A. N. Khokhlov

Stationary Cell Cultures as a Tool for Gerontological Studies

Studies of cellular and molecular mechanisms of aging are currently often carried o u t on cells senescing not only in vivo but also in vitro (Hayflick’s model), that is, altering with increasing of population doubling level in culture. However, some data suggest that in some cases the results obtained with this model are not in accord with those of in vivo aging studies. Furthermore, such experiments, as a rule, are comparable in labor-consuming character with routine studies in laboratory animals. In fact, Hayflick himself suggests that cells in vivo never realize their proliferative potential and never reach phase 111. In other words, an organism never ages because of a cell’s limitation, called “Hayflick’s limit.” It ages, we believe,’ because of an accumulation of various kinds of damage in cells due to restriction of cell proliferation during the formation (the differentiation process) of populations of specialized resting cells or very slowly dividing cells. The rate of any type of damage accumulation in the cell population (not in a single cell!) has to depend on the ratio of the rates of three processes: (1) cell proliferation, ( 2 ) spontaneous appearance of damage, and (3) damage repair. Thus, during cell proliferation a “dilution” of the damage occurs. Our data2 demonstrating a direct relation between the average proliferative activity of a cell line or strain and the average DNA molecular weight support this hypothesis. With all this in mind we suppose that it is more advisable to study cellular aging mechanisms using the “stationary phase aging” model.‘ The model is based on the assumption that in cells of stationary cultures various changes similar to those in cells of aging organisms have to appear. Last year we and other investigators obtained many experimental results confirming this assumption. “Age” changes at different levels (accumulation of DNA breaks and DNA protein cross-links, DNA demethylation, changes in spontaneous sister chromatid exchange level, plasma membrane changes, nuclear structure modulations, and decreased rate of mitogenstimulated cell cycling and of cell colony-forming ability a.0.) were shown to occur in stationary cell cultures. These experiments can be carried out in nearly any type of cell including normal and transformed human and animal cells, plant cells, bacteria, mycoplasmas, yeasts, and the like. In particular, we now study the phenomenon of stationary phase aging and the possibility of modulating this kind of cell aging by geroprotectors (physical or chemical factors that retard aging) and geropromoters (factors that accelerate aging) in cyanobacteria cultures. ‘Thus, an evolutionary approach to analysis of the data is provided. Moreover, changes in stationary cell cultures become detectable very soon, as a rule in 2-3 weeks after beginning the experiment. All of these data suggest that the stationary phase aging model is a good alternative to the Hayflick model.

Does aging need an own program or the existing development program is more than enough

According to author’s concept, the aging process is caused by cell proliferation restriction-induced accumulation of various macromolecular defects (mainly DNA damage) in cells of an organism or cell population. In case of cell cultures, this proliferation restriction is related to either so-called contact inhibition or Hayflick’s limit, and in case of multicellular organisms, to the appearance, in the process of differentiation, of organs and tissues consisting of postmitotic or very slowly dividing cells. Cell proliferation is absolutely incompatible with normal functioning of a macroorganism. Thus, the development program automatically leads to a situation inducing the aging process (martaliry rates increase with age). Therefore, any special program of aging simply becomes senseless. This, however, does not reject for some organisms the reasonability of programmed death, which makes possible the elimination of harmful, from the species’s point of view, individuals. It is also very important to understand that increase or decrease of organism’s life span under the action of various factors are not necessarily related to a modification of the aging process, even though the experimental results in this field are interpreted just in this way.

From Carrel to Hayflick and back, or what we got from the 100-year cytogerontological studies

The history of gerontological experiments on cell cultures is reviewed. Cytogerontological studies and aging theories by Weismann, Carrel, Hayflick, and the author are compared. It is emphasized that the basic notion of aging mechanisms was deeply revised several times within the 20th century. It is concluded that at present the aging of multicellular organisms cannot be satisfactorily explained with the help of cytogerontological studie’s data. Experiments on cell cultures need to be combined with fundamental gerontological studies, including survival curve analysis for humans or experimental animals.

Cytogerontology at the Beginning of the Third Millennium: From “Correlative” to “Gist” Models

For the most part, research in the area of cytogerontology, i.e., investigation of the mechanisms of aging in the experiments on cultured cells, is carried out using the “Hayflicks model”. More than forty years have passed since the appearance of that model, and during this period of time, very much data were obtained on its basis. These data contributed significantly to our knowledge of the behavior of both animal and human cultured cells. Specifically, we already know of the mechanisms underlying the aging in vitro. On the other hand, in my opinion, little has changed in our knowledge of the aging of the whole organism. In all likelihood, this can be explained by that the Hayflicks model is, like many others used in the experimental gerontology, correlative, i.e. based on a number of detected correlations. In the case of Hayflicks model, these are correlations between the mitotic potential of cells (cell population doubling potential) and some “gerontological” parameters and indices: species life-span, donor age, evidence of progeroid syndromes, etc., as well as various changes of normal (diploid) cells during long-term cultivation and during aging of the organism. It is, however, well known that very frequently a good correlation has nothing to do with the essence (gist) of the phenomenon. For example, we do know that the amount of gray hair correlates quite well with the age of an individual but is in no way related to the mechanisms of his/her aging and probability of death. In this case, the absence of cause-effect relationships is evident, which are, at the same time, indispensable for the development of gist models. These models, as distinct from the correlative ones, are based on a certain concept of aging. In the case of Hayflicks model, such a concept is absent: we cannot explain, using the “Hayflicks limit,” why our organism ages. This conclusion was convincingly confirmed by the discovery of telomere mechanism which determines the aging of cellsin vitro. That discovery initiated the appearance of theories attempting to explain the process of aging in vivo also on its basis. However, it has become clear that the mechanisms of aging of the entire organism, located, apparently, in its postmitotic cells, such as neurons or cardiomyocytes, cannot be explained in the framework of this approach. Hence, we believe that it is essential to develop “gist” models of aging using cultured cells. The mechanisms of cell aging in such models should be similar to the mechanisms of cell aging in the entire organism. Our “stationary phase aging” model could be one of such models, which is based on the assumption of the leading role of cell proliferation restriction in the processes of aging. We assume that the accumulation of “senile” damage is caused by the restriction of cell proliferation either due to the formation of differentiated cell populations during development (in vivo) or to the existence of saturation density phenomenon (in vitro). Cell proliferation changes themselves do not induce aging, they only lead to the accumulation of macromolecular defects, which, in turn, lead to the deterioration of tissues, organs, and, eventually, of the entire organism, increasing the probability of its death. Within the framework of our model, we define cell aging as the accumulation in a cell population of various types of damage identical to the damage arising in senescing multicellular organism. And, finally, it is essential to determine how the cell is dying and what the death of the cell is. These definitions will help to draw real parallels between the “genuine” aging of cells (i.e., increasing probability of their death with “age”) and the aging of multicellular organisms.

[Impairment of regeneration in aging: appropriateness or stochastics?].

AbstractThere is a viewpoint that suppression of the proliferative capacity of cells and impairment of the regeneration of tissues and organs in aging are a consequence of specially arisen during evolution mechanisms that reduce the risk of malignant transformation and, thus, protect against cancer. We believe that the restriction of cell proliferation in an aging multicellular organism is not a consequence of implementing a special program of aging. Apparently, such a program does not exist at all and aging is only a “byproduct” of the program of development, implementation of which in higher organisms suggests the need for the emergence of cell populations with very low or even zero proliferative activity, which determines the limited capacity of relevant organs and tissues to regenerate. At the same time, it is the presence of highly differentiated cell populations, barely able or completely unable to reproduce (neurons, cardiomyocytes, hepatocytes), that ensures the normal functioning of the higher animals and humans. Apparently, the impairment of regulatory processes, realized at the neurohumoral level, still plays the main role in the mechanisms of aging of multicellular organisms, not just the accumulation of macromolecular defects in individual cells. It seems that the quality of the cells themselves does not worsen with age as much as reliability of the organism control over cells, organs and tissues, which leads to an increase in the probability of death.

Cytogerontological studies of biological activity of oregano essential oil

In order to clarify possible cytological mechanisms that underlie the beneficial effects of carvacrol-bearing essential oils on health and mental abilities, we studied one of them (oregano essential oil) in experiments on transformed cultured Chinese hamster cells. Possible cytotoxic or mitogenic effects of the preparation at various concentrations were preliminarily estimated by analyzing the cell culture density after 4 days of cultivation. The preparation concentration in the growth medium (on carvacrol basis) varied from 1 × 10−15 up to 5 × 10−4 M (on carvacrol basis). As a result, two concentrations were selected for further experiments, including 2.5 × 10−5 M as the maximal absolutely non-toxic concentration and 2.5 × 10−4 M as the concentration at which the oregano essential oil decreased approximately 2-fold the final cell density of the grown culture. It was found that the preparation at 2.5 × 10−5 M had no effect on either the colony-forming ability of the cells or the saturation density of the culture (which is a marker of its “biological age”) or kinetics of its “stationary phase aging” (degradation of cultured cells in the stationary phase of growth, similar to age-related changes of the cells in aging organism). On the contrary, the oregano essential oil at 2.5 × 10−4 M abruptly diminished colony-forming ability of the cells and influenced as a “pro-aging” factor on the saturation density of the cell culture and kinetics of the cell death induced by “stationary phase aging.” Based on our own concept of aging and the data obtained, we assumed that detected increase in the life span of mice under the influence of the oregano essential oil could be determined by certain functional changes at the organismal level only, but is not associated with any geroprotective (anti-aging) activity of the preparation, which is manifested at the cellular level and improves the cell viability.

Testing of geroprotectors in experiments on cell cultures: Choosing the correct model system

We believe that cytogerontological models, such as the Hayflick model, though very useful for experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both various “age-related” changes in stationary cell cultures and similar changes in the cells of aging multicellular organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, mycoplasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “stationary phase aging” model may prove effective in testing of various geroprotectors (anti-aging factors) and geropromoters (pro-aging factors) in cytogerontological experiments. It should be emphasized, however, that even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this complies with ethical principles of human subject research).

Effect of Gold Nanoparticles on Mouse Spermatogenesis

The response of the mouse male germ cells exposed to gold nanoparticles (∼2.5 nm) was studied. Our investigation demonstrates that treatment with Au nanoparticles for four days does not impair the architecture of the spermatogenic epithelium. Cytogenetic evaluation using micronucleus assay showed that gold nanoparticles can affect the chromosomes of early primary spermatocytes. However, gold nanoparticles did not induce chromosome abnormalities in spermatogonial stem cells. Further, the cauda epididymal sperm was isolated on the 14th day after treatment and was incubated in SDS solution (Na dodecyl sulphate) and then in a solution containing DTT (dithiothreitol) to induce nuclear chromatin decondensation. Observations showed that after four days of treatment of spermiogenic (postmeiotic) cells with gold nanoparticles the decondensation process had no differences from the control. On the contrary, in the experiment with the same cells and period of fixation but with a single exposure to gold nanoparticles, the number of mature gametes with totally decondensed nuclei reached 100% as opposed to 44% in the controls.

A paradoxical effect of hydrated C60-fullerene at an ultralow concentration on the viability and aging of cultured Chinese hamster cells

The effect of an aqueous solution of hydrated C60-fullerene (HyFn) on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during the slowing down of their proliferation within a single passage and the subsequent “aging” in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells was studied. The final calculated concentration of HyFn in the growth medium was 10−19 M. A paradoxical result contrasting the available data on the absence of HyFn cytotoxicity at higher concentrations was obtained in our experiments: namely, HyFn decelerated cell proliferation (estimated by the growth of mass culture, as well as by the efficiency of colony formation) and accelerated the “stationary phase aging” of the cell culture. Moreover, repeated addition of an aqueous solution of HyFn (to the final calculated concentration of 10−19 M) to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. The observed effect of HyFn at ultralow concentration is supposed to arise from the alterations in the properties of the water surrounding the fullerene molecule: namely, water becomes a donor and acceptor of electrons and regulates redox processes (especially those involving oxygen) in aqueous systems. This effect of HyFn at an ultralow concentration may be specific for transformed cells, and, therefore, experiments on normal fibroblasts with limited mitotic potential are planned as a continuation of the present study. It is also possible that the reported antiaging effect of HyFn in experimental animals is due to its anticancer, immunostimulatory, antiviral, and antibacterial properties manifested only at the whole-organism level.

Effects of cholesterol- or 7-ketocholesterol-containing liposomes on colony-forming ability of cultured cells

Experiments with cultured Chinese hamster cells showed that incubation of the cells with (phosphatidylcholine + cholesterol + 7‐ketocholesterol)‐containing liposomes (4:3:1 by weight) during two hours led to a decrease in the colony‐forming ability of cells down to zero, while (phosphatidylcholine + cholesterol)‐containing liposomes (1:1 by weight) reduce this parameter by 90%. Furthermore, the cholesterol‐containing liposomes (without 7‐ketocholesterol) induce a decrease in the number of the maximal‐site colonies accompanied by the corresponding increase in the number of the middle‐size colonies.