• 한국동물생명공학회지
• /
• 제37권2호
• /
• pp.149-154
• /
• 2022

Busulfan is the most commonly used drug for preconditioning during the transplantation of hematopoietic stem cells and male germ cells. Here, we describe side effects of high doses of busulfan in male mongrel dogs. Busulfan was intravenously administered to three groups of dogs at doses of 10, 15, and 17.5 mg/kg body weight. The total white blood cell, neutrophil, eosinophil, lymphocyte, monocyte, and platelet counts steadily reduced in a dose-dependent manner following busulfan treatment. The white blood cell, neutrophil, and monocyte counts recovered after 6 weeks of busulfan treatment, however, the eosinophil, lymphocyte, and platelet counts remained unaltered. Additionally, there was one fatality in the each of the groups that were administered 15 and 17.5 mg/kg busulfan. The gross lesions included severe hemorrhage in the stomach, intestinal tracts, mesentery and urinary bladder. Microscopic investigation revealed severe pulmonary edema and hemorrhage in the lungs, and severe multifocal to coalescing transmural hemorrhage in the intestines and urinary bladder. These results indicated that treatment with busulfan at doses higher than 15 mg/kg initiates severe bleeding in the internal organs and can have fatal results.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.21-21
• /
• 2003

Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. We examined the change of gene expression in detail using cDNA microarray analysis of mouse testis treated with busulfan. A subtoxic dose of busulfan (40mg/kg of body weight) transiently increased 228 mRNA levels among of the 8000 genes analyzed. TagMan analysis confirmed that the mRNA levels such as defensive protein, support protein, enzymatic protein, transport protein, and hormonal protein were rapidly increased. These results were re-confirmed by real-time PCR analysis. However, the expression levels of these genes induced by busulfan treatment were significantly reduced in control testis, indicating that both of male germ cells and somatic cells after busulfan treatment induces self-defense mechanism for protection of testicular cell death. Among them, we conclude that defense proteins play a key role in testis injury induced by busulfan.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.4-4
• /
• 2002

Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. Spontaneous apoptosis of germ cells was observed in the testis of adult mice and experimentally induced busulfan treated mice increased this apoptosis to such an extent that there was a decrease in the weight of the testis. (omitted)

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.21-21
• /
• 2001

일반적으로 세포는 방사능이나 항암제 등의 자극에 의해 DNA가 손상받았을 때 DNA를 합성하기 전, DNA변이를 복구하기 위해 cell cycle을 정지시키게 된다. pRB(retinoblatoma protein)는 이러한 cell cycle의 조절기작에서 중요한 역할을 담당하는 것으로 알려져 있다. G1기에서 S기로 진행하는 것을 조절하는 단백질인 pRB 은 E2F(cell cycle transcription factor)와 상호작용하여 cell cycle 진행에 필요한 전사활성을 억제, PCNA (proliferating cell nuclear antigen)의 합성을 저해한다. 또한, E2F와 결합된 pRB는 apoptosis를 제어하는 유전자를 조절하는 것으로 알려져 있다. Cell cycle에 영향을 미치는 항암제의 일종인 busulfan을 처리하면, 정소 내에 존재하는 대부분의 생식세포들은 사멸되고 spermatogonia만 남는 것으로 알려져 있다. 그러나 그 기작에 대해서는 자세히 연구된 바가 없다. 본 연구에서는 busulfan처리시 spermatogonial stem cell이 어떤 기작에 의해 손상받지 않고 유지되는지를 알아보고자 실험을 수행하였다. Busulfan을 처리한 마우스 (항암제 투여 후 5주)와 정상적인 13주령의 마우스의 정소로부터 각각 세포를 분리하였다. LSC (laser scanning cytometry)를 이용하여 처리군(busulfan treated mice)과 대조군(normal mature mice)에 대해 각각 DNA함량을 비교ㆍ분석한 결과 G0/G1(2N)에 머물러 있는 세포비율이 처리군에서 현저하게 증가했다 (79.3$\pm$5.5%:8.1$\pm$1.3%). Cell cycle의 G1/S check point인 pRB와 PCNA 발현을 Western blot과 면역조직학적인 방법(immunohisto-chemistry)을 이용하여 조사하였다 PCNA는 대조군과 비교해, 처리군에서 매우 낮은 수준으로 발현되었다. 면역염색된 정소단면을 살펴보면, 대조군에서는 모든 세정관에서 PCNA를 발현하는 세포가 높은 비율로 검출되었고, 처리군에서는 소수의 세정관에서 세포들이 낮은 수준으로 검출되었다. 반면에, pRB의 경우 PCNA와는 상반된 결과를 나타내어, 대조군에서는 거의 발현이 되지 않는 반면, 처리군에서는 대부분의 세정관내, 기저막을 따라 위치한 세포들에서 발현되었다. 이상의 결과는 busulfan에 의해 pRB의 인산화가 억제, pRB 와 결합된 E2F는 전사 활성이 억제되어, PCNA 합성을 저해하는 것으로 설명되어질 수 있다. 결론적으로, 인산화가 억제된 pRB (underphosphorylated RB protein)이 quiescent spermatogonial stem cell에서만 특이하게 발현하는 단백질이며, 이러한 pRB의 발현은 apoptosis를 제어하는 역할을 담당해 busulfan처리에 의해 손상받지 않고 남아있는 것으로 시사된다.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.63-65
• /
• 2002

Identification of spermatogonial stem cell-specific surface molecules is important in understanding the molecular mechanisms underlying the maintenance and differentiation of these cells. We have found that spermatogonia from busulfan treated mice expressed an autoantigen that distinguishes between undifferentiated and differentiated spermatogonia. Four to six weeks after busulfan treatment, germ cells located in the basal compartment of seminiferous epithelium show isotype-specific IgG deposits that form due to autoimmunity. Before busulfan treatment, the level of testicular IgG was very low but IgG levels began to increase after week 4 and peaked at week 6. When cells from the busulfan treated testis were analyzed using laser scanning cytomeoy (LSC), the frequency of cells positive for IgG deposits, 6-integrin, and 1-integrin were 16.5${\pm}$3.8%, 11.8${\pm}$2.6%, and 9.0${\pm}$ 1.4%, respectively. Immunofluorescent staining suggested that most, if not all of the cells with IgG-deposits isolated from a laminin-coated dish, were also positive for a spermatogonial stem cell marker \ulcorner6-integrins as well as for a germ cell-specific marker TRA 98. We determined serum and intratesticular IgG levels and the soundness of seminiferous tubule basement membrane from busulfan treated mice using electron microscopy, in order to study the mechanism responsible for IgG deposits in spermatogonia. We found that the basement membranes of seminiferous tubules from busulfan treated mice were severely impaired when compared to those of normal adult, neonates and w/wv mice. Furthermore, new blood cells were observed in the surface of the damaged basement membrane along the seminiferous tubules. These results suggest that the IgG in spermatogonial stem cells accumulates from circulating blood through the impaired basement membranes induced by busulfan treatment. Taken together, our study suggests that IgG can be used as a new marker for undifferentiated spermatogonia cells.

• Clinical and Experimental Reproductive Medicine
• /
• 제40권2호
• /
• pp.60-66
• /
• 2013

Objective: Impairment of spermatogenesis has been identified as an inevitable side effect of cancer treatment. Although estrogen treatment stimulates spermatogenic recovery from the impaired spermatogenesis by suppressing the intra-testicular testosterone (ITT) level, side effects of estrogen are still major impediments to its clinical application in humans. Soybeans are rich in genistein, which is a phytoestrogen that binds to estrogen receptors and has an estrogenic effect. We investigated the effects of genistein administration on ITT levels, testis weight, and recovery of spermatogenesis in rats treated with a chemotherapeutic agent, busulfan. Methods: Busulfan was administered intraperitoneally to rats, and then a GnRH agonist was injected subcutaneously into the back, or genistein was administered orally. Results: The weight of the testes was significantly reduced by the treatment with busulfan. The testis weight was partially restored after busulfan treatment by additional treatment with either the GnRH agonist or genistein. Busulfan also induced atrophy of a high percentage of the seminiferous tubules, but this percentage was decreased by additional treatment with either the GnRH agonist or genistein. Treatment with genistein was effective at suppressing and maintaining ITT levels comparable to that in the GnRH agonist group. Conclusion: Genistein effectively suppressed ITT levels and stimulated the recovery of spermatogenesis in rats treated with a chemotherapeutic drug. This suggests that genistein may be a substitute for estrogens, for helping humans to recover fertility after cancer therapy without the risk of side effects.

• Journal of Ginseng Research
• /
• 제39권3호
• /
• pp.243-249
• /
• 2015

Background: Anticancer agents induce a variety of adverse effects when administered to cancer patients. Busulfan is a known antileukemia agent. When administered for treatment of leukemia in young patients, busulfan could cause damage to the male reproductive system as one of its adverse effects, resulting in sterility. Methods: We investigated the effects of Korean Red Ginseng extract (KRGE) on busulfan-induced damage and/or dysfunction of the male reproductive system. Results: We found that administration of busulfan to mice: decreased testis weight; caused testicular histological damage; reduced the total number of sperm, sperm motility, serum testosterone concentration; and eventually, litter size. Preadministration of KRGE partially attenuated various busulfan-induced damages to the male reproductive system. These results indicate that KRGE has a protective effect against busulfan-induced damage to the male reproduction system. Conclusion: The present study shows a possibility that KRGE could be applied as a useful agent to prevent or protect the male reproductive system from the adverse side effects induced by administration of anticancer agents such as busulfan.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.38-38
• /
• 2001

Spermatogonial stem cells은 sperrnatogenesis에서 중요한 역할을 하며, 곡세정관의 기저막에 위치하고 있는 것으로 알려져 있다. 그러나, 그 동안 이 세포에 특이하게 발현되는 marker가 거의 알려져 있지 않아 spermatogonial stem cell의 연구에 많은 어려움을 가져왔다. 최근 일반적인 stem cell이 갖는 특성 중, 기저막과 상호작용을 하는 surface protein으로 integrin이 존재한다는 사실을 이용하여, anti-$\alpha$$_{6}$/ 또는 anti-$\beta$$_1$ integrin항체로 germ cell을 선발하여 정소에 이식한 결과, 높은 효율로 이식세포유래의 정자발생이 가능하다는 결과가 보고되었다 (Shinohara et al., 1999). 한편, 항암제의 일종인 busulfan을 마우스에 투여(40mg/kg)한 후 4-5주가 경과하면 세정관의 기저막에 위치하는 spermatogonia를 제외하고 대부분의 생식세포는 소멸한다 본 실험의 목적은 이러한 사실들을 이용하여 spermatogonial stem cell의 특성을 밝히고, 이 생식세포를 보다 간편하고 손쉽게 선발할 수 있는 시스템을 확립하는데 있다. Busulfan처리 후 5주가 경과된 마우스와 정상적인 13주령의 마우스 testis로부터 세포를 분리한 후 FITC-conjugated anti-IgG를 이용한 면역형광법으로 측정.분석한 결과, 형광표식된 세포비율이 대조군과 비교하여 busulfan을 처리한 경우에서 유의적인 증가를 보였다.(17$\pm$3.8%. 0.7$\pm$0.3% busulfan vs control). 또한, IgG와 결합한 단백질이 존재하는 이들 세포들은 곡세정관의 기저막을 따라 위치하며, 단백질과 복합체를 형성한 IgG는 anti-Ig $G_{2a}$와 반응하지 않는다는 사실을 관찰했다. 이러한 IgG 복합체를 형성한 세포들의 특성을 이용하여, IgG와 반응을 하지 않는 것으로 확인된 이차 항체인 an1i-Ig $G_{2}$와 일차 항체인 anti-$\alpha$$_{6}$ 또는 anti-$\beta$$_1$ integrin항체를 이용하여 측정.분석하였다. Busulfan을 처리한 마우스 정소에서 분리한 세포를 다시 laminin으로 코팅된 dish에서 선발.회수해서, anti-lgG, anti-$\alpha$$_{6}$ 또는 anti-$\beta$$_1$ integrin항체로 각각 표식된 세포비율을 비교하였다. Laminin으로부터 선발.회수한 세포에서는 IgG복합체가 $\alpha$$_{6}$ 또 는 $\beta$$_1$integrin과 거의 같은 수준에서 높은 비율로 표식되었다. 결론적으로, busulfan에 의해 유도된 IgG와 결합가능한 단백질은 $\alpha$$_{6}$나 $\beta$$_1$ integrin과 마찬가지로 immunoglobulin G를 이용하여 spermatogonial stem cell의 선발을 가능하게 했다. 따라서, busulfan처리시 IgG는 미분화된 정조세포의 선발을 위한 하나의 marker로서 사용가능함을 시사한다.다.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.22-22
• /
• 2002

Identification of spermatogonial stern cell-specific surface molecules is important in understanding the molecular mechanisms underlying the maintenance and differentiation of these cells. We have found that spermatogonia from busulfan treated mice expressed an autoantigen that distinguishes between undifferentiated and differentiated spermatogonia. Four to six weeks after busulfan treatment, germ cells located in the basal compartment of seminiferous epithelium show isotype-specific IgG deposits that form due to autoimmunity. (omitted)

• The Korean Journal of Physiology and Pharmacology
• /
• 제20권3호
• /
• pp.245-251
• /
• 2016

The objective of this study was to externally validate a new dosing scheme for busulfan. Thirty-seven adult patients who received busulfan as conditioning therapy for hematopoietic stem cell transplantation (HCT) participated in this prospective study. Patients were randomized to receive intravenous busulfan, either as the conventional dosage (3.2 mg/kg daily) or according to the new dosing scheme based on their actual body weight (ABW) ($23{\times}ABW^{0.5}mg\;daily$) targeting an area under the concentration-time curve (AUC) of $5924{\mu}M{\cdot}min$. Pharmacokinetic profiles were collected using a limited sampling strategy by randomly selecting 2 time points at 3.5, 5, 6, 7 or 22 hours after starting busulfan administration. Using an established population pharmacokinetic model with NONMEM software, busulfan concentrations at the available blood sampling times were predicted from dosage history and demographic data. The predicted and measured concentrations were compared by a visual predictive check (VPC). Maximum a posteriori Bayesian estimators were estimated to calculate the predicted AUC ($AUC_{PRED}$). The accuracy and precision of the $AUC_{PRED}$ values were assessed by calculating the mean prediction error (MPE) and root mean squared prediction error (RMSE), and compared with the target AUC of $5924{\mu}M{\cdot}min$. VPC showed that most data fell within the 95% prediction interval. MPE and RMSE of $AUC_{PRED}$ were -5.8% and 20.6%, respectively, in the conventional dosing group and -2.1% and 14.0%, respectively, in the new dosing scheme group. These findings demonstrated the validity of a new dosing scheme for daily intravenous busulfan used as conditioning therapy for HCT.