**4.** *Future prospect***: Nuclear spin as factor of reliability in cell nanoreactors**

Thus, our data have documented, for the first time, the magnetic-isotope effect of magnesium-25 *in vivo*. Factual evidence of MIE, on its own, indicates that there is a spinselective "bottle-neck" of the process under investigation. Ions of Mg2+ perform not only the cofactor functions in synthesis and hydrolysis of ATP. In addition, they have the impact on

It has been known that kinetics of recovery of yeast cells from radiation injuries may be represented by a function representing the reduction of the effective radiation dose, *D*eff,

 *Deff(t)=Do[k+(1-k)exp(-βt)]*  In this model of A. Novick and L. Szilard, *D*0 is the radiation dose, *t* is time of post-radiation

irreversible injuries (Grodzinsky et al., 1987; Koltover et al., 1980; Novick & Szilard, 1949). Table 2 represents values of the kinetics parameters resulting from these experiments. While the fraction of irreparable injuries remained almost the same, the value of the rate constant

of the post-radiation recovery was twice higher for the cells enriched with 25Mg than for the cells enriched with 24Mg. This is decisive evidence that the magnetic isotope of magnesium essentially more effectively promotes the recovery of cells from radiation damages. Thus, we have, for the first time, documented the magnetic-isotope effect in radiation biology

24Mg 0.032 ± 0.003 0.70 ± 0.14 25Mg 0.058 ± 0.004\* 0.61 ± 0.12 Table 2. Effect of magnetic 25Mg isotope on postradiation recovery of *S. cerevisiae*, diploid yeast cells, after short wave UV irradiation. \*Difference between the means is significant at

One might suggest that the observed effects in our experiments with bacteria and yeast cells were caused by different levels of impurities in the growth media complemented with different isotopes of magnesium. However, it could hardly be the case. First, according to the mass-spectrometry data, amounts of contaminant elements in the stock solutions of the isotopes did not exceed 20-30 ppm, be it sulphate of 24Mg, 25Mg, or 26Mg. Second, amounts of the contaminants that were administered in the liquid growth media with glucose and other basic components have significantly exceeded amounts of the same contaminants administered with much less additions of the isotope stock solutions. Besides, the impurities that were administered into the growth media from the basic components, as well as the element contents of the solid nutrient agar media, were obviously the same in all experiments, independently of the magnesium isotopes. Hence, one can disregard impurities as a possible reason of higher efficiency of magnetic 25Mg than that of nonmagnetic 24Mg and 26Mg. It is apparent that the cells in the above cited experiments perceive the difference between magnetic and non-magnetic isotopes of magnesium, i.e.,

**4.** *Future prospect***: Nuclear spin as factor of reliability in cell nanoreactors**  Thus, our data have documented, for the first time, the magnetic-isotope effect of magnesium-25 *in vivo*. Factual evidence of MIE, on its own, indicates that there is a spinselective "bottle-neck" of the process under investigation. Ions of Mg2+ perform not only the cofactor functions in synthesis and hydrolysis of ATP. In addition, they have the impact on

, *h*<sup>1</sup> *k*

is the recovery rate constant, and *к* is the fraction of

with time:

recovery in the nutrient-free water,

(Grodzinsky et al., 2011).

*P* = 0.02 (Grodzinsky et al., 2011).

they perceive the nuclear spin's magnetic field of 25Mg.

the structure-functional properties of RNA, RNA-polymerase, ribonuclease, and so on. Besides, there are the specialized proteins which regulate homeostasis and transport of Mg2+ in living cells (Romani, 2011). Moreover, ions of Mg2+ may work as the intracellular second messengers (Li et al., 2011). Up to date, however, there have not been findings of MIE except for the enzyme synthesis of ATP in the above cited papers (Buchachenko, et al., 2005, 2008, 2011). The similar MIE of 25Mg is assumed to be in our experiments. Indeed, adaptation of cells to novel growth conditions requires a large variety of stress proteins to be synthesized and ATP, as the main source of energy in microbial cells, is most likely to be the limiting substrate for the adaptation metabolic processes. Similarly, a large variety of biosynthesis is required for recovery of cells from radiation injuries. It is reasonable to suggest that the kinetics of post-radiation recovery is also limited by spin-selective synthesis of ATP as the "bottle-neck". The post-radiation recovery proceeds with higher rate when the cell nanoreactors run on the magnetic isotope of magnesium, because the nuclear spin of 25Mg catalyzes the ATP synthesis, hereby supplying the cells with more amount of ATP.

The lower level of superoxide dismutase (SOD) activity in the *E. coli* cells enriched with 25Mg, by about 40 per cent when compared to the cells that were grown on nonmagnetic 24Mg (Bogatyrenko et al., 2009a,b), can be also flow from the beneficial effect of the magnetic isotope on the ATP synthesis. The cell nanoreactors of oxidative phosphorylation have very ancient evolutionary origin and, hence, seem to be ones of the most reliable biomolecular machines. But yet their reliability ("robustness") characteristics are not perfect because these molecular machines experience conformational fluctuations (Grodzinsky et al., 1987; Koltover, 1997). It is well known that normal elementary acts of electron transfer on the electron transport chains, be it mitochondria or prokaryotes, alternate with random malfunctions when an electron, rather than waits for transport to the next enzyme of the electron-transport chain, goes directly to an adjacent oxygen molecule. Such an electron leakage results in production of О2●─. Chemical products of О<sup>2</sup> ●─, the so-called reactive oxygen species (ROS), are toxic and initiate free-radical damages in the biopolymer nanoreactors (see, e.g., Chance et al., 1979; Koltover, 2009, 2010a). SOD catalyzes the reaction of dismutation of О<sup>2</sup> ●─ into hydrogen peroxide (H2O2) and oxygen, thus protecting cell structures from О2●─ and its toxic chemical products. It makes its defense "job" in cooperation with two other specific enzymes, catalase and glutathione peroxidase, which catalyze decomposition of H2O2 into nontoxic reagents, namely H2O and O2 (see, e.g., Nelson & Cox, 2008, and references therein).

As a rule, the level of the SOD activity is adjusted to the intracellular level of О<sup>2</sup> ●─. If SOD activity decreases or increases, it normally reflects the relevant decrease or increase in the production of О2●─ as faulty by-products of the electron-transport nanoreactors of oxidative phosphorylation (Imlay & Fridovich, 1991; Koltover, 2010a, 2010b, 2011). Hence, the lower level of SOD activity testifies the lower production of О<sup>2</sup> ●─ in the case when the cells are supplied with the magnetic isotope. As cited above, oxidative phosphorylation of ADP proceeds faster with 25Mg by comparison with 24Mg (Buchachenko et al., 2005). Since 25Mg is more effective cofactor of oxidative phosphorylation, it transpires that the ATP synthase operates faster with the magnetic magnesium nucleus by comparison with the nonmagnetic ones. Meanwhile, under normal coupled conditions, the electron transport is subjected to the "backpressure" of the respiration-generated transmembrane electrochemical H+ gradient. The partial dissipating of this gradient via the acceleration of ADP

Stable Magnetic Isotopes as a New Trend in Biomedicine 119

Apart from magnesium, there are many other elements which have both kind of stable isotopes, nonmagnetic and magnetic ones, amongst them – carbon, oxygen, calcium, iron and zinc (see Table 1). In passing, nuclear spin of 17O should lift the spin ban over the reaction of the mitochondrial ubisemiquinone radicals with oxygen (see Fig. 1c), thereby

and more free-radical damages due to 17O in comparison with nonmagnetic 16O and 18O. The pro-oxidant action of 17O can reveal itself as the selective enrichment of free-radical peroxidation products with this unfavorable magnetic isotope as compared with ordinary

Factual evidence of magnetic isotope effect, on its own, indicates that the "bottle-neck" of the process under investigation is a free-radical or ion-radical reaction. Within the scope of free radical research, MIE can serve as the unique indicator to elucidate if the reaction under study proceeds through a free-radical or ion-radical pair as the key operand of the reaction. Up to date, however, there have been no efforts to detect magnetic-isotope effects for other

Our experimental data have documented, for the first time, the beneficial magnetic-isotope effects of 25Mg *in vivo*. Although the detailed mechanisms of the ability of the living cell to perceive magnetic properties of the atomic nuclei require further investigations, the "nuclear spin catalysis", as such, always and unambiguously indicates that the reaction under study is a spin-selective process with participation of paramagnetic intermediates, such as free radical pair, ion-radical pair or triplet state that undergo the spin conversion. Along this line the general principles of spin chemistry, amongst them – control of biochemical reactivity in living cells by the selective modification with stable magnetic isotopes, hold considerable promise. In part, the preventive antioxidant effect of 25Mg opens the ways toward the novel biomedicine of anti-stress anti-aging drugs enriched with the magnetic-isotopes. The discovery of the magnetic-isotope effect in radiation biology opens up the way to the development of novel radio-protectors, based on the magnetic isotopy. Furthermore, inasmuch as the electron and nuclear spin moments can be changed by external magnetic fields, it makes possible to exert control over efficiency and reliability of biomolecular nanoreactors with the help of relevant magnetic and electromagnetic fields.

Financial support from Russian Foundation for Basic Research, projects no. 10-03-01203a and 10-04-90408-Ukr\_a, is greatly acknowledged. I am grateful to L.V. Avdeeva-Tumanova, T.N. Bogatyrenko, & E.A. Kudryashova (Institute of Problems of Chemical Physics, RAS, Chernogolovka, Moscow Region), V.L. Berdinsky, D.G. Deryabin, E.A. Royba, & U.G. Shevchenko (Orenburg State University, Orenburg), T.A. Evstykhina & V.G. Korolev (Saint-Petersburg Institute of Nuclear Physics, RAS, Gatchina, Leningrad Region), and D.M. Grodzinsky & Y.A. Kutlakhmedov (Institute of Cell Biology and Genetic Engineering, Ukraine Academy of Sciences, Kyiv, Ukraine) for their fruitful collaboration. I am especially appreciated to Kathryn S. Tarasevich-Laukhina for her excellent assistance at the very

●─. As a result, one can expect for more reactive oxygen radicals

catalyzing formation of О<sup>2</sup>

**5. Conclusions and outlook** 

**6. Acknowledgments** 

beginning of this work.

elements, except magnesium, in biopolymer nanoreactors.

metabolites.

phosphorylation has been shown to result in the decreased production of О<sup>2</sup> ●─ (see, e.g., Mailloux et al., 2011). Hence, the higher rate of oxidative phosphorylation with magnetic 25Mg, in comparison with nonmagnetic 24Mg, through the reduction of the H+-gradient "backpressure" should decrease the false electron leakage onto oxygen, thereby reducing the yield of free radicals О<sup>2</sup> ●─ as by-products of the electron transport.

From the point of view of chemical kinetics, with decrease in the rate of oxidative phosphorylation, there is the retardation of electron transport in the sites of the electrontransport chains which are coupled with phosphorylation of ADP. Inasmuch as the input of electron-transport nanoreactors becomes overflowed with electrons ("electron-transport jam"), the probability of electron leakage on oxygen increases. The more is acceleration of the ATP synthesis, the less is probability of the "jam". Hence, the yield of О<sup>2</sup> ●─ as the byproducts of electron transport is bound to be much lower with 25Mg-ADP by comparison with 24Mg-ADP or 26Mg-ADP.

Thus, with magnetic 25Mg, the biopolymer nanoreactors of oxidative phosphorylation operate not only more effective but more reliable too, in comparison with their operation on non-magnetic isotopes 24Mg and 26Mg. Downgrading production of О2●─, the magnetic isotope of magnesium produces, actually, a beneficial preventive antioxidant effect. This antioxidant effect of the nuclear spin, that 25Mg favors the less production of reactive oxygen species, should obviously increase longevity of the electron-transport nanoreactors. Therefore, it can reveal itself in nature as the kinetic nuclear-spin selection of the favorable isotope, namely, the kinetic isotope enrichment with magnetic 25Mg in the processes of recycling and regeneration of the electron-transport nanoreactors. For example, one can expect for the enrichment with the favorable magnetic 25Mg in recycling and regeneration of cell mitochondria with aging of the cells (Koltover, 2007, 2010b).

Contrary, in the case of photosynthetic nanoreactors, one can predict undesirable prooxidant effect of the magnetic nucleus of 25Mg. Indeed, it is known that the function of the vast majority of chlorophyll molecules (Chl), as the derivatives of the magnesiumprotoporphyrin complexes, is to absorb light energy and transfer it to the specific energy sinks, the so-called reaction centers of the photosynthetic nanoreactors (Nelson & Cox, 2008). While performing this energy-transfer function, the light-exited Chl molecules are in the singlet state (1Chl\*, electron spin *S*=0). However, there is probability of the radiationless relaxation into the triplet state (3Chl, *S*=1) followed by formation of singlet oxygen, 1O2, the molecules of which are substantially more reactive by comparison with usual triplet O2 molecules and, thereafter, produce oxidative damages. As nuclear spin of 25Mg can catalyze the conversion of 1Chl\* into the triplet 3Chl, one can expect for the higher yield of 1O2 and, thereafter, more photodynamic damages in the cells with the chlorophyll molecules containing 25Mg instead of the spinless 24Mg or 26Mg. Correspondingly, it is beyond reason to hope for selection of the magnetic 25Mg, in the case of algae or green plants. Besides, the functional disadvantage of 25Mg should be followed by increased synthesis of carotenoids and other natural antioxidants. Indeed, measurements of magnesium isotopic composition of the chlorophylls extracted from cyanobacteria and similar analysis of the chlorophyll forms in the leaves of English Ivy (*Hedera helix L*.) have revealed the isotope distribution following usual classical mass-isotope effect with no evidence for depletion or enrichment of 25Mg (Black et al., 2007).

Apart from magnesium, there are many other elements which have both kind of stable isotopes, nonmagnetic and magnetic ones, amongst them – carbon, oxygen, calcium, iron and zinc (see Table 1). In passing, nuclear spin of 17O should lift the spin ban over the reaction of the mitochondrial ubisemiquinone radicals with oxygen (see Fig. 1c), thereby catalyzing formation of О<sup>2</sup> ●─. As a result, one can expect for more reactive oxygen radicals and more free-radical damages due to 17O in comparison with nonmagnetic 16O and 18O. The pro-oxidant action of 17O can reveal itself as the selective enrichment of free-radical peroxidation products with this unfavorable magnetic isotope as compared with ordinary metabolites.
