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Mutation Research, 56 (1978) 319--327 © Elsevier/North-Holland Biomedical Press
CYTOGENETIC E F F E C T S IN THE MOUSE OF 17 CHEMICAL MUTAGENS AND CARCINOGENS E V A L U A T E D BY THE MICRONUCLEUS TEST
DIETER WILD
Zentrallaboratorium fiir Mutagenita'tspriifung der Deutschen Forschungsgemeinschaft, Freiburg (Germany) (Received 10 August 1977) (Accepted 3 October 1977)
Summary 2 dialkylnitrosamines, 4 oxazaphosphorines, 6 aryldialkyltriazenes, urethane, N-hydroxyurethane, 4-nitroquinoline-l-oxide, procarbazine (natulan) and the inorganic carcinogen potassium chromate were studied for cytogenetic activity in the micronucleus test on mouse bone marrow. Except diethylnitrosamine, all chemicals were active. The results are compared with those known from studies in other mammalian and sub-mammalian test systems. The results of the micronucleus test correlate well with results from other mutagenicity tests and with the carcinogenicity of the chemicals. The lack of an effect of N-nitrosodiethylamine (DENA) is discussed with regard to the short life-time of the ultimate mutagen.
Introduction The increasing demand for protection against environmental mutagens and carcinogens creates a need for reliable short-term tests for detection of genotoxic effects of chemicals in mammals. A recently developed short-term screening test for cytogenetic damage in mammalian somatic cells in vivo is the micronucleus test. Micronuclei, the endpoint of mutagenic processes observed in this test, are nuclear fragments that result from chromosome breaks or chromosome losses; they can most easily be detected in bone-marrow erythrocytes. The present study was undertaken to further validate the micronucleus test and to collect data on the correlation between chromosome-breaking effects in 4-Bromo-PDMT, 1-(4-bromophenyl)-3,3-dimethyltriazene; 4-Chloro-PDMT, 1-(4chlorophenyl)-3,3-dimethyltriazene; DENA, N-nitrosodiethylamine; DMNA, N-nitrosodlmethylamine; 4-NQO, 4-nitroquinoline-l-oxide; PDET, 3,3-diethyl-l-phenyltriazene; PDMT, 3 03-dimethyl1-phenyltriazene; PyDMT, 3,3-dimethyl-l-(pyridyl-3)-triazene; 2,4,6-Trichloro-PDMT, 3,3-dimethyl-l-(2,4,6-trichlorophenyl)-triazene. Abbreviations:
320 mammals in vivo and other mutagenic and carcinogenic effects of chemicals. For this purpose, chemical mutagens and/or carcinogens representing several widely different chemical classes were selected: N-nitrosoamines, oxazaphosphorines, aryldialkyltriazenes, carbamates, a nitroquinoline oxide, a hydrazine derivative, and an inorganic chromium compound. Most of these compounds have been described as mutagens in microbial tests with added liver microsomes. On the other hand, cytogenetic data from mammalian tests in vivo have been reported for only few of these compounds. ' Materials and methods Chemicals The following chemicals were studied: DMNA, DENA, cyclophosphamide, ifosfamide, trofosfamide, sufosfamide, PDMT, 4-chloro-PDMT, 4-bromoPDMT, 2,4,6-trichloro-PDMT, PyDMT, PDET, urethane, N-hydroxyurethane, 4-NQO, procarbazine, potassium chromate. The origin of these chemicals is indicated in Table 1. Animals NMRI mice (supplier: Ivanovas, Kisslegg) weighing 26--35 g were used. They were fed on pelleted standard diet (Herilan HAN-MR3) and water ad libitum. Micronucleus test For each dose, 4 mice of both sexes were treated identically by two intraperitoneal injections, separated by a 24-h interval; volumes of 0.3 ml per 30 g mouse weight were injected. 6 h after the second injection the animals were killed by cervical dislocation, and bone-marrow smears were prepared. After being stained, the slides were coded, 1000 polychromatic erythrocytes per mouse were screened and the number of polychromatic erythrocytes with one or more micronuclei counted; normochromatic micronucleus~ontaining erythrocytes were also recorded. The techniques of bone marrow preparation and staining were performed according to the detailed description of Schmid [34,35]. Results
Table 1 presents the compounds tested, the vehicles used for the injections, doses and results of the micronucleus tests. Some of the present results have been shortly reported previously [43]. The doses applied were all non-lethal within the duration of the experiment. Toxic effects on the bone marrow were, however, noted with some compounds and doses. These manifested themselves as declined frequency of nucleated cells and as declined proportion of polychromatic among total (polychromatic plus normochromatic) erythrocytes. In each experiment, control animals were treated with the vehicle only. The cytogenetic effect of the chemicals is expressed by the frequency of micronucleated polychromatic erythrocytes; this parameter is a more sensitive indicator for micronucleus induction by alkylating mutagens than the frequency of micronucleated total erythrocytes because micronucleated n o r m o -
321 chromatic erythrocytes disappear from the bone marrow [42]. The frequency of micronucleated normochromatic erythrocytes was n o t increased in these experiments. All chemicals tested, with the exception of DENA, produced dose 0.05) and the 2-fold and 4-fold doses did not produce comparable effects. Discussion
Mutagens of several chemical classes were studied: N-nitrosoamines, oxazaphosphorines, aryldialkyltriazenes, carbamates, nitroquinoline oxide, a hydrazine derivative, and an inorganic chromium c o m p o u n d . The carcinogenic and mutagenic effects of DMNA and DENA depend on the biotransformation by mammalian e n z y m e s - primarily in the l i v e r - and the production of unstable electrophilic alkylating agents, possibly alkyldiazonium ions. (For review see ref. 27.) Owing to the instability of these ultimate mutagens, their concentration in organs remote from their site of production cannot exceed a compound-specific, relatively low value. The results of the micronucleus tests can be interpreted as a consequence of this instability: DMNA, compared with other alkylating mutagens, is weakly active, and DENA was inactive in the bone marrow. Similar results were obtained by the use of other tests in the same and different target organs in mice: sister-chromatid exchange in bone marrow and specific-locus mutations in embryonic pigment-cell precursors were induced by DMNA b u t not by DENA [2,9]. All these data are compatible with the assumption that (an} ultimately mutagenic metabolite(s) of DMNA b u t not of DENA is (are) sufficiently stable to leave the metabolizing organ (primarily liver) and to produce gene and chromosomal mutations in remote organs which, themselves, do not or do not significantly produce the metabolites. On the other hand, in Drosophila germ cells, DENA induces recessive lethal mutations, b u t it fails to produce heritable structural chromosomal mutations [40]. Therefore, it had been argued, this mutation-type specificity -- if applicable to m a m m a l s - could explain previously reported lack of dominant-lethal [33] or cytogenetic [43] effects of DENA in mice. However, the extreme specificity observed in Drosophila does not appear in mammalian cells; the cytogenetic activity of DENA has been unequivocally shown under such conditions where the mammalian target cells are actually exposed even to short-lived metabolites. Thus, karyotypic alterations were reported in liver cells of DENAtreated rats [13], and chromosomal aberrations and sister-chromatid exchanges are clearly induced in mammalian cells in vitro in the presence of DENA and active liver microsomes [30]. Possible reasons for the difference between Drosophila and mammals are discussed by Natarajan et al. [30]. The absence of micronucleus induction in bone marrow by DENA cannot, therefore, be satisfactorily explained by a mutation t y p e specificity b u t more likely by the instability of the active metabolite(s) and thereby resulting organ-specificity of effects.
322
TABLE 1 R E S U L T S OF M I C R O N U C L E U S TESTS Compound (vehicle)
Origin o f compound
Intraperitoneal doses mg/kg mmoles/kg
Dialkylnitrosamines DMNA ( 0 . 9 % NaC1)
A
2× 2 × 2X 0
A
2 2 2 2 0
B
2 × 140 2 X 70 2 X 28 0
2×0.5 2X0.25 2X0.1 0
79.1 a 65.8 a 20.9 a 1.6
Ifosfamide (Hanks's solution)
B
2X 140 2 × 70 2 X 35 0
2×0.54 2X0.27 2X0.13 0
37.6 a 24.4 a 13.8 a 1.9
Trofosfamide (olive oil)
B
2 × 160 2 X 80 2 X 40 0
2×0.5 2X0.25 2×0.125 0
47.6 a 56.7 a 33.8 a 2.4
Sufosfamide (Hanks's solution)
B
2 × 0
80
2 X 0.25 0
62.6 a 2.5
A
2 2 2 2 0
25 12.5 6.25 3.12
2 2 2 2 0
4-Chloro-PMDT (olive oil)
C
2 X 100 2 X 50 2 × 25 0
2×0.55 2X0.27 2X0.14 0
95.4 a 30.6 a 22.8 a 2.6
4-Bromo-PDMT (olive oil)
C
2 X 100 2 × 5O 2 × 25 0
2×0.44 2X0.22 2X0.11 0
50.4 a 15.8 a 2.7 1.7
2,4,6-Trichloro-PMDT (olive oil)
C
2 × 100 2 × 50 2 X 25 0
2X0.40 2×0.20 2×0.10 0
48.2 a 12.9 a 7.4 a 2.2
PyDMT (olive oil)
D
2 X 25 2 × 12.5 2 × 6.25 0
2X0.16 2×0.08 2X0.04 0
32.1 a 33.9 a 7.3 a 2.4
PDET (olive oil)
D
2 2 2 2 0
2X0.4 2×0.3 2X0.2 2XO.1 0
16.9 a 22.4 a 9.1 a 5.2 3.2
DENA ( 0 . 9 % NaCI)
Oxazaphosphorines Cyclophosphamide (Hanks's solution)
A r y ldialk y l t r i a z e n e s PDMT (olive oil)
X × × ×
× × × ×
X X × ×
Micronucleated polychromatic e r y t h r o c y t e s (%0)
37 18.5 9.2
2×0.5 2×0.25 2X0.125 0
14.9 a 10.0 a 3.4 2.4
100 50 25 12.5
2X0.98 2X0.49 2X0.25 2×0.125 0
2.6 3.4 6.1 2.9 4.2
71 53.2 35.5 17.7
X 0.168 X 0.084 X 0.042 × 0.021
58.5 53.6 47.3 21.1 2.9
a a a a
323 TABLE 1 (continued)
mg/kg
mmoles/kg
Micronucleated polychromatic e r y t h r o c y t e s (%0)
E
2×356 2 × 178 2 X 89 0
2X4 2× 2 2 X1 0
18.5 a 15.4a 4.4 3.2
F
2 × 250 2 × 125 0
2 × 2.38 2 X 1.19 0
18.8 a 6.8 a 1.9
A
2 X 95 2 X 38 2 X 19 0
2 X 0.5 2 X 0.2 2X0.1 0
33.4 a 19.2 a 5.6a 1.9
Procarbazine (Hanks's solution,
G
2 2 2 2 0
2 2 2 2 0
34.1 28.3 30.4 8.8 3.3
Potassium chromate ( 0 . 9 % NaCI)
H
2 X 48.5 2 X 24.25 2 X 12.12 0
Compound (vehicle)
Carbamates Urethane (Hanks's solution)
N-Hydroxyurethane ( 0 . 9 % NaCI)
Various c o m p o u n d s 4-NQO (3% g u m a r a b i c )
A B C D E F G H
Origin o f compound
Intraperitoneal doses
X 51.6 X 25.8 X 12.9 × 6.45
× 0.2 X 0.1 X 0.05 X 0.025
2 X 0.25 2 X 0.125 2 X 0.062 0
a a a a
15.0 a 9.6 a 4.8 3.1
Schuchardt, Munich, Prof. N. B r o c k , A s t a - W e r k e , Bielefeld. Dr. G. K o l a r , G e r m a n C a n c e r R e s e a r c h C e n t e r , H e i d e l b e r g . S y n t h e s i z e d a c c o r d i n g to ref. 37. Merck, Darmstadt. S y n t h e s i z e d a c c o r d i n g to ref. 11. H o f f m a n - L a R o c h e , Basle, S w i t z e r l a n d . R i e d e l - d e Hai~n, H a n n o v e r .
a Significantly d i f f e r e n t f r o m t h e c o n t r o l ( a ~ 0 . 0 1 ) (ref. 16).
Recently, Friedman and Staub [10] also reported on the effect of DMNA in micronucleus tests on mice. These authors find very weak DMNA effects and do n o t indicate the m e t h o d used for determining the statistical significance of these effects. However, triethylene melamine used for the positive controls also induces only feeble effects with questionable significance. This is in contrast with other published data [23] and deserves further study. The mutagenicity of cyclophosphamide has been studied in numerous submammalian and mammalian test systems (for review see ref. 26) including the micronucleus test [21,23,28]; it was included in this study as a reference mutagen for inter-laboratory comparison, and a good agreement between the present and previous data is evident. For the first time the chromosome-breaking potential of ifosfamide and trofosfamide in mammals is demonstrated here. Qualitatively, the results agree very well with those gained with the Basc test in Drosophila [40], the host-mediated and urinary assays with yeast [36] and from activation and mutation studies on E.coli in vitro [7]. Q u a n t i t a t i v e - admittedly very r o u g h - dose comparisons show that gene mutations in E.coli in
324 vitro and micronuclei in mouse bone marrow are induced by similar doses of these agents, whereas higher doses are required to obtain effects in host-mediated assays [8,36]. Sufosfamide (previously designated "Asta 5122", ref. 4), a methane sulfonate oxazaphosphorine, has not so far been tested for mutagenic properties. It is about equally effective in the micronucleus test as cyclophosphamide, followed by trofosfamide and ifosfamide. In contrast with nitrosamines, oxazaphosphorines are converted into stable alkylating, mutagenic metabolites, which are transported throughout the organism and can even be detected in urine [36]. They can, therefore, efficiently induce mutations not only near the site of metabolism but also in other tissues. Aryldialkyltriazenes are potent carcinogens [32] and mutagens in microbes (those studied here only in the presence of mammalian liver enzymes) and in Drosophila [ 19,22,41]. Two potential mechanisms of the carcinogenic and mutagenic effects of these compounds have been discussed by Preussmann et al. [32]. (1) Spontaneous (non-enzymic) hydrolysis of triazenes under formation of aryldiazonium salts; these can possibly arylate nucleic acids. This pathway appears less important for the triazenes studied here, since they are not easily hydrolyzable. (2) Enzymic oxidative dealkylation under formation of monoalkyltriazenes, known as alkylating agents. The second mechanism is supported by the detection of methylated nucleic acid components after treatment of rats with PDMT [17,20]. PDMT is the only triazene so far studied for genetic activity in mammals in vivG; it induces micronuclei and sister-chromatid exchanges in mouse bone marrow [2]. These results are extended by the present data on 5 additional aryldialkyltriazenes. It is shown here that these carcinogens induce cytogenetic damage in mammals with high efficiency. Of all mutagens in this study, PDMT is the most potent in terms of the lowest dose required for significant effects. PDET is the diethyl homolog of PDMT and probably acts via an ethylating agent. The results with PDET and PDMT clearly show that ethylating as well as methylating agents can produce cytogenetic damage in the bone marrow and that a quantitative difference exists, PDET being less effective than PDMT. The dose--effect curves of the dimethyl compounds follow two different patterns and suggest a correlation with the chemical structure: the triazenes with an unsubstituted aromatic ring, PDMT and PyDMT, are effective at rather low doses and exhibit a saturation at higher doses. On the other hand, the 4-chloro- and 4-bromo-substituted triazenes are only weakly active at these low doses, their effect increasing proportionally with the dose. Based on the assumption that oxidative dealkylation is a prerequisite for the genetic effects, it therefore appears likely that an additional reaction involving the 4-position of the aromatic ring modifies the dose--effect relationship. This reaction could be ring hydroxylation in the 4-position which is in fact a major metabolic reaction occurring on PDMT [ 18]. This reaction is probably inhibited in 4-chloro-PDMT, 4-bromo-PDMT, and 2,4,6-trichloro-PDMT, whereas it is possible in PDMT and PyDMT. Urethane and its metabolite N-hydroxyurethane have been well d o c u m e n t e d as carcinogens [15]. At equimolar doses, both induce micronuclei at comparable frequencies. Compared with the other mutagens in this study, these carbamates are weakly active even at high doses. These data confirm previous reports
325 on induction of chromosomal aberrations in mammalian cells in vitro and in vivo. (For review see ref. 1.) The dominant-lethal test in the mouse and the Salmonella/microsome test, however, failed to detect a genetic activity of urethane [1,24,25]. 4-NQO is carcinogenic and highly mutagenic in numerous organisms. (For review see ref. 29.) It is a representative of the nitroheterocyclic mutagens which are activated metabolically in microbial and mammalian cells by reduction of the nitro group. This activation mechanism and the ultimate mutagen are different from those of indirect alkylating agents, and it is n o t e w o r t h y that the micronucleus test is a sensitive indicator for this type of mutagen as well. The cytostatic hydrazine derivative procarbazine (natulan ~!)) is a p o t e n t inducer of micronuclei: a significant effect was observed at a total dose of 12.9 mg/kg. The single doses equal the upper recommended single therapeutic dose for adults and are therefore "realistic" under the aspect of human exposure. In the dominant-lethal test and in the specific locus test on mice, significant effects of procarbazine were found at doses of 400 and 600 mg/kg, well above those in the micronucleus test [5,6]. It was found inactive ("false negative") in the Salmonella/microsome test [24,25], whereas it follows PDMT as the most p o t e n t mutagen of those studied here. Chromates and dichromates are mutagenic in bacteria [12,31,39] and yeast [3] in absence of mammalian e n z y m e s ; they induce chromosomal aberrations in mammalian cells in culture [38]. It is shown here that potassium chromate is genetically active also in the intact mammal; similar results were found with sodium chromate (unpublished data}. The mutagenic as well as the carcinogenic effects of inorganic chromium c o m p o u n d s are restricted to compounds of hexavalent chromium (chromates, dichromates) whereas trivalent chromium c o m p o u n d s are inactive [14]. Hexavalent chromium c o m p o u n d s are electrophilic and oxidizing agents; specific oxidations by these c o m p o u n d s could, therefore, be responsible for the observed effects. The 17 chemicals studied here represent 7 chemical classes of mutagens and carcinogens (see Table 1). All these c o m p o u n d s except 4-NQO and potassium chromate are indirect mutagens, i.e. in mammals b u t not in bacteria they are metabolically converted into the ultimate mutagens. The actual mutagens are electrophilic alkylating agents in the case of the nitrosamines, oxazaphosphorines and triazenes. The remaining direct and indirect mutagens comprise compounds with different molecular structures and different less well known mutagenic mechanisms. The data reported demonstrate within the group of selected chemicals a high correlation between chromosome-breaking effects in vivo, revealed by the micronucleus test, and otherwise mutagenic effects as determined by sub-mammalian tests. A similar correlation emerges between chromosome-breaking and carcinogenic effects. Since only a small group of mutagens and carcinogens was studied here, further studies on additional classes of carcinogen are required for a more general assessment of the latter correlation. As a tool for such a screening of cytogenetic {chromosome-breaking and spindle inhibiting} effects the micronucleus test can be r e c o m m e n d e d on the basis of previous data and of those presented here and because it is fast by comparison with classical cytogenetic tests in vivo.
326
Potential exceptions deserve especially careful consideration; DENA, for example, is an exception if the results of the micronucleus test and other mammalian tests on bone marrow alone are considered. It is, however, not a "false" negative and does not contradict a correlation of carcinogenic and chromosome-breaking effects, but rather illustrates the critical role of a characteristic property of a mutagen and demonstrates the necessity to take such characteristics into account. References 1 B a t e m a n , A~I., T h e m u t a g e n i c a c t i o n o f u r e t h a n e , M u t a t i o n R e s . , 3 9 ( 1 9 7 6 ) 7 5 - - 9 6 . 2 B a u k n e c h t , T h . , W. V o g e l , U. B a y e r a n d D. Wild, C o m p a r a t i v e in vivo m u t a g e n i c i t y t e s t i n g b y S C E a n d m i c r o n u c l e u s i n d u c t i o n in m o u s e b o n e m a r r o w , H u m a n G e n e t . , 3 5 ( 1 9 7 7 ) 2 9 9 - - 3 0 7 . 3 B o n a t t L S., M. M e i n i a n d A. 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Mutation Research, 56 (1978) 319--327 © Elsevier/North-Holland Biomedical Press
CYTOGENETIC E F F E C T S IN THE MOUSE OF 17 CHEMICAL MUTAGENS AND CARCINOGENS E V A L U A T E D BY THE MICRONUCLEUS TEST
DIETER WILD
Zentrallaboratorium fiir Mutagenita'tspriifung der Deutschen Forschungsgemeinschaft, Freiburg (Germany) (Received 10 August 1977) (Accepted 3 October 1977)
Summary 2 dialkylnitrosamines, 4 oxazaphosphorines, 6 aryldialkyltriazenes, urethane, N-hydroxyurethane, 4-nitroquinoline-l-oxide, procarbazine (natulan) and the inorganic carcinogen potassium chromate were studied for cytogenetic activity in the micronucleus test on mouse bone marrow. Except diethylnitrosamine, all chemicals were active. The results are compared with those known from studies in other mammalian and sub-mammalian test systems. The results of the micronucleus test correlate well with results from other mutagenicity tests and with the carcinogenicity of the chemicals. The lack of an effect of N-nitrosodiethylamine (DENA) is discussed with regard to the short life-time of the ultimate mutagen.
Introduction The increasing demand for protection against environmental mutagens and carcinogens creates a need for reliable short-term tests for detection of genotoxic effects of chemicals in mammals. A recently developed short-term screening test for cytogenetic damage in mammalian somatic cells in vivo is the micronucleus test. Micronuclei, the endpoint of mutagenic processes observed in this test, are nuclear fragments that result from chromosome breaks or chromosome losses; they can most easily be detected in bone-marrow erythrocytes. The present study was undertaken to further validate the micronucleus test and to collect data on the correlation between chromosome-breaking effects in 4-Bromo-PDMT, 1-(4-bromophenyl)-3,3-dimethyltriazene; 4-Chloro-PDMT, 1-(4chlorophenyl)-3,3-dimethyltriazene; DENA, N-nitrosodiethylamine; DMNA, N-nitrosodlmethylamine; 4-NQO, 4-nitroquinoline-l-oxide; PDET, 3,3-diethyl-l-phenyltriazene; PDMT, 3 03-dimethyl1-phenyltriazene; PyDMT, 3,3-dimethyl-l-(pyridyl-3)-triazene; 2,4,6-Trichloro-PDMT, 3,3-dimethyl-l-(2,4,6-trichlorophenyl)-triazene. Abbreviations:
320 mammals in vivo and other mutagenic and carcinogenic effects of chemicals. For this purpose, chemical mutagens and/or carcinogens representing several widely different chemical classes were selected: N-nitrosoamines, oxazaphosphorines, aryldialkyltriazenes, carbamates, a nitroquinoline oxide, a hydrazine derivative, and an inorganic chromium compound. Most of these compounds have been described as mutagens in microbial tests with added liver microsomes. On the other hand, cytogenetic data from mammalian tests in vivo have been reported for only few of these compounds. ' Materials and methods Chemicals The following chemicals were studied: DMNA, DENA, cyclophosphamide, ifosfamide, trofosfamide, sufosfamide, PDMT, 4-chloro-PDMT, 4-bromoPDMT, 2,4,6-trichloro-PDMT, PyDMT, PDET, urethane, N-hydroxyurethane, 4-NQO, procarbazine, potassium chromate. The origin of these chemicals is indicated in Table 1. Animals NMRI mice (supplier: Ivanovas, Kisslegg) weighing 26--35 g were used. They were fed on pelleted standard diet (Herilan HAN-MR3) and water ad libitum. Micronucleus test For each dose, 4 mice of both sexes were treated identically by two intraperitoneal injections, separated by a 24-h interval; volumes of 0.3 ml per 30 g mouse weight were injected. 6 h after the second injection the animals were killed by cervical dislocation, and bone-marrow smears were prepared. After being stained, the slides were coded, 1000 polychromatic erythrocytes per mouse were screened and the number of polychromatic erythrocytes with one or more micronuclei counted; normochromatic micronucleus~ontaining erythrocytes were also recorded. The techniques of bone marrow preparation and staining were performed according to the detailed description of Schmid [34,35]. Results
Table 1 presents the compounds tested, the vehicles used for the injections, doses and results of the micronucleus tests. Some of the present results have been shortly reported previously [43]. The doses applied were all non-lethal within the duration of the experiment. Toxic effects on the bone marrow were, however, noted with some compounds and doses. These manifested themselves as declined frequency of nucleated cells and as declined proportion of polychromatic among total (polychromatic plus normochromatic) erythrocytes. In each experiment, control animals were treated with the vehicle only. The cytogenetic effect of the chemicals is expressed by the frequency of micronucleated polychromatic erythrocytes; this parameter is a more sensitive indicator for micronucleus induction by alkylating mutagens than the frequency of micronucleated total erythrocytes because micronucleated n o r m o -
321 chromatic erythrocytes disappear from the bone marrow [42]. The frequency of micronucleated normochromatic erythrocytes was n o t increased in these experiments. All chemicals tested, with the exception of DENA, produced dose 0.05) and the 2-fold and 4-fold doses did not produce comparable effects. Discussion
Mutagens of several chemical classes were studied: N-nitrosoamines, oxazaphosphorines, aryldialkyltriazenes, carbamates, nitroquinoline oxide, a hydrazine derivative, and an inorganic chromium c o m p o u n d . The carcinogenic and mutagenic effects of DMNA and DENA depend on the biotransformation by mammalian e n z y m e s - primarily in the l i v e r - and the production of unstable electrophilic alkylating agents, possibly alkyldiazonium ions. (For review see ref. 27.) Owing to the instability of these ultimate mutagens, their concentration in organs remote from their site of production cannot exceed a compound-specific, relatively low value. The results of the micronucleus tests can be interpreted as a consequence of this instability: DMNA, compared with other alkylating mutagens, is weakly active, and DENA was inactive in the bone marrow. Similar results were obtained by the use of other tests in the same and different target organs in mice: sister-chromatid exchange in bone marrow and specific-locus mutations in embryonic pigment-cell precursors were induced by DMNA b u t not by DENA [2,9]. All these data are compatible with the assumption that (an} ultimately mutagenic metabolite(s) of DMNA b u t not of DENA is (are) sufficiently stable to leave the metabolizing organ (primarily liver) and to produce gene and chromosomal mutations in remote organs which, themselves, do not or do not significantly produce the metabolites. On the other hand, in Drosophila germ cells, DENA induces recessive lethal mutations, b u t it fails to produce heritable structural chromosomal mutations [40]. Therefore, it had been argued, this mutation-type specificity -- if applicable to m a m m a l s - could explain previously reported lack of dominant-lethal [33] or cytogenetic [43] effects of DENA in mice. However, the extreme specificity observed in Drosophila does not appear in mammalian cells; the cytogenetic activity of DENA has been unequivocally shown under such conditions where the mammalian target cells are actually exposed even to short-lived metabolites. Thus, karyotypic alterations were reported in liver cells of DENAtreated rats [13], and chromosomal aberrations and sister-chromatid exchanges are clearly induced in mammalian cells in vitro in the presence of DENA and active liver microsomes [30]. Possible reasons for the difference between Drosophila and mammals are discussed by Natarajan et al. [30]. The absence of micronucleus induction in bone marrow by DENA cannot, therefore, be satisfactorily explained by a mutation t y p e specificity b u t more likely by the instability of the active metabolite(s) and thereby resulting organ-specificity of effects.
322
TABLE 1 R E S U L T S OF M I C R O N U C L E U S TESTS Compound (vehicle)
Origin o f compound
Intraperitoneal doses mg/kg mmoles/kg
Dialkylnitrosamines DMNA ( 0 . 9 % NaC1)
A
2× 2 × 2X 0
A
2 2 2 2 0
B
2 × 140 2 X 70 2 X 28 0
2×0.5 2X0.25 2X0.1 0
79.1 a 65.8 a 20.9 a 1.6
Ifosfamide (Hanks's solution)
B
2X 140 2 × 70 2 X 35 0
2×0.54 2X0.27 2X0.13 0
37.6 a 24.4 a 13.8 a 1.9
Trofosfamide (olive oil)
B
2 × 160 2 X 80 2 X 40 0
2×0.5 2X0.25 2×0.125 0
47.6 a 56.7 a 33.8 a 2.4
Sufosfamide (Hanks's solution)
B
2 × 0
80
2 X 0.25 0
62.6 a 2.5
A
2 2 2 2 0
25 12.5 6.25 3.12
2 2 2 2 0
4-Chloro-PMDT (olive oil)
C
2 X 100 2 X 50 2 × 25 0
2×0.55 2X0.27 2X0.14 0
95.4 a 30.6 a 22.8 a 2.6
4-Bromo-PDMT (olive oil)
C
2 X 100 2 × 5O 2 × 25 0
2×0.44 2X0.22 2X0.11 0
50.4 a 15.8 a 2.7 1.7
2,4,6-Trichloro-PMDT (olive oil)
C
2 × 100 2 × 50 2 X 25 0
2X0.40 2×0.20 2×0.10 0
48.2 a 12.9 a 7.4 a 2.2
PyDMT (olive oil)
D
2 X 25 2 × 12.5 2 × 6.25 0
2X0.16 2×0.08 2X0.04 0
32.1 a 33.9 a 7.3 a 2.4
PDET (olive oil)
D
2 2 2 2 0
2X0.4 2×0.3 2X0.2 2XO.1 0
16.9 a 22.4 a 9.1 a 5.2 3.2
DENA ( 0 . 9 % NaCI)
Oxazaphosphorines Cyclophosphamide (Hanks's solution)
A r y ldialk y l t r i a z e n e s PDMT (olive oil)
X × × ×
× × × ×
X X × ×
Micronucleated polychromatic e r y t h r o c y t e s (%0)
37 18.5 9.2
2×0.5 2×0.25 2X0.125 0
14.9 a 10.0 a 3.4 2.4
100 50 25 12.5
2X0.98 2X0.49 2X0.25 2×0.125 0
2.6 3.4 6.1 2.9 4.2
71 53.2 35.5 17.7
X 0.168 X 0.084 X 0.042 × 0.021
58.5 53.6 47.3 21.1 2.9
a a a a
323 TABLE 1 (continued)
mg/kg
mmoles/kg
Micronucleated polychromatic e r y t h r o c y t e s (%0)
E
2×356 2 × 178 2 X 89 0
2X4 2× 2 2 X1 0
18.5 a 15.4a 4.4 3.2
F
2 × 250 2 × 125 0
2 × 2.38 2 X 1.19 0
18.8 a 6.8 a 1.9
A
2 X 95 2 X 38 2 X 19 0
2 X 0.5 2 X 0.2 2X0.1 0
33.4 a 19.2 a 5.6a 1.9
Procarbazine (Hanks's solution,
G
2 2 2 2 0
2 2 2 2 0
34.1 28.3 30.4 8.8 3.3
Potassium chromate ( 0 . 9 % NaCI)
H
2 X 48.5 2 X 24.25 2 X 12.12 0
Compound (vehicle)
Carbamates Urethane (Hanks's solution)
N-Hydroxyurethane ( 0 . 9 % NaCI)
Various c o m p o u n d s 4-NQO (3% g u m a r a b i c )
A B C D E F G H
Origin o f compound
Intraperitoneal doses
X 51.6 X 25.8 X 12.9 × 6.45
× 0.2 X 0.1 X 0.05 X 0.025
2 X 0.25 2 X 0.125 2 X 0.062 0
a a a a
15.0 a 9.6 a 4.8 3.1
Schuchardt, Munich, Prof. N. B r o c k , A s t a - W e r k e , Bielefeld. Dr. G. K o l a r , G e r m a n C a n c e r R e s e a r c h C e n t e r , H e i d e l b e r g . S y n t h e s i z e d a c c o r d i n g to ref. 37. Merck, Darmstadt. S y n t h e s i z e d a c c o r d i n g to ref. 11. H o f f m a n - L a R o c h e , Basle, S w i t z e r l a n d . R i e d e l - d e Hai~n, H a n n o v e r .
a Significantly d i f f e r e n t f r o m t h e c o n t r o l ( a ~ 0 . 0 1 ) (ref. 16).
Recently, Friedman and Staub [10] also reported on the effect of DMNA in micronucleus tests on mice. These authors find very weak DMNA effects and do n o t indicate the m e t h o d used for determining the statistical significance of these effects. However, triethylene melamine used for the positive controls also induces only feeble effects with questionable significance. This is in contrast with other published data [23] and deserves further study. The mutagenicity of cyclophosphamide has been studied in numerous submammalian and mammalian test systems (for review see ref. 26) including the micronucleus test [21,23,28]; it was included in this study as a reference mutagen for inter-laboratory comparison, and a good agreement between the present and previous data is evident. For the first time the chromosome-breaking potential of ifosfamide and trofosfamide in mammals is demonstrated here. Qualitatively, the results agree very well with those gained with the Basc test in Drosophila [40], the host-mediated and urinary assays with yeast [36] and from activation and mutation studies on E.coli in vitro [7]. Q u a n t i t a t i v e - admittedly very r o u g h - dose comparisons show that gene mutations in E.coli in
324 vitro and micronuclei in mouse bone marrow are induced by similar doses of these agents, whereas higher doses are required to obtain effects in host-mediated assays [8,36]. Sufosfamide (previously designated "Asta 5122", ref. 4), a methane sulfonate oxazaphosphorine, has not so far been tested for mutagenic properties. It is about equally effective in the micronucleus test as cyclophosphamide, followed by trofosfamide and ifosfamide. In contrast with nitrosamines, oxazaphosphorines are converted into stable alkylating, mutagenic metabolites, which are transported throughout the organism and can even be detected in urine [36]. They can, therefore, efficiently induce mutations not only near the site of metabolism but also in other tissues. Aryldialkyltriazenes are potent carcinogens [32] and mutagens in microbes (those studied here only in the presence of mammalian liver enzymes) and in Drosophila [ 19,22,41]. Two potential mechanisms of the carcinogenic and mutagenic effects of these compounds have been discussed by Preussmann et al. [32]. (1) Spontaneous (non-enzymic) hydrolysis of triazenes under formation of aryldiazonium salts; these can possibly arylate nucleic acids. This pathway appears less important for the triazenes studied here, since they are not easily hydrolyzable. (2) Enzymic oxidative dealkylation under formation of monoalkyltriazenes, known as alkylating agents. The second mechanism is supported by the detection of methylated nucleic acid components after treatment of rats with PDMT [17,20]. PDMT is the only triazene so far studied for genetic activity in mammals in vivG; it induces micronuclei and sister-chromatid exchanges in mouse bone marrow [2]. These results are extended by the present data on 5 additional aryldialkyltriazenes. It is shown here that these carcinogens induce cytogenetic damage in mammals with high efficiency. Of all mutagens in this study, PDMT is the most potent in terms of the lowest dose required for significant effects. PDET is the diethyl homolog of PDMT and probably acts via an ethylating agent. The results with PDET and PDMT clearly show that ethylating as well as methylating agents can produce cytogenetic damage in the bone marrow and that a quantitative difference exists, PDET being less effective than PDMT. The dose--effect curves of the dimethyl compounds follow two different patterns and suggest a correlation with the chemical structure: the triazenes with an unsubstituted aromatic ring, PDMT and PyDMT, are effective at rather low doses and exhibit a saturation at higher doses. On the other hand, the 4-chloro- and 4-bromo-substituted triazenes are only weakly active at these low doses, their effect increasing proportionally with the dose. Based on the assumption that oxidative dealkylation is a prerequisite for the genetic effects, it therefore appears likely that an additional reaction involving the 4-position of the aromatic ring modifies the dose--effect relationship. This reaction could be ring hydroxylation in the 4-position which is in fact a major metabolic reaction occurring on PDMT [ 18]. This reaction is probably inhibited in 4-chloro-PDMT, 4-bromo-PDMT, and 2,4,6-trichloro-PDMT, whereas it is possible in PDMT and PyDMT. Urethane and its metabolite N-hydroxyurethane have been well d o c u m e n t e d as carcinogens [15]. At equimolar doses, both induce micronuclei at comparable frequencies. Compared with the other mutagens in this study, these carbamates are weakly active even at high doses. These data confirm previous reports
325 on induction of chromosomal aberrations in mammalian cells in vitro and in vivo. (For review see ref. 1.) The dominant-lethal test in the mouse and the Salmonella/microsome test, however, failed to detect a genetic activity of urethane [1,24,25]. 4-NQO is carcinogenic and highly mutagenic in numerous organisms. (For review see ref. 29.) It is a representative of the nitroheterocyclic mutagens which are activated metabolically in microbial and mammalian cells by reduction of the nitro group. This activation mechanism and the ultimate mutagen are different from those of indirect alkylating agents, and it is n o t e w o r t h y that the micronucleus test is a sensitive indicator for this type of mutagen as well. The cytostatic hydrazine derivative procarbazine (natulan ~!)) is a p o t e n t inducer of micronuclei: a significant effect was observed at a total dose of 12.9 mg/kg. The single doses equal the upper recommended single therapeutic dose for adults and are therefore "realistic" under the aspect of human exposure. In the dominant-lethal test and in the specific locus test on mice, significant effects of procarbazine were found at doses of 400 and 600 mg/kg, well above those in the micronucleus test [5,6]. It was found inactive ("false negative") in the Salmonella/microsome test [24,25], whereas it follows PDMT as the most p o t e n t mutagen of those studied here. Chromates and dichromates are mutagenic in bacteria [12,31,39] and yeast [3] in absence of mammalian e n z y m e s ; they induce chromosomal aberrations in mammalian cells in culture [38]. It is shown here that potassium chromate is genetically active also in the intact mammal; similar results were found with sodium chromate (unpublished data}. The mutagenic as well as the carcinogenic effects of inorganic chromium c o m p o u n d s are restricted to compounds of hexavalent chromium (chromates, dichromates) whereas trivalent chromium c o m p o u n d s are inactive [14]. Hexavalent chromium c o m p o u n d s are electrophilic and oxidizing agents; specific oxidations by these c o m p o u n d s could, therefore, be responsible for the observed effects. The 17 chemicals studied here represent 7 chemical classes of mutagens and carcinogens (see Table 1). All these c o m p o u n d s except 4-NQO and potassium chromate are indirect mutagens, i.e. in mammals b u t not in bacteria they are metabolically converted into the ultimate mutagens. The actual mutagens are electrophilic alkylating agents in the case of the nitrosamines, oxazaphosphorines and triazenes. The remaining direct and indirect mutagens comprise compounds with different molecular structures and different less well known mutagenic mechanisms. The data reported demonstrate within the group of selected chemicals a high correlation between chromosome-breaking effects in vivo, revealed by the micronucleus test, and otherwise mutagenic effects as determined by sub-mammalian tests. A similar correlation emerges between chromosome-breaking and carcinogenic effects. Since only a small group of mutagens and carcinogens was studied here, further studies on additional classes of carcinogen are required for a more general assessment of the latter correlation. As a tool for such a screening of cytogenetic {chromosome-breaking and spindle inhibiting} effects the micronucleus test can be r e c o m m e n d e d on the basis of previous data and of those presented here and because it is fast by comparison with classical cytogenetic tests in vivo.
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Potential exceptions deserve especially careful consideration; DENA, for example, is an exception if the results of the micronucleus test and other mammalian tests on bone marrow alone are considered. It is, however, not a "false" negative and does not contradict a correlation of carcinogenic and chromosome-breaking effects, but rather illustrates the critical role of a characteristic property of a mutagen and demonstrates the necessity to take such characteristics into account. References 1 B a t e m a n , A~I., T h e m u t a g e n i c a c t i o n o f u r e t h a n e , M u t a t i o n R e s . , 3 9 ( 1 9 7 6 ) 7 5 - - 9 6 . 2 B a u k n e c h t , T h . , W. V o g e l , U. B a y e r a n d D. Wild, C o m p a r a t i v e in vivo m u t a g e n i c i t y t e s t i n g b y S C E a n d m i c r o n u c l e u s i n d u c t i o n in m o u s e b o n e m a r r o w , H u m a n G e n e t . , 3 5 ( 1 9 7 7 ) 2 9 9 - - 3 0 7 . 3 B o n a t t L S., M. M e i n i a n d A. 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