Mutation Research, 282 (1992) 99-105 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
99
MUTLET 0664
Genotoxicity of N-acryloyl-N'-phenylpiperazine, a redox activator for acrylic resin polymerization M. Taningher
a,b, R.
Pasquini c, M.C. Tanzi d and S. Bonatti
a,e
a National Institute for Cancer Research, b Institute of Oncology CIRC, Unicersity of Genoa, 1-16132 Genoa (Italy), c Unit~ersity Department of Hygiene, 1-06100 Perugia (Italy), a Department of Bioengineering, Polytechnic, 1-20133 Milan (Italy) and e lstituto di Mutagenesi e Differenziamento del CNR, 1-56100 Pisa (Italy) (Received 23 December 1991) (Revision received 31 January 1992) (Accepted 3 February 1992)
Keywords: Biomaterials; DNA damage; Gene mutations; Genomic mutations
Summary N-Acryloyl-N'-phenylpiperazine is a promoter of redox reactions synthesized recently, and proposed as an activator for the polymerization of acrylic resins for biomedical use. The chemical was analyzed for different genotoxicity endpoints, to obtain both information on its possible mutagenic/carcinogenic potential and a model analysis of a tertiary arylamine, which belongs to a class of chemicals commonly used as polymerization accelerators in the biomaterial field. The genotoxicity endpoints considered were: gene mutation in the Salmonella test; structural and numerical chromosome alterations in Chinese hamster V79 cells, evaluated by the micronucleus test together with an immunofluorescent staining specific for kinetochore proteins; in vitro and in vivo DNA damage, evaluated in V79 cells and in mouse liver by the alkaline DNA elution technique. On the whole, the results indicate that N-acryloyl-N'-phenylpiperazine is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed, it was not mutagenic in Salmonella (though its toxicity did not allow testing concentrations over 70/~g/plate), and it was weakly positive in inducing chromosomal fragmentation in vitro (one positive, not dose-related, result out of five different doses tested) and in vivo DNA damage (increases in DNA elution rate never doubling control values). The chemical was, however, clearly positive (with dose-dependent effects up to about 25 times the control value) in causing numerical chromosome alterations, at the maximal non-toxic doses.
Acrylic resins, because of their biocompatibility, mechanical properties and ease of processing, are the materials of choice wherever plastics find Correspondence: Dr. Maurizio Taningher, National Institute for Cancer Research (IST), Viale Benedetto XV No. 10, 1-16132 Genoa, Italy. Tel.: + 39/10/353421; Fax: ÷ 39/10/352999.
application in dental and orthopedic practice, such as in prosthetic device manufacture or in bone cement preparation. In these applications, acrylic resins are generally cured by a free radical initiated polymerization, where the decomposition rate of the initiator (a peroxide or an azo compound) is heat dependent. Therefore, to cure acrylic composites at room or body temperature,
100
CH2=CIH C=O I
Ac r N P P
Fig. 1. Structure of N-acryloyl-N'-phenylpiperazine.
the initiator must in turn be activated, to generate enough free radicals to enable polymerization to proceed at a satisfactory rate for clinical requirements. For this purpose, tertiary aromatic amines are generally used as activators in the so-called redox initiator-accelerator systems. Obviously, the chemicals chosen must not only have appropriate chemico-physical properties, but also be non-toxic, non-irritant and, more generally, completely biocompatible. N-Acryloyl-N'-phenylpiperazine (AcrNPP) is a disubstituted acrylamide recently synthesized by Tanzi et al. (1990), in which the amidic group is associated with an aromatic tertiary amine function in a piperazine cycle (Fig. 1). A compound of this kind is of potential interest for biomaterial polymerization. Indeed, it bears an aromatic tertiary amine group known to be active in redox initiation, but with a toxicity expected to be lower than that of other commonly used aromatic tertiary amines (such as N,N-dimethylaniline and N,N-dimethyl-p-toluidine) for the following reasons: (a) it has a lower migrability in viscous blend or moulding compounds, as well as in final solid manufacts, due to its greater molecular size; (b) the unsaturated group allows it to be incorporated chemically into the final material through copolymerization, thus avoiding any release into the environment; (c) because of its solid state, it should be safer to handle than liquid chemicals used for the same purpose. Considering both the interest in a new compound that, in chemico-physical terms, seems endowed with reduced toxicity and the lack (to our knowledge) of systematic information on the
genotoxicity of other chemicals of similar use and molecular structure, we decided to test AcrNPP, in biological terms, for this specific toxicity aspect. Genotoxicity was analyzed by the following complementary endpoints: gene mutation in Salmonella, structural and numerical chromosome alterations in mammalian V79 cells and in vitro and in vivo DNA damage (evaluated in V79 cells and in mouse liver, respectively). A similar analysis was also performed on N-phenylpiperazine, the starting compound for AcrNPP synthesis. The aim of the research was to detect a possible mutagenic/carcinogenic risk stemming from the chemicals not only in terms of their release from biomaterials used in prosthetic devices, but also in terms of professional exposure during handling and production. Materials and methods
AcrNPP (CAS No. 129401-88-3) was synthesized by reacting N-phenylpiperazine (NPP) with acryloyl chloride in the presence of triethylamine, in anhydrous toluene solution, as previously described (Tanzi et al., 1990). The NPP we analyzed (97% pure, CAS No. 92-54-6) was purchased from Aldrich (Steinheim, Germany). In all the genotoxicity tests the chemicals were assayed after previous solution in dimethyl sulfoxide. The Ames test was performed on Salmonella strains TA97, TA98 and TA100, according to the preincubation test described by Maron and Ames (1983). The revertant colonies were counted 48 h after plating of the treated bacteria on VogelBonner medium. The chemicals were tested both in the absence and in the presence of 10% or 30% postmitochondrial preparations ($9 fractions), which were obtained from the livers of Aroclor-induced male rats or male hamsters. For this purpose the animals were injected i.p. with 500 m g / k g of Aroclor-1254 five days before killing. The micronucleus test was performed on Chinese hamster V79 cells, which were scored 24 or 48 h after the beginning of treatment. Such a time allows not only the possible production of D N A d a m a g e / c h r o m o s o m e breakage, but also the expression of numerical chromosome aberrations after mitosis. Overall, micronuclei were evi-
101 with 10 ml of e l u a n t c o n t a i n i n g 10 m M N a 2 E D T A a n d t e t r a e t h y l a m m o n i u m hydroxide to give a p H value of 12.35. T h r e e 20-min fractions were collected, a n d the D N A c o n t a i n e d in t h e m a n d that r e m a i n i n g o n the filter was t h e n dosed by m e a n s of a m i c r o f l u o r o m e t r i c m e t h o d u s i n g dia m i n o b e n z o i c acid, as p r e v i o u s l y d e s c r i b e d ( P a r o d i et al., 1978).
d e n c e d by s t a n d a r d G i e m s a staining. A n imm u n o l o g i c a l s t a i n i n g with a n t i b o d i e s against k i n e t o c h o r e p r o t e i n s ( C R E S T a n t i b o d i e s ) was u s e d to d i s c r i m i n a t e b e t w e e n structural (CREST-negative micronuclei) and numerical ( C R E S T - p o s i t i v e , k i n e t o c h o r e - c o n t a i n i n g mic r o n u c l e i ) c h r o m o s o m e a b e r r a t i o n s (Niisse et al., 1989). C R E S T antibodies, o b t a i n e d from a scler o d e r m a patient, were a gift from Dr. Solberg (University Hospital of F r a n k f u r t , G e r m a n y ) . T h e alkaline D N A e l u t i o n test was p e r f o r m e d as previously described (Parodi et al., 1978), with m i n o r modifications. I n short, aliquots of 1 - 2 × 10 6 liver nuclei (or V79 cells) were p u t o n cellulose mixed ester N u c l e o p o r e filters (2.5 cm diameter; 1.2 ~,m pore d i a m e t e r ) a n d lysed for 30 m i n at r o o m t e m p e r a t u r e with a solution c o n t a i n i n g 2.0 M NaC1, 20 m M N a 2 E D T A , 0.2% s o d i u m lauroylsarcosinate, a n d 0.5 m g / m l p r o t e i n a s e K ( p H 10). A f t e r washing, s i n g l e - s t r a n d e d D N A was e l u t e d in the dark, for 1 h at r o o m t e m p e r a t u r e ,
Results Both A c r N P P a n d N P P were negative in the A m e s test, u p to 7 0 / ~ g / p l a t e (that is 324 n m o l e / plate a n d 432 n m o l e / p l a t e , respectively). Because of their toxicity, the chemicals could n o t be tested at higher dosages. A s r e p o r t e d in Fig. 2, n o significant differences in r e v e r t a n t colonies per plate were observed b e t w e e n u n t r e a t e d a n d t r e a t e d samples, either in the a b s e n c e or in the p r e s e n c e of liver p o s t m i t o c h o n d r i a l ($9) fractions which were a d d e d to the mixtures in 10% c o n c e n -
TABLE 1 IN VITRO INDUCTION OF MICRONUCLEI, AS EVALUATED BY GIEMSA STAINING OR BY CREST ANTIBODY IMMUNOFLUORESCENT STAINING IN V79 CELLS Chemical and dose
Treatment time (h)
Observation time, after beginning of treatment 24 h CREST +
NPP 0.3 mM 0.9 mM 1.2 mM Controls
48 48 48
AcrNPP 5 mM 10 mM 0.1 mM 0.5 mM 1 mM
0.5 0.5 24 or 48 b 24 or 48 24 or 48
Methylnitrosourea 0.5 mM Colchicine 0.025 ~.M Controls
0.5 48
48 h CREST-
Giemsa-stained 8.91 16.76 ** * 18.70 ** 9.49
4.71 14.83 ** 8.52 ** * 19.74 * 49.00 *
3.32
a
0.90 1.47 3.27 6.58 5.00
16.05 * 29.90 * 49.27 *
2.80
141.32 * 38.67 * 6.96
CREST+ 4.73 8.55 9.69 5.00 7.80 *** 11.92 ** 10.00 * * 45.87 * 67.99 * 28.47 * 35.20 * 2.66
CREST5.26 6.42 7.30 3.02 3.46 5.29 3.31 8.03 * * * 4.66 114.39 * 3.33 2.32
Each reported value is the mean of results obtained in at least 2 independent experiments, in which at least 3000 cells were counted. Giemsa staining and CREST antibody staining concerning the 48-h treatment time were performed in independent experiments. The experiments were performed on V79 cells, which were grown in FCS-supplemented DMEM and treated 16 h after seeding, a The results are expressed as micronuclei observed per 1000 nuclei. b Treatment times of 24 and 48 h refer to observation times of 24 and 48 h, respectively. Significant difference from concurrent control: * p < 0.001, * * p < 0.01, * * * p < 0.05, according to the g 2 test or the Fischer exact test.
102
tration. Negative results for AcrNPP and NPP were also obtained with $9 fractions in 30% concentration (data not reported). Under the same testing conditions, regular positive dose-dependent effects were obtained with potent mutagenic chemicals such as sodium azide, methyl methanesulfonate, 2-nitrofluorene and benzo[a]pyrene. These chemicals were positive with doses starting from 1 /zg/plate, that is approximately 15.4, 9.1, 4.7 and 4.0 n m o l e / p l a t e , respectively. NPP and AcrNPP both proved positive for micronucleus induction, as evidenced by Giemsa staining, with AcrNPP appearing more potent than NPP in producing a dose-dependent re-
NPP
sponse. The micronucleus test results are reported in Table 1. The positive effect of AcrNPP was repeatedly confirmed in separate experiments, in which the presence in micronuclei of centromere structures was verified by the CREST antibody immunofluorescent staining. As reported in Table 1, the AcrNPP-induced micronuclei contained centromeres (CREST-positive), and thus appear to stem from whole chromosomes, rather than from acentric chromosome fragments. Therefore, the effect of AcrNPP appears comparable to that of colchicine (even if, on a molar basis, ~ 20,000 times lower). Conversely, the effect appears different from that of a
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Fig. 2. M u t a g e n i c activity, as e v a l u a t e d by the A m e s test. All s a m p l e s w e r e t e s t e d in d u p l i c a t e in at least two i n d e p e n d e n t e x p e r i m e n t s . T h e c r i t e r i a to d e f i n e m u t a g e n i c activity w e r e the i n d u c t i o n of r e v e r t a n t colonies to at least twice the control level, a n d the o b s e r v a t i o n of a d o s e - d e p e n d e n t effect.
103
D N A - d a m a g i n g / c h r o m o s o m e - b r e a k i n g agent such as methylnitrosourea, which essentially produces CREST-negative micronuclei. Lack of clastogenic activity is in agreement with the results obtained with the alkaline DNA elution test, when performed on the same cells used for the micronucleus test. As reported in Table 2 (in vitro assays), a 24-h exposure of the cells to 1 mM AcrNPP, which is a clearly higher molarity than those already positive for micronucleus induction, did not change the DNA elution rate as compared with control cells. An exposure time of 30 min to 10 mM AcrNPP again failed to produce detectable DNA fragmentation. The only statistically significant effect produced in vitro by AcrNPP was obtained with a 10 mM concentration, and only after a 24-h exposure. This kind of
treatment proved to be very toxic for cells: about 4 × 10 -4 viable cells after treatment (data not reported). By contrast, methylnitrosourea produced a qualitatively different effect, which was dose-dependent and already present after a short observation time. Table 2 also shows results obtained in terms of in vivo DNA damage. As reported, both NPP and AcrNPP produced a weak effect, 24 h after administration to mice. For comparison, results obtained with dimethylnitrosamine (a DNA-damaging agent requiring metabolic activation) are also reported. Overall, the lower DNA fragmentation background observed for controls in the in vivo assays could depend on a less traumatic handling of liver nuclei than of V79 cells. Indeed, while the former were usually prepared in mild conditions
TABLE 2 IN V I T R O A N D IN V I V O D N A D A M A G E , AS E V A L U A T E D BY T H E A L K A L I N E E L U T I O N TEST IN V79 CELLS AND IN M O U S E L I V E R Chemical and dose In vitro assays AcrNPP 10 mM Methylnitrosourea 1 mM 5 mM Controls AcrNPP 1 mM 10 mM Controls In vivo assays NPP 0.05 m m o l e / k g 0.25 m m o l e / k g 0.25 m m o l e / k g Controls AcrNPP 1 m m o l e / k g 2 mmole/kg 1 mmole/kg Controls Dimethylnitrosamine 0.03 m m o l e / k g 0.15 m m o l e / k g 0.15 m m o l e / k g Controls
Treatment time (h) 0.5 0.5 0.5 24 24
2 2 24 2 2 24 2 2 24
Number of experiments
Elution rate a (mean _+SE)
5 4 2 5 12 9 20
7.52_+0.53 16.26_+1.74 ** 24.15_+1.11 * 6.83 _+1.10 5.39 _+0.66 17.95_+2.53 ** 6.94 _+1.10
6 6 6 10 6 9 11 15 7 9 4 10
1.67 _+0.26 1.66 _+0.40 3.14_+0.39 1.70 _+0.21 1.84-+0.19 2.72 _+0.35 3.17_+0.49 1.94_+0.17 3.87 -+ 1.04 12.14-+ 1.30 16.63 _+ 1.56 1.82_+0.15
***
**** *** * *
The in vitro assays were performed on the same cells used for the micronucleus test. The in vivo assays were performed on liver nuclei prepared from male B A L B / c mice (20-25 g body weight) which were treated by intraperitoneal injection 2 or 24 h before being killed. a The D N A elution rate is expressed as: K = - In(DNA fraction remaining on the filter)/(eluted volume in ml) × 102, according to Parodi et al. (1978). Significant difference from concurrent control: * p < 0.001, ** p < 0.005, * * * p < 0.01, * * * * p < 0.05 (one-tailed) according to the nonparametric Mann-Whitney U test.
104
as reported by Cox et al. (1973), the latter were recovered from culture substrates simply by using a cell scraper.
Discussion When tested on different Salmonella strains responsive to point mutations of different nature (i.e., single-base substitution, insertion and deletion) the chemicals studied proved to be devoid of mutagenic activity, even in the presence of metabolic activation and in molar concentrations ~ 20-100 times higher than those positive with potent genotoxic chemicals. However, it should be noted that, because of their bacterial toxicity, AcrNPP and NPP could not be tested over ~ 0.3 and ~ 0.4 /~mole/plate, respectively. These dosages are notably lower than those positive in micronucleus and alkaline elution tests. In the in vivo D N A damage-alkaline elution test, AcrNPP and NPP gave, at the highest nontoxic doses, very weak increases in DNA elution rate, which never doubled the control values. In evaluating the alkaline D N A elution assay as a predictor of carcinogenic/mutagenic potential, Sina et al. (1983) adopted the criterion that a compound was genotoxic only if it induced an elution rate at least 3 times greater than concurrent controls. On that basis, the chemicals studied should not be regarded as genotoxins. It should be remembered however, from a qualitative point of view, that the rate of alkaline D N A fragmentation could depend not only on the number, but also on the relative percentage and type of different adducts produced on D N A by a genotoxic agent. In the micronucleus test, AcrNPP gave a positive effect, with dose-dependent values for treated samples up to about 25 times greater than concurrent controls. The C R E S T antibody immunofluorescent staining revealed that the induced micronuclei were formed from whole chromosomes, and thus that they stemmed from chromosome malsegregation during mitosis. This effect was similar (though many thousands of times lower) to that of colchicine, an agent which interferes with spindle formation, and different from the effect of a clastogenic agent such as methylnitrosourea.
This result is confirmed by the alkaline DNA elution experiments, performed in vitro on the same cell preparations used for the micronucleus test. As reported in the Results, the only increase in D N A elution produced by AcrNPP was observed after a highly toxic treatment and seems attributable to DNA fragmentation preceding cell death (Williams et al., 1974), rather than to real DNA damage. Moreover, the observed effect was qualitatively different from that of methylnitrosourea, which was dose-dependent and already present after a short treatment time. What was obtained with methylnitrosourea in the alkaline elution test is consistent with the action mechanism of this chemical, which produces the maximum amount of DNA adducts detectable as alkaline D N A fragmentation (such as for example N7-methylpurines) after 30 min to 1 h of treatment (Lawley, 1976; Warren et al., 1979). The results obtained suggest that, on the whole, AcrNPP is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed in the experiments reported here, it did not induce gene mutations and was less effective in producing DNA damage or chromosomal fragmentation than in causing numerical chromosome mutations. From a qualitative standpoint, we need hardly mention that chromosome malsegregation, whether germinal or somatic, may cause severe handicaps such as Down syndrome and retinoblastoma. It is, however, difficult to quantify the mutagenic/carcinogenic risk posed by chemicals such as AcrNPP. To do so we would need to know how much of the active chemical reaches the critical cellular targets and to have access to far more data concerning the potencies of various chemicals in inducing aneuploidy-related micronuclei. Indeed, an adequate data base would enable us to draw up a quantitative correlation between aneuploidy-inducing potency and mutagenic and carcinogenic potencies.
Acknowledgements This work was supported by CNR Grant 90.01366.CTll - Special Project 'Biomaterials'. We are grateful to Mr. Thomas Wiley for reviewing the English format of the manuscript.
105
References Cox, R., I. Damianov, S.E. Abanobi and D.S.R. Sarma (1973) A method for measuring DNA damage and repair in the liver in vivo, Cancer Res., 33, 2114-2121. Lawley, P.D. (1976) Methylation of DNA by carcinogens: some applications of chemical analytical methods, in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Screening Tests in Chemical Carcinogenesis, IARC Sci. Publ. No. 12, International Agency for Research on Cancer, Lyon, pp. 181-208. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Niisse, M., S. Viaggi and S. Bonatti (1989) Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethyl sulfate, Mutagenesis, 4, 174-178. Parodi, S., M. Taningher, L. Santi, M. Cavanna, L. Sciab~ and G. Brambilla (1978) A practical procedure for testing DNA damage in vivo, proposed for a pre-screening of chemical carcinogens, Mutation Res., 54, 39-46.
Sina, J.F., CL. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Tanzi, M.C., M. Levi and F. Danusso (1990) Amides from N-phenylpiperazine as low-toxicity activators in radical polymerization, Polymer, 31, 1735-1738. Warren, W., A.R. Crathorn and K.Y. Shooter (1979) The stability of methylated purines and of methylphosphotriesters in the DNA of V79 cells after treatment with N-methyl-N-nitrosourea, Biochim. Biophys. Acta, 563, 8288. Williams, J.R., J.B. Little and W.U. Shipley (1974) Association of mammalian cell death with a specific endonucleolytic degradation of DNA, Nature (London), 252, 754755.
Communicated by S. De Flora
99
MUTLET 0664
Genotoxicity of N-acryloyl-N'-phenylpiperazine, a redox activator for acrylic resin polymerization M. Taningher
a,b, R.
Pasquini c, M.C. Tanzi d and S. Bonatti
a,e
a National Institute for Cancer Research, b Institute of Oncology CIRC, Unicersity of Genoa, 1-16132 Genoa (Italy), c Unit~ersity Department of Hygiene, 1-06100 Perugia (Italy), a Department of Bioengineering, Polytechnic, 1-20133 Milan (Italy) and e lstituto di Mutagenesi e Differenziamento del CNR, 1-56100 Pisa (Italy) (Received 23 December 1991) (Revision received 31 January 1992) (Accepted 3 February 1992)
Keywords: Biomaterials; DNA damage; Gene mutations; Genomic mutations
Summary N-Acryloyl-N'-phenylpiperazine is a promoter of redox reactions synthesized recently, and proposed as an activator for the polymerization of acrylic resins for biomedical use. The chemical was analyzed for different genotoxicity endpoints, to obtain both information on its possible mutagenic/carcinogenic potential and a model analysis of a tertiary arylamine, which belongs to a class of chemicals commonly used as polymerization accelerators in the biomaterial field. The genotoxicity endpoints considered were: gene mutation in the Salmonella test; structural and numerical chromosome alterations in Chinese hamster V79 cells, evaluated by the micronucleus test together with an immunofluorescent staining specific for kinetochore proteins; in vitro and in vivo DNA damage, evaluated in V79 cells and in mouse liver by the alkaline DNA elution technique. On the whole, the results indicate that N-acryloyl-N'-phenylpiperazine is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed, it was not mutagenic in Salmonella (though its toxicity did not allow testing concentrations over 70/~g/plate), and it was weakly positive in inducing chromosomal fragmentation in vitro (one positive, not dose-related, result out of five different doses tested) and in vivo DNA damage (increases in DNA elution rate never doubling control values). The chemical was, however, clearly positive (with dose-dependent effects up to about 25 times the control value) in causing numerical chromosome alterations, at the maximal non-toxic doses.
Acrylic resins, because of their biocompatibility, mechanical properties and ease of processing, are the materials of choice wherever plastics find Correspondence: Dr. Maurizio Taningher, National Institute for Cancer Research (IST), Viale Benedetto XV No. 10, 1-16132 Genoa, Italy. Tel.: + 39/10/353421; Fax: ÷ 39/10/352999.
application in dental and orthopedic practice, such as in prosthetic device manufacture or in bone cement preparation. In these applications, acrylic resins are generally cured by a free radical initiated polymerization, where the decomposition rate of the initiator (a peroxide or an azo compound) is heat dependent. Therefore, to cure acrylic composites at room or body temperature,
100
CH2=CIH C=O I
Ac r N P P
Fig. 1. Structure of N-acryloyl-N'-phenylpiperazine.
the initiator must in turn be activated, to generate enough free radicals to enable polymerization to proceed at a satisfactory rate for clinical requirements. For this purpose, tertiary aromatic amines are generally used as activators in the so-called redox initiator-accelerator systems. Obviously, the chemicals chosen must not only have appropriate chemico-physical properties, but also be non-toxic, non-irritant and, more generally, completely biocompatible. N-Acryloyl-N'-phenylpiperazine (AcrNPP) is a disubstituted acrylamide recently synthesized by Tanzi et al. (1990), in which the amidic group is associated with an aromatic tertiary amine function in a piperazine cycle (Fig. 1). A compound of this kind is of potential interest for biomaterial polymerization. Indeed, it bears an aromatic tertiary amine group known to be active in redox initiation, but with a toxicity expected to be lower than that of other commonly used aromatic tertiary amines (such as N,N-dimethylaniline and N,N-dimethyl-p-toluidine) for the following reasons: (a) it has a lower migrability in viscous blend or moulding compounds, as well as in final solid manufacts, due to its greater molecular size; (b) the unsaturated group allows it to be incorporated chemically into the final material through copolymerization, thus avoiding any release into the environment; (c) because of its solid state, it should be safer to handle than liquid chemicals used for the same purpose. Considering both the interest in a new compound that, in chemico-physical terms, seems endowed with reduced toxicity and the lack (to our knowledge) of systematic information on the
genotoxicity of other chemicals of similar use and molecular structure, we decided to test AcrNPP, in biological terms, for this specific toxicity aspect. Genotoxicity was analyzed by the following complementary endpoints: gene mutation in Salmonella, structural and numerical chromosome alterations in mammalian V79 cells and in vitro and in vivo DNA damage (evaluated in V79 cells and in mouse liver, respectively). A similar analysis was also performed on N-phenylpiperazine, the starting compound for AcrNPP synthesis. The aim of the research was to detect a possible mutagenic/carcinogenic risk stemming from the chemicals not only in terms of their release from biomaterials used in prosthetic devices, but also in terms of professional exposure during handling and production. Materials and methods
AcrNPP (CAS No. 129401-88-3) was synthesized by reacting N-phenylpiperazine (NPP) with acryloyl chloride in the presence of triethylamine, in anhydrous toluene solution, as previously described (Tanzi et al., 1990). The NPP we analyzed (97% pure, CAS No. 92-54-6) was purchased from Aldrich (Steinheim, Germany). In all the genotoxicity tests the chemicals were assayed after previous solution in dimethyl sulfoxide. The Ames test was performed on Salmonella strains TA97, TA98 and TA100, according to the preincubation test described by Maron and Ames (1983). The revertant colonies were counted 48 h after plating of the treated bacteria on VogelBonner medium. The chemicals were tested both in the absence and in the presence of 10% or 30% postmitochondrial preparations ($9 fractions), which were obtained from the livers of Aroclor-induced male rats or male hamsters. For this purpose the animals were injected i.p. with 500 m g / k g of Aroclor-1254 five days before killing. The micronucleus test was performed on Chinese hamster V79 cells, which were scored 24 or 48 h after the beginning of treatment. Such a time allows not only the possible production of D N A d a m a g e / c h r o m o s o m e breakage, but also the expression of numerical chromosome aberrations after mitosis. Overall, micronuclei were evi-
101 with 10 ml of e l u a n t c o n t a i n i n g 10 m M N a 2 E D T A a n d t e t r a e t h y l a m m o n i u m hydroxide to give a p H value of 12.35. T h r e e 20-min fractions were collected, a n d the D N A c o n t a i n e d in t h e m a n d that r e m a i n i n g o n the filter was t h e n dosed by m e a n s of a m i c r o f l u o r o m e t r i c m e t h o d u s i n g dia m i n o b e n z o i c acid, as p r e v i o u s l y d e s c r i b e d ( P a r o d i et al., 1978).
d e n c e d by s t a n d a r d G i e m s a staining. A n imm u n o l o g i c a l s t a i n i n g with a n t i b o d i e s against k i n e t o c h o r e p r o t e i n s ( C R E S T a n t i b o d i e s ) was u s e d to d i s c r i m i n a t e b e t w e e n structural (CREST-negative micronuclei) and numerical ( C R E S T - p o s i t i v e , k i n e t o c h o r e - c o n t a i n i n g mic r o n u c l e i ) c h r o m o s o m e a b e r r a t i o n s (Niisse et al., 1989). C R E S T antibodies, o b t a i n e d from a scler o d e r m a patient, were a gift from Dr. Solberg (University Hospital of F r a n k f u r t , G e r m a n y ) . T h e alkaline D N A e l u t i o n test was p e r f o r m e d as previously described (Parodi et al., 1978), with m i n o r modifications. I n short, aliquots of 1 - 2 × 10 6 liver nuclei (or V79 cells) were p u t o n cellulose mixed ester N u c l e o p o r e filters (2.5 cm diameter; 1.2 ~,m pore d i a m e t e r ) a n d lysed for 30 m i n at r o o m t e m p e r a t u r e with a solution c o n t a i n i n g 2.0 M NaC1, 20 m M N a 2 E D T A , 0.2% s o d i u m lauroylsarcosinate, a n d 0.5 m g / m l p r o t e i n a s e K ( p H 10). A f t e r washing, s i n g l e - s t r a n d e d D N A was e l u t e d in the dark, for 1 h at r o o m t e m p e r a t u r e ,
Results Both A c r N P P a n d N P P were negative in the A m e s test, u p to 7 0 / ~ g / p l a t e (that is 324 n m o l e / plate a n d 432 n m o l e / p l a t e , respectively). Because of their toxicity, the chemicals could n o t be tested at higher dosages. A s r e p o r t e d in Fig. 2, n o significant differences in r e v e r t a n t colonies per plate were observed b e t w e e n u n t r e a t e d a n d t r e a t e d samples, either in the a b s e n c e or in the p r e s e n c e of liver p o s t m i t o c h o n d r i a l ($9) fractions which were a d d e d to the mixtures in 10% c o n c e n -
TABLE 1 IN VITRO INDUCTION OF MICRONUCLEI, AS EVALUATED BY GIEMSA STAINING OR BY CREST ANTIBODY IMMUNOFLUORESCENT STAINING IN V79 CELLS Chemical and dose
Treatment time (h)
Observation time, after beginning of treatment 24 h CREST +
NPP 0.3 mM 0.9 mM 1.2 mM Controls
48 48 48
AcrNPP 5 mM 10 mM 0.1 mM 0.5 mM 1 mM
0.5 0.5 24 or 48 b 24 or 48 24 or 48
Methylnitrosourea 0.5 mM Colchicine 0.025 ~.M Controls
0.5 48
48 h CREST-
Giemsa-stained 8.91 16.76 ** * 18.70 ** 9.49
4.71 14.83 ** 8.52 ** * 19.74 * 49.00 *
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16.05 * 29.90 * 49.27 *
2.80
141.32 * 38.67 * 6.96
CREST+ 4.73 8.55 9.69 5.00 7.80 *** 11.92 ** 10.00 * * 45.87 * 67.99 * 28.47 * 35.20 * 2.66
CREST5.26 6.42 7.30 3.02 3.46 5.29 3.31 8.03 * * * 4.66 114.39 * 3.33 2.32
Each reported value is the mean of results obtained in at least 2 independent experiments, in which at least 3000 cells were counted. Giemsa staining and CREST antibody staining concerning the 48-h treatment time were performed in independent experiments. The experiments were performed on V79 cells, which were grown in FCS-supplemented DMEM and treated 16 h after seeding, a The results are expressed as micronuclei observed per 1000 nuclei. b Treatment times of 24 and 48 h refer to observation times of 24 and 48 h, respectively. Significant difference from concurrent control: * p < 0.001, * * p < 0.01, * * * p < 0.05, according to the g 2 test or the Fischer exact test.
102
tration. Negative results for AcrNPP and NPP were also obtained with $9 fractions in 30% concentration (data not reported). Under the same testing conditions, regular positive dose-dependent effects were obtained with potent mutagenic chemicals such as sodium azide, methyl methanesulfonate, 2-nitrofluorene and benzo[a]pyrene. These chemicals were positive with doses starting from 1 /zg/plate, that is approximately 15.4, 9.1, 4.7 and 4.0 n m o l e / p l a t e , respectively. NPP and AcrNPP both proved positive for micronucleus induction, as evidenced by Giemsa staining, with AcrNPP appearing more potent than NPP in producing a dose-dependent re-
NPP
sponse. The micronucleus test results are reported in Table 1. The positive effect of AcrNPP was repeatedly confirmed in separate experiments, in which the presence in micronuclei of centromere structures was verified by the CREST antibody immunofluorescent staining. As reported in Table 1, the AcrNPP-induced micronuclei contained centromeres (CREST-positive), and thus appear to stem from whole chromosomes, rather than from acentric chromosome fragments. Therefore, the effect of AcrNPP appears comparable to that of colchicine (even if, on a molar basis, ~ 20,000 times lower). Conversely, the effect appears different from that of a
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Fig. 2. M u t a g e n i c activity, as e v a l u a t e d by the A m e s test. All s a m p l e s w e r e t e s t e d in d u p l i c a t e in at least two i n d e p e n d e n t e x p e r i m e n t s . T h e c r i t e r i a to d e f i n e m u t a g e n i c activity w e r e the i n d u c t i o n of r e v e r t a n t colonies to at least twice the control level, a n d the o b s e r v a t i o n of a d o s e - d e p e n d e n t effect.
103
D N A - d a m a g i n g / c h r o m o s o m e - b r e a k i n g agent such as methylnitrosourea, which essentially produces CREST-negative micronuclei. Lack of clastogenic activity is in agreement with the results obtained with the alkaline DNA elution test, when performed on the same cells used for the micronucleus test. As reported in Table 2 (in vitro assays), a 24-h exposure of the cells to 1 mM AcrNPP, which is a clearly higher molarity than those already positive for micronucleus induction, did not change the DNA elution rate as compared with control cells. An exposure time of 30 min to 10 mM AcrNPP again failed to produce detectable DNA fragmentation. The only statistically significant effect produced in vitro by AcrNPP was obtained with a 10 mM concentration, and only after a 24-h exposure. This kind of
treatment proved to be very toxic for cells: about 4 × 10 -4 viable cells after treatment (data not reported). By contrast, methylnitrosourea produced a qualitatively different effect, which was dose-dependent and already present after a short observation time. Table 2 also shows results obtained in terms of in vivo DNA damage. As reported, both NPP and AcrNPP produced a weak effect, 24 h after administration to mice. For comparison, results obtained with dimethylnitrosamine (a DNA-damaging agent requiring metabolic activation) are also reported. Overall, the lower DNA fragmentation background observed for controls in the in vivo assays could depend on a less traumatic handling of liver nuclei than of V79 cells. Indeed, while the former were usually prepared in mild conditions
TABLE 2 IN V I T R O A N D IN V I V O D N A D A M A G E , AS E V A L U A T E D BY T H E A L K A L I N E E L U T I O N TEST IN V79 CELLS AND IN M O U S E L I V E R Chemical and dose In vitro assays AcrNPP 10 mM Methylnitrosourea 1 mM 5 mM Controls AcrNPP 1 mM 10 mM Controls In vivo assays NPP 0.05 m m o l e / k g 0.25 m m o l e / k g 0.25 m m o l e / k g Controls AcrNPP 1 m m o l e / k g 2 mmole/kg 1 mmole/kg Controls Dimethylnitrosamine 0.03 m m o l e / k g 0.15 m m o l e / k g 0.15 m m o l e / k g Controls
Treatment time (h) 0.5 0.5 0.5 24 24
2 2 24 2 2 24 2 2 24
Number of experiments
Elution rate a (mean _+SE)
5 4 2 5 12 9 20
7.52_+0.53 16.26_+1.74 ** 24.15_+1.11 * 6.83 _+1.10 5.39 _+0.66 17.95_+2.53 ** 6.94 _+1.10
6 6 6 10 6 9 11 15 7 9 4 10
1.67 _+0.26 1.66 _+0.40 3.14_+0.39 1.70 _+0.21 1.84-+0.19 2.72 _+0.35 3.17_+0.49 1.94_+0.17 3.87 -+ 1.04 12.14-+ 1.30 16.63 _+ 1.56 1.82_+0.15
***
**** *** * *
The in vitro assays were performed on the same cells used for the micronucleus test. The in vivo assays were performed on liver nuclei prepared from male B A L B / c mice (20-25 g body weight) which were treated by intraperitoneal injection 2 or 24 h before being killed. a The D N A elution rate is expressed as: K = - In(DNA fraction remaining on the filter)/(eluted volume in ml) × 102, according to Parodi et al. (1978). Significant difference from concurrent control: * p < 0.001, ** p < 0.005, * * * p < 0.01, * * * * p < 0.05 (one-tailed) according to the nonparametric Mann-Whitney U test.
104
as reported by Cox et al. (1973), the latter were recovered from culture substrates simply by using a cell scraper.
Discussion When tested on different Salmonella strains responsive to point mutations of different nature (i.e., single-base substitution, insertion and deletion) the chemicals studied proved to be devoid of mutagenic activity, even in the presence of metabolic activation and in molar concentrations ~ 20-100 times higher than those positive with potent genotoxic chemicals. However, it should be noted that, because of their bacterial toxicity, AcrNPP and NPP could not be tested over ~ 0.3 and ~ 0.4 /~mole/plate, respectively. These dosages are notably lower than those positive in micronucleus and alkaline elution tests. In the in vivo D N A damage-alkaline elution test, AcrNPP and NPP gave, at the highest nontoxic doses, very weak increases in DNA elution rate, which never doubled the control values. In evaluating the alkaline D N A elution assay as a predictor of carcinogenic/mutagenic potential, Sina et al. (1983) adopted the criterion that a compound was genotoxic only if it induced an elution rate at least 3 times greater than concurrent controls. On that basis, the chemicals studied should not be regarded as genotoxins. It should be remembered however, from a qualitative point of view, that the rate of alkaline D N A fragmentation could depend not only on the number, but also on the relative percentage and type of different adducts produced on D N A by a genotoxic agent. In the micronucleus test, AcrNPP gave a positive effect, with dose-dependent values for treated samples up to about 25 times greater than concurrent controls. The C R E S T antibody immunofluorescent staining revealed that the induced micronuclei were formed from whole chromosomes, and thus that they stemmed from chromosome malsegregation during mitosis. This effect was similar (though many thousands of times lower) to that of colchicine, an agent which interferes with spindle formation, and different from the effect of a clastogenic agent such as methylnitrosourea.
This result is confirmed by the alkaline DNA elution experiments, performed in vitro on the same cell preparations used for the micronucleus test. As reported in the Results, the only increase in D N A elution produced by AcrNPP was observed after a highly toxic treatment and seems attributable to DNA fragmentation preceding cell death (Williams et al., 1974), rather than to real DNA damage. Moreover, the observed effect was qualitatively different from that of methylnitrosourea, which was dose-dependent and already present after a short treatment time. What was obtained with methylnitrosourea in the alkaline elution test is consistent with the action mechanism of this chemical, which produces the maximum amount of DNA adducts detectable as alkaline D N A fragmentation (such as for example N7-methylpurines) after 30 min to 1 h of treatment (Lawley, 1976; Warren et al., 1979). The results obtained suggest that, on the whole, AcrNPP is to be regarded not so much as a DNA-damaging agent, but as a genomic mutagen. Indeed in the experiments reported here, it did not induce gene mutations and was less effective in producing DNA damage or chromosomal fragmentation than in causing numerical chromosome mutations. From a qualitative standpoint, we need hardly mention that chromosome malsegregation, whether germinal or somatic, may cause severe handicaps such as Down syndrome and retinoblastoma. It is, however, difficult to quantify the mutagenic/carcinogenic risk posed by chemicals such as AcrNPP. To do so we would need to know how much of the active chemical reaches the critical cellular targets and to have access to far more data concerning the potencies of various chemicals in inducing aneuploidy-related micronuclei. Indeed, an adequate data base would enable us to draw up a quantitative correlation between aneuploidy-inducing potency and mutagenic and carcinogenic potencies.
Acknowledgements This work was supported by CNR Grant 90.01366.CTll - Special Project 'Biomaterials'. We are grateful to Mr. Thomas Wiley for reviewing the English format of the manuscript.
105
References Cox, R., I. Damianov, S.E. Abanobi and D.S.R. Sarma (1973) A method for measuring DNA damage and repair in the liver in vivo, Cancer Res., 33, 2114-2121. Lawley, P.D. (1976) Methylation of DNA by carcinogens: some applications of chemical analytical methods, in: R. Montesano, H. Bartsch and L. Tomatis (Eds.), Screening Tests in Chemical Carcinogenesis, IARC Sci. Publ. No. 12, International Agency for Research on Cancer, Lyon, pp. 181-208. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Niisse, M., S. Viaggi and S. Bonatti (1989) Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethyl sulfate, Mutagenesis, 4, 174-178. Parodi, S., M. Taningher, L. Santi, M. Cavanna, L. Sciab~ and G. Brambilla (1978) A practical procedure for testing DNA damage in vivo, proposed for a pre-screening of chemical carcinogens, Mutation Res., 54, 39-46.
Sina, J.F., CL. Bean, G.R. Dysart, V.I. Taylor and M.O. Bradley (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential, Mutation Res., 113, 357-391. Tanzi, M.C., M. Levi and F. Danusso (1990) Amides from N-phenylpiperazine as low-toxicity activators in radical polymerization, Polymer, 31, 1735-1738. Warren, W., A.R. Crathorn and K.Y. Shooter (1979) The stability of methylated purines and of methylphosphotriesters in the DNA of V79 cells after treatment with N-methyl-N-nitrosourea, Biochim. Biophys. Acta, 563, 8288. Williams, J.R., J.B. Little and W.U. Shipley (1974) Association of mammalian cell death with a specific endonucleolytic degradation of DNA, Nature (London), 252, 754755.
Communicated by S. De Flora
