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More than 40 years ago ethyl nitrosoеurea was identified as a powerful mutagen for mammalian germ cells resulting in random point mutations in gamete DNA. This feature allowed the use of this mutagen for genetic studies on the mechanisms of various pathological and physiological processes in model organisms. In our study genome-wide mutagenesis in C3H mice by ethyl nitrosourea followed in generation F3 by selection of animals resistant to acute lethal hepatotoxicity caused by a combination of E. coli lipopolysaccharide (LPS) and D-galactosamine (D-gal). Tumor necrosis factor (TNF) is known to be a critical mediator of this pathology. Exposure to D-galactosamine increases sensitivity of hepatocytes to TNF leading to their necrosis and/or apoptosis. After double LPS/D-gal screening in F3 several mice resistant to LPS/D-gal-induced hepatotoxicity were identified, and became the founders of the corresponding “mutant” families. Using outcrossing to C57BL6 background followed by intercrossing, generations F5 and F7 were obtained. Among families of mutant animals only one family showed the resistance to the combination of LPS and D-gal, but sensitivity to TNF-D-galactosamine. This phenotype showed approximately Mendelian inheritance consistent with the recessive mutation hypothesis. This latter fact was confirmed by the sensitivity of mice from “heterozygous generations” (F4 and F6) to lethal LPS/Dgal hepatotoxicity. Primary bone marrow macrophages obtained from half of the mutant mice showed significantly reduced levels of TNF after LPS stimulation in vitro. At the same time, the serum TNF levels 1 hour after the administration of a non-lethal LPS dose did not differ in the mutant family mice and wild-type mice. These results implicate a recessive mutation either in innate TLR4-mediated signaling pathway, including proteins associated with LPS transfer, adapter molecules, components of kinase signaling cascades, transcription factors, or in enzymes involved in regulation of TLR4 cascades, such as components of the ubiquitin cycle, or in genomic regulatory sequences that control the expression of one of these genes, including the tnf gene.

About the authors

I. V. Astrakhantseva

Scientific and Technological University “Sirius”;
N. Lobachevsky State University

Astrakhantseva I.V., PhD (Biology), Senior Research Associate Russian Federation

L. S. Gladkova

N. Lobachevsky State University

Gladkova L.S., Laboratory Assistant Russian Federation

E. A. Vasilenko

N. Lobachevsky State University

Vasilenko E.A., Lecturer Russian Federation

V. S. Tarabykin

N. Lobachevsky State University;
Institute of Cell and Neurobiology, Charit – Universit tsmedizin


Tarabykin V.S., PhD, MD (Biology), Professor, Head, Brain Development Genetics Laboratory Institute of Biology and Biomedicine


Russian Federation

M. S. Drutskaya

V. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Drutskaya M.S., PhD (Biology), Leading Research Associate Russian Federation

S. A. Nedospasov

Scientific and Technological University “Sirius”;
V. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences;
M. Lomonosov Moscow State University

Author for correspondence.

Nedospasov S.A., PhD, MD (Biology), Professor, Full Member, Russian Academy of Sciences, Head, Laboratory of Molecular Mechanisms of Immunity

Head, Department of Immunology, Faculty of Biology 

Head, Department

Russian Federation


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Copyright (c) 2020 Astrakhantseva I.V., Gladkova L.S., Vasilenko E.A., Tarabykin V.S., Drutskaya M.S., Nedospasov S.A.

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