Use of the DNA-repair host-mediated assay for determining the organ distribution of genotoxic factors in mice treated orally with nitro-aromatic compounds.

The distribution of genotoxic factors in various organs of mice treated orally with nitro-aromatic compounds of actual or potential use as chemotherapeutic (antiprotozoal and anthelminthical) agents was investigated in the DNA-repair host-mediated assay, with mice as host animals and a pair of E. coli K12 strains differing in DNA-repair capacity as indicators of genotoxicity. The test substances were derivatives of nitroimidazole (metronidazole), nitrofuran (SQ 18 506) and nitrodiphenylamine (amoscanate). Animal-mediated assays were performed by injecting mixtures of the two E. coli strains both intravenously and orally into mice, which were subsequently treated with the test chemicals, and from which the differential survival of indicator bacteria present in liver, lungs, spleen, kidneys, stomach, small intestine, colon and the blood stream was determined on selective agar medium. The same strains and selection procedures were used for assessing the genotoxic activity of the compounds in vitro. All three compounds displayed genotoxic activity in vitro, the order of potency on the basis of exposure concentration being SQ 18 506 greater than metronidazole greater than amoscanate. In the animal-mediated assays the same ranking order of genotoxic activity was observed, but the exposure levels required to produce significant genotoxic effects in vivo were (substantially) higher than in the in vitro tests: SQ 18 506 was active at 0.1 mg/kg body weight, metronidazole at 4 mg/kg, and amoscanate at dosages higher than 10 mg/kg. In host-mediated assays the highest genotoxic activity for all three chemicals was observed in organs of the gastro-intestinal tract (usually in the stomach). All three chemicals also induced genotoxic effects in organs remote from the gastro-intestinal tract although with substantially lower activity, the order of potency being again SQ 18 506 greater than metronidazole greater than amoscanate. In the case of SQ 18 506 and metronidazole, dose-dependent genotoxic activities were observed in liver, spleen, lungs, kidneys and the blood stream, with no clear indication of a preferential target or non-target organ, while the minor genotoxic effects of amoscanate were restricted to bacteria present in the blood stream. This can be taken as an indication that the substances (or active metabolites thereof) have been transported from the intestinal tract into the blood stream and distributed evenly in organ tissues, without an indication of organ specific deactivation during the time periods (less than 180 min) presently investigated.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescent probes for cellular hypoxia: lack of transfer of fluorescence between cells in vitro.

Fluorescent nitroheterocycles may be useful as probes for cellular hypoxia. Reductive metabolism of AF-2 (cis 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide) and NFVO (trans-5-amino-3((5-nitro-2-furyl)vinyl)1,2,4-oxadiazole) results in intracellular accumulation of fluorescent molecules; the mean cellular fluorescence has previously been shown to be related to the cellular oxygen content during incubation in vitro. However, factors in addition to oxygen and nitroreductive activity may also affect cellular accumulation of these drugs. The stability of cellular fluorescence and possible diffusion of metabolites were examined by flow cytometric analysis of mouse and hamster fibroblasts exposed to NFVO and AF-2. Incubation of cells with 14C-AF-2 allowed calibration of the flow cytometer for AF-2 fluorescence; 5 X 10(8) molecules/cell resulted in double the spontaneous cellular fluorescence. Cellular fluorescence was stable for days after exposure to AF-2, and no evidence of transfer between exposed and unexposed cells was observed. For concentrations resulting in less than 5 X 10(9) AF-2 adducts/cell, all of the metabolites could be accounted for intracellularly. Therefore, it is unlikely that transfer of reduced nitroheterocycles occurs between cells.

Cellular metabolism of fluorescent nitroheterocycles.

Since nitroheterocycles are preferentially metabolized and bound in hypoxic cells, we have examined more than 2 dozen nitroheterocycles as potential fluorescent probes for hypoxia. Using flow cytometry, several patterns of cellular fluorescence (CF) have been observed; for most nitroheterocycles, CF was several fold higher for anoxic than for aerobic cells (which was not predicted based on comparison of the fluorescence spectra of parent drug and reduced products). CF gradually increased when cells were exposed to 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) or to 4-nitroquinoline-1-oxide (4-NQO), and cells remained fluorescent when the drug was washed off. In contrast, cells exposed to trans-5-amino-3-[5-nitro-2-furyl)vinyl-1,2,4-oxadiazole (NFVO) lost fluorescence with a half-time of 60 minutes. Cells exposed to nitrofurazone (NF-7) reached maximum fluorescence within 30 minutes and then lost fluorescence, even in the presence of the drug. Finally, cells exposed to 3-nitropyrene (NP-3) were initially more fluorescent when incubated under aerobic conditions than anoxic conditions; however, after 2 hours in the presence of NP-3, anoxic cells continued to increase in fluorescence while aerobic cells lost fluorescence. Differences in the patterns of cellular accumulation of fluorescent nitroheterocycles were accompanied by differences in the toxicity and metabolism of these drugs. Therefore, chemical studies alone do not allow us to predict the potential of a compound as a hypoxic probe; studies at the cellular level are also essential.

Carcinogenicity of the antischistosomal nitrofuran trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole.

The antischistosomal and antitrypanosomal drug trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole [(SQ18506) CAS: 28754-68-9; (E)-amino-3-(2-(5-nitro-2-furyl)-vinyl)-1,2,4-oxadiazole] was carcinogenic for both male and female CD-1 mice when it was administered either in the diet or by gastric intubation. Dose-dependent increases in tumors of the forestomach and lymphatic tissues were observed in all groups receiving SQ18506 including mice infected with Schistosoma mansoni. The predominant tumor observed was squamous cell carcinoma of the forestomach. The presence or absence of schistosome infection did not appear to alter the incidence or distribution of tumors at comparable doses of SQ18506. The incidence of bladder tumors was positively correlated with the dose in gastric intubation studies and inversely correlated with the dose in dietary studies. The carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (CAS: 24554-26-5) was fed to male and female CD-1 mice in the diet as a positive control. The predominant tumor observed in these groups was transitional cell carcinoma of the bladder. These data indicate that SQ18506 is unsuitable for use in the treatment of parasitic diseases.

Effects of multiple putative anticarcinogens on the carcinogenicity of trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole.

In an attempt to dissociate the chemotherapeutic from the carcinogenic properties of the antischistosomal and antitrypanosomal nitrovinylfuran trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]1,2,4-oxadiazole [(SQ18506) CAS: 28754-68-9; (E)-5-amino-3-(2-(5-nitro-2-furyl)vinyl)-1,2,4-oxadiazole], potential inhibitors of carcinogenesis were administered to female outbred CD-1 mice before and during exposure to SQ18506. The compounds tested were ascorbic acid, etretinate, butylated hydroxyanisole (BHA), cysteamine hydrochloride, cysteine hydrochloride, dimercaprol, disulfiram, 1,4-dithiothreitol, reduced glutathione, and spermidine phosphate. The primary types of tumors observed were squamous cell carcinomas of the stomach and thymic and nonthymic lymphomas. BHA reduced the incidence of malignant tumors to control levels, whereas cysteine hydrochloride, spermidine phosphate, and disulfiram reduced the incidence of chemically induced tumors by 42, 34, and 32%, respectively. Although cysteamine hydrochloride and disulfiram had no or only a modest effect on the overall incidence of tumors, the data suggested possible tissue-specific anticarcinogenic properties for these agents. Of the 8 antioxidants tested, only 1 had marked anticarcinogenic properties against SQ18506. These data indicate that antioxidant properties alone cannot account for the anticarcinogenic activity of the compounds tested. Coadministration of the anticarcinogen BHA with SQ18506 also blocked the chemotherapeutic effects of this agent on female CD-1 mice infected with Schistosoma mansoni.

Fluorescent nitroheterocycles for identifying hypoxic cells.

Binding of several nitroheterocycles by mammalian cells is a function of the ambient oxygen concentration; anoxic single cells bind up to 10 times as much of these drugs as do aerobic cells. We thus hypothesized that fluorescent nitroheterocyles could be used to quantitate the fraction of hypoxic cells in multicell systems, and this was tested in multicell spheroids using a relatively nontoxic nitrofuran, trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole. Binding of trans-5-amino-3-[(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole, as quantified by flow cytometry, was highly responsive to external oxygen concentration. To assess the relevance of the observed fluorescence, fluorescence-activated cell sorting was used to examine the radiosensitivity of cells as a function of their fluorescence intensity. The most fluorescent were indeed the most radioresistant (i.e., contained the least oxygen). Additional results confirm the general feasibility of using fluorescent nitroheterocycles as hypoxic cell probes but also reveal that cellular binding of these agents is not exclusively dependent upon cellular oxygen content.

Genetic analysis of ad-3 mutants induced by AF-2 and two other nitrofurans in Neurospora crassa.

Our previous studies have shown that the nitrofurans AF-2, SQ18506, and FANFT are potent mutagens in Neurospora crassa. The genetic damage produced by these chemicals at the ad-3 region in N crassa has been characterized by a series of genetic tests. The results of these tests indicate that all three agents induce a high frequency of point mutations and probably a low frequency of multilocus deletions. A comparison of the complementation patterns among the AF-2--induced ad-3B mutants and those induced by other chemical agents indicates that the spectra of intragenic alterations induced by AF-2 in N crassa are similar to those induced by monofunctional alkylating agents.

Comparative study of SQ 18 506 with other nitroheterocyclic compounds on experimental Chagas' disease.

The activity of the 5-nitrovinylfuran, SQ 18 506, is compared in vivo and in vitro with lampit (a 5-nitrofuran), Ro 7-1051 (a 2-nitroimidazole) and metronidazole (a 5-nitroimidazole). The order of activities on a weight basis was: SQ 18 506 greater than lampit greater than Ro 7-1051 greater than metronidazole. Concern about possible mutagenicity and carcinogenicity, however, has precluded the use of SQ 18 506 in clinical trials.

In search of anti-Trypanosoma cruzi drugs: new leads from a mouse model.

Nine of 25 carefully selected compounds (from a stock of more than 200 000 chemical species amassed principally as a result of testing against other parasitic diseases) were found to have significant suppressive activity against the parasites in the blood of a Trypanosoma cruzi mouse model. Eight of these compounds evaluated in this model had suppressive activity equal to or greater than the reference compound, nifurtimox. For the first time, suppressive activity against T. cruzi is reported for a 7-aminoquinoline, a phosphonium salt, and TAC pamoate; The biological model is believed to be able to serve as a means of identifying other new "leads* in seeking drugs broadly effective against T=ruzi infections in man.

Autoradiographic and tissue distribution studies on a nitrovinylfuran derivative (SQ 18506 14C) in S. mansoni infected mice.

Autoradiographic and tissue distribution studies of SQ 18506 14C were carried out on 13 Swiss albino mice. Infection was done by I.P. route with 50 to 60 cercaria of S. mansoni (puerto Rican strain) per animal. The safe single i.v. dose of a solution containing one mg of SQ 18506 dissolved in 0.06 ml dimethylsulfoxide was 0.003 ml/g b.w. of mice. Schistosome autoradiograms were clearly demonstrated one day after that dose injected in each mouse 50 days post-infection. However, hepatic autoradiograms were visible 14 days after 2 doses of our drug to each infected mouse. The higher the concentration of SQ 18506 14C in the culture medium the darker were the schistosomes in the autoradiograms and the greater their total d.p.m/mg. Tissue distribution studies after 2 1/2 doses of our drug/mouse revealed that schistosome total d.p.m/mg dry-weight was 50 times more than that of its liver. The latter d.p.m/g wet-weight was slightly higher than that of one ml of mouse blood.

Genetic activity of niridazole in yeast.

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.

New antimicrobial nitrofuran, trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-delta2-1,2,4-oxadiazole: antibacterial, antifungal, and antiprotozoal activities in vitro.

trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-Delta(2) -1,2,4-oxadiazole, a new antimicrobial nitrofuran, has shown microbial activity in vitro against a wide range of bacteria and fungi, and against several protozoa. The antimicrobial activity of the nitrofuran is not significantly diminished in vitro in the presence of 50% human serum. The compound is not cross-resistant with a number of common antibiotics or synthetic antimicrobial agents; some cross-resistance with furazolidone is encountered. The development of resistance to the compound in vitro, when it occurs, is mixed, but is unrelated to increase in pathogenicity of the test organism.

New antimicrobial nitrofuran, trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-delta2-1,2,4-oxadiazole: antimicrobial efficacy in mice, rats, and guinea pigs.

A new antimicrobial nitrofuran designated SQ 18,506 showed some therapeutic activity when administered orally to mice infected with Escherichia coli, Salmonella schottmuelleri, Shigella flexneri, or Klebsiella pneumoniae. Animals infected parenterally with Streptococcus pyogenes, Proteus mirabilis, Mycobacterium tuberculosis, and Candida albicans, or topically with Trichophyton mentagrophytes, did not respond to therapy with the drug at the dosage levels used. The compound was as effective as metronidazole in the topical treatment of experimental trichomonal infections in mice and in guinea pigs and as effective as nystatin, candicidin, or a sulfanilamide-aminacrine hydrochloride cream in the treatment of a candidal vaginal infection in rats. The chemotherapeutic efficacy of SQ 18,506 in experimental vaginitis caused by Escherichia coli in the rat surpassed that shown by four commercial products available for the treatment of bacterial vaginitis.