Mutagenesis vol. 19 no. 4 pp. 325-330,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Antioxidants modulate thyroid hormone- and noradrenaline-induced DNA damage in human sperm
gorzata M. Dobrzy
ska1,21Department of Biomedical Sciences, University of Bradford, Bradford, UK and 2Department of Radiation Protection and Radiobiology, National Institute of Hygiene, Warsaw, Poland
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Abstract |
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The genotoxic effects of steroidal oestrogens are probably brought about by metabolic changes in their phenolic groups accompanied by the generation of quinones and reactive oxygen species. Although non-steroidal oestrogens and related compounds have not been thoroughly investigated for genotoxicity, some of them also contain phenolic groups that could be involved in redox cycling. Therefore, the aim of the present study was to evaluate the possible DNA-damaging effects of the thyroid hormones triiodothyronine (T3) and L-thyroxine sodium salt (T4) and the neurotransmitter noradrenaline (NA) in human sperm using the Comet assay. They were compared with diethylstilboestrol (DES), a steroidal oestrogen, as a positive control. After dose–response studies, doses of 80 µM T3, 80 µM T4, 300 µM NA and 175 µM DES, which produced DNA damage but retained good cell viability, were chosen for further experiments with the antioxidant catalase and the flavonoids kaempferol and quercetin. Since the scavenging enzyme catalase reduced the DNA-damaging effects of T3, T4 and NA, it can be surmised that these compounds under these conditions induced DNA damage mainly via the production of reactive oxygen species. This was further confirmed by the inhibitory responses produced by the flavonoids, which are known to have antioxidant effects. Therefore, the mechanism of mutagenic action of both steroidal and non-steroidal compounds imply the creation of oxidative stress and subsequent DNA damage due to reactive oxygen species and possibly due to reactive hormone derivatives created during their redox cycling.
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Introduction |
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During the last decade scientists have been concerned with the problem of diminishing male fertility. From 1940, the sperm count in many human populations has decreased by
40–50% and the quality of sperm is diminishing (Carlson et al., 1993
; Irvine, 1994; van Waeleghem et al., 1994; Auger et al., 1995; Medical Research Council, 1995; Sripada et al., 2004). At the same time the risk of testicular cancer has increased >3-fold (Brown et al., 1986; Osterlind, 1986; Boyle et al., 1987).
It has been postulated that the deterioration in human testicular functions, such as malformations in the reproductive system, may be due to environmental exposure to oestrogen-like compounds (Sharpe, 1993; Jensen et al., 1995; Sharpe et al., 1995). Dose levels of these chemicals are low and usually they possess low activity, but chronic low level exposure may disrupt reproduction and development (Schantz et al., 2001; Mendes, 2002).
A decrease in sperm count together with a deterioration in sperm quality reduces the chance of successful reproduction and causes a fundamental problem from the point of view of public health.
One of the main reasons for testicular dysfunction and male infertility in humans seems to be oxidative stress caused by reactive oxygen species (ROS) and this mechanism may also be involved in the induction of testicular cancer (Aitken, 1999). Human spermatozoa contain a high quantity of polyunsaturated fatty acids, which are susceptible to oxygen radical attack (Jones et al., 1979; Aitken, 1999).
Oestrogens appear to produce ROS, especially hydrogen peroxide, at levels that significantly disrupt DNA structure in spermatozoa and in peripheral lymphocytes (Anderson et al., 2003). In contrast to steroidal hormones, there is little information about the role which non-steroidal hormones may play in DNA damage. Endogenous non-steroidal hormones and related compounds, such as neurotransmitters, are present in humans at low concentrations to aid the regulation of physiological processes and have stimulatory effects on metabolic activities.
The thyroid hormones thyroxine (T4) and triiodothyronine (T3) are iodinated amino acid derivatives formed by oxidative coupling of two iodinated tyrosine residues in the thyroid protein thyroglobulin. These hormones play a role in the stimulation of metabolic rate and regulation of growth, development and differentiation (Nunez, 1999). Thyroid hormones appear to regulate the duration of Sertoli cell proliferation, affecting adult Sertoli cell number and hence the capacity of the testis to produce sperm (Buzzard et al., 2000). T3 and T4 influence the duration of proliferation of Sertoli cells (Francavilla et al., 1991; Van Haaster et al., 1992, 1993). Thyroid hormones as well as steroid hormones interact with a superfamily of cytoplasmic ‘zinc finger’ protein receptors (Rush et al., 1999). Neurotransmitters enable transmission from one neuron to the next across synaptic gaps (Rush et al., 1999).
Catalase and flavonoids are known to have antioxidant properties. Catalase is an endogenous enzyme present in the cells of healthy humans. Flavonoids are present in higher plants and plant products, such as seeds, citrus fruits, olive oil, tea and red wine, and are included in the diet of animals and man. Flavonoids, including quercetin and kaempferol, have been observed in human lymphocytes and sperm to modulate effects of food mutagens (Anderson et al., 1997a, 1998) and of oestrogen-like compounds (Cemeli et al., in press).
The aim of this study was to investigate the effects of the thyroid hormones T4 and T3, as well as the neurotransmitter noradrenaline (NA), on the induction of DNA damage in human sperm in the comet assay and modulation of these effects by catalase (CAT), kaempferol (K) and quercetin (Q).
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Materials and methods |
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The hormones L-thyroxine sodium salt (T4) (CAS no. 6106-07-6), triiodo-L-thyronine sodium salt (T3) (CAS no. 55-06-1), diethylstilbestrol (DES) (CAS no. 56-53-1) and NA (CAS no. 51-41-2), as well as CAT (CAS no. 9001-05-2), K (CAS no. 520-18-3) and Q (CAS no. 6151-25-3) were obtained from Sigma Chemical Co. (Poole, Dorset).
Each compound was examined at four or five doses and the influence of CAT, Q and K on the compounds at two doses each. The doses for the last three compounds were chosen from previous studies in this laboratory (Anderson et al., 1997a,b, 2003).
Semen was obtained from a 22-year-old male healthy non-smoker donor according to WHO criteria (World Health Organization, 2000): sperm number 130 x 106, motility 65% and abnormal forms 30%. Four microlitres of sperm from aliquots frozen at –80°C plus each compound used for treating the sperm were made up to 1 ml with RMPI 1640 medium in an Eppendorf tube. Each was treated for 1 h at 37°C in a water bath according to methods used in previous experiments (Anderson et al., 1997a,b). After incubation the sperm were harvested by centrifugation at 3000 r.p.m. (800 g) for 3 min. Cells were checked for viability by Trypan blue exclusion (Pool-Zobel et al., 1993). Then cells were suspended in 100 µl of low melting point agarose for embedding on slides previously covered with 2% normal melting point agarose. Preparation of slides, electrophoresis, neutralization and staining were conducted according to the method described by Singh et al. (1988, 1989; Tice et al., 2000) with further modifications (Anderson et al., 1997a,b). Slides were immersed in lysing solution containing 0.05 mg/ml proteinase K and they were left in an incubator at 30°C overnight.
Slides were scored using an image analysis system (Komet 5.0; Kinetic Imaging, Liverpool, UK) attached to a fluorescence microscope (Leica, Germany) equipped with appropriate filters. The magnification was 200x (20x objective, 10x eyepiece). The parameter taken for sperm was percentage of head DNA (Anderson et al., 1997a,b), because of the high background damage level of 20% in sperm cells. Hughes et al. (1997) suggested that percentage head DNA was more appropriate for statistical analysis. Images from 50 cells (25 from each replicate slide) were analysed.
All experiments were repeated for reproducibility.
Statistical analyses were performed in each study on median values and the mean ± SE. The data required for parametric statistics violated normality so non-parametric statistical analysis was used. Pairwise comparisons of all treatment groups versus the respective controls were performed using the Mann–Whitney test.
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Results |
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The cell viabilites were >80% in all groups in all the Comet assays.
The effects of the non-steroidal hormones DES, NA, T3 and T4 in human sperm are shown in Table Ia–d, respectively. All compounds, including DES, which was used as a positive control substance, induced a decrease in percentage head DNA in the sperm. Dose–response effects were observed in each experiment, but at the highest dose the percentage of head DNA increased or plateaued. This latter occurrence was caused by some precipitation of chemicals at this dose. Therefore, for the experiments with catalase and flavonoids, the doses which produced the highest DNA damage without precipitation were chosen. The doses were 175 µM T4, 300 µM NA, 80 µM T3 and 80 µM T4.
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Catalase inhibition of the DNA-damaging effects of the compounds is shown in Table IIa–d. The per cent of DNA in the head decreased to
57% after treatment with DES (Table IIa), then increased significantly and returned to control values after addition of catalase. A dose of 100 U/ml CAT increased the per cent head DNA by 78% and a dose of 500 U/ml CAT by 95% in comparison with the results produced by DES alone.
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The neurotransmitter NA (Table IIb) reduced the per cent of head DNA by
55%. Addition of CAT enhanced these values by 75% (100 U/ml CAT) and 97% (500 U/ml CAT).
Treatment with T3 (Table IIc) decreased the per cent of head DNA by 60%. Catalase at a dose of 100 U/ml inhibited this effect by 81% and at dose of 500 U/ml by 95%. The addition of 100 U/ml CAT to T4 (Table IId) increased the per cent of head DNA by 75% and 500 U/ml CAT by 98% in comparison with T4 alone.
K modulation of the DNA-damaging effects of the hormones is shown in Table IIIa–c. The addition of 100 µM K to NA-treated cells (Table IIIa) increased the per cent of head DNA by 73% and at a dose of 500 µM K by 97%.
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Treatment of sperm cells with T3 (Table IIIb) reduced the per cent of head DNA by
54%. Combination with the lower dose of K decreased these effects by 77% and with the higher dose of K by 97% in comparison with the results produced by T3 alone.
Exposure to T4 (Table IIIc) decreased the per cent of head DNA to 58%. The dose of 100 µM K inhibited this effect by 80% and the dose of 500 µM K returned the per cent head DNA to the control value.
Q inhibition of the DNA-damaging effects is shown in Table IVa–c. The addition of 100 µM Q to NA-treated cells (Table IVa) increased the per cent of head DNA by 53% and addition of 500 µM Q returned the per cent of head DNA to the control value.
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Addition of the lower dose of Q to T3 (Table IVb) increased the per cent of head DNA by
85% and the higher dose of Q produced a protective effect which diminished the background percentage head DNA below the value found in the control sperm.
Combination of T4 (Table IVc) with 100 µM Q inhibited the effects of T4 alone by 81% and with 500 µM Q returned the results to the control value.
Thus, in all experiments the per cent of head DNA after combined treatment of human sperm with each hormone or neurotransmitter plus the higher dose of CAT, Q and K returned the per cent DNA in the head almost to the levels shown in controls.
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Discussion |
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The human ejaculate contains a heterogeneous population of spermatozoa. Chromatin of the mature spermatozoa nucleus can be abnormally packed and nuclear DNA can be damaged (Manicardi et al., 1995
; Sakkas et al., 2002). It is known that the presence of nuclear DNA damage, such as single- and double-strand DNA breaks, in the mature spermatozoa is associated with poor semen parameters and male infertility (Lopez et al., 1998; Irvine et al., 2000; Morris et al., 2002; Sakkas et al., 2002; Schmid et al., 2003).
There are some reports indicating mutagenic activity of thyroid hormones and noradrenaline (Wilson et al., 1989; Oppenheimer et al., 1996; Tapia et al., 1999; Djelic and Anderson, 2003).
Thyroid hormones increase metabolic activity and oxygen consumption causing oxidative stress in exposed cells (Wilson et al., 1989; Oppenheimer et al., 1996). Also, during the normal catabolic pathway of oxidation of noradrenaline, ROS and hydrogen peroxide are produced (Graham, 1984).
The low levels of compounds such as thyroid hormones and noradrenaline normally present in the human body should not produce any genetic damage. However, unusual concentrations of thyroid hormones in the human body may be caused by the administration of some drugs. For example, chronic treatment of human and experimental animals with amiodarone, a potent anti-arrhythmic drug results in an increased plasma concentration of T4 and a decreased concentration of T3 (Sogol et al., 1983; Hudig et al., 1997). Noradrenaline is known to have a protective effect against tumour necrosis factor 
(TNF) and cycloheximide-induced apopotosis in brown adipocytes in young rats (Nisoli et al., 2001) and as such could alter noradrenaline levels. Iron also plays a role in noradrenaline-mediated damage (Miura et al., 2000). Some variability in levels of damage in sperm may be due to the fact that these hormones could cause chromatin decondensing effects. However, it is known that responses in human samples can depend on lifestyle factors and the physiology of the individuals from whom the samples were taken (Oliveri and Bosi, 1992; Anderson et al., 1997a,b). It is recognized that probably higher than physiological levels have been used in the present in vitro study designed to determine the potential for any genotoxic damage that might be caused. Genotoxins extrapolate through 0 in dose–response relationships so any damage caused at higher doses could be extrapolated to lower ones.
Free radicals can damage DNA and proteins either through oxidation of DNA bases (primarily guanine via lipid peroxyl or alkoxyl radicals) or through covalent binding to malondialaldehyde, resulting in strand breaks and cross-linking (Alvarez et al., 1987). ROS can also induce oxidation of critical SH groups in proteins and DNA, which will alter cellular integrity and function with an increased susceptibility to attack by macrophages. Cellular damage is theoretically the result of an improper balance between ROS generation and intrinsic scavenging activities. This balance can be referred to as oxidative stress status and its assessment may play a critical role in monitoring testicular toxicity and infertility (Sikka, 1997, 1999). ROS are a significant cause of DNA damage and human sperm is potentially highly sensitive to ROS attack due to lack of capacity for DNA repair in sperm as well as to a high content of polysaturated fatty acids.
However, there are various intracellular antioxidant mechanisms for DNA protection, such as scavenging of damaging ROS and iron binding (Yu, 1994). Such systems are also known in spermatozoa. Adequate levels of superoxide dismutase, catalase and, probably, glutathione peroxidase and reductase inhibit DNA damage caused by ROS in spermatozoa (Storey, 1997; Anderson et al., 2003; Braumber et al., 2003). It has also been shown that K and Q can reduce the effects of steroidal oestrogens in human lymphocytes (Cemeli et al., in press).
The results presented in this paper showed that NA, T3 and T4 increased DNA damage in sperm cells in vitro. The effects are similar to those caused by steroidal oestrogens, represented here by DES, and also shown in previous papers (Anderson et al., 1997b, 2003). Also, our results confirmed those obtained in human lymphocytes exposed to T3 and NA (Djelic and Anderson, 2003).
In our study all compounds induced DNA damage measured as a decrease in per cent head DNA. The inhibition of effects caused on per cent head DNA by NA, T3 and T4 by CAT, Q and K confirmed that the results obtained may be caused by oxidative stress. This is also in good agreement with results obtained in human lymphocytes and sperm treated with oestrogen-like compounds (Cemeli et al., in press) and in human lymphocytes treated with T3 and NA (Djelic and Anderson, 2003). In the above papers a clear inhibition caused by the addition of CAT and flavonoids was found.
It is suggested that oxidative stress is linked to the aetiology of chronic diseases like coronary heart disease and cancers (Gramenzi et al., 1990; Block et al., 1992).
Diets rich in fruits and vegetables are associated with protective effects against these diseases (Segasothy and Phillips, 1999). These effects are likely to be modified by antioxidants which employ a series of redox reactions (Szeto and Benzie, 2002; Blokhina et al., 2003).
The present results support the idea that flavonoids and CAT with their antioxidant properties could be important in protection against DNA damage through oxidative stress.
One of the most important applications of antioxidants in the inhibition of the effects produced by oestrogen-like (both steroidal and non-steroidal) compounds could be the prevention of male infertility. Our results have shown that flavonoids and CAT, because of their antioxidant properties, can protect human sperm from ROS-induced DNA damage. This is the first demonstration of DNA damage induce in human sperm by non-steroidal oestrogens, determined using the comet assay, and inhibition of these effects by antioxidants.
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Acknowledgements |
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M.D. received funding from the UK Royal Society for a short-term study visit to the Department of Biomedical Sciences, University of Bradford. A.B. is a Marie Curie fellow in this department (contract no. QLG4-CT-2002-51611).
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Notes |
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3To whom correspondence should be addressed at: Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK. Email: d.anderson1{at}bradford.ac.uk
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