Reproduction impairment following paternal genotoxin exposure in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus)
Introduction
Aquatic ecosystems are known to be the ultimate recipient of an increasing amount and range of anthropogenic chemical compounds. However, the global threat posed by the sublethal effects of pollutants is not yet adequately addressed although attention has been historically drawn since the Rio de Janeiro World Summit in 1992 to the reduction in biodiversity taking place world-wide (Depledge, 1996). A significant part of the man-made contaminants (up to one-third) has been described as being potentially genotoxic for the living organisms and possibly implicated as important causal factors in biodiversity loss (Claxton et al., 1998). Genotoxicants have been standing in the limelight because of their potential to lead to a cascade of adverse changes from the cellular to the population level (Würgler and Kramers, 1992, Bickham et al., 2000, Belfiore and Anderson, 2001). Thus, the interest of genotoxicity assessment in aquatic ecotoxicology has been highlighted both in vertebrates and invertebrates (Depledge, 1998, Jha, 2004). However, if some progress has been made in understanding the consequences of genotoxin exposure on human health, a huge gap remains concerning aquatic species. In aquatic ecotoxicology, DNA damage has been used for a long time as an exposure biomarker but the further biological consequences of this damage are still poorly understood or limited to the individual response. For example, in somatic cells, DNA strand breaks are known to give rise to chromosomal aberrations if they are un- or mis-repaired (Palitti, 1998). Such chromosomal aberrations can lead to detrimental effects, ranging from cell death up to physiological dysfunction of key organs, but such effects remain circumscribed to the individual. A specificity of ecotoxicological studies is that they should assess pollutant effects at the population level since environmental policies attempt to take into account drastic and population long term effects of pollutant pressure on aquatic ecosystems (Newman and Clements, 2008). However, so far, the majority of studies are individual based and there is still a clear need to construe the genotoxic exposure consequences at the population level. However, this level is difficult to work on. Because of its probable consequences on population parameters, reproductive impairment of fish can be considered as one of the most significant effects of aquatic pollution (Kime, 1995). To study this, one possibility is to pay particular attention to the consequences of germ cell DNA damage that could impact negatively progeny outcomes, since genotoxic damage in such cells can be passed on to future generations if not or misrepaired. A recent study carried out on fish has suggested a possible link between the level of DNA damage in spermatozoa of mature male exposed to a genotoxicant and reproduction impairment, but limited to an effect on hatching rate (Zhou et al., 2006). In the current work, we studied the reproductive consequences of a paternal genotoxin exposure by following the link between the sperm DNA integrity level and the occurrence of developmental abnormalities at early and late developmental stages in fish as well as the subsequent effects on juvenile survival and growth. The comet assay was chosen to measure DNA damage in sperm after male exposure to methyl methane sulfonate (MMS) during late spermatogenesis. MMS was used as a model alkylating genotoxicant because alkylating compounds are thought to be the most potent and abundant genotoxic contaminants in aquatic ecosystems (Kuehl et al., 1994). MMS binds covalently with nucleophilic centers of DNA bases. It primarily alkylates ring nitrogens and depurinates DNA, further leading to strand breaks. Among the different assays available for genotoxicity assessment, the comet assay (also called single cell gel electrophoresis assay) is a rapid, sensitive, relatively inexpensive method, requiring only a small number of cells and providing the opportunity to study DNA damage and repair in individual cells of aquatic organisms (Devaux et al., 1997, Devaux et al., 1998, Lee and Steinert, 2003, Jha, 2008). The alkaline version of this technique used in the present study allows the evaluation of a large array of DNA damages including single and double strand breaks, DNA cross-links, alkali-labile sites and incomplete repair sites (Singh et al., 1988). Development of fish progeny was assessed at different stages during 5 months from the fertilization, at embryonic and larval stages, respectively, by measuring fertilization rate, morphological abnormalities, hatching rate and survival. Two freshwater fish species were chosen, based on both ecological and experimental criteria. Arctic charr (Salvelinus alpinus) lives in the deep and oligotrophic lakes in the Alps and its spawning grounds are located in the deeper parts of the lakes. Brown trout (Salmo trutta) occupies many habitats (rivers, lakes, sea shores) in all the northern and middle Europe but it reproduces only in cold and little polluted brooks.
Section snippets
Chemicals
Eugenol was supplied by Cooper (Melun, France). All other reagents were supplied by Sigma–Aldrich (St Quentin Fallavier, France).
Fish exposure
Experiments were carried out on 2-year-old male and 4-year-old female brown trout (S. trutta), and on 3-year-old male and 4-year-old female Arctic charr (S. alpinus). Fish were reared at the INRA hatchery (Thonon les Bains, France) in 6 m3 outdoor tanks continuously supplied with 51 m deep water from Lake Geneva (water temperature 6 ± 1 °C). Animals were fed daily with dry
Sperm DNA damage
The level of DNA damage measured in trout and charr sperm is illustrated in Fig. 1. Results show a significant increase in DNA damage in sperm 3 weeks after MMS injection. The MMS-associated increase in DNA damage (expressed as % tail DNA) was about the same for both species (28% in treated trout vs. 1% in control and 25% in treated charr vs. 2% in control).
Embryo–larval development assessment
Fertilization rates obtained in control trout and charr are in accordance with those commonly observed in the hatchery, charr exhibiting a
Discussion
This work describes how DNA damage in fish sperm cells can impact negatively on progeny outcome. It supports the link between sperm DNA integrity and occurrence of abnormalities at early and late developmental stages, as well as subsequent effects on juvenile survival and growth. In aquatic toxicology, reproduction is currently viewed as being particularly sensitive to toxic chemical exposures as such exposures can have transgenerational effects and thus disturb population growth and
Conclusion
Aquatic ecotoxicology is increasingly being focused on multi-generational effects, especially those effects on population growth and maintenance. Reproduction failure is seen as a crucial point in the hierarchic range of responses linking individual effects and long-term population changes. The interest in linking genotoxic responses and reproductive success in ecotoxicology has been stressed long ago (Anderson and Wild, 1994). Nevertheless, very few studies concerning the consequences of germ
Acknowledgements
The authors are thankful to Marie Carayon and Philippe Laurent for skillful technical assistance at the INRA fish hatchery (Thonon les Bains, France) and to Professor François Meunier (Museum National d’Histoire Naturelle de Paris, France) for helpful advice concerning staining of fish bony structures.
References (49)
- et al.
Origins and consequences of DNA damage in male germ cells
Reprod. Biomed. Online
(2007) - et al.
Community and populations indicators of ecosystem health: targeting links between levels of biological organisation
Aquat. Toxicol.
(1997) - et al.
The distribution of the tail moments in single cell gel electrophoresis (comet assay) obeys a chi-square (χ2) not a Gaussian distribution
Mut. Res.
(1998) - et al.
Effects of contaminants on genetic patterns in aquatic organisms: a review
Mut. Res.
(2001) - et al.
Effects of chemical contaminants on genetic diversity in natural populations: implications for biomonitoring and ecotoxicology
Mut. Res.
(2000) Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes
Aquaculture
(1992)- et al.
Egg and sperm quality in fish
Gen. Comp. Endocrinol.
(2010) - et al.
Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir
Aquat. Toxicol.
(2005) - et al.
Genotoxicity of industrial wastes and effluents
Mut. Res.
(1998) - et al.
A new in vitro method to assess DNA damage in sperm as an alternative to animal testing in reproductive toxicology
Toxicol. Lett.
(2007)
UV radiation in marine ecosystems: molecular effects and responses
Aquat. Toxicol.
Genetic ecotoxicology: an overview
J. Exp. Mar. Biol. Ecol.
The ecotoxicological significance of genotoxicity in marine invertebrates
Mut. Res.
Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P450 1A induction in chub (Leuciscus cephalus)
Mar. Environ. Res.
On the relevance of genotoxicity effects in zebrafish (Danio rerio) exposed to 4-nitroquinoline-1-oxide in a complete life-cycle test
Aquat. Toxicol.
Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility, and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa
Theriogenology
The comet assay: a comprehensive review
Mut. Res.
The comet assay for the evaluation of genotoxic impact in aquatic environments
Mut. Res.
The stress response in gametes and embryos after paternal chemical exposures
Toxicol. Appl. Pharmacol.
Genotoxicological studies in aquatic organisms: an overview
Mut. Res.
Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine
Cryobiology
Identification of potentially mutagenic contaminants in the aquatic environment by liquid chromatographic–thermospray mass spectrometric characterization of in vitro adducts
J. Chromatogr. A
Genotoxicity assessment in the amphipod Gammarus fossarum by use of the alkaline comet assay
Mut. Res.
Use of the cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) organisms
Mut. Res.
Cited by (85)
Ecogenotoxicity assessment with land snails: A mini-review
2023, Mutation Research - Reviews in Mutation ResearchHow age, captivity and cryopreservation affect sperm quality and reproductive efficiency in precocious Atlantic salmon (Salmo salar L. 1758)
2021, AquacultureCitation Excerpt :However, cryopreservation irretrievably leads to cryogenic alterations induced by ice crystal formation, cryoprotectant toxicity and oxidative stress that compromise spermatozoa integrity and functionality (Ciereszko et al., 2014; Figueroa et al., 2016; Pérez-Cerezales et al., 2010a; Xin et al., 2020) Successful cryopreservation depends on high-quality sperm which can only be ensured through the selection and appropriate management of males. Markers based on parameters defining spermatozoa functionality such as plasma membrane integrity, DNA integrity, mitochondrial membrane potential or computer-assisted motility (CASA) could be directly linked to reproductive success in Salmonidae (e.g. Devaux et al., 2011; Figueroa et al., 2015; Labbe et al., 2001; Pérez-Cerezales et al., 2010a, 2010b. Post-cryopreservation spermatozoa damages have widely been described in adult salmon (e.g. Dziewulska et al., 2011; Figueroa et al., 2018; 2016; Figueroa et al., 2015).
Application of the comet assay for the evaluation of DNA damage in mature sperm
2021, Mutation Research - Reviews in Mutation Research