Summary of some EDs affecting male reproduction.
Nowadays, endocrine-disrupting chemicals are considered to be one of the main causes of the ever-increasing occurrence of problems with male fertility. These compounds of natural or anthropogenic origin are omnipresent in the environment and organisms are exposed to them practically nonstop through the air, water, food, and occupationally. Endocrine disruptors have the ability to mimic effects of reproductive hormones and demonstrably can interfere with the endocrine system leading to reproductive disorders at different levels, and considering male reproductive functions, most of the impacts are performed by the breakdown of estrogen- or androgen-mediated processes. A significant body of evidence based upon laboratory or wildlife animal experiments and meta-analysis of semen studies in men indicates that exposure to endocrine disrupting compounds is associated with male reproductive malfunctions, including impairment of spermatogenesis followed by reduced semen quality parameters (sperm concentration, motility, and morphology). Alkylphenols, bisphenol, and phthalates are substantial components of many products with which people come into contact daily. This brief review will emphasize on the possible effects of alkylphenols, bisphenol, and phthalates on the male reproductive system, and current research efforts related to these substances mainly in the context of two main processes taking place in testicular tissues—steroidogenesis and spermatogenesis.
Over the last years, many epidemiological studies have been observing worrisome trends in the incidence of human infertility rates. Increasing prevalence of congenital abnorm-alities such as hypospadias and cryptorchidism has also been confirmed by numerous reports. Male fertility generally relies on the quantity and quality of spermatozoa, sufficient activity of Leydig cells, and a proper hormonal balance. Infertility is a widespread problem defined as the inability to conceive after one year of unprotected intercourse. In many cases, there are no obvious signs of infertility. Substantial part of the problem is the disruption of essential cellular processes responsible for normal reproductive functions [1, 2]. Given the short time, genetic changes cannot explain such alterations. We may assume that they only reflect on persistently adverse changes in the environment or in lifestyle. However, it cannot be ignored that some individuals may be more susceptible or resistant to these adverse effects than others, indicating that genetic factors do play key roles . Enormous production and release of industrial chemicals into the environment has led the scientific community to hypothesize that current pollutants may irrefutably disrupt health conditions, leading to extensive damages to physiological functions. In fact, a huge number of chemicals have been found to interact with the endocrine system of different animals in laboratory studies and there is an increasing number of reports on the endocrine disruption in wildlife . Endocrine disruptors (EDs) are an extremely heterogeneous group of ubiquitous synthetic substances, environmental pollutants, and commercial products. They are able to alter functions of the endocrine system, inhibit critical cellular processes, increase the risk of hormone-dependent malignancies, and may result in a wide array of adverse health effects. The term endocrine disruption has been adopted by the vernacular of scientists, toxicologists, and appears here to stay [5, 6]. There are many varied sources of EDs. Typical human exposure occurs with respect to the environmental contamination of the food chain, contact with contaminated household dust, and during the use of personal care products. Other EDs are used as industrial lubricants, solvents and high amounts of EDs were found in household products, pesticides, herbicides, detergents, beverage and food storage containers, metal cans, epoxy resins, etc. Many textiles contain contaminants, such as flame-retardants, including tetrabromobisphenol A and polybrominated diphenyl ethers . Although a chronic exposure to ED takes place through the skin contact or inhalation, the major source are food products. Some experimental studies assume that plastic packaging is the largest source of EDs in the human diet. Repeated exposure of food – contact materials to UV light, acid or alkaline contents and heat may cause polymers to breakdown into monomers as phtha lates, which then leach into the food or beverages . Other by-products such as alkylphenols, bisphenols, polychlorinated biphenyls, dioxins or phthalates are ubiquitous and there is a growing concern that living in an ED-contaminated environment may initiate adverse health effects. Detection of ED residues in human serum, seminal plasma, and follicular fluid has raised concerns that environmental exposure to EDs may be affecting human fertility . Nowadays, some of EDs have been banned or otherwise removed from the industrial processes years ago. On the other hand, these are persistent in the environment throughout many years. A wide range of industrial PCB compounds may be still found in pronounce quantities in the environment, although their manufacture was banned in 1977 . Indeed, humans and wildlife are continually exposed to copious potentially hazardous substances that are released into the environment at an alarming rate.
2. Male reproductive system as a major target of EDs
In this context, possible adverse effects of EDs have been taken into focus, both regarding the effects of EDs on the male reproductive system and with respect to its differential susceptibility towards these compounds. Although there has been an effort to list and rank all possible EDs, the number of evaluated chemicals remains limited. Such information and associated concerns regarding the ubiquitous presences of EDs in the environment have sparked discussions regarding the need for strategies to assess and regulate chemicals with endocrine disrupting properties in order to protect human and wildlife health. During the last years, some epidemiological studies have been comparing an increase in the incidence of male reproductive disorders in many countries. The results showed that the global average sperm count dropped by half and that the sperm motility/viability significantly decreased. In addition, many types of reproductive tract abnormalities were observed in several countries . Several experimental studies have found associations between poor semen quality and increased levels of EDs in the environment [13, 14]. EDs may disrupt not only spermatogenesis, by interfering with germ cells and sperm-supporting cells, but may also affect steroidogenesis occurring in Leydig cells. Many researchers have focused on the potential sources of EDs and their pathological consequences on reproductive health as well as ethnologies in the environment.
2.1. Alkylphenols and their impact on steroidogenesis and spermatogenesis
As we mentioned before, environmental exposure to EDs may adversely affect human and wildlife reproductive functions. Many environmental contaminants including alkylphenols are widely used in the preparation of agrochemicals, industrial and household detergents, paints, and plastics . Alkylphenol ethoxylates, a class of nonionic surfactants, are microbially degraded into alkylphenol diethoxylates and alkylphenol monoethoxylates. These are subsequently degraded into alkylphenols (4-octylphenol; 4-nonylphenol) and along with other subproducts, are known to persist in the environment for a long time . Alkylphenols are endocrine-disrupting agents with native estrogen-like structure and show estrogenic activity. This activity is mediated through the binding of these environmental estrogens to estrogen receptors. Previous studies suggested that estrogenic activity of alkylphenols is linked to a tertiary branched α-carbon and the length of the side chain at that position. Therefore, many experimental studies have investigated estrogen receptor binding and subsequent pathological changes in male reproductive functions. The mechanism also involves interaction with steroidogenic enzymes, transport proteins, and cell signaling processes. However, little is known about the direct effect of alkylphenols on the steroidogenic enzymes (3β-HSD and 17β-HSD) and gene expression .
One of the most commonly used alkylphenol is nonylphenol (NP). Due to its wide usage, a large amount of nonylphenol is widespread in the environment, especially into water sources. Vazquez-Duhalt et al.  have been convinced that the concentration of 0.1 μg/L evokes a public health risk. Based on this knowledge, several studies have investigated the potential impact of NP on male reproductive functions.
Ying et al.  demonstrated that nonylphenol’s isomers had different effects on the release of steroid hormones in rat Leydig cells. However, all experimental doses had an unfavorable impact. Specifically, the inhibitory effect of p363-NP isomer was found to be as much as 1.26 times higher than the others. The results imply that the effects of different nonylphenol isomers on the testosterone production do not appear to be completely mediated through the estrogen receptor α or β. For the steroidogenesis, ensured by Leydig cells is an essential conversion of cholesterol into various steroid classes, where 3β-HSD, 17β-HSD, and StAR are responsible for the rate-limiting step. PCR analysis showed that the decrease of testosterone production may be explained by the drastic inhibition of StAR and 3β-HSD gene expression. In a recent study, Wu et al.  demonstrated that NP increased testosterone production in rat Leydig cells. The concentration of 127.5 μM NP stimulated the steroidogenic process by elevating the activity of P450scc and stimulating protein expression of StAR. During the same experiment, trypan blue assay was performed. The authors observed the cytotoxic effect of the highest doses of NP (425 μM). Lower experimental doses (42.5–127.5 μM) used in this study had no cytotoxicity until 4 h cultivation. In a previous study, Jambor et al.  evaluated the potential impact of NP on the biosynthesis of steroid hormones, cell viability, and ROS production. The production of steroids, specifically dehydroepiandrosterone, androstenedione, and testosterone was reduced following exposure to NP after 44 h of
NP is considered to be an endocrine disrupting compound which could be involved in declines of both quantity and quality of spermatozoa in adult men [32, 33]. A lot of experiments show an
Numerous reproductive issues such as an increased incidence of testicular cancer, lower spermatozoa activity, and disruption of the steroidogenic process have been related to exposure to alkylphenols. One of the greatly widespread alkylphenols is octylphenol (OP). It is used as a component of emulsifiers, detergents, paints and many other synthetic products. Nowadays, OP is mainly present in sediments, surface waters, and even drinking water. Due to its relative stability and hydrophobic properties, OP is bioaccumulated in various tissues and poses a large health risk for the organism [48, 49, 50]. It has been reported that certain doses of OP may negatively affect cellular processes such as steroidogenesis and spermatogenesis essential for a normal development and functions of the male sex. However, there are still limited information about the mechanism, through which OP affects biosynthesis of steroid hormones. Some experimental studies have hypothesized that OP may directly modulate the activity of steroidogenic enzymes. Murono et al.  documented that exposure to 2000 nM OP affected the testosterone production in rat Leydig cells. In response to the experimental dose, testosterone levels significantly increased after 2, 4, and 8 h cultivation, when compared with the control. Exposure to shorter periods (0.5 and 1 h) were also examined; however, the weak increase at these times was not statistically significant. The increase in hormone production was not associated with changes in cAMP levels and it did not involve the estrogenic activity (binding) to the estrogen receptors. Furthermore, higher testosterone secretion was not the consequence of inhibiting 5α-reductase activity in Leydig cells. Although these results did not describe signaling pathways, it is necessary to identify the potential mechanisms through which intermediate stages of steroidogenesis may be affected. Some epidemiological studies imply that the inhibiting effects of OP on the steroidogenesis are mediated through the potential of OP to generate ROS and inhibit testosterone secretion. Cytochrome P450scc and P450c17 are essential in converting cholesterol to testosterone in Leydig cells. During the steroidogenic process, ROS are produced by electron leakage outside the electron transfer chains and these radicals may cause lipid peroxidation to inactivate P450 enzymes . Several reports evaluated the potential effects of OP on the steroid hormone synthesis [51, 53]. According to Kotula-Balak et al. , independently of the incubation time, high doses of OP significantly inhibited the progesterone production in mice MA-10 cells. Inhibition in progesterone levels was significantly higher in the experimental groups cultivated with OP for 3 h than in cells incubated for 12 h. This can be related to the restoration of Leydig cell steroidogenic function within the time of culture. Decreased progesterone production could be mediated through the inhibition of 3β-HSD since it was reported that estradiol inhibits the progesterone level via the disruption of the 3β-HSD function. Murono et al.  investigated the impact of OP on the biosynthesis of steroid hormones in rat Leydig cells
2.2. Bisphenols and their impact on steroidogenesis and spermatogenesis
Exposure to xenoestrogens such as bisphenols has been shown to cause adverse effects on male reproductive system in humans and numerous animal species. As typical endocrine disruptors, bisphenols are one of the most studied xenoestrogens in the field of male reproductive system. A survey of the Pubmed database provides more than 10,000 articles on the topic, including epidemiological as well as experimental studies. The overwhelming majority of bisphenols is used as stable components of household products, epoxy resins, inner surface of food metallic cans, dental sealants, and for myriad additional synthetic products. Many of us are mostly confronted by bisphenols through gastrointestinal exposure (food packaging) and dermal exposure (paper money and paper products). It is well known that increased concentration of bisphenols was detected in urine, milk or sweat and over 90% of human population is daily exposit to bisphenol A. Subsequent bioaccumulation and kinetic properties may adversely affect the overall health [70, 71]. Nowadays, bisphenols have been associated with a variety of human diseases, specifically kidney and cardiovascular diseases, obesity, developmental defects, and reproductive disorders. Recent studies indicate a direct link between the incidence of male reproductive dysfunction and rising concentrations of bisphenols in the environment. A decrease in semen quality was the first reported alteration and from this moment on an informative expansion was launched on the potential consequences of bisphenol exposure . Several reports demonstrate a direct effect of bisphenols on the biosynthesis of steroid hormones. Negative effects of bisphenol A (BPA) have been reported in both
2.2.1. Bisphenol A
Lan et al.  evaluated the effects of BPA on two steroidogenic enzymes (
Nowadays, there are many epidemiological studies which evaluated the effect of bisphenols on the spermatozoa or spermatogenesis. Observable changes were recorded in the spermatozoa motility, spermatozoa viability, and DNA integrity.
2.2.2. Bisphenol alternatives
More stringent global regulations of BPA production and the use have led to the development of alternative bisphenol compounds . A few years ago, researchers have begun to deal with potential properties of 4,4′-dihydroxydiphenylsulphone (BPS) or 4,4′-dihydroxydiphenlymethane (BPF). Both are presently not regulated and are used without restriction. Additionally, currently available toxicological data are scarce and the information about their potential impact is limited. Nowadays, studies reported the effects of BPS via genomic mechanisms using extremely high concentrations but there are still no studies evaluating the
Effect of BPS exposure on oxidative stress, generation of ROS, and impairment of DNA integrity of rat sperm cells under the
2.3. Phthalates and their impact on steroidogenesis and spermatogenesis
Numerous environmental contaminants have hormonal or anti-hormonal actions that interfere with endocrine homeostasis of individuals. As we mentioned above, the group of endocrine disruptors is very heterogeneous and phthalates, as ubiquitous chemical compounds are widely used as plasticizers in children’s plastics toys, food packaging, medical tubing, certain cosmetics, shampoos, soaps, and many others household products . Early experimental studies found a low level of phthalate toxicity in rodents, but nowadays, a high extent of carcinogenicity, teratogenicity or testicular atrophy has been widely confirmed. Recent studies have verified that phthalates are capable to affect many physiological mechanism and functions, especially within the reproductive system. Moreover, disorders linked to reproductive toxicity may appear in early life stages, puberty, and some of them may manifest in adulthood. The Department of Health and Human Services estimated that daily human consumption of commonly used phthalates diethylhexyl phthalate (DEHP) revolves around 5.8 mg and monoethylhexyl phthalate (MEHP) ranges from 3.26 to 4.15 in males and 2.93 to 3.51 in females. On the other hand, DEHP is metabolized by intestinal lipases to MEHP, which is glucuronized and excreted from the organism with minimum tissue accumulation [97, 98]. According to its toxicological profile, MEHP seems to be 10-fold more potent in its toxicity to Leydig and Sertoli cells in comparison to DEHP, suggesting that DEHP is the pretoxin which acts via metabolizing into MEHP . Several toxicological reports suggest that DEHP and MEHP disrupt reproductive development and now it is established that these phthalates inhibit the biosynthesis of steroid hormones in Leydig cells at different developmental stages.
2.3.1. Diethylhexyl phthalate (DEHP)
Akingbemi et al.  investigated the ability of DEHP to affect the biosynthesis of steroid hormones in rat Leydig cells. Pubertal rats were exposed to 1, 10, 100, and 200 mg/kg/day DEHP for 2 weeks. The highest experimental dose (200 mg/kg/day) DEHP caused a 77% decrease in the activity of 17β-HSD and reduced the testosterone production to 50% of the control. Paradoxically, prolonged time of cultivation to 28 days resulted in significant increases in the testosterone secretion capacity and in serum LH levels. A few years later, Akingbemi et al.  evaluated the potential effects of DEHP on isolated rat Leydig cells
2.3.2. Monoethylhexyl phthalate (MEHP)
Dees et al.  reported that MEHP inhibits androgen production in MA-10 Leydig cells. By using different MEHP concentrations over a longer time interval (24 and 2 h), the authors have demonstrated that even at low experimental doses MEHP inhibits the steroid production (a 50% inhibition was observed at 10 μM), induces morphological changes such as mitochondrial swelling and vesiculation of the Golgi apparatus. Conversely, at 100 and 300 μM doses, this inhibition was not seen. Thus, it is possible that the absence of any effect may be mediated through an unidentified mechanism, distinct to the mechanisms responsible for the inhibition of steroid production. In the next
Numerous studies have evaluated the testicular toxicity of phthalates in different experimental models and showed that spermatozoa and spermatogenesis were one of the main targets of their actions. Kasahara et al.  indicate associations between DEHP administration and increased production of ROS and selectively decreased GSH and ascorbic acid in the testis with a consequent induction of rat sperm cell apoptosis leading to testicular atrophy after
3. Future directions and recommendations
Probably, research is just at the very start of a long journey to refine understanding of the principal mechanisms of toxicity related to endocrine disruptive compounds and the range of influence of these hormonally active substances to the human and environmental health in the context of male reproduction. Society will definitely continue to use these materials because of their undeniable benefits and primary we have to aim future investigation on testing and development of chemicals to maintain healthier, safe, and more sustainable world for next generations and on evolve suitable strategies of remediation of EDs. Progress in the experimental area of endocrine disruptors effects provides rich lessons that can be usable in other fields of science, as well as in the future missions in toxicology and environmental health.
This still controversial and live topic has already improved research of toxicology and risk assessment and has moved it into certain radically different trends. Further improvement in this field including reproductive biology rests in modern technology, such as toxicogenomics, which can study precursor changes on the level of cells and biological molecules and thus offer understanding of dose and time-dependent responses in more detail. Moreover, the increased usage of human, rather than animal, cell models keep a promise for intensify issues of human relevance. However, reality is that new questions are asked while previous issues associated with impact of EDs on male reproductive organs and behavior persist. The most important fields of investigation for better understanding of how EDs affect functions of tissues involved in male reproductive physiology are associated especially with questions such as why are some tissues, time periods, and even organisms more resistant to EDs exposure; how EDs effect in model organisms and cells translates to human exposure to EDs. There is also need for more studies with aim on syndromes and EDs contribution to development of multiple symptoms at once. The summary of some EDs affecting male reproductive system is presented in Table 1. There is also necessity to interpret specific cell culture responses in the context of whole-organism physiology, ideally that of humans. It is well known that endocrine system mediates reactions on distant tissues and cells. Therefore, research that focuses only on isolated components of endocrine system or target tissues may provide incomplete information. Essential principles of toxicokinetics should be part of key studies related to impact of EDs on specific structures of organisms.
|Aldrin||Competitive binding to androgen receptors; ↓weight of testes; ↓ 3β-HSD and 17β-HSD; ↓spermatozoa MOT;||Insecticide, groundwater||Lemaire et al. |
Chatterjee et al. 
Das Neves et al. 
|Alachlor||Competitive binding to estrogen and progesterone receptors; no effects on testosterone production;|
↓ spermatozoa MOT and viability;
|Herbicide||Mikamo et al. |
Gizard et al. 
|Bisphenols||Estrogenic and anti-androgenic affinity; ↓ 3β-HSD and 17β-HSD; ↑apoptosis; ↓ sperm MOT, viability and concentration;||Plasticizers, epoxy resins, dental sealants,||Eladak et al. |
Lukacova et al. 
Akingbemi et al. 
|DDT and metabolites||Competitive binding to androgen receptors, activation of androgen-sensitive cells proliferation;|
↓ expression of steroidogenic enzymes;
↓ testosterone, estradiol, progesterone production;
|Pesticides, insecticide||Tapiero et al. |
Tesier and Matsumura 
Castellanos et al. 
|Mono/Di-(2-ethylhexyl) phthalate||↓17β-HSD; ↓ StAR expression,|
↑ mitochondrial damages; ↑ ROS;
↓ antioxidant defense; ↑spermatozoa apoptosis;
|Plasticizers, cosmetics, food packaging||Akingbemi et al. [100, 101]|
Svechnikov et al. 
Dees et al. 
|Alkylphenols||↓3β-HSD, 17β-HSD, StAR; ↑ ROS production; ↓ cell viability;|
↑ apoptosis; ↓ spermatozoa MOT and viability; ↑ DNA fragmentation,
|Cosmetics, pesticides, paints, food packaging’s,||Jambor et al. [21, 57]|
Lukacova et al. [69, 82]
Diemer et al. 
Haavisto et al. 
In recent years, a growing incidence of EDs has led scientific community to show how these substances may affect the male reproductive system. The
The study was supported by the Slovak Research and Development Agency Grant no. APVV-16-0289, APVV-15-0543, APVV-15-0544, VEGA 1/0539/18, and KEGA 010SPU-4/2018.
Conflict of interest
The authors declare no conflicts of interest.
|AhR||aryl hydrocarbon receptor|
|cAMP||cyclic adenosine monophosphate|
|PCR||polymerase chain reaction|
|ROS||reactive oxygen species|