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Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes
Mutation Research, 206 (1988) 91-102 Elsevier
91
MTR 01318
Induction of unscheduled D N A synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes Karen L. Steinmetz, Carol E. Green, James P. Bakke, Dana K. Spak and Jon C. Mirsalis Department of Cellular and Genetic Toxicology, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025 (U. S.A.) (Received 8 May 1987) (Revision received 14 March 1988) (Accepted 31 March 1988)
Keywords: Unscheduled DNA synthesis; Species variation; Primary hepatocyte cultures; Hepatocyte metabofism
Summary Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B 1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded < 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.
Interspecies variations in hepatic metabolism have been shown to be important in the relative toxicity of xenobiotics (Green et al., 1984) and
Correspondence: Karen L. Steinmetz, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025 (U.S.A.). Tel. (415) 859-6549.
may contribute to the observed variation in chemical carcinogenicity between species (Haseman et al., 1984). Because many industrial and governmental toxicology screening programs rely on laboratory animals to provide toxicology data which is ultimately used to evaluate the risk of chemicals to humans, it would be useful to demonstrate the validity of these animal models to human, or other primate, models.
0165-1218/88/$03.5001988 Elsevier Science Publishers B.V. (Biomedical Division)
92 Various techniques have been reported for comparison of interspecies differences in hepatocyte metabolism. Green et al. (1984) found isolated hamster hepatocyte cultures more susceptible to toxicity resulting from acetaminophen exposure than isolated dog, rabbit, or rat hepatocyte cultures by measuring covalently bound adducts, L D H release, and detachment of monolayer cell cultures. These results correlate well with results obtained from the in vivo toxicity of acetaminophen (Davis et al., 1974). Similar species differences have been demonstrated by measuring cytochrome P450 activity in untreated hepatocyte cultures (Maslansky and Williams, 1982) or in cultures treated with amphetamine (Green et al., 1986). Many investigators have examined variations in mutagen activation using microsomal fractions derived from different species (Oberley et al., 1982). However, the main limitation in the use of microsomal fractions is the loss of conjugation and detoxification enzymes present in intact cells (Williams, 1981). Co-cultivation of hepatocytes with cell lines provides a more representative profile of metabolism (Langenbach et al., 1983); however, a limitation of this approach is that some highly reactive metabolites may react with hepatocyte macromolecules and never reach the target cell. The in vitro unscheduled D N A synthesis (UDS) assay in rodent hepatocytes is currently used in a number of laboratories. This assay is well characterized and has a large rat hepatocyte data base which can be used as a basis for interspecies comparisons (Probst et al., 1980; Williams et al., 1982). This assay is often used to help predict the potential carcinogenicity of compounds in humans, yet few studies have utilized human hepatocyte cultures. Some limitations in the in vitro UDS assay have been reported. This assay is generally nonresponsive to many heptocarcinogenic nitro and azo compounds that have been detected using the in vivo UDS assay (Doolittle et al., 1983; Kornbrust and Barfknecht, 1985; Mirsalis et al., 1982). However, there are currently no feasible alternatives to in vitro systems for studying UDS in primates and humans. At least two laboratories have reported techniques for the measurement of UDS in human hepatocyte cultures (Butterworth et al., 1982, 1983;
Strom, 1982). The tissue they used was usually obtained from patients undergoing surgical procedures to remove liver tumors. We were able to obtain the entire liver from healthy organ transplant donors between the ages of 20 and 35 years. In the experiments presented in this paper, we have treated isolated hepatocyte cultures with compounds representing several chemical classes of carcinogens that require metabolic activation. We have determined their species-specific genotoxic activity by measuring UDS in hepatocyte cultures from rat, mouse, hamster, monkey, and human. We have also assessed the metabolic capabilities of these cells by measuring a variety of indicators of hepatocyte function. Materials and methods
Chemicals Dimethylnitrosamine (DMN, > 99% pure, Gold Label), 2-acetylaminofluorene (AAF), 4aminobiphenyl (4AB), and aflatoxin B~ (AFB) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Benzidine (BNZ) was obtained from Radian Corporation (Austin, TX). Benzo[a] pyrene (BAP) was obtained from K & K Laboratories (Irvine, CA). DMSO (J.T. Baker Co., Phillipsburg, N J) was the solvent used for AAF, AFB, BNZ, and BAP. D M N was diluted in Williams' medium E (WE, Gibco). Animals Rodents. Male Fischer-344 rats (175-310 g), B6C3Fa mice (24-30 g), and Syrian golden hamsters (100-150 g) were received from the following sources: rats and hamsters from Simonsen Laboratories (Gilroy, CA); rats from Hilltop Laboratory Animals (Chatsworth, CA), and rats and mice from Charles River Laboratories (Wilmington, MA). All animals were housed in polypropylene cages with hardwood-chip bedding in rooms maintained on a 12-h light-dark cycle. Rats were housed 3 per cage, mice were housed 10 per cage, and hamsters were housed 2 per cage. All animals received Purina Rodent Chow No. 5002 (Ralston Purina Co., St. Louis, MO) and deionized, and charcoal filtered (0.5 t~m pore size) tap water ad libitum.
93
Humans. Human liver tissue was received from organ transplant donors at Stanford University Hospital (Stanford, CA). All donors were between the ages of 20 and 35 years and were nonsmoking, cerebral accident victims reported to be in excellent health at the time of the accident. All donors were Caucasian males except for 1 Caucasian female donor. Prior to organ removal, all victims were maintained on life-support systems, and a minimum dose of vasopressin was used to prevent antidiuresis.
and filtered through sterile gauze. Hepatocyte viability was determined by Trypan blue exclusion and ranged from 80 to 95% for rats, monkeys, and humans and from 70 to 85% for hamsters and mice. Approximately 5 × 105 viable cells were seeded into each well of a Falcon (Oxnard, CA) 9.6-cm 2, 6-well tissue culture plate. Each well contained a 25-mm round Thermanox coverslip (BioLabs, Northbrook, IL) and 4 ml of WE supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA). All perfusion and culture solutions were supplemented with 50/~g/ml gentamicin sulfate (Sigma). Cells were incubated for 1.5-2 h at 37 ° C in a 95% air : 5% CO 2 incubator to allow attachment of the cells to the coverslips. Cultures were then washed once with 37 ° C WE supplemented with L-glutamine (WEI) and incubated in 2 ml WEI containing 10 /~Ci/ml [3H]-thymidine (approximately 80 Ci/mmole, New England Nuclear, Boston, MA) and the test compound for 17-21 h.
Hepatocyte isolation and culture
Autoradiography
Hepatocyte cultures were prepared from rats and mice as previously described (Mitchell and Mirsalis, 1984). Livers were perfused in situ for approximately 2 min with a solution of 0.5 mM ethyleneglycolbis(fl-amino ethyl ether)-N,N'-tetraacetic acid (EGTA, Sigma Chemical Co., St. Louis, MO) in Hanks' balanced salt solution without Ca 2+ or Mg 2+ (Gibco, Santa Clara, CA), followed by 12 min in a 3 7 ° C solution of collagenase type I (Sigma, 100 U / m l ) in WE. All perfusion and culture solutions were maintained at pH 6.8-7.0. Hamster hepatocytes were isolated using essentially the same techniques, except that 15% less collagenase and a longer perfusion time (17 min) at a lower flow rate were used, as reported by Green et al. (1984) to be optimal. Monkey and human livers were perfused using the biopsy perfusion method previously described (Reese and Byard, 1981). Perfusion times for human and monkey liver sections ranged from 30 to 50 min. A single-cell suspension of hepatocytes was obtained by combing out cells from the perfused liver into a petri dish containing 37 ° C collagenase solution. Cells were collected by a 5-min centrifugation at 50 × g, resuspended in cold medium,
Cultures were washed 3 times with WEI followed by 10 min in 1% sodium citrate to swell the cells, three 10-min washes in 1 : 3 glacial acetic acid/ethanol, and 6 washes with deionized water. Coverslips were mounted to microscope slides using Permount and dipped in Kodak NTB-2 nuclear-track emulsion diluted 1 : 1 with deionized water. Slides were exposed for 7 days at - 2 0 ° C, then developed and stained as previously described (Mitchell and Mirsalis 1984).
Monkeys. Male cynomolgus monkeys (3.0-4.9 kg) were received from Primate Imports (Port Washington, NY) and were housed singly in a room maintained on a 12-h light-dark cycle. Animals were fed Certified Monkey Chow (Purina) supplemented with fruit, peanuts, vitamins, and tap water ad libitum. Liver sections were obtained from untreated or vehicle control-treated monkeys that were sacrificed for other studies.
Measurement of UDS Quantitative autoradiographic grain counting was accomplished as previously described (Mitchell and Mirsalis, 1984). An area of a slide was randomly selected, and 30 morphologically unaltered cells were counted using an A R T E K Model 880 or 980 colony counter interfaced to a VAX 8800 computer (Digital Equipment Corp.). The highest of two nuclear-size areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). 3 slides were scored for each concentration for a total of 90 cells/ concentration. The percentage of cells in repair is defined as those exhibiting at least 5 NG.
94
Hepatocyte characterization assays Hepatocyte characterization assays were performed using freshly isolated liver cells in suspension culture, 1 × 1 0 6 cells/ml. The functional integrity of the isolated cells was determined by measuring the rate of urea synthesis using 10 mM NH4CI as the substrate in the presence of 10 mM ornithine over a 60-min incubation period (Story et al., 1983). Urea was analyzed by the method previously reported by Foster and Hochholzer (1971). BAP hydroxylase, p-nitroanisole-O-demethylase (pNAD), and p-nitrophenol conjugation (pNPC) were measured to assess the activities of the xenobiotic metabolizing systems of hepatocytes, pNAD activity was calculated from the quantity of pNPC measured after deconjugation (Moldeus et al., 1976). pNPC conjugation was determined by calculating the difference between the quantity of pNPC measured in a deconjugated sample and pNPC in a sample that was not treated with deconjugating enzymes. BAP hydroxylase activity was determined using 0.1 mM [3H]-BAP incubated with cells for 30 min. Samples of incubation medium and cells were partitioned between hexane and an alkaline (0.25 N NaOH) aqueous solution containing 40% ethanol (Steward and Byard, 1981). The percentage of BAP metabolized was calculated by determining the quantity of radioactivity in the aqueous phase. Reduced glutathione (GSH) was measured by the method reported by Hissin and Hill (1976). DNA content was determined by the method reported by Richards (1974). Incubation for the urea synthesis and BAP hydroxylase assays were car-
ried out in Waymouth's 752/1 medium supplemented with hormones, fatty acids, and bovine serum albumin (Green et al., 1983). For the pNAD and pNPC conjugation assays, cells were incubated in Krebs-Ringer bicarbonate buffer plus 2% BSA (Modeus et al., 1976). Results
Chemical carcinogens from 4 chemical classes were examined for their abilities to induce UDS in primary hepatocyte cultures derived from rat, mouse, hamster, monkey, or human livers. Experiments with human hepatocytes were characterized by sex, age, and UDS response to culture medium or 74 /~g/ml (1.0 mM) DMN (Table 1). Both quantitative and qualitative differences were observed in the responses between species (Table 2). Medium control and 1% DMSO control values for all cultures yielded < 0 NG (< 8% IR, Table 3). Exposure of hepatocyte cultures to DMN (Fig. 1) or AFB (Table 4) yielded dose-related increases in UDS in all species; however, variation in the magnitude of response was noted between species. Exposure of isolated rat or human hepatocyte cultures to 4AB yielded positive UDS responses for both species (Table 5). Following exposure to BNZ, rat, hamster, and human hepatocyte cultures yielded dose-related increases that peaked at 1.0-5/xg/ml, followed by a decrease in NG (Fig. 2). Conversely, mouse and monkey hepatocyte cultures yielded < 0 NG following exposure to BNZ. Exposure of rat, hamster, and human
TABLE l C H A R A C T E R I Z A T I O N OF T H E H U M A N H E P A T O C Y T E E X P E R I M E N T S BY SEX, AGE, A N D UDS RESPONSE TO C U L T U R E M E D I U M OR D M N Exp.
Sex
No.
1 2 3 4 5
M M M M F
Age
UDS control compounds
(years)
Medium
20 20 35 28 26
74 t l g / m l D M N
NG
% IR
NG
% IR
- 1.5 - 5.6 -4.5 - 9.0 - 6.4
0 8 2 1 3
24.3 23.8 28.0 22.6 21.2
96 91 91 90 88
NG, net gr ain s/n ucleus; % IR, percentage of cells in repair.
95 TABLE 2 SUMMARY OF UDS RESPONSES BETWEEN SPECIES FOLLOWING
Chemical
Human Carcinogen
Rat
Mouse
DMN AAF BNZ 4AB BAP AFB
P S P P
++++ + + + + + + + + + + +
++++ NT + + +
(37) a (5) (1.0) (1.0) (100) + + + + (0.1)
S P
(37) (10) (20) (20) (1.0)
IN VITRO TREATMENT
Hamster
Monkey
Human
++++ (3.7) + + + + (0.1) + + + + (1.0) NT + (20) NT
+++ (740) (50) (10) NT + + + + (10) + + (0.05)
++++ (3.7) ++++ (5) + + (1.0) + + + + (20) + + + (0.5) ++++ (1.0)
a The number in parentheses is the concentration required to elicit the maximum positive U D S response. P, known human carcinogen; S, suspected human carcinogen. -, <0NG; +-, < 0 N G ( % I R > 1 5 % ) ; +,0-5NG; ++,5-10NG; +++,10-20NG; ++++, >20NG;NT,
j"
//
5C
~" Homster
4(
i 30
" ...%Lg"
i!
p..Humcn
NG
,' ,'
2C
"- - . . . .
/ , Rat
_~ ,~..~..:~,~--..-:.CT. d i
"*- -J " , ~ ' ~ . . . . . . . . . . . .
2~.:1
Y ," ~-.-%use L ,;
lc
i
~
),
_ .....
/
/,
/:
,~
/ / !
//
o
0 05
I
I
05
5
[ DisN]m~
Fig. 1. Induction of U D S in rat, mouse, hamster, monkey, or human hepatocytes following in vitro exposure to D M N .
hepatocyte cultures to AAF yielded dose-related increases in NG; however, mouse and monkey hepatocyte cultures yielded < 0 N G following
nottested.
exposure to AAF (Table 6). Exposure of monkey and human hepatocyte cultures to BAP yielded dose-related increases in NG; however, BAP induced a very weak U D S response in hamster hepatocyte cultures, an equivocal response in mouse hepatocyte cultures, and failed to induce UDS in cultures of rat hepatocytes (Table 7). Isolated hamster, monkey, and human hepatocytes were characterized and compared by measuring several parameters of cell function (Table 8) that have been used in previous studies with rat hepatocytes (Green et al., 1983). The rate of urea synthesis, a hepatocyte-specific function, was assessed as a general indicator of cell viability and organelle integrity (Story et al., 1983). Two cytochrome P450-associated activities, BAP hydroxylase and pNAD, were determined to obtain a measure of the ability of the cells to perform phase I oxidations of xenobiotics and pNPC, a phase II reaction, was assayed. Glutathione, an important
TABLE 3 I N D U C T I O N O F U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D F R O M R A T , M O U S E , H A M S T E R M O N K E Y , O R H U M A N L I V E R F O L L O W I N G N O C H E M I C A L T R E A T M E N T O R E X P O S U R E T O 1% D M S O
Species
Control/untreated
Rat Mouse Hamster Monkey Human
-8.2+--0.9 - 11.6 + 1.6 - 3.5 +- 0.6 - 4.8 + 0.8 - 6.1 +- 0.8
N G + S.E.
1% D M S O % IR (N) 2 0 2 2 5
(7) (2) (1) (2) (5)
N G + S.E. -9.3+--0.8 - 7.3 +- 2.0 - 7.4 +__1.0 - 4.3 + 1.0 - 8.4 + 0.3
% IR (N) 1 1 0 3 2
(7) (3) (1) (2) (5)
N G , net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair; N, number of Expts.
96 TABLE 4 I N D U C T I O N OF U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D H U M A N F O L L O W I N O E X P O S U R E TO A F L A T O X I N B~ Species
Concentration
N G _+ S.E.
% IR
Rat
0.~05 0.001 0.005 0.01 0.1 1.0
- 3 . 1 - + 2.2 0.4_+ 5.2 5.6_+ 0.6 20.9±16.5 28.2_+ 3.2
12 22 52 85 99
Mouse
0.0005 0.0l 0.05 0.1 1.0
-6.9_+ -9.1_+ -6.5± -2.2_+ 9.3_+
0.9 1.0 1.8 1.0 2.2
2 1 3 12 68
Mo~ey
0.001 0.005 0.01 0.05
-4.9_+ -3.4_+ -2.3_+ 7.6_+
0.1 1.1 1.0 1.5
3 7 11 66
Human
0.~05 0.001 0.~5 0.01 0.1 1.0
-6.3_+ -9.9± -6.4_+ 3.0_+ 15.0_+ 53.8_+
1.4 1.3 0.7 5.7 5.1 5.6
2 3 8 47 77 100
(#g/ml)
Expt. 1
F R O M RAT, MOUSE, M O N K E Y , OR
Expt. 2 N G -+ S.E. -3.4-+1.5 -3.0±1.5 0.8-+3.4 1.2-+2.7 11.1±5.1 16.8±3.2
% IR 4 12 25 7 64 83
NG, net grains/nucleus; S.E. Slide-to-slide variation; % IR, percentage of cells in repair.
component in the cellular defense systems against active metabolites and D N A content were also measured.
The total D N A content was higher in monkey and human hepatocytes than in hamster hepatocytes. Glutathione content was the same for all
TABLE 5 I N D U C T I O N O F U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D F R O M R A T O R H U M A N LIVER F O L L O W I N G E X P O S U R E TO 4 - A M I N O B I P H E N Y L Species
Rat
Human
Concentration
Expt. 1
(/~g/ml)
N G -+ S.E.
% IR
Expt. 2
0.1 0.5 1
1.4_+0.5 15.9_+9.1 16.6_+3.7
31 69 86
0.1 0.5 1.0 5 10 20
19.7_+3.3 38.1 _+6.0 36.5 _+5.3 31.5_+2.1 34.1 + 2.9 22.5 -+ 3.7
92 100 100 98 99 96
N G , net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair.
N G _+S.E.
% IR
19.9_+2.4
96
30.6 _+8.6
98
10.2___6.9
61
97 TABLE 6 INDUCTION OF UDS BY PRIMARY HEPATOCYTE CULTURES DERIVED FROM RAT, MOUSE, HAMSTER, MONKEY OR HUMAN LIVER FOLLOWING EXPOSURE TO 2-ACETYLAMINOFLUORENE Species
Concentration
Rat
Mouse
Expt. 1
(/~g/ml)
NG + S.E.
% IR
0.05 0.1 0.5 1.0 5 10
NS 14.1 + 6.2 20.1+ 1.3 20.4 + 12.5 29.5 4-13.9 27.5 + 1.3
76 96 69 91 99
0.1 0.5 1.0 5 10 50
AFB a
Expt.
-7.6.
1.3
1
-3.9--- 2.6
15
- 8.3 +- 2.5 T
10
11.3 ___12.5 53.2 5:21.5 36.5 +- 12.8
31 97 91
1.0
Hamster
0.05 0.1 0.5
Monkey
0.05 0.1 0.5 1 5 50
DMN a
- 5.4 +- 5.5 +_ -4.9+-4.2+ -6.7---
0.05 0.1 0.5 1.0 5 10
N G +_S.E.
% IR
15.2+4.9
63
28.5 +4.8
85
-7.3+0.9 -6.2+2.9 -9.6+1.5 - 9.9 + 1.2
6 7 2 0
9.3 + 2.2
68
1.1 0.6 0.8 2.3 0.8
0 0 1 0
-6.5+1.3
4
-5.7+0.2 -2.8+0.5
2 6
18.3 +_ 5.5
90
15.6 + 1.1
84
- 5.3 5 : 0 . 6 - 3.3 5 : 4 . 2 12.2_+ 7.6 16.7 +- 2.8 7.8--- 4.3 19.3 + 2.6
11 22 67 88 51 90
19.6+2.0
89
20.1+6.5
87
1480
Human
2
1
NG, net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair; NS, not scored; T, toxic. a Data is shown for concurrent positive control.
5O i
Hamstep
4 0 ¸,
Rat
A
[",I N.G
/ . J Human
]01
o 10
~
......... !':-4, ~ "--.
/
~onk~zj..~...
. . . . . . . 0[ /,,
',
---~.}_ . . . . . . . . . . . "~'--~ ~-.---,t
01I
, 10
110 210 35
[BNZ] u g / m ]
Fig. 2. Induction of UDS in rat, mouse, hamster, monkey, or human hepatocytes following in vitro exposure to BNZ.
species tested. BAP hydroxylase activity was higher i n h a m s t e r a n d m o n k e y h e p a t o c y t e c u l t u r e s (4.43 a n d 2.92 n m o l e s / 3 0 m i n X 10 6 cells) a n d w a s sign i f i c a n t l y l o w e r i n h u m a n h e p a t o c y t e c u l t u r e s (0.73 nmole/30 r a i n × 10 6 cells), p N A D activity was higher in isolated hamster and monkey hepatoc y t e s (34.7 a n d 39.0 n m o l e s / 3 0 r a i n x 10 6 cells) t h a n i n h u m a n h e p a t o c y t e c u l t u r e s (14.2 n m o l e s / 30 m i n × 10 6 cells), p N P C a c t i v i t y w a s a p p r o x i m a t e l y t h e s a m e f o r all s p e c i e s . T h e r e w e r e l a r g e standard deviations in urea synthesis rates be-
98 TABLE 7 I N D U C T I O N OF UDS BY PRIMARY HEPATOCYTE CULTURES D E R I V E D FROM RAT, MOUSE, HAMSTER, M O N K E Y OR H U M A N LIVER F O L L O W I N G EXPOSURE TO BENZO[a]PYRENE
Species
Concentration (/~g/ml)
Rat
AAF a
Mouse
AFB a
Hamster
DMN a Monkey
0.1 0.5 1.0 5 10 20 100
Expt. 2
N G ± S.E. -7.2+ -7.2_+ -5.4_+ -5.4_+ -3.9_+ - 3.2-+
0.17 1.1 1.1 3.2 0.4 0.8
% IR 1 3 2 11 2 1
0.5 5
20.8 _+ 5.0
88
0.5 1.0 5 10 20
- 12.2_+ 0.6 - 10.5 -+ 1.9 -0.7-+ 3.8 -3.9-+ 1.2 - 1.1_+ 2.1
1 4 33 19 29
1.0
9.3_+ 2.2
68
0.1 0.5 1.0 10 20
1.3 0.3 1.6 2.5 0.8
1 0 6 21 26
0.1
30.9_+ 13.6
97
0.1 0.5
-3.9-+ 0.9 - 2 . 5 ± 0.4 0.4± 1.3 4.0-+ 1.7 20.3 ± 2.8 17.2± 3.3
0 6 20 33 94 100
1 5 10 20
Human
Expt. 1
0.1 0.5 1.0 5 10 20
-7.8_+ -6.6_+ -4.7+ -0.8-+ 1.1 -+
1.1± 22.0-+ 11.9± 13.0± 21.0-+
0.2 3.1 3.3 5.4 4.4
34 94 78 77 88
NG_+ S.E.
-8.4_+ 0.4
% IR
2
-2.6-+ 0.1
8
42.8± 8.5
97
-6.0-+ 1.3
2
3.6-+ 0.5
38
29.3_+ 10.9
95
-8.2+
0.8
1
5.4-+ 4.4
49
7.8-+ 7.2
52
NG, net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair.
a Data is shown for concurrent positive control.
tween individual hamsters or humans, indicating individual variation with respect to this parameter. Discussion Studies have shown that considerable interspecies variation exists in hepatocyte metabolism (Caldwell et al., 1978; Green et al., 1984; Maslan-
sky and Williams, 1982). In order to assess the relevance of the information obtained from an in vitro endpoint, it would be useful to evaluate the endpoint differences between the test species and humans. Few comparative studies using hepatocytes from different species, including primates, have been performed, probably because largeanimal hepatocyte isolation techniques have only
99 TABLE 8 F U N C T I O N A L CHARACTERISTICS OF ISOLATED HEPATOCYTES
DNA (/~g/106 cells) Glutathione (nmoles/106 cells) Benzo[ a ]pyrene hydroxylase (nmoles/30 min. 106 cells) p-Nitroanisole-O-demethylase (nmoles/30 rain. 106 cells) p-Nitrophenol conjugation (nmoles/30 min. 106 cells) Urea synthesis (nmoles/min. 106 cells)
Hamster (n = 4)
Monkey (n = 2)
Human (n = 4)
10.10_+0.5 22.30+_4.9
15.5 18.1 a
15.6 _+3.6 21.4 +2,6
4.43 -+ 1.1
2.92
34.70_+2.8 7.80 _+4.3 9.11 + 5.22
39.0 a 5.00 a 4.10 a
0.73 -+ 0.44 14.2 _+4.0 4.09 + 1.2 2.75 _+2.6
aN=l.
recently been developed (Green et al., 1983; Reese and Byard, 1981), and fresh primate tissue is often difficult to obtain. The in vitro UDS assay using rodent hepatocytes has been shown to be useful for assessing the genotoxic potential of compounds from a wide variety of chemical classes (Kornbrust and Barfknecht, 1985; Probst et al., 1980; Williams et al., 1982). Extending these techniques to include in vitro UDS in hepatocytes from primates provides a means for validating this rodent model. In these experiments, we have examined 6 chemicals representative of 4 chemical classes in the in vitro UDS assay using hepatocyte cultures derived from rat, mouse, hamster, monkey, and human liver. These chemicals include DMN (nitrosamine), AFB (mycotoxin), BAP (polycyclic aromatic hydrocarbon). BNZ, 4AB, and AAF (aromatic amines), all of which are known or suspect human carcinogens (IARC, 1973, 1978, 1982). The metabolic assays were conducted to demonstrate the viability and functional capabilities of the isolated hepatocytes from different species. These data were also examined to determine whether there was a correlation between a positive UDS response and metabolic activity. Although the glutathione content, BAP hydroxylase activity, pNAD activity, pNPC activity, and urea synthesis rates indicated that the cultures were metabolically competent at the time of chemical exposure, no direct relationship between metabolism and UDS response was observed for any compound
tested. This is not surprising considering the complexity of the metabolic pathways and the number of different isozymes that are involved in the biotransformation of carcinogens. In the reported studies, changes in cytochrome P450 and associated activities during the culture period were not determined. However, other investigators have reported species-related differences in the stability of P450 of cultured hepatocytes (Blaauboer et al., 1985; Malansky and Williams, 1982) which would affect the UDS response observed, although the kinetics of P450 loss and rate of metabolic activation have not been assessed. Additionally, differences in DNA repair, rate of DNA adduct formation, or detoxification of reactive intermediates could also affect the UDS response between species. Dose-related increases in UDS were observed in all hepatocyte cultures treated with DMN. Hamster hepatocyte cultures yielded the strongest response to DMN exposure (Fig. 1). The extreme responsiveness of hamster hepatocyte cultures to DMN in this assay has also been described by Kornbrust and Barfknecht (1984) and by Katoh et al. (1982), who measured cytotoxicity and mutation rate in V79 cells cultured with rat or hamster hepatocytes. They found that the mutation frequencies were substantially higher in cells co-cultured with hamster hepatocytes than with rat hepatocytes. Data from McQueen et al. (1983) does not demonstrate the extreme responsiveness of hamster hepatocytes to DMN exposure.
100 AFB treatment of primary hepatocyte cultures isolated from rat, mouse, human, or monkey liver yielded dose-related increases in UDS (Table 4). Human hepatocyte cultures yielded the strongest response to AFB exposure, which correlates well with the reported epidemiological evidence for human liver cancer (Shank et al., 1972). Conversely, mouse hepatocyte cultures were the least responsive to AFB exposure. One possibility for this weak response may be that mouse hepatocytes are more efficient in AFB detoxification (Decad et al., 1979). Striking qualitative differences were observed between species following exposure to aromatic amines. Human, rat, and hamster hepatocyte cultures yielded dose-related increases following 4AB (Table 5; human and rat only) or AAF exposure (Table 6), and positive responses following exposure to BNZ (Fig. 2), whereas mouse and monkey hepatocyte cultures yielded < 0 N G following exposure to AAF or BNZ. Our findings with BNZ agree with those reported by Hill and Probst (personal communication) and McQueen and Williams (1983), who demonstrated an inhibition of DNA repair at high BNZ concentrations which explains the observed decrease in UDS. The response of mouse hepatocyte cultures to AAF exposure discussed in this paper contrasts with the findings of McQueen et al. (1983), who report an average of 26.3 N G at 10 -3 M, and of Probst and Hill (personal communication), who report between - 1 . 2 5 and 11.31 N G in B6C3F~ mice. In contrast, our findings agree with those of Butterworth and Smith-Oliver (1987) and Millner et al. (1987), who also report a negative UDS response for AAF in B6C3F 1 mouse hepatocytes. Sakai et al. (1976) demonstrated that in vitro covalent binding of N-OH-AAF to D N A in rat hepatocyte nuclei was twice that observed for the mouse. One of the pathways postulated for AAF metabolism to one of its ultimate carcinogenic metabolites involves sulfotransferase (DeBaun et al., 1970). It has been shown that rat liver has greater N-hydroxy-AAF sulfotransferase activity than mouse or hamster hepatocytes. This may account for the response by mouse hepatocyte cultures to AAF; however, hamster hepatocyte cultures exhibited the strongest UDS response following exposure to this chemical. This difference has also been ob-
served in vivo, since rats treated with AAF show significant increases in UDS, whereas B6C3F~ mice do not show increases in UDS following in vivo exposure to AAF (Mirsalis et al., 1985). Disher and Randerath (1987) report that 7--12 times more adducts were formed in rat liver than in mouse liver following AAF exposure. AAF is a potent hepatocarcinogen in rats but is only weakly carcinogenic in mice (Tisdel et al., 1984). Isolated rat hepatocyte cultures consistently yielded negative responses following exposure to BAP when tested to the limit of solubility (Table 7). This response contrasts with data reported by several authors (McQueen et al., 1983; Mitchell et al., 1983; Probst et al., 1980). A number of factors may affect the outcomes of genetic toxicology tests, including diet, animal supplier, bedding, and disease state. We are confident that the lot of BAP used or technical problems were not responsible for the negative UDS response, since human and monkey hepatocyte cultures yielded strong positive responses. Although mouse hepatocyte cultures yielded < 0 N G following exposure to BAP, the 70 IR was very high, with a maximum of 33% IR. Therefore, we consider treatment with BAP to yield an equivocal response in mouse hepatocyte cultures. BAP was weakly genotoxic in hamster hepatocytes treated in vitro. Initial BAP hydroxylase activity did not correlate with the genotoxicity of BAP in hepatocyte cultures, probably because this assay measures total BAP metabolism making no distinction in species differences in activation and detoxification reactions. Quantitative and qualitative differences in UDS responses were observed between all 5 species. The appropriate species to use for testing may depend on the class of chemical examined. In the case of nitrosamines, mycotoxins, and aromatic amines, rat hepatocyte cultures yielded results similar to human hepatocyte cultures, whereas BAP treatment yielded contrasting results in rat and human hepatocyte cultures. Although specific differences between chemical classes may occur, rat hepatocyte cultures appear to be the closest model for human hepatocyte cultures. In general, mouse and monkey hepatocyte cultures seem to be much less responsive to several of the chemicals selected for this study compared with human hepatocyte cultures. Therefore, these species may
101 b e less s u i t a b l e f o r s c r e e n i n g c h e m i c a l s i n t h i s a s s a y w h e n t h e e s t i m a t i o n o f h u m a n r i s k is t h e major objective.
References Blaauboer, B.J., I. van Holsteijn, J. van Graft and A.J. Paine (1985) The concentration of cytochrome P-450 in human hepatocyte culture, Biochem. Pharmacol., 34 2405-2408. Butterworth, B.E., and T. Smith-Oliver (1987) Comparison of the response of carcinogen/non-carcinogen pairs in the rat and mouse hepatocyte DNA repair assays in vivo and in vitro, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin and M.D. Shelby (Eds.), Progress in Mutation Research, Vol. 6, Evaluation of Short-Term Tests for Carcinogens, Report of the International Program on Chemical Safety's Collaborative Study on In Vivo Assays, in press. Butterworth, B.E., D.J. Doolittle, P.K. Working, S.C. Strom, R.L. Jirtle and G. Michalopoulos (1982) Chemically induced DNA repair in rodent and human cells, in: B.A. Bridges, B.E. Butterworth and I.B. Weinstein (Eds.), Banbury Report 13, Indicators of Genotoxic Exposure, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Butterworth, B.E., L.L. Earle, S. Strom, R. Jirtle and G. Michalopoulos (1983) Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene, Mutation Res., 122, 73-80. Caldwell, J., R.T. Williams, B. Olumbe and M.R. French (1978) Drug metabolism in "exotic" animals, Eur. J. Drug. Metab. Pharmacokin., 2, 61-66. Davis, D.C., W.Z. Potter, D.J. Jollow and J.R. Mitchell (1974) Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen, Life Sci., 14, 2099-2109. DeBaun, J.R., E.C. Miller and J.A. Miller (1970) N-Hydroxy2-acetylamino-fluorene sulfotransferase: its probable role in carcinogenesis and in protein-(methion-S-yl) binding in rat fiver, Cancer Res., 30, 577-595. Decad, G.M., K.K. Dougherty, D.P.H. Hsieh and J.L. Byard (1979) Metabolism of aflatoxin B1 in cultured mouse hepatocytes: comparison with rat and effects of cyclohexene oxide and diethyl maleate, Toxicol. Appl. Pharmacol., 50, 429-436. Disher, R.M., and K. Randerath (1987) 32p-Postlabeling assay for covalent DNA binding in rodent tissues, in: J. Ashby, F.J. de Serres, M.D. Shelby, B.H. Margolin, M. Ishidate Jr. and G.C. Becking (Eds.), Progress in Mutation Research, Vol. 3, in press. Doolittle, D.J., J.M. Sherrill and B.E. Butterworth (1983) Influence of intestinal bacteria, sex of the animal, and the position of the nitro group on the hepatic genotoxicity of nitrotoluene isomers in vivo, Cancer Res., 43, 2836-2842. Foster, L.B., and J.M. Hochholzer (1971) A single reagent manual method for directory determining urea nitrogen in serum, Clin. Chem., 17, 921-925. Green, C.E., J.E. Dabbs and C.A. Tyson (1983) Functional
integrity of isolated rat hepatocytes prepared by whole liver vs. biopsy perfusion, Anal. Biochem., 129, 269-276. Green, C.E., J.E. Dabbs and C.A. Tyson (1984) Metabolism and cytotoxicity of acetaminophen in hepatocytes isolated from resistant and susceptible species, Toxicol. Appl. Pharmacol., 76, 130-149. Green, C.E., S.E. LeValley and C.A. Tyson (1986) Comparison of amphetamine metabolism using isolated hepatocytes from five species including human, J. Pharmacol. Exp. Ther., 237, 931-936. Haseman, J.K., D.D. Crawford, J.E. Huff, G.A. Boorman and E.E. McConnell (1984) Results from 86 two-year carcinogenicity studies conducted by the National Toxicology Program, J. Toxicol. Environ. Health, 14, 621-639. Hissin, P.J., and F. Hilf (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues, Anal. Biochem., 74, 214-226. International Agency for Research on Cancer (1973) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemical to Man, Vol. 3, Certain Polycyclic Aromatic Hydrocarbons and Heterocyclic Compounds, IARC, Lyon, pp. 91-136. International Agency for Research on Cancer (1978) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemical to Humans, Vol. 17 Some N-Nitroso Compounds, IARC, Lyon, pp. 125-175. International Agency for Research on Cancer (1982) IARC Monographs on the Evaluation of Carcinogenic Risk of the Chemical to Humans, Vol. 1-29, Suppl. 4, Chemicals, Industrial Processes and Industries Associated with Cancer in Humans IARC, Lyon. Katoh, Y., M. Tanaka and S. Takayama (1982) Higher efficiency of hamster hepatocytes than rat hepatocytes for detecting DMN and DEN in hepatocyte-mediated Chinese hamster V79 cell mutagenesis assay, Mutation Res, 105, 265-269. Kornbrust, D.S., and T.R. Barfknecht (1984) Comparison of rat and hamster hepatocyte primary culture/DNA repair assays, Environ. Mutagen., 6, 1-11. Kornbrust, D., and T. Barfknecht (1985) Testing of 24 food, drug, cosmetic and fabric dyes in the in vitro and the in vivo/in vitro rat hepatocyte primary culture/DNA repair assays, Environ. Mutagen., 7, 101-120. Langenbach, R., C. Hix, L. Oglesby and J. Allen (1983) Cell-mediated mutagenesis of Chinese hamster V-79 cells and Salmonella typhimurium, Ann. N.Y. Acad. Sci., 407, 258-166. Maslansky, C.J., and G.M. Williams (1982) Primary cultures and the levels of cytochrome P450 in hepatocytes from mouse, rat, hamster and rabbit liver, In Vitro, 18(8), 683-693. McQueen, C.A., and G.M. Williams (1983) The use of cells from rat, mouse, hamster, and rabbit in the hepatocyte primary culture/DNA-repair test, Ann. N.Y. Acad. Sci., 407, 199-130. McQueen, C.A., D.M. Kreiser and G.M. Williams (1983) The primary hepatocyte culture/DNA repair assay using mouse or hamster hepatocytes, Environ. Mutagen., 5, 1-8.
102 Millner, G., J.G. Shaddock, R.D. Harbison and D.A. Casciano (1987) Comparative evaluations of unscheduled DNA synthesis (UDS) in hepatocytes from various mouse strains, Environ. Mutagen., 9, Suppl. 8, 73. Mirsalis, J.C., T.E. Hamm. J.M. Sherrill and B.E. Butterworth (1982) Role of gut flora in the genotoxicity of dinitrotoluene, Nature (London), 295, 322-323. Mirsalis, J.C., C.K. Tyson, E.N. Loh, K.L. Steinmetz, J.P. Bakke, C.M. Hamilton, D.K. Spak and J.W. Spalding (1985) Induction of hepatic cell proliferation and unscheduled DNA synthesis in mouse hepatocytes following in vivo treatment, Carcinogenesis, 6(10), 1521-1524. Mitchell, A.D., and J.C. Mirsalis (1984) UDS as an indicator of genotoxic exposure, in: A. Ansari and F.J. de Serres (Eds.), Single-Cell Mutation Monitoring Systems, Plenum, New York. Mitchell, A.D., D.A. Casciano, M.L. Meltz, D.E. Robinson, R.H.C. San, G.M. Williams and E.S. Von Halle (1983) Unscheduled DNA synthesis tests, a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 123, 363-410. Moldeus, P., H. Vadi and M. Berggren (1976) Oxidative and conjugative metabolism of p-nitroanisole and p-nitrophenol in isolated rat liver cells, Acta Pharmacol. Toxicol., 39, 17-32. Oberley, T,J., C.E. Piper and D.S. McDonald (1982) Metabolic activation capabilities of $9 liver fractions from 3 species in the L5178Y mouse-lymplioma assay, Mutation Res., 105, 439-444. Probst, G.S., L.E. Hill and BJ. Bewsey (1980) Comparison of three in vitro assays for carcinogen-induced DNA damage, J. Toxicol. Environ. Health, 6, 333-349. Reese, J.A., and J.L. Byard (1981) Isolation and culture of adult hepatocytes from liver biopsies, In Vitro, 17(11), 935-940.
Richards, B.M. (1974) Modification of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA, Anal. Biochem., 57, 369 376. Sakai, S., C.E. Reinhold, P.J. Wirth and S.S. Thorgeirsson (1976) Mechanism of in vitro mutagenic activation and covalent binding of N-hydroxy-2-acetylaminofluorene in isolated liver cell nuclei, Cancer Res., 38, 2058-2067. Shank, R.C., J.E. Gordon and G.N. Wogan (1972) Dietary aflatoxins and human liver cancer, Ill. Field survey of rural Thai families for ingested aflatoxins, Food Cosmet. Toxicol., 10~ 71-84. Steward, A.R., and J.L. Byard (1981) Induction of benzo[a]pyrene metabolism by 2,3,7,8-tetrachlorodibenzop-dioxin in primary cultures of adult rat hepatocytes, Toxicol. Appl. Pharmacol., 59, 603-616. Story, D.L., S.J. Gee, C.A. Tyson and D.H. Gould (1983) Response of isolated hepatocytes to organic and inorganic cytotoxins, J. Toxicol. Environ. Health, 11,483-501. Strom, S.C., D.L. Noviki, A. Novotny, R.L. Jirtle and G. Michalopoulos (1983) Human hepatocyte-mediated mutagenesis and DNA repair activity, Carcinogenesis, 4, 683-686. Tisdel, M.O., R.H. Weltman and R.J. Ryzia (1984) Two-year feeding studies with 2-acetylaminofluorene: comparative histopathology in Sprague-Dawley rats and CD-1 mice, Toxicologist, 4, 36, Williams, G.M. (1981) Mammalian cell culture systems for the study of genetic effects of N-substituted aryl compounds, Natl. Cancer Inst. Monogr., 58, 237-242. Williams, G.M., M.F. Laspia and V.C. Dunkel (1982) Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and non-carcinogens, Mutation Res., 97, 359-370.
91
MTR 01318
Induction of unscheduled D N A synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes Karen L. Steinmetz, Carol E. Green, James P. Bakke, Dana K. Spak and Jon C. Mirsalis Department of Cellular and Genetic Toxicology, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025 (U. S.A.) (Received 8 May 1987) (Revision received 14 March 1988) (Accepted 31 March 1988)
Keywords: Unscheduled DNA synthesis; Species variation; Primary hepatocyte cultures; Hepatocyte metabofism
Summary Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B 1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded < 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.
Interspecies variations in hepatic metabolism have been shown to be important in the relative toxicity of xenobiotics (Green et al., 1984) and
Correspondence: Karen L. Steinmetz, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025 (U.S.A.). Tel. (415) 859-6549.
may contribute to the observed variation in chemical carcinogenicity between species (Haseman et al., 1984). Because many industrial and governmental toxicology screening programs rely on laboratory animals to provide toxicology data which is ultimately used to evaluate the risk of chemicals to humans, it would be useful to demonstrate the validity of these animal models to human, or other primate, models.
0165-1218/88/$03.5001988 Elsevier Science Publishers B.V. (Biomedical Division)
92 Various techniques have been reported for comparison of interspecies differences in hepatocyte metabolism. Green et al. (1984) found isolated hamster hepatocyte cultures more susceptible to toxicity resulting from acetaminophen exposure than isolated dog, rabbit, or rat hepatocyte cultures by measuring covalently bound adducts, L D H release, and detachment of monolayer cell cultures. These results correlate well with results obtained from the in vivo toxicity of acetaminophen (Davis et al., 1974). Similar species differences have been demonstrated by measuring cytochrome P450 activity in untreated hepatocyte cultures (Maslansky and Williams, 1982) or in cultures treated with amphetamine (Green et al., 1986). Many investigators have examined variations in mutagen activation using microsomal fractions derived from different species (Oberley et al., 1982). However, the main limitation in the use of microsomal fractions is the loss of conjugation and detoxification enzymes present in intact cells (Williams, 1981). Co-cultivation of hepatocytes with cell lines provides a more representative profile of metabolism (Langenbach et al., 1983); however, a limitation of this approach is that some highly reactive metabolites may react with hepatocyte macromolecules and never reach the target cell. The in vitro unscheduled D N A synthesis (UDS) assay in rodent hepatocytes is currently used in a number of laboratories. This assay is well characterized and has a large rat hepatocyte data base which can be used as a basis for interspecies comparisons (Probst et al., 1980; Williams et al., 1982). This assay is often used to help predict the potential carcinogenicity of compounds in humans, yet few studies have utilized human hepatocyte cultures. Some limitations in the in vitro UDS assay have been reported. This assay is generally nonresponsive to many heptocarcinogenic nitro and azo compounds that have been detected using the in vivo UDS assay (Doolittle et al., 1983; Kornbrust and Barfknecht, 1985; Mirsalis et al., 1982). However, there are currently no feasible alternatives to in vitro systems for studying UDS in primates and humans. At least two laboratories have reported techniques for the measurement of UDS in human hepatocyte cultures (Butterworth et al., 1982, 1983;
Strom, 1982). The tissue they used was usually obtained from patients undergoing surgical procedures to remove liver tumors. We were able to obtain the entire liver from healthy organ transplant donors between the ages of 20 and 35 years. In the experiments presented in this paper, we have treated isolated hepatocyte cultures with compounds representing several chemical classes of carcinogens that require metabolic activation. We have determined their species-specific genotoxic activity by measuring UDS in hepatocyte cultures from rat, mouse, hamster, monkey, and human. We have also assessed the metabolic capabilities of these cells by measuring a variety of indicators of hepatocyte function. Materials and methods
Chemicals Dimethylnitrosamine (DMN, > 99% pure, Gold Label), 2-acetylaminofluorene (AAF), 4aminobiphenyl (4AB), and aflatoxin B~ (AFB) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Benzidine (BNZ) was obtained from Radian Corporation (Austin, TX). Benzo[a] pyrene (BAP) was obtained from K & K Laboratories (Irvine, CA). DMSO (J.T. Baker Co., Phillipsburg, N J) was the solvent used for AAF, AFB, BNZ, and BAP. D M N was diluted in Williams' medium E (WE, Gibco). Animals Rodents. Male Fischer-344 rats (175-310 g), B6C3Fa mice (24-30 g), and Syrian golden hamsters (100-150 g) were received from the following sources: rats and hamsters from Simonsen Laboratories (Gilroy, CA); rats from Hilltop Laboratory Animals (Chatsworth, CA), and rats and mice from Charles River Laboratories (Wilmington, MA). All animals were housed in polypropylene cages with hardwood-chip bedding in rooms maintained on a 12-h light-dark cycle. Rats were housed 3 per cage, mice were housed 10 per cage, and hamsters were housed 2 per cage. All animals received Purina Rodent Chow No. 5002 (Ralston Purina Co., St. Louis, MO) and deionized, and charcoal filtered (0.5 t~m pore size) tap water ad libitum.
93
Humans. Human liver tissue was received from organ transplant donors at Stanford University Hospital (Stanford, CA). All donors were between the ages of 20 and 35 years and were nonsmoking, cerebral accident victims reported to be in excellent health at the time of the accident. All donors were Caucasian males except for 1 Caucasian female donor. Prior to organ removal, all victims were maintained on life-support systems, and a minimum dose of vasopressin was used to prevent antidiuresis.
and filtered through sterile gauze. Hepatocyte viability was determined by Trypan blue exclusion and ranged from 80 to 95% for rats, monkeys, and humans and from 70 to 85% for hamsters and mice. Approximately 5 × 105 viable cells were seeded into each well of a Falcon (Oxnard, CA) 9.6-cm 2, 6-well tissue culture plate. Each well contained a 25-mm round Thermanox coverslip (BioLabs, Northbrook, IL) and 4 ml of WE supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA). All perfusion and culture solutions were supplemented with 50/~g/ml gentamicin sulfate (Sigma). Cells were incubated for 1.5-2 h at 37 ° C in a 95% air : 5% CO 2 incubator to allow attachment of the cells to the coverslips. Cultures were then washed once with 37 ° C WE supplemented with L-glutamine (WEI) and incubated in 2 ml WEI containing 10 /~Ci/ml [3H]-thymidine (approximately 80 Ci/mmole, New England Nuclear, Boston, MA) and the test compound for 17-21 h.
Hepatocyte isolation and culture
Autoradiography
Hepatocyte cultures were prepared from rats and mice as previously described (Mitchell and Mirsalis, 1984). Livers were perfused in situ for approximately 2 min with a solution of 0.5 mM ethyleneglycolbis(fl-amino ethyl ether)-N,N'-tetraacetic acid (EGTA, Sigma Chemical Co., St. Louis, MO) in Hanks' balanced salt solution without Ca 2+ or Mg 2+ (Gibco, Santa Clara, CA), followed by 12 min in a 3 7 ° C solution of collagenase type I (Sigma, 100 U / m l ) in WE. All perfusion and culture solutions were maintained at pH 6.8-7.0. Hamster hepatocytes were isolated using essentially the same techniques, except that 15% less collagenase and a longer perfusion time (17 min) at a lower flow rate were used, as reported by Green et al. (1984) to be optimal. Monkey and human livers were perfused using the biopsy perfusion method previously described (Reese and Byard, 1981). Perfusion times for human and monkey liver sections ranged from 30 to 50 min. A single-cell suspension of hepatocytes was obtained by combing out cells from the perfused liver into a petri dish containing 37 ° C collagenase solution. Cells were collected by a 5-min centrifugation at 50 × g, resuspended in cold medium,
Cultures were washed 3 times with WEI followed by 10 min in 1% sodium citrate to swell the cells, three 10-min washes in 1 : 3 glacial acetic acid/ethanol, and 6 washes with deionized water. Coverslips were mounted to microscope slides using Permount and dipped in Kodak NTB-2 nuclear-track emulsion diluted 1 : 1 with deionized water. Slides were exposed for 7 days at - 2 0 ° C, then developed and stained as previously described (Mitchell and Mirsalis 1984).
Monkeys. Male cynomolgus monkeys (3.0-4.9 kg) were received from Primate Imports (Port Washington, NY) and were housed singly in a room maintained on a 12-h light-dark cycle. Animals were fed Certified Monkey Chow (Purina) supplemented with fruit, peanuts, vitamins, and tap water ad libitum. Liver sections were obtained from untreated or vehicle control-treated monkeys that were sacrificed for other studies.
Measurement of UDS Quantitative autoradiographic grain counting was accomplished as previously described (Mitchell and Mirsalis, 1984). An area of a slide was randomly selected, and 30 morphologically unaltered cells were counted using an A R T E K Model 880 or 980 colony counter interfaced to a VAX 8800 computer (Digital Equipment Corp.). The highest of two nuclear-size areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). 3 slides were scored for each concentration for a total of 90 cells/ concentration. The percentage of cells in repair is defined as those exhibiting at least 5 NG.
94
Hepatocyte characterization assays Hepatocyte characterization assays were performed using freshly isolated liver cells in suspension culture, 1 × 1 0 6 cells/ml. The functional integrity of the isolated cells was determined by measuring the rate of urea synthesis using 10 mM NH4CI as the substrate in the presence of 10 mM ornithine over a 60-min incubation period (Story et al., 1983). Urea was analyzed by the method previously reported by Foster and Hochholzer (1971). BAP hydroxylase, p-nitroanisole-O-demethylase (pNAD), and p-nitrophenol conjugation (pNPC) were measured to assess the activities of the xenobiotic metabolizing systems of hepatocytes, pNAD activity was calculated from the quantity of pNPC measured after deconjugation (Moldeus et al., 1976). pNPC conjugation was determined by calculating the difference between the quantity of pNPC measured in a deconjugated sample and pNPC in a sample that was not treated with deconjugating enzymes. BAP hydroxylase activity was determined using 0.1 mM [3H]-BAP incubated with cells for 30 min. Samples of incubation medium and cells were partitioned between hexane and an alkaline (0.25 N NaOH) aqueous solution containing 40% ethanol (Steward and Byard, 1981). The percentage of BAP metabolized was calculated by determining the quantity of radioactivity in the aqueous phase. Reduced glutathione (GSH) was measured by the method reported by Hissin and Hill (1976). DNA content was determined by the method reported by Richards (1974). Incubation for the urea synthesis and BAP hydroxylase assays were car-
ried out in Waymouth's 752/1 medium supplemented with hormones, fatty acids, and bovine serum albumin (Green et al., 1983). For the pNAD and pNPC conjugation assays, cells were incubated in Krebs-Ringer bicarbonate buffer plus 2% BSA (Modeus et al., 1976). Results
Chemical carcinogens from 4 chemical classes were examined for their abilities to induce UDS in primary hepatocyte cultures derived from rat, mouse, hamster, monkey, or human livers. Experiments with human hepatocytes were characterized by sex, age, and UDS response to culture medium or 74 /~g/ml (1.0 mM) DMN (Table 1). Both quantitative and qualitative differences were observed in the responses between species (Table 2). Medium control and 1% DMSO control values for all cultures yielded < 0 NG (< 8% IR, Table 3). Exposure of hepatocyte cultures to DMN (Fig. 1) or AFB (Table 4) yielded dose-related increases in UDS in all species; however, variation in the magnitude of response was noted between species. Exposure of isolated rat or human hepatocyte cultures to 4AB yielded positive UDS responses for both species (Table 5). Following exposure to BNZ, rat, hamster, and human hepatocyte cultures yielded dose-related increases that peaked at 1.0-5/xg/ml, followed by a decrease in NG (Fig. 2). Conversely, mouse and monkey hepatocyte cultures yielded < 0 NG following exposure to BNZ. Exposure of rat, hamster, and human
TABLE l C H A R A C T E R I Z A T I O N OF T H E H U M A N H E P A T O C Y T E E X P E R I M E N T S BY SEX, AGE, A N D UDS RESPONSE TO C U L T U R E M E D I U M OR D M N Exp.
Sex
No.
1 2 3 4 5
M M M M F
Age
UDS control compounds
(years)
Medium
20 20 35 28 26
74 t l g / m l D M N
NG
% IR
NG
% IR
- 1.5 - 5.6 -4.5 - 9.0 - 6.4
0 8 2 1 3
24.3 23.8 28.0 22.6 21.2
96 91 91 90 88
NG, net gr ain s/n ucleus; % IR, percentage of cells in repair.
95 TABLE 2 SUMMARY OF UDS RESPONSES BETWEEN SPECIES FOLLOWING
Chemical
Human Carcinogen
Rat
Mouse
DMN AAF BNZ 4AB BAP AFB
P S P P
++++ + + + + + + + + + + +
++++ NT + + +
(37) a (5) (1.0) (1.0) (100) + + + + (0.1)
S P
(37) (10) (20) (20) (1.0)
IN VITRO TREATMENT
Hamster
Monkey
Human
++++ (3.7) + + + + (0.1) + + + + (1.0) NT + (20) NT
+++ (740) (50) (10) NT + + + + (10) + + (0.05)
++++ (3.7) ++++ (5) + + (1.0) + + + + (20) + + + (0.5) ++++ (1.0)
a The number in parentheses is the concentration required to elicit the maximum positive U D S response. P, known human carcinogen; S, suspected human carcinogen. -, <0NG; +-, < 0 N G ( % I R > 1 5 % ) ; +,0-5NG; ++,5-10NG; +++,10-20NG; ++++, >20NG;NT,
j"
//
5C
~" Homster
4(
i 30
" ...%Lg"
i!
p..Humcn
NG
,' ,'
2C
"- - . . . .
/ , Rat
_~ ,~..~..:~,~--..-:.CT. d i
"*- -J " , ~ ' ~ . . . . . . . . . . . .
2~.:1
Y ," ~-.-%use L ,;
lc
i
~
),
_ .....
/
/,
/:
,~
/ / !
//
o
0 05
I
I
05
5
[ DisN]m~
Fig. 1. Induction of U D S in rat, mouse, hamster, monkey, or human hepatocytes following in vitro exposure to D M N .
hepatocyte cultures to AAF yielded dose-related increases in NG; however, mouse and monkey hepatocyte cultures yielded < 0 N G following
nottested.
exposure to AAF (Table 6). Exposure of monkey and human hepatocyte cultures to BAP yielded dose-related increases in NG; however, BAP induced a very weak U D S response in hamster hepatocyte cultures, an equivocal response in mouse hepatocyte cultures, and failed to induce UDS in cultures of rat hepatocytes (Table 7). Isolated hamster, monkey, and human hepatocytes were characterized and compared by measuring several parameters of cell function (Table 8) that have been used in previous studies with rat hepatocytes (Green et al., 1983). The rate of urea synthesis, a hepatocyte-specific function, was assessed as a general indicator of cell viability and organelle integrity (Story et al., 1983). Two cytochrome P450-associated activities, BAP hydroxylase and pNAD, were determined to obtain a measure of the ability of the cells to perform phase I oxidations of xenobiotics and pNPC, a phase II reaction, was assayed. Glutathione, an important
TABLE 3 I N D U C T I O N O F U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D F R O M R A T , M O U S E , H A M S T E R M O N K E Y , O R H U M A N L I V E R F O L L O W I N G N O C H E M I C A L T R E A T M E N T O R E X P O S U R E T O 1% D M S O
Species
Control/untreated
Rat Mouse Hamster Monkey Human
-8.2+--0.9 - 11.6 + 1.6 - 3.5 +- 0.6 - 4.8 + 0.8 - 6.1 +- 0.8
N G + S.E.
1% D M S O % IR (N) 2 0 2 2 5
(7) (2) (1) (2) (5)
N G + S.E. -9.3+--0.8 - 7.3 +- 2.0 - 7.4 +__1.0 - 4.3 + 1.0 - 8.4 + 0.3
% IR (N) 1 1 0 3 2
(7) (3) (1) (2) (5)
N G , net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair; N, number of Expts.
96 TABLE 4 I N D U C T I O N OF U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D H U M A N F O L L O W I N O E X P O S U R E TO A F L A T O X I N B~ Species
Concentration
N G _+ S.E.
% IR
Rat
0.~05 0.001 0.005 0.01 0.1 1.0
- 3 . 1 - + 2.2 0.4_+ 5.2 5.6_+ 0.6 20.9±16.5 28.2_+ 3.2
12 22 52 85 99
Mouse
0.0005 0.0l 0.05 0.1 1.0
-6.9_+ -9.1_+ -6.5± -2.2_+ 9.3_+
0.9 1.0 1.8 1.0 2.2
2 1 3 12 68
Mo~ey
0.001 0.005 0.01 0.05
-4.9_+ -3.4_+ -2.3_+ 7.6_+
0.1 1.1 1.0 1.5
3 7 11 66
Human
0.~05 0.001 0.~5 0.01 0.1 1.0
-6.3_+ -9.9± -6.4_+ 3.0_+ 15.0_+ 53.8_+
1.4 1.3 0.7 5.7 5.1 5.6
2 3 8 47 77 100
(#g/ml)
Expt. 1
F R O M RAT, MOUSE, M O N K E Y , OR
Expt. 2 N G -+ S.E. -3.4-+1.5 -3.0±1.5 0.8-+3.4 1.2-+2.7 11.1±5.1 16.8±3.2
% IR 4 12 25 7 64 83
NG, net grains/nucleus; S.E. Slide-to-slide variation; % IR, percentage of cells in repair.
component in the cellular defense systems against active metabolites and D N A content were also measured.
The total D N A content was higher in monkey and human hepatocytes than in hamster hepatocytes. Glutathione content was the same for all
TABLE 5 I N D U C T I O N O F U D S BY P R I M A R Y H E P A T O C Y T E C U L T U R E S D E R I V E D F R O M R A T O R H U M A N LIVER F O L L O W I N G E X P O S U R E TO 4 - A M I N O B I P H E N Y L Species
Rat
Human
Concentration
Expt. 1
(/~g/ml)
N G -+ S.E.
% IR
Expt. 2
0.1 0.5 1
1.4_+0.5 15.9_+9.1 16.6_+3.7
31 69 86
0.1 0.5 1.0 5 10 20
19.7_+3.3 38.1 _+6.0 36.5 _+5.3 31.5_+2.1 34.1 + 2.9 22.5 -+ 3.7
92 100 100 98 99 96
N G , net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair.
N G _+S.E.
% IR
19.9_+2.4
96
30.6 _+8.6
98
10.2___6.9
61
97 TABLE 6 INDUCTION OF UDS BY PRIMARY HEPATOCYTE CULTURES DERIVED FROM RAT, MOUSE, HAMSTER, MONKEY OR HUMAN LIVER FOLLOWING EXPOSURE TO 2-ACETYLAMINOFLUORENE Species
Concentration
Rat
Mouse
Expt. 1
(/~g/ml)
NG + S.E.
% IR
0.05 0.1 0.5 1.0 5 10
NS 14.1 + 6.2 20.1+ 1.3 20.4 + 12.5 29.5 4-13.9 27.5 + 1.3
76 96 69 91 99
0.1 0.5 1.0 5 10 50
AFB a
Expt.
-7.6.
1.3
1
-3.9--- 2.6
15
- 8.3 +- 2.5 T
10
11.3 ___12.5 53.2 5:21.5 36.5 +- 12.8
31 97 91
1.0
Hamster
0.05 0.1 0.5
Monkey
0.05 0.1 0.5 1 5 50
DMN a
- 5.4 +- 5.5 +_ -4.9+-4.2+ -6.7---
0.05 0.1 0.5 1.0 5 10
N G +_S.E.
% IR
15.2+4.9
63
28.5 +4.8
85
-7.3+0.9 -6.2+2.9 -9.6+1.5 - 9.9 + 1.2
6 7 2 0
9.3 + 2.2
68
1.1 0.6 0.8 2.3 0.8
0 0 1 0
-6.5+1.3
4
-5.7+0.2 -2.8+0.5
2 6
18.3 +_ 5.5
90
15.6 + 1.1
84
- 5.3 5 : 0 . 6 - 3.3 5 : 4 . 2 12.2_+ 7.6 16.7 +- 2.8 7.8--- 4.3 19.3 + 2.6
11 22 67 88 51 90
19.6+2.0
89
20.1+6.5
87
1480
Human
2
1
NG, net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair; NS, not scored; T, toxic. a Data is shown for concurrent positive control.
5O i
Hamstep
4 0 ¸,
Rat
A
[",I N.G
/ . J Human
]01
o 10
~
......... !':-4, ~ "--.
/
~onk~zj..~...
. . . . . . . 0[ /,,
',
---~.}_ . . . . . . . . . . . "~'--~ ~-.---,t
01I
, 10
110 210 35
[BNZ] u g / m ]
Fig. 2. Induction of UDS in rat, mouse, hamster, monkey, or human hepatocytes following in vitro exposure to BNZ.
species tested. BAP hydroxylase activity was higher i n h a m s t e r a n d m o n k e y h e p a t o c y t e c u l t u r e s (4.43 a n d 2.92 n m o l e s / 3 0 m i n X 10 6 cells) a n d w a s sign i f i c a n t l y l o w e r i n h u m a n h e p a t o c y t e c u l t u r e s (0.73 nmole/30 r a i n × 10 6 cells), p N A D activity was higher in isolated hamster and monkey hepatoc y t e s (34.7 a n d 39.0 n m o l e s / 3 0 r a i n x 10 6 cells) t h a n i n h u m a n h e p a t o c y t e c u l t u r e s (14.2 n m o l e s / 30 m i n × 10 6 cells), p N P C a c t i v i t y w a s a p p r o x i m a t e l y t h e s a m e f o r all s p e c i e s . T h e r e w e r e l a r g e standard deviations in urea synthesis rates be-
98 TABLE 7 I N D U C T I O N OF UDS BY PRIMARY HEPATOCYTE CULTURES D E R I V E D FROM RAT, MOUSE, HAMSTER, M O N K E Y OR H U M A N LIVER F O L L O W I N G EXPOSURE TO BENZO[a]PYRENE
Species
Concentration (/~g/ml)
Rat
AAF a
Mouse
AFB a
Hamster
DMN a Monkey
0.1 0.5 1.0 5 10 20 100
Expt. 2
N G ± S.E. -7.2+ -7.2_+ -5.4_+ -5.4_+ -3.9_+ - 3.2-+
0.17 1.1 1.1 3.2 0.4 0.8
% IR 1 3 2 11 2 1
0.5 5
20.8 _+ 5.0
88
0.5 1.0 5 10 20
- 12.2_+ 0.6 - 10.5 -+ 1.9 -0.7-+ 3.8 -3.9-+ 1.2 - 1.1_+ 2.1
1 4 33 19 29
1.0
9.3_+ 2.2
68
0.1 0.5 1.0 10 20
1.3 0.3 1.6 2.5 0.8
1 0 6 21 26
0.1
30.9_+ 13.6
97
0.1 0.5
-3.9-+ 0.9 - 2 . 5 ± 0.4 0.4± 1.3 4.0-+ 1.7 20.3 ± 2.8 17.2± 3.3
0 6 20 33 94 100
1 5 10 20
Human
Expt. 1
0.1 0.5 1.0 5 10 20
-7.8_+ -6.6_+ -4.7+ -0.8-+ 1.1 -+
1.1± 22.0-+ 11.9± 13.0± 21.0-+
0.2 3.1 3.3 5.4 4.4
34 94 78 77 88
NG_+ S.E.
-8.4_+ 0.4
% IR
2
-2.6-+ 0.1
8
42.8± 8.5
97
-6.0-+ 1.3
2
3.6-+ 0.5
38
29.3_+ 10.9
95
-8.2+
0.8
1
5.4-+ 4.4
49
7.8-+ 7.2
52
NG, net grains/nucleus; S.E., slide-to-slide variation; % IR, percentage of cells in repair.
a Data is shown for concurrent positive control.
tween individual hamsters or humans, indicating individual variation with respect to this parameter. Discussion Studies have shown that considerable interspecies variation exists in hepatocyte metabolism (Caldwell et al., 1978; Green et al., 1984; Maslan-
sky and Williams, 1982). In order to assess the relevance of the information obtained from an in vitro endpoint, it would be useful to evaluate the endpoint differences between the test species and humans. Few comparative studies using hepatocytes from different species, including primates, have been performed, probably because largeanimal hepatocyte isolation techniques have only
99 TABLE 8 F U N C T I O N A L CHARACTERISTICS OF ISOLATED HEPATOCYTES
DNA (/~g/106 cells) Glutathione (nmoles/106 cells) Benzo[ a ]pyrene hydroxylase (nmoles/30 min. 106 cells) p-Nitroanisole-O-demethylase (nmoles/30 rain. 106 cells) p-Nitrophenol conjugation (nmoles/30 min. 106 cells) Urea synthesis (nmoles/min. 106 cells)
Hamster (n = 4)
Monkey (n = 2)
Human (n = 4)
10.10_+0.5 22.30+_4.9
15.5 18.1 a
15.6 _+3.6 21.4 +2,6
4.43 -+ 1.1
2.92
34.70_+2.8 7.80 _+4.3 9.11 + 5.22
39.0 a 5.00 a 4.10 a
0.73 -+ 0.44 14.2 _+4.0 4.09 + 1.2 2.75 _+2.6
aN=l.
recently been developed (Green et al., 1983; Reese and Byard, 1981), and fresh primate tissue is often difficult to obtain. The in vitro UDS assay using rodent hepatocytes has been shown to be useful for assessing the genotoxic potential of compounds from a wide variety of chemical classes (Kornbrust and Barfknecht, 1985; Probst et al., 1980; Williams et al., 1982). Extending these techniques to include in vitro UDS in hepatocytes from primates provides a means for validating this rodent model. In these experiments, we have examined 6 chemicals representative of 4 chemical classes in the in vitro UDS assay using hepatocyte cultures derived from rat, mouse, hamster, monkey, and human liver. These chemicals include DMN (nitrosamine), AFB (mycotoxin), BAP (polycyclic aromatic hydrocarbon). BNZ, 4AB, and AAF (aromatic amines), all of which are known or suspect human carcinogens (IARC, 1973, 1978, 1982). The metabolic assays were conducted to demonstrate the viability and functional capabilities of the isolated hepatocytes from different species. These data were also examined to determine whether there was a correlation between a positive UDS response and metabolic activity. Although the glutathione content, BAP hydroxylase activity, pNAD activity, pNPC activity, and urea synthesis rates indicated that the cultures were metabolically competent at the time of chemical exposure, no direct relationship between metabolism and UDS response was observed for any compound
tested. This is not surprising considering the complexity of the metabolic pathways and the number of different isozymes that are involved in the biotransformation of carcinogens. In the reported studies, changes in cytochrome P450 and associated activities during the culture period were not determined. However, other investigators have reported species-related differences in the stability of P450 of cultured hepatocytes (Blaauboer et al., 1985; Malansky and Williams, 1982) which would affect the UDS response observed, although the kinetics of P450 loss and rate of metabolic activation have not been assessed. Additionally, differences in DNA repair, rate of DNA adduct formation, or detoxification of reactive intermediates could also affect the UDS response between species. Dose-related increases in UDS were observed in all hepatocyte cultures treated with DMN. Hamster hepatocyte cultures yielded the strongest response to DMN exposure (Fig. 1). The extreme responsiveness of hamster hepatocyte cultures to DMN in this assay has also been described by Kornbrust and Barfknecht (1984) and by Katoh et al. (1982), who measured cytotoxicity and mutation rate in V79 cells cultured with rat or hamster hepatocytes. They found that the mutation frequencies were substantially higher in cells co-cultured with hamster hepatocytes than with rat hepatocytes. Data from McQueen et al. (1983) does not demonstrate the extreme responsiveness of hamster hepatocytes to DMN exposure.
100 AFB treatment of primary hepatocyte cultures isolated from rat, mouse, human, or monkey liver yielded dose-related increases in UDS (Table 4). Human hepatocyte cultures yielded the strongest response to AFB exposure, which correlates well with the reported epidemiological evidence for human liver cancer (Shank et al., 1972). Conversely, mouse hepatocyte cultures were the least responsive to AFB exposure. One possibility for this weak response may be that mouse hepatocytes are more efficient in AFB detoxification (Decad et al., 1979). Striking qualitative differences were observed between species following exposure to aromatic amines. Human, rat, and hamster hepatocyte cultures yielded dose-related increases following 4AB (Table 5; human and rat only) or AAF exposure (Table 6), and positive responses following exposure to BNZ (Fig. 2), whereas mouse and monkey hepatocyte cultures yielded < 0 N G following exposure to AAF or BNZ. Our findings with BNZ agree with those reported by Hill and Probst (personal communication) and McQueen and Williams (1983), who demonstrated an inhibition of DNA repair at high BNZ concentrations which explains the observed decrease in UDS. The response of mouse hepatocyte cultures to AAF exposure discussed in this paper contrasts with the findings of McQueen et al. (1983), who report an average of 26.3 N G at 10 -3 M, and of Probst and Hill (personal communication), who report between - 1 . 2 5 and 11.31 N G in B6C3F~ mice. In contrast, our findings agree with those of Butterworth and Smith-Oliver (1987) and Millner et al. (1987), who also report a negative UDS response for AAF in B6C3F 1 mouse hepatocytes. Sakai et al. (1976) demonstrated that in vitro covalent binding of N-OH-AAF to D N A in rat hepatocyte nuclei was twice that observed for the mouse. One of the pathways postulated for AAF metabolism to one of its ultimate carcinogenic metabolites involves sulfotransferase (DeBaun et al., 1970). It has been shown that rat liver has greater N-hydroxy-AAF sulfotransferase activity than mouse or hamster hepatocytes. This may account for the response by mouse hepatocyte cultures to AAF; however, hamster hepatocyte cultures exhibited the strongest UDS response following exposure to this chemical. This difference has also been ob-
served in vivo, since rats treated with AAF show significant increases in UDS, whereas B6C3F~ mice do not show increases in UDS following in vivo exposure to AAF (Mirsalis et al., 1985). Disher and Randerath (1987) report that 7--12 times more adducts were formed in rat liver than in mouse liver following AAF exposure. AAF is a potent hepatocarcinogen in rats but is only weakly carcinogenic in mice (Tisdel et al., 1984). Isolated rat hepatocyte cultures consistently yielded negative responses following exposure to BAP when tested to the limit of solubility (Table 7). This response contrasts with data reported by several authors (McQueen et al., 1983; Mitchell et al., 1983; Probst et al., 1980). A number of factors may affect the outcomes of genetic toxicology tests, including diet, animal supplier, bedding, and disease state. We are confident that the lot of BAP used or technical problems were not responsible for the negative UDS response, since human and monkey hepatocyte cultures yielded strong positive responses. Although mouse hepatocyte cultures yielded < 0 N G following exposure to BAP, the 70 IR was very high, with a maximum of 33% IR. Therefore, we consider treatment with BAP to yield an equivocal response in mouse hepatocyte cultures. BAP was weakly genotoxic in hamster hepatocytes treated in vitro. Initial BAP hydroxylase activity did not correlate with the genotoxicity of BAP in hepatocyte cultures, probably because this assay measures total BAP metabolism making no distinction in species differences in activation and detoxification reactions. Quantitative and qualitative differences in UDS responses were observed between all 5 species. The appropriate species to use for testing may depend on the class of chemical examined. In the case of nitrosamines, mycotoxins, and aromatic amines, rat hepatocyte cultures yielded results similar to human hepatocyte cultures, whereas BAP treatment yielded contrasting results in rat and human hepatocyte cultures. Although specific differences between chemical classes may occur, rat hepatocyte cultures appear to be the closest model for human hepatocyte cultures. In general, mouse and monkey hepatocyte cultures seem to be much less responsive to several of the chemicals selected for this study compared with human hepatocyte cultures. Therefore, these species may
101 b e less s u i t a b l e f o r s c r e e n i n g c h e m i c a l s i n t h i s a s s a y w h e n t h e e s t i m a t i o n o f h u m a n r i s k is t h e major objective.
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