Mutagenic activation of CL64,855, an anti-Trypanosoma cruzi nitroderivant, by bacterial nitroreductases

Mutagenic activation of CL64,855, an anti-Trypanosoma cruzi nitroderivant, by bacterial nitroreductases

Marcos Antonio de Morais Jr.¹, Rita de Cássia Café Ferreira² and Luiz Carlos de Souza Ferreira²

¹ Departamento de Genética e Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Av. Morais Rego, s/n, Cidade Universitária, 50732-970 Recife, PE, Brasil. Send correspondence to M.A.M.Jr. Fax: [phone]. E-mail: [e-mail]

² Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Bloco G, Cidade Universitária, 21849-900 Rio de Janeiro, RJ, Brasil. E-mail: [e-mail]

ABSTRACT

CL64,855 is a nitroimidazole-thiodiazole derivate with high anti-Trypanosoma cruzi activity. CL64,855-induced mutagenesis in the Salmonella/microsome test was detected by TA98 and TA98dnp₆ strains, but not by the nitroreductase I-deficient TA98nr strain. The lack of mutagenic response of TA98nr was connected with its extreme resistance to the killing effect of the drug. Presence of S9 mix did not restore mutagenic activity of CL64,855 to the TA98nr strain. Additionally, CL64,855 was reduced in vitro by the nitroreductase I-proficient TA98 strain, mainly in the presence of oxygen, but not by the TA98nr strain. Mutagenic activity was detected in serum samples of treated guinea pigs by nitroreductase-proficient strains TA98 and TA98dnp₆, but not by nitroductase-deficient strain TA98nr. In the case of urine, mutagenic activity was observed with all three tested strains, suggesting an in vivo metabolic activation of the drug by a distinct metabolic pathway.

INTRODUCTION

It is well known that nitroreductases, which are conserved from bacteria to mammals, can reduce the nitro group of nitroderivatives into mutagenic and carcinogenic metabolites (Blumer et al., 1980; Rosenkranz et al., 1982; Biaglow et al., 1986; Vance et al., 1988; Hooberman et al., 1994; Jurado et al., 1994; Raffi et al., 1994). At least three distinctive prokaryotic nitroreductase activities have been identified, based on their sensitivity to oxygen, substrate and cofactor specificity (Rosenkranz et al., 1982). "Classical" or type I nitroreductases are oxygen-insensitive enzymes involved in the reduction of nitrocompounds (Bryant et al., 1981), including drugs with antiparasite effects as metronidazole (Rosenkranz and Speck, 1976).

Two nitrocompounds, nifurtimox (nitrofuran) and benznidazole (nitroimidazole), have been largely used in the chemotherapy of Chagas' disease in South America (Marr and Docampo, 1986; De Castro, 1993). Unfortunately, serious undesirable effects have been observed in patients under treatment (De Castro and Toranzo, 1988), and the mutagenic activity of both drugs has been demonstrated (Ohnish et al., 1980, 1983; Ferreira and Ferreira, 1986a; Ferreira et al., 1988a,b). Berkelhemer and Asato (1968) synthesized a nitroimidazole-thiodiazole derivate, called CL64,855, with a broad-spectrum anti-bacterial and anti-parasite activity (Figure 1). High curative efficiency of CL64,855 was shown in mice infected with two strains of Trypanosoma cruzi, compared to drugs normally used in the treatment of Chagas' disease (Filardi and Brener, 1982).

Figure 1
- Chemical structure of CL64,855 [2-amino-5(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole].

A previous study showed that CL64,855 has mutagenic activity and can be detected by the Ames test using TA98 and TA102 indicator strains, irrespective of S9 mix use (Ferreira and Ferreira, 1986b). Results indicated that CL64,855 can cause frameshift mutations. In this study, the mutagenic activation of CL64,855 by bacterial and mammalian enzymes has been evaluated. Results show that "classical type I" bacterial nitroreductases are responsible for activation of CL64,855 and that mammalian enzymes do not seem to be involved in the mutagenic activation of the drug.

MATERIAL AND METHODS

Bacterial strains and chemicals

Salmonella typhimurium strain TA98 is currently used in the Ames test (Maron and Ames, 1983). Strain TA98nr, isolated from a clone resistant to niridazol, is deficient in type I "classical" nitroreductase (Rosenkranz et al., 1982). Strain TA98dnp₆, isolated from a clone resistant to 1.8 dinitropiren, is deficient in esterificase that acetylates 1.8 dinitropiren nitroderivate radicals and their isomers (McCoy et al., 1983).

CL64,855 was kindly provided by Dr. Z. Brener, Department of Zoology and Parasitology, UFMG, Brazil. Daunorrubicin was provided by the Institute of Antibiotics, UFPE, Brazil. Nitrofurazone and 2-aminofluorene were purchased from Sigma Chemical Co. (St. Louis, MO). All drugs were dissolved in DMSO (Sigma).

Mutagenic assay

The mutagenicity test was performed as described by Maron and Ames (1983). For the anaerobiosis experiments, plates were placed into a desiccator, and air was replaced with nitrogen. Rat liver S9 microsomal fraction was prepared from Aroclor 1254-treated animals, as previously described (Maron and Ames, 1983). 2-aminofluorene was used as a metabolic activated mutagen, while daunorrubicin was used as a direct acting mutagen. Survival curves were obtained by platting diluted cell suspensions onto minimal plates containing histidine (40 ml/ml) and different concentrations of CL64,855. Each experiment was performed at least twice, with triplicates for each dose. Results represent mean values.

Enzymatic reduction assays

Bacterial enzymatic reduction was done according to McCalla et al. (1975) with 25 mg/ml of CL64,855 final concentration. The cells were air bubbled with a pump and samples were withdrawn at indicated intervals. Anaerobiosis experiments were carried out with a continuously bubbled nitrogen stream. The supernatant optical density (O.D.) was measured at 340 nm. Microsomal drug reduction was carried out in 10 ml S9 mix containing 1 mg rat liver protein. The supernatant O.D. was measured at 400 nm to avoid NADP interference.

In vivo metabolic activation

For in vivo metabolic activation, guinea pigs from both sexes weighing around 300 g were given by gavage a single oral dose (100 mg/kg) of CL64,855 dissolved in corn oil. Animals were fed normally before and after drug administration. Blood and urine samples were collected as described previously (Ferreira et al., 1988b). Four animals were tested with pooled blood and urine samples. Aliquots of 100 ml were used for mutagenicity tests.

RESULTS

Mutagenic and toxic effects of CL64,855 in bacteria

Figure 2 shows that increasing doses of CL64,855 induce linear reverse his⁺ mutations in TA98 and TA98dnp₆ strains up to 0.5 mg/ml, both in aerobiosis (Figure 2A) and anaerobiosis (Figure 2B). Presence of S9 mix did not change the mutagenic response of the drug. On the other hand, no mutagenic effect was observed in the strain TA98nr under both aerobic and anaerobic conditions. The presence of S9 mix did not restore mutagenic activity of CL64,855 for the TA98nr strain (Figure 2). Efficiency of the S9 mixture was tested with 2-aminofluorene as a positive control (data not shown).

Figure 2
- Mutagenic activity of CL64,855 in plate incorporation assays. Frequency of reverse his⁺ mutation in the strains TA98 (circles), TA98dnp₆ (triangles) and TA98nr (squares) induced by different concentrations of CL64,855 in aerobiosis (A) or anaerobiosis (B). Cells were treated in the presence (closed symbols) or absence (open symbols) of S9 mix.

The toxic effect of CL64,855 was measured from the survival curves of the indicator strains, either in aerobiosis or anaerobiosis (Figure 3). The excision-deficient TA98 strain was significantly sensitive to the drug, and survival fraction was approximately 0.02% at the dose of 10 mg/plate. The TA98nr strain, although excision-deficient, was resistant to the toxic effect of CL64,855, with a survival fraction of 90% measured at the concentration of 10 mg/plate (Figure 3). In the range used for the mutagenic test (0.05 to 0.5 mg/plate), all strains maintained approximately 95% cell survival.

Figure 3
- Survival curves of the Salmonella indicator strains in different concentrations of CL64,855. The strains TA98 (circles) and TA98nr (squares) were assayed under aerobiotic (open symbols) or anaerobiotic (closed symbols) environments.

Enzymatic reduction of CL64,855

Figure 4A shows reduction of CL64,855 mediated by bacterial nitroreductases under different environments regarding oxygen content. TA98 strain showed a two-phase enzymatic drug reduction. The first 20 min in aerobiosis were marked by a significant reduction of the drug, followed by a slower reduction phase as the incubation continued to 60 min. In anaerobiosis, a faster drug reduction was observed up to 10 min after nitrogen bubbling, but reduction had completely stopped by this time (Figure 4A). On the other hand, TA98nr cells were not able to reduce CL64,855 neither in aerobiosis nor in anaerobiosis conditions (Figure 4A). Microsomal enzymes present in S9 fraction were also proficient in reducing CL64,855, mainly under aerobic condition (Figure 4B). The reduction promoted by bacterial and mammalian nitroreductases was confirmed with nitrofurazone as a positive control (data not shown).

Figure 4
- Enzymatic reduction of CL64,855 by bacterial and mammalian enzymes. A, Bacterial strains TA98 (circles) and TA98nr (squares) were incubated with the drug for the time indicated, and O.D. was measured from the supernatants. B, Rat liver S9 mix was incubated with the drug for the time indicated and O.D. was measured from the supernatants. The experiments were performed under aerobiotic (open symbols) or anaerobiotic (closed symbols) conditions.

In vivo mutagenic activation of CL64,855

Mutagenic activity in serum and urine from guinea pigs treated with CL64,855 was evaluated at two and 48 h after ingestion. Thirty minutes after administration, CL64,855-induced mutagenesis in the TA98 and TA98dnp₆ strains was very high in serum samples, reaching a peak 60 min after administration (Table I). On the other hand, no mutagenic activity was detected in the serum samples by TA98nr strain over a period of 2 h after ingestion (Table I). Similar mutagenicity values were obtained under anaerobic conditions (Table I).

Analysis of urine samples revealed that the peak of CL64,855-induced mutagenesis for both TA98 and TA98dnp₆ strains was reached in urine 9 h after ingestion (Table II). Smaller, but nevertheless significant, mutagenicity values were detected in urine samples harvested 18 and 24 h post-injection using the same indicator strains. Incubation of testing plates in anaerobic conditions did not significantly change the results (Table II). Moreover, a significant CL64,855-induced mutagenicity was also observed in TA98nr strain urine samples harvested 9, 18 and 24 h after drug administration. Mutation frequency in this strain was half of that found for the nitroreductase proficient isogenic TA98 strain (Table II). The same response was observed under anaerobic conditions.

DISCUSSION

In contrast to other drugs with a curative action on T. cruzi, such as the base substitution mutagens nifurtimox and benznidazole, CL64,855 was shown to preferentially induce frameshift mutation in the Ames test (Ferreira and Ferreira, 1986a,b; Figure 1, this work). The uvr excision repair pathway seems to protect cells from the killing effect of CL64,855, since the excision-proficient strain TA102 has proven to be more resistant to this drug than the excision-deficient strain TA98 (Ferreira and Ferreira, 1986b).

The present results show that "classical" bacterial nitroreductase is the major bacterial enzyme involved with the mutagenic activation of CL64,855. It has also been pointed out for other nitroderivates with antiparasite effects (Rosenkranz et al., 1982; Rosenkranz and Mermelstein, 1985; Hooberman et al., 1994; Jurado et al., 1994). No mutagenic response was detected by the type I nitroreductase deficient TA98nr strain; furthermore, no enzymatic reduction of CL64,855 was carried out by this strain. The same responses were observed for experiments in anaerobiosis, a condition in which type-II bacterial nitroreductase was intact in TA98nr strains (McCalla et al., 1975). Lastly, a greater drug reduction by TA98 was observed under aerobiosis conditions which inhibit type II nitroreductase. The role of oxygen in the reductive metabolization pathway of nitrocompounds has been outlined elsewhere (De Castro and Toranzo, 1988). Survival data show that lack of enzymatic activation of CL64,855 is accompanied by an increased resistance to the toxic effect of the drug by the TA98nr strain.

Addition of the S9 mix to the assay did not restore mutagenic activity of CL64,855 for the TA98nr strain, both in aerobiosis or anaerobiosis. Microsomal enzymes in the S9 mix were able to significantly reduce CL64,855, mainly in aerobiosis. Therefore, liver mammalian enzymes did not seem to be involved in the mutagenic activation of CL64,855, but rather in some metabolic reduction which may lead to the formation of non-mutagenic metabolites. It has been previously shown that oxygen-sensitive and oxygen-insensitive nitroreductases can reduce nitrofurazone leading to superoxide formation and cyan derivatives, respectively, in both Escherichia coli and rat hepatic microsomes (Peterson et al., 1978). Nonetheless, it has been reported that benznidazole-induced mutagenicity was partially restored in TA100nr indicator strain under anaerobic conditions, and the presence of S9 mix increased this response (Ferreira and Ferreira, 1988a). The results demonstrate that mutagenic activation of different nitrocompounds by oxygen-insensitive bacterial and mammalian reductases occurs.

The TA98dnp₆ strain, which is defective in further acetylation of the reduced nitro derivatives, shows the same mutagenic response observed in TA98 to CL64,855, indicating that this step is not essential for activation of this drug.

Mutagenic activation in vivo of CL64,855 by the mammalian metabolism was achieved by evaluating serum and urine samples of orally treated animals. Similar to the in vitro experiments using liver microsomes, no mutagenic response was observed in TA98nr with serum samples either in aerobiosis or anaerobiosis. This result indicates that mutagenic metabolites of CL64,855 in the blood stream are not generated by mammalian liver enzymes. The mutagenic activity detected by TA98 may therefore represent the unmetabolized fraction of the drug in the serum samples. Urine samples of treated animals were analyzed, and a peak of mutagenic induction was found 9 h after administration in the TA98 indicator strain. Surprisingly, mutagenic activity in the urine samples collected between 9 and 24 h after drug administration could also be detected in the TA98nr strain, both in aerobiosis or anaerobiosis (Table II). It must be speculated that at least part of this drug had been metabolized by host enzymes.

Based on previous findings, metabolization of nitrocompounds into mutagenic derivatives requires strict anaerobic conditions (Docampo and Stoppani, 1979; Ferreira et al., 1988a). Possible activation of CL64,855 by mammalian enzymes would suggest that oxygen-sensitive specific nitroreductases may be found in some specific tissues such as the kidney. Mutagenic activation of aromatic amines other than liver microsomes has already been detected (Fouarge et al., 1984). Moreover, in vitro differential mutagenic activation of carcinogens by hepatocytes from different mammalian species demonstrates an alternative pathway for enzymatic drug activation (Snyder et al., 1987). Higher concentrations of unmetabolized CL64,855 excreted in the urine samples of treated animals could however lead to nonspecific enzymatic activation of the drug by the TA98nr strain. This may occur by a new metabolic pathway or by a residual activity of the mutated type I oxygen-insensitive nitroreductase in this strain.

ACKNOWLEDGMENTS

We thank Dr. A. Furtado, Centro de Pesquisas Aggeu Magalhães/FIOCRUZ, Recife, for providing laboratory facilities during the experiments. Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).

RESUMO

O derivado nitroimidazol-tiodiazol CL64,855 tem uma alta atividade anti-Trypanosoma cruzi. O CL64,855 induziu uma resposta mutagênica nas linhagens TA98 e TA98dnp₆, mas não na linhagem deficiente em nitrorredutase do tipo I TA98nr. Esta perda da ativação mutagênica foi acompanhada pelo aumento da sobrevivência celular aos efeitos citotóxicos da droga. A presença da fração microssomal S9 não restaurou a resposta mutagênica da TA98nr. Adicionalmente, o CL64,855 foi reduzido in vitro pela nitrorredutase I proficiente TA98, mas não pela TA98nr. Atividade mutagência foi detectada em amostras de soro pelas linhagens proficientes em nitrorredutase TA98 e TA98dnp₆, mas não pela linhagem deficiente TA98nr. Em amostras de urina, a atividade mutagênica foi observada por todas as três linhagens testadas, sugerindo uma ativação metabólica in vivo do CL64,855 por diferentes rotas metabólicas.

Jurado, J., Alejandre-Duran, E. and Pueyo, C. (1994). Mutagenicity testing in Salmonella typhimurium strains possessing both the His reversion and Ara forward mutation systems and different levels of classical nitroreductases or O-acetyltransferase activities. Environ. Mol. Mutagen. 23: 286-293.

(Received November 7, 1997)

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Publication Dates

• Publication in this collection
  01 Mar 1999
• Date of issue
  Dec 1998

History

• Received
  07 Nov 1997