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Abstract

Bisphenol A (BPA), a carbon-based synthetic polymer compound, was newly classified as an environmental toxicant and an endocrine-disrupting chemical leading to abnormalities in cell proliferation, apoptosis, or migration that contributes to cancer development and progression. This study aims to evaluate the effect of the elevation of γ- radiation dose and BPA on the liver and ovaries of female rats. In this study, eighty female albino rats (130–150 g) were used in this work. Rats in this experiment received BPA in ethanol (50 mg/kg b. wt.) for 30 days, day after day, and in the irradiated groups, animals were administered BPA and then exposed to γ- radiation in doses (2, 4, and 6 Gy) one shot dose. Several members of the cytochrome family were examined. Exposure to γ-radiation and BPA showed an increase in cytochrome P450 and b5 fold change. Further, BPA and γ-radiation activate α and β estrogen receptors and also downregulate aromatase (CYT19) fold change. The current results also revealed that BPA and/or γ-radiation regulate the protein expression of the PI3K/Akt signaling pathway. The steroidogenic acute regulatory protein (StAR) appeared to be targeted by BPA and γ-radiation and its relative expression was elevated significantly by raising the γ-radiation dose. In conclusion, exposure to BPA, an endocrine-disrupting chemical, leads to marked toxicity. Additionally, toxicity is heightened by increasing the γ-radiation dose, either alone or in combination with BPA.

Graphical Abstract

Keywords

Bisphenol A gamma radiation liver toxicity PI3K/Akt ovary toxicity

Introduction

Bisphenol A (2, 2-bis-(4-hydroxyphenyl)-propane; BPA) is a monomer widely used to manufacture polycarbonate plastic and epoxy resins.^1,2 It can be found in many daily consumable items such as plastic bottles, beverage containers, medical devices, dental sealants, eyeglass lenses, PVC gloves, recycled paper, printing inks, consumer electronics, and the surface of internal water pipes.³BPA is a well-characterized endocrine-disrupting chemical (EDC)⁴ that can affect hormone synthesis, metabolism, and function.⁵ BPA is a derivative of diphenylmethane comprising two hydroxyphenyl groups that make its structure similar to estrogen,⁶ it is known as a xenoestrogen as it shows estrogenic actions by binding to estrogen receptors, and thereby, disrupts endocrinological roles in living organisms, including humans.¹ Pupo et al.⁷ reported that BPA activates the GPER/ERK1/2/EGFR signaling pathway in cancer cells by inducing target gene expression and coupling with the G protein receptor. Beyond the classical role played by the liver in detoxification, liver enzymes are responsible for glucuronidation (the main route of BPA metabolism) mediated by the UDP-glucuronosyltransferase (UGT) enzymes and sulfation by sulfotransferase,⁸ which produces BPA glucuronide, and BPA sulfate, respectively, both conjugated metabolites with little or no estrogenic activity. Ionizing radiation (IR) is one of the harmful external environmental factors. The living cells absorb IR that makes biological and chemical alterations,⁹ either directly by ionization and formation of DNA adduct or indirectly via water radiolysis and then generation of free radicals and reactive oxygen species (ROS), such as hydroxyl radical OH•, the superoxide anion (O2•−), nitric oxide (NO), hydrogen peroxide (H2O2)and peroxyl radicals ( HO2•) and causing cellular damage and attack all the cell constituents; DNA, proteins, and lipids^10,11 Exposure to BPA and different doses of γ-radiation as two environmental stressors is strongly related to liver and ovarian toxicities.

Aims of the current work

This study aims to examine the harmful role of different doses of γ-radiation combined with BPA on the liver and ovary of female rats.

Material and methods

Chemicals

Bisphenol A (BPA) from Sigma Chemical Co., Cairo, Egypt was obtained in the form of rutin hydrate. All other chemical substances and reagents used in this analysis were of analytical grade. BPA was dissolved in ethanol,³ and then complete the volume by corn oil (ethanol is 5% of the total volume).

Animals

Eighty female Wister rats (120–150 g) were used, got from the National Centre for Radiation Research and Technology (NCCRT), Cairo, Egypt. Rats were kept in an environmentally controlled clean-air room with standard temperature and maintained a 12 h light/dark cycle. They were permitted to adapt to the environmental conditions for 1 week before beginning the experiment and were fed on standard food pellets containing all nutritive elements and liberal water ad libitum. All animal procedures complied with the guidelines of the National Centre for Radiation Research and Technology, Egypt, and with guidance for the proper treatment and use of laboratory animals (NIH Publication No. 85–23, updated 1985), and managed by the animal facilities, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority (Serial No.21A/20).

Irradiation facility

Whole-body ionizing gamma-irradiation (IR) was carried out at the National Centre for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority, Cairo, Egypt, using Canadian Gamma Cell-40 biological irradiator (137 Caesium) produced by Canada Limited Atomic Energy, Ontario, Canada. During the time of exposure, the radiation dose was 0.61 Gy/min. The cumulative dose of radiation was 2.0, 4.0, and 6.0 Gy as a single dose.

Experimental design

Animals were randomly classified into eight groups (n = 10) as follows control(C); female rats were supplied distilled water by gastric intubation daily for 4 weeks. 2- Bisphenol A (BPA) positive control; rats received bisphenol A (50 mg/kg b.wt)for 30 days day after day, for 4 weeks (BPA) according to Pinafo et al³ and Kobroob et al.¹² 3- The two Gray irradiated group (R 2); rats were exposed to a single dose of one shot (2 Gy) γ-radiation.4- The four Gray irradiated group (R4); rats were exposed to a single dose of one shot (4 Gy) γ-radiation. 5-The six Gray irradiated group (6 Gy); Rats were irradiated with a single dose of one-shot γ-radiation (R6).6- BPA + R2 group; rats were orally administrated BPA as group 2 and then exposed to a single dose of IR as group 3. 7- BPA + R4 group; rats were orally treated with BPA as group 2 and then irradiated as group 4. 8-BPA + R6 group; Rats were orally treated with BPA as group 2 and then exposed to IR group 5. After radiation, rats fasted overnight. Intracardiac blood samples were collected, and serum was separated by centrifugation at 3000 r/min for 15 min and stored at −80°C until analysis. The ovary and liver tissues were immediately dissected, rinsed in ice-cold saline, plotted to dry, and weighed. Ovary and liver tissue’s left lobes were fixed in 10% formalin prepared in phosphate-buffered saline (PBS) histopathological examination. A weighted part of each ovary and liver was homogenized with ice-cooled PBS to prepare 20% w/v homogenate. The homogenate was centrifuged at 4000 r/min for 5 min, at 4°C. The aliquots were then kept at −80°C until use.

Biochemical investigation

Serum estrogen (ES), progesterone (PGS), Follicle-stimulating hormone (FSH), and Luteinizing hormone (LH) levels were studied according to standard methods using available commercial kits.

Quantitative real-time PCR

Real-time PCR (Promega, Madison, WI) was used to quantify cytochromeP4502E1 (CYT-P450 2E1), aromatase (CYT19), cytochrome b5 CYT-B5, and StAR expression using specific primers for each gene and β- actin as a housekeeping gene (Table 1). Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1 1.7 Sequence Detection Software from PE Biosystems (Foster City, CA). Relative expression was calculated using the comparative threshold cycle method for each target gene. Variations in the mRNA target content relative to the normalized glyceralde-hyde-3-phosphate dehydrogenase gene¹³

Table 1.

Primer sequences used for RT-PCR.^14,15

CYP P450-2E1	F:5- TACCCCATGAAGCAACCAGA-3
	R:5-CCTGCAGAAAATGCCTTGAA-3
Aromatase (CYP19)	F: 5- GCCTGTCGTGGACTTGGT-3
	R:5, - GGTAAATTCATTGGGCTTGG-3
StAR	F: 5 - GGGCATACTCAACAACCAG-3
	R:5- ACCTCCAGTCGGAACACC-3
Cytb5	F:5-AGTTTCCGGAGGCTTGTTTAACT
	R:5-GATGTCGGGCACTCTACAGATG
β-actin	F: 5, - TCGTGCGTGACATTAAAGAG-3
	R:5 - ATTGCCGATAGTGATGACCT-3

Western blot analysis

The protein expression of AKT (Cat. No.: ab65786, ∼60 kDa) and PI3k (Cat No: ab302958∼126 kDa) was protein assessed in the ovary and liver tissues homogenate. β-actin was used as a housekeeping arbitrary unit.

Histopathological examination

Liver and ovaries were collected from the different experimental groups and routinely processed. The paraffin-embedded blocks were sectioned at the 5-micron thickness and stained with Hematoxylin and Eosin¹⁶ for histopathological examination by a light microscope (Olympus BX50, Japan).

Grading of histopathological alterations

Histopathological alterations of the liver and ovaries were graded as (0) indicating no changes, (+), (++), (+++), and (++++) indicating mild, moderate, less severe, and severe changes, respectively¹⁷

Statistical analysis

The statistical package SPSS (Statistical Program for Social Science) version 20 was used to apply a one-way ANOVA test followed by a post hoc test for multiple comparisons. All data expressed SE (n = 6) and the difference between means was considered significant at p < .05.

Results

Exposure to ionizing radiation and BPA triggers toxicity in many tissues. The liver and ovaries are among the most sensitive tissues that have been affected. The present study showed a marked increase in FSH and LH hormone levels in rats administrated BPA to the respective control. Increasing the radiation dose leads to an elevation in both hormones, alone or combined with BPA (Figure 1(a) and (b)). Similarly, the estradiol level increased by increasing the radiation dose from 2 to 6 Gy compared to the control. There was a significant elevation in estradiol levels in groups that received BPA and were exposed to γ-radiation related to radiation groups (BP + R2, BP + R4, and BP + R6) (Figure 1(c)). Meanwhile, the study of progesterone levels revealed a marked rise in the BPA group linked to control, while there was no significant change between the radiation groups. A combination of BPA and γ-radiation showed a significant elevation in PGS levels compared to the corresponding radiation groups (Figure 1(d)).

Figure 1.

Gamma radiation and BPA impact on female hormones: (a) LH, (b) FSH, (c) estradiol, and (d) progesterone in examined rat groups. Each value represents the mean ± SE (n = 6). Values with different superscript letters are considered significantly different at p < .05.

For exploring the toxic effect of BPA and IR on the liver and ovarian tissues, mRNA expression of phase one monooxygenase enzymes CYT P450-2E1 was estimated (Figure 2). The data in this work revealed that CYT P450-2E1 mRNA expression was significantly elevated in the BPA group corresponding to the control group. Also, there was a potent increase in mRNA expression in rats exposed to radiation regarding the control and BPA group (R2, R4,&R6). Interestingly, by elevating the radiation dose combined with BPA, CYT P450-2E1mRNA expression increased (BP + R2, BP + R4, & BP + R6) in both tissues.

Figure 2.

Effect of different doses of gamma radiation and/or BPA in different female rat groups (a) ovary cytochrome p450-2E1 and (b)liver cytochrome p450-2E1. Each value represents the mean ± SE (n = 6). Values with different superscript letters are considered significantly different at p < .05.

CYT19 and CYTB5 are other members of the cytochrome P450 family, their mRNA expression is illustrated in Figure 3. They exhibited different attitudes CYTB5 m-RNA expression increased by radiation, while declined by BPA administration. Otherwise, aromatase mRNA expression was significantly diminished in the BPA group compared to control. Furthermore, there was a potent decrease in the group administrated BPA and exposed to IR compared to the BPA or IR alone.

Figure 3.

Impact of different doses of γ-radiation and BPA in different tested groups (a) cytochrome B 5 and (b) aromatase. Each value represents the mean ± SE (n = 6).). Values with different superscript letters are considered significantly different at p < .05.

The mRNA expression of StAR (steroidogenic acute regulatory protein) was calculated and represented in Figure 4. Administration of BPA triggered a significant increase in StAR mRNA expression (BPA other) compared to the control. Additionally, rats exposed to different doses of radiation either alone (R2, R4, & R6) or in combination with BPA (BP + R2, BP + R4, & BP + R6) showed a significant elevation regarding control.

Figure 4.

Different doses of γ-radiation and BPA influences StAR in different tested rat groups. Each value represents the mean ± SE (n = 6). Values with different superscript letters are considered significantly different at p < .05.

To discover the impact of increasing the dose of γ-radiation on the liver and ovary, AKT/PI3K pathway protein expression was investigated (Figure 5). Remarkably, there was harmony elevation in this pathway in both tissues in the BPA group compared to the control. Likewise, γ-radiation exposure showed a significant increase regarding corresponding control. The combination exposure to both BPA and IR exhibited a potent augmentation comparison to their corresponding R groups.

Figure 5.

Protein expression of AkTand PI3K in the ovary and liver tissues of different examined female rats. Each value represents the mean ± SE (n = 6). Values with different superscript letters are considered significantly different at p < .05.

Histopathological findings

Liver from the control negative group showed normal hepatic parenchyma with polygonal hepatic cells, normal blood sinusoids, central veins, and portal areas, (Figure 6(a)). In contrast, the liver from the BPA group revealed moderate vacuolar degenerated hepatocytes and congested central vein (Figure 6(b)). Furthermore, livers from the R2 exposed group showed mild hepatocyte swelling with cytoplasmic granulation (Figure 6(c)), moderate vacuolar degeneration was replaced with focal areas of necrosis was reported in the R4 exposed group (Figure 6(d)), and less severe vacuolar degeneration replaced with multifocal areas of necrosis were noted in the R6 exposed group (Figure 6(e)). Moreover, Bisphenol A and Radiation exposed groups showed variant damage degree in hepatic parenchyma, there were moderate, less severe, and severe vacuolar degenerated hepatocytes with focal, multifocal, and diffuse necrotic areas in the BPA + R2 group (Figure 6(f)), BPA + R4 exposed group (Figure 6(g)), and BPA + R6 exposed group (Figure 6(h)) respectively.

Figure 6.

Photomicrographs of liver from different experimental groups stained with H&E X400 showing: (a) Control negative group with normal polygonal hepatic cells, normal blood sinusoids, central veins, and portal areas (0), (b) BP intoxicated group with moderate vacuolar degenerated hepatocytes (*) and congested central vein (arrow) (++), (c) R2 exposed group with mild hepatocytes swelling and cytoplasmic granulation (*) (+), (d) R4 exposed group with Moderate vacuolar degeneration replaced with focal areas of necrosis (arrow) (++), (e) R6 exposed group with less severe vacuolar degeneration replaced with multi focal areas of necrosis (arrow) (+++). (f) BP + R2 exposed group with Moderate vacuolar degeneration replaced with focal areas of necrosis (arrow) (++), (g) Bp + R4 exposed group with less severe vacuolar degeneration replaced with multi focal areas of necrosis (arrow) (+++), (h) BP + R6 exposed group with Severe Vacuolarly degenerated hepatocytes replaced with diffuse areas of necrosis (arrow) (++++).

Ovaries from control negative group showed normal ovarian follicles and interstitial tissue (Figure 7(a)), while ovaries from Bisphenol A intoxicated group revealed moderate atretic follicles with fragmentation of granulose cell layer with interstitial tissue reaction and congested blood vessels (Figure 7(b)). Ovaries from R2 exposed group showed mild atretic follicles with nuclear pyknosis (Figure 7(c)), Moderate atretic follicles with fragmentation of granulose cell layer and focal areas of clusters of pale interstitial cells interspersed among follicles were reported in R4 exposed group (Figure 7(d)), and less severe atretic follicles with fragmentation of granulose cell layer and multi focal areas of clusters of pale interstitial cells interspersed among follicles were reported in R6 exposed group (Figure 7(e)). Bisphenol A and Radiation exposed groups showed variant damage degree in ovaries, there were moderate, less severe, and severe ovarian damage with focal, multifocal, and diffuse areas of clusters of pale interstitial cells interspersed among follicles in Bisphenol A + R2 exposed group (Figure 7(f)), Bisphenol A + R4 exposed group (Figure 7(g)), and Bisphenol A + R6 exposed group (Figure 7(h)) respectively.

Figure 7.

Photomicrographs of ovaries from different experimental groups stained with H&E X200 showing: (a) control negative group with normal ovarian follicles and interstitial tissue (0), (b) BP intoxicated group with moderate atretic follicles with fragmentation of granulose cell layer and interstitial tissue reaction (*), and congested blood vessels (arrow) (++), (c) R2 exposed group with mild atretic follicles and nuclear pyknosis (*) (+), (d) R4 exposed group with Moderate atretic follicles and fragmentation of granulose cell layer, and focal areas of clusters of pale interstitial cells interspersed among follicles (*) (++), (e) R6 exposed group with less severe atretic follicles and fragmentation of granulose cell layer, and multi focal areas of clusters of pale interstitial cells interspersed among follicles (*) (+++). (f) BP + R2 exposed group with moderate atretic follicles with fragmentation of granulose cell layer and congested blood vessels (arrow) (++), (g) BP + R4 exposed group with less severe atretic follicles and fragmentation of granulose cell layer, and multi focal areas of clusters of pale interstitial cells interspersed among follicles (*) (+++), (h) BP + R6 exposed group with Severe atretic follicles with fragmentation of granulose cell layer and diffuse areas of interspersed clusters of pale interstitial cells among follicles (*) (++++).

Histopathological lesion scoring

All the recorded lesions in the liver and ovaries were scored as shown in Table 2.

Table 2.

Histopathological criteria and grading of the lesions found in liver.

Liver	Grade
Normal parenchyma with no histological changes	(0)
Mild hepatocytes swelling with cytoplasmic granulation	(+)
Moderate vacuolarly degenerated hepatocytes and congested central vein and/or replaced with focal areas of necrosis	(++)
Less severe vacuolarly degenerated hepatocytes replaced with multi-focal areas of necrosis	(+++)
Severe vacuolarly degenerated hepatocytes replaced with diffuse areas of necrosis	(++++)

Discussion

Endocrine-disrupting (Tables 3) chemicals (EDCs) can disturb hormone synthesis, metabolism, and function. Exposure to EDCs happens by ingestion of water, food, and dust, inhalation of gases, or by dermal absorption. Subsequently, they accumulate in fat tissue for a long time, and then their metabolites harm the organism.⁵ Bisphenol A (BPA) is one of these compounds that used in manufacturing for decades.¹⁸ Steroidogenesis is the process in which the wanted forms of steroids are produced by the transformation of other steroids including progesterone, androgens, estrogens, corticoids, cortisol, and aldosterone.¹⁹ Many researchers have found that the level of γ-radiation exposure is directly proportional to the health risks associated with it. Elbakry²⁰ and his team have highlighted that high doses of ionizing radiation can trigger a state of oxidative stress, which impairs various essential biochemical mechanisms. Additionally, the combination of BPA and γ-radiation can lead to significant harmful changes in antioxidant levels. Furthermore, the combination of BPA and R can interfere with cytoplasmic Erα and ERβ nuclear translocation in the brain and cause neurodegenerative changes.¹¹ In this study, BPA impact was investigated either alone or combined with γ- radiation; rats administrated BPA showed a significant elevation in female hormones (FSH, LH, ES, and PGS) as the unconjugated BPA was released. In the liver, phase II conjugation of BPA occurred through the transformation to BPA-Glucoronide (BPA-G) by uridine-5-diphospho-glucouronosyl transferase (UGTs). The UGT enzyme abnormalities cause the increase in the unconjugated BPA levels which is responsible for the vital disturbances in many pathways causing moderately acute toxicity.²¹ Moreover, the gonadotrophin-resealing hormone release (GnRH) from the hypothalamus stimulates the LH and FSH release from the anterior segment of the pituitary. These gonadotrophs at the ovarian level, act on ovarian follicles to release PGS and ES. E2 is the estrogen responsible for LH and GnRH production by a negative feedback action; BPA can mimic E2 action and bind to ER and then disturb the hypothalamus and pituitary functions.²² Patel et al²³ documented BPA-induced disruption in steroidogenesis. In addition, BPA significantly increased PGS levels by increasing cytochrome P450 steroidogenic enzyme as 17-a hydroxylase (Cyp17a1), cholesterol side chain cleavage enzyme (Cyp11a1).⁶ Based on data observation, the increase of γ- radiation dose (2,4–6 Gy) causes hypersecretion of FSH and LH hormones that may be a result of the hypothalamic-pituitary axis activation.²⁴ This result was consistent with El-Tawill et al²⁵ who reported that FSH and LH levels increased by γ- radiation exposure.

Table 3.

Grading of histopathological lesions in the liver and ovaries in all groups.

Organs		Grades of the lesions
Organs		0 (Negative)	+ (Mild)	++ (Moderate)	+++ (Less Severe)	++++ (Severe)
Control group	Liver	√
Control group	Ovaries	√
Bp group	Liver			√
Bp group	Ovaries			√
R2 group	Liver		√
R2 group	Ovaries		√
R4 group	Liver			√
R4 group	Ovaries			√
R6 group	Liver				√
R6 group	Ovaries				√
BpR2 group	Liver			√
BpR2 group	Ovaries
BpR4 Group	Liver				√
BpR4 Group	Ovaries				√
BpR6 group	Liver					√
BpR6 group	Ovaries					√

The Cytochrome superfamily is a set of membrane-bounded hemoproteins, that induce a series of crucial biological reactions in all living organisms.²⁶ CYP450s have central roles in some endogenous compounds’ biosynthesis; like steroids, fatty acids, vitamins, and eicosanoids²⁷. Besides, they play a vital role in xenobiotics detoxification such as drugs.²⁸ On the other hand, they have a pivotal function in the bioactivation of many carcinogens.²⁹ The CYP450 family is categorized into two groups depending on their ability to substrate recognition. The first group stimulates specific reactions on specific endogenous substrates, and the other contains enzymes evolved in xenobiotic metabolism.³⁰ In this study, several members of the cytochrome family have been investigated. The mRNA expression of CYP450 2E1 in the tissues of the liver and ovary showed a marked increase in the BPA group. As well-known, BPA metabolized to 2,2-bis(4-hydroxyphenyl) propanol, o-hydroxy bisphenol, or bisphenol-o-quinone products by P450 oxidative action.³¹ Also, Wang et al³² emphasized that BPA toxicity is mitigated by the action of CYP450. Consequently, the role of γ-radiation was understandable by the significant elevation of P450 2E1 in both hepatic and ovarian tissues after the radiation exposure, which is in agreement with Chung et al,³³ who documented that relatively high irradiation-induced CYP2E1in the liver with an associated increase in its mRNA. Further, Chandra et al³⁴ stated that the induced activity of the cytochrome P450 system might aid in metabolizing the chemically reactive species produced from free-radical reactions. Interestingly, this significant elevation was confirmed by the action of sex hormones explored, this is confirmed by Lee et al³⁵ who stated that sex and growth hormones regulate the drug-metabolizing enzyme expression including CYP450. There is a complex relationship between CYP450 and CYb5; CYb5 can stimulate P450, not affect or even prohibit its action,²⁷ so in this study, P450 was not affected by CYb5 (Figures 2 and 3). Furthermore, the γ-radiation effect was clear in Figure 3(a) that it elevates CYb5 activity either alone or with BPA. Previously, it was documented by Rendic et al³⁶ that the γ-radiation increases CYb5 activity in a dose-dependent manner (up to 6 Gy). Aromatase (CYP19) is another member of the CYP450 family that stimulates the aromatization of androgens into estrogen which plays a crucial role in reproductive development.²⁴ The present data demonstrate that BPA administration declines aromatase mRNA levels in the ovary, which may be due to the activation of PARP γ which downregulates aromatase enzyme through NF-κB inhibition.³⁷ This results in harmony with Huang and Leung³⁸ who reported that BPA downregulates the mRNA expression of CYP19 in the placental cell. Looking at our data, CYP19 expression was also declined by exposure to γ-radiation, maybe through decreasing aromatase promoter II mRNA expression and COX-1 and COX-2 expression.³⁹ El Bakary et al⁴⁰ also reported that low doses of radiation can suppress CYP19 mRNA expression in breast cancer-bearing mice. Mukhopadhyay et al²¹ suggested that exposure to BPA CYP19A1 downregulates that affect the conversion of androgen to E2.

Studying StAR protein production is very important because it represents a rate-limiting step in steroidogenesis. The data of this study revealed that BPA elevates StAR mRNA expression.

Zhou¹⁸ and his colleagues demonstrated that BPA caused an elevation in theca-interstitial cell testosterone levels, which was related to increased pregnenolone and E2 concentrations and increased expression of P450 c17, P450 from, P450scc, and StAR mRNA. Mukhopadhyay et al²¹ emphasized that BPA administration causes an elevation in StAR protein that promotes progesterone production.

PI3K/Akt signaling is encompassed in the cell function regulation including cell proliferation, cell survival, and cell cycle progression.⁴¹ Ionizing radiation (IR) can induce the abnormal activation of numerous signaling pathways.⁴² Based on the aforementioned data, the current results revealed a remarkable increase in PI3K/Akt signaling in hepatic and ovarian tissues by BPA or γ -irradiation administration. The previous study stated that BPA activates primordial follicle progression mainly through the PI3K/Akt pathway.⁴³ Additionally, Xia et al⁴⁴ documented that BPA induces ROS production which stimulates the HIF-1a/VEGF/PI3K/AKT axis to promote the migration and invasion of human colon cancer cells. The estrogen elevation obtained from the present study also can induce the PI3K/Akt pathway, this is in the same context as⁴¹ who indicated that estrogen stimulates the growth signal PI3K/Akt pathway through the mediation of its specific receptor (ERα) in breast cancer cells. Moreover, current results manifested that exposure to γ -irradiation stimulates PI3K/Akt signaling. Many previous studies documented that γ -irradiation induces both MAPK and PI3K/AKT pathways except ERK.⁴⁵ Furthermore, Zingg et al⁴⁶ postulated that the elevation of the PI3K/Akt signaling pathway by radiation occurring via ErbB receptor stimulation in tumor cells is activated through growth factor- and radiation-induced ErbB receptor stimulation. Additionally, Suman et al⁴⁷ suggested that radiation increases PI3K/Akt through the insulin growth factor/insulin receptor substrate pathway (IGF1/IRS1).

Conclusion

Although assessing every combination of stressors is unrealistic, we should not ignore the interaction of physical (γ-radiation) and chemical (bisphenol A) pollutants that share similar modes of action. For these reasons, these findings could provide support for enhancing knowledge and developing in silico models. Moreover, the current study reveals the risks resulting from exposure to radiation while chronically exposed to a pollutant like BPA. Both stressors can cause disruptions in female hormone levels and make changes in many members of the cytochrome family. Furthermore, they trigger the PI3K/Akt signaling pathway in hepatic and ovarian tissues, which can prevent apoptosis and potentially lead to the development of cancer. Further research is necessary to fully comprehend their combined impact on apoptosis prevention and cancer development.

Footnotes

Author contributions

A. A. Hassan (corresponding author): Validation. Methodology. Writing – review & editing.

S. Z. Mansour: Conceptualization. Supervision. Resources

S.S. Abdelgayed: Visualization.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Asmaa A Hassan

Data Availability Statement

Data is available as a request.

References

Asahi

Kamo

Baba

, et al. Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes. Life Sci 2010; 87: 431–438.

Mathew

Mahalingaiah

. Do prenatal exposures pose a real threat to ovarian function? Bisphenol A as a case study. Reproduction 2019; 157: R143–R157.

Pinafo

Benedetti

Gaiotte

, et al. Effects of Bauhinia forficata on glycaemia, lipid profile, hepatic glycogen content and oxidative stress in rats exposed to Bisphenol A. Toxicol Rep 2019; 6: 244–252. DOI: 10.1016/j.toxrep.2019.03.001.

Zeng

Chen

Liu

, et al. Bisphenol A analogues in associations with serum hormone levels among reproductive-aged Chinese men. Environ Int 2022; 167: 107446. DOI: 10.1016/j.envint.2022.

Pivonello

Muscogiuri

Nardone

, et al. Bisphenol A: an emerging threat to female fertility. Reprod Biol Endocrinol 2020; 18: 22. DOI: 10.1186/s12958-019-0558-8.

Khan

Correia

Adiga

, et al. A comprehensive review on the carcinogenic potential of bisphenol A: clues and evidence. Environ Sci Pollut Res Int 2021; 28: 19643-19663. DOI. 10.1007/s11356-021-13071-w.

Pupo

Pisano

Lappano

, et al. Bisphenol A Induces gene expression changes and proliferative effects through GPER in breast cancer cells and cancer-associated fibroblasts. Environ Health Perspect 2012; 120: 1177–1182. DOI: 10.1289/ehp.1104526

Iwano

Inoue

Nishikawa

, et al. Biotransformation of Bisphenol A and its adverse effects on the next generation. In: Ahmed

(ed) Endocrine disruptors. InTech, 2018.

Mozdarani

Taheri

Haeri

, et al. Assessment of the radioprotective effects of amifostine on human lymphocytes irradiated in vitro by gamma-rays using cytokinesis-blocked micronucleus assay. Int J Radiat Res 2007; 5: 9–16.

10.

Santos

Tsutsumi

Vieira

, et al. Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with (60) Co gamma-radiation. Mutat Res Genet Toxicol Environ Mutagen 2014; 762: 17-23. DOI: 10.1016/j.mrgentox.2013.11.004.

11.

Abdel-Rafei

Thabet

. Modulatory effect of methylsulfonylmethane against BPA/γ-radiation induced neurodegenerative alterations in rats: influence of TREM-2/DAP-12/Syk pathway. Life Sci 2020; 260: 118410. DOI: 10.1016/j.lfs.2020.118410.

12.

Kobroob

Peerapanyasut

Chattipakorn

, et al. Damaging effects of Bisphenol A on the kidney and the protection by melatonin: emerging evidences from in vivo and in vitro studies. Oxid Med Cell Longev 2018; 2018: 3082438. DOI: 10.1155/2018/3082438.

13.

Livak

Schmittgen

. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4): 402–408.

14.

Havelock

Rainey

Bradshaw

, et al. The post-menopausal ovary displays a unique pattern of steroidogenic enzyme expression. Hum Reprod 2006; 21(1): 309–317. DOI: 10.1093/humrep/dei373.

15.

Marghani

Ateya

Saleh

, et al. Bisphenol A down-regulates the mRNA expression of steroidogenic genes and induces histopathological changes in testes of rats. J Vet Healthc 2018; 1: 32–44. DOI: 10.14302/issn.2575-1212.jvhc-18-.2012

16.

Bancroft

Stevans

Turner

. Theory and practice of histological techniques. 4th ed. Edinburgh: Churchill Livingstone, 2013.

17.

Arsad

Esa

Hamza

. Histopathologic changes in liver and kidneys tissues from male Sprague Dawley rats treated with Rhaphidophora decursiva (Roxb.) Schott extract. J Cytol Histol S4 2014: 1

18.

Czubacka

Wielgomas

Klimowska

, et al. Urinary bisphenol A concentrations and parameters of ovarian reserve among women from a fertility clinic. Int J Environ Res Public Health 2021; 18: 8041. DOI: 10.3390/ijerph18158041.

19.

Kim

Han

Kim

, et al. Bisphenol A-induced aromatase activation is mediated by cyclooxygenase-2 up-regulation in rat testicular Leydig cells. Toxicol Lett 2010; 193: 200–208. DOI: 10.1016/j.toxlet.2010.01.011.

20.

Elbakry

MMM

Mansour

Helal

, et al. Nattokinase attenuates bisphenol A or gamma irradiation-mediated hepatic and neural toxicity by activation of Nrf2 and suppression of inflammatory mediators in rats. Environ Sci Pollut Res Int 2022; 29(49): 75086–75100. DOI: 10.1007/s11356-022-21126-9.

21.

Mukhopadhyay

Prabhu

Kabekkodu

, et al. Review on bisphenol A and the risk of polycystic ovarian syndrome: an insight from endocrine and gene expression. Environ Sci Pollut Res Int 2022; 29(22): 32631–32650. DOI: 10.1007/s11356-022-19244-5.

22.

Patel

Zhou

Rattan

, et al. Effects of endocrine-disrupting chemicals on the ovary. Biol Reprod 2015; 93(20): 1–9.

23.

Zhou

Liu

Liao

, et al. Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells. Mol Cell Endocrinol 2008; 13(283): 12–18. DOI: 10.1016/j.mce.2007.10.010.

24.

Dueland

Guren

Olsen

, et al. Radiation therapy induced changes in male sex hormone levels in rectal cancer patients. Radiother Oncol 2003; 68(3): 249–253. DOI: 10.1016/s0167-8140(03)00120-8.

25.

El-Twill

Fatih

Montaser

, et al. Antioxidant and anti infertility potency of pomegranate (punica granatum L.) in Î³ irradiated rats. Egyptian Journal of Radiation Sciences and Applications 2020; 33(2): 133–142. 10.21608/ejrsa.2021.52462.

26.

Liu

Zhang

, et al. Characteristics and roles of cytochrome b5 in cytochrome P450-mediated oxidative reactions in Locusta migratoria. J Integr Agric 2020; 19(6): 1512–1521. DOI: 10.1016/S2095-3119(19)62827-3.

27.

Bart

Scott

. Structural and functional effects of cytochrome b₅ interactions with human cytochrome P450 enzymes. J Biol Chem 2017; 292(292): 20818–20833. DOI: 10.1074/jbc.RA117.000220.

28.

Esteves

Rueff

Kranendonk

. The central role of cytochrome P450 in xenobiotic metabolism—a brief review on a fascinating enzyme family. J Xenobiot 2021; 11: 94–114. DOI: 10.3390/jox11030007.

29.

Reed

Mrizova

Barta

, et al. Cytochrome b ₅ impacts on cytochrome P450-mediated metabolism of benzo[a]pyrene and its DNA adduct formation: studies in hepatic cytochrome b ₅/P450 reductase null (HBRN) mice. Arch Toxicol 2018; 92(4): 1625–1638. DOI: 10.1007/s00204-018-2162-7.

30.

Di Nardo

Zhang

Marcelli

, et al. Molecular and structural evolution of cytochrome P450 aromatase. Int J Mol Sci 2021; 22: 631. DOI: 10.3390/ijms22020631.

31.

Nakamura

Tezuka

Ushiyama

, et al. Ipso substitution of bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of estrogenic activity. Toxicol Lett 2011; 203(203): 92–95. DOI: 10.1016/j.toxlet.2011.03.010.

32.

Wang

Qin

, et al. Bisphenol A degradation pathway and associated metabolic networks in Escherichia coli harboring the gene encoding CYP450. J Hazard Mater 2020; 388: 121737. DOI: 10.1016/j.jhazmat.2019.121737.

33.

Chung

Kim

Lee

, et al. Mitochondrial dysfunction by γ-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver. Toxicology 2001; 161: 79–91. DOI: 10.1016/S0300-483X(01)00332-8.

34.

Chandra

Kale

. Influence of gamma-rays on the mouse liver cytochrome P450 system and its modulation by phenothiazine drugs. Int J Radiat Biol 1999; 75(3): 335-349. DOI: 10.1080/095530099140519.

35.

Lee

Yun

, et al. Expression of hepatic and ovarian cytochrome P450 during estrous cycle in rats. Arch Toxicol 2012; 86: 75–85. DOI: 10.1007/s00204-011-0730-1.

36.

Rendic

Guengerich

. Summary of information on the effects of ionizing and non-ionizing radiation on cytochrome P450 and other drug metabolizing enzymes and transporters. Curr Drug Metab 2012; 13(6): 787–814. DOI: 10.2174/138920012800840356.

37.

Kwintkiewicz

Nishi

Yanase

, et al. Peroxisome proliferator-activated receptor-gamma mediates bisphenol A inhibition of FSH-stimulated IGF-1, aromatase, and estradiol in human granulosa cells. Environ Health Perspect 2010; 118: 400–406. DOI: 10.1289/ehp.0901161.

38.

Huang

Leung

. Bisphenol A downregulates CYP19 transcription in JEG-3 cells. Toxicol Lett 2009; 189(3): 248–252.

39.

González-González

García Nieto

González

, et al. Melatonin modulation of radiation and chemotherapeutics-induced changes on differentiation of breast fibroblasts. Int J Mol Sci 2019; 13(20): 3935. DOI: 10.3390/ijms20163935.

40.

Bakary

Azab

Hanafy

Calcitriol revises aromatase gene expression in ehrlich solid tumor bearing mice exposed to low dose gamma radiation. Cancer Research and Cellular Therapeutics 2021; 5(1). Doi:10.31579/2640-1053/074.

41.

Lee

Park

, et al. Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells. Biochem Biophys Res Commun 2005; 336: 1221–1226.

42.

Liu

Zhao

, et al. 1,8-cineole alleviates bisphenol A-induced apoptosis and necroptosis in bursa of Fabricius in chicken through regulating oxidative stress and PI3K/AKT pathway. Ecotoxicol Environ Saf 2021; 226: 112877. DOI: 10.1016/j.ecoenv.2021.112877.

43.

Zhao

Sun

, et al. Exposure to Bisphenol A at physiological concentrations observed in Chinese children promotes primordial follicle growth through the PI3K/Akt pathway in an ovarian culture system. Toxicol Vitro 2014; 28(8): 1424–1429. DOI: 10.1016/j.tiv.2014.07.009.

44.

Xia

Guo

Zhang

, et al. Bisphenol A promotes the progression of colon cancer through dual-targeting of NADPH oxidase and mitochondrial electron-transport chain to produce ROS and activating HIF-1a/VEGF/PI3K/AKT Axis. Front Endocrinol 2022; 13: 933051. DOI: 10.3389/fendo.2022.933051.

45.

Park

Kim

Shim

, et al. Radiation-activated PI3K/AKT pathway promotes the induction of cancer stem-like cells via the upregulation of SOX2 in colorectal cancer. Cells 2021; 10(10): 135. DOI: 10.3390/cells10010135.

46.

Zingg

Riesterer

Fabbro

, et al. Pathway by ionizing radiation in tumor and primary endothelial cells. Cancer Res 2004; 1(64): 5398–5406. DOI: 10.1158/0008-5472.CAN-03-3369.

47.

Suman

Kallakury

Fornace

, et al. Protracted upregulation of leptin and IGF1 is associated with activation of PI3K/Akt and JAK2 pathway in mouse intestine after ionizing radiation exposure. Int J Biol Sci 2015; 20(11): 274–283. DOI: 10.7150/ijbs.10684.

Liver and ovarian toxicities boosted by bisphenol and gamma radiation in female albino rats

Abstract

Keywords

Introduction

Aims of the current work

Material and methods

Chemicals

Animals

Irradiation facility

Experimental design

Biochemical investigation

Quantitative real-time PCR

Western blot analysis

Histopathological examination

Grading of histopathological alterations

Statistical analysis

Results

Histopathological findings

Histopathological lesion scoring

Discussion

Conclusion

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

Data Availability Statement

References