Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

Abstract

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4',6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Figure 1

Photographs of mouse MI and II spermatocytes after DAPI staining (A–C) and first-cleavage (1-Cl) zygotes after hybridization with chromosome-specific painting probes for chromosomes 1–3 and X (labeled with biotin and signaled with FITC) and chromosome Y [labeled with digoxigenin and signaled with rhodamine (D–F)]. Images were taken by using a Vysis (Downers Grove, IL) QUIPS Imaging Analysis System, and the final composite figure was made in Adobe PHOTOSHOP. (A) Normal MI spermatocyte. (B) MI spermatocyte with multiple chromosome structural aberrations. (C) MII spermatocyte with chromatid acentric fragments (one is indicated by the arrow, the second is in the center of the metaphase). (D) Normal 1-Cl zygote metaphase with the Y-bearing sperm-derived chromosomes on the left. (E) 1-Cl zygote with a centric fragment in the paternal chromosomes (arrow). (F) Hyperploid 1-Cl zygote. Note the presence ofa an extra chromosome (green) in the X-bearing sperm-derived chromosomes.

Figure 2

Comparison of the proportions of zygotes with unstable chromosomal aberrations and/or aneuploidy vs. proportions of dead implants. d, days.