83
Mutation Research, 263 (1991) 83-92 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$03.50 ADONIS 01657992910005IM MUTLET 0494
Further investigations on the clastogenicity of paracetamol and acetylsalicylic acid in vitro Lutz Miiller, Peter Kasper and Stephan Madle* Institutefor Drugs. Federal Health Agency, 1)-1000 Berlin 65 (F.R.G.) (Received 13 December 1990) (Accepted 28 January1991)
Keywords: Clastogenieity in vitro; Paraeetamol; Acetylsalieylicacid; $9 mix; Hepatocytes
Summary Paracetarnol (PCM) and acetylsalicylic acid (ASA), both widely used analgesics, were tested for their clastogenicity in V79 cells in vitro. Rat liver $9 mix and primary rat hepatocytes (PRH) were used as external activation systems. ASA was found to be negative with and without activation system in concentrations up to 10-2 M. In contrast PCM induced concentration-dependent chromosomal aberrations with and without activation system within the range of 3 x 10-3 and 10-2 M. The greatest effects were observed following continuous treatment with PRH activation and without external metabolization. Pulse treatments without external metabolization, with $9 mix and PRH were less effective. The clastogenic potency of PCM seems to be partly independent of metabolic activation. Although clastogenic effects in vitro were observed only in very high concentrations pharmacokinetic data and other published mutagenicity data indicate that there might be a risk for human use. Peak plasma levels of more than 10 -4 M have been reported (Forrest et al., 1982) and 2 groups of investigators (Kocisova et al., 1988; Hongslo et al., 1990) found PCM to be weakly clastogenic in human lymphocytes in vivo in the maximum human therapeutic dose range.
Paracetamol (PCM, CAS No. 103-90-2) and acetylsalicylic acid (ASA, CAS No. 50-78-2) are extensively used over the counter analgesics. Both compounds were studied in several mutagenicity as well as carcinogenicity studies. ASA was noncarcinogenic to rodents even in doses that induced Correspondence: Dr. Lutz MfiUer, Institute for Drugs, Federal Health Agency, Seestr. 10, D-1000 Berlin 65 (F.R.G.). * Present address: Max-von-Pettenkofer-Institute, Federal Health Agency, Thielallee 88-92, D-1000 Berlin 33 (F.R.G.).
chronic gastric irritation and ulceration but exhibited cocarcinogenic effects when given together with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Chang et al., 1983). ASA was found to be negative in several gene mutation assays with bacteria without and with $9 mix of different species (Bruce and Heddle, 1979; Camus et al., 1982; Kadotani et al., 1984; Kawachi et al., 1980; King et al., 1979) and in a recessive lethal test with Drosophila (King et al., 1979). Several tests for induction of chromosomal aberrations in vitro with fibroblasts or lymphocytes
84
revealed weakly positive or positive results (Ishidate, 1983; Karaseva, 1974; Kawachi et al., 1980; Meisner and Inhorn, 1972). Mauer and Weinstein (1970) quoted negative results in human lymphocytes in vitro and also in lymphocytes of several volunteers who had been taking 2.4 g ASA per day for 1 month. ASA induced no chromosomal aberrations in rat embryos (Tsuruzaki, 1982) and human lymphocytes in vivo (Mauer and Weinstein, 1970); no increase in bone marrow micronucleus frequencies of rats and mice was observed by Heddle et al. (1979) and King et al. (1979). Kawachi et al. (1980) report a positive result for chromosomal aberrations in rat bone marrow without giving any quantitative data. PCM treatment in long-term rodent bioassays resulted in hepatic toxicity. In 1 study carcinogenic effects were observed in the liver (Flaks and Flaks, 1983), 3 others were negative (Hiraga and Fuji, 1985; Hagiwara and Ward, 1986; NTP, 1990). Like ASA, PCM was negative in several gene mutation assays with bacteria with and without $9 mix of several species (Dybing, 1977; Bartsch et al., 1980; Kawachi et al., 1980; Wirth et al., 1980; Dybing et al., 1984; Nohmi et al., 1985; NTP, 1990). A recessive lethal test with Drosophila (King et al., 1979) and a gene mutation assay at the ouabain locus in Chinese hamster cells (Sasaki et al., 1980) were negative. Several in vitro unscheduled DNA ~ynthesis (UDS) assays were carried out by Dybing et al. (1984), Holme et al. (1986) and Milam and Byard (1985) with hepatocytes of different species (rat, mouse, guinea pig). Weakly positive results were observed with mouse hepatocytes only. Alkaline elution assays were negative with PCM (Nordenskj61d and Moldeus, 1983; Dybing et al., 1984; Hongslo et al., 1988) but clearly positive with Nacetyl-p-benzoquinone-imine (NAPQI), which is believed to be the ultimate hepatotoxic metabolite (Dybing et al., 1984). Repeatedly positive results were published for PCM in in vitro tests for clastogenicity with human lymphocytes and rodent cell lines (Kawachi et al., 1980; Ishidate et al., 1980, 1988; Sasaki et al., 1980, 1983; Yoshida et al., 1980; Watanabe, 1982;
Shimane, 1985; Dunn et al., 1987). With the exception of the data published by Yoshida (1980) and NTP (1990) no external metabolization systems were used. Aberration rates varied between weakly and strongly positive. In vivo bone marrow assays (chromosomal aberrations and micronuclei) yielded conflicting results with some negatives (King et al., 1979; Kawachi et al., 1980; Yoshida et al., 1980) and positives (Yoshida et al., 1980; Sicardi et al., 1987). Kocisova et al. (1988) and Hongslo et al. (1990) reported weakly positive effects in human lymphocytes in healthy volunteers after oral treatment with 3 x 1 g PCM within 8 h. Many of the mutagenicity tests reported for ASA and PCM are out of date with respect to the state of the art (e.g., in vitro tests without external metabolization system, too few animals in in vivo tests, only few cells per culture or animal evaluated) or badly documented (e.g., no quantification of results, types of chromosomal aberrations not specified). But for both compounds the published results point towards a clastogenic potential. We investigated whether such a clastogenic potential in vitro depends on the concentration used, the exposure period or is influenced by external metabolization systems such as rat liver $9 mix and primary rat hepatocytes (PRH). Especially for PCM it is clear that hepatic cytochrome P450 forms are major determinants of bioactivation in humans (Raucy et al., 1989) and formation of electrophilic intermediates like NAPQI may account for hepatic toxicity. Therefore metabolic activation has to be considered also when assessing the mutagenic properties of this compound, For assessing toxic effects of PCM metabolites and evaluation of antidotes primary hepatocytes have been shown to be a useful experimental model (Boobis et al., 1986). Materials and m e t h o d s
Test compounds and treatment concentrations
The positive controls mitomycin C (MMC) and cyclophosphamide (CP) were purchased from
85 Sigma (Munich, F.R.G.). P.a.-grade paracetamol and acetylsalicylic acid were purchased from Merck (Darmstadt, F.R.G.). Both compounds were tested in concentrations ranging from 10 -4 to 10 -2 M. Stock solutions were set up in DMSO immediately before use. Final concentrations of DMSO in the cultures did not exceed 1°7o.
106 hepatocytes were seeded per 25-cm2 plastic flask that had been seeded with 2 × 105 V79 cells 24 h earlier. After 1.5 h the medium was replaced by FCS-free Williams E medium (WEM, Flow). Cells treated for 2.5 h or 18.5 h were harvested 18.5 h after beginning of the treatment. All experiments were run with 2 cultures in parallel; experiments with pulse treatment were carried out twice.
Controls In all experiments the following controls were set up: a control without any treatment and a control with DMSO and the metabolizing system to be tested. All controls were set up in duplicate or triplicate.
Experiments system
without
external
metabolization
As target cells for evaluation of chromosomal aberrations V79 cells were used. Experiments without external metabolization system were carried out with 4 different treatment schedules (harvest time in parentheses): 6 h (6 h), 2 h (12 h), 12 h (12 h), and 24 h (24 h). All experiments were run with 2 cultures in parallel. Experiments with 12-h continuous treatment were carried out twice.
Preparation of metaphases:, microscopic analysis For arresting of mitoses in the metaphase, cultures were incubated for 2 h with Colcemid (80 ng/ml). Mitotic cells were shaken off and standard techniques were used. The cells were stained in Giemsa (5070, pH 6.88). All slides were coded prior to analysis. 100 metaphases were analyzed for chromosomal aberrations per culture if possible. Chromatid-type aberrations (chromatid breaks or fragments, chromatid exchanges) and chromosome-type aberrations (chromosome breaks or fragments, dicentrics, and rings) were scored. Metaphases showing multiple aberrations were recorded separately. Gaps and isogaps (achromatic lesions not greater than the width of a chromatid) were scored but were not included in the calculation of damaged cells.
Experiments with $9 mix The $9 fraction was prepared from phenobarbital-pretreated rats. All experiments were carried out with the same batch of $9 fraction which was stored in liquid nitrogen. The $9 mix was prepared and added to the V79 cultures as previously reported (Madle et al., 1986). $9 concentration in cultures was 107o. Treatments with $9 mix were carried out for 2 h. The cells were then washed twice, reincubated for 10 h and then harvested. All experiments were run with 2 cultures in parallel and were carried out twice.
Experiments with hepatocyte/ V79 cocultures Primary rat hepatocytes (PRH) were isolated from 8-10-week-old Wistar rats by the 2-step collagenase perfusion technique as previously reported (Madle et al., 1986). Hepatocytes were used only if the perfusion yielded more than 75070 viable cells (determined by trypan blue exclusion).
Statistics The results were analyzed by the non-parametric H-test (Kruskal and Wallis, 1952), a 1-way analysis of variance for independent data and different sample sizes. P-values below 0.01 were considered to indicate significant differences. Results
Controls The positive controls MMC and CP induced significant incidences of chromosomal aberrations within the range of historical data from our laboratory. The data for CP indicate that $9 mix as well as hepatocytes were able to activate this premutagen. Mean spontaneous aberration rates range from 0.407o to 4.007o between experiments without differences between DMSO-treated and non-treated cultures.
86
ASA Following A S A treatment no significantly elevated chromosomal aberration rates were observed in the whole range of concentrations
tested (Tables 1-3). No influence of a metabolic activation system was registered. A S A was considered to be not clastogenic under the employed test conditions.
TABLE 1 C H R O M O S O M A L A B E R R A T I O N S IN V79 C E L L S I N D U C E D BY A S A A N D P C M W I T H O U T E X T E R N A L M E T A B O L I Z A TION T r e a t m e n t (mole/l)
Harvest time 6 h, 0 (control) D M S O (control) MMC 3.16 ASA 1 ASA 3.16 ASA 1 ASA 3.16 ASA 1 PCM 1 PCM 3.16 PCM 1 PCM 3.16 PCM 1
Cells scored
continuous treatment 300 300 x 10 -6 200 x 10 -2 200 x 10 -3 200 x 10- 3 200 x 10 -4 200 X 10 -4 200 x 10- 2 163 X 10 -3 124 × 10- 3 200 x 10 -4 200 x 10 -4 200
Harvest time 12 h, continuous 0 (control) D M S O (control) MMC 3.16 x 10 -7 ASA 1 x 10 -2 ASA 3.16 x 10 -3 ASA 1 x 10 -3 ASA 3.16 x 10 -4 ASA 1 X 10 - 4 PCM 1 X 10 -2 PCM 3.16 × 10 -3 PCM 1 x 10- 3 PCM 3.16 × 10 -4 PCM 1 x 10 -4
treatment 700 451 100 300 300 400 400 400 52 193 400 400 400
Harvest time 12 h, 2-h treatment 0 (control) 200 D M S O (control) 200 MMC 3.16 × 10 -5 146 PCM 1 × 10 -2 200 PCM 3.16 × 10 -3 200 PCM 1 × 10- 3 200 PCM 3.16 x 10 -4 200 PCM 1 × 10 -4 200
Aberrations per 100 metaphases
Aberrant metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
%
(SEM)
1.3 3.3 14.0 0.5 3.0 2.5 2.0 0.0 24.4 5.1 3.0 6.5 1.5
0.7 1.3 4.5 1.0 0.5 1.0 0.0 0.0 17.4 7.3 1.0 3.0 0.0
0.0 0.3 3.0 0.0 0.0 0.0 0.0 0.0 0.6 0.6 0.0 0.5 0.0
1.0 1.3 6.0 0.0 0.0 0.0 1.0 0.0 19.8 5.1 1.5 2.0 1.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0
0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
4 8 21 2 1 2 1 0 43 17 5 10 2
1.3 2.7 10.5" 1.0 0.5 1.0 0.5 0.0 26.4* 13.7" 2.5 5.0 1.0
(0.7) (0.9) (2.2) (0.7) (0.5) (0.7) (0.5) (0.0) (3.4) (3.1) (1.1) (1.5) (0.7)
2,0 1.1 5.0 3.3 3.0 2.8 3.3 2.8 5.8 10.9 10.0 6.8 3.7
0.7 0.2 4.0 0.3 1.0 0.3 0.5 0.8 13.5 13.0 4.0 1.3 0.5
0.0 0.0 8.0 0.3 0.3 0.3 0.0 0.0 3.9 5.2 3.5 0.0 0.0
0.1 0.0 1.0 0.7 0.7 0.3 0.3 0.3 15.4 8.8 1.3 2.0 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 0.0 0.0
5 2 13 4 5 3 3 4 15 28 31 11 4
0.7 0.4 13.0" 1.3 1.7 0.8 0.8 1.0 28.9* 14.5" 7.8* 2.8* 1.0
0.3) (0.3) (3.6) (0.7) (0.8) (0.5) (0.5) (0.5) (6.3) (2.5) (1.3) 0.8) (0.5)
0.0 0.0 4.1 0.0 1.0 0.0 0.0 0.5
2.0 1.0 15.8 2.5 5.5 0.0 0.0 0.5
0.0 0.0 6.2 0.5 4.5 0.5 0.0 0.0
3.5 1.5 13.0 0.5 3.0 1.5 0.0 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0
8 4 24 5 13 4 0 2
4.0 2.0 16.4" 2.5 6.5* 2.0 0.0 1.0
(1.4) (1.~ (3.1) (1.1) (1.7) (1.0) (0.0) 0.7)
87 T A B L E 1 (continued) T r e a t m e n t (mole/l)
Harvest time 24 h, continuous 0 (control) D M S O (control) MMC 3.16 × 10 - s ASA 1 x 10 -2 ASA 3.16 × 10 -3 ASA 1 x 10 - 3 ASA 3.16 × 10 -4 ASA 1 x l0 -4 PCM 1 × l0 -2 PCM 3.16 × 10 -3 PCM 1 x l0 -3 PCM 3.16 x l0 -4 PCM 1 >( 10 -4
Cells scored a
treatment 300 300 50 100 100 100 200 200 n.a. n.a. 100 68 200
Aberrations per 100 metaphases
Aberrent metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
%
(SEM)
3.0 2.0 26.0 4.0 1.0 3.0 1.5 0.5
0.7 0.7 50.0 0.0 0.0 1.0 0.0 1.0
0.0 0.0 30.0 0.0 1.0 0.0 0.0 0.0
0.0 1.0 26.0 0.0 0.0 0.0 0.0 0.5
0.0 0.0 2.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 8.0 0.0 0.0 0.0 0.0 0.0
2 2 26 0 1 1 0 2
0.7 0.7 52.0* 0.0 1.0 1.0 0.0 1.0
(0.5) (0.5) (7.1) (0.0) (1.0) (1.0) (0.0) (0.7)
3.0 8.8 3.5
2.0 1.5 0.5
0.0 0.0 0.0
0.0 0.0 0.5
0.0 0.0 0.0
0.0 0.0 0.0
2 1 2
2.0 1.5 1.0
(1.4) (1.5) (0.7)
n.a., not analyzable. G, gaps; B', chromatid breaks a n d fragments; RB', chromatid exchanges; B " , c h r o m o s o m e breaks and fragments; dic/ring, dicentrics a n d rings; M_A, multiple aberrations. * Significant difference vs. negative control at p < 0.01.
PCM PCM was clearly clastogenic in high concentrations with and without external metabolization system. 6-h treatment without external metabolization led to high aberration incidences in concentrations higher than 10- 3 M (Table 1). Slightly higher effects were observed following 12-h exposure to PCM (Table 1). Here 10 -9 M PCM significantly increased the aberration rates to 7.8070 aberrant metaphases. The greatest effects were obtained at 10 -2 M with 28.9070 aberrant metaphases. A 2-h treatment for this harvest time resulted in only weakly increased aberration rates (Table 1). A continuous treatment of 24 h was negative in concentrations up to 10-3 M (Table 1). Treated cultures with concentrations higher than 10-3 M were not analyzable due to lack of metaphases. Addition of $9 mix as external metabolization system for 2 h resulted in a less pronounced increase in the percentage of aberrant metaphases compared to data with V79 cells alone (Table 2). At concentrations higher than 10 -9 M positive responses were obtained with 8.8070 (3.16 x 10-3 M
PCM) and 6.5°70 aberrant metaphases (10 -2 M PCM). Lower concentrations were negative. Cocultivation of V79 cells and primary rat hepatocytes with a treatment period of 2.5 h led to high aberration rates in concentrations above 10-3 M (Table 3). Concentrations up to 10 -9 M were negative. Continuous treatment during the cocultivation period of 18.5 h yielded aberration rates of more than 40070 following treatment with 3.16x 10 -9 M PCM, but only few metaphases could be analyzed due to cytotoxicity (Table 3). Again, concentrations up to I 0 - 3 M were negative.
Discussion ASA was negative in all experiments with and without external metabolization. This is in contradiction to some of the published data, which show clastogenicity of ASA (Ishidate, 1983; Kawachi et al., 1980; Karaseva et al., 1974; Meisner and Inhorn, 1972). Most of these data were generated without using external metabolization, were not reproduced, results were not quan-
88 TABLE 2 C H R O M O S O M A L ABERRATIONS IN V79 CELLS P H E N O B A R B I T A L - I N D U C E D RAT LIVER $9 MIX Treatment (mole/l)
Cells scored
I N D U C E D BY ASA A N D
PCM
Aberrations per 100 metaphases
IN THE
PRESENCE
OF
107o
Aberrant metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
070
(SEM)
3.0 5.2 13.3 3.0 4.0 3.5 4.3 3.8 2.3 5.3 4.8 3.3 4.0
1.0 1.5 12.9 2.3 1.0 0.5 1.8 1.0 3.0 9.3 3.8 1.5 2.0
0.0 0.0 7.6 0.0 0.0 0.3 0.0 0.0 4.5 6.5 2.0 0.0 0.0
0.7 0.3 6.0 0.8 1.0
0.2 0.0 0.2 0.0 0.0
0.0 0.0 7.8 0.0 0.0
7 11 101 11 3
1.2 1.8 23.2* 2.8 0.8
(0.5) (0.5) (2.0) (0.8) (0.5)
0.0
0.0
0.0
3
0.8
(0.5)
0.3 0.3 0.8 4.0 0.5 0.5
0.0 0.0 0.0 0.0 0.0 0.0
0.3 0.3 0.5 2.0 0.3 0.0
6 6 26 35 14 8
1.5 1.5 6.5* 8.8* 3.5 2.0
(0.6) (0.6) (1.2) (1.4) (0.9) (0.7)
0.7
0.0
0.0
4
1.3
(0.7)
Harvest time 12 h, 2-h treatment 0 (control) DMSO (control) CP 1 ASA 1 ASA 3.16 ASA 1 ASA 3.16 ASA 1 PCM 1 PCM 3.16 PCM 1 PCM 3.16 PCM 1
+ × × x × x × x × x x x
$9 10 -5 10- 2 10 -3 10- 3 10 -4 10 -4 10 -z 10 -3 10- 3 10 -4 10 -4
600 600 435 400 400 400 400 400 400 400 400 400 300
For abbreviations see Table 1. *Significant difference vs. negative control at p < 0.01.
tified, or evaluation criteria are quite unclear (e.g., no discrimination of different types of aberrations). Ishidate (1983) found high incidences of chromosomal aberrations in CHL cells 48 h after treatment with and without $9 mix, but no increase in the 24-h harvest time. The range of concentrations was the same as in the present investigation. The reasons for this time dependence, i.e., the observation of high incidences of chromosomal aberrations after 2 or more cell cycles and no aberrations after 1-2 cell cycles, are quite unclear and still have to be investigated. PCM was clearly clastogenic with and without external activation. Strongly positive responses in all experiments were obtained with concentrations higher than 10-3 M, weak effects were observed when cells were treated with 10- 3 M PCM. Shortterm exposure with and without $9 mix resulted in weakly elevated chromosomal aberration rates only whereas cocultivation with PRH led to much more pronounced effects. Continuous exposure to PCM was generally strongly cytotoxic in high concentrations but clearly elevated chromosomal aber-
ration rates were already observed at concentrations without any marked cytotoxicity. The data suggest that PCM is clastogenic independent of metabolization but this effect could be intensified by cocultivation with PRH. Aspects of these 2 principles have been discussed in the literature. Hongslo et al. (1988) found SCE induction by PCM to be independent of cocultivation of V79 cells with or without primary mouse hepatocytes and suggested that PCM induces chromosomal alterations such as SCE via an indirect mechanism independent of activation: PCM inhibits the ribonucleotide-reductase which results in a reduction of the ribonucleotide pool and thus leads to reduced DNA synthesis and possibly chromosomal alterations (Hongslo et al., 1989a,b). On the other hand P450-mediated activation of PCM leads to electrophilic species such as Nacetyl-p-benzoquinone-imine (NAPQI) (for review of PCM metabolism see Hinson, 1983). Reactive metabolites such as NAPQI may also covalently bind to DNA and could well take part in the clastogenicity induced via external metabolization.
89
TABLE 3 C H R O M O S O M A L ABERRATIONS IN V79 CELLS I N D U C E D W I T H A S A A N D PCM: C O C U L T I V A T I O N W I T H PRIMARY RAT HEPATOCYTES (PRH)
Treatment (mole/l)
Cells scored
Aberrations per 100 metapbases
G Harvest time 18.5 h, 2.5-h treatment 0 control 400 DMSO (control) + PRH 400 CP 1 x 10 -4 200 ASA 1 x 10 -2 400 ASA 3.16 x 10 -3 400 ASA 1 x l0 -~ 400 ASA 3.16 x 10 -4 350 ASA 1 X l0 -4 400 PCM 1 X l0 -2 387 PCM 3.16 × l0 -~ 400 PCM 1 x l0 -3 300 PCM 3.16 x l0 -4 400 PCM 1 x 10 -4 400
2.4 2.3 13.5 3.0 2.8 2.8 2.3 6.1 2.6 2.0 3.7 3.3 2.8
Harvest time 18.5 h, continuous treatment 0 (control) 200 1.5 DMSO (control) + PRH 200 0.5 CP 1 x 10 -4 200 6.0 PCM 1 x 10 -z n.a. PCM 3.16 x 10 -3 57 8.8 PCM 1 x 10- 3 200 1.5 PCM 3.16 x 10 -4 200 1.5 PCM 1 x 10 -4 200 1.0
B'
RB'
B"
Aberrant metaphases excl. gaps dic/ring
MA
n
o70
(SEM)
1.8 2.5 4.0 0.8 2.5 2.3 1.4 2.8 7.8 6.3 1.0 1.5 2.8
0.0 0.0 1.5 0.0 0.0 0.5 0.3 0.0 7.8 9.3 0.0 0.3 0.0
0.0 0.8 6.0 0.5 0.5 0.3 0.3 0.3 2.8 3.3 1.0 0.5 0.8
0.0 0.3 0.5 0.3 0.0 0.3 0.0 0.0 0.3 0.3 0.3 0.5 0.0
0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 6.7 6.5 0.0 0.0 0.0
4 10 15 6 9 13 5 12 59 57 7 7 7
1.0 2.5 7.5* 1.5 2.3 3.3 1.4 3.0 15.2" 14.3" 2.3 1.8 1.8
(0.5) (0.8) (1.9) (0.6) (0.8) (0.9) (0.6) (0.9) (1.8) (1.8) (0.9) (0.7) (0.7)
0.5 0.5 40.5
0.0 0.0 19.0
2.5 0.5 27.5
0.0 0.0 -
0.0 0.0 6.0
5 2 95
2.5 1.0 42.5*
(1.1) (0.7) (3.5)
24.6 0.5 0.5 0.0
15.8 1.5 0.0 0.0
28.1 1.5 1.5 0.0
0.0 0.0 0.0 0.0
17.5 0.0 0.0 0.0
26 7 4 0
45.6* 3.0 2.0 0.0
(6.6) (1.2) (1.0) (0.0)
For abbreviations see Table 1. * Significant difference vs. negative control at p < 0.01
The reactive metabolites of PCM preferentially bind to proteins and glutathione in the liver (Garle and Fry, 1989; Bartolone et al., 1989). The latter reaction is an efficient protective mechanism against the toxic properties of PCM. However, in high doses the glutathione pool is depleted and free toxic metabolites occur. Glutathione depletion as reported in animals has been observed in liver ceils of humans in vitro (Tee et al., 1987) and in vivo following an intake of 0.5-3 g PCM (Slattery et al., 1987). But marked differences in cytotoxicity of PCM and covalent binding of PCM to microsomal proteins were demonstrated for hepatocytes of different species in vitro (Tee et al., 1987). Hamster and mouse hepatocytes were more sensitive than
primary rat hepatocytes and human hepatocytes, which turned out to be almost equally resistant. Tee et al. (1987) attribute these findings to different concentrations required for glutathione depletion and different conversion rates of PCM to its reactive metabolite NAPQI, which was equally effective in hepatocytes of all species used. PCM was also more effective in inducing UDS in primary mouse hepatocytes than in PRH (Holme and Soederlund, 1986). These data suggest that PRH used in our experiments are a good model for the human situation. The indirect action of PCM via reduction of the nucleotide pool and the direct interaction with DNA via reactive metabolites that can be trapped
90 with glutathione up to its depletion suggest the existence of threshold mechanisms. Our in vitro results are in good agreement with this theory: PCM acted as a clastogen with and without external metabolization only in concentrations of 10-3 M and greater. An additional effect in the same concentrations was observed when hepatocytes were used as cocultivation system. This should be the result of hepatocyte-generated DNA-damaging reactive metabolites. From our results a no observed effect level of 3.16 x l0 -4 M could be deduced. Other data from UDS tests (Dybing et al., 1984; Holme and Soederlund, 1986), alkaline elution (Hongslo et al., 1988), SCE analysis (Hongslo et al., 1988, 1990), LDH leakage, cytotoxicity, and intracellular glutathione depletion (Dybing et al., 1984; Tee et al., 1987) are in line with this interpretation, because these effects were also not observed below this concentration. UDS data (Holme and Soederlund, 1986) and our test results show also that PCM was generally strongly cytotoxic in the same concentration range where genotoxic effects were observed. Therefore it was suggested that cytotoxic effects of PCM may partly dominate its genotoxic properties (Holme and Soederlund, 1986; Holme et al., 1988). Results of in vivo bone marrow mutagenicity tests with PCM in mice and rats were predominantly negative although not sufficiently documented (King et al., 1979; Kawachi et al., 1980; Yoshida et al., 1980). In 1 test with rats slightly elevated micronucleus incidences in bone marrow were found at doses of 100 mg/kg and 150 mg/kg i.p. In 2 studies with human volunteers elevated incidences of chromosomal aberrations in lymphocytes following uptake of 3 × 1 g PCM within 8 ~h were reported (Kocisova et ai., 1988; Hongslo et al., 1990). Since PCM is almost uniformly distributed in the body, comparison of in vivo blood plasma levels with in vitro concentrations should give additional information. However, in the studies with human lymphocytes, no data on plasma levels were presented. A therapeutic dose of about 1 g PCM leads to average blood plasma levels of 20/~g/ml (Forrest et al., 1982), giving a concentration of
1.3x 10 - 4 M. It is apparent that human plasma concentrations are not far below the high concentrations, which yielded clastogenic effects in vitro. Although in vivo/in vitro comparisons are only approximative and in vitro data suggest a threshold mechanism, the weakly positive in vivo data on chromosome mutations in human lymphocytes are in line with our in vitro data. Since glutathione-mediated detoxification of PCM plays a major role in the present evaluation individual differences in glutathione levels in the human population are of interest. Recently published results show that lymphocytes from persons defective in glutathione S-transferase were more susceptible to epoxide-induced genetic damage than lymphocytes from persons having this enzyme (Wiencke et al., 1990). Indiviuals lacking the enzyme are also at high risk for smokinginduced lung cancer (Seidegard et al., 1986). For example about 50°70 of all caucasians are genetically deficient in this isozyme. Data on genetic differences between the volunteers of the 2 in vivo studies with PCM are not available, but the observation that individual data showed more pronounced effects for some volunteers whereas others were negative (personal communication) gives indications that there could be an association with glutathione S-transferase activities. Taking in vitro and in vivo data together, it cannot be excluded that therapeutic use of PCM gives rise to genotoxic effects, but the mechanisms of action still have to be elucidated and risk assessment has to be done.
Acknowledgements The authors greatly appreciate the expert technical assistance of Mrs. G. Kaufmann and Mrs. H. Madle in scoring the slides and preparing the tables and statistics.
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91 extrahepatic proteins: an in vivo and in vitro analysis, Toxicol. Appl. Pharmacol., 99, 240-249. Bartsch, H., C. Malaveille, A.-M. Camus, G. Martel-Planche, G. Brun, A. Hautefeuille, N. Sabadie, A. Barbin, T. Kuroki, C. Drevon, C. Piccoli and R. Montesano (1980) Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutation Res., 76, 1-50.
Boobis, A.R., L.B.G. Tee, C.E. Hampden and D.S. Davies (1986) Freshly isolated hepatocytes as a model for studying the toxicity of paracetamol, Food Chem. Toxicol., 24, 731-736. Bruce, W.R., and J.A. Heddle (1979) The mutagenic activity of 61 agents as determined by the micronucleus, Salmonella and sperm abnormality assays, Can. J. Genet. Cytol., 21, 319-334. Camus, A.-M., M. Friesen, A. Croisy and H. Bartsch (1982) Species-specific activation of phenacetin into bacterial mutagens by hamster liver enzymes and identification of Nhydroxyphenacetin O-glucuronide as a promutagen in the urine, Cancer Res., 42, 3201-3208. Chang, T.H., Y.C. Lee, K.Y. Lee, C.H. Sun and Y.P. Chang (1983) Cocarcinogenic action of aspirin on gastric tumors induced by N-nitroso-N-methylnitroguanidine in rats, J. Natl. Cancer Inst., 70, 1067-1075. Dunn, T., R.A. Gardiner, G.J. Seymour and M.F. Lavin (1987) Genotoxicity of analgesic compounds assessed by an in vitro micronucleus assay, Mutation Res., 189, 299-306. Dybing, E. (1977) Methyldopa binding to cells in culture, Acta Pharmacol. Toxicol., 40, 161-168. Dybing, E., J.A. Holme, W.P. Gordon, E.J. Soederlund, D.C. Dahlin and S.D. Nelson (1984) Genotoxicity studies with paracetamol, Mutation Res., 138, 21-32. Flaks, A., and B. Flaks (1983) Induction of liver cell tumours in IF mice by paracetamol, Carcinogenesis, 4, 363-368. Forrest, J.A.H., J.A. Clements and L.F. Prescott (1982) Clinical pharmacokinetics of paracetamol, Clin. Pharmacokinet., 7, 93-107. Garle, M.J., and J.F. Fry (1989) Detection of reactive metabolites in vitro, Toxicology, 54, 101-110. Hagirawa, A., and J.M. Ward (1986) The chronic hepatotoxic, tumor-promoting, and carcinogenic effects of acetaminophen in male B6C3FI mice, Fund. Appl. Toxicol., 7, 376-386. Hinson, J.A. (1983) Reactive metabolites of phenacetin and acetaminophen: a review, Environ. Health Perspect., 49, 71-79. Hiraga, K., and T. Fuji (1985) Carcinogenicity testing of acetaminophen in F344 rats, Jpn. J. Cancer Res. (Gann), 76, 79-85. Holme, J.A., and E. Soederlund (1986) Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes, Mutation Res., 164, 167-175.
Holme, J.A., J.K. Hongslo, Ch. Bjoernstad, P.J. Harvison and S.D. Nelson (1988) Toxic effects of paracetamol and related structures in V79 Chinese hamster cells, Mutagenesis, 3, 51-56. Hongslo, J.K., T. Christensen, G. Brunborg, Ch. Bjoernstad and J.A. Holme (1988) Genotoxic effects of paracetamol in V79 Chinese hamster cells, Mutation Res., 204, 333-341. Hongslo, J.K., C. Bjoernstad, P.E. Schwarze and J.A. Holme (1989a) Inhibition of replicative DNA synthesis by paracetamol in V79 Chinese hamster cells, Toxicol. in Vitro, 3, 13-20. Hongslo, J.K., C. Bjoerge, P.E. Schwarze, A. Brogger, G. Mann, L. Thelander and J.A. Holme (1989b) Paracetamol inhibits replicative DNA synthesis and induces sister chromatid exchange and chromosomal aberrations by inhibition of ribonucleotide reductase, Mutagenesis, 5, 475-480. Hongslo, J.K., A. Br6gger, C. Bj6rge and J.A. Holme (1990) Increased frequency of chromosomal aberrations in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol, Pharmacol. Toxicol., 66, Suppl. V, Abstract PI4. Ishidate, M. Jr. 0983) Chromosomal Aberration Test in Vitro, Realize Inc., Tokyo, p. 158. lshidate, M. Jr., and K. Yoshikawa (1980) Chromosome aberration tests with Chinese hamster cells in vitro with and without metabolic activation - a comparative study on mutagens and carcinogens, Arch. Toxicol., 4, 41-44. Ishidate, M. Jr., M.C. Harnois and T. Sofuni (1988) A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures, Mutation Res., 195, 151-213. Kadotani, S., M. Arisawa and H.B. Maruyama 0984) Mutagenicity examination of several non-steroidal antiinflammatory drugs in bacterial systems, Mutation Res., 138, 133-136. Karaseva, N.M. (1974) Cytogenetic action of aspirin in a human lymphocyte culture, Bull. Exp. Biol. Med., 78, 1194-1195. Kawachi, T., T. Komatsu, T. Kada, M. lshidate, M. Sasaki, T. Sugiyama and Y. Tazima (1980) Results of recent studies on the relevance of various short-term screening tests in Japan, Appl. Methods Oncol., 3, 253-267. King, M.-T., H. Beikirch, K. Eckhardt, E. Gocke and D. Wild (1979) Mutagenicity studies with X-ray-contrast media, analgesics, antipyretics, antirheumatics and some other pharmaceutical drugs in bacterial, Drosophila and mammalian test systems, Mutation Res., 66, 33-43. Kocisova, J., P. Rossner, B. Binkova, H. Bavorova and R.J. Sram (1988) Mutagenicity studies on paracetamol in human volunteers. 1. Cytogenetic analysis of peripheral lymphocytes and lipid peroxidation in plasma, Mutation Res., 209, 161-165. Kruskal, W.H., and W.A. Wallis (1952) Use of ranks in oncecriterion variance analysis, J. Am. Statist. Ass., 47, 583-621. Madle, E., G. Tiedemann, St. Madle, A. Ott and G. Kaufmann (1986) Comparison of $9 mix and hepatocytes as external
92 metabolizing systems in mammalian cell cultures: cytogenetic effects of 7,12-dimethylbenzanthracene and aflatoxin B1, Environ. Mutagen., 8,423-437. Mauer, I., and D. Weinstein (1970) Acetylsalicylic acid: no chromosome damage in human leukocytes, Science, 169, 198-200. Meisner, L.F., and S.L. Inhorn (1972) Chemically-induced chromosome changes in human cells in vitro, Acta Cytol., 16, 41-47. Milam, K.M., and J.L. Byard 0985) Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures, Toxicol. Appl. Pharmacol., 79, 342-347. Nohmi, T., M. Ishidate Jr., A. Hiratsuka and T. Watabe (1985) Mechanism of metabolic activation of the anaigetic bucetin to bacterial mutagens by hamster liver microsomes, Chem. Pharm. Bull., 33, 2877-2885. Nordenskj61d, M., and P. Mold6us (1983) Induction of DNAstrand breaks in cultured human fibroblasts by reactive metabolites, Ann. NY Acad. Sci., 407,460-462. NTP (1990) Technical Report 394 on the Toxicology and Carcinogenesis Studies of Acetaminophen in F344/N Rats and B6C3F Mice, NIH Publication No. 90-2849, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, MD. Raucy, J.L., J.M. Lasker, Ch.S. Liebert and M. Black (1989) Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2, Arch. Biochem. Biophys., 271, 270-283. Sasaki, M., K. Sugimura, K. Misuaki, A. Yoshida and S. Abe (1980) Cytogenetic effects of 60 chemicals on cultured human and Chinese hamster cells, Kromosomo, II-20, 574-584. Sasaki, M., S. Yoshida and K. Hiraga (1983) Additional effect of acetaminophen on the mutagenicity and clastogenicity of N-methyl-N'-nitro-N-nitrosoguanidine in cultured Chinese hamster CHO-K1 cells, Mutation Res., 122, 367-372.
Seidegard, J., R.W. Pero, D.G. Miller and E.J. Bea(tie (1986) A glutathione transferase in human leukocytes as a marker for the susceptibility to lung cancer, Carcinogenesis, 7, 751-753. Sicardi, S.M., J.L. Martiarena and M.T. Iglesias 0987) Acci6n clastog6nica in vivo de fenacetina y paracetamol, Acta Farm. Bonaerense, 6, 71-75. Shimane, Y. (1985) Mutagenicity of acetaminophen on Chinese hamster, Shigaku, 72, 1175-1187. Slattery, J.T., J.M. Wilson, T.F. Kalhorn, B.S. Nelson and S.D. Nelson (1987) Dose-dependent pharmacokinetics of acetaminophen: evidence of glutathione depletion in humans, Clin. Pharmacol. Ther., 413-418. Tee, L.B., D.S. Davies, C.E. Seddon and A.R. Boobis (1987) Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its cytotoxic metabolite, Biochem. Pharmacol., 36, 1041-1052. Tsuruzaki, T. (1982) The effects of aspirin and acetaminophen during pregnancy on chromosomal structure in rat fetuses, Jpn. J. Hyg., 37, 787-796. Watanabe, M. (1982) The cytogenetic effects of aspirin and acetaminophen on in vitro human lymphocytes, Jpn. J. Hyg., 37, 673-684. Wiencke, J.K., K.T. Kelsey, R.A. Lamela and W.A. Toscano Jr. (1990) Human glutathione S-transferase deficiency as a marker of susceptibility to epoxide-induced cytogenetic damage, Cancer Res., 50, 1585-1590. Wirth, P.J., E. Dybing, Ch. von Bahr and S.S. Thorgeirsson (1980) Mechanism of N-hydroxyacetylarylamine mutagenicity in the Salmonella test system: metabolic activation of Nhydroxyphenacetin by liver and kidney fractions from rat, mouse, hamster, and man, Mol. Pharmacol., 18, 117-127. Yoshida, S., S. Nawai and K. Hiraga (1980) Cytogenetic studies of acetaminophen and its related compounds, Ann. Rep. Tokyo Mete Res. Lab. PH, 31, 102-108. Communicated by H. Greim
Mutation Research, 263 (1991) 83-92 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$03.50 ADONIS 01657992910005IM MUTLET 0494
Further investigations on the clastogenicity of paracetamol and acetylsalicylic acid in vitro Lutz Miiller, Peter Kasper and Stephan Madle* Institutefor Drugs. Federal Health Agency, 1)-1000 Berlin 65 (F.R.G.) (Received 13 December 1990) (Accepted 28 January1991)
Keywords: Clastogenieity in vitro; Paraeetamol; Acetylsalieylicacid; $9 mix; Hepatocytes
Summary Paracetarnol (PCM) and acetylsalicylic acid (ASA), both widely used analgesics, were tested for their clastogenicity in V79 cells in vitro. Rat liver $9 mix and primary rat hepatocytes (PRH) were used as external activation systems. ASA was found to be negative with and without activation system in concentrations up to 10-2 M. In contrast PCM induced concentration-dependent chromosomal aberrations with and without activation system within the range of 3 x 10-3 and 10-2 M. The greatest effects were observed following continuous treatment with PRH activation and without external metabolization. Pulse treatments without external metabolization, with $9 mix and PRH were less effective. The clastogenic potency of PCM seems to be partly independent of metabolic activation. Although clastogenic effects in vitro were observed only in very high concentrations pharmacokinetic data and other published mutagenicity data indicate that there might be a risk for human use. Peak plasma levels of more than 10 -4 M have been reported (Forrest et al., 1982) and 2 groups of investigators (Kocisova et al., 1988; Hongslo et al., 1990) found PCM to be weakly clastogenic in human lymphocytes in vivo in the maximum human therapeutic dose range.
Paracetamol (PCM, CAS No. 103-90-2) and acetylsalicylic acid (ASA, CAS No. 50-78-2) are extensively used over the counter analgesics. Both compounds were studied in several mutagenicity as well as carcinogenicity studies. ASA was noncarcinogenic to rodents even in doses that induced Correspondence: Dr. Lutz MfiUer, Institute for Drugs, Federal Health Agency, Seestr. 10, D-1000 Berlin 65 (F.R.G.). * Present address: Max-von-Pettenkofer-Institute, Federal Health Agency, Thielallee 88-92, D-1000 Berlin 33 (F.R.G.).
chronic gastric irritation and ulceration but exhibited cocarcinogenic effects when given together with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Chang et al., 1983). ASA was found to be negative in several gene mutation assays with bacteria without and with $9 mix of different species (Bruce and Heddle, 1979; Camus et al., 1982; Kadotani et al., 1984; Kawachi et al., 1980; King et al., 1979) and in a recessive lethal test with Drosophila (King et al., 1979). Several tests for induction of chromosomal aberrations in vitro with fibroblasts or lymphocytes
84
revealed weakly positive or positive results (Ishidate, 1983; Karaseva, 1974; Kawachi et al., 1980; Meisner and Inhorn, 1972). Mauer and Weinstein (1970) quoted negative results in human lymphocytes in vitro and also in lymphocytes of several volunteers who had been taking 2.4 g ASA per day for 1 month. ASA induced no chromosomal aberrations in rat embryos (Tsuruzaki, 1982) and human lymphocytes in vivo (Mauer and Weinstein, 1970); no increase in bone marrow micronucleus frequencies of rats and mice was observed by Heddle et al. (1979) and King et al. (1979). Kawachi et al. (1980) report a positive result for chromosomal aberrations in rat bone marrow without giving any quantitative data. PCM treatment in long-term rodent bioassays resulted in hepatic toxicity. In 1 study carcinogenic effects were observed in the liver (Flaks and Flaks, 1983), 3 others were negative (Hiraga and Fuji, 1985; Hagiwara and Ward, 1986; NTP, 1990). Like ASA, PCM was negative in several gene mutation assays with bacteria with and without $9 mix of several species (Dybing, 1977; Bartsch et al., 1980; Kawachi et al., 1980; Wirth et al., 1980; Dybing et al., 1984; Nohmi et al., 1985; NTP, 1990). A recessive lethal test with Drosophila (King et al., 1979) and a gene mutation assay at the ouabain locus in Chinese hamster cells (Sasaki et al., 1980) were negative. Several in vitro unscheduled DNA ~ynthesis (UDS) assays were carried out by Dybing et al. (1984), Holme et al. (1986) and Milam and Byard (1985) with hepatocytes of different species (rat, mouse, guinea pig). Weakly positive results were observed with mouse hepatocytes only. Alkaline elution assays were negative with PCM (Nordenskj61d and Moldeus, 1983; Dybing et al., 1984; Hongslo et al., 1988) but clearly positive with Nacetyl-p-benzoquinone-imine (NAPQI), which is believed to be the ultimate hepatotoxic metabolite (Dybing et al., 1984). Repeatedly positive results were published for PCM in in vitro tests for clastogenicity with human lymphocytes and rodent cell lines (Kawachi et al., 1980; Ishidate et al., 1980, 1988; Sasaki et al., 1980, 1983; Yoshida et al., 1980; Watanabe, 1982;
Shimane, 1985; Dunn et al., 1987). With the exception of the data published by Yoshida (1980) and NTP (1990) no external metabolization systems were used. Aberration rates varied between weakly and strongly positive. In vivo bone marrow assays (chromosomal aberrations and micronuclei) yielded conflicting results with some negatives (King et al., 1979; Kawachi et al., 1980; Yoshida et al., 1980) and positives (Yoshida et al., 1980; Sicardi et al., 1987). Kocisova et al. (1988) and Hongslo et al. (1990) reported weakly positive effects in human lymphocytes in healthy volunteers after oral treatment with 3 x 1 g PCM within 8 h. Many of the mutagenicity tests reported for ASA and PCM are out of date with respect to the state of the art (e.g., in vitro tests without external metabolization system, too few animals in in vivo tests, only few cells per culture or animal evaluated) or badly documented (e.g., no quantification of results, types of chromosomal aberrations not specified). But for both compounds the published results point towards a clastogenic potential. We investigated whether such a clastogenic potential in vitro depends on the concentration used, the exposure period or is influenced by external metabolization systems such as rat liver $9 mix and primary rat hepatocytes (PRH). Especially for PCM it is clear that hepatic cytochrome P450 forms are major determinants of bioactivation in humans (Raucy et al., 1989) and formation of electrophilic intermediates like NAPQI may account for hepatic toxicity. Therefore metabolic activation has to be considered also when assessing the mutagenic properties of this compound, For assessing toxic effects of PCM metabolites and evaluation of antidotes primary hepatocytes have been shown to be a useful experimental model (Boobis et al., 1986). Materials and m e t h o d s
Test compounds and treatment concentrations
The positive controls mitomycin C (MMC) and cyclophosphamide (CP) were purchased from
85 Sigma (Munich, F.R.G.). P.a.-grade paracetamol and acetylsalicylic acid were purchased from Merck (Darmstadt, F.R.G.). Both compounds were tested in concentrations ranging from 10 -4 to 10 -2 M. Stock solutions were set up in DMSO immediately before use. Final concentrations of DMSO in the cultures did not exceed 1°7o.
106 hepatocytes were seeded per 25-cm2 plastic flask that had been seeded with 2 × 105 V79 cells 24 h earlier. After 1.5 h the medium was replaced by FCS-free Williams E medium (WEM, Flow). Cells treated for 2.5 h or 18.5 h were harvested 18.5 h after beginning of the treatment. All experiments were run with 2 cultures in parallel; experiments with pulse treatment were carried out twice.
Controls In all experiments the following controls were set up: a control without any treatment and a control with DMSO and the metabolizing system to be tested. All controls were set up in duplicate or triplicate.
Experiments system
without
external
metabolization
As target cells for evaluation of chromosomal aberrations V79 cells were used. Experiments without external metabolization system were carried out with 4 different treatment schedules (harvest time in parentheses): 6 h (6 h), 2 h (12 h), 12 h (12 h), and 24 h (24 h). All experiments were run with 2 cultures in parallel. Experiments with 12-h continuous treatment were carried out twice.
Preparation of metaphases:, microscopic analysis For arresting of mitoses in the metaphase, cultures were incubated for 2 h with Colcemid (80 ng/ml). Mitotic cells were shaken off and standard techniques were used. The cells were stained in Giemsa (5070, pH 6.88). All slides were coded prior to analysis. 100 metaphases were analyzed for chromosomal aberrations per culture if possible. Chromatid-type aberrations (chromatid breaks or fragments, chromatid exchanges) and chromosome-type aberrations (chromosome breaks or fragments, dicentrics, and rings) were scored. Metaphases showing multiple aberrations were recorded separately. Gaps and isogaps (achromatic lesions not greater than the width of a chromatid) were scored but were not included in the calculation of damaged cells.
Experiments with $9 mix The $9 fraction was prepared from phenobarbital-pretreated rats. All experiments were carried out with the same batch of $9 fraction which was stored in liquid nitrogen. The $9 mix was prepared and added to the V79 cultures as previously reported (Madle et al., 1986). $9 concentration in cultures was 107o. Treatments with $9 mix were carried out for 2 h. The cells were then washed twice, reincubated for 10 h and then harvested. All experiments were run with 2 cultures in parallel and were carried out twice.
Experiments with hepatocyte/ V79 cocultures Primary rat hepatocytes (PRH) were isolated from 8-10-week-old Wistar rats by the 2-step collagenase perfusion technique as previously reported (Madle et al., 1986). Hepatocytes were used only if the perfusion yielded more than 75070 viable cells (determined by trypan blue exclusion).
Statistics The results were analyzed by the non-parametric H-test (Kruskal and Wallis, 1952), a 1-way analysis of variance for independent data and different sample sizes. P-values below 0.01 were considered to indicate significant differences. Results
Controls The positive controls MMC and CP induced significant incidences of chromosomal aberrations within the range of historical data from our laboratory. The data for CP indicate that $9 mix as well as hepatocytes were able to activate this premutagen. Mean spontaneous aberration rates range from 0.407o to 4.007o between experiments without differences between DMSO-treated and non-treated cultures.
86
ASA Following A S A treatment no significantly elevated chromosomal aberration rates were observed in the whole range of concentrations
tested (Tables 1-3). No influence of a metabolic activation system was registered. A S A was considered to be not clastogenic under the employed test conditions.
TABLE 1 C H R O M O S O M A L A B E R R A T I O N S IN V79 C E L L S I N D U C E D BY A S A A N D P C M W I T H O U T E X T E R N A L M E T A B O L I Z A TION T r e a t m e n t (mole/l)
Harvest time 6 h, 0 (control) D M S O (control) MMC 3.16 ASA 1 ASA 3.16 ASA 1 ASA 3.16 ASA 1 PCM 1 PCM 3.16 PCM 1 PCM 3.16 PCM 1
Cells scored
continuous treatment 300 300 x 10 -6 200 x 10 -2 200 x 10 -3 200 x 10- 3 200 x 10 -4 200 X 10 -4 200 x 10- 2 163 X 10 -3 124 × 10- 3 200 x 10 -4 200 x 10 -4 200
Harvest time 12 h, continuous 0 (control) D M S O (control) MMC 3.16 x 10 -7 ASA 1 x 10 -2 ASA 3.16 x 10 -3 ASA 1 x 10 -3 ASA 3.16 x 10 -4 ASA 1 X 10 - 4 PCM 1 X 10 -2 PCM 3.16 × 10 -3 PCM 1 x 10- 3 PCM 3.16 × 10 -4 PCM 1 x 10 -4
treatment 700 451 100 300 300 400 400 400 52 193 400 400 400
Harvest time 12 h, 2-h treatment 0 (control) 200 D M S O (control) 200 MMC 3.16 × 10 -5 146 PCM 1 × 10 -2 200 PCM 3.16 × 10 -3 200 PCM 1 × 10- 3 200 PCM 3.16 x 10 -4 200 PCM 1 × 10 -4 200
Aberrations per 100 metaphases
Aberrant metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
%
(SEM)
1.3 3.3 14.0 0.5 3.0 2.5 2.0 0.0 24.4 5.1 3.0 6.5 1.5
0.7 1.3 4.5 1.0 0.5 1.0 0.0 0.0 17.4 7.3 1.0 3.0 0.0
0.0 0.3 3.0 0.0 0.0 0.0 0.0 0.0 0.6 0.6 0.0 0.5 0.0
1.0 1.3 6.0 0.0 0.0 0.0 1.0 0.0 19.8 5.1 1.5 2.0 1.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0
0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
4 8 21 2 1 2 1 0 43 17 5 10 2
1.3 2.7 10.5" 1.0 0.5 1.0 0.5 0.0 26.4* 13.7" 2.5 5.0 1.0
(0.7) (0.9) (2.2) (0.7) (0.5) (0.7) (0.5) (0.0) (3.4) (3.1) (1.1) (1.5) (0.7)
2,0 1.1 5.0 3.3 3.0 2.8 3.3 2.8 5.8 10.9 10.0 6.8 3.7
0.7 0.2 4.0 0.3 1.0 0.3 0.5 0.8 13.5 13.0 4.0 1.3 0.5
0.0 0.0 8.0 0.3 0.3 0.3 0.0 0.0 3.9 5.2 3.5 0.0 0.0
0.1 0.0 1.0 0.7 0.7 0.3 0.3 0.3 15.4 8.8 1.3 2.0 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 0.0 0.0
5 2 13 4 5 3 3 4 15 28 31 11 4
0.7 0.4 13.0" 1.3 1.7 0.8 0.8 1.0 28.9* 14.5" 7.8* 2.8* 1.0
0.3) (0.3) (3.6) (0.7) (0.8) (0.5) (0.5) (0.5) (6.3) (2.5) (1.3) 0.8) (0.5)
0.0 0.0 4.1 0.0 1.0 0.0 0.0 0.5
2.0 1.0 15.8 2.5 5.5 0.0 0.0 0.5
0.0 0.0 6.2 0.5 4.5 0.5 0.0 0.0
3.5 1.5 13.0 0.5 3.0 1.5 0.0 0.5
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0
8 4 24 5 13 4 0 2
4.0 2.0 16.4" 2.5 6.5* 2.0 0.0 1.0
(1.4) (1.~ (3.1) (1.1) (1.7) (1.0) (0.0) 0.7)
87 T A B L E 1 (continued) T r e a t m e n t (mole/l)
Harvest time 24 h, continuous 0 (control) D M S O (control) MMC 3.16 × 10 - s ASA 1 x 10 -2 ASA 3.16 × 10 -3 ASA 1 x 10 - 3 ASA 3.16 × 10 -4 ASA 1 x l0 -4 PCM 1 × l0 -2 PCM 3.16 × 10 -3 PCM 1 x l0 -3 PCM 3.16 x l0 -4 PCM 1 >( 10 -4
Cells scored a
treatment 300 300 50 100 100 100 200 200 n.a. n.a. 100 68 200
Aberrations per 100 metaphases
Aberrent metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
%
(SEM)
3.0 2.0 26.0 4.0 1.0 3.0 1.5 0.5
0.7 0.7 50.0 0.0 0.0 1.0 0.0 1.0
0.0 0.0 30.0 0.0 1.0 0.0 0.0 0.0
0.0 1.0 26.0 0.0 0.0 0.0 0.0 0.5
0.0 0.0 2.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 8.0 0.0 0.0 0.0 0.0 0.0
2 2 26 0 1 1 0 2
0.7 0.7 52.0* 0.0 1.0 1.0 0.0 1.0
(0.5) (0.5) (7.1) (0.0) (1.0) (1.0) (0.0) (0.7)
3.0 8.8 3.5
2.0 1.5 0.5
0.0 0.0 0.0
0.0 0.0 0.5
0.0 0.0 0.0
0.0 0.0 0.0
2 1 2
2.0 1.5 1.0
(1.4) (1.5) (0.7)
n.a., not analyzable. G, gaps; B', chromatid breaks a n d fragments; RB', chromatid exchanges; B " , c h r o m o s o m e breaks and fragments; dic/ring, dicentrics a n d rings; M_A, multiple aberrations. * Significant difference vs. negative control at p < 0.01.
PCM PCM was clearly clastogenic in high concentrations with and without external metabolization system. 6-h treatment without external metabolization led to high aberration incidences in concentrations higher than 10- 3 M (Table 1). Slightly higher effects were observed following 12-h exposure to PCM (Table 1). Here 10 -9 M PCM significantly increased the aberration rates to 7.8070 aberrant metaphases. The greatest effects were obtained at 10 -2 M with 28.9070 aberrant metaphases. A 2-h treatment for this harvest time resulted in only weakly increased aberration rates (Table 1). A continuous treatment of 24 h was negative in concentrations up to 10-3 M (Table 1). Treated cultures with concentrations higher than 10-3 M were not analyzable due to lack of metaphases. Addition of $9 mix as external metabolization system for 2 h resulted in a less pronounced increase in the percentage of aberrant metaphases compared to data with V79 cells alone (Table 2). At concentrations higher than 10 -9 M positive responses were obtained with 8.8070 (3.16 x 10-3 M
PCM) and 6.5°70 aberrant metaphases (10 -2 M PCM). Lower concentrations were negative. Cocultivation of V79 cells and primary rat hepatocytes with a treatment period of 2.5 h led to high aberration rates in concentrations above 10-3 M (Table 3). Concentrations up to 10 -9 M were negative. Continuous treatment during the cocultivation period of 18.5 h yielded aberration rates of more than 40070 following treatment with 3.16x 10 -9 M PCM, but only few metaphases could be analyzed due to cytotoxicity (Table 3). Again, concentrations up to I 0 - 3 M were negative.
Discussion ASA was negative in all experiments with and without external metabolization. This is in contradiction to some of the published data, which show clastogenicity of ASA (Ishidate, 1983; Kawachi et al., 1980; Karaseva et al., 1974; Meisner and Inhorn, 1972). Most of these data were generated without using external metabolization, were not reproduced, results were not quan-
88 TABLE 2 C H R O M O S O M A L ABERRATIONS IN V79 CELLS P H E N O B A R B I T A L - I N D U C E D RAT LIVER $9 MIX Treatment (mole/l)
Cells scored
I N D U C E D BY ASA A N D
PCM
Aberrations per 100 metaphases
IN THE
PRESENCE
OF
107o
Aberrant metaphases excl. gaps
G
B'
RB'
B"
dic/ring
MA
n
070
(SEM)
3.0 5.2 13.3 3.0 4.0 3.5 4.3 3.8 2.3 5.3 4.8 3.3 4.0
1.0 1.5 12.9 2.3 1.0 0.5 1.8 1.0 3.0 9.3 3.8 1.5 2.0
0.0 0.0 7.6 0.0 0.0 0.3 0.0 0.0 4.5 6.5 2.0 0.0 0.0
0.7 0.3 6.0 0.8 1.0
0.2 0.0 0.2 0.0 0.0
0.0 0.0 7.8 0.0 0.0
7 11 101 11 3
1.2 1.8 23.2* 2.8 0.8
(0.5) (0.5) (2.0) (0.8) (0.5)
0.0
0.0
0.0
3
0.8
(0.5)
0.3 0.3 0.8 4.0 0.5 0.5
0.0 0.0 0.0 0.0 0.0 0.0
0.3 0.3 0.5 2.0 0.3 0.0
6 6 26 35 14 8
1.5 1.5 6.5* 8.8* 3.5 2.0
(0.6) (0.6) (1.2) (1.4) (0.9) (0.7)
0.7
0.0
0.0
4
1.3
(0.7)
Harvest time 12 h, 2-h treatment 0 (control) DMSO (control) CP 1 ASA 1 ASA 3.16 ASA 1 ASA 3.16 ASA 1 PCM 1 PCM 3.16 PCM 1 PCM 3.16 PCM 1
+ × × x × x × x × x x x
$9 10 -5 10- 2 10 -3 10- 3 10 -4 10 -4 10 -z 10 -3 10- 3 10 -4 10 -4
600 600 435 400 400 400 400 400 400 400 400 400 300
For abbreviations see Table 1. *Significant difference vs. negative control at p < 0.01.
tified, or evaluation criteria are quite unclear (e.g., no discrimination of different types of aberrations). Ishidate (1983) found high incidences of chromosomal aberrations in CHL cells 48 h after treatment with and without $9 mix, but no increase in the 24-h harvest time. The range of concentrations was the same as in the present investigation. The reasons for this time dependence, i.e., the observation of high incidences of chromosomal aberrations after 2 or more cell cycles and no aberrations after 1-2 cell cycles, are quite unclear and still have to be investigated. PCM was clearly clastogenic with and without external activation. Strongly positive responses in all experiments were obtained with concentrations higher than 10-3 M, weak effects were observed when cells were treated with 10- 3 M PCM. Shortterm exposure with and without $9 mix resulted in weakly elevated chromosomal aberration rates only whereas cocultivation with PRH led to much more pronounced effects. Continuous exposure to PCM was generally strongly cytotoxic in high concentrations but clearly elevated chromosomal aber-
ration rates were already observed at concentrations without any marked cytotoxicity. The data suggest that PCM is clastogenic independent of metabolization but this effect could be intensified by cocultivation with PRH. Aspects of these 2 principles have been discussed in the literature. Hongslo et al. (1988) found SCE induction by PCM to be independent of cocultivation of V79 cells with or without primary mouse hepatocytes and suggested that PCM induces chromosomal alterations such as SCE via an indirect mechanism independent of activation: PCM inhibits the ribonucleotide-reductase which results in a reduction of the ribonucleotide pool and thus leads to reduced DNA synthesis and possibly chromosomal alterations (Hongslo et al., 1989a,b). On the other hand P450-mediated activation of PCM leads to electrophilic species such as Nacetyl-p-benzoquinone-imine (NAPQI) (for review of PCM metabolism see Hinson, 1983). Reactive metabolites such as NAPQI may also covalently bind to DNA and could well take part in the clastogenicity induced via external metabolization.
89
TABLE 3 C H R O M O S O M A L ABERRATIONS IN V79 CELLS I N D U C E D W I T H A S A A N D PCM: C O C U L T I V A T I O N W I T H PRIMARY RAT HEPATOCYTES (PRH)
Treatment (mole/l)
Cells scored
Aberrations per 100 metapbases
G Harvest time 18.5 h, 2.5-h treatment 0 control 400 DMSO (control) + PRH 400 CP 1 x 10 -4 200 ASA 1 x 10 -2 400 ASA 3.16 x 10 -3 400 ASA 1 x l0 -~ 400 ASA 3.16 x 10 -4 350 ASA 1 X l0 -4 400 PCM 1 X l0 -2 387 PCM 3.16 × l0 -~ 400 PCM 1 x l0 -3 300 PCM 3.16 x l0 -4 400 PCM 1 x 10 -4 400
2.4 2.3 13.5 3.0 2.8 2.8 2.3 6.1 2.6 2.0 3.7 3.3 2.8
Harvest time 18.5 h, continuous treatment 0 (control) 200 1.5 DMSO (control) + PRH 200 0.5 CP 1 x 10 -4 200 6.0 PCM 1 x 10 -z n.a. PCM 3.16 x 10 -3 57 8.8 PCM 1 x 10- 3 200 1.5 PCM 3.16 x 10 -4 200 1.5 PCM 1 x 10 -4 200 1.0
B'
RB'
B"
Aberrant metaphases excl. gaps dic/ring
MA
n
o70
(SEM)
1.8 2.5 4.0 0.8 2.5 2.3 1.4 2.8 7.8 6.3 1.0 1.5 2.8
0.0 0.0 1.5 0.0 0.0 0.5 0.3 0.0 7.8 9.3 0.0 0.3 0.0
0.0 0.8 6.0 0.5 0.5 0.3 0.3 0.3 2.8 3.3 1.0 0.5 0.8
0.0 0.3 0.5 0.3 0.0 0.3 0.0 0.0 0.3 0.3 0.3 0.5 0.0
0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 6.7 6.5 0.0 0.0 0.0
4 10 15 6 9 13 5 12 59 57 7 7 7
1.0 2.5 7.5* 1.5 2.3 3.3 1.4 3.0 15.2" 14.3" 2.3 1.8 1.8
(0.5) (0.8) (1.9) (0.6) (0.8) (0.9) (0.6) (0.9) (1.8) (1.8) (0.9) (0.7) (0.7)
0.5 0.5 40.5
0.0 0.0 19.0
2.5 0.5 27.5
0.0 0.0 -
0.0 0.0 6.0
5 2 95
2.5 1.0 42.5*
(1.1) (0.7) (3.5)
24.6 0.5 0.5 0.0
15.8 1.5 0.0 0.0
28.1 1.5 1.5 0.0
0.0 0.0 0.0 0.0
17.5 0.0 0.0 0.0
26 7 4 0
45.6* 3.0 2.0 0.0
(6.6) (1.2) (1.0) (0.0)
For abbreviations see Table 1. * Significant difference vs. negative control at p < 0.01
The reactive metabolites of PCM preferentially bind to proteins and glutathione in the liver (Garle and Fry, 1989; Bartolone et al., 1989). The latter reaction is an efficient protective mechanism against the toxic properties of PCM. However, in high doses the glutathione pool is depleted and free toxic metabolites occur. Glutathione depletion as reported in animals has been observed in liver ceils of humans in vitro (Tee et al., 1987) and in vivo following an intake of 0.5-3 g PCM (Slattery et al., 1987). But marked differences in cytotoxicity of PCM and covalent binding of PCM to microsomal proteins were demonstrated for hepatocytes of different species in vitro (Tee et al., 1987). Hamster and mouse hepatocytes were more sensitive than
primary rat hepatocytes and human hepatocytes, which turned out to be almost equally resistant. Tee et al. (1987) attribute these findings to different concentrations required for glutathione depletion and different conversion rates of PCM to its reactive metabolite NAPQI, which was equally effective in hepatocytes of all species used. PCM was also more effective in inducing UDS in primary mouse hepatocytes than in PRH (Holme and Soederlund, 1986). These data suggest that PRH used in our experiments are a good model for the human situation. The indirect action of PCM via reduction of the nucleotide pool and the direct interaction with DNA via reactive metabolites that can be trapped
90 with glutathione up to its depletion suggest the existence of threshold mechanisms. Our in vitro results are in good agreement with this theory: PCM acted as a clastogen with and without external metabolization only in concentrations of 10-3 M and greater. An additional effect in the same concentrations was observed when hepatocytes were used as cocultivation system. This should be the result of hepatocyte-generated DNA-damaging reactive metabolites. From our results a no observed effect level of 3.16 x l0 -4 M could be deduced. Other data from UDS tests (Dybing et al., 1984; Holme and Soederlund, 1986), alkaline elution (Hongslo et al., 1988), SCE analysis (Hongslo et al., 1988, 1990), LDH leakage, cytotoxicity, and intracellular glutathione depletion (Dybing et al., 1984; Tee et al., 1987) are in line with this interpretation, because these effects were also not observed below this concentration. UDS data (Holme and Soederlund, 1986) and our test results show also that PCM was generally strongly cytotoxic in the same concentration range where genotoxic effects were observed. Therefore it was suggested that cytotoxic effects of PCM may partly dominate its genotoxic properties (Holme and Soederlund, 1986; Holme et al., 1988). Results of in vivo bone marrow mutagenicity tests with PCM in mice and rats were predominantly negative although not sufficiently documented (King et al., 1979; Kawachi et al., 1980; Yoshida et al., 1980). In 1 test with rats slightly elevated micronucleus incidences in bone marrow were found at doses of 100 mg/kg and 150 mg/kg i.p. In 2 studies with human volunteers elevated incidences of chromosomal aberrations in lymphocytes following uptake of 3 × 1 g PCM within 8 ~h were reported (Kocisova et ai., 1988; Hongslo et al., 1990). Since PCM is almost uniformly distributed in the body, comparison of in vivo blood plasma levels with in vitro concentrations should give additional information. However, in the studies with human lymphocytes, no data on plasma levels were presented. A therapeutic dose of about 1 g PCM leads to average blood plasma levels of 20/~g/ml (Forrest et al., 1982), giving a concentration of
1.3x 10 - 4 M. It is apparent that human plasma concentrations are not far below the high concentrations, which yielded clastogenic effects in vitro. Although in vivo/in vitro comparisons are only approximative and in vitro data suggest a threshold mechanism, the weakly positive in vivo data on chromosome mutations in human lymphocytes are in line with our in vitro data. Since glutathione-mediated detoxification of PCM plays a major role in the present evaluation individual differences in glutathione levels in the human population are of interest. Recently published results show that lymphocytes from persons defective in glutathione S-transferase were more susceptible to epoxide-induced genetic damage than lymphocytes from persons having this enzyme (Wiencke et al., 1990). Indiviuals lacking the enzyme are also at high risk for smokinginduced lung cancer (Seidegard et al., 1986). For example about 50°70 of all caucasians are genetically deficient in this isozyme. Data on genetic differences between the volunteers of the 2 in vivo studies with PCM are not available, but the observation that individual data showed more pronounced effects for some volunteers whereas others were negative (personal communication) gives indications that there could be an association with glutathione S-transferase activities. Taking in vitro and in vivo data together, it cannot be excluded that therapeutic use of PCM gives rise to genotoxic effects, but the mechanisms of action still have to be elucidated and risk assessment has to be done.
Acknowledgements The authors greatly appreciate the expert technical assistance of Mrs. G. Kaufmann and Mrs. H. Madle in scoring the slides and preparing the tables and statistics.
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