In vitro genotoxicity of nitroimidazoles as a tool in the search of new trypanocidal agents.

BACKGROUND: Only benznidazole (Bnz) (1) and nifurtimox (Nfx) (2) are licensed for the treatment of Chagas disease although their safety and efficacy profile are far from ideal. Farmanguinhos from Fiocruz has developed seven nitroimidazole compounds (4-10) analogs of megazol (3). OBJECTIVES: To evaluate whether the genotoxic effect of 3 was abolished in the seven nitroimidazoles (4-10) analogs using the in vitro alkaline comet assay (CA) and the in vitro cytokinesis-block micronucleus assay (CBMN) in whole human blood cells (WHBC) and correlate this effect with their trypanocidal activity using bloodstream trypomastigote forms of Trypanosoma cruzi. METHODS: The toxicity of 3-10 to WHBC in the in vitro CA was determined using the fluorescein diacetate/ethidium bromide assay. DNA damage in the in vitro CA was evaluated according to tail size in four classes (0-3) and methyl methane-sulfonate (MMS) was used as a positive control. The cytotoxicity of 3-10 to WHBC in the CBMN was measured using the cytokinesis-block proliferation index and the replication index. The number of the micronucleate cells in 2,000 binucleate cells by experimental group was determined. Mitomycin C and N-deacetyl-N-methylcolchicine were used as positive controls. FINDINGS: Compound 3 showed a significant DNA strand break effect through the in vitro CA and highly significant clastogenic and/or aneugenic effect in the CBMN. Compounds 5, 6, 8, 9 and 10 showed negative results in the CBMN and positive results in the in vitro CA, while the inverse effect was observed for 4 and 7. MAIN CONCLUSIONS: Compound 10 was the most promising to proceed with the development as a drug candidate in the treatment of Chagas disease showing absence of chromosomal cytogenetic damage and high activity against T. cruzi, about two times higher than 3 and the clinical drug 1.

Toxicity of nifurtimox as second-line treatment after benznidazole intolerance in patients with chronic Chagas disease: when available options fail.

OBJECTIVE: To describe the tolerability and rate of nifurtimox discontinuation when administered as a second-line treatment to patients with previous treatment interruptions due to adverse reactions with benznidazole. METHODS: We studied a prospective cohort study of adult patients with chronic Chagas disease in a referral centre in Spain treated from July 2007 to July 2017. We analysed the tolerability profile and treatment interruption rate due to adverse reactions (ARs) to nifurtimox in patients previously incompletely treated (less than 30 days) with benznidazole due to ARs. RESULTS: A total of 472 patients initiated treatment with benznidazole during the study period. Of these, 118 (25%) developed ARs that led to treatment discontinuation before 30 days of therapy. Fifty-three (44.9%) of 118 initiated nifurtimox as second-line treatment; most were women (79.3%), were of Bolivian origin (98.1%) and had a median age of 37.3 years (interquartile range, 29.8-43.2). The most common ARs with nifurtimox were cutaneous hypersensitivity (24.1%), digestive disorders (22.2%), fever (12.9%), neurologic disturbances (11.1%), depression, anxiety or insomnia (9.2%), dyspnoea (7.4%), myalgia (5.5%), and dizziness, asthenia or malaise (7.4%). Twenty-six (49.1%) of 53 patients discontinued nifurtimox due to ARs, all of them before the required minimal therapy duration of 60 days. There were no deaths. CONCLUSIONS: Treatment of chronic Chagas disease relies on two drugs with a poor tolerability profile. In our cohort, 12.3% of the patients who initiated benznidazole and subsequently nifurtimox in case of nontolerance developed ARs that led to permanent treatment discontinuation. Most were women of childbearing age, a group for whom therapy has the added benefit of interrupting vertical transmission.

Effects of astaxanthin in mice acutely infected with Trypanosoma cruzi.

During Trypanosoma cruzi infection, oxidative stress is considered a contributing factor for dilated cardiomyopathy development. In this study, the effects of astaxanthin (ASTX) were evaluated as an alternative drug treatment for Chagas disease in a mouse model during the acute infection phase, given its anti-inflammatory, immunomodulating, and anti-oxidative properties. ASTX was tested in vitro in parasites grown axenically and in co-culture with Vero cells. In vivo tests were performed in BALB/c mice (4-6 weeks old) infected with Trypanosoma cruzi and supplemented with ASTX (10 mg/kg/day) and/or nifurtimox (NFMX; 100 mg/kg/day). Results show that ASTX has some detrimental effects on axenically cultured parasites, but not when cultured with mammalian cell monolayers. In vivo, ASTX did not have any therapeutic value against acute Trypanosoma cruzi infection, used either alone or in combination with NFMX. Infected animals treated with NFMX or ASTX/NFMX survived the experimental period (60 days), while infected animals treated only with ASTX died before day 30 post-infection. ASTX did not show any effect on the control of parasitemia; however, it was associated with an increment in focal heart lymphoplasmacytic infiltration, a reduced number of amastigote nests in cardiac tissue, and less hyperplasic spleen follicles when compared to control groups. Unexpectedly, ASTX showed a negative effect in infected animals co-treated with NFMX. An increment in parasitemia duration was observed, possibly due to ASTX blocking of free radicals, an anti-parasitic mechanism of NFMX. In conclusion, astaxanthin is not recommended during the acute phase of Chagas disease, either alone or in combination with nifurtimox.

Effect of chronic alcohol drinking on rat liver microsomal nitroreductive metabolism of nifurtimox and benznidazole.

Nifurtimox (Nfx) and benznidazole (Bz) have serious toxic side effects. Manufacturers warn about significant adverse effects when simultaneous alcohol consumption is being made, but its mechanism is not known. The levels and toxicity of these drugs are linked to their liver microsomal nitroreduction to reactive metabolites. In this study, we analyzed whether alcohol drinking enhanced those nitroreductive processes. Male and female Sprague-Dawley rats, 5-6 weeks old (125-150 g body weight) were used. They were fed ad libitum for 28 days with Lieber and De Carli control or alcohol regular liquid diets. The rats were separated into two dietary groups: ethanol and control group. Both were pair fed with the respective diet. Their liver microsomes were isolated and the nicotinamide adenine dinucleotide phosphate-dependent nitroreduction of Nfx and Bz were determined. Alcohol drinking significantly induced microsomal nitroreduction of these drugs in male rats (11% for Nfx and 41% for Bz) but not in females. The activity observed in the alcohol-induced male rats was 100% inhibited by diphenyleneiodonium and attributable to P450 reductase. Inductive effects of alcohol drinking on nitroreductive activation of both drugs might be only partially involved in the harmful interactions described.

5-Nitro-2-furyl derivative actives against Trypanosoma cruzi: preliminary in vivo studies.

Ten 5-nitro-2-furyl derivatives, with good to excellent in vitro anti-Trypanosoma cruzi activity, and nifurtimox were tested oral and intraperitoneally on healthy animals for its acute toxicity on murine models. According to animals' survival percentage, organ histological results, biochemical and haematological findings, three new derivatives, with toxicity like nifurtimox, were selected to test in vivo as antichagasic agents. Clearly, dependences between chemical structure and both acute toxicity and in vivo anti-T. cruzi activity were observed. 4-Hexyl-1-[3-(5-nitro-2-furyl)-2-propenylidene]semicarbazide displayed good profile as anti-T. cruzi agent and better acute toxicity profile than nifurtimox.

Early nifurtimox-induced biochemical and ultrastructural alterations in rat heart.

Nifurtimox (Nfx) and Benznidazole (Bz) are being used for the treatment of the acute phase of Chagas' disease. Recently, they were also considered for use in the indeterminate phase. Both the nitroheterocyclic drugs have serious toxic side effects. The mechanism of Nfx toxicity is associated with the formation of reactive oxygen species (ROS) generated during nitroreduction. Potential effects on cardiac function have not been established yet, despite the well-known cardiopathy often produced by the disease itself. We describe experiments testing some acute effects of Nfx on the male Sprague Dawley rat heart. Nifurtimox was present in the heart at 1, 3 and 6 h after intragastric (i.g) treatment. In vitro studies on Nfx microsomal and cytosolic nitroreductase activities showed that only the microsomal fraction had the ability to nitroreduce it. Cytochrome P450 and cytochrome P450 reductase would be involved in the process as suggested by their response to specific inhibitors. Nifurtimox increased the cardiac protein carbonyl content at 1 and 3 h and decreased the protein sulfhydryl content at 3 h. In addition, 24 h after treatment ultrastructural alterations such as marked cytoplasmic vacuolization, separation and loss of myofibrils and mitochondrial swelling were observed. Results suggest that Nfx administration might aggravate pre-existing adverse cardiac conditions. Human & Experimental Toxicology (2007) 26, 781 -788.

Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of Nifurtimox, an antitrypanosomiasis drug.

The chronic toxicity and carcinogenicity of Nifurtimox (NFX), a 5-nitrofuran derivative used in the treatment of American trypanosomiasis, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity bioassay designed to assess potential carcinogenic activity of a test substance in critical organs and tissues of rodents in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder (UB), mammary gland (MG), bone marrow, spleen, kidneys, colon, stomach and any grossly observed lesions. NFX is a genotoxin which has been reported previously to exert a variable degree of carcinogenic activity in rat liver, kidney, UB and MG. The present study was undertaken to assess whether NFX has initiating activity in these four named target sites. In the initiation phase, groups of 20 Wistar rats were given NFX daily in the diet at 0.2% for the first 12 weeks of the study to assess initiating activity, followed by promoters (PROs) for four organs for an additional 24 weeks. NFX was compared to the following known initiators (INs) for each of these four tissues: diethylnitrosamine (DEN) for liver and kidney, N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) for UB and 7,12-dimethylbenz(a)anthracene (DMBA) for MG. PROs included phenobarbital (PB) for liver and kidney, nitrilotriacetic acid (NTA) for UB, and diethylstilbestrol (DES) for MG. NFX was also administered continuously without PROs for 40 weeks. At the end of dosing (40 weeks) and at the end of recovery (52 weeks), animals were sacrificed and subjected to complete gross and histopathological examinations, along with evaluations of body weight gain over time and terminal body weights. Mortality was highest with DEN+PB (group 6) (40%), followed by BBN+NTA (group 7) (15%) and NFX+DES (group 5) and DMBA+DES (group 8) (10% each). The same groups also showed significant reductions in body weight gain over time and terminal body weights at sacrifice. In these groups, the expected preneoplastic, neoplastic and metastatic neoplastic lesions were produced, demonstrating the sensitivity of the model. In groups given NFX+PROs (groups 3-5), either no neoplasms occurred (group 4) or only single neoplasms (groups 3 and 5). In contrast, the PROs all elicited tumors in groups given INs (groups 6-8). Also, NFX given alone for 40 weeks did not produce any chronic toxicity, preneoplastic or neoplastic lesions. Thus, in this study, NFX did not demonstrate chronic toxicity or carcinogenicity. Moreover, in four target sites, i.e., liver, kidney, UB and MG, it exhibited no neoplastic initiating activity manifested by PROs for these four target sites.

Liver microsomal biotransformation of nitro-aryl drugs: mechanism for potential oxidative stress induction.

Toxic effects of several nitro-aryl drugs are attributed to the nitro-reduction that may be suffered in vivo, a reaction that may be catalysed by different reductases. One of these enzymes is NADPH-cytochrome P450 reductase, which belongs to the cytochrome P450 oxidative system mainly localized in the endoplasmic reticulum of the hepatic cell. This system is responsible for the biotransformation of oxidative lipophilic compounds, so that oxidative and reductive metabolic pathways of lipophilic nitro-aryl drugs can take place simultaneously. Because of the affinity of nitro-aryl drugs (xenobiotics) for the endoplasmic reticulum, we propose this subcellular organelle as a good biological system for investigating the toxicity induced by the biotransformation of these or another compounds. In this work we used rat liver microsomes to assess the oxidative stress induced by nitro-aryl drug biotransformation. Incubation of microsomes of rat liver with nifurtimox and nitrofurantoin in the presence of NADPH induced lipoperoxidation, UDP-glucuronyltransferase activation and an increase in the basal microsomal oxygen consumption. Nitro-aryl-1,4-dihydropyridines did not elicit these prooxidant effects; furthermore, they inhibited lipoperoxidation and oxygen consumption induced by Fe3+/ascorbate. Nifurtimox and nitrofurantoin modified the maximum absorption of cytochrome P450 oxidase and inhibited p-nitroanisole O-demethylation, an oxidative reaction catalysed by the cytochrome P450 system, signifying that oxidation may proceed in a similar way to that described for nitro-aryl-1,4-dihydropyridines. Thus the balance between lipophilic nitro-aryl drug oxidation and reduction may be involved in the potential oxidative stress induced by biotransformation.

Reactions of nifurtimox with critical sulfhydryl-containing biomolecules: their potential toxicological relevance.

Nifurtimox (Nfx) is a drug used in the treatment of Chagas' disease, an endemic parasitic disease from Latin American countries. It produces undesirable side-effects in patients, frequently forcing the treatment to be stopped. Its toxic mechanism is not fully understood. In this work we describe purely chemical reactions of Nfx with relevant cellular sulfhydryl (SH) compounds. The compounds tested were glutathione (GSH), cysteine (RSH), lipoic acid (LA) and coenzyme A (CoA). All reacted with Nfx to give nitrite (NO(-) (2)). The relative reaction rates were CoA>LA>GSH>RSH. In studies with GSH and RSH the formation of nitrite was accompanied by decreases in Nfx concentration and increases in the formation of a reaction product revealed by HPLC. We failed to show the presence of liver cytosolic GST (GSH transferase activity)-mediated formation of NO2- from Nfx. These NO(-) (2)-releasing processes occurred under in vivo conditions in Nfx-treated Sprague-Dawley male rats (240-260 g body weight) at a dose of 100 mg Nfx kg(-1) p.o. In urine samples NO(-) (2) excretion was accompanied by unchanged drug and two unidentified more polar metabolites detectable by HPLC. The Nfx reactions with critical SH from molecules such as GSH, RSH, LA and CoA, and potentially others containing SH residues (e.g. enzymes or structural proteins), might have toxicological relevance not only for the Nfx side-effects but also for the chemotherapeutic effects on Trypanosoma cruzi. In addition, Nfx reactions with GSH might be crucial in Nfx detoxification.

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative.

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.

Ultrastructural alterations induced by nifurtimox and another nitro derivative on epimastigotes of Trypanosoma cruzi.

We report the ultrastructural alterations induced on epimastigotes by nifurtimox and 5-nitro-2-thienyl-malononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity. Parasites treated with concentrations of nifurtimox lower than usually employed for this kind of study showed vacuolisation, alterations of the mitochondria, the nucleus and the ribosomes. 5NO2TM caused the same kind of damage, but to a greater degree. This result correlates with the fact that cultures treated with this compound experienced a greater loss of viability.

Synthesis and antichagasic properties of new 1,2,6-thiadiazin-3,5-dione 1,1-dioxides and related compounds.

In a search for antichagasic drugs, 14 new 4-(nitroarylidene)-1,2,6-thiadiazin-3,5-dione 1,1-dioxide derivatives were synthesized and tested in vitro against the epimastigote form of Trypanosoma cruzi and some of them showed important antiprotozoan activity. Attempts to synthesize new 4-(nitroarylidene)-3,5-diamino-1,2,6-thiadiazine 1,1-dioxides were unsuccessful. The antichagasic properties of nitroarylidene-malononitrile and nitroarylidene-cyanoacetamide derivatives, thus obtained, were also tested. The cytotoxic properties against Vero cells of compounds which showed remarkable in vitro antichagasic activity were evaluated. All compounds tested exhibited high toxicity percentages at 100 micrograms/ml. However, compound 3c showed in vitro antichagasic and cytotoxic properties such as nifurtimox at the dose of 10 micrograms/ml.

Chagas' disease: carcinogenic activity of the antitrypanosomal nitroarenes in mice.

The carcinogenic activity of antitrypanosomal 2-nitroimidazole, 5-nitroimidazole and 5-nitrofuran derivatives was assessed in female Swiss mice of the same age group. A statistically significantly higher incidence of growths was seen in mice into which 2-nitro had been injected than in mice receiving 5-nitro derivatives intraperitoneally. A histologic type of lymphoblastic lymphoma that invades lymph nodes, spleen, liver, lungs and lymphatic tissue elsewhere was frequently found in nitroarene-treated mice. Further, it is shown that the potency of the drug, rather than the duration of its administration, was usually associated with the growth of lymphomas. The 2-nitro derivative which induced the highest incidence of lymphomas significantly decreased the survival of treated mice; this probably occurred because it undergoes enzymatic reduction of the nitro group more efficiently than the 5-nitro compounds used. The differences of incidence of lymphomas in mice receiving any of these nitroarenes and in control mice that received daily injections of 0.15 M saline were statistically significant (alpha = 0.05). The indiscriminate use of these nitroarenes to treat Trypanosoma cruzi infections in man could therefore induce a significant number of lymphomas.

Genetic differences between the standard Ames tester strains TA100 and TA98.

The standard Ames tester strains of Salmonella typhimurium are separated by many steps in their pedigree, some involving mutagen treatments, and contain independently isolated uvrB-bio-gal deletions and rfa mutations. In this work the araD531 mutation was introduced into the Ames tester strains TA100 and TA98. The responsiveness of the resulting strains (BA15 and BA14) to a number of chemical mutagens was then assessed by monitoring the induction of forward mutations to L-arabinose resistance (Ara test). Here we have shown that these two strains of the Ames test differ greatly in their responses to mutagens, in ways that are not associated with the mutagenic specificities of the original his mutations. In general, the genetic background of strain TA100 appears to be more sensitive to the killing effects of chemicals than that of TA98. The greatest differences were found with nifurtimox (NFX) and its analogue, compound 1K. The Ara test responded to the mutagenic effects of these two nitrofurans when carried out in the genetic background of strain TA98 but not in that of TA100. A higher sensitivity to the lethal effects of NFX and 1K together with the greater nitro-reduction capability of strain TA100 as compared with TA98 might explain the differences. In conclusion, our results indicate that the standard Ames S. typhimurium tester strains are not isogenic and that genetic differences at loci other than his might be significant for mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial chemistry. VI. Biological activities and cytotoxicity of 1,3-dihydro-2H-indol-2-one derivatives.

The biosynthetic pigment from Chromobacterium violaceum BB-78, 1,3-dihydro-2H-indol-2-one and its derivatives exhibit biological activities such as antimicrobial action, low hemolytic effects on red blood cells and in vitro trypanocide activity. A relatively high cytotoxicity on V-79 hamster fibroblast cells of the biosynthetic pigment was found, although with the methylol derivative the toxicity was almost eliminated. The methylol derivative exhibited similar toxicity as Nifurtimox, a known, commercial trypanocide compound.

Bacterial chemistry. VI. Biological activities and cytotoxicity of 1, 3-dihydro-2H-indol-2-one derivatives

The biosynthetic pigment from Chromobacterium violaceum BB-78, 1,3-dihydro-2H-indol-2-one and its derivatives exhibit biological activities such as antimicrobial action, low hemolytic effects on red blood cells and in vitro trypanocide activity. A relatively high cytotoxicity on V-79 hamster fibroblast cells of the biosynthetic pigment was found, although with the methylol derivative the toxicity was almost eliminated. The methylol derivative exhibited similar toxicity as Nifurtimox, a known, commercial trypanocide compound

Thirteenfold increase of chromosomal aberrations non-randomly distributed in chagasic children treated with nifurtimox.

Chromosomal aberrations were analyzed from cultures of peripheral lymphocytes in 2 groups of chagasic children, before and after treatment with nifurtimox. The mean incidence of chromosomal aberrations increased from control values of 1.75 +/- 1.39 (8 patients) to 23.55 +/- 9.55 (6 patients) at a significance of P less than 0.0001. G-banding analysis of chromosomal aberration sites revealed that treated patients present coincidence in the chromosome regions affected: 1p11, 1q11-12, 9q11-13, 17q11-21, 2p21, 2q23, 2q31, 2q33, 6p21, 6p21, 7q32, 13q14, 13q22, 15q22. These data indicate a non-random distribution of chromosomal aberrations induced by nifurtimox therapeutic treatment.

Ultrastructural alterations in ovaries from nifurtimox or benznidazole-treated rats: their relation to ovarian nitroreductive biotransformation of both drugs.

Chagas' disease is a parasitic chronic condition affecting several million people in Latin America. Two drugs are used in the chemotherapy of Chagas' disease: nifurtimox (Nfx) and benznidazole (Bz). Both are nitroderivatives whose deleterious effects are related to their reductive biotransformation. In this work we report that rat ovaries exhibited Bz and Nfx nitroreductase activity. The Bz nitroreductase was only found in the mitochondrial fraction and was partially inhibited by CO. The Nfx nitroreductase activity was maximal in ovarian mitochondria but was also present in microsomes and in the cytosol. The microsomal enzyme was completely inhibited by CO while that in mitochondria was only partially inhibited by CO. The cytosolic activity only proceeded using hypoxanthine as substrate and was inhibited by allopurinol. The cytosolic activity was able to proceed in part under oxygen. All the other Bz or Nfx nitroreductases were completely inhibited by atmospheric oxygen. The potential participation of cytochrome P450, flavoenzymes, iron-sulfur-protein, and xanthinooxidase in both nitroreductive processes is discussed. The administration of either Nfx or Bz to female rats produced ultrastructural degenerative effects in the different cell types of ovaries. Specific alterations such as swelling, disruption, disorganization, and loss of matrix components were observed in ovarian mitochondria. These alterations occurred irrespectively of the ovarian cycle stage. The potential reproductive toxicological consequences of Bz or Nfx administration are analyzed.

Activation of anti-Trypanosoma cruzi drugs to genotoxic metabolites promoted by mammalian microsomal enzymes.

Salmonella typhimurium TA100 and its nitroreductase-deficient derivative, TA100 NR, were used to reevaluate the mutagenic activities of benznidazole and nifurtimox. Mutagenicity and toxicity of nifurtimox were abolished in the TA100 NR tester strain under aerobic or anaerobic conditions and addition of rat liver extracts did not alter the results. However, benznidazole showed a significant mutagenicity and toxicity to the nitroreductase-deficient strain TA100 NR under hypoxic conditions. Addition of rat liver extracts enhanced the observed mutagenicity and toxicity of benznidazole even more. In the presence of O2 the genotoxic activities of benznidazole to the TA100 NR tester strain were eliminated. These results lead us to conclude that bacterial enzymes were responsible for the previously observed genotoxic effects of nifurtimox and benznidazole on S. typhimurium TA100. Moreover, under anaerobic conditions, only benznidazole could be metabolized by mammalian nitroreductases into a mutagenic derivative.