Busulfan is an alkylating agent that is commonly used as a preconditioning drug in the clinical transplantation of hematopoietic stem cells, and is also traditionally used method for chemical sterilization (McCune and Holmberg, 2009; Hur et al., 2017). The side effects of busulfan include bone marrow suppression, pulmonary fibrosis, and skin pigmentation. Previous studies have demonstrated that preconditioning using busulfan is more effective than irradiation during human B cell differentiation (Krivoy et al., 2008; Hayakawa et al., 2009). Additionally, busulfan is most commonly used for chemical sterilization during the transplantation of male germ cells. Busulfan depletes male germ cells and disrupts the junctions between Sertoli cells (Bucci and Meistrich, 1987).

Numerous studies have reported that the dose of busulfan necessary for sterilization varies widely among species. The dose of busulfan necessary for sterilization in pigs, monkeys, coyotes, mice, rats and chickens is 40-100 mg/kg (Kim et al., 1997), 8-12 mg/kg (Hermann et al., 2012), 4-12 mg/kg (Stellflug et al., 1985), 20-40 mg/kg (Xie et al., 2020), two injections of 10 or 15 mg/kg (Ogawa et al., 1999) and 40 mg/kg (Kim et al., 2021), respectively. We have previously reported the successful preparation of recipient testis during the transplantation of spermatogonial stem cells in dogs by treatment with 15-17.5 mg/kg of busulfan (Hur et al., 2017), based on the results of another study (Deeg et al., 1999).

In this study, we report the side effects of busulfan in dogs when administered at high doses for chemical sterilization. Hematological analysis was performed every week according to the dose at which busulfan was administered. We additionally performed autopsy and histological analysis, and two fatalities occurred after receiving busulfan at high doses. This report is the first to describe the side effects of busulfan in dogs for the preparation of recipient testes for the transplantation of male germ cells.

Animals and experimental design

One-year-old mongrel dogs (n = 10), weighing approximately 15-20 kg, were randomly divided into four groups: control (Con, n = 1), 10 mg/kg (n = 3), 15 mg/kg (n= 3), and 17.5 mg/kg (n= 3). Experimental dogs received acepromazine (0.1 mg/kg; IM) for premedication. Busulfan (Sigma Aldrich, B2635) was dissolved in dimethyl sulfoxide and injected into the vein along with 50 mL of saline according to previous study (Deeg et al., 1999). The changes in the body weights of the mongrels were measured and whole-blood samples were collected every week to 6weeks from busulfan treatment. The experimental procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Animal Science in Republic of Korea (approval no. 2016-853).

Analysis of behavior changes

In order to assess the behavioral changes of the animals in the experimental groups, the body weights and behavioral attributes of the animals were evaluated on a daily basis. The food and water intake of the animals, their activity, depression, crunching, diarrhea, anorexia, and lethargy were evaluated.

Blood cell analysis

The whole blood samples were collected in EDTA-coated tubes and inverted several times for preventing coagulation. The white blood cell, neutrophil, lymphocyte, eosinophil, basophil, monocyte, and platelet counts were compared among the treatment groups and control. The samples were immediately analyzed using a multi-species analyzer (HEMAVET® 950, Drew Scientific Inc., USA), according to the manufacturer’s instructions.

Autopsy and organ preparation

Postmortem analyses were performed by a veterinarian and the internal organs of the deceased animals were collected. The organs were then approximately cut into pieces of 1 × 1 × 1 cm³ size, and fixed overnight in Bouin’s solution at 4℃. The fixed samples were subsequently washed with 70-100% (v/v) ethanol, washed in xylene, embedded in paraffin, sliced into 5-µm-thick sections using a microtome (Thermo Fisher Scientific, Waltham, MA, USA), and mounted on glass slides. The mounted tissues were rehydrated with xylene and by passing through a gradient of 100-0% ethanol. The rehydrated specimens of testes were stained with hematoxylin and eosin (H&E).

Statistical analysis

The one and three animals used each control and busulfan treatment groups. Additionally, 3 biological and 3 technical replicates were performed for blood analysis. The data were analyzed by one-way analysis of variance using the SPSS statistical package, version 21.0, for Windows. A t-test was performed for comparing the control and experimental groups. All the data are presented as the mean ± standard deviation. The null hypothesis was rejected when p < 0.05.

Analysis of behavior changes

The behavioral changes of the animals in the experimental groups are summarized in Table 1. The two animals that expired did not display any abnormal behavior until after 9 days of treatment with busulfan. In the mongrel that received 15 mg/kg of busulfan, depression was noted on day 10, crunching with anorexia on day 13, and fatality on day 16. In the mongrel that received 17.5 mg/kg of busulfan, depression was noted on day 13, hematuria, melena, and anorexia on day 14, and fatality on day 15. No side effects were observed in the animals that received 10 mg/kg of busulfan and those in the control group (Table 1).

Table 1. Clinical signs in mongrel dogs after high-dose busulfan therapy.

Dosage mg/kg	Days after busulfan treatment
	0	1	7	8	9	10	11	12	13	14	15	16
10	N	N	N	N	N	N	N	N	N	N	N	N
15	N	N	N	N	N	D	D/C	D	A	A/C	A/L	death
17.5	N	N	N	N	N	N	N	N	A	L/M/H	death
Control	N	N	N	N	N	N	N	D	D	D	N	N

N, normal behavior; D, depression; C, crouching; A, anorexia; L, lethargy; M, melena; H, hematuria..

Analysis of hematological changes

We analyzed the complete blood count for determining whether busulfan treatment affects the population of blood cells. The total white blood cell counts were significantly reduced after 3 weeks of treatment with 10-17.5 mg/kg of busulfan. Additionally, the number of neutrophils, eosinophils, and lymphocytes among the total number of white blood cells significantly reduced in the busulfan-treated groups. The total counts of white blood cells and neutrophils recovered after 6 weeks of treatment with busulfan, however, the eosinophil and lymphocyte counts did not undergo recovery. Neutrophils primarily play a role in acute inflammation (Fig. 1), and the neutrophil-to-lymphocyte ratio is a measure of the balance between the neutrophil and lymphocyte counts, with a high neutrophil-to-lymphocyte ratio indicating systemic inflammation (Zahorec, 2001). In this study, busulfan was used to induce inflammation. The monocyte counts significantly decreased after 2 weeks of treatment and recovered after 6 weeks of treatment with busulfan. The platelet counts significantly decreased after 3 weeks of treatment with busulfan. The platelets were exhausted in the groups that were treated with 15-17.5 mg/kg of busulfan. Platelet production principally occurs in the bone marrow by the process of thrombopoiesis. During thrombopoiesis, the hematopoietic progenitor cells differentiate to form megakaryocytes, which terminally differentiate to release platelets from their long cytoplasmic processes (Perdomo et al., 2017). In the present study, the counts of most of the blood cells were significantly reduced following treatment with busulfan. These results were consistent with those of a previous study (Deeg et al., 1999). Another study described that 20 mg/kg of busulfan, administered either in a single intravenous infusion or in increments of 5 mg/kg/day for four consecutive days, consistently induced marrow ablation with severe neutropenia and thrombocytopenia in dogs (Storb et al., 1977).

Figure 1.Number of white blood cells (WBC), granulocytes, monocytes and platelet following treatment with busulfan. (A) white blood cells, (B) Neutrophils, (C) Eosinophils, (D) Lymphocyte, (E) Monocytes, and (F) platelets are presented as the number of total white blood cells. A statistical analysis was conducted to determine differences in cell numbers between the 0 mg/kg (control) group and each busulfan treatment group in the same week 5 (*p < 0.05, n = 9).

Of the dogs that received high doses of busulfan, two dogs exhibited depression, crunching, and anorexia after 10 days of treatment with busulfan. The animals also exhibited thrombocytopenia during this time. The abnormalities noted in this study were similar to those reported in a previous study in which a pig developed similar abnormalities following the administration of busulfan at a high dose. The animal suffered from hematuria, poor appetite, and decreased activity after approximately 10 days of busulfan treatment, and the platelet count diminished to nearly zero by day 11 (Abe et al., 2016). This suggests that the thrombocytopenia that was observed 10 days after treatment with busulfan was due to myeloablation.

Gross and microscopic lesion

Following treatment with 15 mg/kg of busulfan, gross lesions were noted in various organs. These included severe petechiations in the lungs, blood clots in the abdominal cavity, and ecchymoses in the mesentery, stomach, intestinal tract, and urinary bladder (Fig. 2A). The administration of 17.5 mg/kg of busulfan was accompanied by the development of gross lesions, including severe, multifocal petechiations in the lungs, mesentery, stomach, and large intestines. Additionally, there was pronounced hemorrhage in the urinary bladder (Fig. 3A).

Figure 2.Gross lesions present in various organs and histological analysis following treatment with 15 mg/kg of busulfan. (A) Gross lesions, a. lung, b. liver, c. stomach, d. duodenum, e. rectum, f. urinary bladder. (B) Hematoxylin & Eosin staining analysis, a. lung (200x), b. liver (50x), c. stomach (50x), d. duodenum (200x), e. rectum (50x), f. urinary bladder (50x). The scale bars indicate 100 μm.

Figure 3.Gross lesions present in various organs and histological analysis following treatment with 17.5 mg/kg of busulfan. (A) Gross lesions, a. lung, b. liver, c. stomach, d. duodenum, e. rectum, f. urinary bladder. (B) Hematoxylin & Eosin staining analysis, a. lung (50x), b. liver (50x), c. stomach (50x), d. duodenum (50x), e. rectum (50x), f. urinary bladder (50x). The scale bars indicate 100 μm.

The administration of 15 mg/kg of busulfan was accompanied by the development of microscopic lesions, including severe hemosiderosis and the presence of red blood cells in the sinuses and lymph nodes, severe pulmonary edema and hemorrhage in the lungs, moderate submucosal hemorrhage in the stomach, and severe multifocal to coalescing transmural hemorrhage in the intestinal tract (Fig. 2B). The administration of 17.5 mg/kg of busulfan was accompanied by the development of microscopic lesions, including severe hemorrhage in the urinary bladder, congestion and hemorrhage in the intestinal tract, and severe pulmonary edema and hemorrhage in the lungs (Fig. 3B).

The gross and microscopic lesions that were noted in the two fatalities were consistent with the severe hemorrhage observed therein (Fig. 2 and 3). This suggested that the intestinal damage following the administration of busulfan at a high dose is not restricted to the mucosal surface, but affects the deeper compartments as well (Storb et al., 1977). The dog that received 17.5 mg/kg of busulfan had hematuria and melena, which correlated with the microscopic lesions in the bladder and intestines, observed during necropsy (Fig. 3). These findings were slightly different from those observed for the dog that was administered 15 mg/kg of busulfan. The intestinal mucosal hemorrhage that was observed in this study was consistent with that reported by Deeg and coworkers, in which the dogs were treated with high doses of busulfan (Deeg et al., 1999). The slight differences in the lesions observed between the two cases in this study indicated that the side effects associated with the use of busulfan in dogs are dose-dependent.

In this study, the gross lesions that were observed in the two cases included severe hemorrhagic abnormalities in various organs, including the stomach, bladder, and mesentery. The microscopic lesions included severe hemorrhages of various degrees in all the internal organs. These lesions indicated that treatment with busulfan at a high dose leads to cytotoxicity in various organs and can have lethal consequences in dogs.

We thank Dr. Soyoung Chun for caring and treating experimetal dogs.

Conceptualization, T-Y.H., S.L., S-A.O.; methodology, H.J.; writing—original draft preparation, T-Y.H.; writing—review and editing, W-Y.L.

This study was supported by Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration, Republic of Korea (grant number PJ011865).

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No potential conflict of interest relevant to this article was reported.