Revealing possible genetoxic effects on human and other live organisms of various food products, as well as medicinal and other preparations

1

Размещено на http://www.allbest.ru/

Mendeleyev Russian Research Institute for Metrology

REVEALING POSSIBLE GENETOXIC EFFECTS ON HUMAN AND OTHER LIVE ORGANISMS OF VARIOUS FOOD PRODUCTS, AS WELL AS MEDICINAL AND OTHER PREPARATIONS

V.S. Sibirtsev

A.V. Garabadzhiu

Recently, as a result of active development of pharmacology and various biotechnology methods, as well as increase of common level of anthropogenic workload on environment, rather impotant becomes the problem is enough fast valuation of opportunity any genetoxic effects from new or modificated food products, medicinal preparations and etc. - and even as at isolated action of them on live organism, as in a combination of them to other food products, medicinal preparations, various environment factors, specific for that region, and etc. And besides is frequently important problem is reasonably fast and complex valuation of ecological nonwell-being degree of any region - and even in case, when the standard methods do not there fix excess of the norms on one of possible factors; however in a combination with one another they can make damages on the people and other live organismes.

Above mentioned problems are resolved can be different ways… but the most acceptable use for these purposes standard biosystems (which under characteristics are close to human organism, are cheap, are accessible in mass quantity and have relatively short life cycle)… one of major parameters, describing condition of which, are changes in a structure them cell genome... which, in turn, is the most simple and quickly, in our opinion, can be revealed with the help of synthetic fluorophores, capable to interact with DNA including directly in a cell nucleus and sensitive to presence even rather small quantities of nucleic acids… owing to that, DNA-specific fluorophores can be used, besides mentioned, as medicinal preparations, in research, diagnostic purposes and etc.

Pursuant to above said, purpose of the present work has become:

- to develop with the help of DNA-specific fluorophores a method of the analysis not only nucleic acids total, but also changes in a cell genome structure;

- to develop on the basis of above-stated, as well as using standard biosystems, method of valuation of possible genetoxical effects of various food products, medicinal preparations, damaging factors of environment (including at joint their action on live organismes);

- as well as to consider opportunities of application of DNA-specific fluorophores in other methods of diagnostics and monitoring, based on the quantitative and qualitative analysis of a genetic material in cells.

DNA analysis with the help of system from two dyes with agreed properties

At all variety of above mentioned potential opportunities for use флуорофоров the spectrum of methods particular, practical application of such substances so far more far from so is great, as it would be necessary. It is result, in particular, that the "strong selective linkage with substrat, as a rule, requires from ligand of one structural features, and availability of properties suitable for registration - other. And one of the best ways of the sanction of this contradiction we consider use for DNA analysis of systems including several nucleotide specific fluorophores, which are indirectly linked together (as far as the separate fragments of large complex substance can not only mutually supplement, but also to interfere one another) via the common for them substrate, but share concerted complex forming and spectral properties.

As example of one of such systems by us were chosen two widely used (on separateness) commercial DNA-specific fluorophores: Hoechst-33258 (2-[2-(4-hydroxyphenyl)-5(6)-benzimidazoyl]-5(6)-(1-methyl-4-piperazinyl)benzimidazole) (Ht) and ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinum bromide) (EB). First of which (EB) is an intercalator that lies between two base pairs of the polynucleotide double helix; it exhibits mod erate preference to GC-rich sites, but basically specific to secondary and higher orders of DNA molecule organization [1]. Whereas second (Ht) joins a DNA molecule "outside" and is preferablly specific for sites of polynucleotide double helix containing three sequentially positioned AT and only GC base pairs [2] (see fig.1). And besides at joint sorption of Ht and EB on a polynucleotide between molecules these dyes a fluorescent resonant non-radiative energy transfer (FRET) can occur.

The FRET is the phenomenon, when at fluorescence excitation of energy donor (in our case this is Ht) molecules begin to shine not only they, but also energy acceptor (in our case this is EB) molecules. FRET can occur only, when the fluorescent emission wavelength region of energy donor is largely crossed with the fluorescence excitation wavelength region of energy acceptor (as takes place in the case of Ht and EB) (see fig.2). And the FRET efficiency rather considerably depends on distance between chromophore groups of the energy donor and acceptor [3,4]. fluorescent nucleic dye genome

Thus obviously, that at the combined use of Ht and EB it is possible to evaluate not only DNA total in a sample, but also change degree of a cell genome structure. Below we consider a practical example of one possible protocol of genetic material analysis using the system "Ht+EB".

1) Mix 0.1ml sample (e.g., whole blood) with 1ml of the water lysing mixture - used that to infringe integrity of cell walls, as well as external cell and nuclear membranes, and thus to ensure access of dye from "exter-cellular" solution to "inter-cellular" DNA, and containing 2M NaCl + 0.1M Na2EDTA (ethylenediamine tetraacetate, disodium salt) + 0.01M Tris (2-amino-2-hydroxymethyl-1,3-propane diol) + 0.5% (v/v) Triton X-100 (4-octyl-[2,4,6,8,10-decapentol]hydroxybenzene) (pH 8.0). Incubate resultant mixture for 5min. Then, take 0.05ml aliquot and mix it with 1ml of standard water buffer - containing 0.01M NaCl + 0.01M Na2EDTA + 0.01M Tris (pH 7.4). As a result, we receive a solution 1, for which we measure background fluorescence intensities: IFH (at excitation and emission wavelengths: 350 and 455 nm - appropriate to complex of Ht with DNA) and IFE (at excitation and emission wavelengths: 520 and 605 nm - appropriate to complex of EB with DNA).

2) Add 0.05ml of Ht dissolved in a standard water buffer in concentration 10µg/ml to 1.05ml of solution 1. As a result, we receive a solution 2, for which at excitation and emission wavelengths 350 and 455 nm we measure fluorescence intensity IH. Then, add 0.02ml of standard DNA dissolved in a standard water buffer in concentration 60µg/ml to 1.1ml of solution 2. As a result, we receive a solution 3, for which at the same wavelengths, as earlier we measure fluorescence intensity IDH. Then, we calculate a DNA total in a sample as: CDH=g·CDS·(IH-IFH)/(IDH-IH) - where g=246 is analysed DNA dilution in solution 3 compared with the whole blood, and CDS=3.3·10-6M is concentration of standard DNA in solution 3.

3) Add 0.05ml of EB dissolved in a standard water buffer in concentration 200µg/ml to 1.05ml of solution 1. As a result, we receive a solution 4, for which at excitation and emission wavelengths 520 and 605 nm we measure fluorescence intensity IE. Then, add 0.02ml of standard DNA dissolved in a standard water buffer in concentration 60µg/ml to 1.1ml of solution 4. As a result, we receive a solution 5, for which at the same wavelengths, as earlier we measure fluorescence intensity IDE. Then, we calculate a structural factor: Ks=CDE/CDH (where CDE=g·CDS·[Iе-IFE]/[IDе-Iе]) - change of significance of which should, in particular, to reflect changes in character of the tertiary and higher orders of organization of genome structure in a sample (as far as EB, as was already marked, to it is essentially more specific, than Ht).

4) At first, we measure IFHе - fluorescence intensity of solution 2 at excitation and emission wavelengths 520 and 605 nm. Then, add 0.02ml of EB dissolved in a standard water buffer in concentration 200µg/ml to 1.12ml of solution 2. As a result, we receive a solution 6, for which at excitation and emission wavelengths 520 and 605 nm we measure fluorescence intensity IHE. Then, we calculate a factor of effectiveness of energy transfer: Kt=(IHE-IFHE)/CDH - the reduction of significance of which should, in particular, as shown in fig.2, in a certain measure to reflect reduction of a length of genome telomere sites… which for all vertebrates, for example, represent fragments "TTAGGG" repeated on the ends of DNA molecules… the quantity of which decreases at each cell division (due to DNA replication problem), and accordingly determines the maximal limit of live organism age… executing also some other, in particular regulating, function [5,6].

Valuation of genetoxic effects of various products, preparations and environment factors

Being based on above-stated us were developed: method for valuation of possible genetoxical effects of various food products and medicinal preparations on live organismes (including at joint action on these organismes of other products, preparations or environment factors, specific for any region)… and method for valuation of possible genetoxical effects of low dozes radiating, chemical, microbiological and other damaging factors of environment att joint their action on live organismes. These methods are based that group of animals (for example, rats, the life cycle of which proceeds about in 20 times faster, than at the people) during a certain time feed with a analyzed product (or enter to him a appropriate preparation, or subject to effect of damaging factor)… in a combination, if necessary, with other products, preparations or environment factors. At it samples of blood from animals regularly select. Then process by it special lysing mixture (its contents see above). Then evaluate a DNA total and genome structure in sample by special dye (or system from several dyes - such as Ht and EB, for example)… ration them on quantity of leucocytes (as far as erythrocytes in a normal condition in mammal blood do not contain of DNA)… compare received parameters to similar values, determined for control animal group, as well as for all animals, participating in analysis, prior to the beginning its realization. And on the basis of this comparison come to the conclusion about availability of any genetoxic effect of analyzed products or factors on live organisms.

Valuation of "microbial pollution (seeding)" of liquid environments

The new method for express valuation of "general microbial pollution (seeding)" of liquid environments (which is one of important parameters at valuation of quality for a water and food products, at the monitoring of various biotechnological manufactures and etc.) was developed us also. Standard methods, used for this purpose at present (and including: seeding of analyzed material on medium; incubation it then at constant temperature, in steril conditions during from one till several days and subsequent count of number of formed microbial colones), require for realization of a significant time and specialized stationary conditions. And our method permits to execute the similar analysis practically in a "real time mode" and directly on those places, where samples were taken, and consists in following.

Mix 1ml analysed sample with 0.1ml of the water lysing mixture (its contents see above). Then received solution we agitate, incubate during several minutes (at to=253oC - up to establishment of stable indications on fluorimeter). Then we measure background fluorescence intensity of sample IF1. Then to analyzed solution we add also 0,1ml solution (the background fluorescence intensity which IF2 is defined beforehand), containing by 10?g/ml Ht (or other DNA-specific fluorophor in other concentration) in a standard water buffer (its contents see above). And as well as earlier, we determine new fluorescence intensity ID. Then, we add also 0.02ml of standard DNA (for example, thymus DNA dissolved in a standard water buffer in concentration 60µg/ml) to analyzed solution. And as well as earlier, we determine new fluorescence intensity IDS. Then we calculate microbe total in a sample as: Cm=а0+а1(ID-IF1-IF2)/(IDS-ID) - where empirical factors а0 and а1 are defined on a data of calibration with use standards with the known microbe contents.

This method (on which we received the patent [13]), using a ТКО-100 portable microfluorimeter, has allowed to define microbe total in sample since 20cl/ml, with error not more than 30%, expending not more than 10min on one measurement. Besides in particular, if instead of lysing mixture in above-described method to use any antibiotic, forming channel only in external wall and membrane of cell - DNA-specific fluorophor will paint probablly only prokaryotes. And if to deduct these data from microbe total in a sample (determined as is described above), it will be possible to evaluate also quantity of eukaryotes, contained in analyzed sample. And on manufactures, realized in stationary conditions, for registration of staining of DNA it is possible to use besides already not portable microfluorimeter and stationary flow cytofluorimeter - that will allow not only to execute valuation of "microbial pollution" really in a "real time mode", but also essentially to increase sensitivity of such valuation.

Valuation of individual radiating sensitivity of organism

At last, we developed a method of valuation of individual radiating sensitivity of animals and people, based that at cancer diseased, for example, prior to the beginning radiation therapy take sample of blood (it is enough 1ml), adulterate by a standard buffer (its contents see above) and divide on two parts, one of which then subject to action of X-ray and other (control) - leave as is. Then each of these samples incubate during 3 hours at 370C (for reparation of DNA primary damages) and process then by a lysing mixture. Then valuation of DNA total and genome structure in a sample by special dye (or system from several dyes - as for example Ht and EB) and fluorimeter… and ration received values on quantity of leucocytes. At last, comparing received values among themselves, judge a degree of damaging effect, which will render probablly radiation therapy in a chosen doze on this particular diseased.

Fig.1. The schemes of DNA structure ДНК and interaction of intercalators (EB) and nonintercalators (Ht) with DNA. "hb" are designated the hydrogen links between complementary nucleotides of the first and second chains of DNA.

Fig.2. The scheme of fluorescent resonant energy transfer (FRET) between molecules Ht and EB. I are designated the fluorescence intensity. hv1 and hv2 are designated the light quantums, absorbed Ht and emited EB, accordingly.

References

1. A.R.MORGAN, J.S.LEE, D.E.PULLEYBLANK, N.L.MURRAY, D.H.EVANS: Nucl. Acids Res. 7, 547-571 (1979)

2. P.E.PJURA, K.GRZESKOWIUK, R.E.DICKERSON: J. Mol. Biol. 197, 257-271 (1987)

3. V.S.SIBIRTSEV: Biochemistry (Moscow). 70 (4), 449-457 (2005)

4. S.SPEISER: Chem. Rev. 96, 1953-1976 (1996)

5. A.K.MIKER, D.S.KOFFI: Biochemistry (Moscow). 62 (11), 1547-1557 (1997)

6. E.X.BLEKBERN: Biochemistry (Moscow). 62 (11), 1400-1406 (1997)

7. S.D.IVANOV, V.S.SIBIRTSEV: Method of valuation general microbiological pollution. Russia patent №2079138, bulletin №13 (1997).

Abstracts

New protocol of DNA analysis (a using system from two fluorescent nucleic-specific dyes with agreed properties) are considered - which enables to evaluate not only DNA total in a sample, but also change degree of a cell genome structure. Number of new biotesting methods, developed on the basis of above mentioned DNA analysis protocol, are considered also - which enables to evaluate: "general microbial pollution (seeding)" of liquid environments; possible genetoxical effects of various food products, medicinal preparations, damaging factors of environment (including at joint their action on live organismes); individual radiating sensitivity of organism and etc.

Key words: biotesting, DNA analysis, fluorescent dyes on DNA

Размещено на Allbest.ru