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SCE induction in human lymphocytes by combined treatment with aniline and norharman
Mutation Research, 101 (1982) 165-172 Elsevier Biomedical Press
165
SCE induction in human lymphocytes by combined treatment with aniline and norharman S. Takehisa and N. Kanaya Department of Biology. Keio University. Yokohama 223 (Japan)
(Received 18 July 1981) (Revision received 22 October 1981) (Accepted 26 October 1981)
Summa~ In human blood lymphocytes, aniline was unable to induce an increase of SCEs in vitro with or without metabolic activation by $9 mix. p-Aminophenol, one of the C-hydroxylation metabolites of aniline in the body, however, increased the SCE frequencies of lymphocytes at a concentration of 10-4 M. The addition of norharman to aniline plus $9 mix increased the SCE frequencies. This increase, however, was due to the SCE-inducing activity of norharman. These data show that the addition of norharman, which enhances the sensitivity of the Salmonella/microsome test, does not produce an enhancement of the sensitivity of the SCE test.
Abe and Sasaki (1977) reported that aniline induced SCEs in cultured Chinese hamster cells in vitro without any metabolic activation system. Since SCEs are an excellent indicator of mutagenic and carcinogenic activity, it appears that aniline is a potential mutagen/carcinogen. In the Ames Salmonella test, however, aniline was not mutagenic in vitro even with metabolic activation with $9 mix (McCann et al., 1975; Nagao et al., 1977b). Only with co-incubation with norharman in the presence of $9 mix, was aniline found to be mutagenic (Nagao et al., 1977b). These results prompted us to determine whether aniline could induce SCEs in other cell types. Here we report the results obtained with a human peripheral blood lymphocyte system. In brief, aniline with or without $9 mix did not induce SCEs. Cells treated with norharman and aniline did have an increased level of SCEs. This increase, however, was due to the SCE-inducing activity of norharman.
Materials and methods Samples of peripheral blood were drawn from a normal healthy male and heparinized (40 units heparin/ml of blood). About 0.4 ml of the heparinized whole 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
166
blood was added to a culture bottle containing 5 ml of McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml) and streptomycin (100/~g/ml). Phytohemagglutinin (0.1 ml, PHA-M, Gibco) and bromodeoxyuridine (BrdUrd, 20/~M final concentration) were added at the beginning of culture to each bottle, which was then gassed with air containing 5% CO z. The bottles were tightly capped and incubated at 37°C for 24 or 48 h in the dark. The test chemicals used were aniline hydrochloride (Wako Pure Chem.), paminophenol (Wako Pure Chem.), cyclophosphamide (Sionogi), mitomycin C (P-L Biochem., Milwaukee), and norharman (Aldrich, purity 98%). They were dissolved immediately before use in sterilized H20, or (norharman) in dimethyl sulfoxide (DMSO). Cyclophosphamide and mitomycin C were used as positive controls. For treatment in vitro without metabolic activation, the cells were exposed to the test chemicals after 24 h of culture. After the addition of the test chemicals to the culture bottles, the bottles were gassed again with 5% CO 2 in air and incubated for an additional 48 h. When treated in vitro with a metabolic activation system, the cells grown without chemicals were collected after 48 h of culture by centrifugation at 1500 rpm for 5 min. The supernatant was removed by aspiration and the medium was replaced by 5 ml serum-free McCoy's 5A medium containing streptomycin (100 t~g/ml) and penicillin (100 units/ml). The $9 mix (0.3 ml unless otherwise stated), and subsequently the test chemicals, were added to the medium. The final concentration of D M S O in the medium was 1% for all cell cultures including the controls. The cells were exposed for 2 h at 37°C in the dark. During exposure, the culture bottles were shaken gently for several seconds every 30 min. After this the cells were washed 3 times with McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml) and streptomycin (100 /~g/ml). Cells were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml), streptomycin (100/xg/ml), and 20/~M BrdUrd for an additional 24 h as described above. The control cell populations were treated in the same way except for the addition of the test chemicals and $9 mix. The $9 mix consisted of i0% (by volume) $9 rat-liver extract (purchased from Oriental Yeast Co., Tokyo; protein content, 15.2 rag/S9 ml), which was prepared from male Sprague-Dawley rats injected with phenobarbital and 5,6-benzoflavone according to the procedure described by Ames et al. (1975) and 8 × 10 3 M MgC12; 3.3X 10 2M KC1; 5 × 10 3M glucose 6-phosphate; 4 X 10 3M N A D P ; and 10 i M N a 2 H P O 4 - N a H 2 P O 4 (pH 7.4). For the last 3 h of culture, Colcemid (5.4 × 10 7 M) was added. The cells were collected by centrifugation, treated with 0.075 M KCI for 15 min, and then fixed in methanol-acetic acid (3: 1). Slides were processed by a modification of the F P G technique (Perry and Wolff, 1974; Takehisa and Wolff, 1978). Usually 25-50 metaphases were scored from each culture.
167
Results The blood samples used in these experiments were obtained 15 times from 1 person over 1 year. The background level of SCE frequency ranged from 5.0 to 8.8 SCEs per cell. In preliminary experiments the lymphocyte cultures treated with aniline at concentrations of 2 × 10 3 and 10-2 M for the last 24h of culture showed a very slight increase in SCE frequency above the control level. (In 4 separate experiments the SCE/cell values in treatments with aniline ranged from 6.1 -+ 0.7 to 7.4 ± 0.9, those in the control from 5.0 -+ 0.4 to 5.7 - 0.6.) Effective concentrations of mitomycin C necessary to produce a significant increase of SCEs began at 10 ng/ml. (SCE/cell values in mitomycin C: 80 n g / m l , 25.1 -+ 1.3; 20 n g / m l , 17.2 ± 1.2 19.8 ± 2.0; 10 n g / m l , 8.7 ± 0.6; those in the control, 5.0 ± 0.4-5.7 ± 0.6.) This was the positive control. Next, it was decided to treat the blood cultures with chemicals for the last 48 h of culture and to adopt a 20 n g / m l concentration of mitomycin C as a control. In the treatments for 48 h with concentrations of aniline ranging from 10-2 to 1 0 - 4 M (Table 1), few or no cells were found at the second mitosis after treatment with a concentration of 10 2 M. This was the same result as reported for Chinese hamster cells (Abe and Sasaki, 1977). At 10 3 and 10-4 M an increase of SCEs was not apparent, although the SCE frequency was occasionally slightly higher than that of the control (Table 1). Aniline is metabolized in the body by liver microsomes to p-aminophenol, a C-hydroxylated metabolite of aniline, or to phenylhydroxylamine, an N-hydroxylated metabolite of aniline (Kiese, 1966; Uehleke, 1971). We examined the effect of p-aminophenol on the incidence of SCEs. p-Aminophenol increased the yield of SCEs at a concentration of 1 0 - 4 M (Table 1). Treatment with p-aminophenol at above 10-4 M was not tried, because the treatment at 10 4 M produced few cells at the second mitosis, suggesting mitotic inhibition. Because the results obtained with p-aminophenol seemed to suggest that the present blood culture system was not able to activate aniline, the in vitro metabolic activation system, $9 mix, was added to the cultures. After 48 h of culture, the cells were exposed to aniline at 10 3 - 1 0 - 5 M for 2 h in the presence of $9 mix. As shown in Table 2, the effectiveness of the addition of $9 mix in producing an active metabolite of cyclophosphamide was made evident by an increase of SCEs in cells exposed to cyclophosphamide at 10 -5 M (column 11 in Table 2). However, treatment with aniline combined with $9 mix did not result in an increase in SCEs (columns 1-3 in Table 2). Nagao et al. (1977b), using Salmonella typhimurium TA98 in the Ames mutation test, found that aniline did not show any mutagenic activity even with co-incubation with $9 mix, and that addition of norharman to the co-incubation mixture of aniline with $9 mix resulted in a dose-dependent mutagenicity of aniline on TA98. Norharman itself did not display any mutagenic activity. Therefore, the effect of the addition of norharman to blood culture treated with aniline in the presence of $9 mix was examined here. As shown in Table 2, the treatment with aniline plus norharman in the presence of $9 mix increased the SCE frequencies (columns 4 and 5 in Table 2). This did not imply simply that aniline has
10 4 M 10 S M 10 6 M I0 7 M
20ng/ml
p-Aminophenol
MitomycinC 5.2 ~ 0.4 (25)
17.6+1.3"(25)
N.M. 6 . 7 + 0 . 6 ** (25) 6 . 6 ~ 0 . 6 " * (25)
I
SCEs per cell
7.6-+0.7 (25)
13.6+0.7"(25)
7 . 8 ÷ 0 . 5 (50) 7 . 8 - 0 . 4 (50)
I1
AFTER
* (]6) (39} (24) (47)
C.F.
11.9; 1 . 0 * ( l l )
11.3 + 0 . 9 7.1 + 0 . 5 6.6÷0.5 6.5+0.4
TO
ANILINE,
7.3 + 0 . 6 (25)
15.7 ~ 0 . 9 " ( 2 5 }
10.1 ~ 0.8 * (21)
IV
EXPOSURE
8.3 ~ 0.5 ** (50) 6 . 5 - 0 . 4 (30)
III
LYMPHOCYTES
7 . 4 + 0 . 5 (25)
16.2 ' 0.8 * (25)
11.7 ~ 1 . 5 . * (1(}) 6.6+-0.4 (50)
v
p-AMINOPHENOL
AND
Each R o m a n numeral denotes a separate experiment. Each entry represents the m e a n ~ S.E. In parentheses, the n u m b e r of cells c o n t r i b u t i n g to this average. * ** t test for the S C E / c e l l value: *, significant at 1% level: ** significant at 5'~: level, c o m p a r e d with the control values of each experiment. N.M., no mitotic cell: C.F., culture failed. In Expt. lII, the S C E / c e l l value of the trcatment with p - a m i n o p h e n o l at 10 " M was taken as thc control value for the I test.
Control
10 2 M 10-3M 10 4 M
Aniline
Concentration
SCE F R E Q U E N C I E S IN H U M A N P E R I P H E R A L B L O O D M I T O M Y C I N C F O R T H E LAST 48 h O F C U L T U R E
TABLE I
12.5 + 1.0 (32) *
7.7-+0.5 (25) 8.6--+0.5 (34) 7.3 -+0.5 (25)
25.5+0.9 * (50) 10.0+0.4 ** (50) 8.6+0.5 (32)
14.6±0.7 * (50)
11.9:"0.5 * (49) 14.4+0.8 * (50)
7.6+0.6 (36)
II
* (50) * (50) ** (50) (50) (50)
8.3 + 0.4 (50)
43.7-+2.1 * (39)
15.5±0.7 20.9 + 1.4 10.0±0.5 9.0-+0.5 9.0+0.4
14.1 +0.6 * (50) 15.3+0.9 * (50)
III
Each Roman numeral denotes a separate experiment. Each entry represents the mean + S.E, In parentheses, the number of cells contributing to this average. * ** the same as in Table 1; * P<0.01; ** P<0.05. a The proportion of the $9 mix was increased here; I ml $9 mix to 4 ml McCoy's 5A medium.
8.8+0.6 (25)
7.3 +0.7 (22)
mix mix ( 4 × ) a mix mix
3 M) and $9 mix 3 M) and $9 mix
7.9+0.5 (40) 7.6±0.5 (28) 6.8+0.3 (25)
14. Control ($9 mix alone) 15. Control (DMSO plus $9 mix) 16. Control
plus $9 plus $9 plus $9 plus $9 alone
$9 mix $9 mix $9 mix norharman (10 norharman (10
19.6-+ 1.0 * (50) 7.5+0.4 (41) 6.1 +0.5 (26)
(10 -3 M) (10 -3 M) (10 4 M) (10 5 M) (10 3 M)
plus plus plus plus plus
I
SCEs per cell
11. CP (10 -5 M) plus $9 mix 12. CP (10-6 M) plus $9 mix 13. CP (10 -5 M) alone
Norharman Norharman Norharman Norharman Norharman
6. 7. 8. 9. 10.
(10-3 M) (10-4 M) (10 5 M) (10 3 M) (10 -4 M)
Aniline Aniline Aniline Aniline Aniline
1. 2. 3. 4. 5.
Treatments done at 48 h of culture.
CYCLOPHOSPHAMIDE (CP) IN THE PRESENCE OF $9 MIX
FREQUENCIES IN H U M A N PERIPHERAL BLOOD LYMPHOCYTES AFTER A 2-h EXPOSURE TO ANILINE A N D / O R N O R H A R M A N , A N D
TABLE 2
,.G
4 M) 3 M) 4 M) 4 M) 1 3 . 0 + 0 . 6 * (44) 11.9 " 1.0 (49)
EXPOSURE
parentheses,
7.9: + 0.5 (27)
13.6 ~ 0,7 * (25)
N.M. 1 0 . 7 t 0 . 6 * (50) 7 . 8 - 0.4 (50)
10.9 ' 0.5 * (50) 10,1 ~ 0.5 * (50)
II
AFTER
E a c h R o m a n n u m e r a l d e n o t e s a s e p a r a t e e x p e r i m e n t . E a c h e n t r 5 r e p r e s e n t s the m e a n * S.E. In • ** T h c s a m e as in T a b l e I; * P < 0 . 0 1 : ** P < 0 . 0 5 . N . M . , n o mitosis.
9.6 • 1.0 (14)
(10 (10 (10 (10
I 1.9 + 1.0 * ( I 1)
aniline aniline aniline aniline
I
M i t o m y c i n C (20 n g / m l )
3 M) 4 M) s M)
N o r h a r m a n (10 N o r h a r m a n (10 N o r h a r m a n (10
plus plus plus plus
LYMPHOCYTES
S C E s p e r cell
BLOOD
Control (DMSO)
3 M) 4 M) 4 M) s M)
(10 (10 (10 (10
Norharman Norharman Norharman Norharman
SCE FREQUENCIES IN HUMAN PERIPHERAL A N I L I N E F O R T H E L A S T 48 h O F C U L T . U R E
TABLE 3 WITH
OR
WITttOUT
the n m n b e r of cells c o n t r i b u t i n g to this a,,crage.
8.4 ' 0.5 (50)
16.2:- 0.8 * (25)
N.M. 10.4 * 0,7 ** (42)
11.4= 0.5 * 150)
N.M.
Ill
TO NORtlARMAN
171
intrinsic mutagenic potential. There remained the possibility that norharman itself might have SCE-inducing activity. Treatment of cells with norharman plus $9 mix also induced an increase in SCEs which indicated that norharman has some SCE-inducing capability (columns 6-9 in Table 2). Table 2 shows that the incidence of SCEs decreased with increasing concentrations of aniline in the treatments with aniline plus norharman plus $9 mix (columns 4 and 5 in Table 2). Thus, aniline seems to counteract the SCE-inducing activity of activated norharman. The frequency of SCEs induced by norharman with $9 mix was dose-dependent and increased with increasing concentration of $9 mix in the medium (columns 6-9 in Table2). The SCE-inducing activity of norharman was apparent from its single application for 48 h, as shown in Table 3. Simultaneous application of norharman and aniline for 48 h did not affect the norharman-induced SCE frequencies (Table 3). Discussion
Abe and Sasaki (1977) reported that aniline could induce SCEs in Chinese hamster cells. The results obtained here showed that aniline, even after treatment in vitro with metabolic activation by $9 mix, did not significantly increase the SCE frequencies in human lymphocytes. Aniline is considered to be inert by itself. (~niline is known to be metabolized in the body, and its oxidized metabolites have been correlated with the induction of methemoglobinemia (Kiese, 1966; Uehleke, 1971). In the usual Ames Salmonella/microsome test, aniline did not show any mutagenic potential (McCann et al., 1975; Nagao et al., 1977b). The degree of covalent binding of aniline to DNA in vivo, as expressed with a CBI (covalent-binding index), is low (Lutz, 1979). Taking these facts into consideration, the present results indicate that, in human whole blood cells, the degree of metabolic activation is lower than that in Chinese hamster cells and is too low to allow for the production of SCE-producing metabolites of aniline. Aniline is metabolized by microsomal mixed-function oxidases to the Chydroxylated or the N-hydroxylated metabolites (Uehleke, 1971). In the present experiments, p-aminophenol, one of the C-hydroxylated metabolites of aniline, increased the SCE frequencies at a concentration of 10 4M (Table 1). It should be noted that p-aminophenol is regarded as a non-carcinogen and that the Ames Salmonella/microsome test showed it to be non-mutagenic (McCann et al., 1975). Since Nagao et al. (1977b) reported that aniline was mutagenic in their norharmanenhanced Salmonella/microsome test, the SCE-inducing property of p-aminophenol found here may imply that aniline is a promutagen/procarcinogen. The $9 mix used here had aniline hydroxylase activity (108.6x 10 -9 moles p - a m i n o p h e n o l / m g protein/h), but treatment with aniline plus $9 mix did not affect the incidence of SCEs (Table2). Under the present experimental conditions, the amount of p-aminophenol produced was less than 2 X 10-5 M / 2 h/culture bottle. This was a borderline concentration in inducing an increase in SCEs, as shown in Table 1. Because human blood lymphocytes were insensitive to induction of SCEs after
172 t r e a t m e n t with aniline alone or with aniline plus $9 mix, n o r h a r m a n was a d d e d to the t r e a t m e n t with aniline plus $9 mix. This a d d i t i o n of n o r h a r m a n might be e x p e c t e d to p r o d u c e the same results as in the S a l m o n e l l a test of N a g a o et al. (1977b); i.e., e n h a n c e m e n t of the n u m b e r of SCEs detected. Such a t r e a t m e n t indeed increased the S C E f r e q u e n c y ( T a b le 2 ) . H o w e v e r , it appears that this increase of S C E s was due to the S C E - i n d u c i n g activity of n o r h a r m a n itself (Tables 2 and 3). Th e S C E - i n d u c i n g activity of n o r h a r m a n was also o b s e r v e d in the b l o o d of a n o t h e r h e a l t h y male ( S C E / c e l l , 12.8 at 10 4 M , exposure time 48 h; control S C E / c e l I , 9.4: P < 0.01). In the e x p e r i m e n t s of N a g a o et al. (1977b), n o r h a r m a n itself did not show a n y mutagenicity, and their n o r h a r m a n - t r e a t e d S a l m o n e l l a / m i c r o s o m e test because m o r e sensitive than the original S a l m o n e l l a / m i c r o s o m e test in d et ect i n g the potential m u t a g e n i c i t y of s o m e classes of chemicals ( N a g a o et al., 1977a,b, 1978: W a k a b a y a s h i et al., 1981). However, the results o b t a i n e d here suggest that the same pri n ci p l e does not work in the m a m m a l i a n S C E test. In addition, because carboline derivatives such as T r p - P - l , Trp-P-2, and 2 - a m i n o - ~ - c a r b o l i n e are able to induce S C E s in h u m a n l y m p h o b l a s t o i d cells ( T 0 h d a et al., 1980), n o r h a r m a n , a carboline derivative, may also be e x p e c t e d to induce SCEs in o t h er cell lines.
Acknowledgement We thank Dr. Sheldon W o l f f of the U n i v e r s i t y of California, San Francisco, for critically r ead i n g our manuscript.
References Abe, S., and M. Sasaki (1977) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals, J. Natl. Cancer Inst., 58, 1635-1641. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-363. Kiese, M. (1966) The biochemical production of ferrihemoglobin-forming derivatives from aromatic amines, and mechanisms of ferrihemoglobin formation, Pharmacol. Rev., 18. 1091 1161. Lutz, W.K. (1979) In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis, Mutation Res., 65, 289-356. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carinogens as mutagens in the Satmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Nagao, M., T. Yahagi, T. Kawachi, T. Sugimura, T. Kosuge, K.Tsufi, K. Wakabayashi, S. Mizusaki and T. Matsumoto (1977a) Comutagenic action of norharman and harman, Proc. Jpn. Acad., 53, 95-98. Nagao, M., T. Yahagi, M. Honda, Y. Seino, T. Matsushima and T. Sugimura (1977b) Demonstration of mutagenicity of aniline and o-toluidine by norharman, Proc. Jpn. Acad., 53(B), 34-37 Nagao, M., T. Yahagi and T. Sugimura (1978) Differences in effects of norharman with various classes of chemical mutagens and amount of S-9, Biochem. Biophys. Res. Commun., 83. 373 378. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Takehisa, S., and S. Wolff (1978) Sister-chromatid exchanges induced in rabbit lvmphocytes by 2aminofluorene and 2-acetylaminofluorene after in vitro and in vivo metabolic activation, Mutation Res., 58, 321-329. Tohda, H., A. Oikawa, T. Kawachi and T. Sugimura (1980) Induction of sister-chromatid exchanges by mutagens from amino acid and protein pyrolysates, Mutation Rcs., 77, 65 69. Uehleke, H. ( 1971 ) N-Hydroxylation, Xenobiotica, 1, 327-338. Wakabayashi, K., M. Nagao, T. Kawachi and T. Sugimura (1981) Co-mutagcnic effect of norharman with N-nitrosamine derivatives, Mutation Res.. 80, I-7.
165
SCE induction in human lymphocytes by combined treatment with aniline and norharman S. Takehisa and N. Kanaya Department of Biology. Keio University. Yokohama 223 (Japan)
(Received 18 July 1981) (Revision received 22 October 1981) (Accepted 26 October 1981)
Summa~ In human blood lymphocytes, aniline was unable to induce an increase of SCEs in vitro with or without metabolic activation by $9 mix. p-Aminophenol, one of the C-hydroxylation metabolites of aniline in the body, however, increased the SCE frequencies of lymphocytes at a concentration of 10-4 M. The addition of norharman to aniline plus $9 mix increased the SCE frequencies. This increase, however, was due to the SCE-inducing activity of norharman. These data show that the addition of norharman, which enhances the sensitivity of the Salmonella/microsome test, does not produce an enhancement of the sensitivity of the SCE test.
Abe and Sasaki (1977) reported that aniline induced SCEs in cultured Chinese hamster cells in vitro without any metabolic activation system. Since SCEs are an excellent indicator of mutagenic and carcinogenic activity, it appears that aniline is a potential mutagen/carcinogen. In the Ames Salmonella test, however, aniline was not mutagenic in vitro even with metabolic activation with $9 mix (McCann et al., 1975; Nagao et al., 1977b). Only with co-incubation with norharman in the presence of $9 mix, was aniline found to be mutagenic (Nagao et al., 1977b). These results prompted us to determine whether aniline could induce SCEs in other cell types. Here we report the results obtained with a human peripheral blood lymphocyte system. In brief, aniline with or without $9 mix did not induce SCEs. Cells treated with norharman and aniline did have an increased level of SCEs. This increase, however, was due to the SCE-inducing activity of norharman.
Materials and methods Samples of peripheral blood were drawn from a normal healthy male and heparinized (40 units heparin/ml of blood). About 0.4 ml of the heparinized whole 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
166
blood was added to a culture bottle containing 5 ml of McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml) and streptomycin (100/~g/ml). Phytohemagglutinin (0.1 ml, PHA-M, Gibco) and bromodeoxyuridine (BrdUrd, 20/~M final concentration) were added at the beginning of culture to each bottle, which was then gassed with air containing 5% CO z. The bottles were tightly capped and incubated at 37°C for 24 or 48 h in the dark. The test chemicals used were aniline hydrochloride (Wako Pure Chem.), paminophenol (Wako Pure Chem.), cyclophosphamide (Sionogi), mitomycin C (P-L Biochem., Milwaukee), and norharman (Aldrich, purity 98%). They were dissolved immediately before use in sterilized H20, or (norharman) in dimethyl sulfoxide (DMSO). Cyclophosphamide and mitomycin C were used as positive controls. For treatment in vitro without metabolic activation, the cells were exposed to the test chemicals after 24 h of culture. After the addition of the test chemicals to the culture bottles, the bottles were gassed again with 5% CO 2 in air and incubated for an additional 48 h. When treated in vitro with a metabolic activation system, the cells grown without chemicals were collected after 48 h of culture by centrifugation at 1500 rpm for 5 min. The supernatant was removed by aspiration and the medium was replaced by 5 ml serum-free McCoy's 5A medium containing streptomycin (100 t~g/ml) and penicillin (100 units/ml). The $9 mix (0.3 ml unless otherwise stated), and subsequently the test chemicals, were added to the medium. The final concentration of D M S O in the medium was 1% for all cell cultures including the controls. The cells were exposed for 2 h at 37°C in the dark. During exposure, the culture bottles were shaken gently for several seconds every 30 min. After this the cells were washed 3 times with McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml) and streptomycin (100 /~g/ml). Cells were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum, penicillin (100 units/ml), streptomycin (100/xg/ml), and 20/~M BrdUrd for an additional 24 h as described above. The control cell populations were treated in the same way except for the addition of the test chemicals and $9 mix. The $9 mix consisted of i0% (by volume) $9 rat-liver extract (purchased from Oriental Yeast Co., Tokyo; protein content, 15.2 rag/S9 ml), which was prepared from male Sprague-Dawley rats injected with phenobarbital and 5,6-benzoflavone according to the procedure described by Ames et al. (1975) and 8 × 10 3 M MgC12; 3.3X 10 2M KC1; 5 × 10 3M glucose 6-phosphate; 4 X 10 3M N A D P ; and 10 i M N a 2 H P O 4 - N a H 2 P O 4 (pH 7.4). For the last 3 h of culture, Colcemid (5.4 × 10 7 M) was added. The cells were collected by centrifugation, treated with 0.075 M KCI for 15 min, and then fixed in methanol-acetic acid (3: 1). Slides were processed by a modification of the F P G technique (Perry and Wolff, 1974; Takehisa and Wolff, 1978). Usually 25-50 metaphases were scored from each culture.
167
Results The blood samples used in these experiments were obtained 15 times from 1 person over 1 year. The background level of SCE frequency ranged from 5.0 to 8.8 SCEs per cell. In preliminary experiments the lymphocyte cultures treated with aniline at concentrations of 2 × 10 3 and 10-2 M for the last 24h of culture showed a very slight increase in SCE frequency above the control level. (In 4 separate experiments the SCE/cell values in treatments with aniline ranged from 6.1 -+ 0.7 to 7.4 ± 0.9, those in the control from 5.0 -+ 0.4 to 5.7 - 0.6.) Effective concentrations of mitomycin C necessary to produce a significant increase of SCEs began at 10 ng/ml. (SCE/cell values in mitomycin C: 80 n g / m l , 25.1 -+ 1.3; 20 n g / m l , 17.2 ± 1.2 19.8 ± 2.0; 10 n g / m l , 8.7 ± 0.6; those in the control, 5.0 ± 0.4-5.7 ± 0.6.) This was the positive control. Next, it was decided to treat the blood cultures with chemicals for the last 48 h of culture and to adopt a 20 n g / m l concentration of mitomycin C as a control. In the treatments for 48 h with concentrations of aniline ranging from 10-2 to 1 0 - 4 M (Table 1), few or no cells were found at the second mitosis after treatment with a concentration of 10 2 M. This was the same result as reported for Chinese hamster cells (Abe and Sasaki, 1977). At 10 3 and 10-4 M an increase of SCEs was not apparent, although the SCE frequency was occasionally slightly higher than that of the control (Table 1). Aniline is metabolized in the body by liver microsomes to p-aminophenol, a C-hydroxylated metabolite of aniline, or to phenylhydroxylamine, an N-hydroxylated metabolite of aniline (Kiese, 1966; Uehleke, 1971). We examined the effect of p-aminophenol on the incidence of SCEs. p-Aminophenol increased the yield of SCEs at a concentration of 1 0 - 4 M (Table 1). Treatment with p-aminophenol at above 10-4 M was not tried, because the treatment at 10 4 M produced few cells at the second mitosis, suggesting mitotic inhibition. Because the results obtained with p-aminophenol seemed to suggest that the present blood culture system was not able to activate aniline, the in vitro metabolic activation system, $9 mix, was added to the cultures. After 48 h of culture, the cells were exposed to aniline at 10 3 - 1 0 - 5 M for 2 h in the presence of $9 mix. As shown in Table 2, the effectiveness of the addition of $9 mix in producing an active metabolite of cyclophosphamide was made evident by an increase of SCEs in cells exposed to cyclophosphamide at 10 -5 M (column 11 in Table 2). However, treatment with aniline combined with $9 mix did not result in an increase in SCEs (columns 1-3 in Table 2). Nagao et al. (1977b), using Salmonella typhimurium TA98 in the Ames mutation test, found that aniline did not show any mutagenic activity even with co-incubation with $9 mix, and that addition of norharman to the co-incubation mixture of aniline with $9 mix resulted in a dose-dependent mutagenicity of aniline on TA98. Norharman itself did not display any mutagenic activity. Therefore, the effect of the addition of norharman to blood culture treated with aniline in the presence of $9 mix was examined here. As shown in Table 2, the treatment with aniline plus norharman in the presence of $9 mix increased the SCE frequencies (columns 4 and 5 in Table 2). This did not imply simply that aniline has
10 4 M 10 S M 10 6 M I0 7 M
20ng/ml
p-Aminophenol
MitomycinC 5.2 ~ 0.4 (25)
17.6+1.3"(25)
N.M. 6 . 7 + 0 . 6 ** (25) 6 . 6 ~ 0 . 6 " * (25)
I
SCEs per cell
7.6-+0.7 (25)
13.6+0.7"(25)
7 . 8 ÷ 0 . 5 (50) 7 . 8 - 0 . 4 (50)
I1
AFTER
* (]6) (39} (24) (47)
C.F.
11.9; 1 . 0 * ( l l )
11.3 + 0 . 9 7.1 + 0 . 5 6.6÷0.5 6.5+0.4
TO
ANILINE,
7.3 + 0 . 6 (25)
15.7 ~ 0 . 9 " ( 2 5 }
10.1 ~ 0.8 * (21)
IV
EXPOSURE
8.3 ~ 0.5 ** (50) 6 . 5 - 0 . 4 (30)
III
LYMPHOCYTES
7 . 4 + 0 . 5 (25)
16.2 ' 0.8 * (25)
11.7 ~ 1 . 5 . * (1(}) 6.6+-0.4 (50)
v
p-AMINOPHENOL
AND
Each R o m a n numeral denotes a separate experiment. Each entry represents the m e a n ~ S.E. In parentheses, the n u m b e r of cells c o n t r i b u t i n g to this average. * ** t test for the S C E / c e l l value: *, significant at 1% level: ** significant at 5'~: level, c o m p a r e d with the control values of each experiment. N.M., no mitotic cell: C.F., culture failed. In Expt. lII, the S C E / c e l l value of the trcatment with p - a m i n o p h e n o l at 10 " M was taken as thc control value for the I test.
Control
10 2 M 10-3M 10 4 M
Aniline
Concentration
SCE F R E Q U E N C I E S IN H U M A N P E R I P H E R A L B L O O D M I T O M Y C I N C F O R T H E LAST 48 h O F C U L T U R E
TABLE I
12.5 + 1.0 (32) *
7.7-+0.5 (25) 8.6--+0.5 (34) 7.3 -+0.5 (25)
25.5+0.9 * (50) 10.0+0.4 ** (50) 8.6+0.5 (32)
14.6±0.7 * (50)
11.9:"0.5 * (49) 14.4+0.8 * (50)
7.6+0.6 (36)
II
* (50) * (50) ** (50) (50) (50)
8.3 + 0.4 (50)
43.7-+2.1 * (39)
15.5±0.7 20.9 + 1.4 10.0±0.5 9.0-+0.5 9.0+0.4
14.1 +0.6 * (50) 15.3+0.9 * (50)
III
Each Roman numeral denotes a separate experiment. Each entry represents the mean + S.E, In parentheses, the number of cells contributing to this average. * ** the same as in Table 1; * P<0.01; ** P<0.05. a The proportion of the $9 mix was increased here; I ml $9 mix to 4 ml McCoy's 5A medium.
8.8+0.6 (25)
7.3 +0.7 (22)
mix mix ( 4 × ) a mix mix
3 M) and $9 mix 3 M) and $9 mix
7.9+0.5 (40) 7.6±0.5 (28) 6.8+0.3 (25)
14. Control ($9 mix alone) 15. Control (DMSO plus $9 mix) 16. Control
plus $9 plus $9 plus $9 plus $9 alone
$9 mix $9 mix $9 mix norharman (10 norharman (10
19.6-+ 1.0 * (50) 7.5+0.4 (41) 6.1 +0.5 (26)
(10 -3 M) (10 -3 M) (10 4 M) (10 5 M) (10 3 M)
plus plus plus plus plus
I
SCEs per cell
11. CP (10 -5 M) plus $9 mix 12. CP (10-6 M) plus $9 mix 13. CP (10 -5 M) alone
Norharman Norharman Norharman Norharman Norharman
6. 7. 8. 9. 10.
(10-3 M) (10-4 M) (10 5 M) (10 3 M) (10 -4 M)
Aniline Aniline Aniline Aniline Aniline
1. 2. 3. 4. 5.
Treatments done at 48 h of culture.
CYCLOPHOSPHAMIDE (CP) IN THE PRESENCE OF $9 MIX
FREQUENCIES IN H U M A N PERIPHERAL BLOOD LYMPHOCYTES AFTER A 2-h EXPOSURE TO ANILINE A N D / O R N O R H A R M A N , A N D
TABLE 2
,.G
4 M) 3 M) 4 M) 4 M) 1 3 . 0 + 0 . 6 * (44) 11.9 " 1.0 (49)
EXPOSURE
parentheses,
7.9: + 0.5 (27)
13.6 ~ 0,7 * (25)
N.M. 1 0 . 7 t 0 . 6 * (50) 7 . 8 - 0.4 (50)
10.9 ' 0.5 * (50) 10,1 ~ 0.5 * (50)
II
AFTER
E a c h R o m a n n u m e r a l d e n o t e s a s e p a r a t e e x p e r i m e n t . E a c h e n t r 5 r e p r e s e n t s the m e a n * S.E. In • ** T h c s a m e as in T a b l e I; * P < 0 . 0 1 : ** P < 0 . 0 5 . N . M . , n o mitosis.
9.6 • 1.0 (14)
(10 (10 (10 (10
I 1.9 + 1.0 * ( I 1)
aniline aniline aniline aniline
I
M i t o m y c i n C (20 n g / m l )
3 M) 4 M) s M)
N o r h a r m a n (10 N o r h a r m a n (10 N o r h a r m a n (10
plus plus plus plus
LYMPHOCYTES
S C E s p e r cell
BLOOD
Control (DMSO)
3 M) 4 M) 4 M) s M)
(10 (10 (10 (10
Norharman Norharman Norharman Norharman
SCE FREQUENCIES IN HUMAN PERIPHERAL A N I L I N E F O R T H E L A S T 48 h O F C U L T . U R E
TABLE 3 WITH
OR
WITttOUT
the n m n b e r of cells c o n t r i b u t i n g to this a,,crage.
8.4 ' 0.5 (50)
16.2:- 0.8 * (25)
N.M. 10.4 * 0,7 ** (42)
11.4= 0.5 * 150)
N.M.
Ill
TO NORtlARMAN
171
intrinsic mutagenic potential. There remained the possibility that norharman itself might have SCE-inducing activity. Treatment of cells with norharman plus $9 mix also induced an increase in SCEs which indicated that norharman has some SCE-inducing capability (columns 6-9 in Table 2). Table 2 shows that the incidence of SCEs decreased with increasing concentrations of aniline in the treatments with aniline plus norharman plus $9 mix (columns 4 and 5 in Table 2). Thus, aniline seems to counteract the SCE-inducing activity of activated norharman. The frequency of SCEs induced by norharman with $9 mix was dose-dependent and increased with increasing concentration of $9 mix in the medium (columns 6-9 in Table2). The SCE-inducing activity of norharman was apparent from its single application for 48 h, as shown in Table 3. Simultaneous application of norharman and aniline for 48 h did not affect the norharman-induced SCE frequencies (Table 3). Discussion
Abe and Sasaki (1977) reported that aniline could induce SCEs in Chinese hamster cells. The results obtained here showed that aniline, even after treatment in vitro with metabolic activation by $9 mix, did not significantly increase the SCE frequencies in human lymphocytes. Aniline is considered to be inert by itself. (~niline is known to be metabolized in the body, and its oxidized metabolites have been correlated with the induction of methemoglobinemia (Kiese, 1966; Uehleke, 1971). In the usual Ames Salmonella/microsome test, aniline did not show any mutagenic potential (McCann et al., 1975; Nagao et al., 1977b). The degree of covalent binding of aniline to DNA in vivo, as expressed with a CBI (covalent-binding index), is low (Lutz, 1979). Taking these facts into consideration, the present results indicate that, in human whole blood cells, the degree of metabolic activation is lower than that in Chinese hamster cells and is too low to allow for the production of SCE-producing metabolites of aniline. Aniline is metabolized by microsomal mixed-function oxidases to the Chydroxylated or the N-hydroxylated metabolites (Uehleke, 1971). In the present experiments, p-aminophenol, one of the C-hydroxylated metabolites of aniline, increased the SCE frequencies at a concentration of 10 4M (Table 1). It should be noted that p-aminophenol is regarded as a non-carcinogen and that the Ames Salmonella/microsome test showed it to be non-mutagenic (McCann et al., 1975). Since Nagao et al. (1977b) reported that aniline was mutagenic in their norharmanenhanced Salmonella/microsome test, the SCE-inducing property of p-aminophenol found here may imply that aniline is a promutagen/procarcinogen. The $9 mix used here had aniline hydroxylase activity (108.6x 10 -9 moles p - a m i n o p h e n o l / m g protein/h), but treatment with aniline plus $9 mix did not affect the incidence of SCEs (Table2). Under the present experimental conditions, the amount of p-aminophenol produced was less than 2 X 10-5 M / 2 h/culture bottle. This was a borderline concentration in inducing an increase in SCEs, as shown in Table 1. Because human blood lymphocytes were insensitive to induction of SCEs after
172 t r e a t m e n t with aniline alone or with aniline plus $9 mix, n o r h a r m a n was a d d e d to the t r e a t m e n t with aniline plus $9 mix. This a d d i t i o n of n o r h a r m a n might be e x p e c t e d to p r o d u c e the same results as in the S a l m o n e l l a test of N a g a o et al. (1977b); i.e., e n h a n c e m e n t of the n u m b e r of SCEs detected. Such a t r e a t m e n t indeed increased the S C E f r e q u e n c y ( T a b le 2 ) . H o w e v e r , it appears that this increase of S C E s was due to the S C E - i n d u c i n g activity of n o r h a r m a n itself (Tables 2 and 3). Th e S C E - i n d u c i n g activity of n o r h a r m a n was also o b s e r v e d in the b l o o d of a n o t h e r h e a l t h y male ( S C E / c e l l , 12.8 at 10 4 M , exposure time 48 h; control S C E / c e l I , 9.4: P < 0.01). In the e x p e r i m e n t s of N a g a o et al. (1977b), n o r h a r m a n itself did not show a n y mutagenicity, and their n o r h a r m a n - t r e a t e d S a l m o n e l l a / m i c r o s o m e test because m o r e sensitive than the original S a l m o n e l l a / m i c r o s o m e test in d et ect i n g the potential m u t a g e n i c i t y of s o m e classes of chemicals ( N a g a o et al., 1977a,b, 1978: W a k a b a y a s h i et al., 1981). However, the results o b t a i n e d here suggest that the same pri n ci p l e does not work in the m a m m a l i a n S C E test. In addition, because carboline derivatives such as T r p - P - l , Trp-P-2, and 2 - a m i n o - ~ - c a r b o l i n e are able to induce S C E s in h u m a n l y m p h o b l a s t o i d cells ( T 0 h d a et al., 1980), n o r h a r m a n , a carboline derivative, may also be e x p e c t e d to induce SCEs in o t h er cell lines.
Acknowledgement We thank Dr. Sheldon W o l f f of the U n i v e r s i t y of California, San Francisco, for critically r ead i n g our manuscript.
References Abe, S., and M. Sasaki (1977) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals, J. Natl. Cancer Inst., 58, 1635-1641. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-363. Kiese, M. (1966) The biochemical production of ferrihemoglobin-forming derivatives from aromatic amines, and mechanisms of ferrihemoglobin formation, Pharmacol. Rev., 18. 1091 1161. Lutz, W.K. (1979) In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis, Mutation Res., 65, 289-356. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carinogens as mutagens in the Satmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Nagao, M., T. Yahagi, T. Kawachi, T. Sugimura, T. Kosuge, K.Tsufi, K. Wakabayashi, S. Mizusaki and T. Matsumoto (1977a) Comutagenic action of norharman and harman, Proc. Jpn. Acad., 53, 95-98. Nagao, M., T. Yahagi, M. Honda, Y. Seino, T. Matsushima and T. Sugimura (1977b) Demonstration of mutagenicity of aniline and o-toluidine by norharman, Proc. Jpn. Acad., 53(B), 34-37 Nagao, M., T. Yahagi and T. Sugimura (1978) Differences in effects of norharman with various classes of chemical mutagens and amount of S-9, Biochem. Biophys. Res. Commun., 83. 373 378. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Takehisa, S., and S. Wolff (1978) Sister-chromatid exchanges induced in rabbit lvmphocytes by 2aminofluorene and 2-acetylaminofluorene after in vitro and in vivo metabolic activation, Mutation Res., 58, 321-329. Tohda, H., A. Oikawa, T. Kawachi and T. Sugimura (1980) Induction of sister-chromatid exchanges by mutagens from amino acid and protein pyrolysates, Mutation Rcs., 77, 65 69. Uehleke, H. ( 1971 ) N-Hydroxylation, Xenobiotica, 1, 327-338. Wakabayashi, K., M. Nagao, T. Kawachi and T. Sugimura (1981) Co-mutagcnic effect of norharman with N-nitrosamine derivatives, Mutation Res.. 80, I-7.