Jack Bishop

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

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.

Chemically induced mutations observed as mosaics in Drosophila melanogaster

Abstract This study shows that 2 commonly held explanations for mosaics induced by treating postmeiotic spermatozoa with chemical mutagens are incosistent with an analysis of the percent of mutant tissue produced in the F 1 . The first model tested assumes that mosaics are the results of altering one strand of the DNA double helix so that there will be an equal number of mutant and nonmutant nuclei following semiconservative replication. The second model assumes that alkylation of a base in DNA will cause a fixed probability of base pairing mistakes and may produced a mutation at any replication. In phage the probability of mispairing following alkylation is low so that a mutation likely occurs only once in 8 replications; therefore, most of the mutations are fixed after the first 2 divisions. If Drosophila is similar to phage, the mean frequency of mutant nuclei and the frequency distribution of mosaics by class of percent mutant tissue will be distinctly different for the 2 models. Males were fed ethyl methanesulfonate and mutations were detected in the F 1 by the specific locus method at the yellow ( y ) and white w loci. Agreement between this experiment and previous work with mosaics produced by chromosome aberrations was found in that the germ line developed from a small sample of poorly mixed nuclei that was independent of the phenotype of head and thorax. The latter finding allowed a selection of mosaics to be made independent of the germ line, and a method of analysis was developed for flies selected because they were mosaic in the head or thorax. Analyses of results from the eye mosaics in this experiment and from 3 experiments with the dumpy (dp) locus previously reported by others but not analyzed in this was were inconsistent with both original models. The average percent of mutant phenotype among the yellow mosaic observed in this experiment was also found inconsistent with both original models, and a frequency distribution of mosaics classified by percent of mutant tissue was inconsistent with both original models and a combination of the two; therefore, both of our initial models and a combination of the two were rejected. The data are consistent with delayed mutation in which an alkylated base has about a 50% probability of mispairing at each replication and producing a fixed mutation or with a multistranded model in which the postmeiotic eukaryote chromosome contains 2 parallel DNA molecules for each locus.

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair.

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the eggs DNA repair machinery producing chromosomally abnormal offspring. These findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.

Chromosome breakage in Drosophila melanogaster induced by a monofunctional alkylating agent (EMS)

Abstract Ethyl methanesulfonate (EMS)-induced mutations involving the yellow locus of Drosophila melanogaster were studied, both when the dominant alleles for yellow ( y + ) and closely linked achaete ( ac + ) were adjacent to euchromatin in their normal position on the left end of the X chromosome and when adjacent to constitutive heterochromatin on the Y chromosome. A yellow mutation was considered intragenic if the adjacent achaete locus was not affected. The intragenic mutation frequency did not change with the position of the yellow locus in the genome; however, the total mutation frequency was three-fold higher when the yellow locus was positioned adjacent to consititutive heterochromatin. Results show that an additional class of mutations resulting from chromosome breakage accounts for the increased mutational frequency when the yellow locus is adjacent to heterochromatin.

Chapter 18:Advances in the Direct Measurements of Partial Chromosomal Duplication, Deletions and Breaks in Human and Murine Sperm by Sperm FISH

Abnormal pregnancy outcomes are relatively common in humans and defects reaching birth can be both traumatic and expensive for family and society.1,2 Every year in the United States, about 2 million pregnancies are lost before the 20th week of gestation, about 7% of newborns have low birth weight, a...