Sperm chromatin damage associated with male smoking

doi:10.1016/S0027-5107(98)00242-5

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 423, Issues 1–2, 25 January 1999, Pages 103-111

https://doi.org/10.1016/S0027-5107(98)00242-5 Get rights and content

Abstract

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3′-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.

Introduction

Due to the mutagenicity of cigarette smoke, numerous studies have investigated the impact of male smoking on sperm, which have been reviewed several times 1, 2, 3, 4. Smoking is associated with an overall reduction in semen quality, specifically a reduction in sperm count and motility, and an increase in the number of abnormal cells 5, 6, 7, 8. Additionally, smoking has been associated with increased levels of aneuploidy in human sperm [9], lower seminal plasma anti-oxidant levels, and increased oxidative damage to sperm DNA 10, 11.

Certain epidemiological studies have also implied that paternal pre-conceptual smoking may be associated with adverse effects on offspring. Sorahan et al. 12, 13, 14found paternal pre-conceptual daily cigarette consumption produced an overall increase in all childhood cancers. Zhang et al. [15]also reported an overall elevated risk of birth defects in offspring of paternal smokers.

This study analysed the DNA of sperm from smoking and non-smoking subjects using flow cytometric methods. The sperm chromatin structure assay (SCSA), which has been correlated with exposure to several mutagenic compounds 16, 17, 18, measured the susceptibility of sperm cells to acid denaturation. The terminal deoxynucleotidyl transferase assay (TdTA) allowed the measurement of DNA strand breaks, by addition of the biotinylated nucleotide dUTP to 3′-OH ends of DNA, sites of DNA breakage [19], using the enzyme terminal deoxynucleotidyl transferase (TdT). The biotinylated nucleotide is labelled with fluoresceinated avidin (FITC-avidin) and its emissions determined by flow cytometry to ascertain the proportion of cells containing strand breaks. The purpose of this paper is to report that more damage is present in the nuclear material of sperm from smokers compared to that of non-smokers.

Section snippets

Semen samples

Normal semen samples were obtained from patients being screened for fertility at the Royal United Hospital, Bath, UK (only those classified as fertile as defined by the World Health Organization [20]were included in the study). All samples were collected by masturbation after 3 days of abstinence and following liquefaction stored at −20°C prior to use. The smoking status of each donor was recorded and the samples used in the study characterised as either non-smokers or smokers of more than 10

SCSA

Fig. 1 depicts typical SCSA flow cytometric data from a non-smoker, before (Fig. 1a) and after (Fig. 1b) acid induced denaturation. Fig. 2 shows typical SCSA data from a smoker, before (Fig. 2a) and after (Fig. 2b) acid treatment. The scatter plots were gated to eliminate cellular debris emissions from the data acquired, and show emissions at 530 nm (dsDNA) versus 630 nm (ssDNA). Frequency histograms were then produced representing the ratio of emissions at 630:530 nm (ssDNA:dsDNA). Before acid

Discussion

Some epidemiological studies have suggested that paternal cigarette smoking may predispose offspring to an increased risk of birth defects and childhood cancers 12, 13, 14, 15. In this study, we collected evidence to suggest that smoking adversely effects sperm nuclear material, which could potentially produce sperm DNA mutations and lead to the above observations.

Results from this study demonstrate that sperm DNA from smokers is more sensitive to acid induced denaturation and contains more

Acknowledgements

This work was supported by the University of Bath. We also thank D. Bamford, former consultant gynaecologist at the Royal United Hospital, Bath, UK, for help in several aspects of the study.

References (30)

D.R Mattison
The effects of smoking on fertility from gametogenesis to implantation
Environ. Res.
(1982)
J Little et al.
Mutagenic lifestyles? A review of evidence of associations between germ-cell mutations in humans and smoking, alcohol consumption and use of `recreational' drugs
Mutation Res.
(1994)
N Sofikitis et al.
Effects of smoking on testicular function, semen quality and sperm fertilizing capacity
J. Urol.
(1995)
M.F Vine et al.
Cigarette smoking and semen quality
Fertil. Steril.
(1996)
C.G Fraga et al.
Smoking and low antioxidant levels increase oxidative damage to sperm DNA
Mutation Res.
(1996)
H.M Shen et al.
Detection of oxidative DNA damage in human sperm and the association with cigarette smoking
Reprod. Toxicol.
(1997)
T.P Sorahan
Childhood cancer and parental use of alcohol and tobacco
Ann. Epidemiol.
(1995)
D.P Evenson et al.
Glutathione depletion potentiates ethyl methanesulfonate-induced damage to sperm chromatin structure
Reprod. Toxicol.
(1993)
C.M Hughes et al.
Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay
Mutation Res.
(1997)
G.N Rivrud
Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix
Mutation Res.
(1988)

R.J Stillman et al.

Smoking and reproduction

Fertil. Steril.

(1986)

M.F Vine

Smoking and male reproduction: a review

Int. J. Androl.

(1996)

R Pacifici et al.

Nicotine, cotinine, and trans-3-hydroxycotinine levels in seminal plasma of smokers—effects on sperm parameters

Ther. Drug Monit.

(1993)

M.F Vine et al.

Cigarette smoking and sperm concentration: a meta-analysis

Fertil. Steril.

(1994)

A.J Wyrobek et al.

Smokers produce more aneuploid sperm than non-smokers

Am. J. Cytogenet.

(1995)

Cited by (178)

The sperm chromatin structure assay does not detect alterations in sperm chromatin structure induced by hydrogen peroxide
2022, Animal Reproduction Science
The objective of this study was to investigate the effects of hydrogen peroxide (H₂O₂) on the chromatin structure of sperm. For this purpose, 44 cryopreserved bovine ejaculates were analyzed immediately post-thaw (control sperm, CON S), after 1 h of post-thaw incubation (non-oxidized sperm, NOX S), and after 1 h of post-thaw incubation in different concentrations of H₂O₂ (OX S; 50, 100 µM, 1000 µM H₂O₂). Sperm motility was determined using computer-assisted sperm analysis. Sperm plasma membrane and acrosome integrity were assessed by flow cytometry after staining with propidium iodide and fluorescein isothiocyanate-conjugated peanut agglutinin. Chromatin damage was assessed using the sperm chromatin structure assay (SCSA), and DNA damage was evaluated using the single cell gel electrophoresis (Comet) assay. Sperm motility and plasma membrane and acrosome integrity decreased while chromatin damage and DNA damage increased after 1 h of incubation (P < 0.05). The addition of H₂O₂ adversely affected all parameters (P < 0.05) except for chromatin structure. The role of H₂O₂ in sperm chromatin damage was investigated by supplementation of 10 IU catalase, which reversed the damage (P < 0.05). Interestingly, the chromatin decondensation induced by 10 mM dithioreitol, evidenced by an increase in chromatin damage in the SCSA (P < 0.05), was reversed by H₂O₂. In conclusion, H₂O₂ causes alterations in sperm functional status and DNA integrity. However, our results show that high concentrations of H₂O₂ can induce chromatin condensation, and thus the use of the SCSA for the assessment of H₂O₂-induced chromatin damage should be carefully considered.
From sperm to offspring: Assessing the heritable genetic consequences of paternal smoking and potential public health impacts
2017, Mutation Research - Reviews in Mutation Research
Citation Excerpt :
Tobacco smoke has also been linked to DNA strand breaks as demonstrated using several methods to assess DNA fragmentation in sperm [98]. The majority of human studies (63%) that used the TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labeling) [99] show that the sperm of smokers have 1.2–4.3 times the amount of DNA fragmentation of non-smokers [80,100–103]. The majority of results (75%) obtained using the comet assay in humans and rodents [58,60,104–107] are concordant with these findings, indicating that smoking increases DNA damage in humans by 1.3–5.0 fold [88,108] and 1.1–9.2-fold in rodents [58,60,106].
Individuals who smoke generally do so with the knowledge of potential consequences to their own health. What is rarely considered are the effects of smoking on their future children. The objective of this work was to review the scientific literature on the effects of paternal smoking on sperm and assess the consequences to offspring. A literature search identified over 200 studies with relevant data in humans and animal models. The available data were reviewed to assess the weight of evidence that tobacco smoke is a human germ cell mutagen and estimate effect sizes. These results were used to model the potential increase in genetic disease burden in offspring caused by paternal smoking, with specific focus on aneuploid syndromes and intellectual disability, and the socioeconomic impacts of such an effect. The review revealed strong evidence that tobacco smoking is associated with impaired male fertility, and increases in DNA damage, aneuploidies, and mutations in sperm. Studies support that these effects are heritable and adversely impact the offspring. Our model estimates that, with even a modest 25% increase in sperm mutation frequency caused by smoke-exposure, for each generation across the global population there will be millions of smoking-induced de novo mutations transmitted from fathers to offspring. Furthermore, paternal smoking is estimated to contribute to 1.3 million extra cases of aneuploid pregnancies per generation. Thus, the available evidence makes a compelling case that tobacco smoke is a human germ cell mutagen with serious public health and socio-economic implications. Increased public education should be encouraged to promote abstinence from smoking, well in advance of reproduction, to minimize the transmission of harmful mutations to the next-generation.
Mutagenicity and carcinogenicity
2017, Reproductive and Developmental Toxicology
Endocrine disruptors are chemicals that produce various toxicities such as developmental or reproductive toxicity, carcinogenicity, mutagenicity, immunotoxicity, and neurotoxicity (Choi et al., 2004). Mechanisms of carcinogenesis caused by exposure to endocrine disruptors may be associated with free radical generation and biomolecule (e.g., DNA, protein, lipid) damage after the dysregulation of hormones such as estrogen, testosterone, thyroid, and insulin. The hormonal effect on carcinogenesis is receptor dependent or receptor independent, and there is often cross-talk between hormones and complex signaling cascades may also be involved. During multistage carcinogenesis {i.e., initiation, promotion [stage I (reversible), stage II (irreversible)], progression}, genotoxic and epigenetic events are critically associated, which may lead to mutation and cancer. Although genotoxic events are fundamentally the limiting step in carcinogenesis and reproductive disorders, the transgenerational epigenetic role of carcinogens could also be important. Given the unavoidable human exposure to endocrine disruptors or carcinogens, the application of a chemopreventive strategy that either blocks or retards the process of carcinogenesis might be helpful for public health.
Paternal smoking and germ cell death: A mechanistic link to the effects of cigarette smoke on spermatogenesis and possible long-term sequelae in offspring
2016, Molecular and Cellular Endocrinology
Citation Excerpt :
A comprehensive review by Ramlau-Hansen et al. (2007) and Mostafa (2010) have provided a thorough overview on cigarette smoking by men and associated abnormalities in sperm count, motility, and morphology, as well as other qualitative and quantitative measures of sperm characteristics. Male smokers exhibit several seminal anomalies including increased levels of oxidative DNA damage (Fraga et al., 1996; Shen et al., 1997), sperm DNA strand breaks (Potts et al., 1999), DNA adducts (Horak et al., 2003), chromosomal abnormalities (Robbins et al., 1997; Rubes et al., 1998), and decreased viability, and fertility (Kunzle et al., 2003). Exposure to cigarette smoke (CS) results in decreased sperm membrane permeability and activity of acrosin (Sofikitis et al., 2000).
Paternal exposure to constituents of cigarette smoke (CS) is reportedly associated with infertility, birth defects and childhood cancers even though the mechanism behind this relationship is still unclear. Chronic cigarette smoking by men leads to poor sperm quality and quantity mainly through oxidative stress and also direct assault by CS metabolites. Among several carcinogenic and teratogenic components of cigarette smoke condensate (CSC), polycyclic aromatic hydrocarbons (PAHs) display a preeminent role in accelerating germ cell death via the cytoplasmic transcription factor, aryl hydrocarbon receptor (AHR) that is present across all stages of spermatogenesis. Activation of AHR by growth factors though benefits normal cellular functions, its mediation by CSC in a spermatocyte cell line [Gc2(spd)ts] adversely affects the expression of a battery of genes associated with antioxidant mechanisms, cell proliferation and apoptosis, and cell cycle progress. Besides, the CSC-mediated cross talk either between AHR and NRF2 or AHR-NRF2 and MAPKs pathways inhibits normal proliferation of the spermatogenic GC-2spd(ts) cells in vitro and cell death of spermatocytes in vivo. Pharmacological inactivation of CSC-induced AHR but not its genetic manipulation seems preventing DNA and cell membrane damage in Gc2(spd)ts. Data from recent reports suggest that the cigarette smoke affects both the genomic and epigenomic components of the sperm and attributes any associated changes to developmental defects in the offspring. Thus, the studies discussed here in this review shed light on possible mechanistic factors that could probably be responsible for the paternally mediated birth defects in the offspring following exposure to the toxic constituents of cigarette smoke.
Interaction of exposure concentration and duration in determining the apoptosis of testis in rats after cigarette smoke inhalation
2016, Saudi Journal of Biological Sciences
The effects of differences in smoke concentration and exposure duration in Sprague Dawley rats to determine variation in type and severity of the testis apoptosis were evaluated. The daily dosages were 10, 20 and 30 non-filter cigarettes for a period of 2, 4, 6, 8 and 12 weeks. Mainstream smoke exposure suppressed body weight gain in all regimens. A dose-related increase in plasma nicotine concentration was observed in smoke-exposed groups for 4, 6, 8 and 12 week regimens. Histopathological examination of the exposed groups showed disturbances in the stages of spermatogenesis, tubules atrophying and these appeared to be dose-related. Cytoplasmic caspase-3 immunostaining was detected both in Sertoli cells and germ cells in smoke-exposure groups. An increase in TUNEL-positive cells of testicular cells was observed after 6 weeks of cigarette exposure. The results indicate that cigarette exposure concentration and duration have interaction effect to induce apoptosis in the rat testes.
In vitro fertilization using frozen-thawed feline epididymal spermatozoa from corpus and cauda regions
2016, Theriogenology
Citation Excerpt :
This phenomenon might be explained by the immature stage of the corpus spermatozoa and the difference in DNA condensation between sperm from different regions of the epididymis. Previous studies found that immature human sperm did not have an increase in the level of DNA fragmentation [14], but the immature sperm could have a negative effect on pregnancy rate, likely due to the difference in ratio of protamine nucleoproteins [15]. During sperm maturation in the epididymis, nuclear histones are replaced by disulphide-rich protamines, which makes the mature sperm more resistant to DNA cleavage and denaturation.
Epididymal sperm preservation offers a potential for rescuing genetic material from endangered or valuable animals after injury or death. Spermatozoa from corpus, as well as from cauda, have the capability to be motile and to undergo capacitation and can thus potentially be preserved for assisted reproductive technologies. In the present study, feline frozen-thawed epididymal spermatozoa from corpus and cauda regions were investigated for their ability to fertilize homologous oocytes and further embryo development in vitro. Epididymal spermatozoa from corpus and cauda of seven cats were cryopreserved and used for IVF. Cumulus–oocyte complexes (n = 419) were obtained from female cats after routine spaying. Frozen-thawed corpus epididymal spermatozoa showed similar properties of acrosome integrity, membrane integrity, and chromatin integrity as frozen-thawed spermatozoa from cauda except corpus spermatozoa showed lower motility (P < 0.05). The fertilizing capacity of frozen-thawed corpus epididymal spermatozoa was confirmed by similar number of embryos developing to the two- and four-cell stages compared with sperm from cauda (32.03% vs. 33.33%). However, oocytes fertilized with corpus spermatozoa had lower potential to develop to the blastocyst stage (6.79%) and had lower cell numbers compared to oocytes fertilized with cauda spermatozoa (14.08%). In conclusion, spermatozoa from corpus epididymis had a similar capability to fertilize homologous oocytes in vitro as sperm from cauda but resulted in fewer embryos developing to the blastocyst stage compared to spermatozoa from the cauda.

View all citing articles on Scopus

View full text

Sperm chromatin damage associated with male smoking

Abstract

Introduction

Section snippets

Semen samples

SCSA

Discussion

Acknowledgements

Environ. Res.

Mutation Res.

J. Urol.

Fertil. Steril.

Mutation Res.

Reprod. Toxicol.

Ann. Epidemiol.

Reprod. Toxicol.

Mutation Res.

Mutation Res.

Smoking and reproduction

Fertil. Steril.

Smoking and male reproduction: a review

Int. J. Androl.

Nicotine, cotinine, and trans-3-hydroxycotinine levels in seminal plasma of smokers—effects on sperm parameters

Ther. Drug Monit.

Cigarette smoking and sperm concentration: a meta-analysis

Fertil. Steril.

Smokers produce more aneuploid sperm than non-smokers

Am. J. Cytogenet.