title: Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.
abstract: A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW, Australia.
authors.forename: L E
authors.initials: LE
authors.lastname: Bennetts
authors.forename: G N
authors.initials: GN
authors.lastname: De Iuliis
authors.forename: B
authors.initials: B
authors.lastname: Nixon
authors.forename: M
authors.initials: M
authors.lastname: Kime
authors.forename: K
authors.initials: K
authors.lastname: Zelski
authors.forename: C M
authors.initials: CM
authors.lastname: McVicar
authors.forename: S E
authors.initials: SE
authors.lastname: Lewis
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020552:Comet Assay; D003432:Cross-Linking Reagents; D018736:DNA Adducts / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004967:Estrogens / Q000494:pharmacology*; D004996:Ethidium / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004967:Estrogens; D017382:Reactive Oxygen Species; C067883:dihydroethidium; D004996:Ethidium