Werner Buselmaier

Standard protocol for the dominant lethal test on male mice set up by the work group “dominant lethal mutations of the ad hoc Committee Chemogenetics”

AbstractThe members of the work group “Dominant Lethal Mutations of the ad hoc Committee Chemogenetics” jointly carried out experimental studies in the period from November 1972 until February 1976. On the basis of the results obtained and the experience gained, they worked out on February 27, 1976, a standard protocol for the dominant lethal test (DLT) on male mice. The recommendations are:1.The mating period should preferentially be as short as possible to obtain information about germ cell-stage specific actions of chemical mutagens. Its selection should be based on the objectives of individual investigators. For screening purposes, and with respect to a high fertilization rate, a 4-day mating period is recommended.2.The total test period should cover at least 12 mating periods, i.e. the whole spermatogenic cycle. A limitation of the DLT to certain “critical” mating periods of high sensitivity is only permissible in repeat tests, or if the parts of gametogenesis concerned, e.g. spermatogoniogenesis or spermatocytogenesis, are studied by cytogenetical tests.3.The mode of mating should preferentially be 1∶1.4.The number of test animals to be used depends on various biological and statistical factors; no generally valid statement can be made concerning the number of test animals. By rule of thumb in the order of 50 fertilized females per group per mating interval is recommended.5.The individual dosing of the substance in terms of mg/kg body weight is to be preferred. Otherwise, the weights of the animals should not deviate more than 5% from the mean value.6.Results obtained from ill animals or those that died in the course of the trial should not be included in the evaluation.7.The sensitivity of the mouse strain used against a standard dose of a known mutagen should be regularly checked; this evidence should be presented in publications.8.For the following test conditions each investigator should make an optimal choice: animal strain, animal age and housing conditions. The investigator has also to decide whether a preliminary mating, in order to check the fertility of the animals to be used, and vaginal-plug evidence are useful in a given case.9.The autopsy of the dams is best carried out a fortnight from the middle of the mating interval.10.The results should be documented as completely as possible in the investigational report. The following data were also considered as essential, serving a direct comparison of the test groups: number of mated females (absolute), number of females with implantation (absolute and in %), number of implantations and of live and dead implants (absolute and per female). If the corpora lutea graviditates were counted, their number as well as the pre-implantation loss (absolute and per female) are to be stated. These data are also desirable for publications.11.For the biological evaluation the following formula can be used for the calculation of dominant lethal factors: FL% = [1-live implants per female of the test group/live implants per female of the control group]×10012.Statistical evaluation is an essential part of the DLT and various methods are known. For the statistical evaluation it is decisive which biological model and which statistical criteria are most important for the investigator. The DLT must be carried out according to the requirement of the model chosen.

Comparative investigations on the chemical induction of point mutations and dominant lethal mutations in mice.

SummaryA selection of mono- and polyfunctional alkylating agents as well as a folic acid antagonist and an acridine derivate were tested with the host-mediated assay, and as far as not known from the literature, with the dominant lethal test for mutagenic activity in mice. In the host-mediated assay system the indicator organisms Salmonella typhimurium G46 His−, Serratia marcescens a 13 His−and a 21 Leu− were used as back mutation systems and E. coli 343 as a forward mutation system. We found indications that polyfunctional alkylating agents induce dominant lethal mutations to a larger extent, whereas monofunctional alkylating agents revealed more mutagenic activity on the molecular level. No definite mutagenicity could be observed for amethopterine, which is mutagenic in cytogenetic investigations. Trypaflavin which is known to be mutagenic in the dominant lethal test, did not induce point mutations in our indicator strains. We conclude that the spectra of mutations, which can be recognized by these two methods, overlap only partially.

The influence of metabolism on mutagenic activity in the host-mediated assay

The problem to be examined is the extent to which induced changes in metabolism influence the mutagenic activity of several N-nitroso-compounds in the host-mediated assay. After pretreatment with carbon tetrachloride it is impossible to induce mutations with dimethylnitrosamine in the host-mediated assay system. Pretreatment with phenobarbital does not influence the mutagenic activity of this compound. Methyl-nitroso-nitroguanidine has mutagenic effects only when applied parenterally, but not when given orally. Methyl-nitrosourea induces mutations when injected subcutaneously and when given orally. These results may be explained by differences of absorption.ZusammenfassungEs wird untersucht, inwieweit induzierte Veränderungen des Metabolismus die mutagene Wirksamkeit einiger N-Nitrosoverbindungen im host-mediated assay beeinflussen können. Eine Vorbehandlung der Maus mit Tetrachlorkohlenstoff macht es unmöglich, mit Dimethylnitrosamin im host-mediated assay Mutationen auszulösen. Die Vorbehandlung mit Phenobarbital hat keinen Einfluß auf die mutagene Wirkung dieser Verbindung. Methyl-nitroso-nitroguanidin wirkt nur nach parenteraler, nicht nach oraler Applikation, mutagen. Methylnitrosoharnstoff induziert Mutationen bei subcutaner Injektion und oraler Gabe. Die Verschiedenheiten werden auf eine unterschiedliche Resorption zurückgeführt.

Über die mutagene Wirkung des Methylmethansulfonats (MMS) in vitro und im „Host-Mediated Assay“

The concentration- and time-dependent genetic activity of methyl methanesulfonate (MMS) was investigated in vitro and in the host-mediated assay using four different indicator organisms. Dose/effect curves achieved with the different indicator organisms are similar.ZusammenfassungDie konzentrations-und zeitabhängige genetische Wirksamkeit des Methylmethansulfonats (MMS) wurde unter Einsatz von vier verschiedenen Indikatororganismen sowohl in vitro als auch im „host-mediated assay“ untersucht. Die Dosis/EffektKurven zeigen eine weitgehende Übereinstimmung zwischen den verschiedenen Indikatororganismen.AbstractThe concentration- and time-dependent genetic activity of methyl methane-sulfonate (MMS) was investigated in vitro and in the host-mediated assay using four different indicator organisms. Dose/effect curves achieved with the different indicator organisms are similar.

Zur Mutagenität des INH

Isoniazid showed mutagenic activity in submammalian test systems. If these results were also applicable to man, the mutational load of the population due to isoniazid would be rather large, because this is a widely used agent. We, therefore, carried out mutation experiments in mice, using two different test systems: the dominant lethal test and the host-mediated assay with Salmonella typhimurium G 46 as indicator organism. These methods were chosen because we intended to cover two different spectra of mutations: point mutations and dominant lethal mutations which are thought to be the result of chromosome aberrations.Isoniazid was mutagenic in the host-mediated assay after a total dose of 25–150 mg/kg s. c., but did not induce dominant lethal mutations in mice.In vitro, isoniazid did not show mutagenic activity in this microbial strain, whereas hydrazine was strongly mutagenic in vitro as well as in vivo. The mutagenicity of isoniazid in the host-mediated assay may be explained by the formation of hydrazine in the mouse organism.Although in the host-mediated assay the mammalian metabolism is taken into account, the mutations are induced in microorganisms. Thus we have only indications but no real proof that isoniazid is mutagenic in mammals. These findings do, however, call for profound and critical studies to elucidate the question of whether isoniazid could be mutagenic for man.ZusammenfassungIsoniazid erhöhte in der intra-animalen Kultur (host-mediated assay) mit Salmonella typhimurium G 46 als Indicatororganismus die Mutationsfrequenz, löste dagegen nach Applikation an männliche Mäuse keine dominanten Letalmutationen aus. In stärkerem Maß erzeugte Hydrazin Mutationen in der intra-animalen Kultur. Im host-mediated assay werden die Mutationen nur an Mikroorganismen induziert und nachgewiesen; der Befund kann aber nur Hinweis auf eine mögliche mutagene Wirkung am Menschen sein. Da die Substanz jedoch weiten Bevölkerungskreisen verabreicht wird, ist eine gründliche Untersuchung der mutagenen Eigenschaften des INH erforderlich.

Grundzüge der Ökologie

Die Okologie gibt uns Aufschluss daruber, wie Individuen, Populationen oder Arten in Beziehung stehen. Da der Mensch – genau wie seine Krankheitserreger – Teil dieses komplexen Gefuges ist, ist es lohnenswert, sich die Grundlagen der Okologie klarzumachen.

Das entschlüsselte Morsealphabet des Lebens

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Das ideologisierte Ernährungsproblem

Wie hat sich das Ernahrungsverhalten von der Steinzeit an bis heute verandert? Wie sind aktuelle Ernahrungstrends wie Probiotica, Genfood, Superfood und Smoothies zu bewerten? Auf diese und weitere Fragen gibt das vorliegende Kapitel Aufschluss.

Das Modulsystem Mensch

Wenn man bedenkt, dass das menschliche Metagenom, das zentrales Thema dieses Kapitels ist, nur zu 10% aus menschlicher DNA aufgebaut wird, ist schnell klar, dass der Mensch ein System mehrerer biologischer Module darstellt. Waren viele der den Mensch bevolkernden Bakterien nicht seine standigen Begleiter, wurde er viel weniger resistent gegen bestimmte Erkrankungen sein. Auch ist ein groser Teil unseres Genoms viraler Herkunft.

Verändern Medizin und Gesellschaft unser Genom

Welchen Einfluss haben Medizin und gesellschaftliche Veranderungen auf unser Erbgut? In diesem Kapitel werden Beispiele dafur gegeben, wie und warum es zu einer Verminderung oder Vermehrung von Genhaufigkeiten kommen kann, und welche Auswirkungen Veranderungen im Reproduktionsverhalten haben konnen.