Effects of Rufloxacin in Salmonella typhimurium Infection in Mice

Summary

This study was undertaken to investigate the efficacy of rufloxacin, a new quinolone which is interesting due to its pharmacokinetics characterized by a long plasma half-life, in the treatment of systemic salmonella infections in the mouse typhoid model. Innately susceptible BALB/c and resistant CBA mice were used to investigate the efficacy of rufloxacin in controlling systemic salmonella infections when given for brief or prolonged periods. The present study shows that rufloxacin is not only very effective on both mouse strains, but can completely eradicate the salmonellae from livers and spleens when given early in the infection of CBA resistant mice.     

Airborne mutagens bioassayed in Salmonella typhimurium.

Particulate airborne pollutants, collected in Buffalo, New York, and Berkeley, California, were asayed for mutagenic activity in the Ames Salmonella typhimurium test system. Mutagens requiring liver enzymes for activation, as well as direct acting mutagens, were readily detected in the Buffalo sample. By contrast, only direct acting mutagens were detected in the Berkeley sample.     

Mutagenicity of nitrosourea compounds for Salmonella typhimurium.

The nitrosourea derivatives 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC-409962), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; NSC-79037), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Me-CCNU; NSC-95441), and 1-methyl-1-nitrosourea (MNU; NSC-23909) were screened for mutagenic potential with Salmonella typhimurium strains G46 and TA1530 in vitro and in vivo. Alu were also mutagenic in the host-mediated assay. Four additional chemotherapeutic nitrosoureas were tested in vitro, three of which were mutagenic. The lack of activity of the fourth agent was probably a reflection of its stability in solution rather than a true indication of mutagenic potential. All eight agents were tested in a repair test with S. typhimurium strains TA1978 and TA1538. Results of this test were negative, reflecting the insensitivity of these strains to ethylating and methylating agents. The insensitivity of the host-mediated assay is also discussed.     

Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice

Background:

An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue.

Methods:

We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL.

Results:

After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2 Gy γ-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%.

Conclusion:

These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.     

Antimutagenic Effects of Black Tea in the Salmonella typhimurium Reverse Mutation Assay

Black tea (Camellia sinensis) is one of the most widely consumed beverages worldwide. Its chemopreventive ‍effects are well documented in the literature. In the present set of investigations antimutagenic effects of aqueous ‍black tea extract (ATE) and black tea polyphenols (BTP) were evaluated in the Ames test using Salmonella typhimurium ‍tester strains TA 98 and TA 100. Addition of benzo(a)pyrene (BaP) and cyclophosphamide (CP), two well known ‍mutagens, at the concentrations of 20 and 15ìg/plate, respectively, in an S-9 metabolically activated system resulted ‍in significant induction of his+ revertant colonies. However, addition of 500 ìl 1, 2 and 4% ATE to the BaP and CP ‍treated plates resulted in a dose dependent inhibition in the number of his+ revertant colonies. Furthermore in ‍another set of experiments, supplementation with BTP at the concentrations of 100, 200 and 400 ìg/plate also led to ‍a significant inhibition in BaP and CP induced colony formation. The antimutagenic activity of BTP was found to be ‍higher than that of ATE, which may be attributable to the higher amount of polyphenolic ingredients. Hence the ‍study revealed that black tea has a protective efficacy in suppressing BaP and CP induced mutagenicity in a microbial ‍test system.     

Tissue-mediated mutagenicity of vinylidene chloride in Salmonella typhimurium TA1535.

Vinylidene chloride is weakly positive in the Salmonella typhimurium TA1535 test, mediated by kidney and liver post-mitochondrial supernatant (S-9 mix) from normal mice, but strongly positive with the S-9 mix from the induced animals. In the case of mediation by rat tissue, only liver S-9 mix from induced animals affords a significant positive response. These findings agree with the greater availability in treated mice than in rats of reactive vinylidene chloride metabolites, 1,1-dichloroethylene oxide and chloroacetyl chloride [5], and with the vinylidene carcinogeneicity found in mice but not in rats [9]. Exploratory tissue-mediated testing of vinylidene chloride involving liver S-9 mix from marmosets and man suggests a trend in the generation of alkylating metabolites and their reactions with bacterial DNA for these primates which resembles rats more than mice.     

Inhibition of 2-fluorenamine-induced mutagenesis in Salmonella typhimurium by vitamin A.

Vitamin A alcohol (retinol) completely inhibited the mutagenicity of the carcinogen 2-fluorenamine (2-FA) in Salmonella typhimurium TA98 when the mutagen was activated by liver microsomes from CFN rats. The mutagenicity of 2-FA activated by 9,000Xg rat liver supernatant S9 was inhibited by retinol to a lesser degree. The decline in the number of his+ revertants was not an artifact due to bacterial killing, inasmuch as retinol was not toxic to the bacteria at levels that totally inhibited mutagenesis by 2-FA. Mutagenesis induced by adriamycin, an antibiotic that does not require metabolic activation for mutagenic potential, was unaffected by vitamin A. These results indicate that retinoids inhibit the metabolic activation of some chemical carcinogens to forms that can interact with DNA. Our findings are consistent with the hypothesis that retinoids may exert anticancer activity by inhibiting carcinogen activation, thereby inhibiting tumor induction. In addition to the more widely accepted role of retinoids in modulating the proliferation of epithelially derived neoplasms.     

Mutagenicity of five cyclic N-nitrosamines: assay with Salmonella typhimurium.

The mutagenicity of five cyclic N-nitrosamines was studied with the use of Salmonella typhimurium TA1535 in vitro with and without microsomal activation. The carcinogens nitrosopiperidine and nitrosopyrrolidine required metabolic activation before manifesting mutagenic activity. Nitrosoproline and nitrosohydroxyproline, noncarcinogens, were not mutagenic. Nitroso-3-pyrrolidinol was mutagenic in the absence of microsomes, thereby suggesting a role of hydroxylation in the metabolic activation of nitrosopyrrolidine to an ultimate carcinogenic species.     

Non-mutagenicity of 3-dimethylamino-psi-saccharin to Salmonella typhimurium

3-Dimethylamino-psi-saccharin has been confirmed as non-mutagenic to Salmonella typhimurium when evaluated in vitro in the presence of induced rat liver S9 mix.     

Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice

We report here a modified auxotrophic strain of Salmonella typhimurium that can target and cure breast tumors in orthotopic mouse models. We have previously reported development of a genetically modified strain of S. typhimurium, selected for prostate tumor targeting and therapy in vivo. The strain, termed S. typhimurium A1, selectively grew in prostate tumors in xenograft models causing tumor regression. In contrast, normal tissue was cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. A1 is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support only from tumor tissue. The ability to grow in viable tumor tissue may account, in part, for the unique antitumor efficacy of the strain. In the present report, to increase tumor-targeting capability of A1, the strain was reisolated after infection of a human colon tumor growing in nude mice. The tumor-isolated strain, termed A1-R, had increased targeting for tumor cells in vivo as well as in vitro compared with A1. Treatment with A1-R resulted in highly effective tumor targeting, including viable tumor tissue and significant tumor shrinkage in mice with s.c. or orthotopic human breast cancer xerographs. Survival of the treated animals was significantly prolonged. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. These results suggest that amino acid auxotrophic virulent bacteria, which selectively infect and attack viable tumor tissue, are a promising approach to cancer therapy.     

Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice

Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.     

Mutagenicity of some congeners of benzidine in the Salmonella typhimurium assay system.

The analogs of benzidine were assay for mutagenicity using Salmonella typhimurium TA-98 and TA-100 and a mouse liver enzyme preparation. Only 4-aminobiphenyl produced both frameshift and base pair substitution mutations and 3,3'-dichlorobenzidine was the only compound which was mutagenic without the mammalian enzyme factor. When hydrochloride salts of the parent compounds were made to improve their stability for animal feeding experiments, the mutagenicity for the Salmonella tester strains was reduced except for 3,3'-dimethylbenzidine. Animal feeding trials in mice are underway to determine the dose response relationship of tumor incidence and molecular configuration.     

The effects of bay-region methyl substitution on 6-nitrochrysene mutagenicity in Salmonella typhimurium and tumorigenicity in newborn mice

The mutagenic activities in Salmonella typhimurium and tumorigenicactivities in newborn mice of 6-nitrochrvsene (6-NC), 5-methyl-6-nitrochrysene(5-Me-6-NC), 11-methyl-6-nttrochrysene (11-Me-6-NC) and 5-methylchrysene(5-MeC) were compared. In S.typhimurium TA100 in the absenceof rat liver 9000 g supernatant, 11-Me-6-NC was the most activecompound followed by 6-NC; 5-Me-6-NC and 5-MeC were inactive.In the assays conducted in the presence of rat liver 9000 gsupernatant, the order of activity was 11-Me-6-NC < 6-NC< 5-Me-6-NC {small tilde} 5-MeC. In S.typhimurium TA98 asimilar trend was observed. For the tumorigenicity studies,groups of mice were treated with the appropriate compounds inDMSO by i.p. injections on the 1st, 8th and 15th day of life.At a dose of 100 nmol/mouse 6-NC induced significantly morelung tumors than 5-MeC, which in turn was more active than 11-Me-6-NCand 5-Me-6-NC. All compounds induced significant numbers ofliver tumors in treated males compared to controls; the orderof activity was the same as that observed for lung tumor induction.The results of this study clearly indicate that bay region methylsubstitution can either inhibit (5-position) or enhance (11-position)the mutagenic activity of 6-NC. In contrast, bay region methylsubstitution (5- and 11-positions) inhibited the tumorigenicactivity of 6-NC in newborn mice. Since ring oxidation and nitroreductionare involved in the metabolic activation of 6-NC in newbornmice, bay region methyl substitution may either inhibit thenitroreduction pathway or hinder the formation of the appropriatebay region diol epoxide. Sterk factors may be important in determiningthe tumorigenicity of methylated nitrochrysenes.     

Mutagenicity for Salmonella typhimurium of urine obtained from humans receiving nitrofurantoin.

Urine samples from 12 humans receiving oral therapeutic doses of nitrofurantoin were mutagenic for Salmonella typhimurium strain TA 100 and nonmutagenic for strain TA 100-FR1. Mutagenic activity of the urine was not increased by treatment with beta-glucuronidase. Spot mutation assay of the chromatogram of urine revealed that the mutagenic activity of the urine was mainly due to unmetabolized nitrofurantoin.     

Alkyl methane sulfonate mutation of diploid human lymphoblasts and Salmonella typhimurium.

Concentration dependence of mutation in equigenerational exposures to methyl, ethyl, propyl, and butyl methanesulfonates has been determined in diploid human lymphoblasts and Salmonella typhimurium. Forward mutation was measured at the hypoxanthine guanine phosphoribosyltransferase locus in human lymphoblasts and at the putative guanine phosphoribosyltransferase locus in S. typhimurium. Reverse mutation at the his G46 locus was also measured in S. typhimurium. This analysis and previous reports support the conclusion that S. typhimurium and mammalian cells are essentially equisensitive to the mutagenic effects of ethyl methanesulfonate when concentration and exposure time are taken into account. Comparison of forward and reverse mutation assays in S. typhimurium reveals no important differences in sensitivities for the four compounds studied.     

Effects of acetyl salicylate and ibuprofen on fluoroquinolone MICs on Salmonella enterica serovar typhimurium in vitro

In this study, the effects of acetylsalicylate and ibuprofen at 2, 4 and 8 mM concentration were investigated on ofloxacin, ciprofloxacin, levofloxacin and pefloxacin minimum inhibitory concentrations (MICs) for 14 Salmonella enterica serovar typhimurium clinical isolates, one standard strain (SZH KUEN 557), SH7616 (acr mutant), SH5014 (parent strain of acr mutant) and PP120 (soxRS mutant) strains. All isolates were susceptible to the 4 fluoroquinolones. In the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen, 2- to 8-fold increases were observed in fluoroquinolone MICs. This rise was higher, especially in the presence of acetylsalicylate. In spite of this rise, none of the MICs were in the range of resistance limits in vitro. Except for a 2-fold increase in levofloxacin MICs, we did not observe any difference in MICs of ofloxacin, ciprofloxacin, and pefloxacin in the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen for SH7616 and PP120 strains. According to the in vitro results of this study, it can be suggested that use of acetylsalicylate or ibuprofen together with clinical treatment of bacteria, especially bacteria which show intermediate resistance, will cause resistance. However, since clinical data are insufficient, further studies are needed.     

Mutagenicity of 3,4-diphenyl-5-nitrofuran analogs in Salmonella typhimurium

A new series of chemicals comprising eight different 3, 4-di-phenylsubstitutedfuran analogs, namely, methyl-3, 4-di-phenyl-2-furoate, methyl-3,4-diphenyl-5-nitro-2-furoate, 3, 4-diphenyl-5-nitro-2-furoicacid, 3, 4-diphenyl-5-nitro-2-acetylfuran, 3, 4-diphenyl-5-nitro-2-bromoacetylfuran,2-amino-4-(3, 4-diphenyl-5-nitro-2-furyl)thiazole, 2-acetyl-amino-4-(3,4-diphenyl-5-nitro-2-furyl)thiazole and 2-formyl-amino-4-(3,4-diphenyl-5-nitro-2-furyl)thiazole were synthesized and theirmutagenic activities tested in Salmonella typhi-murium. Thestructure—activity relationship studies revealed thatfor mutagenic activity the nitro group is essential and thatthe potency of activity is greatly altered by the nature ofthe substituent at the 2-position of the furan ring. The mutagenicactivities of these chemicals were generally much higher inTA100 compared to TA98. The relative order of activities for2-substituted, 3, 4-diphenyl-5-nitrofurans were COOCH₃ >COCH₂BR > COCH₃ > COOH in S. typhi-murium TA100. 3, 4-Diphenyl-5-nitro-2-bromoacetylfuranwas equally active in nitroreductase-proficient (TA98, TA100)and in nitroreductase-deficient (TA98NR, TA100NR) strains. Incontrast, the acetyl and carboxymethyl ester analogs were relativelyless active in nitroreductase-deficient strains. Mutagenic activitiesof 3, 4-diphenyl-substituted furylthiazoles in comparison withthe unsubstituted analogs of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide,N-[4-(5-nitro-2-furyl)-2-thiazolyl]-acetamide and 2-amino-4-(5-nitro-2-furyl)thiazolerevealed that the phenyl groups drastically reduced their mutagenicactivities. However, the relative order of activities formylamino<< acetylamino > amino were the same between phenyl-substitutedand unsubstituted analogs.