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Human Defense from Highly Toxic Agents

#1040


New Principle for Protection of Humans and Development of Protectors Against Highly Toxic Agents

Tech Area / Field

  • MED-DID/Diagnostics & Devices/Medicine
  • BIO-SFS/Biosafety and BioSecurity/Biotechnology

Status
3 Approved without Funding

Registration date
09.07.1997

Leading Institute
State Research Center of Virology and Biotechnology VECTOR, Russia, Novosibirsk reg., Koltsovo

Supporting institutes

  • Inter-Institute Laboratory, Russia, Novosibirsk reg., Novosibirsk\nEngelhardt Institute of Molecular Biology, Russia, Moscow\nState Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk

Collaborators

  • Argonne National Laboratory (ANL), USA, IL, Argonne

Project summary

At our days the mankind faces the problem of chemical weapons destruction. These weapons accumulated during a long period of time are represented mainly by Yperite (Mustard-Gas, [MG]). Hundreds of thousands tons of Yperite are stored in the warehouses at the territories of the former USSR, USA, Great Britain, France and Germany. It was used by Iraq against Iran and even against their own people. There are reports on injuring of US soldiers during the Gulf War. The Tokyo incident opened the door to new form of terrorism. MG is a low-cost and easily manufactured compound thus being suitable for terrorists. Hundreds of thousands tons of military ammunition loaded with Yperite and Lewisite were buried in the Baltic Sea after the World War II. The metallic parts of this ammunition will be naturally destroyed within 10 - 15 years. Incidents where fishermen are injured by MG are occurring. It is known that even prolonged storage in the static layer of water does not lead to any perceptible decrease of damaging impact of these substances. The destruction of chemical weapons is thus becoming a necessary and significant branch of modem industry all over the world. During the destruction of the chemical weapons and degassing of the area, significant number of personnel and population will come into contact with these agents, mostly in small concentration. Biological action of MG and Lewisite in low doses is mostly mutagenic, MG being a classic carcinogen. It is necessary to emphasize that the DNA initial lesions increase the risk of tumor formation in present and cause genetical diseases in the following generations. The problem of personnel and environmental security becomes urgent. There are no tests for MG exposure. The pensions and lawsuits can be expensive. It is necessary (and quite real) to select the personnel on the basis of the genetic resistance towards the definite concentration of the specified agents.

The general aim of the proposal is the ensuring the genetic safety of personnel and population's contact with DNA damaging agents in low doses.

The main goals are following: the development of the principles for human genome defence from the highly toxic agents (Yperite and some other alkylating agents, g-rays, radiomimetics); elaboration of substances for human cell protection from the damaging effect of definite types of toxic agents; creation of complex methods for reliable testing of biological effect of toxic agents; creation of scientific basis for a system of the genetic suitability selection for the work with toxic agents of certain types.

Yperite is a bifunctional alkylating agent with the following mechanism of action on the genetic apparatus. On the first stage it forms a covalent bond with a nitrogen base (Guanidine) of one of the DNA chains, forming monoadduct (MA). The second stage is a physicochemical process of monoadduct transformation into diadduct resulting in interstrand DNA cross-link (ICL) formation. During the last 15 years we have been studying the mechanisms of DNA damage and defence. We use such crosslinking agents (CLA) as 8-metoxypsoralen and its derivatives, as well as g-radiation and Xrays as models. The range of chromosome aberrations caused by bifunctional CLA was determined. The specific biological test for efficiency of scavenging of target radical has been worked out. The method of detection of primary DNA damages through several cell cycles after appearance based on biological effect has been worked out; this allowing the detection of the contact with CLA after a long period of time. Our data indicate that the efficiency of the ICL realization in a mutation and/or cytotoxic effect is 103 higher than that of MA. The reparation system was shown to be active towards ICL and the way of increasing the reparation activity was found. However, the possibilities of this system remain limited. According to our indirect estimations, the effectivity of the radical scavenging system in genome defense from ICL within high mutagenic rates is by 102 higher than that of the reparation system. Thus the main application point of the genome defence from the CLA is the process of the ICL formation. Blocking of the transformation of MA into ICL is crucial for genome protection against CLA. We have demonstrated the principal possibility of this blocking. We have discovered a powerful endogenous inducible protective system, which scavenges the MA as target radical. The protection effect is connected not with the ICL reparation but with the prevention of the CLA formation without influence on the MA induction. We have demonstrated that MA increase the occurrence of translocations in the line of cell generations and demonstrated the principal possibility of defence from MA formation. We have suggested performing biological monitoring of the contact with low doses of Yperite and other agents basing on the control of genetic apparatus' damage. The sensitivity of this methodological approach is significantly (103 - 104) higher than conventional clinical and physiological methods. The ideology of the search for the ways for the prophylactics and the treatment of consequences of the contacts with CLA has been worked out. The principles of the search for protectors should take into account the following levels of an interaction between toxic agents and cells or DNA: transport through the membrane; formation of radicals; primary DNA damage; mutation formation; induction of "gene-protectors".

Study of the genetical apparatus response to the contact with toxic agents and the dose-effect curves evaluation, searching for the reliable inductors of the endogenous protective system, isolation and characterization of inducible proteins, specific to definite types of the DNA initial lesions, localization of their genes, study of the mechanisms of cells resistance towards highly toxical agents are the main stages of this work. This would lead to the development of the methods of stimulation of organism's protective properties and to the elaboration of human cell protectors against definite chemicals.

Technical Approach and Methodology

This research involves cytogenetical and molecular-biological experiments.

Cultivated human and animal blood lymphocytes are used as standard cell models.

Study of the genetical apparatus response to the contact with toxic agents and the dose -effect curves evaluation are based on cytogenetical analysis of the structural chromosome rearrangements: Sister Chromatid Exchanges, Chromosome Aberrations and Micronuclei Formation.

Quantitative and qualitative evaluation of the DNA initial lesions includes alcalin gel-electrophoresis, chromatography on HAP, electron microscopy, our biological method for evaluation of antimutagenic effect concerning definite type of primary damage, etc. Free radicals are registered using chemoluminesce, electroparamagnetic resonance, our biological test for scavenging of target radical and other methods.

The search for inductors of the endogenous protector system is based on their antimutagenic effect. The induced proteins are isolated using one-and double dimension electrophoresis; their specificity for definite radicals is determined using affine chromatography with DNA-toxin complex. After sequencing of the protein ends the oligonucleotide probes are synthesized and used for genes searching; they are cloned, sequenced and expressed. Methods of cDNA transfection, differential phage display, displacement elution, differential RNA display, expression cloning, etc are used. Fluorescent in situ hybridization is used for gene localization on chromosomes; molecular hybridization with cDNA libraries of the appropriate chromosomes leads to precise localization of the protector genes. Computerized Image analyzers are used in cytogenetical and biochemical studies. The elaborated protectors and inductors will be trialed preclinically.

The method of personnel selection for biological suitability is based on determination of the "group of risk" (people with defects in the reparation and protection systems) and will include cytogenetical investigation of the inpidual's cells resistance towards definite agents; reparative DNA synthesis estimation; study of the reparative ability in the "viral host cell reactivation" system; our test for specific defence from target radical. As well as "biological dosimetry", this protocol can include different combinations of these and other methodics.

The main scientific result:

Evaluation of the mechanisms and capacities of inducible endogenous system protecting human cells from damaging impact of highly toxic agents.

The applied and commercial products:

Effective means of prophylactics and treatment of the consequences of contact with toxicals: the biological protectors, protocols of biological monitoring and professional suitability selection; the results of preclynical trials.

References


1. Makedonov G. P., Evgrafov O. V., Fedotov A. R. (1980). A Quantitative evaluation of monoadducts and intercrosslinks induced by 8-Methoxypsoralen in eucariotic chromosome DNA. In: "Organism sensivity to mutagenic factors and mutations formation". Vilnius, p. 5.
2. Makedonov G. P., Safronov V. V., Osipova R. G., Tarasov V. A., Dong H. H. (1982). Comparative efficiency of Sister chromatid exchanges and Chromosome aberrations upon the induction of Psoralen DNA Monoadducts and Intercrosslinks. In: Molecular mechanisms of genetic processes. Molecular genetics, evolution and genitic engineering. Moscow, "Nauka", pp. 718-24.
3. Evgrafov O. V., Fedotov A. R., Makedonov G. P. (1983). DNA intercrosslinks efficiency in Sister chromatid exchanges formation in Chinese hamster cells cultivated in vitro. In: "Molecular mechanisms of genetic proccesses. Moscow, "Nauka", pp. 24.7.
4. Evgrafov O. V., Makedonov G. P., Tinyakov Yu. G., Fedotov A. R. A method for DNA monoadducts and diadducts evaluation. Domestic patent N 1028719 (1983).
5. Makedonov G.P., Chekova V.V, Yakubovskaya E.L. and G.D. Zasukhina (1990). Modification of DNA repair by human interferons. Acta Biologica Hungarica 41 (1-3), pp. 187-197.
6. Makedonov G.P., Yakubovskaya E.L., Zasukhina G.D. (1990). A differential effect of the interferons antimutagenic action towards initial DNA lesions: The genetic risk of 8-Methoxypsoralen DNA monoadducts is higher than that of interstrand DNA crosslinks. Dokladi Akademii Nauk USSR, v. 314, N 3, pp. 372-375.
7. Yakubovskaya E.L., G.P.Makedonov, I.V. Kolonina, S.A. Solopov. New mechanism of antimutagenic effect of interferon. (1996). Mutation Research, in press (REG-4558).
8. Makedonov G.P., Yakubovskaya E.L., Zasukhina G.P. (1989). Interferon protects human cells against 8-Methoxy-Psoralen-induced interstrand cross-links by postreplicative repair. Genetika (Rus), v. XXV, N 3, pp. 552-554.
9. Yakubovskaya E.L., Makedonov G.P., Kolonina I.V., Solopov S.A. New mechanism of antimutagenic effect of interferon. (1993). Radiobiol. Radioecol. v. 33 is 2 (5), pp. 665-673.
10. Makedonov G.P., AlekhinaN.I., Tskhovrebova L.V., Zasukhina G.D. (1993). Comparison of expression of anticlastogenic action of interferon and radioadaptive response. Radiobiology, v. 33, is. 2, pp. 259-264.
11. Makedonov G. P., Tskhovrebova L. V. (1994). Cytogenetic investigation of radioadaptive response in repair-defective human cells. Genetica (Rus), v. 30, suppl., p. 94.
12. Tskhovrebova L.V., Makedonov G.P., Lvova G.N., Chekova V.V., Maksimova T.N., Zasukhina G.D. (1995). Radioadaptive response in lymphocytes of patients suffering by schizophrenia. Radiobiol. Radioecol. v. 35, is. 5, pp. 665-669.
13. Makedonov G. P., Tarasov V. A. (1995). In: T. M. Turpaev, ed. Mechanisms of the Production of Chromosomal Aberrations in Eucariotic Cells. Harwood Academic Publishers, pp. 30-33.
14. Makedonov G. P., Tskhovrebova L. V., Yakubovskaya E. L. (1992). A modifued micronucleus test in human lymphocytes. "Ecological problems of mountain territories. "The First international conference, Vladicaucasus, p. 336.
15. Fedotov A. R., Makedonov G. P. (1994). Human genome response on specific genotoxic agents’ action: Damage mechanisms and defense. Int. Conference "Molecular Biology at the Border of XXI Century. Genome Structure and Functional Analysis. Moscow, p. 10.
16. Makedonov G.P., Bobileva L.A., Chekova V.V., Zasukhina G.D. (1995). Study of anticlastogenic effect of ascorbic acid in relation to damage induced by photomutagenic action of 8-Methoxypsoralen and ammonium molibdenat in human lymphocytes. Radiobiol. Radioecol. v. 35, is. 5, pp. 726-729.


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