An in vivo evaluation of induction of abnormal sperm morphology by some anthelmintic drugs in mice.

Using the murine sperm-head abnormality test, the mutagenicity of pyrantel pamoate, levamisole, albendazole, mebendazole and niridazole was evaluated. Pyrantel pamoate and niridazole induced increases in sperm-head abnormalities statistically significant over the negative controls at all the dose levels that were considered; the induction was dose-dependent indicating that both drugs might be mutagenic. Levamisole, albendazole, mebendazole and thiabendazole, all were unable to induce statistically significant increases in sperm-head abnormalities over the negative controls at all the dose levels tested; there was no correlation between dose level of administered drugs and incidence of abnormal sperms, indicating that the drugs might not be mutagenic.

The role of oxygenation in embryotoxic mechanisms of three bioreducible agents.

Many xenobiotics used in the treatment of hypoxic pathogens and tumors require reductive bioactivation under anaerobic conditions for maximal effectiveness and/or toxicity. A number of agents of this type have been shown to be teratogenic in vitro and/or in vivo. Early conceptuses may be vulnerable to these agents because they exist in a relatively anaerobic environment and have the capacity to perform reductive metabolism. It has been hypothesized that the single electron redox potential of bioreducible agents plays a dominant role in the capacity to induce anomalies. We examined the in vitro embryotoxicity in rats of three bioreducible drugs of similar redox potential under normoxic and hypoxic conditions as well as the capacity of those drugs to redox cycle and to damage DNA in embryonic tissue. Adriamycin, mitomycin C, and niridazole were shown to have differential embryotoxic responses in vitro to altered oxygenation. Studies of the bases of drug action showed (1) Adriamycin induces DNA strand breaks at concentrations that correlate well with embryolethality; (2) Mitomycin C does not induce strand breaks, but its dysmorphogenicity is increased by hypoxia; and (3) Niridazole does not produce DNA damage but appears to induce asymmetric malformations by depleting embryonic oxygen through redox cycling. Together the studies show that dysmorphogenic and embryolethal effects may result from separate mechanisms and that oxygenation plays an important role in those mechanisms.

Mutagenic effects of niridazole in animal-mediated and in liquid suspension assays using Escherichia coli K-12 as an indicator.

The mutagenic effects of the antischistosomal drug niridazole (1-(5-nitro-2-thiazolyl)-2-imidazolidinone) were investigated in liquid suspension and intrasanguineous animal-mediated assays with mice. As indicator strains Escherichia coli K-12 343/113 (Nir(S)) and a newly constructed niridazole nitroreductase-deficient derivative (Escherichia coli K-12 343/113 Nir(r) 200) were used. With the parental strain (Nir(S)) induction of nalidixic acid- and valine-resistant mutants was observed under in vivo conditions in the liver and, to a lesser extent, in the spleen. Positive results were also found when intestinal homogenates, blood sera, and urine samples of niridazole-treated animals were tested in vitro with the wild-type strain. With Escherichia coli K-12 343/113 Nir(r) 200 no clear-cut positive results were obtained in animal-mediated assays. In liquid suspension assays positive results were restricted to the urine samples. These findings indicate that the positive results obtained with the wild-type strain are due to nitroreduction and that the concentrations of mutagenic metabolites formed by activation processes in the living animal are too low to enable their detection in inner organs, intestines, and the blood with the reductase-deficient strain. In agreement with our present findings showing increased genotoxicity in urine, niridazole causes tumors in rodents preferentially in the kidneys and in the bladder.

Asymmetric development of mitochondrial activity in rat embryos as a determinant of the defect patterns induced by exposure to hypoxia, hyperoxia, and redox cyclers in vitro.

Previous study has shown that midorganogenesis-stage rat embryos exposed to strong redox cyclers under moderate hypoxia in vitro develop severe necrotic defects on the right side. Similar effects can be produced by exposure to severe hypoxia alone. Studies presented here indicate that exposure to severe but survivable hyperoxia induces comparable necrotic degeneration on the left sides of all embryos. We hypothesize that the basis of these axially asymmetric defects is relatively precocious mitochondrial maturity on the left side of the embryo. In order to investigate this hypothesis, we compared mitochondrial oxygen utilization (NADH oxidase activities) on either side of rat embryos between days 11 and 14 of gestation. Activities were consistently higher on the left side during this period and significantly higher on day 11. We also found that the asymmetric embryotoxicity induced by niridazole, a strong redox cycler, could be attenuated by prior culture under hyperoxic conditions. We propose that mitochondrial immaturity on the right results in inadequate energy generation under hypoxic conditions, either directly or as a result of redox cycling. On the other hand, necrosis associated with hyperoxic conditions results from "leakage" of superoxide from functionally mature mitochondria on the left side.

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding.

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.

On the capacity of nitroheterocyclic compounds to elicit an unusual axial asymmetry in cultured rat embryos.

A series of nitroheterocyclic compounds with antimicrobial and radiosensitizing properties was tested for embryotoxicity in cultured Sprague-Dawley rat embryos, and their effects were compared with various other five-membered heterocycles. Nifuroxime, furazolidone, nitrofurazone, niridazole, 2-nitroimidazole, and ronidazole each elicited a striking malformation characterized by asymmetrical, right-sided hypoplasia when coincubated with embryos for 26 hr. Minimum concentrations required to elicit this unusual defect ranged from 0.01 mM with nifuroxime and furazolidone to 0.5 mM with ronidazole and were roughly correlated with single electron redox potentials; i.e., agents with relatively high redox potentials were generally more effective than those with low potentials. Nitrofurantoin failed to elicit this unusual malformation but exhibited an extremely steep dose-response curve for embryolethality. Metronidazole and 4-nitroimidazole, nitroheterocyclic agents with relatively low redox potentials, did not produce the asymmetric abnormality nor were they highly embryotoxic, even at concentrations up to 2 mM. 2-Amino-5-nitrothiazole and 4'-methylniridazole also failed to evoke the asymmetric malformation but produced significant embryotoxicity as manifested by decreased growth parameters and elicitation of other kinds of malformations. Heterocyclic compounds not bearing a nitro group (furosemide, 2-aminothiazole, and 2-aminoimidazole) failed to elicit axial asymmetry at concentrations up to 1.0 mM but produced other signs of embryotoxicity at the highest concentrations tested. The results suggest that the presence of a reducible nitro group is critical for generation of the unusual malformation and that the single-electron redox potential of the nitro group plays a dominant but not exclusive role in the capacity of these chemicals to evoke axial asymmetry in cultured rat embryos.

The toxicity of niridazole in rat embryos in vitro.

The antischistosomal drug niridazole (NDZ) was found to be teratogenic in a concentration-dependent manner from 10 to 50 micrograms/ml (47-233 microM) in rat embryos cultured from day 10 to day 11. A striking malformation consisting of axial asymmetry in which the right side of the embryo showed severe necrosis was the predominant malformation. The drug showed significantly greater dysmorphogenic activity at low (5%) compared to high (20%) oxygen tensions in cultures. Coincubation of embryos and NDZ with an exogenous source of metabolic enzymes and cofactors (NADPH) failed to modify teratogenicity. Inclusion of CO in the culture atmosphere significantly reduced the malformation incidence as did preincubation of the drug with S-9 and cofactors in medium with low O2 tension. Treatment of gravida with NDZ up to and including the maternal-lethal dose failed to result in observable malformations despite the use of several routes of exposure. These data lead us to hypothesize that rat embryos are capable of performing the reductive activation of NDZ in a fashion analogous to the schistosome but that reductive extraembryonic metabolism may result in teratogenic bioinactivation.

The effect of oxygen concentration on the teratogenicity of salicylate, niridazole, cyclophosphamide, and phosphoramide mustard in rat embryos in vitro.

Comparisons were made of rat embryos cultured at 5% or 20% oxygen in the presence of salicylate (SAL), cyclophosphamide (CP), niridazole (NDZ), or phosphoramide mustard (PM). Multiple regression analyses were used to compare the effects of drug concentration, oxygen concentration, and the product of drug times oxygen concentration on malformation incidence, viability, and protein content of embryos cultured for 24 hours. Drug concentration significantly affected malformation incidence or severity and protein content (P less than 0.001) for the four drugs tested. Oxygen concentration significantly affected protein content for the four compounds (P less than 0.001) but affected malformation incidence only with NDZ. Furthermore, the interaction of oxygen concentration and drug concentration significantly affected the malformation incidence in the presence of NDZ (P less than 0.001), and protein content (P less than 0.001) and viability (P less than 0.001) in the presence of CP. The pattern of significant effects of the independent variables (drug concentration, oxygen concentration, and drug times oxygen concentration) is consistent with the hypotheses of oxygen-dependent metabolism (or lack of metabolism) of the drugs in question. NDZ, which is thought to be converted to reactive intermediates by an oxygen-inhibited nitroreductase, was more toxic at reduced oxygen tension. CP, which is activated by an oxygen-dependent P-450 system, was more toxic with increased oxygen tension. Significant effects of the independent variables on embryos exposed to SAL or PM were consistent with the effects on control embryos, notably, increased protein content with increased oxygen.

Covalent binding and glutathione depletion in the rat following niridazole (ambilhar) pretreatment.

In vivo and in vitro studies with rats have shown that (14C) niridazole (Ambilhar) binds covalently to tissue proteins, but not to nucleic acids. In the in vitro experiments, binding required the presence of NADPH in the incubation medium, suggesting the production of an active metabolite via a cytochrome P-450-mediated reaction. Niridazole also caused significant dose-dependent decreases in liver and kidney glutathione levels, even though it had no apparent effect on blood glutathione. Alteration of tissue glutathione availability by pretreatment with chloracetamide or cysteine respectively either increased or decreased the NADPH-dependent covalent binding. Pretreatment with phenobarbital, 3-methylcholanthrene or cobaltous chloride, which change the rate of metabolism of (14C) niridazole, similarly altered the extent of protein binding. It is shown that the decrease in tissue glutathione concentration is not due to an effect of the drug on the activities of either glucose-6-phosphate dehydrogenase or glutathione-S-transferases. However, there is a significant reduction in glutathione reductase activity in all the tissues studied. The possible relationships between the results obtained and the cytotoxic effects of niridazole have been discussed.

4-Keto niridazole: a major niridazole metabolite with central nervous system toxicity different than niridazole.

4-Keto niridazole, isolated by high-pressure liquid chromatography, was identified by high resolution electron impact mass spectral analysis as a major drug metabolite of niridazole in the serum or plasma of rats and mice treated orally or i.p. with niridazole. This metabolite has a pKa of 5.8 and is approximately 40% bound at physiologic pH to serum proteins of mice receiving therapeutic doses of niridazole. After i.p. injection of niridazole (160 mg/kg), peak serum levels of 4-keto niridazole (10.4 micrograms/ml) were reached within 6 hr in DBA/2J mice. The acute LD50 for 4-keto niridazole i.p. was 55 mg/kg in DBA/2J mice and 51 mg/kg in C57BL/6J mice; the comparable value for niridazole was 220 mg/kg in DBA/2J mice. Signs of acute 4-keto niridazole toxicity were different from those of niridazole toxicity and consisted of profound sedation and labored, irregular breathing terminating in respiratory arrest. Daily i.p. injection of 30 mg/kg of 4-keto niridazole for 5 days into DBA/2J mice resulted in no evidence of cumulative toxicity. The serum and brain concentrations of 4-keto niridazole after a 70-mg/kg i.p. LD90 dose of this compound were 93 micrograms/ml and 7.5 micrograms/g just before death. If an LD90 dose of niridazole (285 mg/kg) was injected into DBA/2J mice, the serum and brain concentrations of 4-keto niridazole just before death were 15 and 5%, respectively, of those found after an LD90 dose of 4-keto niridazole. Thus, 4-keto niridazole does not appear to account for the central nervous system toxicity of niridazole.

Immunosuppression in canine renal allografts using niridazole.

The immunosuppressive activity of niridazole in unmatched kidney allografts in nephrectomized dogs was studied. The drug alone was not effective, but improved survival when used with azathioprine and prednisolone or azathioprine alone. However, niridazole was toxic in dogs in the doses used (25 or 50 mg/kg/day).

Toxicogenetics of niridazole in inbred mice.

The lethal potency of the antischistosomal agent niridazole (NDZ) was compared in C57BL/6J (B6) and DBA/2J (D2) mice and in their F1 hybrid, backcross and F2 progeny. A daily i.p. dosage range was chosen so that the lethal effect, ascribed to central nervous system toxicity, did not occur before 4 to 5 days. Death was always preceded by a generalized tonic-clonic seizure which terminated in respiratory arrest. In B6 mice the LD50 was 202 mg kg-1 day-1 while in D2 mice the LD50 was 146 mg kg-1 day-1; the LD50 for NDZ in similarly treated F1 hybrid mice was found to be the arithmetic mean of the LD50 values for the parental strains (172 mg kg-1 day-1). Determination of the level of NDZ in the plasma and brains of B6 and D2 mice treated subacutely with the same daily dose of NDZ failed to reveal any strain differences. Moreover, there was no evidence of in vivo accumulation of NDZ with subacute treatment which suggests that a NDZ metabolite is responsible for the observed toxicity. An association between susceptibility to the lethal effects of NDZ and the Ah locus is suggested by experiments in backcross and F2 mice. The incidence of death observed after subacute treatment with 162 mg/kg-1 day-1 of NDZ matched that predicted on the basis of genotype, i.e., it was lethal to 72% of nonresponsive and 38% of aromatic hydrocarbon responsive mice.

Kidney tumors induced in rats by the antischistosomal drug niridazole.

An increased incidence of kidney tumors was found in MRC rats fed the antischistosomal drug niridazole at four dose levels in the diet. Histologically, the adenomas and adenocarcinomas were solid papillary, clear cell, and tubular types, with the latter type predominating. Seven mesenchymal tumors were found among the 107 renal epithelial neoplasms. Severe nephrosclerosis occurred in both treated and control rats and has been suggested as important in renal carcinogenesis. Niridazole is considered a potent inducer of epithelial kidney tumors.

Mutagenicity and mutagenic specificity of metronidazole and niridazole in Neurospora crassa.

Mutagenicity and mutagenic specificity of niridazole and metronidazole, two chemotherapeutic agents used in the treatment of human parasitic diseases, were studied with the ad-3 test system of Neurospora crassa. The results show that neither compound is mutagenic in resting conidia. In growing vegetative cells, however, both compounds are mutagenic in Neurospora crassa. Genetic analysis of the mutants indicate that niridazole induces predominantly base-pair substitution mutations. None of the niridazole-induced mutants resulted from multilocus deletions. The spectra of genetic alterations induced by metronidazole are similar to those induced by the monofunctional alkylating agents, EI, EMS and ICR-177. It is suggested, therefore, that the mechanism of mutation induction by metronidazole in Neurospora is similar to that of monofunctional alkylating agents.

Evaluation of niridazole as a suppressant of cellular immunity in chickens.

Niridazole was administered by gavage to 5-week-old chickens at dosage levels of 50, 100, and 200 mg/kg of body weight. The 2 larger dosage levels were toxic and resulted in the death of many of the recipients. The 50 mg/kg dosage level was not lethal, but depressed weight gain. Cellular immune functions were measured by responses of lymphocytes in whole blood to T-cell mitogens and by delayed hypersensitivity reactions to tuberculin. Blastogenic responses to phytohemagglutinin and concanavalin A were reduced in niridazole-treated chickens with responses of controls. Wattle tests measuring delayed hypersensitivity were positive only in control chickens; reactions in niridazole-treated chickens did not differ from nonimmunized controls.

Carcinogenic effects of niridazole in rats.

Niridazole was administered in the diet to rats at levels of 0.1, 0.05 and 0.025%. The drug induced adenomas and adenocarcinomas of the kidneys and forestomach papillomas.

Niridazole as an immunosuppressive agent.

The immunosuppressive properties of niridazole, an antihelminthic drug, have been investigated in rats. When given orally at a dosage of 50 mg/kg it extended the median survival of cardiac allografts from 7 to 20 days. The immunosuppressive effect was not increased by giving either azathioprine or prednisolone concurrently, but when all three drugs were combined the immunosuppression was profound, and only 2 of 8 grafts were rejected. Drug combinations incorporating niridazole at a lower dosage or for a shorter period were less effective, and azathioprine and prednisolone on their own or together prolonged graft survival only marginally in this model.