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ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 2  |  Page : 351-358

The protective effect of pumpkin seed oil on L-thyroxine-induced rat testicular oxidative stress


Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission17-Sep-2020
Date of Decision15-Oct-2020
Date of Acceptance01-Nov-2020
Date of Web Publication27-Jul-2022

Correspondence Address:
Nader Galal Zaki
Shebin El-Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_333_20

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  Abstract 


Objectives
This work aimed to study the effect of exogenously administered L-thyroxine on adult rat testis and to clarify the possible protective effect of pumpkin seed oil.
Background
L-thyroxine is a manufactured form of the thyroid hormone thyroxine. It is used for the treatment of cases of thyroid hormone deficiency. Recently, infertility among men has become a major concern. Pumpkin is an annual herbaceous plant and has been safely used as a natural remedy in many countries. It has a wide range of pharmacological activities and a proposed antioxidant effect.
Materials and methods
A total of 42 adult male albino rats were divided into the control group, pumpkin seed oil-treated group (4 ml/kg) orally for 10 days, L-thyroxine-treated group (0.3 mg/kg) intraperitoneal for 15 days, and both pumpkin seed oil and L-thyroxine-treated group. Specimens from the testes were processed for light microscopy. An immunohistochemical study was carried out using caspase 3.
Results
Specimens from L-thyroxine-treated rats showed a statistically significant decrease in both tubular diameter and thickness of the epithelium of seminiferous tubules. Germinal epithelium showed cellular degeneration, vacuolated cytoplasm, and also there were apoptotic and multinucleated giant cells.
The immunohistochemical study of the L-thyroxine-treated group showed intense positive caspase 3 immunostaining. In contrast, minimal changes were observed in rats treated concomitantly with both pumpkin seed oil and L-thyroxine-treated rats, with minimal change in immunoreaction compared with the control group.
Conclusion
Exogenously administered L-thyroxine induced significant testicular damage and germ cell apoptosis, which could be ameliorated by concomitant treatment with pumpkin seed oil.

Keywords: oxidative stress, pumpkin, testis, thyroxine


How to cite this article:
Zaki NG, Shabana FM, El-sherif NM, El-Habeby MM. The protective effect of pumpkin seed oil on L-thyroxine-induced rat testicular oxidative stress. Menoufia Med J 2022;35:351-8

How to cite this URL:
Zaki NG, Shabana FM, El-sherif NM, El-Habeby MM. The protective effect of pumpkin seed oil on L-thyroxine-induced rat testicular oxidative stress. Menoufia Med J [serial online] 2022 [cited 2024 Mar 28];35:351-8. Available from: http://www.mmj.eg.net/text.asp?2022/35/2/351/352196




  Introduction Top


L-thyroxine is used to treat the cases of thyroid hormone deficiency[1].

It is a manufactured form of the thyroid hormone, thyroxine (T4). The exact mechanism of action occurs by genomic and nongenomic effects. They exert their genomic effects by diffusing into the cell nucleus and binding to thyroid hormone receptors in DNA regions[2]. Adverse effects are generally caused by incorrect dosing. Long-term suppression of thyroid-stimulating hormone (TSH) levels below normal values will frequently cause cardiac adverse effects and contribute to decreases in bone mineral density (low TSH levels are also well known to contribute to osteoporosis)[3].

A dual regulatory role of thyroid hormone on Leydig cell functions was proposed by examining the effects of T3 on steroidogenesis and luteinizing hormone (LH) cell receptors in Leydig tumor cell lines[4].

However, information on its role in regulating the various physiological and biochemical functions of the adult testis in general and antioxidant defenses, in particular, is inadequate[5].

In recent years, there is a growing interest in the use of natural products, especially those derived from plants.

Fluted pumpkin, known as Telfairia occidentalis, is a species of the Cucurbitaceae family in the tropics and largely consumed in Nigeria, Ghana, and the Sierra[6].

Pumpkin seed and seed oil are rich natural sources of phytosterols[7], proteins, polyunsaturated fatty acids[8], antioxidant vitamins, carotenoids, tocopherols[9], and various elements[10].

Fluted pumpkin seed oil (FPSO) has been reported to possess some essential properties (vitamin A, tannins, linoleic acid, oleic acid, and alkaloids) that suppress lipid peroxidation, hence, improving testicular function[11].

The present study aimed to assess the effect of exogenously administered L-thyroxine on adult rat testis and to clarify the possible protective effect of pumpkin seed oil.


  Materials and methods Top


The procedure was approved by the ethics committee on the animal experiment of the Faculty of Medicine, Menoufia University, Egypt, by the international regulations on care and use of laboratory animals.

This experimental prospective study was done on 42 adult male albino rats from October 2019 to November 2019 at Theodor Bilharz Research Institute. Inclusion criteria were male albino rats, 150–200 g, with age from 1 month to 1 year. Exclusion criteria included female rats, weight less than 150 g, black rats, and age less than 1 month or more than 1 year.

Randomization was achieved by numbering the rats from 1 to 42, then dividing them randomly into four groups. There was no selection bias because we followed the inclusion criteria. Blinding was achieved by nontelling the investigator about the division of groups.

Sample size

Sample size was based on previous studies. Elfiky et al.[12] studied T4-induced stress and its possible prevention by catechin and reported that T4 induced significant alteration of testis weight (TW) in T4-administered rats by 1.457 ± 0.019 g% of body weight, whereas alteration of TW in the control group was 1.108 ± 0.015 g% of body weight. The calculated sample size using the sample size calculation sheet is 42 adult male rats.

Chemicals

Pumpkin seed oil was obtained from EL Captin Company (Al Obour City, Cairo, Egypt). Eltroxin (100 μg) was purchased from Al Menoufia Pharmacy (Shibin El-Kom, Menoufia, Egypt).

Study design

Forty-two adult male albino rats were used in this study; each of them weighed from 150 to 200 g. All animals were kept in clean properly ventilated cages with free access to a balanced laboratory diet and water. They were acclimatized for 2 weeks before the experiment.

The animals were divided into four groups. Group I (control group) included 12 rats subdivided into two subgroups (six rats each) as follows: subgroup IA (normal control subgroup) was not injected by the drug or distilled water, and subgroup IB (sham control subgroup) was injected intraperitoneally with distilled water. Group II (pumpkin seed oil-treated) consisted of 10 rats receiving a daily dose of 4 ml/kg of pumpkin seed oil by oral gavages for 10 consecutive days[13].

Group III (L-thyroxine treated) consisted of 10 rats receiving a daily dose of 0.3 mg/kg of eltroxin intraperitoneally for ∼15 days[14].

Group IV (L-thyroxine and pumpkin seed oil treated) consisted of 10 rats receiving a daily dose of 4 ml/kg of pumpkin seed oil by oral gavages for 10 days in addition to 0.3 mg/kg of eltroxin intraperitoneally for 15 days.

At the end of the determined period of each group, the animals from all groups were anesthetized using ether inhalation. Blood samples were collected from the medial canthus, and then, a median abdominal incision was achieved to expose the testes. The two testes of each rat were carefully dissected.

Biochemical analysis was conducted as follows: the collected blood samples were used for measurement of the total serum level of testosterone hormone and superoxide dismutase (SOD) enzyme. For the histological study, tissue samples were fixed in 10% formalin, then the specimens were dehydrated, cleared, and embedded in paraffin blocks. Serial coronal sections were cut 5–7-μm thick and stained with the routine hematoxylin and eosin, periodic acid- Schiff (PAS), and Van Gieson stain, and then examined by light microscope. For immunohistochemistry, 5-μm-thick testicular sections were deparaffinized, rehydrated, and washed in phosphate-buffered saline. The primary antibody (monoclonal mouse caspase 3 antibody) was applied by dropping on the tissue sections, then incubated horizontally in the humidity chamber for an hour, at room temperature. Biotinylated goat-polyvalent secondary antibody was applied for 10 min at room temperature. Sections were incubated in preformed streptavidin peroxidase for 10 min at room temperature and rinsed three times in phosphate-buffered saline. The immunoreactivity was visualized using 3, 3'diaminobenzidine–hydrogen peroxide as a chromogen.

Quantitative study was done on Image J analyzer (Leica Microscope DML, Image J analyzer version 1.4308; National institute of health in Bethesda City, Washington DC state Institutes of Health, USA) at Central Research Lab, Faculty of Medicine, Menoufia University, Egypt. Using hematoxylin and eosin-stained sections of different groups, morphometric measurements in the form of seminiferous tubular diameter and seminiferous epithelial thickness were measured (under light microscopy using an objective lens 40, scale bar 20). Using PAS and Van Gieson-stained sections of different groups, the color intensity of fibrosis was measured using an objective lens 20, scale bar 50. Using caspase 3-stained sections of different groups, the number of caspase 3-positive cells was measured using an objective lens 40, scale bar 20.

Statistical analysis

Data were analyzed using an IBM-compatible personal computer with Statistical Packages for the Social Science (SPSS), version 23 (2015; IBM Corporations, Armonk, New York, USA) where the following statistics were applied:

Descriptive statistics, in which quantitative data were presented in the form of the mean and SD.

Analytic statistics used were as follows: analysis of variance test was used for comparison of quantitative variables between more than two groups of normally distributed data. Kruskal–Wallis test was used for comparison of quantitative variables between more than two groups of not normal distributed data. Differences were regarded as significant if P value was less than 0.05.


  Results Top


No animal little mortalities replacing (no animal deaths) deaths were reported throughout the experiment, except in the L-thyroxine group with little mortalities.

General results

A significant decrease in body and TW was observed in L-thyroxin-treated group (group III) (P < 0.001) as compared with the control group; however, there was a significant increase in body and TW in pumpkin seed oil and L-thyroxin-treated group (group IV) (P < 0.001) as compared with the L-thyroxin group (group III).

Biochemical results

Regarding total serum testosterone level, a significant decrease in total serum testosterone level was observed in L-thyroxin-treated group (group III) (P < 0.001) as compared with the control group (group I). However, there was a significant increase in its level in pumpkin seed oil and L-thyroxin-treated group (group IV) (P < 0.001) as compared with the L-thyroxin group (group III).

Regarding the total serum SOD level, a significant decrease in total serum SOD level was observed in L-thyroxin-treated group (group III) (P < 0.001) as compared with the control group I. However, there was a significant increase in its level in pumpkin seed oil and L-thyroxin-treated group (group IV) (P < 0.001) as compared with the group III.

Histological results

Hematoxylin and eosin stain

Group I (control): sections of this group showed a normal histological picture of rat testis. The testis showed many seminiferous tubules with fine connective tissue trabeculae in-between [Figure 1]a. The tubules were bounded by basal laminae and lined by a complex stratified epithelium that varied from one tubule to another showing different stages of spermatogenic cells. The spermatogenic cells [Figure 1]b formed of spermatogonia (curved arrow) that appeared as small rounded cells resting on a basement membrane, primary spermatocytes (arrowhead) with rounded dark nuclei that were the largest, and spermatids which appeared near the lumen. Secondary spermatocytes (notched arrow) were smaller than the primary spermatocytes and nearer to the lumen. However, they were hardly recognized as they usually have a very short life span. Sertoli cells (arrow) with triangular nuclei were observed in between the germ cells. The lumen of the tubules appeared filled with sperms (S) [Figure 1]a and [Figure 1]b.
Figure 1: Representative hematoxylin and eosin staining of rat testes of different experimental groups. (a) The testes of control rats showed normal structure of seminiferous tubules containing spermatogenic cells with normal interstitial tissue in-between. (b) The spermatogenic cells included spermatozoa in the lumen (S), spermatogonia (curved arrow), primary spermatocyte (arrow head), secondary spermatocyte (notched arrow), and sertoli cell (arrow). (h) In the testes of L-thyroxine-treated group, spermatogenic cells appeared disorganized with homogenous acidophilic exudates in-between the tubules. (d) No sperms in lumen of tubules. (e) Some tubules showed replacement of spermatogenic cells with cytoplasmic vacuolations and showed giant cells in the lumen. (f) Cells with pyknotic nucleus. (g) Apoptotic cells. (i) Pumpkin and L-thyroxine-treated group showed some degenerated tubules side by side to normal ones.

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Group II (pumpkin seed oil-treated group): sections of the testes of this group revealed the same normal histological architecture as the control group. The seminiferous tubules appeared normal in appearance and arrangement and contained different germ cells (arrow). Several mature spermatozoa appeared in the lumen, and fine interstitial tissue (I) was seen in-between the seminiferous tubules [Figure 1]c.

Group III (L-thyroxin-treated group): sections of this group showed massive destructive changes including distortion of the seminiferous tubules with loss of spermatozoa in the lumen [Figure 1]d. Some tubules showed degeneration of its cells, which were replaced with cytoplasmic vacuolations [Figure 1]e. The damaged spermatogenic cells were exfoliated in the lumen [Figure 1]h, leaving thin disorganized germinal epithelium. Giant cells appeared in the lumen of some tubules [Figure 1]e. The pyknotic nucleus with vacuolar cytoplasm and karyorrhectic nuclei were noticed in the seminiferous tubules [Figure 1]f. The early sequences of apoptosis were detected, as apoptotic cells could be seen surrounded by a clear halo [Figure 1]g. Widening of the interstitium with the appearance of acidophilic homogenous exudates was seen [Figure 1]h.

Group IV (pumpkin seed oil and L-thyroxin-treated group): the testes of the rats of this group showed some seminiferous tubules which were nearly similar to control. Some degenerated tubules (curved arrow) were observed beside normal ones (arrow) [Figure 1]i.

Periodic acid-Schiff stain

Control group: sections from the control animals showed moderate positive PAS reaction in the well-circumscribed basal lamina of seminiferous tubules [Figure 2]a.
Figure 2: Representative PAS and Van Gieson staining of rat testes of different experimental groups. (a) The control group showed moderate PAS reaction in basal laminae of seminiferous tubules. (b) L-thyroxine-treated group showed strong positive PAS reaction in basal lamina and around blood vessels. (c) Pumpkin seed oil and L-thyroxine-treated group showed moderate positive PAS reaction. (d) The control group showed normal seminiferous tubule surrounded by basal lamina. (e) L-thyroxine-treated group showed seminiferous tubule with moderate fibrosis (red color) around the basal lamina and dilated congested blood vessel surrounded with dense fibrosis (red color). (f) Pumpkin seed oil and L-thyroxine-treated group showed minimal fibrosis (tinge of red color) around basal lamina of seminiferous tubules.

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Group III (L-thyroxin-treated group): sections from the L-thyroxin-treated group showed strong positive PAS reaction in the basement membrane of seminiferous tubules and around blood vessels [Figure 2]b.

Group IV (pumpkin seed oil and L-thyroxin-treated group): sections from this group showed moderate positive PAS reaction in the basement membrane of seminiferous tubules with moderate PAS-positive material deposition in-between seminiferous tubules [Figure 2]c.

Van Gieson stain

Group I (control group): sections of this group showed normal seminiferous tubules, surrounded by basal lamina, with normal interstitial tissue in-between [Figure 2]d.

Group III (L-thyroxin-treated testis): the sections showed seminiferous tubule with minimal fibrosis around the basal lamina and dilated congested blood vessel surrounded with dense fibrosis)[Figure 2]e).

Group IV (pumpkin seed oil and L-thyroxin-treated testis): there was mild fibrosis around basal lamina of seminiferous tubule)[Figure 2]f).

Immunohistochemical results

In group I (control), sections showed negative immunostaining for caspase 3 [Figure 3]a. In group III (L-thyroxin-treated testis), sections showed intense positive caspase 3 (deep brown) cytoplasmic immunostaining [Figure 3]b. In group IV (pumpkin seed oil and L-thyroxin-treated testis), sections showed moderate cytoplasmic caspase 3 immunostaining in some germ cells [Figure 3]c.
Figure 3: Representative caspase 3 immunostaining of rat testes of different experimental groups. (a) The control showed negative cytoplasmic caspase 3 immunostaining. (b) L-thyroxine-treated group showed intense positive caspase 3 immunostaining. (c) Pumpkin seed oil and L-thyroxine-treated group showed moderate positive caspase 3 immunostaining.

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Morphometric and statistical results

The mean tubular diameter of seminiferous tubules and thickness of seminiferous epithelium showed a statistically significant decrease in L-thyroxine-treated group (group III) compared with the control group. However, there was a significant increase in their values in pumpkin seed oil and L-thyroxin-treated group (group IV) (P < 0.001) as compared with L-thyroxin group (group III) [Table 1].
Table 1: Means±SE of tubular diameter of seminiferous tubule and tubular thickness of seminiferous epithelium

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The color intensity of fibrosis and the mean number of caspase 3-positive cells showed a statistically significant increase in L-thyroxine-treated group (group III) compared with the control group. However, there was a significant decrease in their values in pumpkin seed oil and L-thyroxin-treated group (group IV) as compared with L-thyroxin group (group III) [Table 2].
Table 2: Means±SE of color intensity of fibrosis and number of caspase 3-positive cells

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  Discussion Top


The thyroid hormone is considered as one of the important physiological regulators of the tissue. The deficiency of this hormone in the body leads to a decrease in several cellular activities such as basal metabolic activity and respiration, which are restored to normal levels by the supplementation of the hormone[15].

A dual regulatory role of thyroid hormone on Leydig cell functions was proposed by examining the effects of T3 on steroidogenesis and LH cell receptors in Leydig tumor cell lines[4].

However, information on its role in regulating the various physiological and biochemical functions of the adult testis in general and antioxidant defenses, in particular, is inadequate[5].

Fluted pumpkin, known as T. occidentalis is a species of the Cucurbitaceae family in the tropics and largely consumed in Nigeria, Ghana, and Sierra[6].

Fluted pumpkin seed oil has been reported to possess some essential properties (vitamin A, tannins, linoleic acid, oleic acid, and alkaloids) that suppress lipid peroxidation, hence improving testicular function[11].

Accordingly, this research aimed to study the effect of exogenously administered L-thyroxine on adult rat testis and to clarify the possible protective effect of pumpkin seed oil by biochemical, histopathological, immunohistochemical, and morphometric studies.

After reviewing the light microscopic results of this study, it could be demonstrated that treatment with L-thyroxine has altered the histological structure of the testis and caused marked destruction of seminiferous tubules with sloughing of their epithelial cells. The remaining epithelial cells were vacuolated and degenerated having pyknotic nuclei. These results were in agreement with Rockett et al.[16], who noticed variably sized vacuoles in the seminiferous tubules.

Rai et al.[17] explained the presence of the vacuoles in the seminiferous tubules by the intratesticular lipid deposition. L-thyroxin-treated group also showed multinucleated giant cells in the lumen of seminiferous tubules. This is in agreement with the results of the studies conducted by Ghoneim et al.[18].

Hess and Nakai[19] explained the appearance of these multinucleated giant cells as a result of the widening of the intercellular bridges between the adjacent spermatids, leading to the fusion of two or more cells.

In the present work, a light microscopic study showed the appearance of apoptotic germ cells near the basal lamina.

This was in agreement with Yazawa et al.[20] who reported that the predominant cell types undergoing apoptosis included spermatogonia.

In this study, widening of the interstitium with the appearance of homogenous acidophilic exudates was noticed in L-thyroxin-treated group.

This is in harmony with the results of the study conducted by Abdellatief et al.[21].

The widening of the interstitium was in line with the results of Elgawish and Abdelrazek[22], who accounted for the widening of peritubular tissue owing to the withdrawal of gonadotrophic stimulation as occurring after hypophysectomy.

Hasanin et al.[23] reported that the inter-tubular homogenous acidophilic material detected in this study could be attributed to excess lymphatic exudates oozing from degenerated lymphatic vessels or owing to an increase in vascular permeability.

Add to this, there was marked degeneration of germinal epithelium as demonstrated by the morphometric results, which revealed a statistically significant decrease in both the tubular thickness of seminiferous epithelium and diameter of seminiferous tubules. These results were in harmony with the clinical results of some authors who postulated that injection of L-T4 0.3 mg/kg for about 15 days induced an increase in serum T3 and T4 levels associated with a reduction in serum TSH levels, and a significant decrease in LH, follicle-stimulating hormone, and testosterone serum levels was reported[24].

The results of the present study demonstrated the modulation of the antioxidant defense system of adult rat testis by thyroid hormone as evidenced by Manna et al.[4], who attributed the suppressed steroidogenesis associated with hyperthyroidism to the reduction of cholesterol supply to the mitochondrial inner membrane caused by inhibition of StAR (steroidogenic acute regulatory protein) synthesis.

The generation of superoxide radicals takes place owing to the incomplete reduction of oxygen molecules during cellular respiration[24]. Superoxide radicals are converted to hydrogen peroxide by the enzyme SOD.

In the present study, there was a decrease of SOD in the testes in response to hyperthyroidism. This may be attributed to an increase in oxidative stress that exceeds cellular antioxidant enzyme activities[25].

The explanation for decreased SOD serum level is that the thyroid hormone may cause downregulation and up-regulation of SOD and catalase enzyme genes, respectively[26].

This was in harmony with Shiva et al.[27] who found that increase in reactive oxygen species formation owing to lipid peroxidation and compromised antioxidant defense system in hyperthyroid rats is associated with mid-piece abnormalities and decreased sperm count and motility.

In this study, there was a marked deposition of collagen fibers in the basement membrane of seminiferous tubules, tunica albuginea, and around blood vessels in L-thyroxin-treated group.

The marked deposition of collagen fibers in the testicular capsule, the interstitial tissues, and in the basal lamina, in addition to the presence of strong PAS-positive materials in basal lamina and in between the seminiferous tubules was attributed to the oxidative stress induced by L-thyroxin. This explanation coincided with the finding of Sciarra and Toscano[28], who reported that the formation of reactive oxidizing molecules led to lipid peroxidation, causing damage to proteins and nucleic acids. The results of these reactions led to an increase in collagen fibers and ground substance formation.

The present study showed that L-thyroxin treatment induced apoptosis in both germ and Leydig cells, as indicated by increased expression of activated caspase 3 in the L-thyroxin-treated group compared with the control group. Therefore, it is likely that the reduction in testosterone levels occurred as a result of the increased Leydig cell death, thereby affecting endocrine function.

In this study, pumpkin seed oil was used for protection at a dose of 4 ml/kg by oral gavages for 10 consecutive days. According to Elfiky et al.[12], pumpkin seed oil is found to be very effective in protection from L-thyroxin-induced toxicity in the testes.

In the present study, the histological study of the testis of pumpkin seed oil and L-thyroxin-treated group showed that most of the seminiferous tubules appeared normal with normal spermatogenic cells and a lumen full of sperms. Only a few tubules were affected, with no sperms in the lumen. Some congestion of blood vessels and some increase in collagen fibers in tunica albuginea, in the interstitium, and around the blood vessels were present.

The improvement of histopathological findings in this group compared with L-thyroxin-treated group may be owing to the antioxidant effect of pumpkin seed oil which ameliorates the oxidative stress caused by L-thyroxin.

This study goes with Hashemi[29], who found that oral pretreatment of rats with pumpkin seed oil ameliorated the effect of quinine-induced testicular damage and significantly increased the weight of testes; normalized serum levels of testosterone, follicle-stimulating hormone, and LH; improved semen quality and quantity; and ameliorated the testicular degenerative damage induced by oxidative stress. There was also a significant decrease in lipid peroxidation and an increase in activities of antioxidant enzymes in the testicular tissue.

The mechanism of pumpkin seed oil's protective role was explained by Procida et al.[30], who reported that pumpkin seed oil is rich in many antioxidants and has beneficial nutritional components such as essential fatty acids, amino acids, phytosterols, B-carotenes, lutein, and selenium.


  Conclusion Top


In conclusion, exogenous administration of L-thyroxin caused significant testicular damage and germ cell apoptosis, which probably affected reproductive functions.

Pretreatment of L-thyroxin-treated rats with pumpkin seed oil significantly protected testicular histology and function owing to antioxidative properties.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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