Specific Effects of Superlow Doses of Biologically Active Substances and Low-Level Physical Factors

E.B. Burlakova

In 1983, a joint team of researchers of our Institute and the Institute of Psychology studied the effect of antioxidants on the electric activity of an isolated neuron of the grape snail and obtained an unexpected result. A primary dose (10^-3 M ) of agent was not only active but also toxic for the neuron, therefore a less concentrated solution was used. To our surprise, the dose that was four orders of magnitude lower than the primary one was not only less toxic but also more effective. A further decrease in concentration resulted in enhancement of the effect: it reached a maximum at l0^-15 M and then decreased (at 10^-17 M ) almost to the level of the control results.¹ Similar trends were recorded in later experiments with animals that were given injections of cholinomimetic arecoline.²

We studied the discovered effect using a variety of active factors: anti-tumor and anti-metastasis agents, radiation protectors, inhibitors and stimulants of plant growth. neurotropic preparations of various classes, honnones, adaptogens, immunomodulators, detoxicants, antioxidants, and physical factors - ionizing radiation and others.³ Levels of biological organization at which the effect of superlow doses (SLD) of biologically active substances were studied, were also varied - from macromolecules, cells. organs. and tissues to animals, plants, and even populations. This does not mean that the effect was observed for SLD of any biologically active substance or for any biological object. This means, it should be emphasized, that the effect of substances in concentrations of l0^-13 - 10^-17 M and lower cannot be attributed to any specific structure of a substance or a a level of biological organization.

The obtained results led us to believe that we are dealing here nol with a peculiar effect of a certain substance or a response of a unique biological object but with some principally new interaction of biological objects with SLD of biologically active substances. Each of the substances may have its own target. mechanism of amplification, and pattern of metabolism; however, SLD of these substances exhibit a number of common features.³ In recent years, more scientists have been addressing the problem of SLD effects; the gamut of bioobjects under investigation has been extended, as well as a variety of chemical substances and physical factors, for which SLD activity was discovered.

Let us dwell on the definition of the term of superlow doses. With all the quantitative differences in the existing definitions of a boundary that differentiates SL doses from conventionally applied ones, there is a common opinion that SLD of biologically active substances are those whose effectiveness cannot be consistently explained in the context of traditional theories and, hence, novel mechanisms should be explored.

Various approaches to define SLD are presented by investigators in the issue of Russian Chemical Journal, V. XLIII, #5, 1999. For instance, F.S. Dukhovich et al.⁴ take a concentration of 10^-11 M as the absolute boundary based on the number of cell receptors in an organism and the affinity ofligands to them. For agents showing a low affinity to receptors, higher values, e.g., 10^-9 - 10^-10 M , may be considered as SL concentrations (doses). According to this approach, even in cases of a hypothetically higher (above 10¹² M ) affinity of ligands to receptors, the boundary cannot be lower than 10^-11 M .

We share the opinion that the boundary is determined by the number of molecules of a biologically active substance per cell. As a substance is introduced into an organism in doses of 10^-12 - 10^-13 M , a cell will contain at least one to ten molecules of this substance. Consequently, we consider concentrations of 10^-12 and lower as SLD. Similar conclusions were drawn by researchers who determined the SLD boundary on the basis of a maximum affinity of ligands to receptors and take 10^-13 M and lower as SL doses (concentrations) of biologically active substances.

As for SLD of physical factors, no common definition has been formulated yet. In each particular case involving any particular physical agent separate definitions should be provided. Thus, the United Nations Scientific Committee on Atomic Energy recommends that doses below 250 mGy (25 R) and dose-rates of 1.5 mGy/min and lower be regarded as low doses of ionizing radiation and low dose-rates respectively. However, such definitions cannot be adequately applied to cases of radiation resistant organisms (bacteria and eukaryote protozoa) and plant cells. Therefore, in some published works on radiobiology, low doses of radiation are frequently defined as doses responsible for initiating a change of sign of the effect, e.g., transition from inhibition to stimulation of cell growth.⁵

Phenomenological features of SLD effects

Based on results of our studies and those cited in the literature, a conclusion may be drawn that, with reference to cell metabolism, SLD of biologically active substances and low-level physical factors exhibit many common features relevant both to fonnal indices (effect dependent on the dose) and indications of biological activity.

The nature of this phenomenon may be associated with a common character of critical targets, e.g., cellular and subcellular membranes, and with specific features of the kinetics of reactions, in which weak interactions play an important role. Among the characteristic features of SLD effects, note the following ones:

- A nonmonotonic, nonlinear, and polymodal dose-effect dependence. In most cases, maxima of the activity are observed nithin certain dose ranges separated by a “dead zone.”

- Modification in sensitivity (enhancement, as a rule) of bioobjects to the simultaneous action of various agents in SLD, both endogenous and exogenous.

- Occurrence of kinetic paradoxes, i.e., when an SLD effect ofa biologically active substance can be discerned even if a cell or organism contains the same substance in concentrations several orders of magnitude higher, the substance affects the receptors when applied in doses several orders of magnitude lower than the constants of dissociation of ligand-receptor complexes.

- Dependence of the sign of the effect on the initial condition of an object.

- Separation of properties of biologically active substances: as their concentrations are decreased, the primary activity may remain while side effects vanish;

The common feature of SLD action most drastically manifests in the study of dose-effect dependence. In some cases the dependence is bimodal: the effect increases at SLD of agents; as the dose increases, the effect decreases and passes into a “dead zone,” then it increases again (Fig. 1). In one of our previous studies on the effect of a herbicide of the class of hydroperoxides on a plant cell culture it was found that the agent exhibits an identical activity in doses differing by six orders of magnitude (10^-13 and 10^-7 M); within the range of intermediate concentrations the effect was absent⁶. Sometimes the dose-effect dependence has a zone with a reversed sign of the effect, i.e., with the increase in concentration the inhibiting effect changes into the stimulating effect and then again into inhibiting effect. There are cases when the effect is dose-independent within a wide range of concentrations. This kind of dependence was observed for a wide variety of biologically active substances.^3,7,8,9 It can be added that similar dependencies were obtained for antimetastasis agents ephazole and lonidamine,¹⁰ for pyracetamll as well as for thyroliberine,¹² phenosan,¹³ and phorbolic ester¹⁴ in a study of the viscous properties of membranes. Such complexity of dose-effect dependencies with their dead zones may account for the fact that the activity of SLD of agents had not been discovered earlier. The results obtained for a dose range before the beginning of a dead zone did not encourage researchers to diminish the dose further or anticipate any SLD effects.

Another specific feature of SLD effects - change in sensitivity to a variety of factors after introduction of SLD of substances - also seems universal. It provided a basis for obtaining such effects as synergism in the action of two antitumor agents^15,16 and enhancement of the activity of herbicide preparations above the additive leve^13,17 when, as in both cases, one of the agents is introduced in SLD .

Information related to the observed kinetic paradoxes and some suggested explanations are reported by I.P. Ashmarin et al.¹⁸ and S.Y. Zaitsev et al.¹⁹

Regarding the sign of the effect: it is known that the sign of the effect depends on the initial condition of the bioobject. For example, it was found that the effect of an antioxidant in SLD acted in opposite directions on isolated neurons with different activity potentials: the antioxidant reduced a high potential and increased a low initial potential.³ Similar trends were observed for the effect of electromagnetic radiation on a calcium receptor,²⁰ the effect of antioxidants on the oxidizing activity of erythrocyte membranes with different initial levels thereof,³ and in the effect of ionizing radiation on the activity of enzymes.^21,22

Separation of properties and elimination of side-effects with a decrease in concentration of a biologically active substance were demonstrated by the study on the effect of phenazepam in a wide range of concentrations.²³ High doses of phenazepam produce a soporific effect, therefore it is approved and prescribed as a night tranquilizer. Taken in SLD phenazeman retains its tranquilizing activity but is devoid of its sedative and myorelaxant effects.^24,25,26 This allowed the authors to patent the SLD phenazepam as a daytime tranquilizer.

Biological effect of low dose ionizing radiation

The main specific features of the action of low-level physical factors will be considered by an example of ionizing radiation. The studies carried out in the last few years showed unambiguously that low-dose irradiation induces multiple structural changes in cells that persist for a long time after irradiation and result in modification in the functional activity of cells.

We have carried out a series of biochemical and biophysical studies of cells of organs in animals (mice) exposed to low-level γ-irradiation (¹³⁷Cs). We studied the rate of alkali elution of lymphocyte and spleen DNA, the rates of neutral elution and adsorption of spleen DNA on nitrocellulose (NC) filters, structural properties (with an EPR spin probe technique) of nuclear, mitochondrial, synaptic, erythrocytic, and leukocytic membranes.

As for the functional activity of cells, we studied (i) the activity and isoforms of enzymes aldolase and lactate dehydrogenase, (ii) the activity of acetylcholinesterase, superoxide dismutase, glutathione peroxidase, (iii) the rate of formation of superoxide anion-radicals, (iv) composition and anti oxidizing activity of lipids of the above-mentioned membranes, (v) sensitivity of cells, membranes, DNA, and organism to the action of other damaging factors.^27,28,29,30

For all the parameters under study a bimodal dose dependence was determined, i.e., the effect increased at low doses, reached its (low-dose) maximum, and decreased (in some cases, the sign of the effect reversed); then, the effect increased again with an increase in dose. A common feature of dose dependences in studied cases is the shift of maximum toward lower doses with the decrease of radiation intensity. It was determined that lowdose irradiation results in (1) elevation of the degree of hemolysis of erythrocytes in experimental animals (mice), (ii) modification in sensitivity of the central nervous system to neuromediators, agonists, and antagonists, and (iii) modification in response of cells to regulatory effects, repeated irradiation, introduction of radiosensitizers and radioprotectors.^31,32

We suggest an explanation for the nature of a nonlinear, bimodal dose-effect dependence based on a concept that there exists a gap between doses that induce damages in bioobjects and doses that trigger the systems of repair of these damages. In this context, the bioeffect increases with dose until the reparative (or adaptive) systems start functioning adequately. As the reparative processes build up, the effect may persist or diminish to elimination, or it may change its sign and increase again with an increase in dose when damages in a bioobject prevail over repair.

Thus, the effects of low-level physical factors, particularly ionizing radiation, exhibit the features characteristic of SLD of biologically active substances: a nonlinear, nonmonotonic, bimodal dose-effect dependence, zones of silence, modification in sensitivity of endogenous and exogenous factors, and an inverse dependence on dose-rate. Similar results were obtained for nonionizing radiation.^33,34,35

SLD effect on enzymes' activity

As noted above, an anomalous dose-dependent response to SL concentrations of biologically active substances was recorded not only for cells or whole organisms but also for individual biomacromolecules. Experiments were performed on isolated enzymes as well as on enzymes in cells and organisms with subsequent assessment of their activity. In experiments with protein kinase C - an enzyme isolated from animals' heart - an inhibiting effect of tocopherol was studied. Protein kinase C is a complex, lipid-dependent, calcium-requiring enzyme that has several active sites interacting allosterically with each other. According to published works, tocopherol produces an inhibiting effect on the activity of protein kinase C at concentrations of 10^-4 to 10^-5 M.³⁶ In our studies performed for a wide range of concentrations (10^-18 - 10^-5 M) we found a bimodal dose-effect dependence: tocopherol actively inhibits protein kinase C at concentrations of 10^-16 to 10^-12 M and 10^-4 to 10^-5 M, but at intermediate concentrations, the effect is considerably lower.

Similar results were obtained for protein kinase C preactivated with phorbolic ester. Inhibition of such enzyme with low concentrations of tocopherol is lower than for the nonactivated enzyme; its maximum is observed at higher concentrations of tocopherol, but a bimodal character of the dependence persists³⁷ (see Fig. 1).

It should be noted that although inhibition of protein kinase C with SLD and standard doses of tocopherol is almost identical, the kinetic parameters of this enzyme, particularly the Michaelis constant, and the degree of the enzyme cooperation with substrate vary differently. At a tocopherol concentration of 10^-14 M, the enzyme affinity to substrate remains unchanged, whereas the affinity reduces by half at a dose of 10^-4 M. An inverse dependence is observed for the Hill coefficient that determines the kinetic cooperation. A degree of cooperation under the action of SLD of tocopherol is twice as high as in the control and in the experiment with 10^-4 M tocopherol. To some extent, it may be accounted for by modification in the enzyme structure. In the above study, a unidirectional pattern of the effect of tocopherol in SL and standard doses was determined: inhibition was observed in both cases. Results of this study are reported in detail in the article by N.P. Palmina et al.³⁸

Also, the effect of another antioxidant, phenozan, on the activity of protein kinase C was studied.39 In previous experiments, it was detennined that phenozan exhibits pronounced activating properties both toward isolated enzymes⁴⁰ and at the cellular level. In no concentration range studied an inverse effect - inhibition of the activity - was observed. As with tocopherol, a maximum effect of phenozan was observed within two concentration ranges: from 10^-8 to 10^-4 M for normal cells (from 10^-6 to 10^-4 M for tumor cells) and from 10^-16 to 10^-18 M. Between these concentrations, the effect was scarcely observed. The ratio of phenozan molecules (for the range of 10^-16 - 10^-18 M) to the number of cells under the effect is from 3 to 300 (molecules per cell); as for protein kinase C molecules, the ratio is 1-100 phenozan molecules per 10⁷ protein kinase C molecules. The details of these studies are also reported by Palmina et al.³⁸

E.M. Molochkina et al studied the activity of acetylcholinesterase under the effect of SLD of antioxidant phenozan.^41,42,43 Of particular importance of these works is that the studies were perfonned in parallel: in vitro on an isolated enzyme, and with the enzyme as a constituent of synaptosomes isolated from animals' brain after intra-abdominal injection of phenozan. The effect of phenozan on the soluable enzyme and membrane-bound enzyme in vivo resulted in reduction in the enzyme's activity (and accordingly, its activity with “real” physiological concentrations of acetylcholine), mainly, via an increase in the Michaelis constant K_M. A dose dependence of the kinetic parameters exhibits two maxima for concentrations separated by six orders of magnitude. Sizes of the effect of standard and SL doses of phenozan (differing in concentration by a factor of 10⁶ to 10⁸) are comparable.

A similar series of studies with intra-abdominal injection of acetylcholine into mice showed an activation of acetylcholinesterase due to a decrease of the Michaelis constant K_M and an increase of the maximum rate of acetylcholine hydrolysis. The effect for K_M was identical both in the low and high dose ranges.⁴⁴

The effect of acetylcholine on the soluble enzyme resulted in a stage-by-stage modification of the acetylcholinesterase kinetic parameters. The effectiveness of the enzyme exhibited no significant changes. For the membrane-bound enzyme significant changes in the kinetic parameters were observed at acetylcholine concentration of 10^-13 M. An in-depth study of the effect of phenozan and acetylcholine (exogenous) is described in Molochkina et al.⁴³

M.G. Sergeeva et al.⁴⁵ studied the effect of brufene (analgetic ibuprophene) on the activity of enzyme prostaglandin synthetase. Authors observed an increase in the rate of yield of products involving the enzyme when concentrations ofbrufene ranged from 10^-11 to 10^-14 M. At the concentration of 10^-6 M brufene acted as an inhibitor. However, the effect of SLD of brufene manifested itself only for cells but not for the isolated enzyme. This supported the authors' conclusion that low doses of brufene accelerate the escape of prostaglandin synthetase from cells into the reaction medium. The ratio of brufene molecules to the number of cells was 6:1. As in the experiments with protein kinase C and acetylcholinesterase, the activity of prostaglandinsynthetase depended on the brufene dose (a maximum was observed at 10^-12 M); a "dead zone" was also observed at concentrations from 10^-9 to 10^-6 M in the transition from stimulating to inhibiting effect.

G. Harrish and I. Dittmash⁴⁶ studied the effect of various ligands on the activity of enzymes in vivo and in vitro in cell cultures and cell-free media. A nonlinear and nonmonotonic dose-effect dependence was observed for all modifications. Effects of SLD were observed for a wide variety of enzymes: urate oxidase, acid phosphatase, cytochrome P-450 reductase, xanthine oxidase, dehydrogenase, and glutathione-S-transferase. Unfortunately, no quantitative data on the ratio of enzyme molecules to ligands is cited.

Similar bimodal dependences on a dose were discovered for the effect of antioxidant phenosan on the activity of regulatory enzymes aldolase and lactate dehydrogenase.¹³ It was shown that changes in the enzyme activity proceed simultaneously with the modification of viscous properties of membrane lipids.

As seen from results of the studies on the activity of protein kinase C and acetylcholinesterase, the effect of antioxidants in standard (10^-3 - 10^-5 M) and SL (10^-9 - 10^-18 M) concentrations is of the same type. If tocopherol acts as an inhibitor of protein kinase C at a concentration of 10^-4 M, it also inhibits the activity of this enzyme at 10^-15 to 10^-17 M; if phenozan activates protein kinase C at doses of 10^-5 to 10^-6 M, it also activates it at a dose of 10^-18 M; and ifphenozan is an inhibitor of acetylcholinesterase at doses of 10^-4 to 10^-5 M, it is also an inhibitor of this enzyme at doses of 10^-13 to 10^-14 M.

The above data might have lead to a conclusion that there is a common mechanism by which ligands influence the activity of enzymes at standard and SL doses. However, the obtained results cannot be explained in terms of classical biochemistry. The ligand! enzyme ratio that is on the average one ligand molecule per 10⁴ - 10⁹ enzyme molecules rules out the possibility of explaining the nature of SLD effects in the context of formation of ligand-enzyme complexes. Biochemical mechanisms of the response enhancement, for instance via systems of regulation of cyclic nucleotide or via a phosphoinositide cycle, applicable to effects at the cellular level, cannot be invoked to explain the effects in model systems.

The question is which other mechanisms can explain the effect of biologically active substances in concentrations of 10^-11 -10^-18 M on the enzymes' activity.

On the mechanism of effects of SLD of biologically active substances and low-level physical factors

To understand a mechanism of SLD effects on a biological object, it is necessary to explain the feasibility of reactions of such minute amounts of molecules with their targets from the kinetics standpoint. At concentrations of 10^-15 M and lower the van't Hoff's law of mass action is no longer valid and the term "concentration" to some degree loses its meaning.

L.A. Blumenfeld, A.Yu. Grosberg, and A.N. Tikhonov⁴⁷ used the Smoluchowski's equation to describe reactions between molecules and minute but macroscopically closed vesicles in terms of the statistical physics. It was shown that the law of mass action is violated when vesicle volume and/or the reaction's equilibrium constant are relatively small and an average number of free particles within a vesicle is on the order of one or less. In case of biological vesicles of a size 10² - 10³A fluctuations become very important.

Blumenfeld proposed a concept of parametric resonance as a possible mechanism of the effect of SL concentrations of biologically active substances at the level of cellular or subcellular organization.⁴⁸ According to the concept, a parametric resonance results from a coincidence of temporal parameters of intracellular processes initiated by biologically active substances and a characteristic time of a substance approaching its target. As a result of an active substance binding with its target, an enzyme (receptor) transforms into a conformationally nonequilibrium state which, at a certain stage of relaxation, ensures its maximum activity.

At very high concentrations of a biologically active substance, when a characteristic time of approach to a target is short and the approach frequency is high, the enzyme will be in a lowactivity nonequilibrium state. At a very low concentration, when a characteristic time of approaching the target by the substance is very long, almost the entire enzyme (receptor) remains in its initial low-activity nonequilibrium state. Only for doses at which the characteristic time of approaching the target and temporal parameters of intracellular processes, initiated by the substance, nearly coincide, one can expect an intermediate active nonequilibrium conformation at a maximum stationary concentration.

Calculations indicate that the peak activity of enzymes corresponds to substance doses of 10^-11 to 10^-15 M. At higher and lower doses the activity will be significantly lower. An equal or higher activity is predicted with an increase in concentration to 10^-5 - 10^-4 M, when another mechanism holds as a result of saturation of enzyme (receptor) sites with ligands. In the context of this concept, a biologically active substance may interact with its targets even if the constant of dissociation of its complex with the target will be considerably higher (several orders of magnitude) as compared with applied concentrations. In terms of this concept, the observed reduction in enzyme activity with an increase in agent dose can be explained.

We suggested another approach to the problem of the kinetic paradoxes⁴⁹ based on the concept of allosteric interactions of catalytic sites in enzyme molecules.

Assume that an enzyme or a receptor contains several sites with different affinity to the substrate, for example, the constant of dissociation for one site is 10^-13 M and for another 10^-8 M. After introduction of a low dose of a substance, its molecules bind predominantly with a high-activity site of the enzyme. With an increase in the dose, another site of the enzyme comes into play. It interacts allosterically with tile first site and thus reduces its affinity to the substrate. As a result, all molecules bound with the first site "leave" it. They can bind with it again only after the substance concentration approaches a constant of dissociation of the complex of ligand with the first site that is induced by the second site. This concept is used, in particular, to explain the response of olfactory receptors to a change in substrate dose.

The main problem with this and similar hypotheses lies in the explanation of the primary act of interaction of individual molecules with biotargets. In our studies, we discovered that an introduction of SLD of a biologically active substance into animal organism, cell culture, or model system containing a membrane suspension always results in some modification of the membrane structural properties. In turn, modification of the membrane structure may result in a modification of the functional state of the cell. Polymodality of the response may be attributed to switching of the mechanism depending on the substance's concentration range. However, how can one explain the initial act of interaction of a biologically active substance with a protein or a lipid of the membrane, if the ratio of the substance molecules to that of protein is 1 to 10⁶ - 10⁹?

There are two alternative approaches to the interpretation of mechanisms of SLD action. Some researchers believe that the common trends in the dose-effect dependence, modification in sensitivity of biological objects to a broad spectrum of factors (internal as well as external), and the heterogeneity of responses to SLD - all these testifY to the outward similarity only. In each particular case, its own intrinsic mechanism, targets, means of signal amplification, etc. should be sought. Other researchers, not denying the specificity of reaction in each particular case, advance a concept of a common character in response formation of biological objects to SLD and of a systemic modification in metabolism in response to signals from environment.

We share the second viewpoint, i.e., we attempt to determine the mechanisms that would rationally explain the effects of SLD not only at the cell and organism levels but also at the level of isolated biopolymers. This leads us first of all to look into the structural changes in water under the influence of SLD of physical factors and chemical substances.

The role of water and its possible structural modifications

Numerous (predominantly theoretical) studies on the role of structure of water in its biological activity may be divided into two groups. According to some researchers, long-lived clusters exist in a mere water; others believe that water clusters are induced after introduction of biologically active substances. In the context of these two viewpoints, we present a brief review of concepts regarding structural formations in water and their role in effecting biological systems as follows.

S.I. Aksenov and coauthors⁵⁰ point to a complex effect of water on the structure ofbiopolymers and biomembranes considering factors such as hydration of polar groups, competition of water molecules for hydrogen bonds in these structures, hydrophobic interactions, different dielectric permeability of free and bound water, etc. Analysis of experimental data enabled the authors to determine four stages of hydration associated with corresponding changes in the structure, dynamics, and function of photosynthetic membranes. The above processes are very sensitive to the influence of various factors even in SLD. According to the authors, SLD do not induce new stable structures in water but only affect the interactions of water with biopolymers and thus modify their functional activity. Arad et al. hold similar views.⁵¹

N.A. Bulienkov,^52,53 on the basis of the modular generalization of crystallographic data, developed all possible types of algorithms producing formation of hierarchic structures of bound water that correspond to morphological patterns widespread in animate nature. The hierarchic modular structures of bound water may playa role in forming spatial structures of biopolymers and biosystems on their basis. Instead of the conventional model of direct interaction of ligands with biotargets the author proposes a model oftheir interaction along the system-forming patterns where hydrogen bonds produce helix-shaped molecular carcasses. The effect of SLD of biologically active substances is also mediated through their action on carcasses of bound water molecules.

S.V Zenin, VI. Lobyshev, et aI.,^54,55,56 suggest that long-lived structures are ever present in pure water. Some conclusions about the structure of water and its solutions have been drawn on the basis of water luminescence studies.⁵⁶ The adsorption spectrum of distilled water exhibits two peaks: at 280 and 310 nm; the emission spectrum exhibits peaks at 360 and 410 nm. The luminescence intensity depends on the time of water storage and the presence of impurities that mayor may not have luminescence properties on tlleir own.

Luminescence spectra indicate that the structure of water in dilute solutions undergoes modification so that it takes it several days after preparation to come to the equilibrium. This process may be monotonic or alternating. The intensity of luminescence is sensitive to weak electromagnetic fields and the reaction strongly depends on the state of the solution at the moment of imposition of the field - it is at maximum when the system is far from equilibrium. Authors⁵⁶ consider structures of water and aqueous solutions being primary targets of dissolved substances (at low concentrations) and weak fields. The modification in water properties results in modification ofbiomembrane properties and, hence, in modification of the cell's functional activity.

To verifY the clathrate model of water and aqueous solutions, dielectric and differential scanning calorimetry techniques were invoked.⁵⁷ With tile former technique, it was confirmed that highly dilute solutions contain molecules bound in a form of clathrates. The latter technique made it possible to determine that phase transitions are accounted for by dissociation of clathrates at a certain temperature that is specific for each substance. By surrounding a molecule of a biologically active substance, clatluates are "impressed" by its structure and these impressions are fairly longlived (days to months).

S. V Zenin et aI.,^{54,55 assume that water has a uniform structure. A dissolved substance brings about certain "defects" in this structure that can persist for a long time and survive in successive dilutions up to complete absence of the substance.}

D. Arad et al.,⁵¹ and many other researchers, attach particular significance to hydration of protein molecules and disturbances in water-protein interactions by dissolved substances. The resulting modification in the functional activity of proteins is associated not with their direct interaction with biologically active substances, but with changes in a degree of the protein hydration and, consequently by the modification of its structure and activity.

Thus, many models are suggested in an attempt to explain reaction of bioobjects to SLD of biologically active substances in light of structural properties of water.⁵⁰,^{51,52,53,54,55,56,57,58} However, experimental verification of these models is inadequate and, what is most important, there is no experimental data confirming existence of long-living structural formations in water.

One cannot ignore, however, that many above-mentioned SLD paradoxes can be logically resolved in view of modification of water structure. For example, the fact that the sign or direction of the effect in some cases depends on the initial condition of a bioobject can find a plausible explanation. If an enzyme activity was too high, it would be reduced, and if it was too low - enhanced. It is most remarkable that the level to which the activity is modified is the same. This can be comprehended if the structure of water in a solution of a biologically active substance modifies the protein's structure in a similar way.

Involvement of water resolves another paradox of the SLD effect when its concentration is many orders of magnitude lower than the constant of dissociation of ligand-receptor complexs or the protein concentration.

In conclusion, it should be noted that the current broad interest in SLD effects stimulates study into structure of water and influence on it of various factors. A principally novel view on the mechanism of SLD effects emerged recently. Some authors believe that in the processes of dissolving and potentating a biologically active substance or during exposure of such a solution to electromagnetic fields active oxygen forms in water. This oxygen, and not the direct radiation or a biologically active substance, produce the effect on bioobjects.⁵⁹

There is a probability that new ways of explaining the SLD effect in the context of water structure may emerge based on studies of substances similar in structure and activity at doses of 10^-5 - 10^-4 M but differing in their SLD effects, i.e., when one substance has a SLD zone of activity while the other has not. ^59,60 A quantum-chemical study of such substances will reveal differences between them. However, whether these differences are essential for SLD effects and whether they can be associated with the effect of these substances on water structure remains unclear.

Examples of practical application of SLD

In the articles by T.A. Voronina et al.²³ and by LA. Yamskov et al.⁶¹ data are cited on physiological effects of medicines in SLD that are already approved (adhelon) or are under consideration for approval by the Russian Pharmacologic Committee (phenazepam in SLD). Authors discuss substantial advantages of these kinds of drugs.

Oncologists give special attention to the development of medicinal preparations in SLD. It is well-known that the main obstacle in chemotherapy of malignant tumors is the toxicity of antitumor compounds. Therefore, studies in the field of practical chemotherapy are aimed at development of antitumor preparations, and their application, that ensure their selective damaging effect on malignant cells and tumors and avoiding toxic reactions of normal tissues.

The phenomenon of induction of chromosomal aberrations in malignant tumors with SLD of an antitumor preparation Nitrosomethylurea that we uncovered⁶³ provides an encouragement for such an inquiry. A cytogenetic study of the effect of this preparation on the chromosomal structure of the Ehrlich tumor and L-121O leukosis showed that Nitrosomethylurea in a dose of 10^-17 mol/kg-day induces chromosomal aberrations in cells of both types of tumors with a single as well as multiple introduction. A share of cells with chromosomal restructuring was 15 to 20%, which is comparable with the cytogenetic effect of this substance in the therapeutic dose of 10^-4 mol/kg (25% of damaged cells in a population). A single introduction of Nitrosomethylurea in a dose of 10^-17 mol/kg results in prolongation of the average life span of animals with the L-121O leucosis by 40%.¹⁶

In subsequent studies biological effects of several types of antitumor preparations - Nitrosomethylurea, Cyclophosphane, and Adriamycine - was assessed for a range of SL concentrations differing from conventional therapeutic doses by ten and more orders of magnitude. The tumor model was Lewis carcinoma of lungs. Criteria for the biological effect assessment were changes in the life span and sizes of tumors in test animals. It was found that SLD of all these cytostatic agents exhibit biological effectiveness in the Lewis carcinoma model and that the effect direction depends on the nature of the agent and preparation dose.

Nitrosomethylurea produces both a strong inhibiting (at 10^-5 - 10^-10 M) and stimulating (at 10^-20 M) effects on the tumor. Cyclophosphane in all the studied SL concentrations stimulates while Adriamycine in all cases inhibits growth of tumors.⁶⁴

Thus, it was discovered that cytostatics, Adriamycine in particular, are capable of producing an antitumor effect in SLD (10^-10 - 10^-20 M), comparable with the effect of these preparations in usual therapeutic doses (10^-2 - 10^-3 M). Results of this experimental study provided a basis for the development of a program for a pilot clinical trial on Adriamycine in SLD for treatment of patients with various malignancies. Adjacent to this direction are studies on the antimetastatic effect of some medicinal preparations.^10,65 The obtained results add to the accumulated information on reactions of living systems to very weak interventions and testify to the necessity for further experimental studies in this field.

It can be concluded that even at the present stage of investigation of SLD effects of biologically active substances one can see a broad perspective for practical application of the obtained results in the near future while progress in the understanding of the mechanisms of SLD effects may be expected sometime in the remote future.

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26. Patent RF no. 2102986.