Hycanthone analogs: dissociation of mutagenic effects from antischistosomal effects.

N-Oxidation at the diethylamino group of hycanthone, of lucanthone, and of two chlorobenzothiopyranoindazoles resulted in a marked reduction in mutagenic activity, while antischistosomal activity was retained or even enhanced. Introduction of chlorine into the 8-position of benzothiopyranoindazoles reduced acute toxicity but had no effect on chemnotherapeutic potency. These dissociations of biological activities indicate that safer antischistosomal compounds of this class can be developed.

Hycanthone: a frameshift mutagen.

Rapid spot-test screening of antischistosomal agents reveals that hycanthone is a potent frameshift mutagen while the closely related compound, miracil D, is nonmutagenic in Salmonella. Both hycanthone and miracil D are frameshift mutagens for T4 bacteriophage during growth in Escherichia coli.

Detection of mutagens-carcinogens: carcinogen-induced lesions pinpointed by alkaline phosphatase activity in fixed gastric specimens from rats.

Alkaline phosphatase (AP) activity was not detectable by histochemical staining in the glandular stomachs of normal rats. However, AP activity was present at high levels in the brush borders of the intestine and persisted after fixation of the tissues in Formalin at room temperature. Foci of AP activity were detected in gross Formalin-fixed specimens of glandular stomachs of male and female inbred F344 rats exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and killed over 6 years ago. The incidence of AP-positive foci increased in proportion to the dose of the carcinogen MNNG. Histologically, most of the grossly visible AP-positive foci corresponded to areas of intestinal metaplasia and adenocarcinoma. AP-positive foci localized sites of pathologic significance for microscopic examination and pinpointed gastric sites containing very early tumors that were missed by standard examination.

Aroclor 1254-induced intestinal metaplasia and adenocarcinoma in the glandular stomach of F344 rats.

Ingestion of diets containing Aroclor 1254, a mixture of polychlorinated biphenyls, for 2 years led to a dose-related increase in the incidence of focal lesions in the glandular stomachs of male and female F344 rats. The incidence of stomach lesions was 6% in control specimens and in specimens from rats fed a diet containing 25, 50, or 100 ppm Aroclor 1254, the incidences of stomach lesions were 10, 17, and 35%, respectively. The majority of gastric lesions in treated rats were histologically identified as intestinal metaplasia characterized by an architecture resembling that of intestinal crypts and particularly by goblet cells, which stained with Alcian blue and periodic acid-Schiff reagent. Adenocarcinomas were found in six specimens. Most (88%) of the lesions were located in the pyloric region of the glandular stomach. No multiple lesions were observed among 47 control specimens examined; however, nine cases of multiple lesions were observed in 30 lesion-containing specimens from Aroclor 1254-treated rats. Although the exact relationship between gastric intestinal metaplasia and adenocarcinoma remains to be established, they commonly coexist and may share initiating mechanisms.

Effect of plasma and carboxylesterase on the stability, mutagenicity, and DNA cross-linking activity of some direct-acting N-nitroso compounds.

The effects of mouse plasma, human plasma, and purified porcine liver carboxylesterase on nitrosourea, nitrosamide, and nitrosocarbamate chemical stability, mutagenicity, and DNA cross-linking activity were compared. These three classes of N-nitroso compounds are chemically similar but displayed different biological activities and were affected differently by plasma and carboxylesterase. Nitrosourea stability as well as mutagenicity and DNA cross-linking activity were affected negligibly by esterase or plasma. In contrast, nitrosamide and nitrosocarbamate stability, mutagenicity, and DNA cross-linking activity were rapidly decreased in the presence of plasma or carboxylesterase. For example, chemical half-lives were from 10- to 20-fold shorter for the nitrosamides and nitrosocarbamates in the presence of 5% mouse plasma. Similar decreases were seen for mutagenicity and DNA cross-linking activity. Preliminary studies indicated one active plasma component to be an enzyme, possibly an esterase. Additional factors such as sulfhydryls may also participate. Whereas some nitrosoureas are active antitumor agents, the lack of antitumor activity for analogous nitrosamides and nitrosocarbamates may reside predominantly in their rapid in vivo inactivation. These results may help to account for the high in vitro mutagenicity as compared with the low in vivo activities of nitrosamides and nitrosocarbamates.

Comparison of mutagenicity, antitumor activity, and chemical properties of selected nitrosoureas and nitrosoamides.

A number of nitrosoureas and nitrosoamides have been compared with respect to mutagenicity for Salmonella typhimurium, in vitro cytotoxicity, in vivo toxicity and antitumor activity against murine L1210 leukemia, and chemical properties. Despite chemical similarities between the nitrosoureas and nitrosoamides, they show important differences in biological activity. Some of the nitrosoureas are very active antitumor agents, and they are less mutagenic than are the corresponding nitrosoamides, which lack antitumor activity.

Some improved methods in P22 transduction.

Recent refinements simplify methods for P22 transduction in Salmonella and allow improved recovery of phage-free transductional clones. The methods include use of: integration- and lysis-defective phage mutants, heat-killed bacteria to eliminate free phage, direct plating of phage and bacteria, replica-plating for detection of phage content of individual clones, improved broth for phage growth, and procurement of high titer phage from P22 lysogens.

A fine structure map of the salmonella histidine operator-promoter.

Over 100 regulatory mutations linked to the histidine (his) operon of S. typhimurium have been isolated. They all map in a region estimated to be several hundred base pairs in length located at one end of the his operon ("the hisO region"). The mutations are located at sixteen recombinationally separable sites or are deletions encompassing several sites. Data obtained from pairs of reciprocal three-point tests show that "constitutive" (high enzyme levels) and "promoter-like" (low enzyme levels) hisO mutations are interspersed on the genetic map. In a few crosses, recombination was not observed to occur between markers shown to occupy different sites based on behavior in other recombination tests.

Absence of deoxyuridine and 5-hydroxymethyldeoxyuridine in the DNA from three tissues of mice of various ages.

Mutational damage to DNA may modulate the aging process as well as contribute to the high incidence of cancer in older animals. Uracil (Ura) is the deamination product of cytosine and hydroxymethyluracil (HMU) is an oxidation product of thymine. Ura, when generated from cytosine, induces mutations by mispairing with adenine. Both HMU and Ura are known to be excised from DNA by glycosylases that cleave the respective N-glycosidic bonds. This hydrolysis leaves apyrimidinic sites which are subsequently repaired by excision repair. In this report a sensitive method to detect these altered bases of HPLC separation of the components of DNA hydrolysates is described. Neither deoxyuridine (dU) nor 5-hydroxymethyldeoxyuridine (dHMU) were found in hydrolysates of DNA samples from brain, liver or small intestinal mucosa of mice of different ages.

Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22.

Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

Early years of the Salmonella mutagen tester strains: lessons from hycanthone.

The 1960s witnessed detailed studies on the genetic properties of a large number of histidine-requiring mutants of Salmonella typhimurium. The early 1970s saw development of selected strains, the Ames strains, for use in rapid, cheap, sensitive, and manipulable tests of chemicals and chemical mixtures for genotoxic activities. Our contribution during this latter period was an investigation into the mutagenicity of hycanthone and some of its analogues. Some lessons that this study provided are enumerated. Hycanthone is definitely a liver carcinogen in rodents predisposed by hepatic hyperplasia. Between 1969 and 1975, an estimated total of 100 kg of hycanthone was injected into some 1,000,000 humans with liver hyperplasia caused by infections with parasites. It may now be possible to assess directly the long-term impacts of hycanthone in man.

Breakthrough of ultraviolet light from various brands of fluorescent lamps: lethal effects on DNA repair-defective bacteria.

In a comparative study of 17 pairs of 15 W fluorescent lamps intended for use in homes and purchased in local stores, we detect over 10-fold differences in UVB + UVC emissions between various lamps. This breakthrough of ultraviolet (UV) light is in part correlated with ability of lamps to kill DNA repair-defective recA-uvrB- Salmonella. Relative proficiency of lamps in eliciting photoreactivation of UV-induced DNA lesions also plays a prominent role in the relative rates of bacterial inactivation by emissions from different lamps. Lamps made in Chile, such as Philips brand lamps and one type of General Electric lamp, produce far less UVB + UVC and fail to kill recA-uvrB- bacteria. In contrast, all tested lamps manufactured in the USA, Hungary, and Japan exhibit readily observed deleterious biological effects. When an E. coli recA-uvrB-phr- (photolyase-negative) triple mutant is used for assay, lethal radiations are detected from all lamps, and single-hit exponential inactivation rates rather closely correlate to amount of directly measured UVB + UVC output of each pair of lamps. Although all lamps tested may meet international and United States standards for radiation safety, optimal practices in lamp manufacture are clearly capable of decreasing human exposure to indoor UV light.

Cd2+ tolerance in Escherichia coli and Salmonella typhimurium.

Relative tolerance of Escherichia coli strains to Cd2+ is induced by prior growth in medium containing low levels of either Cd2+ or Hg2+, and maximal induction appears to be dependent upon recA+ function. Biosynthesis of glutathione is not required for induction or for expression of induced resistance. Salmonella typhimurium strains are relatively resistant to Cd2+, and this resistance is essentially constitutive.

Superoxide generation by photomediated redox cycling of anthraquinones.

Anthraquinones (AQs) comprise one important class of secondary metabolites predominantly produced by fungi and higher plants but also produced by a variety of other organisms. Humans orally ingest AQs from environmental sources as well as through direct use as nonprescription laxatives, and some AQ derivatives are used as topically applied antipsoritic agents. Some AQs are mutagenic. We present evidence that aqueous solutions of several AQs in the presence of an appropriate reducing agent and dissolved oxygen generate superoxide when they are illuminated with broad-spectrum light. Redox cycling of AQs could be responsible for some aspects of their toxicity in biological systems.

Antimutagens and anticarcinogens: a survey of putative interceptor molecules.

In this review recent publications are cited for a number of antimutagens. The molecules surveyed are potential or proven "desmutagens" or "interceptors." These are biologically prevalent or synthetic molecules that are most often small metabolites proficient in binding to, or reacting with, mutagenic chemicals and free radicals. Many of this class of "blocking agents" are "soft" and "hard" nucleophiles with consequently varying abilities to react with particular classes of electrophiles, the major classes of direct-acting mutagens. Although they serve as a first line of defense against mutagens and carcinogens, many interceptor molecules are under-investigated with regard to their spectra of activity and their possible relevance to prophylaxis or treatment of human disease states.

Interception of some direct-acting mutagens by ergothioneine.

Ergothioneine, a novel imidazole sulfhydryl/thione compound formed in millimolar amounts by fungi, is a potentially important defense against electrophiles and free radicals. Protection may well occur both in organisms that synthesize ergothioneine and in animals including man that ingest and store ergothioneine in red blood cells, the liver, seminal fluid, and central nervous system. Ergothioneine blocks the mutagenicity for Salmonella strain TA1950 (hisG46 uvrB) of the nitrosation products of spermidine to an extent that is approximately proportional to the ergothioneine concentration. Ergothioneine also alleviates mutagenicity of cumene and t-butyl hydroperoxides but does not react with N-methyl-N'nitro-N-nitrosoguanidine as does the cysteinyl sulfhydryl compound, glutathione.

Molecular models that may account for nitrous acid mutagenesis in organisms containing double-stranded DNA.

Nitrous acid (NA) is often presumed to cause base substitutions in organisms with double-stranded DNA as a direct consequence of oxidative deamination of adenine and of cytosine residues. Here we summarize evidence indicating that other mechanisms are involved in the case of NA-induced G/C-->A/T transition mutations. We present several models for pathways of NA mutagenesis that may account for our experimental results and overlapping data noted in the literature. One model proposes that the base substitution mutations observed are due to DNA alkylation damage mediated via nitrosation of polyamines and/or other ubiquitous cellular molecules. Other models assume that predisposing lesions, such as G-to-G cross-links, are first formed. The cross-links are pictured as leading to perturbations in DNA structure that allow subsequent opportunity for NA-induced deaminations of cytosine residues in their immediate vicinity. The deaminations preferentially result in G/C-->A/T transition mutations at sites highly dependent on adjoining base sequence context (i.e., in NA "mutational hotspots"). A final model proposes that NA-induced G/C-->A/T transition mutations arise mainly from oxidative deamination of guanosine residues and not from deamination of cytosine residues in duplex DNA.

Scavenging of singlet molecular oxygen by imidazole compounds: high and sustained activities of carboxy terminal histidine dipeptides and exceptional activity of imidazole-4-acetic acid.

Singlet molecular oxygen was generated by illumination of phenosafranin in phosphate buffer at pH 7.5. Relative efficiencies of various imidazole compounds to form endoperoxides were assayed by following at 25 degrees C the rate of light- and imidazole-dependent bleaching of N,N-dimethyl-4-nitrosoaniline. Of over 30 imidazole compounds tested, imidazole-4-acetic acid, a major catabolite of histamine in mammals, exhibited the highest activity. L-Carnosine (beta-alanyl-L-histidine), a natural dipeptide prevalent in striated muscle of mammals, possessed several properties important for a physiologically significant scavenger of singlet oxygen. On a molar basis, this readily water-soluble C-terminal histidine dipeptide reacted with singlet oxygen two- to four-fold faster than free L-histidine and approximately two-fold faster than the N-terminal L-histidine dipeptides tested. Furthermore scavenging ability of L-carnosine did not appreciably increase or decrease with time of reaction, in contrast to behaviors exhibited by a number of other imidazole compounds that included some other C-terminal L-histidine dipeptides. The fungal metabolite, ergothioneine, blocked singlet oxygen generation by illuminated phenosafranin.