Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase

Abstract

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. © 2004 Wiley-Liss, Inc. Key words: testis; etoposide; apoptosis; rat; spermatogenesisEtoposide is a podophyllotoxin semiderivative agent that has been used in a variety of chemotherapeutic treatments, including therapy for pediatric tumors. Previous reports have proposed that etoposide antimitotic activity is mediated by its interaction with topoisomerase II, an ATP-dependent nuclear enzyme that regulates DNA topology by transiently breaking and uniting doublestranded DNA (Arnold, 1979;Hande, 1998). Topoisomerase II is essential for DNA replication, transcription, recombination, and chromosomal segregation. Etoposide promotes the formation of a ternary DNA-topoisomeraseetoposide complex; thus, topoisomerase II is not allowed to rejoin the DNA fragment that is produced during the cell cycle (Creaven and Allen, 1975). This action converts topoisomerase II into physiological toxins that induce irreversible damage to DNA by generating insertions, deletions, chromosomal aberrations, and translocations. When a sufficient concentration of permanent DNA breaks is present in the cells, a series of events occurs that culminates in apoptosis (Creaven and Allen, 1975;Russell et al., 1998).Apoptosis is a common physiological phenomenon that occurs in proliferating and constantly renewing tissues, including the seminiferous epithelium (Russell et al., 1987;Kerr, 1992;Brinkworth et al., 1995; Blanco-Rodrí-guez and Martínez-Garcia, 1996). Spontaneous cell death during spermatogenesis is an essential means of maintaining homeostasis in the testis, and is responsible...

Carnitine partially protects the rat testis against the late damage produced by doxorubicin administered during pre‐puberty

“…Indeed, our group has been showing that chemotherapeutic treatments during pre-puberty can lead to irreversible damage to the seminiferous epithelium of rats, although some degree of spermatogenesis restoration is frequently observed (Stumpp et al, 2004;Okada et al, 2009;Vendramini et al, 2010Vendramini et al, , 2012Brilhante et al, 2011Brilhante et al, , 2012. Among the chemotherapeutic drugs studied, etoposide (Stumpp et al, 2004;Okada et al, 2009) and doxorubicin (Vendramini et al, 2010(Vendramini et al, , 2012Brilhante et al, 2011Brilhante et al, , 2012 appear to be particularly aggressive to the reproduction.…”

“…Mammary gland sections supplied with the ApopTag kit were used as positive controls. Cells were considered TUNEL-positive when they showed dark labeling as intense as, or more intense than, those apoptotic cells observed in the positive control slides (Hasegawa et al, 1997;Stumpp et al, 2004). The number of TUNEL-positive cells per cubic millimeter (numerical density) was obtained from 60 fields per rat.…”

“…The number of TUNEL-positive cells per cubic millimeter (numerical density) was obtained from 60 fields per rat. The numerical density of TUNEL-positive cells was calculated according to Stumpp et al (2004).…”

Inter‐laboratory control data for reproductive endpoints required in the OPPTS 870.3800/OECD 416 reproduction and fertility test

“…Morphology is considered a sensitive endpoint with which to detect treatment-related effects because the probability of detecting a doubling of abnormal sperm forms (e.g., 3 to 6%) is 90% when n 5 20 samples/group (Chapin and Conner, 1999). Chapin et al (1997) showed altered fertility when sperm morphology alterations exceeded 10%, but it seems likely that when morphology alterations achieved a certain threshold, decreases in sperm count also could be noted due to multiple apoptotic pathways in the rodent testis responsible for the maintenance of sperm integrity (Sasagawa et al, 2001;Stumpp et al, 2004). Sperm morphology alterations also have been associated with decreased reproductive organ weights, sperm motility changes, and altered testicular histopathology (Chapin et al, 1985;Linder et al, 1988;Gray et al, 1990); however, changes in sperm shape need not be correlated with alterations in other sperm endpoints (Chapin and Conner, 1999;Morrissey et al, 1988).…”

Prenatal and lactation nicotine exposure affects Sertoli cell and gonadotropin levels in rats

“…Two non-consecutive testicular sections per animal were analyzed under !40 objective lens. Fifty fields were randomly analyzed in each testicular section, totaling a 100 fields per rat (Stumpp et al 2004). …”