Plausible effect of hesperetin and nano-hesperetin against bisphenol-A induced hepatoxicity in a rat model

1 Introduction

Natural or synthetic compounds that adversely impact the endocrine system by causing a disarray in the endogenous hormone production and functions have been termed as xenoestrogens or endocrine disruptors (Schug et al., 2016, Ma et al., 2019). Bisphenol A (BPA) is one such xenoestrogen, being globally used in the manufacture various polymers; epoxy resins, plastics etc. From 2016 through 2022, a gross annual growth rate of about 4.8 % has been recorded. The wide range of its use in polycarbonate plastics and epoxy resins which are being used in every industry was also cited as a reason for the output expansion to 10.7 million metric tons by 2020. In 2022, it is predicted that there will be a USD 22.49 billion global demand for BPA (Manzoor et al., 2022). The substance is prevalent in all environmental compartments (air, land, and water), which expands the channels via which people might be exposed to it (Meli et al., 2020). Asia Pacific accounts for about 52 % of the global BPA market, with the United States and Europe producing the remaining 36 % (Almeida et al., 2018). Due to this, it has a plethora of applications in consumer products used in everyday life (Vasiljevic and Harner, 2021, Xing et al., 2022). Additionally, BPA is a common constituent in several consumer products such as food and drinking bottles or cans containers that leads to its leaching out at under high temperatures and acidic or basic conditions adding to its human exposure (Elswefy et al., 2016). Since BPA has a hormone-like feature, it may lead to impaired cellular responses associated with oxidative stress and inflammations that trigger the immune system, which are key elements in its pathogenesis (Jang et al., 2020). The rising incidence of several types of cancers has been associated with BPA exposure (Manzoor et al., 2022). The enhanced estrogenic activity reveals the carcinogenic molecular action of BPA, according to several prior in vivo investigations (such as mice, rats, etc.) (Rubin, 2011). According to a recent study, BPA exposure decreased the amount of the protein Nrf2/Keap1 in male mice more than in female mice, making male mice more vulnerable to the hepatic oxidative stress caused by BPA (Müller et al., 2018).

Additionally, the plasma and colon tissues of BPA-exposed mice had increased amounts of reactive oxygen species (ROS and proinflammatory cytokines (Wang et al., 2021a, 2021b).

As the production and consumption of products containing BPA continues to grow with a concomitant environmental exposure, natural compounds such as flavonoids with a potent antioxidant status can alleviate the BPA toxicity. Several studies have been reviewed and it has been reported that the flavonoids could be potential BPA-detoxifying agents attributed to their wide spectrum of pharmacological benefits such as antioxidant activity, anti-inflammatory, hepatoprotective, anti-apoptotic-modulatory, and their ability to activate the immune system (Amjad et al., 2020). The biological activity of flavonoids is due to functional hydroxyl groups and the ability to catalyze electron transport and scavenge free radicals (Yao et al., 2004, Rahal et al., 2014, Khan et al., 2018). Hesperetin (3′,5,7-Trihydroxy-4′-methoxyflavanone) is one of such flavonoid and is a potent antioxidant in citrus fruits. It belongs to the flavanone class of flavonoids and is extracted mainly from peel of citrus fruits. Several studies have reported the efficacy of hesperetin against certain pathological conditions, exhibiting its anti-inflammatory and antioxidant properties (Khan et al., 2020). However, despite its proven pharmacological actions in humans, its poor physicochemical and biopharmaceutical properties have restricted its status to be recognized, which is mainly due to its limited oral bioavailability from conventional dosage forms (Pollard et al., 2006, Ratnam et al., 2006, Yang et al., 2012). However, nanotechnology has been a promising approach in the therapeutic use of phytochemicals as it enhances the bioavailability of natural antioxidants which is the current trend and forefront of research in nanomedicine. There has been a plethora of studies on the use of natural plant based bioactive compounds in the fabrication of nanoparticles either in conjugation with metals or in pure form (Yadav et al., 2020; Liu et al., 2023). The core concept of green synthesis is the redox reaction, in which an organism's constituent parts or its extract convert metal ions to stable nanoparticles. In addition, the process of reducing particles to the nanoscale, known as “nanonization,” increases the surface area, which raises its saturation solubility and rate of dissolution, subsequently leading to enhanced bioavailability. Several modes of nanonization of phytochemicals have been reported along with their efficacious therapeutic applications (Ghosh et al., 2022; Kapoor-Narula and Lenka, 2022).

Moreover, biogenic synthesis of nanoparticles; based on using microorganisms and phytochemicals; also, being non-metallic provide an environmentally benign, reliable, and sustainable mode of synthesis. In order to enhance the antioxidant activity and provide a targeted distribution of the bio constituents that otherwise exhibit poor cell permeability and internalization, nanoparticles functionalized with natural antioxidants act as the carriers for these phytocompounds (Khalil et al., 2019). There have been several studies on nano formulations of hesperidin and hesperitin, the major bioflavonoids in citrus fruits (Kheradmand et al., 2018,Elmoghayer et al., 2023; Stanisic et al., 2020; Simão et al., 2020). After being nanonized, hesperidin nanoparticles were used to create stable nano emulsions to be used for topical application as a face cream (Stanisic et al., 2020). Furthermore, hesperitin-loaded nano lipid carriers demonstrated remarkable in vitro cytotoxic effect of on glioblastoma cells (Simão et al., 2020). Hence, the present study evaluates the antioxidative efficacy of hesperetin, and it is nano-formulation against bisphenol-A-induced hepatotoxicity in a male rat model. The process of nanonization intended on keeping the essence of green credentials and includes no metallic component and chemical surfactants/ stabilizers. The mode of ‘green synthesis’ employed was granted a patent in 2016 (Elobeid et al., 2016).

2 Materials and methods

3 Results

3.1

3.1 Characterization of hesperetin nanoparticles

3.1.1

3.1.1 Size and morphology

As shown in Fig. 1 the average size of the Hst nanoparticle was 139.9 ± 43.40 nm with a poly-dispersity index (PDI) of 0.362, observed as a single peak.

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Fig. 1

Particle size distribution of the nano-hesperetin.

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Both TEM and SEM microscopy were used to determine the morphology of nano-Hst as shown in Fig. 2. The micrographs display agglomerated particles that are predominantly spherical and rod-shaped and have distinct varied morphotype. The spherical nanoparticles had an average diameter of 69 nm (Fig. 2A, B, C and D).

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Fig. 2

A and B show the micrographs (TEM), C,D and E show the scanning electron microscope (SEM) of the nanoparticles of hesperetin.

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3.1.2

3.1.2 X-ray diffractometer (XRD)

The X-ray diffractograms of the bulk Hst and nano Hst illustrated that the intensity of the peaks was less in nano-Hst, which indicates an amorphous structure of nanoparticles. Furthermore, since the peaks observed for both the bulk and the nano- Hst were at the same position which is attributed to the similar chemical composition of the two samples (Fig. 3).

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Fig. 3

X-ray diffractograms of the bulk and nano-hesperetin.

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3.1.3

3.1.3 Fourier Transform Infrared spectroscopy (FTIR)

According to Fig. 4, the FTIR spectra identified the potential biomolecules of bulk and nano-Hst. The bulk and nano-Hst spectra were remarkably similar, indicating that the nano scaling process did not alter the chemical structure of the compound. Noticeable IR bands were observed for the bulk-Hst at 3497.47 cm⁻¹, 1639.11 cm⁻¹, 1583.13 cm⁻¹, 1305.11 cm⁻¹, 1175.25 cm⁻¹, 818.51 cm⁻¹, and 742.30 cm⁻¹. Identical bands were illustrated for the nano-Hst. In the bulk-Hst spectra, the strong and broad band in the 3550–3200 cm⁻¹ area corresponded to hydrogen-bonded O—H stretching identified alcohol compound. The band at 1648–1638 cm⁻¹ indicate alkene compound with strong C⚌C stretching. Further, peaks between 1600 cm⁻¹ and 1585 cm⁻¹ were indicate C—C stretch were identified as aromatics. The band at 1310–1250 cm⁻¹ with strong C—O stretching was attributed to aromatic ester compound. The band at 1205–1124 cm⁻¹ identified a strong C—O stretching with tertiary alcohol compound. Finally, the bands at 840–790 cm⁻¹ was ascribed to alkene with medium C⚌C bending.

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Fig. 4

FTIR spectra of the bulk and nano-hesperetin.

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3.2

3.2 Bioassays

3.2.1

3.2.1 Concentration of pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α)

In comparison to the control group (42.1 ± 0.6 pg/ml), exposure to BPA significantly (p ≤ 0.05) increased serum levels of IL-6 (56.5 ± 0.3 pg/ml). Both the bulk Hst treatment (45.8 ± 0.7 pg/ml) and the nano-Hst treatment (41.3 ± 2.9 pg/ml) considerably (p ≤ 0.05) decreased the concentrations. Between the control group and the group that received the nano-Hst, there was no discernible change (Fig. 5A). In addition, the serum levels of IL-8 considerably (p ≤ 0.05) increased (52.6 ± 2.5 pg/ml) on exposure to BPA. The serum concentrations of IL-8 were considerably (p ≤ 0.05) decreased with co-administration of both bulk Hst (39.3 ± 0.3 pg/ml) and nano- Hst (37.8 ± 0.4 pg/ml)(Fig. 5B). Additionally, IL-1β was significantly (p ≤ 0.05) elevated on BPA exposure (6.9 ± 0.2 pg/ml). Co-administration of both bulk Hst (4.84 ± 0.192 pg/ml) and nano hesperetin (3.4167 ± 0.0998 pg/ml) showed a significant (p ≤ 0.05) reduction in the concentrations. Nevertheless, nano-Hst was more effective (Fig. 5C). Significantly higher TNF-α levels (111.7 ± 0.7 pg/ml) were observed as compared to the control (99.9 ± 0.7 pg/ml).Co-administration of both bulk Hst (105.5 ± 2.1 pg/ml) and nano- Hst (102.7 ± 1.0 pg/ml) significantly (p ≤ 0.05) declined the TNF-α concentrations (Fig. 5D).Among the treatments, however, there was no discernible difference observed.

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Fig. 5

Mean (±SE) the concentration levels of (A) IL-6 (pg/ml), (B) IL-8 (pg/ml), (C) IL-1β (pg/ml), (D) TNF-α (pg/ml) in serum of rats exposed to BPA and treated with hesperetin (Hst) and nano hesperetin (NHst). Different letters indicate a significant (p ≤ 0.05) difference between the experimental groups.

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3.2.2

3.2.2 mRNA expression of nuclear factor- erythroid 2 (NFE2) -related factor 2 (Nrf2) in liver

A significant fold change in the expression of hepatic mRNA of Nrf-2 in was observed on BPA exposure in comparison to the control. Treatment with nano- Hst (0.6 ± 0.1) and only nano- Hst (0.7 ± 0.1) significantly (p ≤ 0.05) downregulated the mRNA expression of Nrf2 compared to BPA exposed group (1.7 ± 0.3) (Fig. 6).

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Fig. 6

Mean (±SE) mRNA expression of nuclear factor- erythroid 2 (NFE2) -related factor 2 (Nrf2) of rats exposed to BPA and treated with hesperetin (Hst) and nano hesperetin (NHst). Different letters signify a difference that is statistically significant (p0.05) between the experimental groups.

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3.3

3.3 Histopathological investigation

3.3.1

3.3.1 Liver

Untreated control liver sections showed normal appearance with an anastomose network of hepatocytes with abundant central nuclei, also tissue revealed binucleated cells referring to regeneration (Fig. 7A). Additionally, liver of rats treated with nano-Hst displayed the same feature of untreated control liver (Fig. 7B). Whereas, liver sections from the group exposed to BPA exhibited severe steatosis both micro and macro-vesicular with dilatation of sinusoids (Fig. 7C). Meanwhile, sections of rats from the BPA + Hst group showed marked improvement with almost healthy and less steatosis (Fig. 7D). Moreover, rats treated with BPA + nano Hst revealed less steatosis compared to control and a few numbers of inflammatory cells (Fig. 7E). Hepatic pathological score showed increasing levels of liver pathology to severe changes in the BPA group due to increasing ballooning, inflammation and steatosis. Whereas, treated groups; BPA + Hst and BPA + nano Hst revealed less pathological score as changes decreased (Table 3).

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Fig. 7

Photomicrograph of liver sections. (A) Sections from untreated control liver showing normal structure, binucleated cells (green arrow). (B) Sections of rat liver treated with nano hesperetin revealing normal structure, central vein (V). (C) Sections from rat liver treated with bisphenol A exhibiting severe steatosis (black arrows). (D) Sections from rat liver treated with BPA + hesperetin displaying normal tissue, vein (V). (E) Sections of rat liver treated with bisphenol A + nano- hesperetin showing improvement, less steatosis (black arrow), inflammatory cells (red arrows). (H&E-400X)(scale bar 25 µm.).

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Table 3 Hepatic pathological score.

Groups	Ballooning	Inflammation	Steatosis
Control	0	0	0
Nano-Hst	0	0	0
BPA	2	2	3
BPA + Hst	1	1	2
BPA + nano-Hst	1	1	1

3.3.2

3.3.2 Kidneys

Untreated control kidney showed normal renal structure with abundant spherical glomeruli in between proximal and convoluted tubules in the cortex area (Fig. 8A). Additionally, the renal tissue of rats treated with nano- Hst showed the same structure as the untreated control (Fig. 8B). While sections exposed BPA revealed intense degeneration of glomeruli and tubules with increased glomerular space due to the severe degeneration. Additionally, the tubules looked faint and very thin (Fig. 8C). Meanwhile, rats from the BPA + Hst group revealed almost normal renal tissue with healthy glomeruli and tubules (Fig. 8D). Moreover, renal tissue of rats treated with BPA + nano- Hst showed only slight improvement compared to control group (Fig. 8E). The group exposed to BPA displayed increasing of renal pathological score of glomerular and tubular zones as retraction of the glomerular tuft and loss of tubular brush borders besides increasing of inflammation and tubular casts. Meanwhile, the treated groups; BPA + Hst and BPA + nano- Hst exhibited less renal pathological score due to improvement of the renal tissue (Table 4).

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Fig. 8

Photomicrograph of kidney sections. (A) Sections from untreated control renal tissue displaying normal structure, glomerulus (G), proximal convoluted tubules (PCT), distal convoluted tubules (DCT). (B) Sections of rat kidney treated with nano hesperetin showing healthy structure, glomerulus (G), proximal convoluted tubules (PCT), distal convoluted tubules (DCT). (C) Sections of rat kidney treated with bisphenol A revealing severe degeneration, increasing of the glomerular space (double headed arrows). (D) Sections of rat kidney treated with bisphenol A + hesperetin displaying marked improvement, glomerulus (G). (E) Sections from rat kidney treated with bisphenol A + nano hesperetin posting some improvement, glomerulus (G), tubular cast (black arrow). (H&E-400X))(scale bar 25 µm.).

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Table 4 Renal pathological score.

Groups	Glomerular zone	Tubular zone
Control	0	0
Nano-Hst	0	0
BPA	1–2	2–3
BPA + Hst	2	1
BPA + nano- Hst	2	2–3

4 Discussion

A ‘green synthesis’ of hesperetin nanoparticles was successfully reported keeping the ‘clean technology’ as the pivotal point since the technique did not make use of any chemical surfactants and stabilizers. The chemical groups mapped in hesperetin were similar in both the bulk and nano Hst as the mode of synthesis included only nano scaling of the flavonoid. The obtained average size of hesperetin nanoparticles is from 139.9 nm, with mainly spherical and rod-shaped particles, respectively, which is in line with the previous study reported by Elobeid et al. (Elobeid et al., 2016). The XRD analysis showed the crystalline structure of bulk and nano-Hst, with the nano-Hst having low intensity contrary to bulk Hst, which revealed an amorphous state of nano-Hst, which facilitates diffusion and solubility (Lazer et al., 2018). Although there were slight variations in the pattern and intensity of the nano-Hst spectra, the FTIR spectra revealed similar functional groups in both bulk and nano-Hst (Paramita et al., 2022). Previous literature has reported that induction of pro-inflammatory cytokines including IL-6, IL-1β, TNF-α, and IFN-β by the endocrine disrupting chemicals(EDCs) which are defined as substances, either natural or man-made, that have the ability to mimic, inhibit, or interfere with the endocrine system (Popescu et al., 2021). A study in the recent times by Pan et al. (Pan et al., 2023) reported that BPA exposure in porcine intestinal epithelial cells perturbed the oxidant/antioxidant status leading to oxidative stress/damage with a parallel proinflammatory response. In consensus with this, the findings of the present study show that exposure to BPA (250 mg\Kg) elicited the enhanced production of IL-6, IL-8, IL-1β-b, and TNF-α. Studies have reported that cell apoptosis is the major signal pathway involved in the BPA-induced tissue damage, including the brain (Caglayan et al., 2022), liver (Gu et al., 2021), and lung (Liu et al., 2016). Furthermore, Wang et al demonstrated a dose-dependent increase in the TNF-α and IL-6 levels on BPA exposure (Wang et al., 2021a, 2021b). On the contrary, according to Lu et al. (Lu et al., 2019), exposure to a low dose of BPA triggers the expression of pro-inflammatory cytokines such as TNF- α and IL-6.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor-κB (NF-κB) are two important transcription factors that control cellular reactions to oxidative stress and inflammation, respectively. According to research, Nrf2 and NF-κB are important pathways that regulate the equilibrium of cellular redox status and inflammatory stress responses. This molecular cross-talk between the components explains why these pathways are so important (Wardyn et al., 2015). In the current investigation, BPA exposure caused the hepatic Nrf2 mRNA expression to increase, reflecting oxidative damage. A number of toxic insults / diseases that are normally linked to oxidative stress have shown a contributing role of Nrf2, which has emerged as a key regulator of oxidative resistance (Ma, 2013).

Treatment with nano Hst and bulk Hst effectively attenuated and suppressed the Nrf2 expression Nevertheless, the treatments showed no observable difference. A previous study showed that rats treated with hesperetin had no effect on Nrf2 expression, while a decrease in the expression of Nrf2 protein was reported (Miler et al., 2020). The anti-inflammatory effect of hesperetin is re-attributed to reduced ROS overproduction through the Nrf2 pathway (Kubatka et al., 2021). This explains the role of hesperetin in alleviating inflammation which was demonstrated in the present study. Treatment with both bulk Hst and nano Hst significantly lowered the serum levels of pro-inflammatory cytokines. Taken together, the effect of the nano Hst was more pronounced than the bulk Hst. The protective and inhibitory effect of hesperetin is well recognized on inflammation. These findings are in line with other studies on the role of hesperetin in attenuating oxidative damage and inflammatory responses. (Li et al., 2021a, Li et al., 2021b).

The histopathological investigation in the current study demonstrated that the sections of liver exposed to BPA showed severe steatosis and dilatation of sinusoids, which indicates the abnormal retention of lipids within hepatocytes, attributed to the accumulative nature of bisphenol A (Al-Mustafa and Al-Sultan, 2022). Furthermore, exposed to BPA caused hemorrhage in the central vein, degenerated and vacuolized hepatocytes with pyknotic nuclei, and lymphocyte aggregation (Eweda et al., 2019). Besides, treatment with both bulk Hst and nano Hst reversed these effects and alleviated the steatosis, inflammation, degeneration and necrosis of hepatocytes, and function of the liver (Wan et al., 2020, Li et al., 2021a, 2021b). Furthermore, the H&E staining result showed intense degeneration of glomeruli and tubules, with increasing glomerular space due to the severe degeneration in the sections of kidneys from the group exposed to BPA (Koriem, 2022). Additionally, treatment the rats treated with Hst revealed a well maintained renal tissue with healthy glomeruli and tubules. This is in congruence with previous study that showed the similar histopathological alterations in rats induced by BPA (Shi et al., 2021). The ameliorative effect of Hst was also observed cisplatin-induced acute kidney injury (Chen et al., 2019). In addition, the pathological score for both hepatic and renal tissue mirrored the histopathological alterations. The effects of the nanosized Hst were not very conspicuous over the bulk, which could be due to the short period of exposure and treatment. However, the nano-Hst proved to be comparable in efficacy with the bulk and could potentially be a nanosized form with enhanced available to its intended biological destination. There have been several reports in the past few years which have recognized the pharmacological role of the flavonoid, Hst in attenuating oxidative stress, restoring cellular antioxidant status, inflammatory response and apoptosis (Baradaran et al., 2020). In addition, several forms of nanoparticles of the flavonoid with lipid carriers or or metals compounds have been formulated using varied methods of synthesis and their enhanced efficacy has been highlighted both in in vivo and in vitro studies (Baradaran et al., 2020;Khalaj et al., 2018; Kheradmand et al., 2018). The above mentioned studies have primarily reported the neuroprotective effect of nano crystals of Hst against oxido-inflammatory stress in the brain. In congruence with these reports, the key findings of the present study elucidated the counteractive effect of both Hst and nano Hst against BPA toxicity. The nano formulation reported demonstrates an amalgamation of green chemistry and nanoscience using an environmentally acceptable solvent; methanol. Thus, these findings offer a novel therapeutic approach to combat oxidative damage/inflammatory resonse induced by the continual exposure to plasticizers in our daily life in the current times.

5 Conclusion

The cornerstone of green synthesis is the capacity of organic substances such as phytocompounds to stabilize them into nanoparticles. The nano-formulation in the present study does offer a novel green synthesis of nanoparticles with substantial advantages over the traditional methods of nanosization; the prime advantage is being environmentally benign, economical and facile. The pro-inflammatory cytokines (IL-6, IL-8, IL-1b, TNF) were enhanced with a parallel increase in Nrf2 mRNA expression, clearly illustrating an inflammatory response elicited by the induction of oxidative stress by BPA. Treatment with hesperetin and its nano- formulation was efficacious in attenuating these modulations. Considering that hesperetin, a bioflavonoid has been widely recognized for its broad spectrum of pharmacological benefits, its nanonization sans chemicals could be advantageous in therapeutic strategies. Furthermore, the mode of nano formulation followed a sustainable approach making the process facile, safe and environmentally acceptable.