Attenuation of di(2-ethylhexyl)phthalate-induced hepatic and renal toxicity by naringin nanoparticles in a rat model

Malak Abdullah Al-Qahtani; Promy Virk; Manal Awad; Mai Elobeid; Khawlah Sultan Alotaibi

doi:10.1515/gps-2022-8122

Article Open Access

Attenuation of di(2-ethylhexyl)phthalate-induced hepatic and renal toxicity by naringin nanoparticles in a rat model

Malak Abdullah Al-Qahtani , Promy Virk , Manal Awad , Mai Elobeid and Khawlah Sultan Alotaibi

Published/Copyright: May 2, 2023

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From the journal Green Processing and Synthesis Volume 12 Issue 1

Abstract

This study assessed the protective effect of citrus flavanone, naringin (Nar), and its nanoformulation against di(2-ethylhexyl)phthalate (DEHP) toxicity in albino rats. Keeping green nanotechnology as the cornerstone, nanoparticles of Nar were synthesized and characterized using electron microscopy (transmission electron microscopy and scanning electron microscopy), particle size distribution, Fourier transform infrared spectroscopy, and X-ray diffraction. The synthesized nanoparticles were primarily spherical with an average size of 109 nm and a low polydispersity index of 0.175. Mature male albino rats were used for the exposure study. Group I was negative control. Groups II, III, and IV were exposed to (250 mg·kg b·wt⁻¹) DEHP for 3 weeks. Group III was treated with bulk Nar (5 mg·kg b·wt⁻¹), and group IV was treated with non-naringin (NNar) (5 mg·kg b·wt⁻¹). Group V was exposed only to NNar. Exposure to DEHP significantly enhanced serum levels of pro-inflammatory cytokines, interleukin-1β, 6, 8 (IL-1β, IL-6, IL-8), and tumour necrosis factor (TNF-α). In addition, the repression of hepatic mRNA expression of nuclear factor-erythroid 2-related factor 2 was also observed. In addition, marked histopathological alterations were observed in the hepatic and renal tissues. Treatment with both Nar and NNar significantly alleviated the DEHP-induced oxidative stress/inflammatory response along with the associated histological alterations. However, therapeutic utility of NNar was more profound underlining its potential in nutraceutical therapeutics with high green credentials.

Graphical abstract

Effect of naringin and its nanoparticles against DEHP toxicity in a rat.

Keywords: di(2-ethylhexyl)phthalate; naringin; nanoparticles; oxidative stress; inflammation

1 Introduction

Over the past four decades, exposure to phthalates, a class of endocrine disruptors, and their related toxicity and impact on human health have been extensively studied [1]. These compounds have been widely used as plasticizers to impart flexibility, transparency, and durability to plastic materials [2]. Even though the production of phthalates including di(2-ethylhexyl)phthalate (DEHP) was banned in the USA, Canada, and Europe, they continue to impact ecosystems and human health due to their environmental prevalence and persistence. DEHP is commonly used as a plasticizer in polyvinyl chloride (PVC) products [3]. In the current times, owing to its extensive use and high volume of production, it is an environmental concern [4]. Since DEHP is not chemically bound to PVC, it has a continual release into the environment and can thus migrate within the air, soil, water, food, etc. Subsequently, humans are exposed through ingestion, inhalation, or dermal absorption [5,6]. Humans are exposed to phthalates on a daily basis through plastic products, cosmetics, food, children’s toys, personal care products, medical devices, etc. [4,7]. Several in vitro/in vivo studies have highlighted the health hazards of DEHP, such as DNA damage, hepatotoxicity, thyrotoxicity, and carcinogenicity [8]. Further, DEHP-mediated genotoxicity is mostly ushered via the generation of reactive oxygen species (ROS) [9]. It has been postulated that cornerstone mechanisms in phthalate toxicity are the induction and promotion of inflammation, oxidative stress, and apoptosis [10].

Reduction of ROS formation or its detoxification involves various physiological systems that include adaptive mechanisms that are implicated as antioxidant enzymes or antioxidant compounds [11]. The master cellular sensor for oxidative stress is the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) [2]. Nrf2 is a transcription factor of the basic leucine zipper protein family and regulates genes that contain antioxidant response elements in their promoters [12]. Being localized in the cytosol, Nrf2 translocates to the nucleus during oxidative stress and mediates the induction of a series of cytoprotective and antioxidant defence enzymes [13]. Thus, the Nrf2 primarily regulates the coordinated expression of detoxifying antioxidant enzymes and exhibits a protective role against cellular oxidative damage [14].

The etiological contribution of DEHP and its active metabolite monoethyl phthalate (MEHP) also includes immunotoxicity in mammalian species, causing an inhibition of cell proliferation and inflammation, lowered antibody response, and apoptosis [15]. In addition, phthalates also affect the cytokine secretion and influence both innate and adaptive immunity [15]. Both DEHP and MEHP have influenced cytokine secretion from monocytes/macrophages and T cells in a similar pattern, which includes an enhanced secretion of interleukins (IL-6, IL-8, IL-10 monocytes/macrophages) and impaired secretion of tumour necrosis factor (TNF)-α, IL-1, IL-2, IL-4, and interferon-γ by T cells [16,17].

The quest for novel molecules with antioxidant properties is an effective way to promote healthy lifestyle and counteract oxidative stress associated with exposure to environmental pollutants. Naringin (Nar), a flavanone glycoside that is derived from the flavanone naringenin, is one of the main active constituents of Chinese herbal medicines and citrus fruits [18] and imparts a bitter taste to citrus juices [19]. An extensive literature survey has revealed that Nar possesses antioxidant, anti-inflammatory, anti-apoptotic, antiulcer, anti-osteoporotic, and anticarcinogenic properties [18,20,21].

The major challenge in using natural plant extracts is their limited efficacy due to their incompetence in passing through the blood–brain barrier. Despite its wide curative potential, the use of Nar is restricted owing to its susceptibility to oxidation, short half-life, poor water solubility, and dissolution rates that contribute to its low in vivo bioavailability, resulting in slow irregular digestive absorption [22]. To overcome these challenges, several antioxidant functionalized nanoparticles have been derived from biological sources. Attributed to their properties such as biocompatibility, high stability, and sustained release, nanoparticles offer greater prospects as alternative in drug delivery. With this premise, this study was designed to assess the therapeutic efficacy of Nar and its nonmetallic nanoparticles synthesized without chemical surfactants against DEHP-induced toxicity in Wistar rat.

2 Materials and methods

2.1 Chemicals and kits

DEHP of analytical grade was purchased from Tokyo Chemical Industry. Commercially available Nar (purity >98%) powder was purchased from L’eternel World (USA). Commercial ELISA Kits for IL-6, IL-8, and IL-1β, TNF-α were procured from My BioSource Inc. (USA), and the Nrf2 primers were procured from Microgen Inc (Seoul, Korea).

2.2 Synthesis of nanoparticles

The nanoparticles of Nar were synthesized using the nanoprecipitation technique [23]. First, 50 mg of Nar was dissolved in 5 mL of methanol. After 30 min, the solution was sprayed into 50 mL of boiling distilled water (drop by drop) under ultrasonic conditions (50–60 Hz, 40 kHz ± 6%). After 2 h of sonication, the solution was stirred for 1 h to obtain the nanoparticles.

2.3 Characterization of the nanoparticles

The synthesized nanoparticles were characterized based on their average size measured using Zetasizer (Malvern Panalytical Model ZEN3600, UK) based on the dynamic light scattering technique. The shape and morphology were assessed using transmission electron microscope (TEM, JEOL Model JEM 2100F, Japan) and field emission scanning electron microscope (JEOL; Model JSM7600F, Japan). X-ray diffraction (XRD) analysis used a Bruker D8 ADVANCE X-ray diffractometer (Bruker, Billerica, MA, USA) operating at 40 kV and 40 MA with Cu Kα radiation at 1.5418 Å in the 2θ range of 0–80°. The constituent functional groups in the bulk and nanoparticles of Nar were assessed by the Fourier transform infrared (FTIR) spectroscopy using a spectrometer (PerkinElmer, Waltham, USA).

2.4 Experimental design

Adult male albino rats (n = 60), weighing 250 ± 10 g, were procured from the Animal House Facility at King Saud University, Riyadh. The study conformed to the standards within the guidelines of the Institutional Ethics Committee. The rats were acclimated to the laboratory conditions at 22 ± 2°C in metabolic cages and maintained under a 12 h light/dark cycle. The rats were fed commercial diet and given tap water ad libitum. After the period of acclimatization (7 days), the rats were randomly allocated into five groups comprising 10 rats each. The experimental groups were administered the required doses of DEHP, Nar, and the nanoparticles for a period of 21 days as given below.

Group I: control was administered distilled water.
Group II: received an oral dose of DEHP (250 mg·kg b·wt⁻¹) [15].
Group III: received an oral dose of DEHP (250 mg·kg b·wt⁻¹) and bulk Nar (5 mg·kg b·wt⁻¹) [24].
Group IV: received an oral dose of DEHP (250 mg·kg b·wt⁻¹) and nanoparticles of Nar (5 mg·kg b·wt⁻¹) [24].
Group V: received only nanoparticles of Nar (5 mg·kg b·wt⁻¹).

After exposure period of 3 weeks, whole blood samples were collected from all experimental rats. Serum was prepared to assess the levels of pro-inflammatory cytokines. Further, samples of liver were excised for bioassay to assess the mRNA expression of transcription factor, Nrf2. Liver and kidney samples were excised and fixed in 10% formalin for 2 weeks for histopathological investigation. All tissue and serum samples were stored at −80°C till further analysis.

2.5 Determination of inflammatory cytokines

Commercial ELISA kits were used to assess the serum levels of pro-inflammatory cytokines IL-6, IL-8, and IL-1β, and TNF-α in accordance with the manufacturer’s protocol.

2.6 Quantification of mRNA of Nrf-2

Quantification of mRNA of Nrf-2 was performed by the quantitative reverse transcription polymerase chain reaction (PCR) analysis using GAPDH as the housekeeping gene. The assay was performed according to the manufacturer’s instructions. For cDNA synthesis, the Pure Link RNA mini kit Quick Reference was used (Qiagen, Limburg, Netherlands). A two-step process was employed for cytokine mRNA quantification (SYBR Green PCR Master Mix kit, Qiagen). cDNA for liver from all the experimental groups with specific forward and reverse primers for GAPDH (housekeeping gene) and the target gene Nrf-2 (Table 1) were used. The amplifications were carried out in duplicate with a real-time PCR instrument (7500, Applied Biosystems, Grand Island, USA). The ΔCT value was calculated by the subtraction of the GAPDH CT from each Nrf-2 CT. This was followed by the calculation of the ΔΔCT value by subtraction of the control ΔCT from each Nrf-2 ΔCT. The expression relative to control was finally calculated using the 2^−ΔΔCT method.

Table 1

Primers for target and housekeeping gene

Gene name	Description type	Primer sequence
Nrf2	Forward	CAC ATC CAG ACA GAC ACC AGT
	Reverse	CTA CAA ATG GGA ATG TCT CTG C
Gapdh	Forward	AGG TTG TCT CCT GTG ACT TC
	Reverse	CTG TTG CTG TAG CCA TAT TC

Nrf2: Nuclear factor-erythroid 2; Gapdh: Glyceraldehyde 3-phosphate dehydrogenase.

2.7 Statistical analysis

All presented data were expressed as mean ± standard deviation (SD). One-way analysis of variance was performed followed by Tukey’s test to analyse group differences. The data were analysed using SPSS 22.0 statistical software Chicago, IL, USA. The significant level was set to p ≤ 0.05.

3 Results

3.1 Size, distribution, and morphology

The obtained nanoparticles have a size range in nanometre, as aimed. The particle size distribution of the prepared naringin nanoparticles (non-naringin [NNar]) is shown in Figure 1a. The mean particle size observed was 109 nm with a polydispersity index of 0.175, as depicted by a single peak. The morphology of the nanoparticles was determined by both scanning electron microscope (SEM) and TEM images as shown in Figure 1b and c. From the images, it is clear that the particles were agglomerated in nature. There are distinct and visible variable shapes, which were mainly spherical and rod-shaped (bacillary). The micrograph shows that the average diameter of the spherical nanoparticles is 65 nm.

Figure 1

(a) Particle size and distribution of Nar nanoparticles. (b) SEM micrographs of Nar nanoparticles showing the morphology and size. (c) TEM micrographs of Nar nanoparticles.

3.1.1 FTIR

FTIR measurement was carried out to identify the possible biomolecules present both in the bulk and in the NNar. The spectra for both the bulk and nanoparticles were almost similar, showing that there was no major change in the chemical composition of the material on nanosization. Prominent IR bands were observed for both the bulk Nar and the nanoparticles at 3,822.02 and 3,806.01 cm⁻¹, 3,750.11 and 3,752.34 cm⁻¹, respectively, corresponding to the O–H stretching of the alcohols. Both spectra showed a medium C–H stretching at 2,929 cm⁻¹. An additional sharp band ascribed to C–H stretching at 2,888.66 cm⁻¹ was observed only in the spectra for the bulk. On the other hand, for the NNar, a prominent N═C═S stretching was observed at 2,092.53 cm⁻¹. Strong C═C stretching was observed in both spectra within the range of 1,642.51–1,647.69 cm⁻¹. An observable change was noted in the pattern of the spectra for the bulk and the NNar in the frequency ranges between 1,550 and 500 cm⁻¹. The absorption bands for the NNar were fewer with less intensity in comparison with the bulk material. The bands at 1,515.41, 1,516.76, and 1,544.02 cm⁻¹ are ascribed to strong N–O stretching due to the presence of nitro compounds. The band at 1,451.49 cm⁻¹ indicates medium C–H bending in an alkane with methyl group. In addition, the band 1,394.30 cm⁻¹ illustrates the O–H bending of the carboxylic group (Figure 2a).

Figure 2

(a) FTIR spectra. (b) XRD of the Nar (bulk) and NNar powder.

The bands between 1,293.30 and 1,041.41 cm⁻¹ were identified as alcohol and ester groups with strong C–O stretching. A strong S═O stretching was observed between the absorption bands at 1,335–1,372 cm⁻¹ due to the presence of sulphonic acid. The band at 1,293.30 cm⁻¹ exhibited strong C–O stretching due to the presence of aromatic ester. The bands at 1,177.48 cm⁻¹ exhibited C–O strong stretching due to the presence tertiary alcohol. The band at 1,041.41 cm⁻¹ may be attributed to strong and broad CO–O–CO bonding of anhydride. The bands at 887.42, 888.02, 820.33, and 825.45 cm⁻¹ indicate strong C–H bending. The band at 739.97 cm⁻¹ exhibited C═C bonding of alkene. The band at 699.52 cm⁻¹ exhibited a strong C═C bonding of alkene. The bands at 627.33 and 561.12 cm⁻¹ exhibited a strong bond and C–Br stretching attributed to the halo compound (Figure 2a).

3.1.2 XRD

The synthesized nanoparticles and the Nar powder were characterized by XRD. The diffractograms of the bulk Nar and the nanoparticles are shown in Figure 2b. The XRD diffraction patterns of NNar and Nar were identical and located at angles (2 θ ) at 9.62°, 11.44°, 14.37°, 16.27°, 18.49°, 21.29°, 26.08°, and 28.66°, pointing out the high crystallinity of nanoparticles. However, the intensity of the peaks was less in NNar (Figure 2b).

3.2 Bioassays

3.2.1 Inflammatory cytokines

In order to evaluate the impact of DEHP on the production of inflammatory cytokines, the serum levels of TNF-α, IL-6, IL-8, and IL-1β were assessed.

3.2.1.1 Serum concentration of TNF-α

Exposure to 250 mg·kg⁻¹ of DEHP for 21 days significantly (p < 0.001) enhanced the serum levels of pro-inflammatory cytokine, TNF-α (119.38 ± 2.265 pg·mL⁻¹), as compared to the controls (99.99 ± 1.633 pg·mL⁻¹). Treatment with NNar significantly (p = 0.03) decreased the serum TNF-α concentrations (115.63 ± 0.894 pg·mL⁻¹), while the group treated with bulk Nar (116.792 ± 1.635 pg·mL⁻¹) was comparable to the group treated with DEHP only. The group treated with only the NNar showed no significant difference from the controls (Figure 3a).

Figure 3

(a) Mean (±SD) concentration of TNF-α in serum (pg·mL⁻¹). (b) IL-6 in serum (pg·mL⁻¹). (c) IL-8 in serum (pg·mL⁻¹). (d) IL-1β in serum (pg·mL⁻¹) in rats exposed to DEHP and treated with bulk Nar (DEHP + Nar) and NNar (DEHP + NNar). Different letters indicate a significant (p ≤ 0.05) difference between the experimental groups (n = 5).

3.2.1.2 Serum concentrations of IL-1β, IL-6, and IL-8

Exposure to 250 mg·kg⁻¹ of DEHP for 21 days significantly (p = 0.009) enhanced the serum levels of pro-inflammatory cytokine, IL-6 (61.63 ± 16.325 pg·mL⁻¹), as compared to the controls (42.072 ± 1.435 pg·mL⁻¹). Treatment with NNar significantly (p < 0.001) decreased the serum IL-6 concentrations (35.80 ± 2.280 pg·mL⁻¹), while the group treated with bulk Nar showed no significant change. The group treated with only the NNar showed no significant difference from the controls (Figure 3b). The serum IL-8 concentrations were significantly (p < 0.001) increased on exposure to DEHP (50.69 ± 3.292 pg·mL⁻¹). Treatment with both bulk Nar (37.25 ± 2.938 pg·mL⁻¹) and NNar (34.33 ± 1.167 pg·mL⁻¹) significantly (p < 0.001) reduced the serum concentrations of IL-8. However, there was no significant difference observed within the treated groups. Also, the group treated with only the NNar showed no significant difference from the controls (Figure 3c).

The serum concentrations of IL-1β were significantly (p = 0.000) increased on exposure to DEHP (6.4033 ± 0.3672 pg·mL⁻¹) as compared to the control group. Treatment with both bulk Nar (5.17 ± 0.413 pg·mL⁻¹) and NNar (3.44 ± 0.246 pg·mL⁻¹) significantly (p = 0.000, p < 0.001, respectively) reduced the serum concentrations of IL-1β. Further, a significant difference was observed within the treated groups as the NNar was more efficacious. The group treated with only the NNar showed no significant difference from the control (Figure 3d).

3.2.2 mRNA expression of Nrf2

The Gapdh proved to be a good reference gene as its expression was not affected by the experimental factors. The group treated with only the NNar showed no significant difference from the control group in the fold change. Hepatic mRNA expression (fold change) of Nrf-2 was significantly (p < 0.001) downregulated on DEHP exposure. A significantly (p ≥ 0.05) enhanced expression of the mRNA was observed on treatment with NNar, while no significant difference was observed with the bulk Nar (Figure 4).

Figure 4

Mean (±SD) concentration in fold change of mRNA expression of Nrf2 in rats exposed to DEHP and treated with bulk Nar (DEHP + Nar) and NNar (DEHP + NNar). Different letters indicate a significant (p ≤ 0.05) difference between the experimental groups (n = 3).

3.3 Histopathological analysis

3.3.1 Liver

Control liver sections showed a normal tissue organization characterized by central vein surrounded with a connected network of hepatocytes with spherical abundant nuclei. Hepatocytes were observed separated from each other by blood sinusoids (Figure 5a). Additionally, liver sections from the group exposed to only NNar also revealed a similar normal hepatic architectural structure as the control (Figure 5b). However, liver sections from rats exposed to DEHP showed marked hepatic pathological signs such as micro- and macro-steatosis besides great foci of inflammation in addition to dilatation of the sinusoids. Overall, the tissue looked faint due to the general cytoplasmic degeneration (Figure 5c). Meanwhile, liver sections of rats from the group treated with Nar (DEHP + Nar) showed a slight improvement in terms of reduced steatosis and a fewer number of inflammatory cells surrounding the portal area (Figure 5d). Further, sections from the group treated with NNar (DEHP + NNar) showed a conspicuously restored structural organization of the hepatic tissue with healthy hepatocytes (Figure 5e).

Figure 5

Photomicrographs of sections of liver of rats from the experimental groups. (a) Section of liver from the control group showing normal hepatic structure, central vein (V), healthy hepatocytes, and blood sinusoids. (b) Section of liver from the group treated only with NNar revealing healthy hepatic architecture. (c) Section of liver exposed to DEHP exhibiting pathological alterations, inflammatory foci (black arrows), and steatosis (red arrows). (d) Sections of liver exposed to DEHP and treated Nar (DEHP + Nar) displaying less pathological alterations, inflammatory cells (black arrows), and less steatosis (red arrows). (e) Section of liver from the group exposed to DEHP and treated with NNar showing healthy hepatic tissue with a few traces of steatosis (red arrows) (H&E-400X).

3.3.2 Kidneys

Sections of kidney from the control group revealed normal renal structure, and the cortex area showed well-organized glomeruli, proximal and distal convoluted tubules along with the large and wide collecting tubules (Figure 6a). Kidney sections of rats exposed to only NNar also showed healthy renal tissue architecture (Figure 6b). However, sections from kidneys of rats from the group exposed to DEHP displayed dilated renal vessel congested with haemorrhage and hemosiderin granules, and the glomeruli were distorted and also showed atrophy. Tubules revealed cytoplasmic degeneration, with degeneration of proximal tubules with internal inclusions filled with casts (Figure 6c). Renal sections of rats treated with treated Nar (DEHP + Nar) showed a marked improvement in terms of organization of glomeruli and tubules (Figure 6d). Further, sections of kidney from the group treated with NNar (DEHP + NNar) showed a more observable restoration in the structural organization of the glomeruli and renal tissue, which was comparable to the sections from the control group (Figure 6e).

Figure 6

Photomicrographs of sections of kidney of rats from the experimental groups. (a) Section of kidney from the control group displaying normal structure, glomerulus (G), proximal convoluted tubules (PCT), distal convoluted tubules (DCT), and collecting tubules (CT). (b) Section of kidney from the group treated only with NNar revealing healthy renal architecture, G. (c) Section of kidney exposed to DEHP exhibiting atrophied G, dilated vessel congested with haemorrhage and hemosiderin (green arrow), tubular cast (black arrow). (d) Sections of kidney exposed to DEHP and treated Nar (DEHP + Nar) displaying a marked improvement, G, PCT, and DCT. (e) Section of kidney from the group exposed to DEHP and treated with NNar showing healthy and normal renal structure, G, PCT, and DCT (H&E-400X).

4 Discussion

The current boom in the development of green chemistry and nanotechnology has triggered researchers worldwide to explore novel green synthetic methods for synthesizing nanoparticles using natural compounds in plant extracts. These phytoconstituents act as natural stabilizers’ reducing and capping agents. The green synthesis is the cornerstone of sustainable chemistry, which has garnered attention in nanoscience. The plant-extract-mediated methods are advantageous, being low cost, nontoxic, facile scaleup, and environmentally benign, which is highly conducive to a sustainable development in nanomedicine. A successful synthesis of Nar nanoparticles was reported in this study in a nanosize range of 65–110 nm being predominantly spherical in shape. In congruence with these findings, an almost similar-sized distribution (121 nm), low polydispersity (<0.1), and spherical-shaped nanoparticles of Nar and naringenin were reported to be synthesized in a recent study by Cordenonsi et al. [25]. The chemical bonding and crystalline behaviour of the bulk Nar powder and the nanoparticles (NNar) were confirmed by FTIR and XRD analyses. The FTIR spectra of both the Nar and NNar were almost similar with a slight shift in the pattern with a lower intensity observed in the NNar spectra. This reflected the presence of similar constituent groups contributing to their antioxidant status. The XRD analysis of both the bulk Nar and NNar showed the crystallinity with the prominent peaks of NNar being at a lower intensity comparable to bulk Nar. The intensity of the peaks reflects the atomic arrangement in the lattice, which changes with the nanosizing as lowered intensity corresponds to a decreased crystallinity, which is attributed to the small size of the nanoparticles [26]. It has been previously reported that DEHP exposure promotes an inflammatory response, which is primarily mediated via the induction of the nuclear translocation of the nuclear factor (NF)-κB. This subsequently leads to an increased expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, and TNF-α [16,27]. Nrf2 and NF-κB are the major pathways that are instrumental in regulating the balance of cellular redox status and contribute towards the stress and inflammatory response. Both Nrf2 and NF-κB are regulated by redox-sensitive factors where oxidative and nitrosative stress is augmented by the absence/suppression of Nrf2, eventually resulting in enhanced cytokine production. Thus, an activation of NF-κB in an oxidative milieu results in a depletion in Nrf2 [28]. Thus, Nrf2 is a key transcription factor that controls several aspects of cellular homeostasis in response to oxidative stress [29]. The oxidative stress elicited by phthalates is proposed as a key determinant in DEHP toxicity. Considering its role as a peroxisome proliferator-activated receptor, DEHP has been reported to elicit oxidative stress via increasing peroxidase expression and ROS generation [2]. This is in line with the findings of this study where a marked increase in the pro-inflammatory cytokines was observed on exposure to DEHP for 3 weeks with a concomitant inhibition in the expression of the Nrf2 mRNA. Inhibition of the Nrf2 signalling induced on exposure to DEHP led to oxidative damage in the liver and kidney, which was evident from the histopathological alterations observed in these tissues. Previous studies have also reported a similar histopathological pattern in tissues including testis, specifically the Sertoli cells [30], spleen [31], and lung [32] exposed to DEHP. In addition, a recent study by Amara et al. [2] also demonstrated a similar Nrf2 repression attributed to the oxidative stress and apoptosis in mouse kidney tissues. The production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) was significantly increased in rats in this study on exposure to DEHP. In line with these results, exposure to DEHP has been reported to promote the expression of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, and TNF-α [16,17]. A systemic review on the effect of phthalates on cytokine production in monocytes and macrophages by Hansen et al. [33] reported that four out of five studies that investigated DEHP showed an enhanced TNF-α secretion from monocytes or macrophages. A TNF-α is an essential pro-inflammatory cytokine secreted primarily by monocytes and macrophages and also by other cell types and plays a key role in triggering both local and systemic inflammation via the induction of other cytokines (including IL-1β and IL-6) [34]. Furthermore, this study demonstrated that single exposure to DEHP exhibited a marked histological alteration in the liver and kidney with conspicuous abnormalities. In congruence with these findings, an induction of hepatic and renal injury was also reported in previous studies [2,35], which could be mainly attributed to the oxidative damage in the tissues. Treatment with bulk Nar and its nanoformulation, NNar, ameliorated the DEHP-induced oxidative stress and associated inflammation. The expression of Nrf2 was enhanced in the group treated with NNar in comparison with DEHP alone.

Additionally, both the treatments (Nar and NNar) also alleviated the histopathological alterations in both target tissues, liver and kidney. This is well explained considering that Nar is a bioflavonoid that possesses a gamut of biological and therapeutic properties such as anti-apoptotic, antidiabetic, neuroprotective, anti-inflammatory, metal chelating, antimicrobial, antimutagenic, anticancer, free radical scavenging, and antioxidative [36]. The protective effect of Nar and NNar was evident from the reduction in the pro-inflammatory cytokine levels, which were more pronounced in the group treated with the NNar. Furthermore, the repression of the Nrf2 expression elicited by the DEHP-induced oxidative stress was attenuated on treatment with Nar and NNar. However, the NNar had a more profound effect. Also, the hepatocellular injury and renal damage induced by the DEHP single exposure were reduced on treatment with Nar and NNar where the nanoparticles exhibited a more marked protective effect on the cellular architecture. These findings are in congruence with previous studies reporting the ameliorative effect of Nar on oxidative stress [20,36,37]. Additionally, Nar has been reported to downregulate the pro-inflammatory mediators, such TNF-α and IL-6, as observed in this study [35]. Nar acts through diverse mechanisms in different pathological conditions. For instance, a systematic review and meta-analysis by Vishwanatha et al. [20] reported that the neuroprotective effect of the bioflavonoid was attributed to the alleviation of the oxidative stress through a modulation of the nitric oxide pathway, diminishing the gene expression and levels of pro-inflammatory cytokines (TNF-a, IL-6, NF-kB p65 unit, iNOS, IL-1b, COX-2), reducing the expression of apoptotic factors (Cascapase-3, Caspase-9), increased the Bcl-2:Bax ratio and downregulation of JAK2/STAT3 signalling pathway. Taken together, the findings of this study clearly showed that the nanoparticles, NNar, were more efficacious against the DEHP toxicity than the bulk Nar. In agreement with these results, a recent study by Wang et al. [38] on the comparative assessment of the hepatoprotective and antioxidant activity of Nar-loaded solid nanoparticles and free Nar against aflatoxin B1-induced hepatocellular carcinoma showed that although Nar is a promising nutraceutical with a gamut of pharmacological effects and is widely used in Chinese medicine, the major restraints in its use have been its decreased oral bioavailability, low solubility, and increased rate of gastrointestinal degradation. A more targeted and sustained deliverance of the flavonoid has been possible by fabricating its nanoformulations. There have been several studies in recent years that have reported enhanced efficacy of nanoformulations of Nar in several pathological conditions [22,38,39].

5 Conclusion

Taken together, the key findings of this study showed that the flavonoid Nar and its nanosized form were efficacious in alleviating the DEHP-induced hepatotoxicity and renal toxicity in a rat model. The curative effect of the nanoparticles was potent than the bulk, which could possibly be ascribed to an enhanced bioavailability of the flavonoid. Thus, the nanoparticles of Nar, NNar, could be introduced as a prospective nutraceutical to combat human health impacts of the inevitable exposure to plasticizers in the current times.

Acknowledgements

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project no. (IFKSURG-2-1550).

Funding information: The research was funded by the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, project no. IFKSURG-2-1550.
Author contributions: Malak Abdullah Al-Qahtani: methodology, formal analysis, writing – original draft; Promy Virk: conceptualization, project administration, formal analysis, writing – original draft, writing – review and editing; Manal Awad: methodology, formal analysis, writing – original draft; Mai Elobeid: resources, writing – review and editing; Khawlah Sultan Alotaibi: methodology.
Conflict of interest: The authors state no conflict of interest.
Data availability statement: All data generated or analysed during this study are included in this published article.

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Received: 2022-10-04

Revised: 2023-03-12

Accepted: 2023-03-28

Published Online: 2023-05-02

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/gps-2022-8122

Keywords for this article

di(2-ethylhexyl)phthalate; naringin; nanoparticles; oxidative stress; inflammation

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