Toxicity testing in the 21st century—a vision and a strategy

https://www.fertstert.org/article/S0015-0282(13)00616-X/pdf

Impact of chemotherapy and radiotherapy for testicular germ cell tumors on spermatogenesis and sperm DNA: a multicenter prospective study from the CECOS network

Impact of chemotherapeutics and advanced testicular cancer or Hodgkin lymphoma on sperm deoxyribonucleic acid integrity.

STUDY QUESTION Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? SUMMARY ANSWER Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation WHAT IS KNOWN ALREADY There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities STUDY DESIGN, SIZE, DURATION Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 03 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases PARTICIPANTS/MATERIALS, SETTINGS, METHODS F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development F1 males were subsequently mated with chow-fed female mice Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)185 and one delivered and followed postnatally DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E185 placenta and F2 E185 brain cortex MAIN RESULTS AND THE ROLE OF CHANCE F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 005) Post-implantation losses were increased amongst F2 E185 day litters from 20FS exposed F1 males (P < 005) F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 005) Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 005; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 005) While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E185 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups Inter-individual alterations in Peg1 methylation were found in F2 E185 day 10FS group brain cortex (P < 005) LARGE SCALE DATA Not applicable LIMITATIONS REASONS FOR CAUTION The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes WIDER IMPLICATIONS OF THE FINDINGS The findings of this study provide further support for paternally transmitted environmental effects The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm STUDY FUNDING AND COMPETING INTERESTS This study was supported by a grant to JMT from the Canadian Institutes of Health Research (CIHR #89944) The authors declare they have no conflicts of interest

Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation.

The incidence of testicular cancer has increased dramatically over the past 50 years. Advances in treatment, which include the coadministration of bleomycin, etoposide, and cis-platinum (BEP), have brought the cure rate to over 90%. After treatment, most patients go through a temporary period of azoo/oligozoospermia. Although the sperm concentration in approximately 80% of the patients returns to at least 10 million/mL, little is known about the integrity of the chromatin of their germ cells. Using an animal model, we assessed DNA integrity in the spermatozoa of male rats treated for 3, 6 or 9 weeks with BEP at doses, adjusted for surface area, equivalent to 0X, 1/3X, 2/3X, or 1X of the human dose. We did not observe any difference in protamination content, as assessed by the chromomycin A3 (CMA3) assay. After 9 weeks of 1X treatment, the susceptibility of DNA to denaturation evaluated by the sperm chromatin structure assay (SCSA/acridine orange assay (AO) was increased, as well as the number of single and double DNA strand breaks measured by the TUNEL and COMET assays. Parameters obtained from the AO and TUNEL assays were highly correlated with the motility of the spermatozoa, suggesting that conventional sperm analysis parameters can serve as a good indicator of chromatin integrity and vice versa. Correlation studies also suggested that the parameters obtained with the different assays do not overlap, but complement each other. Thus, BEP treatment altered spermatozoal chromatin quality, and these alterations may impact adversely on progeny outcome.

Effects of the chemotherapy cocktail used to treat testicular cancer on sperm chromatin integrity.

Testicular cancer is the most common cancer affecting men of reproductive age. Advances in treatment of the disease, which include the coadministration of bleomycin, etoposide, and cis-platinum (BEP), have brought the cure rate to over 90%. This high cure rate, coupled with the young age of patients, makes elucidation of the impact of the treatment on reproductive function, fertility, and progeny outcome increasingly important. The goal of this study was to determine the effects of BEP, in doses analogous to those given to humans, on the male reproductive system, spermatozoa, fertility, and progeny outcome in an animal model. Male Sprague-Dawley rats were treated daily with BEP for 3 cycles of 3 weeks each, for a total of 9 weeks. After 6 and 9 weeks, males were mated to 2 groups of untreated females. BEP treatment resulted in decreases in testicular and epididymal weights of 52% and 28%, respectively, when compared to control. Decreased testis and epididymis weights were accompanied by impairment of spermatogenesis and by a decrease in spermatozoal count of nearly 90% (11.9 x 10(7) spermatozoa per caput epididymidis in control vs 1.65 x 10(7) in BEP-treated rats). The percent of motile spermatozoa in the treated rats was more than 30% lower than in controls. Defects in the flagella of spermatozoa increased by more than twofold in the midpiece, and by more than sixfold in the principal piece. Paternal BEP treatment, for either 6 or 9 weeks, did not affect fertility, pre- or postimplantation loss, litter size, or sex ratio among progeny on gestation day 21. In contrast, among the pregnancies allowed to proceed to delivery, a significant number of pups sired by males treated with BEP for 9 weeks died between birth and postnatal day 2; this was not observed in pups sired by males treated for 6 weeks. Markers of postnatal development were not affected in the surviving offspring from either group. Thus, despite the dramatic effects of the testicular cancer drug regimen on spermatogenesis, the numbers of spermatozoa, and their motility and morphology, male rats were fertile. While fetal development was apparently normal, early postnatal mortality, which may be associated with a delay in parturition, was elevated among the progeny sired by males exposed to BEP for 9 weeks.

https://onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.05103

Effects of chemotherapeutic agents for testicular cancer on the male rat reproductive system, spermatozoa, and fertility.

Testicular cancer is the most common cancer among young men of reproductive age. A regimen of bleomycin, etoposide, and cisplatin (BEP regimen) is the standard chemotherapy for testicular cancer. BEP has adverse effects on spermatogenic function that pose a long-term reproductive health risk to cancer survivors and their progeny. Using a rat model, we investigated the persistence of the effects of BEP on male reproductive function, fertility, and progeny outcome. Adult male Sprague-Dawley rats received a BEP regimen mimicking human clinical exposure (three 21-day cycles of etoposide and cisplatin on days 1-5 and bleomycin on days 2, 9, and 16, or vehicle). Reproductive and progeny outcome parameters were assessed at the end of BEP treatment and up to 9 weeks post-treatment, at 3-week intervals. BEP treatment reduced testicular weights and impaired spermatogenesis, characterized by abnormal testis histology and germ cell depletion. Germ cell apoptosis increased at least 3-fold in BEP-treated rats compared with controls at the end of treatment; 9 weeks posttreatment, germ cell apoptosis in BEP-treated rats did not differ from controls. BEP-exposed males were fertile; a decrease in litter size and an increase in preimplantation and postimplantation losses were observed. Preimplantation loss remained elevated in litters sired by BEP-treated males up to 9 weeks posttreatment; however, neither postimplantation loss nor litter sizes differed from controls. Thus, both germ cell apoptosis and the postimplantation loss induced by BEP treatment were reversible. The persistence of the elevation in preimplantation loss 9 weeks after BEP treatment suggests that spermatogonia are affected.

https://onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.107.004218

Reversibility of the effects of subchronic exposure to the cancer chemotherapeutics bleomycin, etoposide, and cisplatin on spermatogenesis, fertility, and progeny outcome in the male rat

In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC(50)) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.

https://bioone.org/journals/Biology-of-Reproduction/volume-83/issue-2/biolreprod.110.083568/Development-of-a-Short-Term-Fluorescence-Based-Assay-to-Assess/10.1095/biolreprod.110.083568.pdf

Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro

Spermatogonial stem cells (SSCs) are responsible for the production of spermatozoa throughout adulthood and for the recovery of spermatogenesis following exposure to cytotoxic agents. Previously, we have shown that the combined administration of bleomycin, etoposide, and cisplatin (BEP) used in the treatment of testicular cancer causes impaired spermatogenesis and reduced sperm production in the rat. However, definitive evidence about the potential impact of such chemotherapy on SSCs is still lacking. The objective of this study was to determine whether chronic exposure to BEP treatment causes adverse effects on rat SSC activity. We first investigated the effects of BEP treatment on the clonal organization of undifferentiated spermatogonia by staining whole-mount preparations of rat seminiferous tubules for GFRA1 and ZBTB16 (previously known as PLZF), 2 established markers of undifferentiated spermatogonia. We found that BEP treatment drastically reduced the number of A-aligned spermatogonia while sparing A-single and A-paired cells from the effect. Next, we determined the SSC activity following BEP exposure. Adult transgenic rats carrying EGFP expression in the germ line were treated with BEP for 9 weeks, and SSCs were quantified using spermatogonial transplantation. We found that BEP treatment significantly decreased SSC numbers, which were restored to the control level after a 9-week recovery period. These results demonstrate that BEP treatment transiently affects the activity of rat SSCs.

Effects of chemotherapeutic agents for testicular cancer on rat spermatogonial stem/progenitor cells.

Reversibility of the effects of the chemotherapeutic regimen for non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone, on the male rat reproductive system and progeny outcome.

Ludovic Marcon

Papers

Effects of chemotherapeutic agents for testicular cancer on the male rat reproductive system, spermatozoa, and fertility.

Reversibility of the effects of subchronic exposure to the cancer chemotherapeutics bleomycin, etoposide, and cisplatin on spermatogenesis, fertility, and progeny outcome in the male rat

Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro

Effects of chemotherapeutic agents for testicular cancer on rat spermatogonial stem/progenitor cells.

Reversibility of the effects of the chemotherapeutic regimen for non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone, on the male rat reproductive system and progeny outcome.