Acute restraint stress increases the frequency of vinblastine-induced micronuclei in mouse bone marrow cells

Abstract

Acute physiological stress induces remarkable effects on the nervous, endocrine, and immune systems and also on cellular metabolism and cell division processes. Stress-induced instability of cellular mechanisms might play an important role in increasing cell division disorders. In this study, a relationship between stress and micronucleus (MN) induction in mouse (balb/c) bone marrow cells following vinblastine treatment, or stress or stress and vinblastine treatment in comparison to a non-stressed control group was investigated. In order to test the effects of treatments on MN induction, an in vivo MN assay was performed on bone marrow cells. The results revealed a significantly greater increase in MNs in bone marrow cells (polychromatic erythrocytes) from the stressed/vinblastine treated mice. The data indicate the ability of exposure to an emotional stressor to enhance the damaging actions on bone marrow cells of an aneugenic agent.

Keywords::

• acute restraint stress
• carcinogenesis
• micronucleus assay
• micronucleus induction
• polychromatic erythrocytes
• vinblastine

Introduction

The role of genetic and chromosomal abnormalities in provoking and in the development of cancer has been established (Duesberg 2007). The process of carcinogenesis involves a multi-factorial pathway in which multiple extra- and intra-cellular factors ultimately lead to tumor formation (Hamilton and Meltzer 2006).

Sudden changes in the environment may result in destabilizing and changing various functions of an organism. Stress has been described as circumstances where coping with a variety of actual or perceived stimuli alters the homeostatic state of an organism, including behavioral, endocrine, and immunological systems. Neurophysiological responses induced by different stressors activate the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. The effects of various kinds of stressors on the plasma levels of stress hormones, behavioral responses, and nervous system have been studied in many animal species (Kosten et al. 2007; Mantsch et al. 2007; Weissman et al. 2007). Like other kinds of stressor, acute restraint stress stimulates an immediate increase in plasma levels of glucocorticoids (Bowers et al. 2008). Such hormones will affect the metabolic pathways of proteins, carbohydrates and lipids, and regulate homeostasis. Restraint stress also suppresses the biosynthesis of testosterone (Weissman et al. 2007). Due to the wide range of glucocorticoid-mediated effects on physiological and cellular activities, it is not surprising that stress may enhance the probability of genomic damage and alter the transcriptional regulation of the cell cycle in dividing cells (Flint et al. 2005). Stress could alter the expression of genes for cell cycle regulators, heat shock proteins, proto-oncogenes, and metabolic enzymes (Mizuno et al. 2004). The effects of stress on cellular function via glucocorticoids (Dickmeis et al. 2007) and induction of malfunction of proliferation and cell division might lead to genetic abnormalities in descendant daughter cells. Restraint stress, at the molecular level, could cause changes in expression pattern of genes, which are responsible for cell cycle regulation and apoptosis in mouse T cells (Flint et al. 2005).

Considering the effects of stress on endocrine and metabolic functions and possible induction of damage to cellular genetic status, in this study the effects of acute restraint stress on induction of chromosomal damage were investigated. To enhance chromosomal damage to enable analysis of interaction with environmental stress, the effects of a known aneugen, vinblastine, alone and in stressed mice were also investigated. Vinblastine (C₄₆H₅₈N₄O₉) is a Vinca alkaloid that has been used for chemotherapy of some cancers. Vinblastine at low concentrations (0.1–6 nM), blocks mitosis at the transition from metaphase to anaphase. This potent drug suppresses the dynamic growth and shortening of the ends of microtubules and extends division duration. Furthermore, while vinblastine is a potent mitotic inhibitor, it acts as an aneugen; by interfering with spindle formation it causes chromosome mal-segregation (Brunner et al. 1991; Wendell et al. 1993; Huber et al. 1996).

In this study, a micronucleus (MN) assay was performed. This assay has been used widely to analyze chromosomal abnormalities. In this assay, any structural and/or numerical chromosomal abnormalities, resulting from chromosome loss, can be distinguished by finding a small nucleus in the cytoplasm. Any chromosome left behind or chromosome fragments, from the migration of chromosomes towards the poles in anaphase are surrounded by a nuclear envelope to form a small nucleus, or MN (Countryman and Heddle 1976). An increased number of MNs represent increased chromosomal abnormalities, either chromosomal breaks or loss of chromosomes.

We tested the hypothesis that mouse bone marrow cells would show more MNs after restraint stress, and that this stressor would increase the effects of vinblastine.

Materials and methods

Animals

In this study, 78 healthy male balb/c mice (5–7 weeks of age, 20–35 g body weight) were obtained from the Animal Division of the Razi Vaccine & Serum Research Institute (Mashhad, Iran) and housed under a standard 12 h light: 12 h dark cycle (lights on from 07.00 to 19.00 h) and controlled temperature (20 ± 2°C) conditions for at least a week to adapt. Four mice were kept in each cage. Food and water were available ad libitum. All procedures pertaining to the care and use of experimental animals were performed according to the Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, 1996, and were approved by the local committee for animal care. Mice were divided into groups of 6 for each treatment category.

Vinblastine treatment

Different doses of 1, 2, 3, and 5 mg/kg of body weight of vinblastine (Gedeon Richter LTD, Budapest, Hungary) from a stock solution of 0.4 mg/ml were administered intraperitoneally at 17.00 h. The total amount of injection never exceeded 0.4 ml.

Whenever stress was combined with vinblastine treatment, a vinblastine dose of 2 mg/kg body weight was given 1 h prior to stress exposure.

Stress procedure

Mice were exposed to acute restraint stress as described by Silberman et al. (2003). In brief, they were placed in a well-ventilated polypropylene tube (30 mm diameter × 90 mm length) for 2 h starting at 1 h before dark (18.00 h). The mice were kept in their home cage for 24 h following the stress and then killed by chloroform inhalation. The non-stressed control mice were left undisturbed in their home cages prior to being killed. Mice in all groups were handled gently.

Bone marrow cells: Mitotic index (MI) and MN assay

The bone marrow cells of vinblastine-treated mice were harvested at different time intervals post-treatment, from mice euthanized by chloroform inhalation. Femoral marrow cells were gently flushed out with a 5 ml syringe containing 3 ml fetal bovine serum (Gibco, North Andover, MA, USA) and smeared onto clean slides. The smeared cells were left to air dry for 24 h and fixed with absolute methanol for 5 min and stained according to the May-Grünwald–Gimsa technique. Observations were made within 24 h.

MI was calculated for each vinblastine dose by scoring the mitotic blocked cells in all cells scored. The mitotic blocked cells (Figure 1) were distinguishable by their appearance as they contained visible chromosomal clumps instead of interphase nuclei.

Figure 1.  Mitotic blocked bone marrow cells versus interphase cells.

The MN assay was performed according to Hayashi et al. (1994). The coverslipped slides were blindly scored on coded slides at × 1000 magnification by different viewers. At least 2000 polychromatic erythrocytes (PCEs) with or without MNs (Figure 2) were scored per slide. The ratio of micronucleated polychromatic erythrocytes (MNPCEs) to PCEs was calculated after recording both PCE and MNPCEs on each slide. Normochromatic erythrocytes (NCEs) were also scored on each slide.

Figure 2.  Micronucleated polychromatic erythrocyte (MNPCE).

Statistical analysis

The statistical analysis was performed using software MINITAB. The differences between treated and control groups and also between treated groups were analyzed by one-way analysis of variance (ANOVA).

Results

Selection the optimal dose and time interval for vinblastine treatment

Treatment of mice with different doses of vinblastine clearly led to MN induction in PCEs 6 h post-treatment [F(1,10) = 16.62, p = 0.002 for the lowest dose]. Results in Table I represent the vinblastine-induced frequency of MNPCEs for the four different doses used. A dose of vinblastine of 3 mg/kg body weight induced the highest frequency of MNPCE [F(1,10) = 60.99, p = 0.000], but in the bone marrow cells of mice treated with this dose, vinblastine also increased the MI [F(1,10) = 66.29, p = 0.000]. The dose of vinblastine that induced a significant level of aneuploidy [F(1,10) = 15.15, p = 0.003] with the lowest MI frequency was 2 mg/kg body weight. Hence, a dose of 2 mg/kg body weight was used for aneugenic treatment in the rest of the study. The frequency of MNs in PCE of the mice treated with this dose of vinblastine was scored at 6, 18, 24, and 30 h post-treatment. Results in Table I show the significant time-dependent increase in the frequency of MNPCE to 24 h post-treatment [F(1,10) = 49.70, p = 0.000]. After 30 h of vinblastine treatment, the frequency of MNPCE declined. To detect the maximum frequency of MNPCE, the vinblastine dose and time of harvesting were selected at 2 mg/kg body weight and 24 h post-treatment, respectively, for the main experiment. There were no cells with mitosis blocked at 18, 24 and 30 h post-treatment.

Table I.  Effect of different doses of vinblastine on MNPCE-induction.

Dose of vinblastine (mg/kg BW)	Hours post-vinblastine treatment	MNPCEs in 100 PCEs (means+SEM)	MI in 100 nucleated cells (means+SEM)
0	–	1.12 ± 0.47^*	0.00^*
1	6	2.28 ± 0.64^*,^*^*	0.00^*
2	6	2.44 ± 0.72^*^*	0.39 ± 0.11^*
	18	3.18 ± 1.08^*^*	–
	24	6.24 ± 1.11^*^*^*	–
	30	5.87 ± 1.10^*^*^*	–
3	6	4.31 ± 1.17^*^*^*	4.98 ± 1.78^*^*
5	6	3.01 ± 0.65^*^*,^*^*^*	5.14 ± 2.79^*^*

Cells from both femurs of four mice were analyzed for each treatment. MNPCEs, micronucleated polychromatic erythrocytes; PCEs, polychromatic erythrocytes; MI, mitotic index. ^*,^**,^***Significantly different (p < 0.01).

MN induction in stressed mice

Data in Table II show that stress alone did not increase the frequency of MNPCE. The frequency of MNPCE in mice restrained for 2 h was not significantly different from control (Table II) [F(1,10) = 3.705, p = 0.08]. For the vinblastine-treated mice, the frequency of MNPCE after 24 h was almost twice that in the control and stressed groups of mice [F(1,10) = 128.16, p = 0.000]. The stress+vinblastine treated mice showed the highest frequency of MNPCE. The frequency of MNPCE for this group was nearly sevenfold that in the control and stressed groups [F(1,10) = 107.50, p = 0.000]. There were no statistical differences in the proportion of PCEs in the total population, including NCEs, i.e. in PCE/PCE+NCE, in any group of treated mice compared to control [F(1,10) = 2.85, p = 0.07] (Table II).

Table II.  Effect of restraint stress on MNs induction in bone marrow cells.

	MNPCEs in 100 PCEs (means+SEM)	PCE/PCE+NCE (means+SEM)
Control	1.73 ± 0.23^*	49.98 ± 0.75
Stress	2.93 ± 1.79^*,^*^*	50.23 ± 1.12
Vinblastine treatment	4.07 ± 0.61^*^*	51.12 ± 0.95
Stress+vinblastine treatment	14.26 ± 3.31^*^*^*	48.56 ± 1.27

Cells from both femurs of four mice were analyzed for each treatment. MNPCEs, micronucleated polychromatic erythrocytes; PCEs, polychromatic erythrocytes; NCE, normochromatic erythrocyte. ^*,^**,^***Significantly different (p < 0.01).

Discussion

In this study, the frequency of bone marrow MNPCEs in the control group (1.73%) was similar to the frequency reported by others (Tiku et al. 2004). The increase in frequency of MNPCEs in vinblastine treated mice was expected because of its aneugenic capability, as documented in several reports (Channarayappa and Nath 1992; Huber et al. 1996). The observed MNs contain whole chromosomes delayed and consequently lost in mitosis (Wakata and Sasaki 1987). Vinblastine treatment at 2 mg/kg body weight significantly increased the frequency of MNs at 24 h post-treatment in comparison with the control group, and this dose did not increase MI. The MN assay reveals damage to the genetic material both in structure and chromosome number, seen when cells are dividing. Mitotically blocked cells cannot reveal chromosomal damage in the form of MNs, hence for the main experiment to obtain the highest level of MNs induction the vinblastine dose of 2 mg/kg was used as this did not block mitosis. This confirms previous findings on clastogenic effects of vinblastine (Ramesh et al. 2004).

Stress and its effect on fidelity of cell cycle

There is evidence that stress may damage the cell cycle fidelity and chromosomes (Gorlov and Borodin 1986; Flint et al. 2005, 2007). However, there is no direct evidence of its effects on the chromosomal integrity of the cells in the presence of external stimuli that induce aneuploidy. Cytogenetic effects of stress and effects on cell cycle mechanisms, and direct effects of severe psychogenic stress on chromosomes have been reported (Gorlov and Borodin 1986; Nersesyan et al. 2001; Vostrikova and Butorina 2006).

In this study, the frequency of MNPCE of mice under acute restraint stress was not significantly altered by restraint stress alone, but, when accompanied by aneugenic treatment (vinblastine administration), there was a significantly greater level of induced MNs compared to vinblastine treatment alone. This result indicates the synergistic effects of stress with vinblastine in increasing the frequency of aneuploidy in bone marrow cells. Hence, combined stress and vinblastine treatment resulted in an MNPCE frequency exceeding the expected additive effect of the two individual treatments. The stress itself may not have profound harmful effects on genetic material, but the data suggest that it is capable of sensitizing mouse bone marrow cells to the effects of vinblastine. This could happen by stress-induced malfunctioning of the cellular mechanisms monitoring the genetic integrity (Gorlov et al. 1986; Flint et al. 2007), or perhaps by increased blood pressure as a result of catecholamine release during stress, causing more vinblastine to enter the bone marrow.

However, it is also possible that direct actions of stress hormones mediate the synergistic action of stress on the effects of vinblastine on bone marrow cells. Plasma levels of glucocorticoids are highly increased for a short time after restraint stress. Glucocorticoids have a variety of effects on metabolic procedures and cell proliferation in bone marrow, with a complex role either stimulating or inhibiting various cellular processes (Phillips et al. 2006; Sundberg et al. 2006), and affecting apoptosis and cell death mechanisms (Chen et al. 2005). Flint et al. (2007) have demonstrated the effects of acute stress on provoking DNA damage, alteration in repair mechanisms, gene expression, and transcriptional activation.

In conclusion, the study has shown a remarkable augmentation of the number of MNPCE in bone marrow by combining vinblastine treatment with acute restraint stress, indicating enhancement of the damaging actions of an aneugenic agent by exposure to an emotional stressor.

Acknowledgements

This research was sponsored by a grant from Ferdowsi University of Mashhad-Iran Research Department. We would like to express our deepest gratitude to Dr Moghadam Matin for her detailed revision of the manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.