G.R. Mohn

The role of glutathione conjugation in the mutagenicity of 1,2-dibromoethane

Abstract Two mechanisms for the toxic actions of 1,2-dibromoethane have been postulated, both of which involve biotransformation. The first is oxidation to 2-bromoacetaldehyde, a highly reactive substance, the second a possible direct conjugation to glutathione, giving rise to a reactive half-mustard. It was the purpose of this investigation to determine to what extent these two reactive species are responsible for the mutagenicity of 1,2-dibromoethane. To assess quantitatively the importance of the conjugation to glutathione in vivo, rats were administered single doses of 1,2-dibromoethane; 30–55 per cent of the dose was excreted as mercapturic acid. The conjugation of 1,2-dibromoethane to glutathione was also studied in vitro. Specific activities of the metabolizing systems used in the mutagenicity experiments were determined. The mutagenicity of 1,2-dibromoethane towards Salmonella typhimurium TA100 was considerably enhanced by the addition of 100,000 g supernatant fraction, whereas the addition of microsomes had no effect, indicating that the primary glutathione adduct is responsible for the mutagenic effect. As a model for the mutagenic intermediate, S-2-bromoethyl-N-acetyl-cysteine methyl ester was synthesized. This proved to be a very reactive and highly mutagenic compound, which can be further metabolized and thereby detoxified by glutathione conjugation. A similar phenomenon is likely to occur in the mutagenicity test with 1,2-dibromoethane, where after an initial rise in the number of mutants with increasing amounts of glutathione, the number of mutations decreases again. These results clearly indicate that glutathione conjugation plays an important role in the mutagenicity of 1,2-dibromoethane.

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by single oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, black mutation from arg-56 to arg+ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (1O2) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D2O and H2O media (pH 7.0) and quenching of 1O2 with 1,4-diazabicyclo[2.2.2]octane (DABCO). When photodynamy was performed with the indicator cells suspended in D2O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H2O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D2O itself was not mutagenic under th present experimental conditions. Addition of DABCO in concentrations of 0.1--10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions. From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that 1O2 generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.

5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides.

Abstract The induction of 5-methyltryptophan (5-MT) resistance mutations was assayed as a test system for mutagenic chemicals in Escherichia coli. It is assumed that different premutational alterations in several genes of the Escherichia coli chromosome will lead to 5-MT-resistant mutants. The chemicals used were three monofunctional alkylating agents as reference compounds, namely β-propiolactone (β-PL), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methyl methanesulfonate (MMS), which are all mutagenic in the 5-MT system; of the eight organophosphorus insecticides tested, four have definite mutagenic activity (Dichlorvos, Oxydemetonmethyl, Dimethoate, and Bidrin), one is probably mutagenic (Methylparathion) and the remaining three (Parathion, Malathion and Diazinon) do not induce 5-MT resistance mutations in the conditions used here ( MMS > Dichlorvos > Oxydemetonmethyl, Dimethoate and Bidrin. The concentration-dependent mutagenic activity of all mutagenic compounds is nearly linear when plotted on a log-log scale (with slopes varying from 1.0 to 1.5) and could be taken as an indication that one premutational reaction will be sufficient for the induction of one 5-MT-resistant mutant.

Methodologies for the determination of various genetic effects in permeable strains of E. coli K-12 differing in DNA repair capacity Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal-mediated assays

Derivatives of E. coli K-12 strain 343/113 differing in DNA repair capacity, in permeability to large molecules, and in some metabolizing activities (nitroreductase, glutathione), were constructed for the quantitative determination of the induction of various genetic effects, such as forward and back mutations, lysogenic induction of prophage lambda, and repairable DNA damage. These E. coli strains can be used in assay procedures which allow variation and control over several experimental conditions, such as oxygen tension, time, pH, temperature of incubation and growth phase of the indicator cells. Methods are described for the simultaneous determination of genetic effects and of DNA-adduct formation during mutagen treatment, i.e. by using radio-labeled compounds or by means of an enzyme-linked immunosorbent assay (ELISA). Mammalian biotransformation of xenobiotics can be investigated by including various fractions of mammalian organs in the system. Examples of the relative effectiveness of the activating potential of S9, S100 and isolated hepatocytes for dialkylnitrosamines and other carcinogens are presented. Host-mediated assays, finally, are described which, in addition to gene mutations, can also be used for the determination of repairable DNA damage in bacteria present in different organs, including the liver, spleen, lungs, kidneys, pancreas, and the blood stream of chemically treated mice. It is concluded that quantitative tests in vitro for assessment of induced mutagenic spectrum and genotoxic potency, combined with the host-mediated assay as a monitor, in vivo, of genotoxic factors present in various organs of animals, may become useful in the assessment of genotoxic (and possibly tumor-initiating) properties of chemicals for which long-term in-vivo mutagenicity and/or carcinogenicity data are not yet available.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate. Quantitative comparison of mutation induction in escherichia coli, v79 chinese hamster cells and l5178y mouse lymphoma cells, and some cytological results in vitro and in vivo.

Molecular dosimetry studies were carried out to measure the extent of binding of radio-labeled ethyl groups to the DNA of Escherichia coli, V79 Chinese hamster cells and L5178Y mouse lymphoma cells treated with ethyl methanesulfonate (EMS). The results show that (1) the amount of ethylation of the DNA is similar in these cells when treatment conditions are identical, (2) the relationship between dose to DNA (ethylations per nucleotide) versus exposure (mM applied concentration) is non-linear in the sense that less alkylation of the DNA is observed at the higher exposures than would be predicted on the basis of proportionality between dose to DNA and exposure, and (3) the non-linearity of the genetic response in the bacterial cells is not reflected in a non-linearity of the alkylation of the DNA in those cells. Quantitative comparison of the frequencies of gene mutations in the various systems shows that the mutation frequency per unit of DNA alkylation is heterogeneous among the mammalian cell systems and that the frequencies observed in the bacterial cells fall within the range observed with mammalian cells. Alkylation of the DNA in the bone marrow, testis and liver of Swiss random-bred mice was also measured. The results support the conclusion that the distribution of the compound to the various tissues is rapid and probably uniform. Quantitative assessment of the cytological data (micronuclei, sister-chromatid exchanges, etc.) on the basis of dose was not as useful because of the low efficiency of EMS for inducing cytologically observable damage.

Mutagenic activation of dibromomethane and diiodomethane by mammalian microsomes and glutathione S-transferases

The influence of mammalian metabolizing enzymes on the mutagenic activity of dibromomethane and diiodomethane was investigated by using Salmonella typhimurium strain TA100 as indicator. The 2 compounds are known to be metabolized via an oxidative pathway catalysed by microsomal enzymes as well as through direct enzymatic conjugation with glutathione; both pathways possibly give rise to reactive electrophilic intermediates. In mutagenicity plate assays with pre-incubation, dibromo- and diiodo-methane were directly mutagenic towards strain TA100; their mutagenic activity was enhanced upon incubation either with rat-liver microsomes or with the cytosol fraction of the same organ, containing the glutathione S-transferases. These data can be taken as an indication that both microsomal oxidation and conjugation to glutathione are indeed responsible for the mammalian mutagenic activation of dihalomethanes.

Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens.

Subcellular fractions from Drosophila melanogaster, known to have several xenobiotic-metabolizing enzymatic activities, were investigated with respect to their ability to biotransform compounds that require metabolic activation before exerting mutagenic effects. Nitrofurazone, dimethylnitrosamine, cyclophosphamide and 2-acetylaminofluorene were activated to mutagens upon incubation with Drosophila microsomes or 20000 x g supernatant: mutagenicity was observed in Chinese hamster ovary cells, Escherichia coli strains 343/113/R-9 and 343/113/uvrB, and Salmonella typhimurium TA1538. Under the conditions used, microsomal preparations of Drosophila were not able to activate benzo[a]pyrene to a mutagen for Salmonella typhimurium TA98. The spectrum of mutagenic effects observed shows some correlation with the known mutagenicity of these compounds in vivo in Drosophila melanogaster. Drosophila microsomes appeared to be at least as active as rat-liver microsomes when compared in this type of mutagenicity testing.

The DNA repair host-mediated assay as a rapid and sensitive in vivo procedure for the determination of genotoxic factors present in various organs of mice

The DNA repair host-mediated assay, in which repairable DNA damage is determined in E. coli cells present in various organs of mice exposed to genotoxic agents, was further developed to broaden the range of organs under study and to simplify the procedure of assessing differential bacterial cell survival. A pair of derivatives of E. coli K-12 strain 343/113 was constructed which differed vastly in DNA repair capacity (uvr+/rec+ vs uvrB/recA), as a means of assessing DNA damaging effects; furthermore, the strains differed in their ability to ferment lactose (Δ Lac vs Lac+), so that the individual survival of both strains could be determined on a single agar medium (containing neutral red as pH indicator), on which the strains had different colony colour morphology (red, Lac+ vs white, Lac− colonies). Finally, the strains were made streptomycin-dependent, to prevent uncontrolled growth of the bacterial cells within the various organs and also to inhibit contamination of the survival agar medium by representatives of the normal intestinal microflora.The experimental procedure consisted of injecting mixtures of stationary cells of the two strains (ca. 3–5×108 viable cells per mouse) both intravenously and orally into mice, either pretreated or subsequently treated with test chemicals. Ninety minutes after injection of the bacteria, the liver, spleen, lungs, kidneys, stomach, intestine, colon, and ca. 50 μl blood, were removed, suspended in buffer, homogenized, and the survival of the two strains determined on neutral red agar supplemented with streptomycin.In preliminary experiments in which the mice were treated with intraperitoneal injections of mitomycin C (0–2.0 mg per kg body weight), a dose-dependent increase in DNA damaging activity was induced in bacterial cells present in all organs tested, the lowest effects being observed in kidneys and lungs, and the highest in liver and blood. These results need further confirmation in more extensive tests, but they do nevertheless clearly indicate the possible usefulness of the DNA repair host-mediated assay as a rapid biological dose monitor for obtaining information on the genotoxic activity in vivo of compounds for which long-term mutagenicity and carcinogenicity data are not yet available.

Influence of DNA adenine methylation dam mutation and of plasmid pKM101 on the spontaneous and induced mutability of certain genes in Escherichia coli K12

Abstract The spontaneous and chemically induced mutability of several markers of E. coli K12/343/113 was compared in dam − derivative which are defective in DNA adenine methylation instructed error avoidance (MIEA) and/or strains carrying the error-prone mutator plasmid pKM101. The results show that the plasmid pKM101 and the dam − mutation affect spontaneous mutagenesis differently: the dam − mutation enhances the mutation frequencies of all genetic markers tested, namely, galR , MTR, arg 56 and nad 113 , while pKM101 slightly enhances the mutability of only certain genes ( arg 56 ). In the case of chemically induced mutagenesis the intercalating agent 9-aminoacridine and the phenylating agent methylphenylnitrosamine show greatly enhanced mutagenesis in a dam − background while the alkylating agent methyl methanesulfonate and the cross-linking agent mitomycin C show increased mutagenic efficiency in the pKM101-carrying strain. The strong mutagenecity of methylnitronitrosoguanidine, and that of methyl methanesulfonate, is abolished in strain with dam − background. In the case of ethylmethanesulfonate, mutagenesis is enhanced in both the dam − strain and the pKM101 host. The results presented here demonstrate differences in the mode of action of dam − -enhanced and pKM101-enhanced mutagenesis. Our results, furthermore, confirm the relationship between the lack of correction of mismatched bases in the dam − strains and induction of certain frameshift-type mutations; they also indicate the usefulness of dam − tester strains for the efficient detection of certain types of mutagens, such as some intercalating and phenylating agents.

Actual status of mutagenicity testing with the host-mediated assay

The introduction of foreign cells (e.g. ascites tumor cells) into laboratory mammals and their subsequent recovery after treatment of the host with exogenous chemicals to determine the induction of genetic effects (e.g. chromosomal aberrations) is a technique which has been employed for more than 20 years. The use of bacteria as indicators of induced point mutations was first described by Legator et al. (1969). In their technique which they called a host-mediated assay, the microbes (e.g. Salmonella typhimurium) were injected into the peritoneal cavity of mice and, thus, exposed to potentially mutagenic metabolites of the compound under test. Today a wide variety of genetical changes can be detected in several indicator organisms. In addition to the histidine-requiring strains of Salmonella which allow the detection of different types of back-mutations, some other enterobacteria have proven useful. Auxotrophic strains of Serratia marcescens to detect back mutations, and Escherichia coli bacteria in which both forward and back mutations can be assayed simultaneously in several different genes. With fungi or fungal spores as indicators further effects of genetical importance can be determined, e.g. the consequences of recombination processes such as mitotic gene conversion and mitotic recombination in the yeasts, and of deletions in Neurospora crassa (conidia). After the successful development of methods to measure the induction of point mutations in cultured mammalian cells, it is also possible now to use established animal cell lines (e.g. mouse lymphoma cells) in a host-mediated assay provided isogenic or compatible hosts are available.During the past several years the route of administration of the indicator cells and the distribution of these cells within the animal body have been studied. Efforts have been made to bring the indicators into close contact with the reproductive organs or within the liver because this is the organ where most foreign compound metabolism is known to occur. Depending on the inoculation technique (in situ, intraperitoneal, intravenous) it is now possible to recover indicators out of testes, liver spleen, lungs and peritoneal cavity of treated animals in quantities large enough to perform genetic tests. A further improvement is the introduction of indicators within the intestinal tract of rodents; the first experiments along this line using Salmonella seem promising and have opened the way toward using common representatives of the intestinal flora such as Escherichia coli. In general it appears that the host-mediated assay technique is a useful tool to use in assessing the degree of mutagenicity and the organospecificity of foreign chemicals in living mammals and that it should be used until techniques that allow the detection of genetic events in the cells of the different organs have been developed. In the near future, host-mediated assays will have to be used to assess more quantitatively the mutagenicity of the numerous chemicals that have been found genetically active by using direct microsomal assays.Further improvements of host-mediated assays are necessary. For instance, there is as yet no practical method for retention of microbial indicators long enough within the blood stream of living animals to perform genetic tests. This would be desirable for pharmaceutical reasons, e.g. for determination of the kinetics of appearance and removal of mutagenic factors within the blood stream. Preliminary experiments indicate that bacteriophages might be useful indicators, since they are present in blood in quantities sufficient for genetic analysis more than 24 h after intravenous injection. In contrast to all other indicators used so far, bacteriophages do not show any metabolism outside their bacterial hosts that might interfere with the mammalian metabolism. Studies are presently under way to determine what types of mutations or of genetic alterations are best detected after extracellular phage treatment.ZusammenfassungDas Einbringen von Fremdzellen (z. B. Ascites Tumorzellen) in Laborsäuger, um sie nach Behandlung der Wirtstiere und anschließender Wiedergewinnung auf genetische Effekte (z. B. Chromosomenaberrationen) zu untersuchen, ist eine Technik, die seit mehr als 20 Jahren durchgeführt wird. Die Verwendung von Bakterien als Indikatoren für den Nachweis von Punktmutationen wurde von Legator et al. (1969) erstmals beschrieben. Diese intraanimale Mutagenitätsprüfung („host-mediated assay“), bei der die Keime (Salmonella typhimurium) in die Leibeshöhle von Mäusen injiziert wurden, ist inzwischen in mancher Hinsicht weiterentwickelt worden.Heute kann eine Vielzahl von genetischen Veränderungen mit verschiedenen Indikatororganismen untersucht werden. Zusätzlich zu den Rückmutationen erfassenden, Histidin-auxotrophen Salmonellen haben sich weitere Enterobakterien als brauchbar erwiesen. Neben Stämmen von Serratia marcescens werden Escherichia coli-Bakterien verwendet, in denen das Spektrum der erfaßten Mutationstypen auf Vorwärtsmutationen in mehreren Genen erweitert wurde. Mit Pilzen als Indikatoren können ferner zusätzliche Effekte von genetischer Bedeutung untersucht werden: Rekombinative Prozesse wie mitotische Genkonversion und mitotische Rekombination bei der Hefe Saccharomyces cerevisiae sowie Deletionen und rezessive Letalmutationen bei Neurospora crassa (Konidien). Seit der Entwicklung von Systemen, die es erlauben, auch Punktmutationen in Säugerzellkulturen zu erfassen, ist es möglich, etablierte tierische Zellen (z. B. Lymphoma-Zellen der Maus) in isogenen oder kompatiblen Tieren zu verwenden.In den letzten Jahren wurde die Art der Verabreichung der Indikatorkeime und deren Verteilung innerhalb des Wirtstieres vielfältig variiert. Die Bemühungen gingen dahin, die Indikatorkeime näher an die Reproduktionsorgane zu bringen oder in unmittelbarer Nähe des Organs, in dem die meisten metabolischen Umwandlungen von Fremdstoffen geschehen, nämlich der Leber. Je nach Inokulationsweise (in situ, intraperitoneal, intravenös) ist es inzwischen möglich, genügend Keime für Mutagenitätsprüfungen aus Hoden, Leber, Milz, Lunge und Peritoneum behandelter Säuger zu erhalten. Um die organotrope Wirkung von mutagenen Substanzen umfassender zu untersuchen, wäre es weiterhin wichtig, das Vorhandensein mutagener Faktoren im Darmtrakt zu ermitteln. Erste Experimente deuten an, daß Salmonellen als brauchbare Indikatoren in Frage kommen und ermöglichen die Verwendung von Vertreter der üblichen Darmflora, z. B. Escherichia. Insgesamt kann festgestellt werden, daß sich die Methodik des host-mediated assay bewährt hat, um das Ausmaß der Mutagenität und der organspezifischen Wirkung von Fremdstoffen zu bestimmen und weiter Verwendung finden wird, bis Methoden zum direkten Nachweis von genetischen Veränderungen in Zellen der betreffenden Organe ausgearbeitet sind. Zunächst ist noch im host-mediated assay die Mehrzahl der Substanzen zu prüfen, die sich als aktiv in direkten mikrosomalen Tests erwiesen haben.Weiterentwicklungen des host-mediated assay sind nötig: Bisher gibt es keine brauchbare Methode, um die Indikatorkeime bei solchen Mutagenitätsprüfungen lange genug in der Blutbahn der Wirtstiere zu halten. Dies wäre aus pharmakologischer Sicht wünschenswert, um u. a. den zeitlichen Verlauf des Auftretens und des Ausscheidens mutagener Produkte im Blut zu verfolgen. Erste Experimente mit Bakteriophagen deuten an, daß diese sich als Indikatoren eignen, da sie nach mehr als 24 h noch in genügender Anzahl in der Blutbahn vorhanden sind. Im Gegensatz zu den anderen Indikatoren haben Bakteriophagen keinen Eigenstoffwechsel, der mit dem des Wirtstieres interferieren könnte. Zur Zeit wird untersucht, welche Systeme zur Bestimmung von induzierten Mutationen oder anderen genetischen Effekten in extrazellulären Phagen hierbei geeignet sind.