**6. Perspectives**

*Male Reproductive Health*

infertility [80].

known [89, 90].

affects the mammary glands of mothers [94].

influence public health protection.

of offsprings can be changed [76, 77].

stronger negative impact on Y-chromosome-bearing spermatozoa, and the sex ratio

Many non-genomic methods of ED action lead to inappropriate epigenetic changes of DNA and core histones. Although the sequence of nucleotide remains unaffected, the changes of genome-wide methylation status, as well as silencing or enhancing the individual loci, follow the exposure of EDs. These epimutations result in changed transcriptional activity of the genome with many negative impacts, such as failure of scavenging of reactive oxygen species, DNA damage repair, and/or inadequate mitochondrial biogenesis. These cellular changes lead to clinical manifestations, most of which are diagnosed as "idiopathic." Obviously, exposure to EDs causes obesity [78], type 2 diabetes [79], metabolic disorders, and

While the exposure of somatic cells creates health problems for exposed individuals, influence on gametes leads to an intergenerational effect when the burden is transduced to the next generation of daughters and sons [81]. Indeed, the exposure to bisphenols impairs genome-wide DNA methylation, as well as histone code in oocytes [71, 82], followed by changes in the imprinting of genes in the embryo and placenta [83]. In spermatozoa, DNA methylation [84] is potentially affected by environmental pollutants, leading to aberrant gene imprinting [85, 86]. It can be assumed that the sperm histone code is sensitive to endocrine disruptors, with effect similar to estrogens, as well as to the involvement of estrogen receptors in histone code establishment [15]. Moreover, the negative role of environmental pollutants in the influence on noncoding RNAs in spermatozoa, another tool of epigenetic regulation [87] with ability to drive epigenetic inheritance [88], is well-

The exposure in utero and transplacental transmission of an ED affect DNA demethylation in developing PGCs and result in transgenerational inheritance of this burden. Accordingly, the exposure of pregnant rat females to fungicide vinclozolin [91] or DDT [92] leads to modified epigenome, that is, DNA methylome, histone retention in sperm, and ncRNAs. Translactational exposure, another way of indirect influence with environmental agents, is a reason of changes of male reproduction after lactating female mice were exposed to BPA [93]. Moreover, this type of exposure to bisphenols creates a risk of changed nursing behavior and also

Whereas endocrine-disrupting hypothesis is assumed for very low doses of EDs, there is a relevant phenomenon of interactions of individual EDs. The comprehensive work of T. Pollock and his colleagues produced valuable results, describing cross talk of common EDs. The combined presence of bisphenols is considered to be deleterious [95] as well as the simultaneous presence of triclosan, a soap compound [96, 97]. Degradation of bisphenol is inhibited under other ED exposure, and, obviously, the co-exposure achieves various modes on how to affect the body [98]. In addition to human and mammalian models, there is evidence of interaction of xenobiotics and pesticide residua [99], as well as synergistic interactions of organophosphates and pyrethroids [100], potentially leading to the collapse of honey bee colonies [101]. In contrast to synergic effects leading to the increase of the deleterious impact, competition of some pollutants is known, and, surprisingly, a reverse effect of the synergic activity of pollutants has been described, where one pollutant protects cells against damage caused by another pollutant [102]. The molecular action of interacting pollutants remains to be unexplained in mammalian models, and there is obvious need for further study. Also the results of these studies will

**46**

The aforementioned routes of exposure to EDs, including their interactions, obviously lead to different systemic response as the result of molecular action in tissues and cells. The molecular mode of action seems to be the key for the elimination of EDs' negative effect on the body. Based on already described manifestations of EDs in higher and lower doses, two dose-dependent modes of action are recognized: toxic effect and endocrine disruption. It seems that the current issue of EDs is in extremely low doses without clinical manifestations, leading to "idiopathic" infertility, metabolic syndrome, and other failures with nonspecific symptoms. Moreover, intergenerational and transgenerational inheritances occur because of the change of the epigenetic code of germ cells. The posttranslational modifications of crucial proteins, particularly regulating epigenetic factors, seem to be a common feature of these very low doses. In accordance with this, we can mark this effect to be "posttranslational." The possible contribution of posttranslational modifications of key proteins is indicated in **Figure 1**.

There is an obvious direct impact of EDs on male reproduction due to oral, respiratory, and/or transdermal exposure. Thereafter, both the gonads and accessory glands are affected, leading to the failure of male reproduction, often diagnosed as idiopathic. On the spermatozoon level, direct protein targeting is assumed, including cytosolic proteins as well as sperm histone code. Even protamine PTMs are considered to have a biological role, and, in accordance with the abovementioned importance of acetylated lysines, protamine acetylation seems to be most potent for sperm quality. The impact on DNA and chromatin proteins (i.e., histones and protamines) represent hazardous mode of inter- and transgenerational transmission of ED-driven epigenome.

In addition to the direct impact of EDs, indirect impact is also observed. The exposure of EDs during pregnancy and prenatal life represents the most dangerous exposure method when the germline is affected during gene imprinting erasure and re-imprinting in developing spermatozoa [85] and oocytes [103]. This exposure window allows an ED to affect the health of a generation of grandchildren through transgenerational inheritance [104, 105]. Epigenetic transmission to further generations involves various modifications, such as DNA and histone methylation, histone acetylation, and other PTMs of core histones, as well as epigenetic writers and erasures, translational factors, and others. Obviously, PTMs actually drive the phenomenon of the epigenetic inheritance, and the molecular impact of individual EDs is still unknown, as is their interaction (**Table 1**).

There is a strong need for further study focused on the ED-modulated epigenetic code and its manifestation in the body. In accordance with our "posttranslational" hypothesis of ED action, comprehensive screening of the most crucial PTMs should be taken into account in an assessment of individual EDs. Taken together, biomonitoring has an extremely significant role in the fight against EDs, as does the subsequent testing of EDs in the ascertained doses. Simulation of real exposures to individual EDs and their interactions are appropriate, using both in vitro and in vivo experimental assessments. Finally, advanced screening methods capable of identifying PTMs are needed for qualified recognition of an ED as harmful/harmless.

### **Acknowledgements**

The study of endocrine disruptors is supported by the Czech Health Research Council (NV18-01-00544); H2020 (Human Biomonitoring Initiative HBM4EU); MH CZ-DRO (FNBr, 65269705), project MSMT LTC18059; COST action CellFit CA16119; the Charles University Research Fund (Progres Q39); and the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR); project No. SVV 02690 awarded by MEYS CR; and project No. CZ.02.1.01/0.0/0.0/16\_019/0000787 "Fighting Infectious Diseases," awarded by MEYS CR and financed by the European Regional Development Fund. We would like to thank Ms. Iveta Zimova, Mr. Vaclav Rucka, and all graduate and pregraduate students for their kind help with the experimental work.
