Evaluation of the Escherichia coli K12 inductest for detection of potential chemical carcinogens.

46 chemicals of diverse classes and structures, including 30 known animal carcinogens, were evaluated for prophage-inducing ability using the Escherichia coli inductest with lysogenic strain GY5027 envA - uvrB- and indicator strain GY4015 ampR . The inductest detected 9 of 30 known carcinogens as genotoxic agents, including 3 polycyclic hydrocarbons, 2 aflatoxins, and 2 antitumor antimicrobials. Among the 21 carcinogens ineffective as prophage inducers were 3 aromatic amines (other than 2-aminoanthracene), 3 azo-aminoazo compounds, 2 methanesulfonates, and 2 nitro aromatics. In contrast, 18 and 17 of the 30 animal carcinogens were detected as genotoxic agents in the Salmonella/Ames test and E. coli WP2/ WP100 rec assay, respectively. The threshold sensitivity of the inductest was less than that of the Salmonella/Ames test for chemicals genotoxic in both tests. The ineffectiveness of the inductest as a routine test for detecting potential chemical carcinogens may be related to the nature of the DNA damage lesions formed by various genotoxic agents.

The Escherichia coli WP2/WP100 rec assay for detection of potential chemical carcinogens.

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA- recA-) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test detected 20 of 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little gentoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA- recA- genotype of strain WP100.

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. II. Phenotypic expression in intraspecific and interspecific somatic cell hybrids.

Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24-hour survival, 72-hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide-sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half-selective agents in cell hybridization.

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. I. Comparative effects of four classes of polyene macrolides on mammalian cells.

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.

Properties of morphologically variant haploid frog cells formed by combined treatment with Mengo virus and the polyene antibiotic mediocidin.

Comparisons have been made of cell surface glycoproteins, concanavalin A agglutinability, and cloning efficiencies in liquid media of ICR 2A (haploid frog cells), ICR 2A M (three cloned populations of haploid frog cells resistant to 5 microgram per ml of the polyene antibiotic mediocidin), and ICR 2A M/MV cells (five cloned populations of morphologically variant haploid frog cells produced by exposure of the parental cells to the combined effects of mediocidin and an RNA mammalian virus, Mengo virus). Independently isolated ICR 2A M/MV clones exhibited altered cell surface glycoproteins, increased concanavalin A agglutinability, and enhanced cloning efficiency in liquid media when compared with ICR 2A parental cells. In contrast, ICR 2A M cells had properties similar to ICR 2A cells, with the exception of the former's increased resistance to mediocidin. The differences in properties between ICR 2A M/MV and ICR 2A cells suggest that alterations resembling transformation have occurred in ICR 2A M/MV cells as a consequence of combined treatment with mediocidin and Mengo virus.

Toxicity of nystatin and its methyl ester toward parental and hybrid mammalian cells.

Nystatin methyl ester (NME), the methyl ester derivative of the polyene macrolide antibiotic nystatin, is known to be effective against fungi and is now found to be relatively less toxic than the parent antibiotic nystatin (NYS) to animal cells in culture as measured by 51Cr release, cell survival at different posttreatment periods and cell growth. NYS and NME were tested on TK- mouse (B82) and hamster (B1) cells, HGPRT- mouse (RAG) cells, and on lysolecithin-fused cells selected in HAT medium and confirmed as B82-RAG and B1-RAG hybrids by chromosomal analysis plus polyacrylamide gel electrophoresis of lactate dehydrogenase. NME was less toxic and caused less immediate membrane damage than NYS when tested in all five cell systems. However, differences in innate polyene sensitivity were evident between the three parental cell types. B82 and B1 cells were more resistant than RAG cells to NYS and NME. B82-RAG hybrids reflected the higher level resistance of B82 parental cells, and B1-RAG hybrids reflected the higher level resistance of B1 cells. Where one parental cell type is relatively more polyene sensitive, the use of polyenes in the future may be applicable as selective agents in cell hybridization.

Suppression of amphotericin B methyl ester induced growth stimulation in intraspecific mouse somatic cell hybrids.

Amphotericin B methyl ester (AME), the chemically modified derivative of amphotericin B, induced a concentration-dependent growth stimulatory effect on B82 mouse cells as indicated by increased 24- and 72-hour viable cell number, growth rate and DNA and RNA synthesis. In contrast, AME was not growth promoting toward RAG mouse cells or B82-RAG somatic cell hybrids, while hybrid cells exhibited the increased AME resistance pattern of B82 parental cells. A dissociation between the phenotypic expression of growth stimulation and polyene sensitivity was demonstrated in intraspecific mouse hybrids.

The effect of fetal bovine serum on polyene macrolide antibiotic cytotoxicity and antifungal activity.

The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers of 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth of Saccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminating Candida albicans, Saccharomyces cerevisiae, Aspergillus niger or Fusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems.

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture.

The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.

Stimulatory effect of amphotericin B methyl ester on the grwoth of L-M and Vero cells.

The chemically modified polyene macrolide antibiotic, amphotericin B methyl ester (AME), exhibited a concentration-dependent growth stimulatory effect on established lines of mouse (L-M) and monkey (Vero) cells. Stimulation was indicated by increases in growth rate, and in the enhanced synthesis of DNA and RNA. In contrast, the parental antibiotic amphotericin B and the desoxycholate complex of amphotericin B, FungizoneR, did not elicit a similar proliferative response in L-M or Vero cells. AME was not growth-promoting toward low passage strains of mouse (PMK 6) and monkey cells (MGK 8).

Toxicity of amphotericin B and its methyl ester toward normal and tumor cell lines.

The toxicity of amphotericin B, amphotericin B methyl ester (AME), and Fungizone toward three "normal" and three tumor-derived human and mouse cell lines was evaluated in monolayer culture. AME was less toxic than amphotericin B and Fungizone to all cell lines, but the sensitivity of the normal and tumor lines was different. The human (HEL-8 and WISH) and mouse (L-M) cells derived from normal tissue were more resistant to AME than the tumor-derived human (KB and HeLa) and mouse (RAG) cell as indicated by: (a) increased 24-hr survival, (b) increased 72-hr viability, and (c) growth rates at higher AME concentrations. In contrast, no pattern of differential sensitivity was observed with amphotericin B and Fungizone.

Isolation, characterization, and genetic analysis of mutator genes in Escherichia coli B and K-12.

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.

Streptomycin-induced phenotypic suppression of hydroxylamine-induced nonsense mutants in the rII A cistron of bacteriophage T4.

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3'-terminal adenine inhibits misreading of a 5'-terminal uracil.

Nonsense Mutants in the rII A Cistron of Bacteriophage T4.

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC --> AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.

Mutator gene of Escherichia coli B.

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.

Mutants of Escherichia coli variably resistant to bacteriophage T1.

Carta, Guy R. (Rutgers, The State University, New Brunswick, N.J.), and Vernon Bryson. Mutants of Escherichia coli variably resistant to bacteriophage T1. J. Bacteriol. 92:1055-1061. 1966.-Mutants resistant to bacteriophage T1 were isolated from ultraviolet (UV)-irradiated cultures of Escherichia coli B/r, a UV-resistant variant. Bacterial populations derived from some of these mutants were partially but not completely resistant to the bacteriophage. Such mutants, designated variably resistant (B/r/1v), could not be obtained from E. coli B. Phage-free mutant populations taken from different stages in growth consisted of significantly different proportions of T1-resistant and T1-sensitive cells. The growth stage-dependent range of variation exceeded 1,000-fold. In broth cultures, the highest proportion of resistant cells consistently appeared at mid-log phase, and the highest proportion of sensitive cells at lag and stationary phases. Comparable evidence for environmentally dependent changes in host-cell phenotype was obtained by efficiency of plating and cloning efficiency analysis tests. Micromanipulation showed that, in clones growing in the presence of phage T1, sensitive bacteria appeared with high frequency and underwent lysis.