Evaluation of sex steroid hormones and reproductive irregularities in diethyl phthalate-exposed premature mice: modulatory effect of raw honey against potential anomalies

Phthalates, plasticizing chemicals, are top-rated environmental contaminants. Diethyl phthalate (DEP), a chief member of this family, was declared a potent endocrine disruptor and carcinogen in animals and humans. The current study was designed to explore the probable reproductive damage induced by DEP and the therapeutic efficacy of raw honey in male albino mice. Four-week-old 50 male mice were randomized equally in five groups, as control (C) received 0.1 ml distilled water; vehicle control (VC) received corn oil (0.1 ml/mouse); DEP (3mg/g/BW) dissolved in corn oil; honey control (HC) administered with honey (0.2 mg/g/day); and phthalate plus honey (P+H) administered with DEP and honey (3mg and 0.2 mg/g/BW/day respectively). Mice were treated through oral gavage for 54 days routinely, acclimatized for 6 days, and dissected. In the first instance, the antioxidant potential and total phenolic contents (TPC) of honey were analyzed through ferric reducing antioxidant power (FRAP) assay and Folin-Ciocalteu assay to confirm the antioxidant capacity of honey. The morphological, morphometric, histological, micrometric, sperm count, and hormonal analyses, and antioxidant capacity test in tissue homogenates were conducted by using tissues (testis, epididymis) and blood samples of mice. Mice exposed to DEP have a significant increase in body weight, LH level, and seminiferous tubule lumen diameter and decrease in the gonado-somatic index, testosterone level, sperm count, and seminiferous tubule diameter. Additionally, histopathology of testes showed interstitial space dilations, exfoliations, Leydig cell atrophy, germ cell degenerations, and spermatid retention in DEP-exposed testes sections. However, concomitant use of honey and DEP had shown a significant improvement in histopathological lesions, steroid hormone levels, and healthy sperm count. By these results, it is concluded that honey possessed antioxidant potential that can efficiently protect DEP-induced anomalies in male mice.


Introduction
In the era of urbanization and industrialization, the general population is unavoidably exposed to various contaminants from diversified sources (Hernandez et al. 2019;Docea et al. 2018). It is highlighted by recent epidemiological and biomonitoring studies that exposure to these contaminants is associated with public health-hazardous that leads to nephrotoxicity, cardiotoxicity, hepatotoxicity, immunotoxicity, nephrotoxicity, and endocrine disruption (Andjelkovic et al. 2019;Buha et al. 2013;Docea et al. 2019;. Some of the already proven and well-established chemicals involved in endocrine disruption are polybrominated diphenyl ethers, polychlorinated biphenyls, heavy metals, microplastics, pesticides, bisphenols, and phthalates (Buha 2018;Djordjevic et al. 2019;Gore et al. 2015). Recently, phthalates are considered top-listed classified endocrine disruptors that disrupt the hormonal balance on excessive exposure through diet, environment, and daily household stuff (Tanner et al. 2020).
Responsible Editor: Mohamed M. Abdel-Daim Phthalates are recognizable environmental pollutants that are frequently used as additives to enhance polymer versatility and malleability in the production of plastics. These are synthetic, colorless, odorless, and lipophilic compounds (Shaha and Pandit 2020). In 1920, phthalates were first time used for commercial applications (Rahman and Brazel 2004). The six most familiar phthalates such as di (2-ethyl hexyl) phthalate, diethyl phthalate, dibutyl phthalate, dibenzyl phthalate, and di-isopropanol phthalate were declared as environmental pollutants by the United States Environmental Protection Agency (Gani and Kazmi 2020;Li et al. 2020;Li 2020).
Diethyl phthalate (DEP) is a widely used co-contaminant of microplastic and proved as an endocrine disruptor. It is structurally composed of alkyl chains with colorless, odorless, and oily liquid nature soluble in the organic solvent Jeong 2020). DEP is commonly used in pharmaceutics, medical bags, shampoo, perfumes, toys, and food packaging (Radke et al. 2020).
Although DEP has non-covalent bonding with plastic but makes it easy to leach in food products as well as the environment (NRC, 2009). When humans are exposed to DEP, it is metabolized into mono-ethyl phthalate (MEP) within 3 to 28 h (half-life) and eliminated from the body through urine (Radke et al. 2020). DEP compared to other phthalate is detected more in the urine sample of the US population. Due to its ubiquity, its enormous and continuous exposure is associated with impaired fertility, shorter anogenital distance, increased oxidative damage to sperm cells, and carcinogenesis (Hauser et al. 2007;Gopalakrishnan et al. 2020). Hence, animal studies are a prerequisite to understanding the potential link between diethyl phthalate exposure and adverse outcomes. These investigations may provide an understanding of the dose-related characteristics of phthalates. Therefore, it is inevitable to evaluate the noxious effects of diethyl phthalate on prepubertal mice's reproductive parameters.
On the other hand, honey is a natural sweetener and utilized enormously for health benefits (Selvaraju et al. 2019). It is made up as a result of regurgitation and evaporation of floral nectar by honeybees (Pipicelli and Tatti 2009). Honey consists of flavonoids, antioxidants, organic acid, minerals, protein, vitamins, and carbohydrates such as fructose, sucrose, raffinose, and glucose (Mosavat et al. 2019). These chemical constituents had made honey a well-known antibacterial, antimicrobial, and antifungal agent (Aswin and Neelusree 2019). Ancient people used honey both for nutritional purposes and for its medicinal aims (Adebolu 2005). Experimental studies support its usage due to its anti-inflammatory, antiviral, antibacterial, antioxidant, and other bioactivities (Murosak et al. 2002). The therapeutic efficacy of honey is acknowledged in various major diseases, diabetes, cancer, and cardiovascular and degenerative diseases (Hossen et al. 2017). Its positive role as a nephroprotective agent in chemical-induced toxicity is also well-established (Ibrahim et al. 2016). Honey supplementation involved in vitro maturation and improvement of sheep oocytes (Kaabi et al. 2020). Additionally, honey was also reported as an effective remedy to improve fertility status in males and females but results are still inconclusive (Meo et al. 2017). The present study aims to explore the therapeutic potential of honey against DEP-induced probable testicular lesions and reproductive disruptions in albino mice.

Ethical statement
All animal trials were executed according to local and worldwide procedures. The nearby way is the Wet op de dierproeven (article 9) of Dutch law (international) and an associated rule planned via the Bureau of Animal Research Licensing, Local University, as detailed in our earlier papers Ali et al. 2020a, b, c, d;Naeem 2021;Tariq 2021;Mughal et al. 2020;Hussain et al. 2020;Ara et al. 2020;Khan et al. 2019;Mumtaz et al. 2019;Mughal et al. 2019;Dar et al. 2019). The rearing and use of mice were carried out using NIH Publication "Guide for the Care and Use of Laboratory Animals" (NRC 2004) and with the approval vide No. D/681/UZ dated 04-04-2019 by the local bioethical committee of the University on animal experimentation.

Chemicals
Diethyl phthalate (99.5% purity, MW: 222.24) was purchased from Sigma-Aldrich. Honey used in this research was multiflora honey collected in April from Apis mellifera colonies were taken from Honeybee Research Farm, University of the Punjab, Lahore, Pakistan. Corn oil with 99.9% purity was purchased from the Akbari market, Lahore. Hormones Diagnostic Kits were purchased from BioVision Company, distributed by Lab Science, Pakistan. Ethanol, hematoxylin, eosin, hydrochloric acid, ferric chloride, and ferrous sulfate were obtained from Merck & Co., Inc. TPTZ (2,4,6-Tris(2pyridyl)-s-triazine) was acquired from Sigma-Aldrich Company. All chemicals used in the present research were of analytical grade.

Animals' rearing
Swiss Webster strain of albino mice Mus musculus was used in the experiment. Mice were raised employing steel cages with well-managed conditions of 12-h light/dark cycle at 26 ±2°C and 45-55% relative humidity in the animal house of the Institute of Zoology, University of the Punjab, Lahore, Pakistan. Mice were fed with commercially available feed (national feed No. 14 by National Feeds industries, Lahore, Pakistan) pellets and water ad libitum.

Experimental design
Four-week-old 50 male mice with body weight (B.W.) 13±2 g were randomly classified into five groups (n=10): group 1: control (C) provided 0.1 ml distilled water; group 2: vehicle control (VC) administered 0.1 ml corn oil; group 3: DEP, treated with 3 mg/g B.W. DEP, dissolved in corn oil in such a way that 0.1 ml contained required concentrations of DEP; group 4: (P+H), DEP (3 mg/g B.W) and honey (0.2 mg/g); group 5: honey control (HC) exposed to honey (0.2 mg/g B.W). Treatments were given through oral gavage for 54 days daily, once a day. Before treatment, antioxidant potential and total phenolic content (TPC) were also assessed separately for raw honey used in this study.

General observations
Behavioral and other physical changes in mice of all groups were noted and recorded twice a day regularly. The body weight of each animal in each group was measured on weekly basis and dose concentrations were adjusted considering their weights accordingly.

Samples' recovery
After 54 days, the animals were acclimatized for 6 days for self-restoration and were euthanized after isoflurane inhalation. Testes and epididymis were successfully recovered for morphometric, histopathologic, and micrometric analyses, sperm count, and antioxidant capacity test. The blood samples were collected by cardiac perfusion for hormonal assays under deep anesthesia.

Morphometric analysis
Mice's body weight and wet weight of the testes were measured with analytical balance (Ax120 SHIMADZU, JAPAN). Furthermore, the size (length/width) of testes was recorded using a digital Vernier caliper.

Histopathology
Testicular tissues were fixed in Bouin's fixative for 40 h at room temperature (33°C), dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin blocks. The thick sections (5μm) were prepared by rotary microtome and stained with eosin and hematoxylin following established protocols (Bancroft and Layton 2013). The sections were observed and digital photographs were captured under a camera-fitted microscope to highlight histopathological defects and to acquire micrometric data.

Micrometry
For micrometric measurements, from photomicrographs taken at 40X, twenty nearly round randomly chosen seminiferous tubules were traced. Seminiferous tubule and lumen diameters were measured through bisecting lines drawn at the circumference of the tubule using ImageJ software at 400 magnifications (Montoto et al. 2012). The cross-sectional area of the seminiferous tubule (STA) was calculated following a geometric constant equation (Mustafa et al. 2019) where r is the tubule radius.
The area of the tubular lumen (LA) was calculated by the equation: where Lr is the luminal radius.
The epithelium area (EA) was obtained by subtracting LA from STA. Results were expressed as square millimeters (mm 2 ).

Smear preparations for sperm count and morphology:
Testes with attached epididymis recovered in saline solution give a midline incision, gently crushed on clean glass with a glass rod and curdy material used for the sperm count and sperm morphological analysis through the chamber counting method. The sperm with complete head and tail counted once in 4 big 1 mm 2 quadrates under a light microscope (Kirkman-Brown and Björndahl 2009).

Hormonal Analysis
Hormonal analysis for testosterone level and luteinizing hormone (LH) was assessed in the mice's blood samples by kits (BioVision). Hormones were assayed following kit instructions in triplicates.

Tissue homogenate preparation
Testis tissues were preserved in 1.15% ice-cold KCl at −80°C for few hours. Testes were weighed and homogenates were prepared using a glass homogenizer with ice-cold 1.15% KCl (10% w/v) to record the antioxidant effect of honey within tissues. Then, homogenates were centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants were collected for further processing (Katalinica et al. 2005).

Ferric reducing antioxidant power in mice's tissues and honey
The antioxidant power in testes tissues and honey was measured following the ferric reducing antioxidant power (FRAP) assay (Katalinica et al. 2005). The antioxidants present in the supernatant were evaluated as a reducer of Fe +3 to Fe +2 , which is chelated by 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) to form the complex and evaluated using the maximal absorption at 593 nm. The results were presented in μM equivalent ascorbic acid/mg sample.

Total phenolic content
Honey was assessed for the presence of phenolic contents following Valverde-Malaver et al. (2015) protocol. Briefly, 1.0 ml of the diluted honey sample was transferred in separate tubes containing 5.0 ml of 1/10 dilution of Folin-Ciocalteu's reagent in water. Then, 4 ml of a sodium carbonate solution (7.5% w/v) was added. The tubes were allowed to stand at room temperature for 60 min before absorbance at 765 nm was measured. The unit was expressed in a Gallic acid equivalent g/100 g sample.

Statistical analysis
Statistical analysis was carried out using SPSS software (IBM, version 21.0). The data were expressed in terms of mean ± SEM and mean differences between all experimental groups were measured by one-way ANOVA followed by post hoc Tukey and Duncan's multiple range tests. All the tabulated data were presented as mean ± standard error of means and value p < 0.05 was considered significant.

Biometric parameters
The morphological and morphometric analyses included mice's body weight, gonado-somatic index (GSI), and testes size (width and length) in all groups, which are presented in Fig. 1 and Table 1. Animals in groups C, VC, and P+H showed normal morphology and morphometric results. However, the DEP group showed a significant (P≤0.05) increase (33.29±3.30 g) and the HC group showed a remarkable decrease (28.34±1.73 g) in body weight gain against the control (30.80±0.34 g). Contrary to these findings, a decrease (P≤0.05) in the testes weight and gonado-somatic index (0.33a±0.006) in the DEP group as compared to the control (0.40b±0.008) was noted. On the other hand, P+H (phthalate co-administered with honey) showed significant improvement in the above-mentioned parameters.

Micrometry and sperm count observations
Micrometric observations showed significant variations (p<0.05) in seminiferous tubule (ST) diameter and luminal diameter of the seminiferous tubules among various treatment groups ( Fig. 2; Table 1). The group DEP showed significantly (p<0.01) reduced ST diameter (204.8±1.92 μm) and increased (p<0.001) lumen diameter (92.0 ± 3.5 μm) as compared to control (243.3± 0.77 and 67.1 ± 1.3 μm). Contrary to this, the P+H group showed less change (p<0.05) in diameter in comparison with the control. The cross-sectional area of seminiferous tubules (STA) decreases (0.033±2.71 mm 2 ) remarkably (p<0.05) in the DEP group contrary to the luminal area (LA) (0.0066 ±1.27 mm 2 ) that increased significantly in the same group against controls (0.046 ±1.40 and 0.0035 ± 1.05 mm 2 , respectively). The results depicted a decline in mature sperms in the said group. STA and LA also showed variations in other groups as compared to the control but the difference is less significant (Fig. 2; Table 1). The above results are further cleared through sperm count, which decreased in a significant number (p<0.001) in the DEP group (24.7 ± 10.5 million/ml) as compared to the control (C) group (41.8 ± 2.11 million/ml) ( Table 2). The normal to abnormal sperm ratio is also comparable to the above results. A novelty found in the HC group (given honey solely), data about micrometric studies as well as sperm count showed more positive results even from the control group ( Fig. 2; Tables 1 and 2).

Hormonal analysis
The results of serum testosterone and luteinizing hormone quantification in all groups are presented in Fig. 3. There was a statistically significant decrease (p<0.001) in testosterone level (0.10 ± 2.05 ng/ml); alternatively increased LH level (2.02 ± 4.15 mlU/ml) was observed in the DEP group as compared to the control, in which testosterone and LH levels were 0.24 ± 3.22 ng/ml and 1.66± 7.38 mlU/ml, respectively. At the same time, the P+H group showed comparable results with the C and VC groups with 0.23 ± 1.26 ng/ml, testosterone level and 1.57 ± 9.03 mlU/ml, respectively. On the other hand, the serum testosterone levels in the HC group again showed a positive impact of honey on steroidogenesis.

Antioxidant capacity test findings in honey and testicular tissues
There was a significant difference in reducing ability in terms of FRAP value among testicular tissues of various groups. FRAP value in DEP-exposed testes tissues (112.54± 2.3 μM ascorbic acid equi/kg) was significantly decreased (p<0.001) as compared to C (247.65± 2.1) and VC (250.67 ± 1.1 μM ascorbic acid equi/kg). Moreover, FRAP indicated the successful enhancement of antioxidant capacity in testes tissues (241.6 ± 1.4) in the P+H group compared to the DEP group (112.54± 2.3). On the another side, testis of the animals who received honey solely (HC group) showed significantly higher FRAP value (282.9 ± 1.9 μM ascorbic acid equi/kg) even than the C group, which has a FRAP value of 247.65± 2.1 (Table 2). Luminal area (mm 2 ) 0 . 0 0 3 5 b ± 0.002 0.0047 c ± 0.004 0.0066 a ± 0.0001 0.0031 b ± 0.0002 0.0034 b ± 0.0002 0.000 Epithelial area ( mm 2 ) 0 . 0 4 2 5 b ±0.001 0.0323 c ± 0.038 0.0264 a ± 0.001 0.0469 b ± 0.006 0.0367 c ± 0.007 0.000 Note: Values are expressed as mean±SEM. N, number of samples analyzed; C, untreated; VC, corn oil treated; DEP, diethyl phthalate exposed; P+H, diethyl phthalate + honey; HC, honey. In rows, different alphabets showed significant difference among groups analyzed through one-way ANOVA followed Tukey's post hoc at level minimally P≤0.05 The findings of honey antioxidant capacity along with the total phenolic content are presented in below. Results indicate a strong correlation (R 2 =0.9109) between total phenolic content (TPC) and antioxidant capacity (FRAP values) of honey (Fig. 4).

Histopathology
Histopathological sections of testes from the control group (C) showed a regular pattern of seminiferous tubules surrounded by well-defined interstitial tissues. Primordial germ cells, spermatogonia, are arranged in concentric layers on the intact basement membrane followed by layers of spermatocytes. The lumina of seminiferous tubules were filled with mature sperms (Fig. 5A1 and A2). Almost similar anatomical layouts were observed in the VC and HC groups and the P+H group to a great extent (Fig. 5D, E, and C1 and C2 respectively). However, the DEP group showed severe kinds of pathological alterations including germ cell declination and degenerations, presence of residual bodies, amyloids, degeneration of seminiferous tubules, exfoliations, pyknotic nuclei, disrupted spermiation, and Leydig cell autolysis. Even in the mitotic zone, spermatogonial cells were scattered and randomized. Wide luminal spaces were a clear indication for the declination of mature sperms ( Fig. 5B1 and B2). However, the P+H group showed the presence of healthy seminiferous tubules well occupied with germ and supporting cells (Sertoli cells) as compared to the DEP group ( Fig. 5C1 and C2 and Table 4).

Discussion
Repeated exposure to DEP interferes with steroidogenesis as well as other reproductive parameters in developing mice. In this study, the mice's body weight gain was increased and they showed sluggish behavior after DEP treatment. Similarly, in another study, multiple phthalate types present in the urine samples resulted in an increased body mass index (BMI) of 5-to 12-year-old children compared to unexposed individuals (Harley 2017). Probably, the mechanism behind this was metabolites of DEP which showed more affinity for PPARγ receptors. It is strongly associated with commencing adipogenesis (Hurst and Waxman 2003;Rodríguez-Carmona et al. 2019). Honey supplementation in the P+H group successfully maintained the bodyweight like that of the control group and acted sometimes as a lipolytic compound. Another study on honey corroborated our findings, which stated that intake of 20% mono-floral honey reduced the body mass of rats (Nemoseck et al. 2011;Terzo et al. 2020).
The testes morphometry of the DEP group in present data showed testicular hypoplasia resulted in a decrease in the testes weight, testes width, and even gonado-somatic index. However, the length was similar to the control group. Several other researchers reported decreased gonadal size . Male mice, which were prenatally exposed to DEP mixture in 20 μg/kg and 500 mg/kg showed Abnormal sperms (%) 32.5 b ± 1.2 30.7 b ± 1.2 40.4 a ± 0.8 20.8 c ± 1.2 30.0 b ± 1.6 0.000 FRAP value (μM ascorbic acid equi/mg) 247.65 c ± 0.0005 250.67 b ± 0.0005 112.54 e ± 0.0005 282.9 a ± 0.0005 241.6 d ± 0.0005 0.000 Note: Values are expressed as mean± SEM. N, number of samples analyzed; C, untreated; VC, corn oil treated; DEP, diethyl phthalate exposed; P+H, diethyl phthalate + honey; HC, honey treated; FRAP, ferric reducing antioxidant power in testes homogenates. In rows, different alphabets showed significant difference among groups analyzed through one-way ANOVA followed Tukey's post hoc at level minimally P≤0.05 Fig. 3 Bar graph showed a statistical comparison of luteinizing hormone and testosterone level in plasma (mlU/ml) between five groups receiving different treatments expressed in mean ± S.E followed by Tukey's post hoc test decreased gonadal weight in a dose-dependent way. In the P+ H group, testes showed improved morphological and morphometric parameters as compared to the DEP group.
Histological sections showed histopathological defects in DEP-exposed testes like Leydig cell atrophy, degenerations, declination of germ cells, exfoliations, spacious lumen, and disrupted spermiation. These findings are correlated with previous evidence in which DEP exposure causes degenerations of seminiferous tubules, thin basement membrane of testes, and azoospermia (Mondal et al. 2019). In the P+H group, the testes histology showed much improvement in interstitial tissues, healthy spermatozoa with more spermatogonia, and Sertoli cells lining the basement membrane.
The micrometric observations revealed a significant reduction in seminiferous tubular diameter and a profound increase in the lumen diameter in the DEP group as compared to the control group. One obvious reason for the increase in luminal diameter is less production or degeneration of germ cells. Besides, honey supplementation increased seminiferous tubule diameter and decreased lumen diameter. Likewise, in another study, the utilization of honey enhanced the number of Leydig cells, seminiferous tubule diameter, and decreased lumen size against cigarette smoke-induced toxicity (Mohamed et al. 2011). The protection against DEP instigated histopathological lesions seems that raw honey contains handsome amounts of flavonoids and other polyphenols which may function as antioxidants and the consumption of antioxidants along with the protection against various pathologies, has a lot of potential health benefits (Vela et al. 2007;Yeung et al. 2019;Blassa et al. 2006).
Our finding showed sperm count as well as percentage of normal sperms was decreased and simultaneously percentage of abnormal sperms was significantly increased in DEP group than the control group. In previously reported data, DEP exposure caused a reduction in sperm motility and sperm density while sperm count has not exhibited any change among treatment and control groups (Mondal et al. 2019). All these findings were a clear indication of diethyl phthalate caused a decrease or reduce fertility rate. Our data indicates that in animals exposed to honey, testosterone level increased above average, while the LH level has decreased in the same mice. Various researches proved that honey maintained the level of testosterone (Banihani 2019). Less adipose tissue and more Leydig cell stimulated the testosterone balance in the body of male mice. Thus, honey is also loaded with a chrysin molecule which acts as the inhibitor of the aromatase enzyme in adipose tissues. The inhibition of the aromatase enzyme resulted in decreased conversion of testosterone into estrogen, hence testosterone level maintained (Jeong et al. 1999). In contrast to our results, honey administration raised the LH level in the blood serum (Kolawole et al. 2015). Similar to our outcomes, honey supplemented to rats in diabetic conditions maintained the LH level in blood serum (Kadiri 2018;Nasrolahi et al. 2013).
Our results clearly showed the direct and strong correlation between antioxidant capacity and total phenolic compounds. The increase in phenolic contents ultimately increased the antioxidant capacity of honey. Another research reported that there was a positive correlation between antioxidant capacity and total phenolic contents of honey analyzed through FRAP and TPC assays, respectively. Honey was enriched with antioxidant compounds and stabilized ROS by enhancing the activity of catalase and superoxide dismutase enzymes (SOD) (Mohamed et al. 2011). Thus, treatment with honey has shown clear histopathologic, morphometric, steroidogenic, and micrometric recoveries against DEP-instigated reproductive deteriorations in mice.

Conclusion
The present research showed that total phenols present in honey act as free radical scavengers and significantly prevent diethyl phthalate-induced reproductive damages in male mice. Multiflora honey from Apis mellifera can efficiently protect testicular lesions and hormonal deviations during the biological development of mice. Our observations revealed the remedial efficacy of honey on the reducing ability of testes, hormone production, and reclamation of germ cells and micrometric dimensions of seminiferous tubules. Based on the current findings, we suggest honey consumption revitalize spermatogenesis and steroidogenesis in mice exposed to environmental contaminants like DEP. Further studies are needed relating oxidative response and mechanism of action of honey against DEP in other mammalian models.