Mutagenesis, Vol. 14, No. 5, 505-512,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
The effect of ascorbate and 
-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa
Department of Obstetrics and Gynaecology, The Queen's University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, UK
The aim of this study was to determine the effects of supplementation with ascorbate and 
-tocopherol, both singly and in combination, during sperm preparation on subsequent sperm DNA integrity, induced DNA damage and reactive oxygen species (ROS) generation. Semen samples with normozoospermic and asthenozoospermic profiles (n = 15 for each control and antioxidant group) were prepared by Percoll density centrifugation (95.0–47.5%) where the medium had been supplemented with these antioxidants to a number of different concentrations, all within physiological levels. Controls were included which had no ascorbate or -tocopherol added. DNA damage was induced using hydrogen peroxide (H2O2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of ascorbate to sperm preparation medium did not affect baseline DNA integrity but did provide sperm with complete protection against H2O2-induced DNA damage. Generation of H2O2-induced ROS was also significantly reduced after treatment with ascorbate, although baseline levels were unaffected by this antioxidant. Supplementation of sperm preparation medium with -tocopherol did not influence baseline DNA integrity but provided sperm with dose-dependent protection against H2O2-induced DNA damage. Generation of H2O2-induced ROS was significantly reduced after treatment with -tocopherol, although baseline ROS levels were unaffected by this antioxidant. Addition of both ascorbate and -tocopherol in combination to sperm preparation medium actually induced DNA damage and intensified the damage induced by H2O2, however, H2O2-induced ROS production was significantly reduced in a dose-dependent manner by supplementation with both vitamins.
1 To whom correspondence should be addressed. Tel: +44 01232 894633; Fax: +44 01232 328247; Email: e.donnelly{at}qub.ac.uk
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