1. Introduction
DNA is the vital carrier of genetic information in all living cells, but its chemical stability is affected by several factors. In fact, DNA is highly susceptible to chemical modifications by exogenous agents such as ionizing radiation and ultraviolet light [
1,
2,
3] and by several environmental contaminants (pesticides, hydrocarbons, and especially heavy metals), which can generate oxidative stress [
4,
5]. Beyond environmental agents, DNA is also subject to oxidative damage from by-products of cellular metabolism (endogenous agents). The consequential alterations of DNA structure are generally incompatible with its crucial role in the maintenance and transmission of genetic information. For this reason, cells respond to DNA oxidative damage by specified DNA repair pathways to physically remove the damages [
6]. In the past 20 years, many papers regarding the involvement of DNA oxidative damage in human infertility have been published [
7]. It is well known that both male and female gametes can be exposed to DNA damage, which may compromise their functionality and their capacity to produce normal embryos. In particular, DNA damage, affecting sperm quality, increases the risk of genetic and epigenetic abnormalities and can lead to some diseases. Although a small amount of Reactive Oxygen Species (ROS) is necessary for some fundamental processes for the physiological function of male gametes, high levels of ROS can cause functional failure [
8]. There is a great interest in having new insights in the mechanisms of DNA oxidative damage, since it can also cause genetic alterations that may result in diseases, such as cancer and neurodegenerative syndromes [
9], and contribute to some features of aging. Despite the numerous studies conducted on the matter [
10], the precise molecular mechanisms that lead to DNA oxidative damage are not yet fully understood. In particular, to the best of our knowledge, no previous study has evaluated the possible involvement of Sperm Nuclear Basic Proteins (SNBP) in DNA oxidative damage. As a rule, histones and protamines, compacting DNA, protect from oxidative damage. Thus, in some stress conditions, possible functional alterations of SNBP properties should be more evident, considering the higher degree of compaction of sperm chromatin compared to that of somatic cells. As a matter of fact, in a previous work, we have demonstrated that some sperm histones, in the presence of specific heavy metals, can participate in DNA oxidative damage [
4], suggesting that in particular stress conditions, their protective rule appears to be reversed. This observation prompted us to investigate through molecular-level analyses the possibility that SNBP from men living in polluted areas might have an involvement in oxidative DNA damage. In fact, we found a higher DNA fragmentation index in the spermatozoa of males recruited in the “Land of Fires”, which is a high environmental impact area of Campania Region (Southern Italy) [
11,
12,
13,
14] in which resident people presented similar values for semen volume, pH, sperm cell count, and morphology, but a significant increase of immotile cells percentage [
11].
To this aim, we have evaluated the protein framework, the DNA binding, and the potentiality to induce the oxidative DNA damage of SNBP from a cohort of men living in the “Land of Fires”. This study was conducted as part of a biomonitoring project “EcoFoodFertility” [
11] (
http://www.ecofoodfertility.it/the-project.html).
3. Discussion
Living organisms are constantly exposed to numerous DNA damaging agents that can impact health and modulate disease states. DNA damage can cause genetic alterations that lead to the development of cancer, may result in cell death, as in neurodegenerative diseases, and could contribute to some features of aging [
2]. However, the highest risk of DNA damage is represented by the effects at the gametes level, since this can jeopardize the possibility of fertilization and consequently the continuity of the species. Indeed, due to their characteristics, male gametes are the most sensitive cells to the accumulation of damaged DNA, considering their continuous production and exposure to environmental agents, such as oxidizing agents [
23,
24,
25,
26]. In this regard, many studies have been performed to understand the mechanisms of oxidative DNA damage, but some aspects are still unknown. We have tried to give new insights on this topic using human spermatozoa as model cell for this study, evaluating the possible involvement of human SNBP in DNA oxidative damage. In fact, we have already reported that in the presence of heavy metals, some SNBP isolated from other organisms can participate in DNA oxidative damage, reversing their canonical protective rule [
4]. However, in vitro studies are not sufficient to describe the complexity of in vivo effects on DNA oxidative damage, because DNA is not free but complexed with proteins to form chromatin in living cells [
27], and it has been observed that Cu (II)/H
2O
2-induced DNA damage increases in the nucleosome compared to isolated DNA [
28]. Therefore, it is of fundamental importance to conduct in vivo studies on DNA oxidative damage, since the results that can be obtained are an order of magnitude more complex than those obtained by treating only DNA in vitro. These observations are in agreement with our in vivo studies on SNBP from mussels exposed to subtoxic concentrations of heavy metals, such as copper or cadmium [
5,
29]. In fact, we found that the heavy metals measured in gonads accumulated mainly in the fraction of SNBP, causing their involvement in DNA oxidative damage [
5,
30]. Therefore, in the present study, we considered it more appropriate to use the spermatozoa of men living in high environmental impact sites, such the “Land of Fires”, where some heavy metals, that participate in Fenton-like reactions, such as copper or chromium, are particularly abundant. In fact, in these subjects, a higher DNA fragmentation index [
12] and alterations of specific bio-markers of DNA oxidative damages have been reported [
11]. In particular, the comparison of subsets of randomly selected subjects from the L- and H-groups showed significantly lower Glutathione-S reductase and Glutathione peroxidase activities in the subset from the H-group (−32% and −25%, respectively;
p < 0.05). Moreover, the mRNA level of γ-Glutamate cysteine ligase was also two-fold lower in the latter subset. In addition, DNA damage was measured in the same subsets, where antioxidant enzymes were assessed, reporting a DNA fragmentation index (DFI) value 2-fold higher in the H-group with respect to the L- one (
p = 0.01) [
11].
In agreement, our molecular analyses indicated an unusual distribution in the electrophoretic profiles of SNBP in men belonging to the H-group. In addition, we observed that all SNBP isolated from this group changed their protective ability, participating in DNA oxidative damage. This result was particularly marked for those samples presenting a not canonical protamine/histone ratio (
Figure 6a). This strong occurrence of DNA oxidative damage in samples from men belonging to the H-group could be explained by an excess of copper and chromium found in the semen of people living in the “Land of Fires” [
11]. In fact, it is well known that copper overload generally leads to oxidative stress, promoting the formation of hydroxyl radicals, which strongly reacts with practically any biological molecule, including DNA, causing severe damage to the cells [
31,
32,
33,
34]. Several studies have also demonstrated that copper can form several binary and ternary complexes with arginine residues [
35,
36,
37], of which human protamines are extremely rich, promoting a site-specific damage at guanine residues of DNA by a selective binding between guanine and arginine [
38]. Moreover, our recent studies have also revealed that Cu(II) interacts with arginine residues of sperm H1 histones, inducing oxidative DNA damage [
4]. Moreover, Human Protamine 2 has a strong Cu(II)-binding amino acid motif at its N-terminus (Arg-Thr-His), which is able to mediate oxidative DNA double-strand scission and the generation of 8-oxo-2′-deoxyguanosine (8-oxo-dG) from free 2′-deoxyguanosine (dG) and from DNA by H
2O
2 [
39,
40]. Keeping in mind this evidence, it would be possible to speculate that these proteins could trap this metal, increasing the availability of Cu(II) ions near the binding surface of DNA. This condition could have as a consequence the promotion of the Fenton reaction in DNA proximity after H
2O
2 addition, determining DNA breakage and explaining the DNA oxidative damage found in CP/Hr samples of men belonging to the H-group. This finding is in accordance with the analyses carried out in the presence of Cu(II) concentrations for the in vitro determination of DNA-binding affinity of protamines and their involvement in DNA breakage. In fact, preliminary experiments indicated an increase in the DNA-binding affinity of CP/Hr proteins belonging to the L-group, in the presence of copper chloride, saturating the DNA at the protein/DNA ratio of 0.3 (lane 5,
Figure S4) instead of 1.2, as observed for the CP/Hr proteins samples of the L-group in the absence of copper chloride (lane 9,
Figure S1). In addition, in the presence of copper chloride, we observed an increase of relaxed DNA plasmid (lanes 4 and 10,
Figure S5), confirming the involvement of protamines in the DNA oxidative damage, as already demonstrated in our previous work on sperm H1 histones [
4]. We found in the literature that also chromium, the other heavy metal found in excess in the semen of people in the H-group, could participate in Fenton-like reactions producing reactive oxygen species and could influence the structure of chromatin by binding to both DNA and histones [
41,
42]. The toxic effect of chromium results in radical-mediated DNA strand breakage and the formation of stable chromium–DNA complexes, including chromium–DNA adducts and protein–chromium–DNA and DNA–chromium–DNA cross-links [
43,
44]. In addition, histones bind chromium through lysine residues [
41,
45] and could determine an “indirect” DNA damage in a similar way as hypothesized for copper. These evidences could explain the effect measured in the samples from men belonging to the H-group, showing the presence of only histones. In fact, in these samples, we observed an extent of damage comparable to that found in the samples belonging to the men of the H-group, showing a canonical protamine/histone ratio (
Figure 6a).
The concomitant presence in the area under study of an excess of these heavy metals, participating in the Fenton reaction and able to bind histones and protamines respectively, can justify the more marked extend of DNA oxidative damage measured in the samples presenting histones and protamines in not canonical ratio (
Figure 6a). The ability to induce DNA breakage, observed in the SNBP of men belonging to the H-group, can also be ascribed to the structural changes of these proteins due to tertiary/quaternary structure interactions. In order to study these conformational changes, we performed fluorescence measurements that are a sensitive tool to obtain information about protein–ligand interactions. However, we could not use the intrinsic fluorescence of these proteins because of their low content in aromatic amino acids. Therefore, we analyzed the features of sperm nuclear basic proteins in the samples showing the canonical ratio protamines/histones in the L and H-groups by using extrinsic fluorescence approaches with a solvatochromic dye such as ANS [
21]. It is known that solvatochromic dyes are powerful tools for monitoring protein conformational changes and proteins interactions with nucleic acids, other proteins, and lipid membranes [
22]. Generally, the increase of ANS fluorescence intensity and a blue shift in the emission maxima are attributed to the binding of the fluorescent probe to the hydrophobic sites on the protein and to its reduced mobility [
21]. However, it has been also reported that ANS could bind arginine and lysine residues on the protein surface through ion pair formation [
46], although the total fluorescence contribution of the ANS bond to these external sites is much less compared to that from the buried sites. In our experiments, we observed a reduced fluorescence intensity of ANS in the sample from the H-group with respect to the ones from the L-group, indicating a different accessibility of the fluorescent probe to the surface of proteins belonging to the two groups. Taking into account the high extend of basic amino acid residues (arginine, lysine, and histidine residues) on the surface of these proteins, we could explain this outcome by a lesser number of arginine residues that could bind ANS in the sample from the H-group, following the binding with heavy metals. Otherwise, we can also hypothesize an indirect effect on the ANS fluorescence of the binding of protamines with the heavy metals. In fact, the increment of the total surface charge of the H-group protamines due to the addition of the positive charges of heavy metals could result in a more hydrophilic dielectric constant of the solution, quenching the fluorescence of the solvatochromic dye. Either of these hypotheses supported possible changes in the function of protamines, being the protein surface altered. In fact, these differences also affected the ability to bind the DNA of sperm protein from samples belonging to the H-group, having measured a not linear fluorescence quenching at the increased DNA concentrations with respect to the ones of the L-group. These differences in DNA binding were more evident from the plot of the band density against the protein/DNA ratio (
Figure 6b). In fact, analyzing the DNA-binding ability of the SNBP from the samples of the H-group, we observed that DNA saturation was reached using a lower amount of proteins with respect to the SNBP from the samples of the L-group. This behavior supports the hypothesis of a possible alteration in the proteins’ surface with an overall increase in the positive charge of the protein mediated by surface ions, determining a strong bond to DNA.
These differences in DNA binding could also explain the behavior of the samples with nCP/Hr (
Figure 6b). The high content in arginine residues of protamines permits the binding both to minor and major DNA grooves, producing the adequate degree of sperm chromatin compactness, while histones interact only with the precise region of sperm DNA, producing a less compact chromatin [
47]. Taking into account that 10–15% of histones are retained in human sperm chromatin [
48,
49,
50,
51], forming a heterogeneous mixture of nucleohistones and nucleoprotamines, we could hypothesize that the presence of both protamines and histones in altered ratios could determine not only an unstable binding to DNA, but also a reduced DNA protection to the external agents, such as heavy metals. In addition, in samples showing only histones, the low degree of compactness of the sperm chromatin could result in a low amount of chromium presented locally to DNA by histones, but at the same time, an increased exposure of DNA to external perturbants. Accordingly, increased concentrations of only histones samples resulted in a decreased degree of DNA breakage (
Figure S6). As regards chromium, it is also important to consider that growing evidence suggests that epigenetic effects may in part be dependable for their genotoxicity and carcinogenicity [
52,
53]. In fact, it has been demonstrated that long-term chromium exposures may cause a significant increase in histone deacetylation. This effect may be particularly relevant in the histones–protamines transition which, as well known, requires histone acetylase activity and then could explain the high percentage of subjects (about 65%) in H areas that presented only histones in spermatozoa. In addition, the increase in histone deacetylation would lead to histone methylation in specific positions involved in gene repression and silencing, such as H3K9 [
54,
55,
56,
57].
In any case, considering that spermatozoa are produced continuously, we have no evidence that the abnormal protein patterns observed in samples isolated from men living in the “Land of Fires” cannot change over time because of the continuous changes in environmental conditions and in the quantity or types of xenobiotics accumulated in gametes.
In conclusion, in this work, we demonstrated for the first time the involvement in DNA oxidative damage of human SNBP from men exposed to pollutants, giving new insights on the toxicity mechanisms of some heavy metals. The potential implications of these findings could provide guidance in the future to better understand many mechanisms related to different diseases and processes in which oxidative DNA damage is implicated.