**3.** *In situ***: Magnetic-isotope catalysis in living cells**

There is a great variety of chemical elements in biomolecular nanoreactors of living cells (see Table 1). Certain of them are only represented by magnetic isotopes, among them – hydrogen, nitrogen, sodium, phosphorus, potassium, manganese and so on. However, there are chemical elements which have both kinds of stable isotopes, nonmagnetic and magnetic ones, among them – carbon, oxygen, magnesium, calcium, iron, zinc and others (Table 1). Correspondingly, these are the elements which are required to search for magnetic-isotope effects in living cells.

In this regard, magnesium is of particular interest. There are three stable isotopes of magnesium, 24Mg, 25Mg and 26Mg with natural abundance about 79, 10 and 11 %. Among them, only 25Mg has the nuclear spin (*I*=5/2) that produces the magnetic field. Two other isotopes are spinless (*I*=0) and, hence, produce no magnetic fields (Grant & Harris, 1996). As the most abundant intracellular divalent cation, Mg2+ is essential to regulate numerous cellular functions and enzymes. Ions of Mg2+ serve as obligatory cofactors in catalytic centers of many enzymes including ATP-synthase as the primary producer of ATP in mitochondria, chloroplasts, bacteria and archaea (Nelson & Cox, 2008). Moreover, a novel role for Mg2+ as an intracellular second messenger has been recently discovered (Li et al., 2011). Besides, the difference in masses between the isotopes of magnesium is much less, in percentage term, than that for the isotopes of carbon, for example, thereby minimizing the classical mass-isotope effect.

Stable isotopes of magnesium, namely nutrient solutions highly enriched with 25Mg or 26Mg, have been used for many years as *in vivo* tracers to determine magnesium absorption in human subjects, animals and plants models (see., e.g., Coudray et al., 2006; Weatherall et al., 2006). It is reasonable that the problem of possible beneficial effects of the magnetic isotope

Stable Magnetic Isotopes as a New Trend in Biomedicine 111

<0.05

59Co 100 7/2 4.6388 Vitamin B12, biosynthesis of heme

2.2206 2.3790

0.8735

0.5333

0.9099

0.9290

127I 100 5/2 2.7939 Structure-functional unit of thyroid

was not posed in the cited papers. There have been attempts to use the non-radioactive isotopes to make the oxidative biomolecules more stable against free-radical oxidation. It was found that deuterated polyunsaturated fatty acids protect yeast cells against the toxic effects of lipid autoxidation products (Hill et al., 2011). Both natural isotopes of hydrogen,

75As 100 3/2 1.4349 Activator of glycerylaldehyde

Biological functions

carboxylase, etc.

dismutase, etc.

proteins, etc.

dehydrogenase,

Microbial Mn-superoxide dismutase, glutamine synthase, rat liver pyruvate

Heme- and non-heme proteins of electron transport, Fe-superoxide

Cytochrome oxidase, Cu,Zn-

laccase, ascorbate oxidase, monoamine oxidase, etc.

phosphate dehydrogenase

Structure-functional unit of glutathione peroxidase

Structure-functional unit of flavoproteins, nitrogenase, nitrate reductase, sulphite oxidase, xantine

oxidase, etc.

hormones

superoxide dismutase, ceruloplasmin,

Cu, Zn-superoxide dismutase, DNApolymerase, carbonic anhydrase, alcohol dehydrogenase, pyruvate

pyruvate carboxylase, aldolase, etc.

Magnetic moment (), in nuclear magnetons (*eh*/4*Mc*)

Nucleus Natural

54Fe 56Fe 57Fe 58Fe

63Cu 65Cu

64Zn 66Zn 67Zn 68Zn

74Se 76Se 77Se 78Se 80Se 81Se

92Mo 94Mo 95Mo 96Mo 97Mo 98Mo 100Mo abundance, in %

> 5.82 91.66 2.245 0.33

> 69.09 30.91

48.6 27.9 4.12 18.8

0.87 9.02 7.58 23.52 49.82 9.19

14.84 9.25 15.92 16.68 9.55 24.13 9.63

Table 1. Stable isotopes in biological systems.

Nuclear spin (*I*), in units of *h*/2

> 3/2 3/2

55Mn 100 5/2 3.4610


2.79270 0.85738

0 0.70216

0.40357 0.28304

0 0.18930 0

19F 100 1/2 2.6273 Structure unit of dental enamel

<sup>0</sup>0.85471

1/2 0.55477

0.64274

0.82089 0.68329

0.39094 0.21453

1.3153

Biological functions

acid, etc.

etc.

Structure unit of water, biomolecules, mitochondrial bioenergetics, etc.

extracellular buffer (HCO3─/H2CO3)

Structure unit of amino acids, nucleic

Main extracellular cation, functional

electrochemical potential, deposit component of bond tissue, etc.

Main intracellular cation, structure and functional unit of chlorophylls in photosynthesis, obligatory cofactor of Mg2+-dependent enzymes, including oxidative phosphorylation, glycolysis, synthesis of DNA and RNA, etc.

Main structure unit of exoskeleton in radiolarian and diatomic algae

phospholipids, Pi as main anion, Pi as regulator factor of transcription and

structure unit of cystine, cysteine and methionine, and glutathione, etc.

Main intracellular cation, functional

Main structure unit of bond tissue (Ca3(PO4)2), regulator of membrane ion channels, Ca2+-dependent myosin

<sup>2</sup>─),

ADP, ATP, nucleic acids,

Main intracellular anion (SO4

Main intracellular anion

unit of transmembrane electrochemical potential, etc.

and other ATPases, etc.

translation, etc.

Structure unit of biomolecules, intracellular cation (HCO3─),

Structure unit of water and biomolecules, biological oxidation,

unit of transmembrane

Magnetic moment (), in nuclear magnetons (*eh*/4*Mc*)

Nucleus Natural

1H 2H

12C 13C

14N 15N

16O 17O 18O

24Mg 25Mg 26Mg

> 28Si 29Si

32S 33S 34S

35Cl 37Cl

39K

40Ca 42Ca 43Ca 44Ca 48Ca

41K 93.08

abundance, in %

> 99.984 0.016

98.89 1.11

99.635 0.365

99.759 0.037 0.204

78.7 10.13 11.17

92.21 4.7

95.02 0.74 4.22

75.4 24.6

6.91

96.97 0.64 0.13 2.06 0.18

Nuclear spin (*I*), in units of *h*/2

> 1/2 1

> 0 1/2

> 1 1/2

> 0 5/2 0

> 0 5/2

> > 0

0 3/2 0

3/2 3/2

3/2 3/2

23Na 100% 3/2 2.2161

31P 100 1/2 1.1305


Table 1. Stable isotopes in biological systems.

was not posed in the cited papers. There have been attempts to use the non-radioactive isotopes to make the oxidative biomolecules more stable against free-radical oxidation. It was found that deuterated polyunsaturated fatty acids protect yeast cells against the toxic effects of lipid autoxidation products (Hill et al., 2011). Both natural isotopes of hydrogen,

Stable Magnetic Isotopes as a New Trend in Biomedicine 113

Another striking effect of the magnetic isotope of magnesium has been detected when measuring activity of superoxide dismutase (SOD), the main antioxidant enzyme of the cells. The cells enriched with 25Mg demonstrate the reduced activity of SOD, about 40 percent, when compared to the cells enriched with 24Mg (Fig. 3b). It is generally known that *E. coli* growing aerobically contain MnSOD and FeSOD (Imlay & Fridovich, 1991; Nelson & Cox, 2008). Inasmuch as the total SOD activity is normally adjusted to the intracellular level of О2●─, the reduced level of SOD in the cells can be considered as evidence for lower

Fig. 3. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on *E. coli*, strain BB (Bogatyrenko et al., 2009a). Left: ability of the cells enriched with magnetic 25Mg or nonmagnetic 24Mg to form colonies on the nutrition agar. Right: activity of

superoxide dismutase in the cells enriched with magnetic 25Mg or nonmagnetic 24Mg. Data are indicated as *m* ± *SD*, *N* = 3. The difference of the mean values (*m*) for magnetic 25Mg *vs*.

Thereafter, these experiments have been replicated in cooperation with the microbiologists of Orenburg State University using another strain of *E. coli*, K12TG1 (Koltover et al., 2012). After cultivation for 24 h in the artificial liquid minimal M9-medium without magnesium (and without tap water), the cells were suspended in the fresh M9-medium supplemented with different isotopes of magnesium as 0.26 g of 24MgSO4, 25MgSO4 or 26MgSO4 per liter, and grown aerobically at 37 °C. To obtain reproducible results, three parallel experiments with each kind of the isotopes have been simultaneously performed, i.e. bacteria supplied with 24Mg, or 25Mg, or 26Mg, three samples each, were simultaneously tested in the same experimental succession. After reaching the stationary phase with OD620 of about 0.5, the cells were tested for their ability to form colonies after inoculation on the agar plate's

From the experimental results, shown on Fig. 4, one can see that the amount of CFU formed by the bacteria, previously grown on magnetic 25Mg, has turned out to be almost twice higher in comparison with the bacteria which were previously grown on nonmagnetic 24Mg and 26Mg. Noteworthy, there has been no significant difference between nonmagnetic 24Mg and 26Mg in their effects on CFU. It gives evidence that there is the magnetic isotope effect of 25Mg rather than a classical mass-dependent isotope effect of the magnesium isotopes.

production of О2●─ as the failure by-product of cell respiration.

nonmagnetic 24Mg is statistically significant at *P* ≤ 0.01.

surfaces.

1H and 2H (D), are magnetic ones, but they have the twofold difference in masses. Consequently, the observed protection should be ascribed to the mass-isotope effect instead of the magnetic isotopy. In the *in vitro* study of cleavage of deuterated DNA by the hydroxyl radical, the value of the kinetic effect was found to be close to 2, just the mass-ratio of hydrogen and deuterium. Similar mass-isotopic effects are, presumably, anticipated for stable isotopes of nitrogen 15N versus 14N and for carbon 13C that is 8% heavier than 12C (Hill et al., 2011).

Recently, the smart methods of labeling of nematodes *Caenorhabditis elegans*, which are commonly used in gerontology, with 13C and 15N have been developed by feeding the worms with heavy isotope–labeled *Escherichia coli* (Fredens et al., 2011; Larance et al., 2011). However, with regards to the ideas of using the stable isotopes and their mass-isotope effects to stabilize cells against free-radical oxidation and, thereby, improve the living conditions and even extend the lifespan, there are doubts if high amounts of 13C and 15N in the numerous proteins, DNA, RNA, and other molecules of living cells will have, eventually, beneficial effects rather than harmful ones.

In searching beneficial isotope effects, including possible distinctions between the effects of magnetic and nonmagnetic isotopes, the very first step should be preparation of the cells enriched with different isotopes of magnesium. With this aim, we used bacteria *E. coli*, the commonly accepted cell model, and the growth media of the identical chemical composition with one exception, that they were supplemented with different isotopes of magnesium, magnetic 25Mg and nonmagnetic 24Mg or 26Mg, as 25MgSO4, 24MgSO4 or 26MgSO4 (Bogatyrenko et al., 2009a, 2009b; Koltover et al., 2012). The oxides of magnesium, 24MgO, 25MgO and 26MgO, with isotope enrichment 99.9, 98.8 and 97.7 atom percent, correspondingly, were purchased from RosAtom, Russia. 24MgSO4, 25MgSO4 and 26MgSO4 were prepared from the relevant oxides by using a standard acidic treatment with analytically pure sulphuric acid.

The pioneering studies have been done in Institute of Problems of Chemical Physics, RAS. Bacteria *E. coli*, strain BB, were cultivated in accordance with the standard design on the artificial liquid minimal M9-media, composed from 8 g of glucose, 2 g of NH4Cl, 12 g of Na2HPO4, 6 g of K2HPO4, 1 g of NaCl in 750 ml of distilled water and 250 ml of tap water as the source of microelements. The growth media were supplemented with the isotopes of magnesium so that the final concentration of 24MgSO4 or 25MgSO4 was 2.2 mM per liter of the media. Cells were grown aerobically at 37 °C with shaking, harvested at the late growth ("stationary" phase at OD600 of about 0.5), and viability of the cells was tested as their ability to form colonies (colony-forming units, CFU) on the solid nutrient BCP agar using standard Petri's dishes.

The experimental data are presented in Fig. 3. The striking effect of the magnetic isotope, 25Mg, has been detected when tallying up the colony forming units. The standard nutrient agar contains all components necessary for normal growth of cells, including magnesium. Nevertheless, the amount of CFU formed by the bacteria, which were previously grown on magnetic 25Mg, has turned out to be about 40 percent higher in comparison with the bacteria, which were previously grown on nonmagnetic 24Mg (Fig. 3a). Thus, the cells which have been previously enriched with the magnetic isotope of magnesium demonstrate the essentially higher viability in comparison to the cells enriched with the nonmagnetic isotope.

1H and 2H (D), are magnetic ones, but they have the twofold difference in masses. Consequently, the observed protection should be ascribed to the mass-isotope effect instead of the magnetic isotopy. In the *in vitro* study of cleavage of deuterated DNA by the hydroxyl radical, the value of the kinetic effect was found to be close to 2, just the mass-ratio of hydrogen and deuterium. Similar mass-isotopic effects are, presumably, anticipated for stable isotopes of nitrogen 15N versus 14N and for carbon 13C that is 8% heavier than 12C (Hill

Recently, the smart methods of labeling of nematodes *Caenorhabditis elegans*, which are commonly used in gerontology, with 13C and 15N have been developed by feeding the worms with heavy isotope–labeled *Escherichia coli* (Fredens et al., 2011; Larance et al., 2011). However, with regards to the ideas of using the stable isotopes and their mass-isotope effects to stabilize cells against free-radical oxidation and, thereby, improve the living conditions and even extend the lifespan, there are doubts if high amounts of 13C and 15N in the numerous proteins, DNA, RNA, and other molecules of living cells will have,

In searching beneficial isotope effects, including possible distinctions between the effects of magnetic and nonmagnetic isotopes, the very first step should be preparation of the cells enriched with different isotopes of magnesium. With this aim, we used bacteria *E. coli*, the commonly accepted cell model, and the growth media of the identical chemical composition with one exception, that they were supplemented with different isotopes of magnesium, magnetic 25Mg and nonmagnetic 24Mg or 26Mg, as 25MgSO4, 24MgSO4 or 26MgSO4 (Bogatyrenko et al., 2009a, 2009b; Koltover et al., 2012). The oxides of magnesium, 24MgO, 25MgO and 26MgO, with isotope enrichment 99.9, 98.8 and 97.7 atom percent, correspondingly, were purchased from RosAtom, Russia. 24MgSO4, 25MgSO4 and 26MgSO4 were prepared from the relevant oxides by using a standard acidic treatment with

The pioneering studies have been done in Institute of Problems of Chemical Physics, RAS. Bacteria *E. coli*, strain BB, were cultivated in accordance with the standard design on the artificial liquid minimal M9-media, composed from 8 g of glucose, 2 g of NH4Cl, 12 g of Na2HPO4, 6 g of K2HPO4, 1 g of NaCl in 750 ml of distilled water and 250 ml of tap water as the source of microelements. The growth media were supplemented with the isotopes of magnesium so that the final concentration of 24MgSO4 or 25MgSO4 was 2.2 mM per liter of the media. Cells were grown aerobically at 37 °C with shaking, harvested at the late growth ("stationary" phase at OD600 of about 0.5), and viability of the cells was tested as their ability to form colonies (colony-forming units, CFU) on the solid nutrient BCP agar using standard

The experimental data are presented in Fig. 3. The striking effect of the magnetic isotope, 25Mg, has been detected when tallying up the colony forming units. The standard nutrient agar contains all components necessary for normal growth of cells, including magnesium. Nevertheless, the amount of CFU formed by the bacteria, which were previously grown on magnetic 25Mg, has turned out to be about 40 percent higher in comparison with the bacteria, which were previously grown on nonmagnetic 24Mg (Fig. 3a). Thus, the cells which have been previously enriched with the magnetic isotope of magnesium demonstrate the essentially higher viability in comparison to the cells enriched with the nonmagnetic

eventually, beneficial effects rather than harmful ones.

analytically pure sulphuric acid.

Petri's dishes.

isotope.

et al., 2011).

Another striking effect of the magnetic isotope of magnesium has been detected when measuring activity of superoxide dismutase (SOD), the main antioxidant enzyme of the cells. The cells enriched with 25Mg demonstrate the reduced activity of SOD, about 40 percent, when compared to the cells enriched with 24Mg (Fig. 3b). It is generally known that *E. coli* growing aerobically contain MnSOD and FeSOD (Imlay & Fridovich, 1991; Nelson & Cox, 2008). Inasmuch as the total SOD activity is normally adjusted to the intracellular level of О2●─, the reduced level of SOD in the cells can be considered as evidence for lower production of О2●─ as the failure by-product of cell respiration.

Fig. 3. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on *E. coli*, strain BB (Bogatyrenko et al., 2009a). Left: ability of the cells enriched with magnetic 25Mg or nonmagnetic 24Mg to form colonies on the nutrition agar. Right: activity of superoxide dismutase in the cells enriched with magnetic 25Mg or nonmagnetic 24Mg. Data are indicated as *m* ± *SD*, *N* = 3. The difference of the mean values (*m*) for magnetic 25Mg *vs*. nonmagnetic 24Mg is statistically significant at *P* ≤ 0.01.

Thereafter, these experiments have been replicated in cooperation with the microbiologists of Orenburg State University using another strain of *E. coli*, K12TG1 (Koltover et al., 2012). After cultivation for 24 h in the artificial liquid minimal M9-medium without magnesium (and without tap water), the cells were suspended in the fresh M9-medium supplemented with different isotopes of magnesium as 0.26 g of 24MgSO4, 25MgSO4 or 26MgSO4 per liter, and grown aerobically at 37 °C. To obtain reproducible results, three parallel experiments with each kind of the isotopes have been simultaneously performed, i.e. bacteria supplied with 24Mg, or 25Mg, or 26Mg, three samples each, were simultaneously tested in the same experimental succession. After reaching the stationary phase with OD620 of about 0.5, the cells were tested for their ability to form colonies after inoculation on the agar plate's surfaces.

From the experimental results, shown on Fig. 4, one can see that the amount of CFU formed by the bacteria, previously grown on magnetic 25Mg, has turned out to be almost twice higher in comparison with the bacteria which were previously grown on nonmagnetic 24Mg and 26Mg. Noteworthy, there has been no significant difference between nonmagnetic 24Mg and 26Mg in their effects on CFU. It gives evidence that there is the magnetic isotope effect of 25Mg rather than a classical mass-dependent isotope effect of the magnesium isotopes.

Stable Magnetic Isotopes as a New Trend in Biomedicine 115

Of special interest are searches for magnetic-isotope effects in the processes of recovery of cells from radiation injuries. The reason is that any factor, capable to influence on efficiency and reliability of cell nanoreactors, shows up itself most vividly under drastic conditions of post-radiation recovery (Koltover et al., 1980; Grodzinsky et al., 1987; Koltover, 1997).

We undertook the investigation of effects of magnetic and nonmagnetic isotopes of magnesium on post-radiation recovery of *S. cerevisiae* (Grodzinsky et al., 2011). The yeast cells (diploid strain MATα ade2Δ248 leu2-3,112 ura3-160,188 trp1 Δ:kanr) were cultivated on the standard nutrient liquid media M3 supplemented with 24MgSO4 or 25MgSO4. After three days of the cultivation under aerobic conditions at 30 0C, the cells were washed from the nutrient liquid and suspended in nutrient-free ("fasting") media, i.e., sterile phosphate buffer, pH 7.0. Then, the cells were irradiated by the short-wave ultraviolet light (*λ* ≈240 nm, the dose ≈190 *J*/m2), whereupon they were left in the nutrient-free water at 30 0C (with shaking) to study kinetics of the post-radiation recovery of the cells. For this kinetics study, the aliquots of the cells were periodically seeded on the standard nutrient (Petri dishes) and

Fig. 5. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on post-radiation recovery of *S. cerevisiae*, diploid yeast cells. The cells enriched with magnetic 25Mg or nonmagnetic 24Mg were irradiated by the short-wave UV-C light (*λ* = 240-260). Survival of cells was estimated as their ability to form colonies on the nutrition agar: 1 - recovery of the cells enriched with 25Mg; 2 – recovery of the cells enriched with 24Mg

The survival of cells transferred to agar immediately after irradiation was not more than a few percent (Fig. 5). In this case the injured genetic structures in most of the cells could not be repaired before the onset of mitosis, and nonviable daughter cells were produced. Incubation in nutrient-free water, in which the cells do not divide, provides sufficient time for repair processes and leads to a corresponding increase in survival. From the kinetics curves represented on this figure we notice that the cells enriched with magnetic isotope of magnesium, 25Mg, are recovered essentially more effectively than the cell enriched with the

the cell survival was monitored by the standard CFU technique.

(Grodzinsky et al., 2011).

nonmagnetic 24Mg.

Fig. 4. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on *E. coli*, strain K12TG1 (Koltover et al., 2012). Left: Ability of the cells enriched with magnetic 25Mg or nonmagnetic 24Mg and 26Mg to form colonies on the nutrition agar. Data are *m* ± s.d., *N* =3. The differences of *m* for magnetic 25Mg *vs*. nonmagnetic 24Mg or 26Mg are statistically significant at P < 0.005. Right: Length of adaptation period (lag-phase) on the liquid media supplemented with magnetic 25Mg or nonmagnetic 24Mg and 26Mg. Data are *m* ± s.d., *N* =3. The differences of *m* for 25Mg *vs*. 24Mg or 26Mg are statistically significant at P ≤ 0.02.

In addition, kinetics of the cell growth was hourly monitored in the cited experiments, using optical density measurements at 620 nm with 96-cavity micro-plate reader "Uniplan" (Picon, Russia). The kinetic curves of cell biomass growth were typical for the bacterial growth involving slow adaptation period ("lag-phase") followed by exponential growth ("logphase") when the cell mass quickly doubled and the stationary phase when the cell growth completed because of lack of the substrates (primarily, glucose). The striking observation for these experiments was that length of the lag-phase has turned out to be essentially shorter in the case when the cells were transferred on the media with magnetic isotope of 25Mg in comparison with nonmagnetic 24Mg and 26Mg (Fig. 4b). Again, the nonmagnetic isotopes, 24Mg and 26Mg, were not differentiated by their effects on the cell growth. It means that adaptation of cells transferred from the pre-incubation media is limited by some enzymatic processes, efficiency of which is increased by magnetic nuclei of 25Mg.

As it was cited above, magnetic isotope of 25Mg more effectively performs the Mg2+ cofactor function for oxidative phosphorylation in the isolated mitochondria in comparison to 24Mg or 26Mg (Buchachenko et al., 2005). Energetic demands of every operation in prokaryotic cells of bacteria, as well as in eukaryotic cells of animals, are met by molecules of ATP. Hence, ATP as the main source of energy in living cells is most likely to be the limiting substrate for adaptation metabolism in the lag-phase. However, for the exponential phase of the cell growth, we have not found any significant differences in the rates of cell growth between magnetic 25Mg and nonmagnetic 24Mg and 26Mg. The time of doubling of the cell mass was approximately the same, regardless of the type of magnesium isotopes (Koltover et al., 2012). This obviously suggests a different "bottle-neck" of metabolism in the exponential phase of growth with other, than ATP, limiting substrate and another limiting reaction independent upon the nuclear spin of magnesium.

Fig. 4. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on *E. coli*, strain K12TG1 (Koltover et al., 2012). Left: Ability of the cells enriched with magnetic 25Mg or nonmagnetic 24Mg and 26Mg to form colonies on the nutrition agar. Data are *m* ± s.d., *N* =3. The differences of *m* for magnetic 25Mg *vs*. nonmagnetic 24Mg or 26Mg are statistically significant at P < 0.005. Right: Length of adaptation period (lag-phase) on the liquid media supplemented with magnetic 25Mg or nonmagnetic 24Mg and 26Mg. Data are *m* ± s.d., *N* =3. The differences of *m* for 25Mg *vs*. 24Mg or 26Mg are statistically significant

In addition, kinetics of the cell growth was hourly monitored in the cited experiments, using optical density measurements at 620 nm with 96-cavity micro-plate reader "Uniplan" (Picon, Russia). The kinetic curves of cell biomass growth were typical for the bacterial growth involving slow adaptation period ("lag-phase") followed by exponential growth ("logphase") when the cell mass quickly doubled and the stationary phase when the cell growth completed because of lack of the substrates (primarily, glucose). The striking observation for these experiments was that length of the lag-phase has turned out to be essentially shorter in the case when the cells were transferred on the media with magnetic isotope of 25Mg in comparison with nonmagnetic 24Mg and 26Mg (Fig. 4b). Again, the nonmagnetic isotopes, 24Mg and 26Mg, were not differentiated by their effects on the cell growth. It means that adaptation of cells transferred from the pre-incubation media is limited by some enzymatic

As it was cited above, magnetic isotope of 25Mg more effectively performs the Mg2+ cofactor function for oxidative phosphorylation in the isolated mitochondria in comparison to 24Mg or 26Mg (Buchachenko et al., 2005). Energetic demands of every operation in prokaryotic cells of bacteria, as well as in eukaryotic cells of animals, are met by molecules of ATP. Hence, ATP as the main source of energy in living cells is most likely to be the limiting substrate for adaptation metabolism in the lag-phase. However, for the exponential phase of the cell growth, we have not found any significant differences in the rates of cell growth between magnetic 25Mg and nonmagnetic 24Mg and 26Mg. The time of doubling of the cell mass was approximately the same, regardless of the type of magnesium isotopes (Koltover et al., 2012). This obviously suggests a different "bottle-neck" of metabolism in the exponential phase of growth with other, than ATP, limiting substrate and another limiting

processes, efficiency of which is increased by magnetic nuclei of 25Mg.

reaction independent upon the nuclear spin of magnesium.

at P ≤ 0.02.

Of special interest are searches for magnetic-isotope effects in the processes of recovery of cells from radiation injuries. The reason is that any factor, capable to influence on efficiency and reliability of cell nanoreactors, shows up itself most vividly under drastic conditions of post-radiation recovery (Koltover et al., 1980; Grodzinsky et al., 1987; Koltover, 1997).

We undertook the investigation of effects of magnetic and nonmagnetic isotopes of magnesium on post-radiation recovery of *S. cerevisiae* (Grodzinsky et al., 2011). The yeast cells (diploid strain MATα ade2Δ248 leu2-3,112 ura3-160,188 trp1 Δ:kanr) were cultivated on the standard nutrient liquid media M3 supplemented with 24MgSO4 or 25MgSO4. After three days of the cultivation under aerobic conditions at 30 0C, the cells were washed from the nutrient liquid and suspended in nutrient-free ("fasting") media, i.e., sterile phosphate buffer, pH 7.0. Then, the cells were irradiated by the short-wave ultraviolet light (*λ* ≈240 nm, the dose ≈190 *J*/m2), whereupon they were left in the nutrient-free water at 30 0C (with shaking) to study kinetics of the post-radiation recovery of the cells. For this kinetics study, the aliquots of the cells were periodically seeded on the standard nutrient (Petri dishes) and the cell survival was monitored by the standard CFU technique.

Fig. 5. The difference in effects of magnetic and nonmagnetic isotopes of magnesium on post-radiation recovery of *S. cerevisiae*, diploid yeast cells. The cells enriched with magnetic 25Mg or nonmagnetic 24Mg were irradiated by the short-wave UV-C light (*λ* = 240-260). Survival of cells was estimated as their ability to form colonies on the nutrition agar: 1 - recovery of the cells enriched with 25Mg; 2 – recovery of the cells enriched with 24Mg (Grodzinsky et al., 2011).

The survival of cells transferred to agar immediately after irradiation was not more than a few percent (Fig. 5). In this case the injured genetic structures in most of the cells could not be repaired before the onset of mitosis, and nonviable daughter cells were produced. Incubation in nutrient-free water, in which the cells do not divide, provides sufficient time for repair processes and leads to a corresponding increase in survival. From the kinetics curves represented on this figure we notice that the cells enriched with magnetic isotope of magnesium, 25Mg, are recovered essentially more effectively than the cell enriched with the nonmagnetic 24Mg.

Stable Magnetic Isotopes as a New Trend in Biomedicine 117

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.

leakage results in production of О2●─. Chemical products of О<sup>2</sup>

of dismutation of О<sup>2</sup>

Nelson & Cox, 2008, and references therein).

level of SOD activity testifies the lower production of О<sup>2</sup>

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

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

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.,

As a rule, the level of the SOD activity is adjusted to the intracellular level of О2●─. 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

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

●─ into hydrogen peroxide (H2O2) and oxygen, thus protecting cell

●─, the so-called reactive

●─ in the case when the cells are

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, with time:

$$D\_{\rm eff}(t) = D\_o[k + (1 \text{-} k) \exp(-\beta t)]$$

In this model of A. Novick and L. Szilard, *D*0 is the radiation dose, *t* is time of post-radiation recovery in the nutrient-free water, is the recovery rate constant, and *к* is the fraction of 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 (Grodzinsky et al., 2011).


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

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