Chapter 1: A Three-generation Study on Male-mediated Developmental Toxicity in Mice Exposed to Bisphenol A and Bisphenol A–Irradiation Combination

People are exposed constantly to physical and chemical environmental and man-made agents. Such exposure may lead to significant consequences from the point of view of public health, particularly regarding reproduction. Spermatogenesis is a crucial process, which takes place within the seminiferous tubules of the testes and leads to development of the male gametes from spermatogonial stem cells through spermatocytes and spermatids to mature sperm (spermatozoa). Human and animal studies suggested that genetic damage after radiation or chemical exposure might be transmitted to the offspring, leading to male-mediated developmental toxicity.¹ 

Exposure of humans to ubiquitous environmental toxicants including endocrine disruptors and ionizing radiation (IR) takes place continuously from fetal life through puberty to adulthood. Such exposure may affect not only the gametes of exposed individuals, but also their reproduction and development of the subsequent generation/generations. As previous studies showed, endocrine disruptors affect the male reproductive organs of animals exposed either perinatally or after puberty.^2,3  Animal studies showed that exposure to toxic agents of immature individuals might be more dangerous than adults, especially in the case of male germ cells. Pre- and postpubertal periods in mammals are critical for the action of endocrine disruptors. Prepubertal exposure to xenoestrogens may influence the secondary sexual development.⁴ 

The first important report regarding male-mediated developmental toxicity was published by Gardner et al. ^5,6  The authors stated that occupational exposure to ionizing radiation of men working at the Sellafield nuclear plant might be linked to increased susceptibility to leukaemia and non-Hodgkin’s lymphoma in their children. Chemical and physical agents may induce reproduction problems like pregnancy loss, congenital malformations, preterm birth, fetal growth retardation and diseases of the progeny at different times after birth.⁷ 

Bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) is one of the most popular chemicals. More than 8 billion pounds of this compound is produced every year.^8,9  BPA is mainly used in the production of polycarbonates (about 65%) and epoxy resins (about 25%).¹⁰  BPA-based plastic is clear and tough and is a component of a variety of common consumer goods, such as water and infant bottles, sports equipment, kitchen appliances, CDs and DVDs, computers, toys, medical devices, and dental sealants. Epoxy resins containing BPA are used to line water pipes, as coatings on the inside of many food and beverage cans and containers, and in making thermal paper used, for instance, in sales receipts.^11–13 

BPA was initially classified as a weak environmental estrogen, but recent research has shown its toxic behavior with multidirectional effects, in some cases with potency equivalent to 17-ß estradiol.¹⁴  The main route of human exposure to BPA seems to be consumption of contaminated food and beverages; however, inhalation and dermal absorption are also possible and seem to be important.^15–17  The daily exposure to BPA in adults is estimated from less than 0.02 µg per kg bw per day to 59 µg per kg bw per day.¹⁸  The World Health Organization underlined the difference of BPA contaminated product consumption depending on the age of people and estimated dietary exposure to BPA for adults from less than 0.01 µg per kg bw per day to 0.40 µg per kg bw per day and for young children and teenagers from 0.1 µg per kg bw per day to 0.5 µg per kg bw per day.¹⁹  In 2015, the EFSA calculated a temporally tolerable daily intake (t-TDI) for BPA as 4 µg per kg per day, but the highest estimates for BPA exposure from both dietary and non-dietary sources are three to five times lower than the t-TDI.²⁰  BPA may release into food, beverages and liquids from can lacquer coatings,²¹  from polycarbonate bottles during autoclaving,²²  from reusable water bottles,²³  from polycarbonate baby bottles²⁴  and from dental sealants into the saliva of patients.²⁵  The level of released BPA increases during repeated use or heating up.^26–29  Due to the ubiquitous presence of BPA in the environment and the grooving concern about its safety, in 2011 the European Union banned the use of BPA in the manufacturing and marketing of baby bottles and other baby products,³⁰  but they may be still produced and used in countries outside the European Union.

This chapter summarizes our long-term research on the effects of preconceptional exposure covering a full spermatogenesis cycle of pubescent or adult male mice to BPA or to a combination of BPA and irradiation on the male gametes and the development of two generations of their offspring.^31–34 

For conducting this study, the authors obtained permission from the IV Local Ethical Commission for Animal Experiments in Warsaw.

Pzh:Sfis outbred male mice were obtained from the laboratory of Animal Breeding “Kołacz”, Warsaw, Poland. They were housed in standard rodent cages in a room with controlled temperature, humidity, and light cycle (12 h dark, 12 h light). Tap water and a rodent diet (“Labofeed”, Factory for Animal Fodder “Morawski”, Kcynia, Poland) were available ad libitum. According to the composition indicated by the manufacturer, the feed was free from phytoestrogens. To prevent the additional exposure to xenoestrogens present in plastic, the water was served in glass bottles.

After one week of acclimatization, males were assigned randomly to either control or exposed groups. Pubescent 4.5 week or adult 8 week old F0 male mice were exposed to BPA dissolved in a small amount (900 µl) of 70% ethyl alcohol and then diluted in drinking water served in glass bottles (150–200 ml) to obtain the following doses: 5 mg per kg bw, 10 mg per kg bw, 20 mg per kg bw or irradiated with X-rays (0.05 Gy) or exposed to a combination of low doses of both irradiation and BPA (0.05 Gy + 5 mg per kg bw BPA). During the whole experiment, control animals had free access to drinking water. A therapeutic Roentgen unit Medicor type THX-250 was used as the X-ray source. It was operated with the following parameters: 175 kV, 10 mA, added filtration 0.5 mm Cu and HVL 0.8 mm Cu. Mice were whole-body-irradiated at a dose rate of 0.20 Gy min⁻¹. Males were exposed to X-rays or BPA or to both irradiation and BPA for 8 weeks, i.e., for the full spermatogenic cycle. Control animals were sham-irradiated and unexposed to BPA.

A dose of 0.05 Gy was chosen on the basis of a previous study as the lowest dose of X-rays that decreased sperm quality without diminishing the sperm count,³⁵  whereas the doses of BPA chosen for this study were based on LOAEL (50 mg per kg bw daily) and NOAEL (5 mg per kg bw daily) values published previously.^18,36 

24 h after the end of the 8-week exposure as well as at 1, 4 and 8 weeks later groups of males were killed to assess the sperm count and quality. Other groups of males of F0 generation from the control as well as all experimental groups were caged for one week with two unexposed, virgin females each. These were checked daily for the presence of a vaginal plug; this determined day 0 of pregnancy. Three quarters of the mated females from each group were euthanized via cervical dislocation 1 day before expected parturition. The remainder of the females from each group were allowed to deliver and rear litters. One of two females mated to the same male was chosen for the postnatal study, whereas the second one was used for the prenatal study.

From each group of F1 generation, 6–8 males at 8–9 weeks of age were sacrificed to check the weights of reproductive organs as well as the sperm count and quality. The remaining males were caged with 2 females each. Females come from the same experimental or control groups, but from different litters for one week to assess the prenatal development of F2 generation.

Groups of 5–7 males were weighed and euthanized at 24 h after the end of 8-week exposure, and 1, 4 and 8 weeks later. Both the testes and epididymides from each male were dissected and weighed. One epididymis was macerated in 0.2 ml of 1% solution of trisodium citrate for 5–8 min and minced. Then the solution was made up to 2 ml and mixed for about 1 min. The sperm suspension was diluted 1 : 1 in 10% buffered formalin. The spermatozoa were counted using an improved Neubauer haemacytometer.^37,38 

The contents of the second epididymis were placed into 0.2 ml of warm (37 °C) physiological saline. An aliquot was placed on a warm (37 °C) microscopic slide and covered with a cover slip. 200 cells per animal were evaluated by the eye under a light microscope for progressive and non-progressive motility in total within 5 min after sacrificing the animal and then the percentage of motile spermatozoa was calculated.³⁹ 

The remaining sperm was distributed evenly in the saline. The study of the frequency of morphologically abnormal spermatozoa was performed according to the procedure described by Wyrobek and Bruce.⁴⁰  Smears were prepared on microscopic slides, air-dried overnight, and stained with eosin Y. Then 1000 spermatozoa per mouse were analyzed using a light microscope, and abnormal spermatozoa morphology (e.g., lacking hook, amorphous, banana-shaped head, two tails, two heads) was recorded.

For the comet assay, the method of tissue preparation described previously was used.⁴¹  Briefly, one testis from each animal was decapsulated and placed in RMPI 1640 medium and minced with scissors. Before using the cells, tubes were swirled so that single cells remained in suspension. 5 µl of cell suspension was mixed in an Eppendorf tube with 75 µl low melting point agarose (LMA) for embedding on slides. The slides were immersed in alkaline lysing buffer (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid, sodium salt Na₂ EDTA, 10 mM Tris, 1% sodium lauroyl sarcosinate, pH 10) overnight at 4 °C. Then they were drained and placed in a gel electrophoresis tank, and left in the solution for 20 min. The electrophoresis was conducted at 4 °C for 20 min using 19 V and 300 mA. After neutralization, the slides were stained with ethidium bromide (EtBr) and examined using a fluorescence microscope (Nikon, Japan). Images of 100 randomly selected cells from each animal were recorded and analyzed using CASP image analysis software.⁴²  Comet tail moment and % tail DNA were chosen to determine the induction of DNA breaks.

One testis from each mouse was fixed in Bouin’s fluid for 40 hours. Prior to placement of the gonad into a fixative, the tunica albuginea was shallowly pierced with a 21-gauge needle to aid penetration of the fixative. Thereafter, the material was briefly washed in tap water and dehydrated with graded ethyl alcohol concentrations and in n-butanol. The testes after embedding in a paraffin block were serially cut into 7 µm-thick sections, stained with hematoxylin and eosin and examined using a Nikon Eclipse E400 microscope.

Immediately after dissection, the testes were placed in a fixative and cut into 1 mm³ fragments. The tissue fragments were fixed for 2 h in 2.5% glutaraldehyde and postfixed for 1 h in 1% OsO₄ both diluted in 0.1 M phosphate buffer pH 7.4. After dehydration in increasing concentrations of ethanol (50–100%) and in propylene oxide, the material was embedded in Poly/Bed 812 (Polysciences, Inc., Warrington, PA, USA) and cut with a diamond knife on an RMC type MTXL ultramicrotome. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined using a JEM 100S (Jeol, Japan) transmission electron microscope.

The protocol for the dominant lethal assay and congenital malformation study described previously⁴³  was used. Females were sacrificed one day before expected parturition.

A male mated with at least one female was defined as fertile. A female with at least one live or dead implantation was defined as pregnant. Females were examined for the number of implantations, the number of live fetuses and the number of early and late post-implantation deaths. Post-implantation deaths were classified as early if the embryo had died and been resorbed, or late if the dead embryo was at a stage beyond the onset of organogenesis.

Live embryos were weighed and analyzed for the presence and type of gross malformations (e.g., exencephaly). Runts were defined as live fetuses having a body weight less than 75% of the mean of their litters.⁴⁴  Malformed fetuses and half that number of randomly selected normal fetuses from each of the exposed and control groups were assessed for skeletal malformations after alcian blue and alizarin red staining (mixture contents: 0.3% alcian blue diluted in 70% ethanol – 1 part + 0.1% alizarin red diluted in 95% ethanol – 1 part + acetate acid – 1 part + 20% ethanol – 17 parts).

Pups of females having parturition were counted and weighed at birth and then weekly up to 8 weeks of age. They were observed for physiological markers and growth parameters.

The appearance of pinna detachment was recorded as the age (days) when pinna of both ears unfolded to a fully erect position. Eye opening was defined as any visible break in the membrane covering the eye. Vaginal opening was defined as any visible break in the membrane when the vaginal lips were gently pulled laterally. Descent of the testes was recorded when the testes descended to lie in the scrotal sac.⁴⁵ 

When males of the F1 generation reach the age of 8–9 weeks, 6–8 animals from each of the control and experimental groups were euthanized to assess their sperm count and quality. All analyses were performed as described in Sections 1.2.2 and 1.2.3.

The remaining F1 males were caged with two females from the sane group but from different litters each to analyse the prenatal development of the F2 generation. The experiments were carried out as described in Section 1.3.1.

Statistical analysis was performed by one-way analysis of variance (ANOVA) with post hoc Tukey’s test and the chi-square test. The significance level was established at p < 0.5.

The effects of the exposure of pubescent or adult male mice to BPA or the BPA plus X-ray combination have been described in the paper of Dobrzynska et al. ³¹  After exposure of both adult and pubescent males, the mean testis weights were significantly decreased at 24 h and 1 week after 0.05 Gy as well as after combined 0.05 Gy and 5 mg per kg bw BPA exposure.³¹ 

In the case of pubescent males, the sperm count was reduced after irradiation and combined exposure to low doses of irradiation and BPA at 24 h and 1 week after the end of exposure and after exposure to 20 mg per kg bw BPA at 1 and 4 weeks post exposure (see Figure 1.1A). There were no differences in the sperm mobility between control and experimental groups (see Figure 1.1B). An increased percentage of abnormal spermatozoa was observed in all experimental groups at 24 h after the end of exposure and in all BPA groups and in the combined group at 1 week post exposure. Moreover, the significantly higher frequency of abnormal spermatozoa was noted at 4 and 8 weeks in groups of 20 mg per kg bw BPA and in the combined group and at 8 weeks in the irradiated group (see Figure 1.1C). A significantly decreased percentage of DNA in the comet tail was observed at 24 h after the end of exposure to irradiation and in the combined group (see Figure 1.1D).

In males exposed as adult, the sperm count was significantly reduced at 4 weeks in groups of males with irradiation and combined exposure to irradiation and BPA (see Figure 1.2A). Sperm mobility was not significantly decreased in all experimental groups (see Figure 1.2B). The percentage of abnormal spermatozoa was increased in groups of 10 and 20 mg per kg bw BPA, 0.05 Gy and the combination of 0.05 Gy and 5 mg per kg bw BPA when samples were taken just after the end of exposure; in groups of 20 mg per kg bw BPA, 0.05 Gy and 0.05 Gy and 5 mg per kg bw BPA 4 weeks after the end of exposure; and in all groups of BPA, and 0.05 Gy plus 5 mg per kg bw BPA after 8 weeks (see Figure 1.2C). There were no significant changes in the % of DNA in the comet tail (see Figure 1.2D).

Only slight morphological changes seen through a light microscope were visible in the gonads of adult male mice exposed to 10 and 20 mg per kg bw of BPA, as well as in those irradiated and exposed to the combination of X-rays and BPA at low doses at 24 h after the end of exposure. In some seminiferous tubules, degenerating spermatogonia and spermatocytes were present. Occasionally, intraepithelial vacuoles were observed. Intracellular vacuoles between Sertoli cells and spermatogonia were observed using an electron microscope in 20 mg per kg bw BPA, irradiated and combined groups. Differences in the morphology of the gonad between control and experimental groups were clearer in pubescent males. The gonads of animals from all experimental groups contained degenerated spermatogonia and primary as well as secondary spermatocytes. Degenerated cells were more commonly observed in animals exposed to BPA. The number of degenerated cells increased with increasing doses of BPA and was the highest at 24 h and 1 week after the termination of exposure. Sometimes, multinucleated giant cells located within the seminiferous epithelium in the testes of males treated with BPA at a dose of 20 mg per kg bw BPA were observed. Single intraepithelial vacuoles were found in males treated with the highest dose of BPA. Such changes were observed at all time points after the end of exposure. Prominent vacuolization and loss of thickness of some seminiferous tubules were noted 1 and 4 weeks post termination of combined exposure to low doses of irradiation and BPA. Spermatogonia and spermatocytes were clearly separated from Sertoli cells in gonads assessed 24 h or 1 week after the end of irradiation. Ultrastructural analysis confirmed the presence of degenerative changes in the spermatogonia of mice exposed to 20 mg per kg bw of BPA. Intercellular vacuoles on the border of Sertoli cells and spermatogonia, as well as spermatogonia and spermatocytes were identified in mice irradiated, exposed to the highest dose of BPA and to a combination of X-rays and BPA. Intracellular vacuoles and distended cisterns of the smooth endoplasmic reticulum occurred in the cytoplasm of spermatocytes and Sertoli cells, especially at 24 h and 1 week after the end of combined exposure. A few spermatids were binucleated or contained double axonemes.³¹ 

The effects of exposure of pubescent male mice on the F1 generation have been described in the paper of Dobrzynska et al. ³³ 

The results of the male fertility, frequency of pregnant females, and of prenatal development of the offspring of pubescent males exposed to BPA alone or in combination with irradiation are shown in Table 1.1. The fertility of males from all experimental groups was not significantly reduced compared to the control. The frequency of pregnant females was significantly decreased in the groups of 0.05 Gy and 0.05 Gy + 5 mg per kg bw of BPA. The mean numbers of total and live implantations in the 0.05 Gy group were significantly reduced compared to the control and to the groups of 5 mg per kg bw and 10 mg per kg bw. The number of live fetuses in the combined group was significantly different from the control group. The numbers of dead implants were the highest, but not statistically significant in the irradiated and combined groups. In all groups, the majority of dead implants were classified as early deaths. The frequency of dominant lethal mutations was the highest in irradiated (44%), combined (29%) and 20 mg per kg bw of BPA (25%) groups.

The results of the body weight and gross and skeletal malformations of surviving fetuses are shown in Table 1.2. The mean body weight of the offspring of pubescent males exposed to 20 mg per kg bw BPA was significantly reduced. In the groups of 0.05 Gy and to 0.05 Gy + 5 mg per kg bw BPA, the mean body weight of living fetuses was significantly increased compared to control and all BPA groups. The incidence of gross malformations was the highest after exposure of F0 males to irradiation and BPA. Runts were the most frequent gross malformation. Skeletal malformations were significantly increased compared to the control after exposure of F0 males to BPA at doses of 10 mg per kg bw and 20 mg per kg bw, as well as in irradiated and combined groups. In BPA treated groups, the frequency of abnormal skeletons increased in a dose-dependent manner. Concavity of parietal bones and the presence of extra ribs were the most frequent skeletal malformations.

The mean litter sizes at birth were the lowest in the group of 10 mg per kg bw of BPA, but not statistically significant. The mean litter size of the offspring at 8 weeks of age was markedly, but not statistically significantly reduced after exposure to 10 mg per kg bw of BPA and after combined exposure to 0.05 Gy and 5 mg per kg bw of BPA compared to the control.³³  The percentages of mortality of the offspring were significantly elevated after exposure of males to 10 and 20 mg per kg bw BPA, after irradiation alone and in the combined group (see Figure 1.3).

Mean male : female ratios were significantly affected after exposure of pubescent males to 5 and 10 mg per kg bw BPA. In the 5 mg per kg bw BPA group, there were 55% of males vs. 45% of females, whereas in the 10 mg per kg bw of BPA group there were 60% of males vs. 40% of females, compared to 42% of males vs. 58% of females in the control group (see Figure 1.4A). There were no significant differences between the control and experimental groups in the time of appearance of physiological markers, such as pinna detachment, fur development, eye opening, vaginal opening and testis descent.³³ 

The results of the postnatal body weight of pups are presented in Figure 1.5A. The mean body weights of the offspring of males exposed to 5 mg per kg bw of BPA daily were significantly elevated from 3 to 8 weeks of age. The mean body weight of the offspring of males that received 10 mg per kg bw BPA was significantly increased from birth to 6 weeks of age. The mean body weights of the offspring of irradiated F0 males were significantly elevated at 1 week of age compared to the control. In the case of the offspring of pubescent F0 males exposed to a combination of X-rays and BPA, the body weights were significantly increased at birth as well as at the 1st and 5th weeks compared to the control, and additionally at 1, 6 and 7 weeks of age compared to 20 mg per kg bw of BPA alone.

There were no significant differences in the body, testis and epididymis weights between the F1 offspring of unexposed and exposed males. The sperm counts were not significantly lower in all experimental groups, with the lowest values observed in the offspring of males irradiated and exposed to 20 mg per kg bw of BPA. The percentages of motile sperm were significantly reduced in the offspring of males from all experimental groups. The frequency of abnormal spermatozoa was significantly increased in the offspring of F0 males exposed to a combination of X-rays and BPA. The DNA damage (comet tail moments) was not significantly increased in the offspring of males irradiated, exposed to 20 mg per kg bw of BPA and with the combination of both irradiation and BPA.³³ 

The light and transmission electron microscope evaluation revealed no changes in the structure of the gonads of the F1 offspring of preconceptionally exposed pubescent males. Gonads from control and experimental groups exhibited a typical arrangement and structure of the seminiferous tubules. The ultrastructures of the spermatogenic and Sertoli cells in all groups were comparable.³³ 

The results of male and female fertility as well as of prenatal development of the offspring of males exposed to BPA or/and irradiation are shown in Table 1.3. The fertility of males exposed for 8 weeks to 5 mg per kg bw of bisphenol A daily was significantly reduced compared to the control. The frequency of pregnant females was significantly decreased in all experimental groups. The mean numbers of total implantations were significantly reduced compared to the control in all exposed groups, except for 20 mg per kg bw BPA, where the number of total implantations was not significantly increased. The numbers of live fetuses per pregnant female were significantly reduced in 5 mg per kg bw BPA, 10 mg per kg bw BPA, 0.05 Gy and 0.05 Gy + 5 mg per kg bw BPA groups. The number of dead implants was the highest in the irradiated group, but without statistical significance. In all groups, the majority of or all dead implants were classified as early deaths. In all groups, except for males exposed to 20 mg per kg bw BPA daily, the frequency of dominant lethal mutations was over 20%.

The results of the body weight and gross and skeletal malformations of surviving fetuses are presented in Table 1.4. The mean body weights of the progeny of males exposed to 5 mg per kg bw BPA and to 0.05 Gy + 5 mg per kg bw BPA were significantly increased compared to the control group. The incidence of gross malformations was rare in the offspring of both exposed and unexposed males. Skeletal malformations were more frequent, but not statistically significant compared to the control. Extra ribs were observed frequently, in exposed as well as control groups.

The mean litter sizes in all groups paternally exposed to BPA and to irradiation were slightly, but not significantly enhanced compared to the control. The mean litter size of the offspring of males exposed to a combination of X-ray irradiation and BPA was not significantly reduced compared to those exposed to BPA alone.³²  The percentages of mortality of the offspring were significantly elevated after exposure of males to 5 mg per kg bw and 20 mg per kg bw BPA, and after combined exposure to both irradiation and BPA (see Figure 1.3). Mean female : male ratios were similar in all groups (see Figure 1.4B). There were no significant differences between control and experimental groups in the time of appearance of physiological markers, such as pinna detachment, fur development, eye opening, vaginal opening and testis descent however, in the combined group the time of testis descent was slightly delayed (4.5 days) compared to the control.³² 

The results of the postnatal body weight of pups are shown in Figure 1.5B. The mean body weights of the offspring of males exposed to 5 mg per kg bw of BPA daily were significantly reduced at 1 and 2 weeks after birth. The mean body weight of the offspring of males that received 20 mg per kg bw BPA was significantly decreased at 1 week and significantly increased at 4 weeks. The mean body weights of the offspring of irradiated males were significantly elevated from the 3rd to 5th week and at the 7th and 8th week of age. In the case of the offspring of males exposed to a combination of X-rays and BPA, the body weights were significantly increased at all time points compared to the control and BPA alone as well as at birth and at the 1st week of age compared to X-rays alone.

There were no significant differences in the body, testis and epididymis weights between the offspring of unexposed and exposed males. The sperm counts were the lowest in the offspring of irradiated and exposed males to 20 mg per kg bw of BPA, however without statistical significance. The percentages of motile sperm were markedly, but not significantly reduced in the offspring of males exposed to BPA alone and in combination with irradiation with statistical significance in the group of 10 mg per kg BPA only. The frequencies of abnormal spermatozoa were slightly, but not significantly increased in the moderate and the highest BPA groups as well as in the combined group. The percentage of DNA in the comet tail was slightly, but not significantly increased in the offspring of males with irradiation and combined exposure.³² 

There were no morphological changes in the gonads of the offspring of adult preconceptionally exposed males, as viewed with light and transmission electron microscopes. The integrity of the basal lamina of the seminiferous epithelium was preserved. In the experimental and control groups, the thickness of the seminiferous epithelium and the quality of the spermatogenic cells were comparable. Spermatogonia and spermatocytes were in close contact with Sertoli cells. Supporting cells were bound together by tight junctions. The ultrastructures of the spermatogenic and Sertoli cells of the experimental groups were similar to those of the control groups. In gonads from the control and experimental groups, degenerating cells were only occasionally visible. The morphology of the stroma of the testes and of Leydig cells in all groups was comparable to that in the control group.³² 

The effects of preconceptional exposure of adult or pubescent males of the F0 generation to BPA alone or in combination with X-rays on the prenatal development of the F2 generation have been described in the paper of Dobrzynska et al. ³⁴ 

In the case of exposure to BPA or/and X-rays of pubescent F0 males, the frequency of fertile F1 males in the group of 10 mg per kg bw of BPA was significantly decreased and reached only 50%. The frequency of pregnant females in the groups of 10 mg per kg bw and 20 mg per kg bw of BPA was significantly lower (23.81% and 47.62%, respectively) as compared to the control group. There were no significant changes in the frequency of total implantations and live and dead fetuses in all experimental groups as compared to the control.

In the case of exposure of adult F0 males to BPA or/and X-rays, the frequency of fertile males among all groups was similar. The frequency of pregnant females in the groups of 5 mg per kg bw and 20 mg per kg bw of BPA was significantly lower (44.83% and 59.14%, respectively) as compared to the control group. There were no significant changes in the frequency of total implantations and live and dead fetuses in all experimental groups as compared to the control.

Several papers published at the end of the 20th century showed that diminished sperm count and quality of male sperms in general population might be the result of increasing human exposure to endocrine disruptors.^46,47  Exposure to endocrine disruptors, including bisphenol A, has significant meaning for public health, especially because such compounds influence the male reproductive system, including decreasing sperm count and quality and increasing frequency of testicular cancer, cryptorchidism and hypospadias.^46,48 

The male reproductive system is the most sensitive target organ. Reduced fertility, embryo loss, birth defects, and childhood cancer are the most common outcomes of exposure to chemical and physical agents. Male germ cells, especially the younger stages of spermatogenesis, are very sensitive. Sperm disorders, including problems in production and maturation of sperm, may cause male infertility. Normal spermatozoa may be produced in abnormally low numbers or sperm produced in adequate numbers can be immature, with poor motility and morphology. All above incidences diminish the chance to fertilization.⁴⁹ 

In males, high urinary BPA levels may be associated with decreased semen quality and increased DNA damage in spermatozoa.⁵⁰  Previous papers reported that BPA causes testicular toxicity and reduces sperm production following exposure of neonatal, pubertal, and adult laboratory rodents.^51–57  Takao et al. ⁵⁸  stated that exposure to BPA around the pubertal period may directly disrupt the male reproductive tract of mice. BPA significantly reduced the epididymal sperm count in pubertal rats following 5-week treatment as a result of lowered plasma testosterone and this might be a reason of the higher susceptibility of younger animals to harmful effects induced by BPA.⁵⁹  In turn, Sakue et al. ⁵²  observed that there is a sub-population of cells that are impacted by BPA, leading to an approximately 40% decrease in daily sperm production. Similarly, a reduced sperm count was observed in this research study at 1 and 4 weeks after the end of 8-week exposure of pubescent males to 20 mg per kg bw BPA. The harmful effect on the germ cells might be associated with the ability of BPA to induce reactive oxygen species generation in the epididymal sperm of rats.⁶⁰  As Anjum et al. ⁶¹  reported, in animals treated with BPA, all antioxidant enzymes showed a reduced activity.

The results of our study showed the higher susceptibility of pubescent male gametes to lethal damage induced by BPA. The significantly reduced sperm count observed in pubescent males exposed to 20 mg per kg bw BPA was associated with the increased incidence of degenerated spermatogonia and spermatocytes observed in BPA exposed groups. Moreover, intraepithelial vacuoles and multinucleated giant cells within the seminiferous epithelium were observed. Multinucleated giant cells with more than 2 micronuclei were observed by Takao et al. ⁵⁸  in the seminiferous tubes of the testis following 8-week exposure to 120 mg per kg per day of BPA. Simultaneously, the appearance of multinucleated giant cells and exfoliation of spermatids as well as incomplete specialization, redundant ectopic specialization and aplasia in Sertoli cells and spermatids was noted by Toyama et al. ⁶²  Similarly, Tan et al. ⁶³  reported that sub-acute exposure to BPA caused degeneration of the germinal epithelium of juvenile male rats. Multinucleated giant cells were present in all animals and some of them did not show any form of the spermatogenic cycle.

The current study showed also that the germ cells of pubescent males exposed to BPA took longer to recover. The enhanced level of abnormal spermatozoa was observed just after the end of exposure in both adult and pubescent males, however one week later in pubescent males only. There was also a slight, but not significant effect of BPA on the induction of DNA damage in pubescent males just after the termination of exposure. Results of other papers showed an association between BPA exposure and an increased level of DNA in the comet tail.^50,64 

As previous studies showed, the targets of BPA within the testis are Sertoli cells and Leydig cells. Overexposure to estrogens may cause inhibition in the proliferation, development, and function of Sertoli cells.²  Moreover, the expression of Connexin 43, a major gap junction component of Sertoli cells, which plays an important role in spermatogenesis,^65,66  is affected by BPA.⁶⁷  Leydig cells produce testosterone which among others supports spermatogenesis and fertility in adulthood. BPA was found to act directly in Leydig cells because it decreased testosterone production after treatment of Leydig cells in vitro.⁶⁸  The proliferation of precursors and adult Leydig cells during the period of pubertal development is important for the establishment of the adult complement of Leydig cells.²  Development of gametes relies on a highly coordinated interaction with Sertoli cells. Germ and Sertoli cells can communicate directly via ligand/receptor mediated interactions or paracrine factors. The production and secretion of many Sertoli cell proteins, which are involved in germ cell development occur in a stage-dependent manner.²  Environmental toxicants are able to increase oxidative stress by down-regulating the production of antioxidant enzymes, leading to male infertility by disrupting the cell junction and adhesion between Sertoli–Sertoli cells and/or Sertoli–germ cells via the phosphatidylinositol 3-kinase (PI3K)/c-Src/focal adhesion kinase signaling pathway.⁶⁹  The decreased number or dysfunction of Leydig and Sertoli cells might be a reason of the diminished sperm count and quality in pubescent males.

The response following irradiation was different in each age group. The diminished sperm count observed in pubescent males after exposure of mature spermatozoa and late spermatids correlated with reduced testis weight. The significantly reduced sperm count observed in males irradiated since 4.5 weeks of age was associated with the occurrence of degenerating cells, which may suggest apoptosis. The reduced sperm count following irradiation is mainly attributed to apoptosis of germ cells with genomic abnormalities initiated likely by Trp53 protein.⁷⁰  On the other hand, reduced DNA damage at 24 h after the end of irradiation as well as combined X-ray and BPA exposure of pubescent males may reflect stimulation of DNA repair following exposure to a low (0.05 Gy) dose of X-rays. It is known that the DNA damage-control biosystem is stimulated by low dose radiation.⁷¹ 

Following 8-week combined exposure to low doses of both irradiation and BPA, significantly reduced testis weight and sperm count compared to the control and the group with an appropriate dose of BPA were observed in adult and pubescent males. The effect observed in pubescent animals was associated with reduced sperm count. After combined X-rays and BPA exposure, the percentage of abnormal spermatozoa was markedly higher than that following treatment with the lowest dose of BPA in adult males. These effects were similar in pubescent males, except for results noted one week after termination of exposure. A significant reduction of the percentage of DNA in the comet tail compared to the results of control and BPA alone groups at 24 h after the termination of combined exposure was noted in pubescent males; however, also in adults a similar tendency was observed. The reduced sperm count and testis weight, and diminished sperm quality after combined exposure were rather caused by X-rays. Combined treatment mainly enhanced the harmful effects induced by BPA in male germ cells. However, in pubescent males protective effects of low doses of radiation on the DNA of male gametes was observed just after the end of exposure. Low doses of radiation, as mentioned above, stimulate physiological mechanisms that may repair the damage induced following combined exposure. A previous study showed significantly diminished sperm count and quality after 2-week combined X-rays and BPA exposure compared to the control and non-significant differences between the results of combined exposure and each agent alone.⁶⁴  Also, in the same study, the levels of DNA damage in germ and somatic cells after combined X-rays and BPA exposure were significantly lower compared to BPA alone.⁶⁴ 

As previous papers showed, the toxic effects of BPA are more evident during the pubertal period, when neuroendocrine mechanisms during the development of reproductive organs and the appearance of secondary sexual characteristics became more vulnerable to the influence of environmental agents.^16,72,73  Recently the effects of BPA on puberty have been critically reviewed by Leonardi et al. ¹⁶  and the authors concluded that studies on the effects of BPA on male puberty are still limited. Puberty is a period of transition between adolescence and adulthood. Full reproductive maturity is reached through the maturation of the gonads, permanent genital development, and appearance of secondary sexual characters during the period of puberty.⁷⁴  The pathologic mechanism of precocious puberty caused by BPA exposure is related to its estrogen-like activities, which triggers a possible feedback process for the activity of the gonadotropin-releasing hormone (GnRH) pulse generator, which increased LH and FSH control secretion despite the weak effects as a xenoestrogen.⁷⁵  Caserte et al. ⁷²  reported in laboratory animals the effects of BPA on the weak hypothalamic–pituitary–gonadal axis through alterations in brain sexual differentiation, higher GnRH secretion, and alterations in the estradiol-induced LH surge.

Development that includes among others the prenatal period appear to be a critical window with increased sensitivity to BPA effects.⁷⁶  Exposure of pubertal rats to BPA is capable of disrupting the vital endocrine system resulting in the reduction of sperm production and quality.⁵⁹ 

The effects of BPA on the male and couple reproductive health were reported by Minguez-Alarcon et al. ⁷⁷  On the basis of the analyzed papers, the authors concluded that an association between BPA exposure and adverse male reproductive health outcomes in humans remains limited and inconclusive.

In the present study, the frequencies of pregnant females were significantly decreased in groups of pubescent males with irradiation and combined exposure to X-rays and BPA, whereas when adult males were exposed, a reduced percentage of pregnant females was noted in all experimental groups. A similar result was described by Al-Hiyasat et al. ⁷⁸  after 30 days exposure of males mated later to unexposed females. They noted that this may reflect induction of mutations in male germ cells, which makes fertilization of eggs impossible or may indicate preimplantation death. In our study, the numbers of total and live fetuses were significantly decreased compared to the control in the groups exposed to 5 and 10 mg per kg bw BPA, 0.05 Gy and 0.05 Gy + 5 mg per kg bw BPA after exposure of adult males and in the irradiated group in the case of pubescent males. The above treatments induced over 25–44% of dominant lethal mutations. A reduced number of implants induced by BPA were observed in rats.⁷⁹  Another study showed the lack of such response in mice.⁸⁰  Our results support the observation described earlier and indicate a rather unsuccessful fertilization process or preimplantation losses, but not postimplantation losses, because the number of implantation deaths, although in the combined group it reached almost 11%, does not significantly differ from the control. On the contrary, in another study, an increased number of resorptions was noted after 30-day exposure of males to BPA.⁷⁸  Results similar to the current study were noted after irradiation with the same dose in adult males.⁸¹  Results of combined exposure support the conclusion that chronic low dose irradiation of F0 males has a stronger effect on the prenatal development of their offspring than exposure to BPA. The above finding confirmed the results of previous studies, which showed that ionizing radiation seems to be the main factor that caused intra-uterine dominant lethality.^81,82  BPA at low doses seems to moderate the harmful effect of radiation during preconceptional exposure. However, combined irradiation and BPA exposure cannot be considered beneficial because of induction of a decreased number of total and live implantations compared to the effect of BPA alone.

The current study shows that neither irradiation nor BPA induces significant gross malformations of surviving fetuses. The significantly higher body weight of living fetuses from irradiated and combined groups may be caused probably by the lower number of fetuses per pregnant female in the above groups. A significantly increased frequency of skeletal malformations was noted in all groups, except for the lowest dose of BPA, but only after preconceptional exposure of pubescent males.

The results regarding litter size in the experiment of postnatal development differ from those obtained from the experiment of prenatal development. After exposure of pubescent males, at birth, only mean litter sizes of pups born to females mated to males exposed to 10 mg per kg bw of BPA were significantly reduced.³³  In turn, after exposure of adult males, no significant differences in the litter size were observed similarly to the study on the litter size for F1/F2 generations in mice.⁸⁰  Contrarily, reduced litter size after three-generation exposure of rats to BPA was noted by Tyl et al. ⁸⁰  In the present study, the percentage of mortality was significantly increased in all experimental groups, except for the offspring of pubescent males exposed to the lowest dose of BPA, with the highest value (almost 30%) observed in the combined group. After exposure of adult males, an increased frequency of pup mortality was noted in 5 and 20 mg per kg BPA groups and in the combined group. A decreased viability of the pups after exposure of pregnant females to BPA was reported previously.⁸³  On the other hand, Tyl et al. ⁸⁰  did not observe such an effect. The highest percentage of mortality, in the combined group after exposure of pubescent males, which is similar to that observed after exposure of adult males, may suggest the synergistic effect of both irradiation and BPA with the effect of irradiation being superior on the induction of postnatal lethality. The highest percentage of mortality in BPA groups may be caused by both big litter size and lethal mutations manifested in young pups. In the combined group, the high mortality was probably due to lethal mutations since the litter size in this group was the lowest.

The ratio of male to female live births is called the secondary sex ratio (SSR), while the ratio of male to female conception is called the primary sex ratio (PSR).^84,85  Preconceptional exposure of pubescent males to BPA at low or medium doses significantly affected the SSR with reduction of females born compared to the control group, but such an effect was not observed after preconceptional exposure of adult males. Previous studies did not show the effects of BPA on the male : female ratio in rats⁷⁹  and in mice.⁸⁰  No significant differences in the male : female ratio in the offspring of mice exposed from gestational day 1 to postnatal day 42 to BPA were also noted by Fang et al. ⁸⁶  Contrary to our results, the prospective cohort study of Bae et al. ⁸⁵  showed that parental preconceptional exposure to BPA and phthalates may decrease the SSR, whereas only maternal preconceptional exposure to those agents may increase the SSR. There is also a hypothesis that postulates that the SSR may be affected by parental hormone levels around the time of conception.^87,88  According to this hypothesis, low parental testosterone and high parental gonadotropin levels are associated with female births, whereas a high maternal estrogen level leads to male births.⁸⁵ 

Exposure of pubescent as well adult males to BPA, irradiation alone or to a combination did not significantly delay the appearance of physiological markers in their offspring.^32,33  Previously, a delay in testis descent was noted in two-generation mice with dietary exposure to BPA.⁸⁰ 

Recently several review papers have been published regarding the connection between BPA exposure and obesity.^76,89–93  According to the results of previous research, BPA should be considered an obesogenic environmental compound. There were no papers regarding obesity of the offspring after preconceptional exposure of males. The majority of previous papers described the effect of exposure to BPA of pregnant females and pubs just after birth.

The present study showed an increase in the body weight of pups in all experimental groups after exposure of pubescent males. In turn, after exposure of adult males, a decrease in the body weight of pups was noted. The higher body weight of the pups of males exposed to irradiation or to a combination of irradiation and BPA was probably related to the lower litter size but not a beneficial effect due to exposure of male mice.

Similar results after pre- or perinatal exposure were noted previously.^9,85–94  Contrarily, other authors did not observe such results.^{51,83,96–98}  Possibly, early BPA exposure can influence several mechanisms important for body weight regulation including adipocyte deposition, glucose uptake and homeostasis, and the development and maturation pathways and circuits important for energy homeostasis.⁹⁸  The explanation for current results might be transgenerational inheritance of obesity. According to the results of previous papers, exposure to endocrine disruptors, including BPA, has been suggested to contribute to obesity in both animals and humans.^99,100  Previous animal studies showed that endocrine disruptors may affect obesity-related pathways by changing hormone levels or altering gene expression.⁹⁹  Adipose tissue is considered an endocrine organ that actively secretes a number of adipokines.⁷⁶  Earlier studies showed that increased BPA exposure is associated with changes of adipokine levels that are involved in regulation of appetite and satiety leading to obesity. So adipose tissue seems to be the target of BPA.^76,101,102  The impact of BPA exposure on the genetic aspect of obesity is not clear. Several studies showed that the locus fat mass and obesity associated (FTO) gene was described as the most associated gene with an increased BMI.^103–106  The regulation of gene expression is mediated by DNA methylation, RNA silencing, histone acetylation, and nucleosome remodeling.^76,107–109  In an embryonic mouse hypothalamus cell line, BPA alters gene expression levels that can be linked to the observed brain-derived neurotrophic factor (BDNF) modification of expression.¹¹⁰  The above results suggest that BPA fetal exposure induces epigenetic alterations in genes involved in the pathophysiology of obesity, leading to the in utero programming of obesity after BPA exposure.¹¹¹  Maybe a similar result is possible after preconceptional exposure.

Also, radiation exposure has been related to obesity in mice^112,113  and in humans,^114–117  but the effect in the offspring of exposed males has not been described so far. The higher body weight of the pups of males exposed to irradiation or to a combination of irradiation and BPA may be related to both the lower litter size or obesity induction. In the case of combined exposure, obesity may be caused by both BPA and irradiation exposure.

There is evidence that pre- or neonatal exposure to BPA reduces the size of the epididymis and decreases the sperm count in male offspring.⁵¹  Two-generation exposure of mice to 3500 ppm of BPA, opposite to the current results, showed reduced testis weight associated with testicular seminiferous tubule hypoplasia.⁸⁰  Kobayashi et al. ¹¹⁸  showed no significant differences in the motility of sperm of F1 males following exposure of pregnant females to BPA. In this study, the sperm counts of the F1 offspring of males exposed to BPA or irradiation alone and to the BPA–irradiation combination were not significantly reduced, whereas sperm motilities were significantly reduced compared to the control. Moreover, after irradiation and combined exposure, significantly increased frequencies of abnormal spermatozoa were noted. After exposure of the adult males, the diminished sperm motility of F1 offspring was observed only at the moderate dose of BPA. There were no pathological changes in the structure of gonads after exposure of both pubescent and adult males. Reduced sperm motility may cause diminished fertility of the F1 generation since immobile spermatozoa are unable to reach and fertilize eggs, whereas abnormal spermatozoa are rather not able to fertilize.

The current results showed that BPA alone and its combination with irradiation may induce genetic or epigenetic changes in male gametes, which may be transferred to the F1 generation. An epigenetic process may reprogram the germ cell leading for example to altered DNA imprinting. As demonstrated by Singh and Li,¹¹⁹  BPA is epigenetically toxic. Heritable changes in gene expression may occur for instance by reprogramming the germ cell without changes in the DNA sequence. Epigenetic mechanisms include DNA methylation, histone modifications and expression of non-coding RNAs; such an epigenetic effect may cause a transgenerational effect on subsequent generations through the germ line.^{100,121–123} 

DNA methylation patterns are established during embryogenesis, mainly during the early developmental period, through the cooperation of DNA methyltransferases and associated proteins. The up/down alterations in gene expression may proceed throughout the lifetime, leading to adverse health effects including infertility.¹¹⁹  Exposure to BPA may have also cumulative adverse effects mediated through epigenetic mechanisms to the future generations via the sperm.^{100,120–123}  Prenatal or neonatal exposure to BPA reduced the size of the epididymis and decreased the sperm count in male offspring.⁵¹  Two-generation exposure of mice to 3500 ppm of BPA caused reduced testis weight associated with testicular seminiferous tubule hypoplasia.¹²⁴  There were no significant differences in the motility of sperm F1 males following exposure to BPA of pregnant females.¹¹⁸ 

The current results showed that exposure of pubescent males to BPA alone or in combination with irradiation may be more dangerous to their offspring than the exposure of adult males. The exposure of pubescent males to BPA and both irradiation and BPA significantly increased the frequency of abnormal skeletons of surviving fetuses, increased the percentage of mortality of pups of the F1 generation, induced obesity, affected the male : female sex ratio and reduced the sperm motility of F1 males. The combined BPA and irradiation exposure of pubescent males additionally reduced the number of total and live implantations.

Exposure of F0 males to BPA may affect the reproductive system, which can also have an adverse impact on subsequent generations. As Karmakar et al. ¹²⁵  observed recently, BPA induces physiological and functional disruption in male germ cells, which may lead to reproductive issues in subsequent generations.

The above effects may lead to diminished fertility or health conditions of future generations. DNA damage induced in any stages of spermatogenesis may be the cause of cell death or defects in the subsequent germ cell stages. For example, an increase in germ cell mutations may lead to dominant lethality, embryonic or fetal deaths, congenital malformations observed at birth, childhood cancers or genetic diseases in subsequent generations.¹²⁶  If the effect persisted in a generation with no direct exposure, this would be considered as a transgenerational effect.¹²⁴ 

The increased body weight of the female offspring of pubescent males exposed to 10 and 20 mg per kg bw of BPA³⁴  confirmed the results of a previous study.³³  After the mature and pubescent F0 males were exposed to BPA, we observed a significantly reduced percentage of F1 pregnant females. In the case of pubescent males, the percentage of fertile males in the moderate BPA group was also drastically reduced. The reason for this may be the inability of sperm cells to fertilize eggs or preimplantation deaths of fertilized eggs. The diminished quality of sperm confirmed our earlier results where the germ cell motility was significantly reduced, while there was no significant reduction in the sperm count and quality parameters.^33,35 

Two multigenerational studies on rodents did not find any reproductive or developmental effects of low BPA doses.^78,126  Other studies did show such effects. Prenatal exposure to BPA as well as to its analogues – bisphenols E and S – induced subsequent reduction in the sperm count and/or motility in F3 males and altered and disrupted spermatogenic progression and gene expression in their testes.¹²⁷  Prenatal exposure to physiologically relevant doses of BPA and its analogues induced transgenerational adverse effects on male reproductive functions, probably due to the results from the abnormal expression patterns of DNA methyltransferases (DNMTs) and histone marks in the testes of F3 males suggest altered DNA and histone methylations that likely can be transgenerationally transferred to the offspring.¹²⁷  Karmakar et al. ¹²⁵  noted that even at the lowest-observed-adverse-effect level (LOAEL) dose, the testicular abnormalities and alterations in seminiferous epithelium staging persisted in F0–F2 generations, although the reduced total spermatogonia count was found only in F0. Abnormalities in the proportions of germ cells were observed until the F2 generation. A high frequency of apoptotic tubules in germ cells in the F0–F2 generations after the 6-week exposure of F0 males to BPA was observed.¹²⁵ 

In conclusion, our results showed some differences in the response of male germ cells to bisphenol A exposure between adult and pubescent males. The harmful effect induced in the gametes of pubescent males by bisphenol A was clearer, suggesting the higher susceptibility of germ cells of adolescent mammals. Results showed that exposure of males to BPA alone or in combination with irradiation for a full cycle of spermatogenesis may cause heritable changes transferable to subsequent generations, which lead to unsuccessful fertilization or preimplantation losses as well as to death of pups after birth. Such exposure may also diminish the sperm quality of the males of the F1 generation leading to unsuccessful fertilization and induce obesity in the F1 offspring of exposed males. Combined treatment mainly intensified the harmful effect induced by BPA in male germ cells. However, in pubescent males, protective effects of low doses of radiation on the DNA of male gametes were observed just after the end of exposure. Transgenerational effects on subsequent generations might involve genetic and epigenetic mechanisms.

Humans need to be aware that the health of their unborn children may be affected by exposure of parents to mutagenic agents, especially of males during their puberty. Limited use of products containing BPA, especially by children and teenagers, is strongly recommended for protection of the public and reproductive health of men in the future. Particularly dangerous is the reuse of BPA-containing bottles or food containers as well as heating up food inside them.