Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity

Abstract

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16-and 24-dayold rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult. (Cancer Res 2005; 65(21): 9999-10005)

Dexrazoxane exacerbates doxorubicin-induced testicular toxicity

“…Spermatogenic cells are highly susceptible to DXR, mainly due to their high mitotic activity. Spermatocytes and spermatogonia are considered predisposed to toxic stimuli, more than other components of the testis (Quiles et al 2002, Hou et al 2005.…”

“…DXR has been shown to adversely affect spermatogenesis as well as the quantity and quality of sperm; it exerts its toxicity via oxidative stress pathways (Sikka 1996, Suominen et al 2003, Hou et al 2005, Ateşşahin et al 2006. Previous studies have demonstrated that the level of malondialdehyde (MDA), which derives from lipid peroxidation, rises after exposure to DXR, whereas the level of GSH declines (Quiles et al 2002, Ateşşahin et al 2006.…”

“…Previous studies have demonstrated that the level of malondialdehyde (MDA), which derives from lipid peroxidation, rises after exposure to DXR, whereas the level of GSH declines (Quiles et al 2002, Ateşşahin et al 2006. Several protectants have been applied to reduce DXR-induced testicular toxicity (Hou et al 2005); none of them was implemented into clinical practice. Though our results showed that DEX reduced the oxidative stress exerted by DXR within the testis, the oxidative stress may not be the dominant pathway to mediate DXR toxicity, because despite the protective effect of DEX in this cellular pathway the tissue damage was more prominent in mice receiving the combined treatment.…”

“…Doxorubicin (DXR), an anthracycline that is a cornerstone of many chemotherapeutic regimens, is used for treating a wide spectrum of malignancies including both hematological and solid tumors. DXR has been previously studied for its deleterious effect on the gonadal epithelium in rodents, where it was shown to cause chromosome aberrations in spermatogonial cells (Au & Hsu 1980, Abraham & Fränz 1983, inhibition of DNA synthesis and induction of apoptosis (Brinkworth et al 1995, Blanco-Rodríguez & Martínez-García 1996, Shinoda et al 1999, Hou et al 2005. DXR has been shown to disrupt sperm maturation, reduce epididymal sperm concentration as well as alter spermatogonial DNA and sperm morphology (Meistrich et al 1990).…”

“…These include amifostin, lycopene and other anti-oxidants with conflicting evidence as for their benefit (Hou et al 2005, Yeh et al 2007, Vendramini et al 2010. Due to the promising role of DEX in reducing DXR-induced cardiac toxicity, our aim was to evaluate its role in reducing DXR-induced testicular toxicity by assessing morphological and biochemical measurements following in vivo administration of DXR with or without DEX.…”