' balanced salt solution buffered with l0 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (Calbiochem, San Diego, CA), and the pH adjusted to 7.35 with 1 N NaOH. The perfusion rate is maintained for 1.5 rain at 8 m/min. Immediately following the start of perfusion with Solution I, the subhepatic inferior vena cava is severed to permit the perfusate to escape, thereby preventing excessive swelling of the liver and accelerating the washout procedure. If the perfusion is adequate, uniform blanching of the liver should be evident. During this first perfusion, the thoracic inferior vena cava is cannulated by puncturing the fight atrium and the perfusate is collected via a return cannula. At this point, the proximal segment of the subhepatic inferior vena cava is clamped to close the system and the pump speed is then increased to 40 ml/min for 2.5 min. After completion of perfusion with Solution I, 250 ml of sterile Solution II at 37 ° is perfused through the liver. This solution contains 100 unit/ml type 1 collagenase (Sigma) in Williams medium E (WME) buffered with l0 mM Hepes, pH adjusted to 7.35. It is perfused for 10 min at 20 ml/min without recirculation of the return perfusate. During this perfusion the liver is covered with sterile gauze and a 40-W bulb is positioned 6 cm above the liver for warming. Following perfusion, the liver is removed to a sterile petri dish containing warm WME and is trimmed of extraneous fat and connective tissues. The liver is then removed to a fresh petri dish containing approx. 30-50 ml of Solution II. The portis hepatis is grasped with forceps and the capsule of the liver is opened at numerous points on the inferior surface with small scissors and removed. Cells are detached by gentle combing with a round tooth stainless steel comb and shaking off of loose cells. After combing completely, hepatocytes will be dissociated into a suspension leaving a fibrous plug of hepatic connective tissue which is discarded. Using a wide bore pipet,. 25-ml aliquots of the hepatocyte suspension are pipetted into 50-ml centrifuge tubes, and the volume brought to 50 ml per tube with WME supplemented with 10% calf serum and 50 #g/ml gentamycin (WMES). Cells are sedimented at 50 × g for 5 min, resuspended in WMES and gently mixed by inverting each tube several times. A 20-fold dilution of cell suspension is prepared and 0.5 ml is added to 0.1 ml of 0.4% trypan blue stain (Gibco, Grand Island, NY) and viability determined using a hemocytometer. This perfusion technique regularly produces average yields of greater than 200 × l 0 6 cells per 100 g body weight (Williams et al., 1977) with viabilities of greater than 90%. Only such preparations are used for the H P C / D N A repair assay. For H P C / D N A repair studies, suspensions containing 5 × l0 s cells/ml in WMES are immediately seeded onto 25-mm round Thermanox coverslips (Lux Scientific, Newbury Park, CA) in 35-mm 6-well dishes (Flow Labs, Hamden, CT) containing a final volume of 2.5 ml of WMES. The plates are placed in a 95% air, 5% CO 2
362 humidified 37°C incubator for 2 h. The medium is then removed and the coverslips are washed with 2 ml of WME leaving only attached viable cells.
HPC/DNA repair assay The HPC/DNArepair test is performed according to the methods developed by Williams (1976, 1977, 1978). After the 2h attachment period, washed HPCs are exposed to up to five concentrations separated by a factor of 5 or l0 of the test compound in 2 ml of serum-free WME containing l0 #Ci/ml (50 or in more recent studies 60-80 Ci/mM) tritiated thymidine ([3H]TdR; New England Nuclear, Boston, MA) for incorporation during repair synthesis. Additionally, appropriate positive eontrols, a solvent control, and an untreated cell control are run in parallel with the test sets. HPCs are incubated at 37°C for 18-20 h in the presence of the test compound and [3H]-TdR. Each coverslip is then removed from its well and washed by dipping it 5 times in each of 3 successive 100-ml washes of phosphate-buffered saline. Each coverslip is then immersed with the cell surface up in 2 ml of 1% Na citrate (Fisher Scientific, Springfield, N J) in a clean 6-weU dish for 10-15 rnin to allow the nuclei to swell to permit better quantification of nuclear grains. Finally, the cells are fixed in three changes of ethanol-glacial acetic acid (3:1) for 30 min each, air dried, and mounted cell surface up on glass slides with Permount (Fisher). Slides are placed in slide grip holders (Peel-a-way Scientific, South El Monte, CA) and in total darkness, dipped, into NTB-2 emulsion (Eastman Kodak, Rochester, NY), prewarmed for 30 rain at 45°C. Slides are dried overnight by suspending the slides, in their holders, from a rack in a light-tight box. Slides are placed in cardboard slide boxes and wrapped in foil and exposed at 4°C. After 7 days, slides are placed in staining racks and developed in D19 (Eastman Kodak) for 4 min, placed in a stop bath of acidified tap water containing a drop of glacial acetic acid for 30 sec, immersed in fixer (Eastman Kodak) for l0 min, washed in tap water for 5 min and air dried. Slides are stained in Harris' Alum hematoxylin (Harleco, Gibbstown, N J) followed by dipping successively in tap water, acid alcohol, tap water, ammonia water and tap water prior to counterstaining in eosin. The slides are then dehydrated throught 100% ethanol, air dried and coverslipped with Permount. The results of the H P C / D N A repair test are quantified by determining the production of nuclear grains of DNA repair synthesis by a test compound. Cells viable at the time of fixation are recognized by their swollen nuclei (as a result of incubation in the hypotonic sodium citrate), and only such cells evenly coated with emulsion are scored. S-phase cells engaged in replicative DNA synthesis are rare in HPCs (Laishes and Williams, 1976). They are recognized by uncountable dense nuclear grains and excluded from the scoring. An Artek Model 880 electronic counter with a microscopic attachment is used for grain counting. This counter, which can count in either a count or area mode, is operated in the area mode. In order to convert area units into grain counts, the count/area ratio is determined by counting several discrete grains in both modes. The conversion factor obtained is entered into a Ti59 calculator that has been programmed to determine a net grain count for each nucleus.
363 Nuclear counts of unscheduled DNA synthesis in viable cells are obtained in the area created by opening the instrument's aperture to coincide with the nuclear area, adjusting the focus and sensitivity until the perimeters of the grains or aggregates of grains are outlined in white indicating detection by the instrument. Cytoplasmic counts of areas of the same size are made by moving the aperture to positions adjacent to each counted nucleus. Digital counts are automatically recorded by an Artek Compu-print 700 and a Ti59 calculator. The counts in the area mode are converted to grain counts and net nuclear grain counts of repair synthesis are obtained by subtracting the highest cytoplasmic area count from the nuclear count. Generally, a minimum of 20 nuclei (50 in the set of reference compounds) in randomly selected cells are counted using some from each quadrant of the coverslip. The number of cells to be scored depends on the nuclear/cytoplasmic ratio of grains required to meet statistical significance (Rogers, 1973). Results of individual experiments are reported as the mean - standard deviation of the average net nuclear grain counts for three coverslips. The test compound is considered positive when the mean net nuclear grain count is statistically greater than that of the controls and above 5 grains per nucleus which is the upper limit of control values (Williams, 1977). Where possible, a dose response profile is developed for positive test compounds. The compound is considered negative in the assay if the net nuclear count is less than 5 at the highest soluble or non-toxic dose in an experiment in which the positive control compound displays its usual activity. Cytotoxicity of the test compound is determined in autoradiographs by the absence of S-phase cells and by general morphology, including pyknotic nuclei. These observations can be supported by studies on release of intracellular enzymes such as lactic dehydrogenase (Williams, 198 I). Coded chemicals Two groups of coded chemicals were provided by the U.S. National Cancer Institute for testing. The first group of 15 was selected from among reference compounds used for the validation of short-term tests. The second group of 15 was composed of compounds that had been tested for carcinogenicity in long-term animal-studies. The chemicals in this latter group were from the same batch used in the in vivo carcinogenicity tests. Each chemical was acquired from the laboratory that had performed the in vivo bioassay and was reanalyzed for purity and chemical stability by Midwest Research Institute, Kansas City, Missouri. Detailed information for each chemical can be found in the respective National Cancer Institute Carcinogenesis Technical report. All compounds were tested at least three times along with a positive control, usually 3,2'-dimethyl-4-aminobiphenyl.
Results
From the group of 15 coded reference compounds, 5 were positive in the H P C / D N A repair test and all of these showed a dose-response effect (Table 1). All
364 TABLE 1 RESULTS IN THE HPC/DNA Coded compound
Propylenimine
REPAIR TEST WITH CODED REFERENCE COMPOUNDS Result a
+
2-Methyl-4-dimethylaminoa~obenzene
+
4,4'-Methylenebis-(2-chloroaniline)
+
4-Dimethylaminoazobenzene
+
Benzo[ a ]pyrene
+
N a t u l a n hydrochloride
-
B e r y l l i u m sulfate tetrahydrate
-
1,2: 3,4-Diepoxybutane, D,L
-
Hydroxylamine hydrochloride
-
Diethylstilbestrol
-
p,p'-DDE
_ c
p-Rosaniline hydrochloride
-
Pyrene
_ c
1,2-Epoxybutane
Benzo[e]pyrene
-
_ c
Dose (mg/ml)
H P C / D N A repair b (grains/nucleus) Expt. l
Expt. 2
1.0 10 - I 10 - 2
Toxic 20.4±2.3 11.9±3.3
NR
1.0 10- l 10 - 2 1.0 10- l 10 - 2 10- l 10- 2 10 - 3 10-1 10 - 2 10.0 1.0 I0 - I 10.0 1.0 10 - l 3 × 10- l 3 X 10 - 2 3 × 10 - 3 2.0 1.0 10- l 10 - 2 10 - 3 10 - 4 2.0 1.0 10 -1 10- 2 10- 3 10 - 4 6 × 10 -1 10 -1 10 - 2 10.0 1.0 10 - I 10-1 10 - 2 10 - 3
Toxic 12.8 -+ 5.3 1.5±0.3 Toxic 18.9 ± 5.4 8.4±2.0 Toxic 11.1 ± 4.2 11.5 4 . 2 ± 1.1 1.4 ± 0.8 0.1±0.2 1.7 ± 1.4 0.0 Toxic 1.1 --+0.2 0.3±0.3 Toxic 0.9 ± 0.6 0.4±0.3 Toxic 0.7±0.3 0.0 3 . 3 ± 1.4 0.2±0.2 Toxic 2.7 ± 0.9 1.0±0.7 1 . 2 ± 1.1 0.5±0.5 0.7±0.1 0.1±0.1 1.2±0.6 0.1
Toxic 18.3 -+ 7.8 17.5±8.3 Toxic 32.6 --+ 1.7 Toxic 22.6 ± 2.4 8.8±2.1 14.0 ± 5.0 6 . 3 ± 1.5 Toxic 1.9 -+ 0.5 0.3±0.1 Toxic 0.5 -+ 0.4 0.5±0.1 Toxic 0.4±0.3 Toxic 1.2 0.2±0.2 Toxic 0.5-+0.2 1.8±0.4 1 . 8 ± 1.3 0.2±0.2 Toxic 2.3 ± 0.9 0.3±0.3 3.3+2.4 0.6±0.3 0.5±0.5 Toxic 1.3±0.9 1.0±0.5 1.2-- 1.0 0.3±0.1 0.0
a ÷ , i n d u c e d H P C / D N A repair; --, failed to i n d u c e H P C / D N A repair. b M e a n ± s t a n d a r d d e v i a t i o n of triplicate coverslips exposed 1.5 h after i n o c u l a t i o n of h e p a t o c y t e s to the test c o m p o u n d plus 3 H - T d R for 18 h. c N e g a t i v e at the highest non-toxic tested dose.
365 TABLE 2 CARCINOGENICITY AND MUTAGENICITY OF CODED REFERENCE CHEMICALS TESTED IN THE HPC/DNA REPAIR TEST Chemical
Carcino genicity
HPC/DNA repair
S. typhimurium
Mutagenicity
Benzo[a]pyrene 4-Dimethylaminoazobenzene 2-Methyl-4-dimethylaminoazobenzene
+ + +
+ + +
+ + +
4,4'-Methylene-bis-(2-ehloroaniline) Propyleneimine 1,2,3,4-Diepoxybutane,D,L Diethylstilbestrol
+
+
+
+ "4+
+ --
+ -~-
p,p'-DDE Natulan hydrochloride (procarbazine)
+ +
-
-
para-Rosaniline
+
--
--
Beryllium sulfate tetra_hydrate 1,2-Epoxybutane Hydroxylamine hydrochloride
+ -
-
-
Pyrene Benzole ]pyrene
-
-
+
a
a Data from McCann et al. (1975).
positive c o m p o u n d s proved to be carcinogens (Table2). With all 4 of the n o n carcinogens n o repair was i n d u c e d in the rat hepatocytes. The 6 r e m a i n i n g comp o u n d s which were negative i n the assay, i n c l u d e d such c o m p o u n d s as diethylstilbestrol, b e r y l l i u m sulfate a n d p , p ' - D D E which have n o t been d e m o n s t r a t e d to be genotoxic. I n tests with the set of coded chemicals that were assayed b y the U.S. N a t i o n a l C a n c e r Institute for carcinogenicity, 7 c o m p o u n d s were positive in the H P C / D N A repair test a n d 8 were negative (Table 3). Again, a d o s e - r e s p o n s e effect was evident for the positive c o m p o u n d s . 5 of the positive chemicals were reported as carcinogenic i n the in vivo bioassay (Table 4) while p - c h l o r o a n i l i n e was j u d g e d suspect a n d 3 - n i t r o p r o p i o n i c acid was associated with a n i n d i c a t i o n of carcinogenicity. 5 of the 8 negative c o m p o u n d s were n o n c a r c i n o g e n s a n d of the 3 that were carcinogenic, at least nitrilotriacetic acid appears n o t to be genotoxic.
TABLE 3 RESULTS IN THE H P C / D N A REPAIR TEST WITH C O D E D BIOASSAY C O M P O U N D S Coded compound
Result
a
H P C / D N A repair b Expt. 1
p-Chloroaniline
+
2-Amino-5 -nitrothiazole Michler's ketone (4,4'-bis(dimethylamino)benzophenone) 3-Nitropropionic acid
+ +
Ethylene dibromide
+
2-Nitroop-phenylene diamine
+/W
4,4'-Methyl-bis-( N', N-dimethyl)benzenamine 3-(Chloromethyl) pyridine hydrochloride 4-Nitro-o-phenylene diamine
+/W
p-Phenylene-diamine dihydrochloride Anilazine
4-Amino-2-nitrophenol N', N-Dicyclohexyl thiourea Nitrilotriacetic acid trisodium salt, monohydrate Lithocholic acid
- ¢
Expt. 2
Dose (mg/ml)
Grains/ nucleus
Dose (mg/ml)
Grains/ nucleus
5XIO 2 10 -2 5 )< 10 -3 5)< 10 - I 10-1 5)<10 -2 5 )< 10 -2 10 -2 5 X 10 -3 1.0 5)<10 -1 10 -1 10-2% 5)<10-3% 10-3%
5X10 -2 10 -2 5X10 -3 5 X I 0 -1 10 - I 5)<10 2 5)<10 -2 10 -2 5)<10 -3 5)<10 -~ 10 - I 5)<10 -2
Toxic 16.6±7.0 2.6-4"1.6 Toxic 10.8±3.9 5.5±1.1 Toxic 45.1±16.3 42.0±8.5 Toxic 8.8±12.5 4.4±5.2
5X10-3% 10-3%
1.0 5X10 - I 10 1 10-2 5X10 -3 5 X 10 -4 10 -2 5XIO -3 10-3 10-1 5 X 10 -2 10 -2 10-t 5XlO -2 10 -2 10 -2 5 X l 0 -3 10-3 10-2 5 X 10 -3 10 -3 10-1 5)<10 -2 1.0 5X 10 -1
Toxic 27.8± 14.3 4.3±3.3 Toxic 10.7±3.3 1.6±2.8 Toxic 43.0-4-16.3 53.6±24.3 Toxic 10.5-+2.3 7.1-4- 1.9 Toxic 24.2--+6.5 23.8±8.5 Toxic 5.2-+3.9 1.5±1.1 Toxic 5.2±5.4 0.1-4-0.1 Toxac 0.2-4-0.2 1.1±1.2 Toxm 0.0 0.5±0.5 Toxic 0.4 0.9±0.8 Toxic 0.0 0.2-+0.4 Toxic 0.5±0.8 0.1-+0.1 0.6-+0.5 0.6±0.5 0.0 1.9± 1.4
10 I 5 X 10 -2 1.0 5XlO -1
Toxic 28.3±7.2 Toxic 13.6-4-7.1 2.3±5.1 Toxic 10.8±5.7 2.3-4"3.1 Toxic 1.8±2.5 2.4±0.5 Toxic 0.6-4-0.5 0.5-4-0.5 Toxic 1.5±2.0 1.4 Toxic 0.8±0.7 0.9±0.4 Toxm 2.2-4-1.5 1.2±1.1 0.9±0.5 1.7±2.2 3.2±3.2 0.5±0.6
10- 2 5 X I 0 -3 10 -3
Toxic 0.1±0.2 1.0-----0.5
10 - 3 5X10 -4 10 - 4
Toxic 0.5±0.4 0.6--4-0.8
1.0 5×10 -l 10
l
10-2 5X10 -3 10-4 10-2 5 X I 0 -3 10-3 10-1 5 X 10 -2 10-2 5×10 2 10-2 5XIO 3 10 -2 5 × 1 0 -3 10 -3 10-2 5 X 10 -3 10 -3
a + , induced H P C / D N A repair; + / W , weakly induced H P C / D N A repair in one or both assays; - , failed to induce H P C / D N A repair. b M e a n ± s t a n d a r d deviation of triplicate coverslips exposed 1.5 h after exposure of hepatocytes to the test compound plus 3H-TdR for 18 h. c Negative at the highest tested dose.
367 TABLE 4 C A R C I N O G E N I C I T Y A N D M U T A G E N I C I T Y O F C O D E D BIOASSAY C H E M I C A L S T E S T E D IN T H E H P C / D N A R E P A I R TEST Chemical
Carcinogenicity
a
Rat b
2-Amino-5-nitrothiazole Michler's ketone (4,4'bis(dimethylamino)benzophenone) Ethylene dibromide
HPC/DNA repair
S. typhimurium
Mutagenicity e
Mouse
Male
Female
Male
Female
+ d +
_ +
_ +
_ +
+ +
+ +
+
+
+
+
+
+
2-Nitro-p-phenylene diamine 4,4'-Methyl-bis-( N', Ndimethyl)benzenamine Nitrilotriacetic acid trisodium salt
-
-
-
+
+/W
+
+
+
-
+
+/W
+/W
+
+
+
-
-
-
4-Amino-2-nitrophenol 3-(Chloromethyl) pyridine hydrochloride p-Chloroaniline
+ +
S -
+
+
-
+ +
S
--
S
S
+
+
+
+
3-Nitropropionic acid c N, N'-Dicyclohexyl thiourea Anilazine
. .
.
.
Lithocholic acid 4-Nitro-o-phenylene diamine p-Phenylenediamine dihydrochloride
. .
.
.
.
.
+
.
.
.
.
.
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a The chemicals tested for carcinogenicity, D N A repair and mutagenicity were all from the same lot. b Fisher F344 rats wer used in all bioassays except ethylene dibromide which was tested in O s b o r n e Mendel rats. c Contains a 5% impurity. d - , negative; + , positive; + / W , weak positive; S, suspect. e Data from Dunkel and Simmon (1980).
Discussion
In tests with coded response
for
carcinogens
compounds,
12 o f 3 0 c o m p o u n d s . (Tables
p-chloroaniline.
2 and
the HPC/DNA Of these
4); t h e o n l y e x c e p t i o n s
In the in vivo bioassays,
repair test registered
12, 10 p r o v e d
were 3-nitropropionic
3-nitropropionic
a positive
to be well-established acid, which
acid and
contained
a
368 5% contamination by a dimeric ester of 3-hydroxypropionic acid, was associated with a dose-related elevated incidence of hepatocellular neoplasms in male rats, but the evidence of carcinogenicity was considered inconclusive. The bioassay results with p-chloroaniline were judged to be suggestive of carcinogenicity based on the production of fibromas and fibrosarcomas of the spleen. Both compounds were also positive in the Ames test (Dunkel and Simmon, 1980) and thus may be carcinogens which were not detected under the conditions of the bioassays. If so, the activity in short-term tests illustrates how evidence for genotoxicity can be used to aid in the clarification of equivocal results obtained in animal testing (Weisburger and Williams, 1981; Williams and Weisburger, 1981). Moreover, the National Toxicology Program is currently retesting p-chloroaniline, which could prove to be unnecessary in light of the present data. Regardless, the specificity of a positive response to unknown compounds in the H P C / D N A repair test corresponds to the high specificity for carcinogens observed in the validation of this test (Williams, 1980a, 1980b, 1981). Of the 9 carcinogens that were negative, three, diethylstilbestrol, p,p'-DDE and nitrilotriacetic acid, have been suggested to be non-genotoxic carcinogens (Weisburger and Williams, 1980), and thus would not be expected to damage DNA. These were also nonmutagenic. Beryllium, as with other metal carcinogens, may be carcinogenic by decreasing the fidelity of DNA polymerases (Sirover and Loeb, 1976) and, likewise, also not directly damaging to DNA. 3 other carcinogens that were negative for DNA repair, 1,2:3,4-diepoxybutane, 4-amino-2-nitrophenol and 3-chloromethyl pyridine HC1, were all positive in the Ames test (Tables 2 and 4), indicating a greater sensitivity of this test for these types of compounds. However, the Ames test was also positive for 3 noncarcinogens. The remaining 2 carcinogens that were not detected in the rat H P C / D N A repair test, natulan and para-rosaniline, were also negative in the standard Ames test using rat liver $9, In separate studies, we have obtained positive results for natulan in mouse and hamster hepatocytes (McQueen and Williams, in preparation) and para-rosaniline was positive in the Ames test using hamster $9 (Dunkel, 1979). Thus the present negative results may reflect a species difference in sensitivity, as has been demonstrated for other carcinogens (Maslansky and Williams, 1981; McQueen et al., 1981). 12 compounds were positive in both the H P C / D N A repair test and the Ames test. 10 were definitely carcinogenic and the other two, 3-nitropropionic acid and p-chloroaniline, gave indications of carcinogenicity in bioassays. These findings support the suggestion (Weisburger and Williams, 1981; Williams, 1980a) that because of the complementary nature of the metabolism and end points of these two tests, a compound that is positive in both is virtually certain to be carcinogenic. In conclusion, we suggest that the reliability of the H P C / D N A repair test makes it suitable for a routine screening test.
Acknowledgement This research was supported by contract NOI-CP-55205 from the U.S. National Cancer Institute.
369
References Andrae, U., and L.R. Schwarz (1981) Induction of DNA repair synthesis in isolated rat hepatocytes by 5-diazouracil and other DNA damaging compounds, Cancer Lett., 13, 187-193. Bradlaw, J.E., J.W. Hauswirth and C.A. Thomas (1981) Induction of unscheduled DNA synthesis (UDS) in primary rat hepatocytes by some hair dye components, in: Program and Abstracts, 12th Annual Meeting of the Environmental Mutagen Society, 166. Dunkel, V.C. (1979) Collaborative studies on the Salmonella/microsome mutagenicity assay, J. Assoc. Off. Anal. Chem., 2, 874-882. Dunkel, V.C., and V.F. Simmon (1980) Mutagenic activity of chemicals previously tested for carcinogenicity in the National Cancer Institute Bioassay Program, in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Molecular and Cellular Aspects of Carcinogen Screening Tests, Lyon (IARC Scientific Publications No. 27), pp. 283-302. Dunkel, V.C., and G.M. Williams (1981) Biological significance of endpoints and in-vitro test systems for carcinogenicity and mutagenicity, in: C.R. Richmond, P.J. Walsh and E.D. Copenhauer (Eds.), Proceedings of the Third Life Sciences Symposium on Health Risk Analysis, Franklin Press, pp. 249-271. Laishes, B.A., and G.M. Williams (1976) Conditions affecting primary cultures of functional adult rat hepatocytes, II. Dexamethasone enhanced longevity and maintenance of morphology, In Vitro, 12, 821-832. Maslansky, C.J., and G.M. Williams (1981) Evidence for an epigenetic mode of action in organochlorine pesticide hepato-carcinogenicity. A lack of genotoxicity in rat, mouse, and hamster hepatocytes, J. Toxicol. Environ. Health, 8, 121-130. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. McQueen, C.A., and G.M. Williams (1981) Characterization of DNA repair elicited by carcinogens and drugs in the hepatocyte primary culture/DNA repair test, J. Toxicol. Environ. Health, 8, 463-477. McQueen, C.A., C.J. Maslansky, S.B. Crescenzi and G.M. Williams (1981) The genotoxicity of 4,4'-methylene-bis-2-chloroaniline in rat, mouse and hamster hepatocytes, Toxicol. Appl. Pharmacol., 58, 231-235. McQueen, C.A., C.J. Maslansky, I.B. Glowinsky, S.B. Crescenzi, W.W. Weber and G.M. Williams (1982) Relationship between the genetically determined acetylator phenotype and DNA damage by hydralazinc and 2-aminofluorene in cultured rabbit hepatocytes, Proc. Natl. Acad. Sci. (U.S.A.), 79, 1269-1272. Probst, G.S., R.E. McMahon, E.E. Hill, C.Z. Thompson, J.K. Epp and S.B. Neal (1981) Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: A comparison with bacterial mutagenicity using 218 compounds, Environ. Mutagen., 3, 11-32. Rogers, A.W. (1973) in: Techniques of Autoradiography, Elsevier, Amsterdam, p. 218. Sirover, M.A., and L.A. Loeb (1976) Metal-induced infidelity during DNA synthesis, Proc. Natl. Acad. Sci. (U.S.A.), 73, 2331-2335. Ved Brat, S., C. Tong and G.M. Williams (1982) Detection of genotoxic airborne chemicals in rat liver culture system, in: R. Tice, D. Costa and K. Schaich (Eds.), The Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 619-631. Weisburger, J.H., and G.M. Williams (1980) Chemical Carcinogens, in: J. Doull, C.D. Klaassen and M.O. Amdur (Eds.), Toxicology, the Basic Science of Poisons, 2nd edn., New York, pp. 84-138. Weisburger, J.H., and G.M. Williams (1981) Carcinogen testing; Current problems and approaches, Science, 214, 401-407. Weisburger, J.H., and G.M. Williams (1982) Metabolism of chemical carcinogens, in: F.F. Becker (Ed.), Cancer: A Comprehensive Treatise, 2nd edn., Plenum, New York, pp. 241-333. Williams, G.M. (1976) Carcinogen,induced DNA repair in primary rat liver cell cultures; a possible screen for chemical carcinogens, Cancer Lett., 1,231-236. Williams, G.M. (1977) The detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures, Cancer Res., 37, 1845-1851.
370 Williams, G.M. (1978) Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: Detection of carcinogenic biphenyl derivatives, Cancer Lett., 4, 69-75. Williams, G.M. (1980a) The detection of chemical mutagens/carcinogens by DNA repair and mutagenesis in liver cultures, in: F.J. de Serres and A. Hollaender (Eds.), Plenum, New York, pp. 61-79. Williams, G.M. (1980) The predictive value of DNA damage and repair assays for carcinogenicity, in: G.M. Williams, R. Kroes, H.W. Waaijers and K.W. van de Poll (Eds.), The Predictive Value of in vitro Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier/North-Holland, Amsterdam, pp. 213-230. Williams, G.M. (1981) Biochemical and biological indices of the toxicity of xenobiotics in liver cell cultures, in: S.S. Brown and D. Davies (Eds.), Proceedings of Symposium on Chemical Indices and Mechanisms of Organ Directed Toxicity, Pergamon, New York, pp. 131-145. Williams, G.M., and M.F. Laspia (1979) The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test, Cancer Lett., 6, 199-206. Williams, G.M., and J.H. Weisburger (1981) Systematic carcinogen testing through the decision point approach, Annu. Rev. Pharmacol. Toxicol., 21,393-416. Williams, G.M., E. Bermudez and D. Scaramuzzino (1977) Rat hepatocyte primary cell cultures, III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium, In Vitro, 13, 809-817. Williams, G.M., H. Moil, I. Hirono and M. Nagao (1980) Genotoxicity of pyrrolizidine alkaloids in the hepatocyte primary culture/DNA repair test, Mutation Res., 79, 1-5. Yager, LD., and J.A. Miller (1978) DNA repair in primary cultures of rat hepatocytes, Cancer Res., 38, 4385-4395.
359
Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens G.M. W i l l i a m s *, M . F . L a s p i a *+ a n d V.C. D u n k e l ** * Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, N Y 10595; and ** U.S. Food and Drug Administration, Bureau of Foods, Washington, DC 20204 (U.S.A.)
(Received 22 December 1981) (Revision received 16 March 1982) (Accepted 25 March 1982)
Summary The hepatocyte primary culture/DNA repair test was evaluated for its reliability using a series of coded samples. Among the 30 chemicals tested, 15 were general reference compounds and 15 were chemicals that had been tested for carcinogenicity in the U.S. National Cancer Institute Bioassay Program. The latter group were from the same lot that had been used for the in vivo testing and had also been tested for mutagenicity in the Ames test. From the group of 15 reference compounds, 5 were positive for DNA repair and all 5 were carcinogens. Of the 10 samples scored as negative, 4 were noncarcinogens and 6 were carcinogens. Among the 6 carcinogens were 3 compounds whose carcinogenicity probably does not involve the production of DNA damage. From the 15 coded chemicals that were tested for carcinogenicity by the NCI in long-term animal studies, 7 were scored as positive. 5 of these were judged carcinogenic in the in vivo bioassays and the other 2, which were also mutagenic in Salmonella, showed some indication of carcinogenicity. Of the 8 compounds that were scored as negative, 5 were noncarcinogenic. Among the 3 carcinogens that were not detected, there was at least one whose carcinogenicity probably does not involve DNA damage. Thus, the results of this study indicate that positive results in the hepatocyte primary culture/DNA repair test are highly specific for carcinogens and that the test is also highly sensitive in the detection of DNA-damaging genotoxic carcinogens.
÷ Current address: Department of Microbiology, New York Medical College, Valhalla, NY 10595. Abbreviation: HPC, hepatocyte primary culture. 0165-1161/82/0000-0000/$02.75 © Elsevier Biomedical Press
360
The hepatocyte primary culture ( H P C ) / D N A repair test (Williams, 1976, 1977, 1978) measures damage to DNA as repair synthesis in freshly isolated nonreplicating hepatocytes. This screening test for carcinogens combines intact cell metabolism in the cell typewith the broadest capability for xenobiotic biotransformation (Weisburger and Williams, 1982) together with a reliable biochemical end-point for genotoxicity (Dunkel and Williams, 1981). The autoradiographic light nuclear labeling of unscheduled DNA synthesis measured in the H P C / D N A repair test can be visually distinguished unequivocally from the dense nuclear labeling of replicative DNA synthesis seen only rarely in HPCs. This unscheduled DNA synthesis has been shown to represent true repair synthesis in parental DNA (McQueen and Williams, 1981), as in other hepatocyte systems (Andrae and Schwarz, 1981; Yager and Miller, 1978). Importantly, autoradiographic measurement of DNA repair permits the use of small samples of cells and allows determination of the fraction of cells which respond to a genotoxin. The H P C / D N A repair test has been shown to respond to carcinogens representing a wide variety of structural classes that require metabolic activation and to be negative for structurally related non carcinogens (Williams, 1980a, 1980b, 1981). It has also detected types of carcinogens that represented problems in bacterial mutagenicity assays (Williams and Laspia, 1979; Williams et al., 1980). The test has a defined protocol (Williams, 1980a) and has been duplicated in other laboratories (Bradlaw et al., 1981; Probst et al., 1981). An important aspect in the confirmation of the reliability of any screening test is its performance with samples of unknown identity. The H P C / D N A repair test reliably detected four coded samples of complex environmental materials (cigarette smoke condensate, roofing tar emission, diesel exhaust and coke oven emission) submitted by the U.S. Environmental Protection Agency (Ved Brat et al., 1982). We now describe the results of tests with coded compounds provided by the U.S. National Cancer Institute. These consisted of selected reference compounds and compounds that had been tested in long-term animal bioassays by the U.S. National Cancer Institute.
Materials and methods
Preparation of hepatocyte primary cultures HPCs are prepared from adult male F344 rats by the procedures of Williams et al. (1977). Conditions for the use of hepatocytes from mice, hamsters and rabbits have also been reported (Maslansky and Williams, 1981; McQueen et al., 1981, 1982). Perfusion is performed with a Bio-Fiber peristaltic pump (BioRad Labs, Richmond, CA). The tubing connected to the pump contains a bubble trap to prevent obstruction of small radicals of the portal vein during perfusion. The complete circulation system is cleaned with hot water and is sterilized by circulation of 70% ethanol followed by sterile water prior to perfusion. Animals are anesthetized with Nembutal Sodium Solution (Abbott Laboratories, North Chicago, IL) given at 50
361 mg/kg body weight i.p. A ventral midline incision is made from the xiphisternum to the pubic bone and the liver exposed. A 21-gauge butterfly needle is inserted in the portal vein and clamped in place with a serrafine forceps (Arista Surgical, New York, NY). Perfusion is then commenced with 200 ml of sterile Solution I at 37°C. This solution consists of 0.5 mM ethylene glycol-bis-(fl-aminoethyl ether) N-N'-tetracetic acid (EGTA) (Sigma, St. Louis, MO) in Ca 2÷ , Mg 2+ free Hanks' balanced salt solution buffered with l0 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) (Calbiochem, San Diego, CA), and the pH adjusted to 7.35 with 1 N NaOH. The perfusion rate is maintained for 1.5 rain at 8 m/min. Immediately following the start of perfusion with Solution I, the subhepatic inferior vena cava is severed to permit the perfusate to escape, thereby preventing excessive swelling of the liver and accelerating the washout procedure. If the perfusion is adequate, uniform blanching of the liver should be evident. During this first perfusion, the thoracic inferior vena cava is cannulated by puncturing the fight atrium and the perfusate is collected via a return cannula. At this point, the proximal segment of the subhepatic inferior vena cava is clamped to close the system and the pump speed is then increased to 40 ml/min for 2.5 min. After completion of perfusion with Solution I, 250 ml of sterile Solution II at 37 ° is perfused through the liver. This solution contains 100 unit/ml type 1 collagenase (Sigma) in Williams medium E (WME) buffered with l0 mM Hepes, pH adjusted to 7.35. It is perfused for 10 min at 20 ml/min without recirculation of the return perfusate. During this perfusion the liver is covered with sterile gauze and a 40-W bulb is positioned 6 cm above the liver for warming. Following perfusion, the liver is removed to a sterile petri dish containing warm WME and is trimmed of extraneous fat and connective tissues. The liver is then removed to a fresh petri dish containing approx. 30-50 ml of Solution II. The portis hepatis is grasped with forceps and the capsule of the liver is opened at numerous points on the inferior surface with small scissors and removed. Cells are detached by gentle combing with a round tooth stainless steel comb and shaking off of loose cells. After combing completely, hepatocytes will be dissociated into a suspension leaving a fibrous plug of hepatic connective tissue which is discarded. Using a wide bore pipet,. 25-ml aliquots of the hepatocyte suspension are pipetted into 50-ml centrifuge tubes, and the volume brought to 50 ml per tube with WME supplemented with 10% calf serum and 50 #g/ml gentamycin (WMES). Cells are sedimented at 50 × g for 5 min, resuspended in WMES and gently mixed by inverting each tube several times. A 20-fold dilution of cell suspension is prepared and 0.5 ml is added to 0.1 ml of 0.4% trypan blue stain (Gibco, Grand Island, NY) and viability determined using a hemocytometer. This perfusion technique regularly produces average yields of greater than 200 × l 0 6 cells per 100 g body weight (Williams et al., 1977) with viabilities of greater than 90%. Only such preparations are used for the H P C / D N A repair assay. For H P C / D N A repair studies, suspensions containing 5 × l0 s cells/ml in WMES are immediately seeded onto 25-mm round Thermanox coverslips (Lux Scientific, Newbury Park, CA) in 35-mm 6-well dishes (Flow Labs, Hamden, CT) containing a final volume of 2.5 ml of WMES. The plates are placed in a 95% air, 5% CO 2
362 humidified 37°C incubator for 2 h. The medium is then removed and the coverslips are washed with 2 ml of WME leaving only attached viable cells.
HPC/DNA repair assay The HPC/DNArepair test is performed according to the methods developed by Williams (1976, 1977, 1978). After the 2h attachment period, washed HPCs are exposed to up to five concentrations separated by a factor of 5 or l0 of the test compound in 2 ml of serum-free WME containing l0 #Ci/ml (50 or in more recent studies 60-80 Ci/mM) tritiated thymidine ([3H]TdR; New England Nuclear, Boston, MA) for incorporation during repair synthesis. Additionally, appropriate positive eontrols, a solvent control, and an untreated cell control are run in parallel with the test sets. HPCs are incubated at 37°C for 18-20 h in the presence of the test compound and [3H]-TdR. Each coverslip is then removed from its well and washed by dipping it 5 times in each of 3 successive 100-ml washes of phosphate-buffered saline. Each coverslip is then immersed with the cell surface up in 2 ml of 1% Na citrate (Fisher Scientific, Springfield, N J) in a clean 6-weU dish for 10-15 rnin to allow the nuclei to swell to permit better quantification of nuclear grains. Finally, the cells are fixed in three changes of ethanol-glacial acetic acid (3:1) for 30 min each, air dried, and mounted cell surface up on glass slides with Permount (Fisher). Slides are placed in slide grip holders (Peel-a-way Scientific, South El Monte, CA) and in total darkness, dipped, into NTB-2 emulsion (Eastman Kodak, Rochester, NY), prewarmed for 30 rain at 45°C. Slides are dried overnight by suspending the slides, in their holders, from a rack in a light-tight box. Slides are placed in cardboard slide boxes and wrapped in foil and exposed at 4°C. After 7 days, slides are placed in staining racks and developed in D19 (Eastman Kodak) for 4 min, placed in a stop bath of acidified tap water containing a drop of glacial acetic acid for 30 sec, immersed in fixer (Eastman Kodak) for l0 min, washed in tap water for 5 min and air dried. Slides are stained in Harris' Alum hematoxylin (Harleco, Gibbstown, N J) followed by dipping successively in tap water, acid alcohol, tap water, ammonia water and tap water prior to counterstaining in eosin. The slides are then dehydrated throught 100% ethanol, air dried and coverslipped with Permount. The results of the H P C / D N A repair test are quantified by determining the production of nuclear grains of DNA repair synthesis by a test compound. Cells viable at the time of fixation are recognized by their swollen nuclei (as a result of incubation in the hypotonic sodium citrate), and only such cells evenly coated with emulsion are scored. S-phase cells engaged in replicative DNA synthesis are rare in HPCs (Laishes and Williams, 1976). They are recognized by uncountable dense nuclear grains and excluded from the scoring. An Artek Model 880 electronic counter with a microscopic attachment is used for grain counting. This counter, which can count in either a count or area mode, is operated in the area mode. In order to convert area units into grain counts, the count/area ratio is determined by counting several discrete grains in both modes. The conversion factor obtained is entered into a Ti59 calculator that has been programmed to determine a net grain count for each nucleus.
363 Nuclear counts of unscheduled DNA synthesis in viable cells are obtained in the area created by opening the instrument's aperture to coincide with the nuclear area, adjusting the focus and sensitivity until the perimeters of the grains or aggregates of grains are outlined in white indicating detection by the instrument. Cytoplasmic counts of areas of the same size are made by moving the aperture to positions adjacent to each counted nucleus. Digital counts are automatically recorded by an Artek Compu-print 700 and a Ti59 calculator. The counts in the area mode are converted to grain counts and net nuclear grain counts of repair synthesis are obtained by subtracting the highest cytoplasmic area count from the nuclear count. Generally, a minimum of 20 nuclei (50 in the set of reference compounds) in randomly selected cells are counted using some from each quadrant of the coverslip. The number of cells to be scored depends on the nuclear/cytoplasmic ratio of grains required to meet statistical significance (Rogers, 1973). Results of individual experiments are reported as the mean - standard deviation of the average net nuclear grain counts for three coverslips. The test compound is considered positive when the mean net nuclear grain count is statistically greater than that of the controls and above 5 grains per nucleus which is the upper limit of control values (Williams, 1977). Where possible, a dose response profile is developed for positive test compounds. The compound is considered negative in the assay if the net nuclear count is less than 5 at the highest soluble or non-toxic dose in an experiment in which the positive control compound displays its usual activity. Cytotoxicity of the test compound is determined in autoradiographs by the absence of S-phase cells and by general morphology, including pyknotic nuclei. These observations can be supported by studies on release of intracellular enzymes such as lactic dehydrogenase (Williams, 198 I). Coded chemicals Two groups of coded chemicals were provided by the U.S. National Cancer Institute for testing. The first group of 15 was selected from among reference compounds used for the validation of short-term tests. The second group of 15 was composed of compounds that had been tested for carcinogenicity in long-term animal-studies. The chemicals in this latter group were from the same batch used in the in vivo carcinogenicity tests. Each chemical was acquired from the laboratory that had performed the in vivo bioassay and was reanalyzed for purity and chemical stability by Midwest Research Institute, Kansas City, Missouri. Detailed information for each chemical can be found in the respective National Cancer Institute Carcinogenesis Technical report. All compounds were tested at least three times along with a positive control, usually 3,2'-dimethyl-4-aminobiphenyl.
Results
From the group of 15 coded reference compounds, 5 were positive in the H P C / D N A repair test and all of these showed a dose-response effect (Table 1). All
364 TABLE 1 RESULTS IN THE HPC/DNA Coded compound
Propylenimine
REPAIR TEST WITH CODED REFERENCE COMPOUNDS Result a
+
2-Methyl-4-dimethylaminoa~obenzene
+
4,4'-Methylenebis-(2-chloroaniline)
+
4-Dimethylaminoazobenzene
+
Benzo[ a ]pyrene
+
N a t u l a n hydrochloride
-
B e r y l l i u m sulfate tetrahydrate
-
1,2: 3,4-Diepoxybutane, D,L
-
Hydroxylamine hydrochloride
-
Diethylstilbestrol
-
p,p'-DDE
_ c
p-Rosaniline hydrochloride
-
Pyrene
_ c
1,2-Epoxybutane
Benzo[e]pyrene
-
_ c
Dose (mg/ml)
H P C / D N A repair b (grains/nucleus) Expt. l
Expt. 2
1.0 10 - I 10 - 2
Toxic 20.4±2.3 11.9±3.3
NR
1.0 10- l 10 - 2 1.0 10- l 10 - 2 10- l 10- 2 10 - 3 10-1 10 - 2 10.0 1.0 I0 - I 10.0 1.0 10 - l 3 × 10- l 3 X 10 - 2 3 × 10 - 3 2.0 1.0 10- l 10 - 2 10 - 3 10 - 4 2.0 1.0 10 -1 10- 2 10- 3 10 - 4 6 × 10 -1 10 -1 10 - 2 10.0 1.0 10 - I 10-1 10 - 2 10 - 3
Toxic 12.8 -+ 5.3 1.5±0.3 Toxic 18.9 ± 5.4 8.4±2.0 Toxic 11.1 ± 4.2 11.5 4 . 2 ± 1.1 1.4 ± 0.8 0.1±0.2 1.7 ± 1.4 0.0 Toxic 1.1 --+0.2 0.3±0.3 Toxic 0.9 ± 0.6 0.4±0.3 Toxic 0.7±0.3 0.0 3 . 3 ± 1.4 0.2±0.2 Toxic 2.7 ± 0.9 1.0±0.7 1 . 2 ± 1.1 0.5±0.5 0.7±0.1 0.1±0.1 1.2±0.6 0.1
Toxic 18.3 -+ 7.8 17.5±8.3 Toxic 32.6 --+ 1.7 Toxic 22.6 ± 2.4 8.8±2.1 14.0 ± 5.0 6 . 3 ± 1.5 Toxic 1.9 -+ 0.5 0.3±0.1 Toxic 0.5 -+ 0.4 0.5±0.1 Toxic 0.4±0.3 Toxic 1.2 0.2±0.2 Toxic 0.5-+0.2 1.8±0.4 1 . 8 ± 1.3 0.2±0.2 Toxic 2.3 ± 0.9 0.3±0.3 3.3+2.4 0.6±0.3 0.5±0.5 Toxic 1.3±0.9 1.0±0.5 1.2-- 1.0 0.3±0.1 0.0
a ÷ , i n d u c e d H P C / D N A repair; --, failed to i n d u c e H P C / D N A repair. b M e a n ± s t a n d a r d d e v i a t i o n of triplicate coverslips exposed 1.5 h after i n o c u l a t i o n of h e p a t o c y t e s to the test c o m p o u n d plus 3 H - T d R for 18 h. c N e g a t i v e at the highest non-toxic tested dose.
365 TABLE 2 CARCINOGENICITY AND MUTAGENICITY OF CODED REFERENCE CHEMICALS TESTED IN THE HPC/DNA REPAIR TEST Chemical
Carcino genicity
HPC/DNA repair
S. typhimurium
Mutagenicity
Benzo[a]pyrene 4-Dimethylaminoazobenzene 2-Methyl-4-dimethylaminoazobenzene
+ + +
+ + +
+ + +
4,4'-Methylene-bis-(2-ehloroaniline) Propyleneimine 1,2,3,4-Diepoxybutane,D,L Diethylstilbestrol
+
+
+
+ "4+
+ --
+ -~-
p,p'-DDE Natulan hydrochloride (procarbazine)
+ +
-
-
para-Rosaniline
+
--
--
Beryllium sulfate tetra_hydrate 1,2-Epoxybutane Hydroxylamine hydrochloride
+ -
-
-
Pyrene Benzole ]pyrene
-
-
+
a
a Data from McCann et al. (1975).
positive c o m p o u n d s proved to be carcinogens (Table2). With all 4 of the n o n carcinogens n o repair was i n d u c e d in the rat hepatocytes. The 6 r e m a i n i n g comp o u n d s which were negative i n the assay, i n c l u d e d such c o m p o u n d s as diethylstilbestrol, b e r y l l i u m sulfate a n d p , p ' - D D E which have n o t been d e m o n s t r a t e d to be genotoxic. I n tests with the set of coded chemicals that were assayed b y the U.S. N a t i o n a l C a n c e r Institute for carcinogenicity, 7 c o m p o u n d s were positive in the H P C / D N A repair test a n d 8 were negative (Table 3). Again, a d o s e - r e s p o n s e effect was evident for the positive c o m p o u n d s . 5 of the positive chemicals were reported as carcinogenic i n the in vivo bioassay (Table 4) while p - c h l o r o a n i l i n e was j u d g e d suspect a n d 3 - n i t r o p r o p i o n i c acid was associated with a n i n d i c a t i o n of carcinogenicity. 5 of the 8 negative c o m p o u n d s were n o n c a r c i n o g e n s a n d of the 3 that were carcinogenic, at least nitrilotriacetic acid appears n o t to be genotoxic.
TABLE 3 RESULTS IN THE H P C / D N A REPAIR TEST WITH C O D E D BIOASSAY C O M P O U N D S Coded compound
Result
a
H P C / D N A repair b Expt. 1
p-Chloroaniline
+
2-Amino-5 -nitrothiazole Michler's ketone (4,4'-bis(dimethylamino)benzophenone) 3-Nitropropionic acid
+ +
Ethylene dibromide
+
2-Nitroop-phenylene diamine
+/W
4,4'-Methyl-bis-( N', N-dimethyl)benzenamine 3-(Chloromethyl) pyridine hydrochloride 4-Nitro-o-phenylene diamine
+/W
p-Phenylene-diamine dihydrochloride Anilazine
4-Amino-2-nitrophenol N', N-Dicyclohexyl thiourea Nitrilotriacetic acid trisodium salt, monohydrate Lithocholic acid
- ¢
Expt. 2
Dose (mg/ml)
Grains/ nucleus
Dose (mg/ml)
Grains/ nucleus
5XIO 2 10 -2 5 )< 10 -3 5)< 10 - I 10-1 5)<10 -2 5 )< 10 -2 10 -2 5 X 10 -3 1.0 5)<10 -1 10 -1 10-2% 5)<10-3% 10-3%
5X10 -2 10 -2 5X10 -3 5 X I 0 -1 10 - I 5)<10 2 5)<10 -2 10 -2 5)<10 -3 5)<10 -~ 10 - I 5)<10 -2
Toxic 16.6±7.0 2.6-4"1.6 Toxic 10.8±3.9 5.5±1.1 Toxic 45.1±16.3 42.0±8.5 Toxic 8.8±12.5 4.4±5.2
5X10-3% 10-3%
1.0 5X10 - I 10 1 10-2 5X10 -3 5 X 10 -4 10 -2 5XIO -3 10-3 10-1 5 X 10 -2 10 -2 10-t 5XlO -2 10 -2 10 -2 5 X l 0 -3 10-3 10-2 5 X 10 -3 10 -3 10-1 5)<10 -2 1.0 5X 10 -1
Toxic 27.8± 14.3 4.3±3.3 Toxic 10.7±3.3 1.6±2.8 Toxic 43.0-4-16.3 53.6±24.3 Toxic 10.5-+2.3 7.1-4- 1.9 Toxic 24.2--+6.5 23.8±8.5 Toxic 5.2-+3.9 1.5±1.1 Toxic 5.2±5.4 0.1-4-0.1 Toxac 0.2-4-0.2 1.1±1.2 Toxm 0.0 0.5±0.5 Toxic 0.4 0.9±0.8 Toxic 0.0 0.2-+0.4 Toxic 0.5±0.8 0.1-+0.1 0.6-+0.5 0.6±0.5 0.0 1.9± 1.4
10 I 5 X 10 -2 1.0 5XlO -1
Toxic 28.3±7.2 Toxic 13.6-4-7.1 2.3±5.1 Toxic 10.8±5.7 2.3-4"3.1 Toxic 1.8±2.5 2.4±0.5 Toxic 0.6-4-0.5 0.5-4-0.5 Toxic 1.5±2.0 1.4 Toxic 0.8±0.7 0.9±0.4 Toxm 2.2-4-1.5 1.2±1.1 0.9±0.5 1.7±2.2 3.2±3.2 0.5±0.6
10- 2 5 X I 0 -3 10 -3
Toxic 0.1±0.2 1.0-----0.5
10 - 3 5X10 -4 10 - 4
Toxic 0.5±0.4 0.6--4-0.8
1.0 5×10 -l 10
l
10-2 5X10 -3 10-4 10-2 5 X I 0 -3 10-3 10-1 5 X 10 -2 10-2 5×10 2 10-2 5XIO 3 10 -2 5 × 1 0 -3 10 -3 10-2 5 X 10 -3 10 -3
a + , induced H P C / D N A repair; + / W , weakly induced H P C / D N A repair in one or both assays; - , failed to induce H P C / D N A repair. b M e a n ± s t a n d a r d deviation of triplicate coverslips exposed 1.5 h after exposure of hepatocytes to the test compound plus 3H-TdR for 18 h. c Negative at the highest tested dose.
367 TABLE 4 C A R C I N O G E N I C I T Y A N D M U T A G E N I C I T Y O F C O D E D BIOASSAY C H E M I C A L S T E S T E D IN T H E H P C / D N A R E P A I R TEST Chemical
Carcinogenicity
a
Rat b
2-Amino-5-nitrothiazole Michler's ketone (4,4'bis(dimethylamino)benzophenone) Ethylene dibromide
HPC/DNA repair
S. typhimurium
Mutagenicity e
Mouse
Male
Female
Male
Female
+ d +
_ +
_ +
_ +
+ +
+ +
+
+
+
+
+
+
2-Nitro-p-phenylene diamine 4,4'-Methyl-bis-( N', Ndimethyl)benzenamine Nitrilotriacetic acid trisodium salt
-
-
-
+
+/W
+
+
+
-
+
+/W
+/W
+
+
+
-
-
-
4-Amino-2-nitrophenol 3-(Chloromethyl) pyridine hydrochloride p-Chloroaniline
+ +
S -
+
+
-
+ +
S
--
S
S
+
+
+
+
3-Nitropropionic acid c N, N'-Dicyclohexyl thiourea Anilazine
. .
.
.
Lithocholic acid 4-Nitro-o-phenylene diamine p-Phenylenediamine dihydrochloride
. .
.
.
.
.
+
.
.
.
.
.
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a The chemicals tested for carcinogenicity, D N A repair and mutagenicity were all from the same lot. b Fisher F344 rats wer used in all bioassays except ethylene dibromide which was tested in O s b o r n e Mendel rats. c Contains a 5% impurity. d - , negative; + , positive; + / W , weak positive; S, suspect. e Data from Dunkel and Simmon (1980).
Discussion
In tests with coded response
for
carcinogens
compounds,
12 o f 3 0 c o m p o u n d s . (Tables
p-chloroaniline.
2 and
the HPC/DNA Of these
4); t h e o n l y e x c e p t i o n s
In the in vivo bioassays,
repair test registered
12, 10 p r o v e d
were 3-nitropropionic
3-nitropropionic
a positive
to be well-established acid, which
acid and
contained
a
368 5% contamination by a dimeric ester of 3-hydroxypropionic acid, was associated with a dose-related elevated incidence of hepatocellular neoplasms in male rats, but the evidence of carcinogenicity was considered inconclusive. The bioassay results with p-chloroaniline were judged to be suggestive of carcinogenicity based on the production of fibromas and fibrosarcomas of the spleen. Both compounds were also positive in the Ames test (Dunkel and Simmon, 1980) and thus may be carcinogens which were not detected under the conditions of the bioassays. If so, the activity in short-term tests illustrates how evidence for genotoxicity can be used to aid in the clarification of equivocal results obtained in animal testing (Weisburger and Williams, 1981; Williams and Weisburger, 1981). Moreover, the National Toxicology Program is currently retesting p-chloroaniline, which could prove to be unnecessary in light of the present data. Regardless, the specificity of a positive response to unknown compounds in the H P C / D N A repair test corresponds to the high specificity for carcinogens observed in the validation of this test (Williams, 1980a, 1980b, 1981). Of the 9 carcinogens that were negative, three, diethylstilbestrol, p,p'-DDE and nitrilotriacetic acid, have been suggested to be non-genotoxic carcinogens (Weisburger and Williams, 1980), and thus would not be expected to damage DNA. These were also nonmutagenic. Beryllium, as with other metal carcinogens, may be carcinogenic by decreasing the fidelity of DNA polymerases (Sirover and Loeb, 1976) and, likewise, also not directly damaging to DNA. 3 other carcinogens that were negative for DNA repair, 1,2:3,4-diepoxybutane, 4-amino-2-nitrophenol and 3-chloromethyl pyridine HC1, were all positive in the Ames test (Tables 2 and 4), indicating a greater sensitivity of this test for these types of compounds. However, the Ames test was also positive for 3 noncarcinogens. The remaining 2 carcinogens that were not detected in the rat H P C / D N A repair test, natulan and para-rosaniline, were also negative in the standard Ames test using rat liver $9, In separate studies, we have obtained positive results for natulan in mouse and hamster hepatocytes (McQueen and Williams, in preparation) and para-rosaniline was positive in the Ames test using hamster $9 (Dunkel, 1979). Thus the present negative results may reflect a species difference in sensitivity, as has been demonstrated for other carcinogens (Maslansky and Williams, 1981; McQueen et al., 1981). 12 compounds were positive in both the H P C / D N A repair test and the Ames test. 10 were definitely carcinogenic and the other two, 3-nitropropionic acid and p-chloroaniline, gave indications of carcinogenicity in bioassays. These findings support the suggestion (Weisburger and Williams, 1981; Williams, 1980a) that because of the complementary nature of the metabolism and end points of these two tests, a compound that is positive in both is virtually certain to be carcinogenic. In conclusion, we suggest that the reliability of the H P C / D N A repair test makes it suitable for a routine screening test.
Acknowledgement This research was supported by contract NOI-CP-55205 from the U.S. National Cancer Institute.
369
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370 Williams, G.M. (1978) Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: Detection of carcinogenic biphenyl derivatives, Cancer Lett., 4, 69-75. Williams, G.M. (1980a) The detection of chemical mutagens/carcinogens by DNA repair and mutagenesis in liver cultures, in: F.J. de Serres and A. Hollaender (Eds.), Plenum, New York, pp. 61-79. Williams, G.M. (1980) The predictive value of DNA damage and repair assays for carcinogenicity, in: G.M. Williams, R. Kroes, H.W. Waaijers and K.W. van de Poll (Eds.), The Predictive Value of in vitro Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier/North-Holland, Amsterdam, pp. 213-230. Williams, G.M. (1981) Biochemical and biological indices of the toxicity of xenobiotics in liver cell cultures, in: S.S. Brown and D. Davies (Eds.), Proceedings of Symposium on Chemical Indices and Mechanisms of Organ Directed Toxicity, Pergamon, New York, pp. 131-145. Williams, G.M., and M.F. Laspia (1979) The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test, Cancer Lett., 6, 199-206. Williams, G.M., and J.H. Weisburger (1981) Systematic carcinogen testing through the decision point approach, Annu. Rev. Pharmacol. Toxicol., 21,393-416. Williams, G.M., E. Bermudez and D. Scaramuzzino (1977) Rat hepatocyte primary cell cultures, III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium, In Vitro, 13, 809-817. Williams, G.M., H. Moil, I. Hirono and M. Nagao (1980) Genotoxicity of pyrrolizidine alkaloids in the hepatocyte primary culture/DNA repair test, Mutation Res., 79, 1-5. Yager, LD., and J.A. Miller (1978) DNA repair in primary cultures of rat hepatocytes, Cancer Res., 38, 4385-4395.