The paper aims to examine the proliferation of bone marrow cell pool in Djungarian hamsters and the subsequent restoration of their genetic stability after the action of thiotepa (TT). The study involved 36 animals, of which 16 were in the control group (injected with 0.25 ml of physiological solution), and 20 in the experimental group (0.25 ml of thiotepa at a dose of 1.5 mg per 1 kg of body weight). The maximum number of cells with CA amounting to 30.0% was observed 13 hours after TT injection (p≤0.05 between the control and experimental groups) and rapidly declined to 5.7% over subsequent periods by the 37^th hour of the experiment (p≤0.05). The results suggest that the restoration of cell pool genetic stability is largely associated with the cell selection mechanisms, which confers an advantage over cell proliferation without chromosome anomalies.

bone marrow cells, cell cycle, cell proliferation, chromosomal aberrations, genetic homeostasis, thiotepa

Studies examining mutagen activity, which can be a variety of chemical compounds, are still highly relevant nowadays (Saxena and Kumar 2020). One of the primary mutagenicity tests is the analysis of chromosomal aberrations in laboratory-preserved chromosomes of simple-toothed rodents (Egoshin et al. 2018; Nin et al. 2020). The advantage of this method is its availability. Similarly, the karyotype of small rodents is dominated by acrocentric chromosomes, complexifying the identification process of such anomalies in quantitative terms (Chen et al. 2020).

In modern medicine, the prevention of inherited or mutagenic diseases is a central focus (Hall 2015). Therefore, studies assessing mutagenesis in cells of small rodents are of great importance, including for medical purposes. Most mutagens are chemicals that can cause changes in both DNA and chromosomes, affecting the hereditary background. In addition to chemical compounds, mutagens also include physical or biological factors, such as ultraviolet light, radioactive radiation, and certain viruses. Among chemical mutagens, drugs used to treat tumors are particularly distinctive (Qin et al. 2019b).

The spectrum of mutations is very broad and can include both somatic and generative cells of the adult body and embryo or fetus (Wei et al. 2018). Mutagenic consequences include infertility, hereditary diseases, various forms of cancer, and birth defects (Deng et al. 2018; Farooqi et al. 2021). Only for Russia each year, according to the Ministry of Statistics, there are about 0.3 to 0.5 million registered cases of cancer and the same volume of disablement due to birth defects. Infertility is observed in one-tenth of all couples in the country. More than half of the embryos fail to implant into the uterus or fully develop through spontaneous abortions (RBC 2019). With the aggravation of environmental conditions caused by exposure to industrial emissions, there is a growing incidence of disease, deaths caused by mutagenic effects. It is important to note that the body has efficient mechanisms that repair genetic disorders in cells or remove cells with such damages. Hence, genetic disturbances occur only when mutant cells persist in the body. Currently, the mechanisms and models of mutant cell elimination, unlike DNA repair, are not well understood.

Bone marrow (BM) is a rapidly renewing tissue based on polypotent stem cells (SCs) that spend most of their lives during the G0 period. Under normal conditions, the reproduction of hematopoietic cells, like other SCs, occurs primarily by asymmetrical mitosis. Afterward, one cell remains a stem cell, and the second – a transient cell – is released from the niche, loses its stem properties, and differentiates in one of the hematopoiesis directions (Wilson et al. 2008; van der Wath et al. 2009; Hérault et al. 2021). In contrast to stem cells, transient cells can actively proliferate, resulting in the replacement of differentiated cells leaving the BM. In various damages, SCs are activated to proliferation for restorative regeneration and replacement of lost cells (Rossi et al. 2007, 2008; Wilson et al. 2008).

The accumulation of mutations in SCs over a lifetime is one of the reasons for their reduction, which diminishes the possibilities of physiological and restorative regeneration and may also cause a variety of diseases, including tumor development (Wilson et al. 2008; Harding et al. 2017). The maintenance of genetic homeostasis in the tissue requires various effective action mechanisms, the main ones being the DNA repair and the processes happening at the control points of the mitotic cycle, which are currently well studied (Beerman et al. 2014; Brown and Geiger 2018; Ovejero et al. 2020).

However, a significant fraction of mutant cells is capable of passing the mitotic cycle and preserving the capacity to proliferate (Kuzin and Stukalov 1998; Pelevina et al. 2009; Neroyev et al. 2013; Li et al. 2020). The laws and selection mechanisms that can eliminate these cells from the pool and replace them with cells without genetic disturbances during proliferation are poorly understood.

Several medications used in tumor chemotherapy are genotoxic. In all SCs retained after treatment by mutagens, the number of genetic abnormalities increases significantly. The proliferation of these cells should not only provide for the reconstitution of cell loss and tissue regeneration but also maintain genetic stability since its further violation may result in disruption of homeostasis and the development of secondary tumors (Brown and Geiger 2018; Li et al. 2011). Under these conditions, the selection mechanisms which eliminate the most damaged mutant cells from the cell pool become especially important.

Test systems capable of detecting mutagenic activity are important in preventing genetically determined conditions (Hall 2015). The choice of a suitable biomodel for the experimental design remains one of the major links determining the effectiveness of the test system used. In current methods of evaluating mutagenic activity, laboratory mice, less commonly rats, dominate as experimental animals (Khan et al. 2019). Moreover, Djungarian hamsters are more appropriate biomodels for studying chromosomal aberrations because they have a convenient set of chromosomes for karyotyping (Sokova and Pogosiants 1974). However, they are not used as frequently as other rodents in laboratory studies. Another important point is the use of chemicals that have a well-known action mechanism. Thiotepa’s formula is SP (NC2H4)3; it is an analog of N,N′,N′′-triethylenephosphoramide. This substance, which belongs to the class of alkylating antineoplastic drugs, does not occur in nature. The history of alkylating drugs application goes back to the times of World War I, when the so-called mustard gas was actively used, on the basis of which the drugs of this group are synthesized. In medicine, nitrogenous analogues of mustard gas began to be used (in oncology). Thus, thiotepa contains three groups of ethylene (Saxena and Kumar 2020). The mutagenic effect of this compound has been well studied in mice, fibroblasts, and human white blood cells. For all of the aforementioned biomodels, a high frequency of chromosomal aberrations under exposure to ТТ was reported (Saxena and Kumar 2020). Meanwhile, such studies have not been conducted on hamsters. In this paper, the authors examined the proliferation of bone marrow cells in Djungarian hamsters at different times after exposure to ТТ. The limits of maximum ТТ effect on the induction of chromosomal aberrations were established, showing the kinetic of cells with various chromosomal damages. Besides, the authors hypothesize that the restoration of cell pool genetic stability is largely specified by the mechanisms of cell selection, which confers an advantage on cell proliferation without chromosome anomalies.

The study aimed to examine peculiar features of proliferation in bone marrow cells of Djungarian hamsters with different levels of chromosomal abnormalities after exposure to ТТ, i.e., a cytostatic agent with an alkylating mutagenic effect.

TT is a cytostatic agent with an effect related to DNA alkylation. The maximum concentration of the drug and its active derivatives is reached immediately after administration and is progressively reduced with metabolism and excretion. The data presented in Table 1 show that TT causes a significant delay in the duration of the mitotic cycle in cells that resisted the drug effect and retained the ability to proliferate. While in the controls, 9% of cells managed to undergo two cycles of replication already by 13 hours. For the experimental group, such cells appeared 6 hours later, by the 19^th hour after TT injection. A similar delay of approximately six hours is observed when cells undergo the third and fourth mitotic cycles. These data indicate that the main inhibition of proliferation occurs in the first cell cycle, i.e., when the drug and its active metabolites are involved. Afterward, the rate of proliferative processes is largely re-established and becomes comparable to the control parameters.

M1, M2, M3, M4 are cells that entered mitosis after passing one, two, three, or four mitotic cycles, respectively. M1-2 are cells that were in the S-period at the beginning of the experiment, passed the rest of the first cycle, and completed the second mitotic cycles. The 2-hour interval for each study term reflects the accumulation of cells in the metaphase during that time with colchicine.

Based on the data obtained, the average time of the proliferation cycle of the bone marrow cells in the control is 10 to 12 hours in the experiment – 13 to 15 hours (p ≤ 0.05 between control and experiment). In animals with no TT injection, 17.7% of the fastest cells were able to complete four mitotic cycles in 31 hours. Therefore, the average cycle lasted about 8 hours (p ≤ 0.01 with the control). After TT injection, 29.6% of the cells with the highest proliferation rate entered their fourth reproduction only by the 37^th hour, i.e., each mitotic cycle took on average 9–10 hours (p ≤ 0.05).

Given the delay of about 6 hours in the first cycle, their passage rate over the next three cycles was roughly the same as in controls (p ≥0.05). In both controls and experiments, there are a very small fraction of cells (about 1%) with an even higher proliferation rate, and the duration of each of the four passed cycles was about 1–2 hours shorter. In contrast, the fraction of cells that entered into 1 or 2 reproduction cycles by the 25^th-31^st hour in the control and 25^th – 37^th hour in the experiment (p ≤ 0.05) have significantly longer cycle durations. However, at least some of them are in the G0 period for a while, so it is difficult to determine by how much is their mitotic cycle prolonged.

It should be noted that the use of BrdU does not make it possible to determine, in what part of the G1 period the cells were at the time of its introduction. Consequently, the exact total duration of the first mitotic cycle can be determined only for those cells that were at the beginning of such period. For cells that have already passed some G1 at the moment of injection, an upward correction is required. For cells in the G0 period, the time to reach the first mitosis is longer than the duration of the mitotic cycle by the amount necessary to activate proliferation and pass from G0 to G1 period.

The data presented in Fig. 2 show that a selected dose of TT has a pronounced mutagenic effect on Djungarian hamsters, leading to CAs in a large proportion of bone marrow cells. The maximum effect is reached 13 hours after injecting TT. The mean number of cells with CA in the control group ranges from 1% for all phases of the experiment. In the experimental group, the number of aberrant cells 13 hours after the drug injection exceeded the control values by more than 30 times (p ≤ 0.001). In subsequent terms, the cell fraction with CA decreases and reaches 5.7% by 37 hours of the experiment, which is more than 5 times less than the values at 13 hours (30.0%, p ≤ 0.05). The data of this study show that the decline in chromosomal abnormalities is associated with the preferential proliferation of CA-free cells. After each mitotic cycle passed, the proportion of cells with CA decreases significantly, with less than 4% in the third or fourth mitosis. At the same time, the number of abnormal cells entering the first mitosis increases even after 13 hours and reaches 39.2% by the 31^st hour (p≤0.05). The proportion of complex chromosome aberrations caused by multiple chromosomes ruptures increases in them (Fig. 2b). A significant decrease in chromosomal abnormalities in cells entering the first mitosis after TT injection begins only after 37 hours. The number of CA decreases to 16.0%, with cells with multiple abnormalities of more than 5 chromosome ruptures per cell completely disappearing (p ≤ 0.05).

Figure 2. 

Changes in the number of metaphases with chromosomal aberrations (a) or the number of chromosome ruptures (b) in the bone marrow of Djungarian hamsters at different periods after the injection of saline (control) or thiophosphamide (experiment). Abscissa axis: time since injection (hour); ordinate axis: number (%) of metaphases with chromosome aberrations (a) or number of chromosome ruptures per 100 metaphases (b) among all cells under division (all mitoses) or those that passed only one mitotic cycle (M1)

TT has been used in cancer medicine for over 60 years. Pharmacokinetics study of the drug showed that the highest concentration of TT and its metabolites is achieved in the blood of mice within one hour after administration and decreases thereafter. The majority of TT and its metabolites are excreted into the urine within 10 hours (Kuzin et al. 1989). Studies on the kinetics of alkylating agents in the blood of monkeys and rabbits have also shown that they reach their maximum concentration in one hour, and their principal effect manifests within the first hours after administration of the medication (Stukalov 1988; Sinai and Kerem 2018). Thus, the effects of TT observed in this study are associated with the action on the cells only during one first mitotic cycle, which is reflected in the scheme of Fig. 1. The exception is the “fastest” cells, which were in S-period at the time of injection and, having completed the first cycle, could have had time to enter the second cycle, when a small amount of alkylating agents was still active. However, according to data of this study, only 1.9% of such cells existed (Table: experiment, M1-2, 11–13 hours).

Bone marrow is a heterogeneous cell system that consists of cell sub-pools, whose degree of maturity and sense of differentiation vary significantly. CAs constitute a very small fraction of cells that seldom replicate (Schneider et al. 1977; Hérault et al. 2021). Consequently, among the mitoses in the series of control, there could only be simple cells. As a result, the findings observed in this study on cell kinetics and cell cycle parameters do not refer to them but to different groups of actively proliferating transient cells of various differentiation directions. Among these heterogeneous cell groups, the duration of the mitotic cycle can be precisely calculated only for those that underwent 3–4 cycles in 31 hours of the experiment. For actively proliferating transient cells, it varies from 8 to 12 hours in monitoring. For cells that enter into first or second mitosis at 25 to 31 hours, there is a high probability that they spent some part of their time in the G0 period. Therefore, the precise duration of their mitotic cycle cannot be determined. For example, cells that have undergone one replicative cycle in the presence of BrdU and entered the first mitosis at 31 hours may have a mitotic cycle duration of only 8 hours, and the remaining 23 hours is the time spent in the G0 period.

However, the data of this study allow determining fairly precise cycle parameters for the slower cells as well. The specifics of chromosomal coloration identified the cells that were in the S-period at the time of BrdU administration. These cells entered the second mitosis at 25 hours of the experiment (M1-2 in the Table), passed a part of the first cycle(½S, G2, M), and then full second mitotic cycle during this time. As a result, the duration of the mitotic cycle for them is approximately 16–18 hours.

The variation in the duration of a mitotic cycle of bone marrow cells between 8 and 18 hours reflects the heterogeneity of its constituent cells. For a small part (1% of the fastest or slowest cells), these values may be lowered or increased by several hours. The mean value of 12 hours is the data known from available literature sources (Sokova and Pogosiants 1974). Significantly higher values characterize the cell cycle rather than mitotic, i.e., include more G0 period time.

Introducing TT significantly alters the kinetics of bone marrow cells. The data obtained shows that these changes primarily affect the first mitotic cycle, i.e., there is a delay in its passage of around 6 hours. Over the next three cycles, the proliferation rate has recovered. Based on the pharmacokinetics of the drug it can be concluded that TT and its metabolites affect cell proliferation at the moment they are in the body. Their action affects mainly the cells that continue to be in the first cycle and insignificantly affects the proliferative processes of the majority of cells that manage to overcome the first mitotic cycle. Obviously, much of this is due to the action of the control point mechanism, which slows down the cells presenting genetic abnormalities for DNA repair.

It should be noted that TT has a significant cytotoxic effect, and the data in this study were obtained only for the fraction of cells that managed to survive and retain the ability to proliferate. Djungarian hamsters are very sensitive to cytostatic and mutagenic agents (Kuzin et al. 1987). The dose of 1.5 mg/kg is comparatively high for these animals. However, it maintains sufficient cell viability to determine cell kinetics and chromosomal anomalies in several successive cycles of division. Preliminary experiments performed specifically to choose the dosage of the drug have shown that the increase to 2–7 mg/kg significantly reduced the number of dividing cells, making it difficult to obtain the number of metaphases necessary for analysis.

Cellular death after cytostatic action causes the activation of preserved SCs and the transition of a significant portion of them into the mitotic cycle. The transition from G0 to proliferation takes one to two days (Wilson et al. 2008; Saakyan et al. 2011; Brown and Geiger 2018). Therefore, it can be assumed that, unlike the control, in the experiment, the number of SCs entering mitosis at an advanced stage significantly increases. The data obtained in this study support this hypothesis on the increase of the first mitosis fraction in the experiment versus the control at 31–37 hours of the experiment. Undoubtedly, some of them are cells with significant damage that require a long time to overcome the mitotic cycle. However, most of these cells are destroyed by apoptosis by 37 hours. A sharp decrease in the CA cells fraction with entering their first mitosis at 37 hours (Fig. 2) indicates the activation and transition to the proliferation of relatively mutagen-resistant SCs that were in the G0 period during TT injection and had relatively few genetic abnormalities.

It has been shown a pronounced mutagenic effect of TT on bone marrow cells in Djungarian hamsters, leading to chromosome damage. The genotoxic effect of TT has been studied in numerous sources of literature and is well documented (Thiotepa 2011; Sinai and Kerem 2018; Lezaja and Altmeyer 2020). It is important to note that the CA observed represents only a small fraction of the visible mutations. Every single cell receives significant genetic damage as a result of TT injection. Meanwhile, after cytostatic action, SC preserved during multiplication should not only provide replenishment for cell loss but also restore genetic homeostasis. Otherwise, increasing genetic instability can result in different pathologies. This is particularly important for intensive chemotherapy, when SC transplantation is not used after treatment and regeneration is ensured by the own preserved cells.

Under the action of high mutagenic doses, including TT, the recovery begins after the increase in the number of genetic disorders, and the number of cells with CA gradually decreases (Stukalov 1988; Sinai and Kerem 2018). Currently, the processes reducing the number of mutations that occur at the molecular genetic level, i.e., DNA repair and apoptosis of cells, including those associated with the mitotic cycle control points are quite well studied. It is generally believed that cells with large genetic abnormalities that have not undergone effective repair and have entered proliferation are delayed in control points, eliminated, and cannot be reproduced. However, this applies only to some fraction of cells. The data obtained demonstrate that even cells with very significant chromosomal abnormalities can pass through and replicate into one mitotic cycle. And the part of the cells in which CA is caused by 1 or 2 ruptures is capable of passing at least 4 mitotic cycles. A number of studies have shown that DNA disruption may not only persist and continue to be repaired in cells that have passed the control points, but also cause extension of DNA replication beyond S-period, i.e., in G2 and even mitosis (Schneider et al. 1977; Mankouri et al. 2013; Beerman et al. 2014; Kuzin 2019). And the study of sister chromatid exchanges indicates that some of the primary DNA damage is preserved and causes recombinant repair even in the third cycle following the action of TT (Li et al. 2020). Thus, mechanisms for repairing genetic homeostasis are not limited to repairing damage to the DNA or apoptosis of mutant cells in a single cell cycle. They are largely associated with cell selection, which occurs at the cell pool level during proliferation.

Furthermore, the decrease in the number of genetic disorders in the BM pool is associated with the predominant reproduction of cells without CA or with minor chromosome damage. With significant genetic abnormalities, cells are slower to prepare for division, which is related to the preservation and even increase in the proportion of such cells by 25^th and especially by 31^st hour of the experiment. They are unable to divide more than once and have a limited lifespan, as shown by a significant decrease in their number at 37 hours. Moreover, at this time only cells with less than 5 chromosome ruptures preserve the ability to division, while at 31 hours of the experiment, cells with 5 or more ruptures account for 36.3% of all aberrant metaphasesq associated not only with the death of the most damaged cells delayed in the mitotic cycle but also with the activated proliferation of SCs that were in the G0 period. Resting cells are known to be less sensitive to genotoxic effects than proliferating cells and exhibit therefore fewer chromosomal abnormalities. Besides, the entry of SCs into proliferation involves additional mechanisms of genetic homeostasis, namely, effective postreplicative repair and mitotic cycle checkpoint filter (Pelevina et al. 2009; Brown and Geiger 2018; Kuzin 2019).

The data we obtained enables presenting the following scheme of bone marrow cell proliferation following mutagenic exposure to TT. Some of the cells most affected by DNA cannot grow and die. An additional part of these cells can undergo a mitotic cycle and division. Their sustainability is limited to about 30 hours, and they can only pass through one cycle. Less damaged cells with 1–2 chromosomal ruptures remain viable throughout the observation and can pass through at least four mitotic cycles. However, after every division, a part of these cells is eliminated. Loss recovery occurs at the expense of reproduction of cells without CAs, including proliferating activated SCs that were dormant before exposure. Genetic disorders lead to a slower proliferation, apparently associated with delayed cells at first control points after the mitotic cycle of cytostatic action. The mean duration of the delay is 6 hours, which is proportional to the number of genetic abnormalities.

The mechanisms of cell selection, which give an advantage in cell reproduction without genetic disorders, play an essential role in maintaining the genetic homeostasis of the cell pool. The importance of such mechanisms is also indicated by the fact that the rate of CA elimination after TT action in the pool of rapidly proliferating BM cells is significantly higher than in blood lymphocytes (Stukalov 1988; Sinai and Kerem 2018). The mechanisms used to select cells maintaining genetic stability are poorly understood. As suggested earlier, one of these mechanisms could be associated with changes in the spatial and temporal organization of mutant cells (Georgiades et al. 2010). It is well known that SCs that changed their location and left the niche lose their stem properties and start differentiating. Similarly to the spatial niche, the delay of mutant cells with genetic disorders in the control points of the mitotic cycle leads to their falling out of the “temporary window” of circadian proliferation rhythm typical for all renewing tissues. That also causes their differentiation. This mechanism preserves the slightly damaged mutant cells, allows them to exercise their functions in the differentiation process, and only afterward removes them from the tissue. The possibility of such a mechanism is confirmed by the data on the second wave of increase in the number of lymphocytes with CA in the blood of rabbits on day 4 after TT administration (Saakyan et al. 2013; Subarkhan et al. 2019). Such an increase cannot be attributed to the mutagenic effect of TT, which is eliminated during the first 11 hours but is well explained by the displacement of differentiated cells with chromosomal abnormalities from the bone marrow by this time. It should be noted that differentiating mutant cells (except generative cells) are usually not harmful. As opposed to stem cells, they have a limited number of divisions and a limited lifetime.

The results of this work allow concluding that the restoration of genetic stability in the BM cell pool after TT injection is only partially performed by the well-known mechanisms of DNA and control points repair, which act mainly in the first mitotic cycle. Then, less studied selection mechanisms begin to play a decisive role. They work through the selective advantage of cells without chromosomal aberrations, which progressively move mutant cells from the pool during proliferation. Studying cell-pool mechanisms of genetic homeostasis will enable to influence the processes of preserving genetic stability of renewing tissues after mutagenic exposure, which is especially important in the treatment of oncological diseases.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors declare that they have no conflict of interests.

Data will be available on request.

Experiments with animals were carried out according to bioethical requirements.

None.