Ameliorative Effects of Sesame Seed Oil Against Penconazole-Induced Testicular Toxicity and Endocrine Disruption in Male Rats

The current study investigated the protective effect of sesame seed oil (SSO) against testicular damage and endocrine disruption induced by the oral intoxication with penconazole (PENZ), a commonly used systemic triazole fungicide, in adult male Wistar rats. Subchronic oral exposure to two different doses of PENZ significantly reduced fertility indices, serum luteinizing hormone, follicle stimulating hormone, triiodothyronine and thyroxine levels, and by contrast a significant increase was recorded in serum estradiol and testosterone levels. Furthermore, PENZ significantly depleted testicular vitamins (A, C and E) level. On the other hand, administration of SSO prior to PENZ intoxication ameliorated most of the fertility indices and minimized PENZ-induced adverse effects on serum sexual, reproductive and thyroid hormones, compared to PENZ-treated animals. Histological data confirmed the biochemical findings. In conclusion, supplementation of SSO might be beneficial against the endocrine-disrupting potential and testicular toxicity induced by PENZ in adult male rats. abstract


Introduction
Penconazole (PENZ), a fungicide that belongs to the triazole class, is commonly used in agriculture, horticulture and forestry industries for the control of powdery mildew [1]. It stops fungal growth by inhibiting lanosterol-14-demethylase (CYP51), which is required for ergosterol biosynthesis [2]. As a result of its in-depth utilization as an agrochemical, PENZ residues remain on treated crops, which may affect the environmental safety and human health [3,4]. Being refractory to degradation because of its long mean halflife value (117 days), PENZ is susceptible to accumulate in soils [5].

Animals
Adult male Wistar rats were obtained from the breeding unit of the Egyptian Stock Holding Company for Biological Products, Vaccines, Sera and Drugs (Cairo, Egypt). The animals were housed in plastic cages (5/cage) with a free access to commercial pelleted diet and tap water for one week before the start of the experiment as an acclimatization period. All animal procedures were performed under protocols approved by the Local Institutional Animal Ethics Committee of the Agricultural Research Center in accordance with the Guide for the Care and Use of Laboratory Animals (17).

Determination of PENZ Median Lethal Dose (LD50)
The median lethal dose (LD50) of PENZ was determined in adult male Wistar rats according to the OECD guideline for the testing of chemicals [18].

Experimental Protocol
In total, 60 adult male Wistar rats were equally allocated into six groups as follows: Normal control group: Rats were supplemented with the basal diet and left without any treatment.
Negative control group (SSO): Rats were supplemented with SSO using an intragastric tube at a dose of 1 ml/kg bw (5 days/week) for three consecutive months [19]. Positive control group I (LD PENZ): Rats were intoxicated with 30 mg/kg bw PENZ (1/20 of the LD50 of PENZ) using an intragastric tube five days per week for three consecutive months. Positive control group II (HD PENZ): Rats were intoxicated with 60 mg/kg bw PENZ (1/10 of the LD50 of PENZ) using an intragastric tube five days per week for three consecutive months. Preventive group I (SSO+LD PENZ): Rats were orally administered with SSO (1 ml/kg bw) 2 h prior to the oral intoxication with PENZ (30 mg/kg bw) 5 days per week for three consecutive months. Preventive group II (SSO+HD PENZ): Rats were orally administered with SSO (1 ml/kg bw) 2 h prior to the oral intoxication with PENZ (60 mg/kg bw) 5 days per week for three consecutive months.

Blood Collection and Tissue Sampling
At the end of the experimental period (90 days), animals were weighed, then blood samples were collected from the abdominal aorta under light ether anesthesia. A volume of 1 ml blood was dispensed into a heparinized tube that was centrifuged at 200g for 5 min and the separated plasma was aliquoted and stored at -40°C. The remainder of blood was collected in a plain centrifuge tube and left to stand for 30 min at room temperature to clot.
The blood was centrifuged at 500g for 15 min and serum was separated, aliquoted and stored at -40°C. At autopsy, the epididymis was immediately removed for the evaluation of fertility indices. The right testis was immediately frozen in liquid nitrogen and kept at -40°C until homogenization, whereas the left one was fixed in 10% phosphate-buffered formalin for histological examination.

Preparation of Testicular Tissue Homogenate
A 10% testicular homogenate was prepared in ice cold 1.17% potassium chloride using a chilled glass-Teflon Potter-Elvehjem tissue Homogenizer. An aliquot of the whole testicular tissue homogenate was used for the estimation of vitamin C concentration by the method of Henry et al. [20].

Extraction of Vitamins A and E From Testicular Tissues
Extraction of vitamins A and E from testicular tissues was done according to the method of Hinds et al. [21]. Testicular vitamin A and E concentrations were evaluated according to the colorimetric methods of Pett & Lepage and Martinek [22,23], respectively.

Fertility Indices
A 10% (w/v) homogenate was obtained by mincing the cauda epididymis of rats in isotonic saline at 37 for the evaluation of semen parameters, including sperm concentration and morphology [24] sperm motility [25] and sperm viability [26].

Biochemical Assays
Serum total testosterone (T) and estradiol (E2) levels were determined using RIA kits (Diagnostic Products Corporation, USA), while serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were analyzed using rat ELISA kits (Kamiya Biomedical Company, USA). Serum triiodothyronine (T3) and thyroxine (T4) levels were assayed using rat ELISA kits purchased from USCN and Cusabio Companies (USA), respectively.

Histological Examination
Testes fixed in formalin were embedded in paraffin and 4 μmthick sections were processed by a microtome. Tissue slides were stained with haematoxylin and eosin (H&E) and examined using a light microscope.

Statistical Analysis
The statistical difference between means was computed using one-way analysis of variance (ANOVA) followed by the posthoc

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orally the same dose of PENZ according to its weight. Toxicity signs of PENZ included paralysis, sedation, congestion in eyes and rough hair. The calculated oral median lethal dose (LD50) of PENZ, according to Weil [27], was 620.71 mg/kg bw.

The Percentage Survival of Rats at the End of the Experiment
No mortalities were recorded in normal control and SSO-administered rats, whereas PENZ treatment of low and high doses caused 20 and 40% deaths, respectively, and by contrast, administration of SSO prior to PENZ intoxication reduced the death percentage to only 10% in both SSO-supplemented groups at the end of the treatment period.

Effect of Treatment with SSO and/or PENZ Intoxication on Body Weight as well as Relative Testis and Epididymis Weights
Administration of SSO and/or PENZ caused non-significant changes in total body weight, as well as absolute and relative testis weights, whereas a significant reduction in absolute and relative epididymis weights (

Effect of Treatment with SSO and/or PENZ on Fertility Indices in Male Rats
Treatment of male rats with low and high doses of PENZ resulted in the deterioration of fertility markers, as evidenced by the significant reduction in sperm count, motility as well as viability and protective groups showed that all the fertility-related parameters were ameliorated with SSO supplementation ( Table 2). Table 1: Effect of treatment with sesame seed oil (SSO) and/or penconazole (PENZ) on body weight, relative and absolute testis and epididymis weights in male rats.

Groups
Body weight (g) Note: Results are mean ± standard deviation (SD). The F-ratio is significant at p<0.05. The letters a, b, c, d, e, and f denote significant change versus the normal control group, SSO, LD PENZ, HD PENZ, SSO+LD PENZ, and SSO+HD PENZ groups, respectively. *, **, *** percentage change versus normal control, LD PENZ and HD PENZ groups, respectively. Note: Results are mean ± standard deviation (SD). The F-ratio is significant at p<0.05. The letters a, b, c, d, e, and f denote significant change versus the normal control group, SSO, LD PENZ, HD PENZ, SSO+LD PENZ, and SSO+HD PENZ groups, respectively. *, **, *** percentage change versus normal control, LD PENZ and HD PENZ groups, respectively.

Effect of treatment with SSO and/or PENZ on Serum Testosterone (T), Estradiol (E2), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels
The intoxication of adult male rats with low and high doses of  (Table 3). Note: Results are mean ± standard deviation (SD). The F-ratio is significant at p<0.05. The letters a, b, c, d, e, and f denote significant change versus the normal control group, SSO, LD PENZ, HD PENZ, SSO+LD PENZ, and SSO+HD PENZ groups, respectively. *, **, *** percentage change versus normal control, LD PENZ and HD PENZ groups, respectively.  Note: Results are mean ± standard deviation (SD). The F-ratio is significant at p<0.05. The letters a, b, c, d, e, and f denote significant change versus the normal control group, SSO, LD PENZ, HD PENZ, SSO+LD PENZ, and SSO+HD PENZ groups, respectively. *, **, *** percentage change versus normal control, LD PENZ and HD PENZ groups, respectively.

Effect of Treatment with SSO and/or PENZ on Testicular Vitamins (A, C and E)
The intoxication of male rats with low and high PENZ  Note: Results are mean ± standard deviation (SD). The F-ratio is significant at p<0.05. The letters a, b, c, d, e, and f denote significant change versus the normal control group, SSO, LD PENZ, HD PENZ, SSO+LD PENZ, and SSO+HD PENZ groups, respectively. *, **, *** percentage change versus normal control, LD PENZ and HD PENZ groups, respectively.

Effect of Treatment with SSO and/or PENZ on Testicular Histology
A normal histological structure of the mature, active seminiferous tubules with complete spermatogenic series was noticed in the testes of normal rats and those supplemented with SSO ( Figure 1A). Treatment of male rats with the low PENZ dose caused testicular degeneration in most of the seminiferous tubules along with increased intertubular space ( Figure 1B).
In addition, the high PENZ dose induced degeneration, atrophy and desquamation in the seminiferous tubules, as well as the absence of spermatogenic series, increased intertubular space and congestion in stromal blood vessels ( Figure 1C). On the other hand, the administration of SSO prior to the low PENZ dose intoxication caused the irregular arrangement of spermatogenic series in some tubules and desquamation in seminiferous tubule ( Figure   1D). By contrast, rats co-administered SSO with high PENZ dose intoxication demonstrated a normal intact histological structure of most of the seminiferous tubules, whereas maturation arrest was noticed in other seminiferous tubules ( Figure 1E).

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Figure 1: Photomicrographs of rat testis sections stained with haematoxylin and eosin (40). The normal histological structure of the mature, active seminiferous tubules with complete spermatogenic series (s) in normal control and SSO-treated animals was shown (A). The testis of a low dose PENZ-treated rat showed degeneration in most of the seminiferous tubules (ds) and increased intertubular space (B). The high dose PENZ-treated rat demonstrated increased testicular intertubular space, degenerated (s) and atrophied (a) seminiferous tubules, absence of spermatogenic series, congestion in stromal blood vessels, hemorrhage (v) inside the seminiferous tubules as well as desquamation (C). The testis of a male rat administered SSO before the low dose PENZ intoxication showed the irregular arrangement of spermatogenic series in some tubules (s) and desquamation (o) in seminiferous tubules (D). A male rat administered SSO before the high dose PENZ intoxication demonstrated almost a normal intact histological structure of most of the seminiferous tubules (s) and maturation arrest in other seminiferous tubules (E).

Discussion
Many chemicals, including pesticides with their widespread presence in the environment, have been suspected over recent decades to be endocrine disruptors [28]. Also, there is much concern that exposure to pesticides may cause reproductive toxicity through impairment of sperm motility, decreasing sperm counts, reducing fertilization ability and producing abnormal sperms [29]. Reproductive toxicity of PENZ and other triazole fungicides had been previously reported [1,16,30]. Therefore, abnormalities

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showed degeneration and atrophy in the seminiferous tubules and low luminal spermatozoa concentration. It is therefore reasonable to suggest that the decrease in the sperm count is an ultimate outcome of spermatogenic arrest following PENZ intoxication. Also, abnormalities in sperm morphology could be attributed to a direct toxic injury of PENZ, at both doses, to seminiferous tubules.It is well known that antioxidants enhance fertility, since antioxidant-rich plants improve sperm morphology and increase sperm motility and counts [33]. Therefore, the observed ameliorative effect of SSO on the fertility indices of PENZ-treated rats could be attributed to its high contents from free radical scavenger antioxidants [34].
In agreement with our finding, Ukwenya et al. [35] studied the effect of the alcoholic extract of sesame seed on the fertility of male Sprague Dawley rats and recorded an elevation in sperm motility.
Thyroid hormones are essential for normal spermatogenesis and regulate testicular function in rats of all ages [36]. Thus far, the literature contains no evidence regarding the effect of PENZ on thyroid hormones. We reported a dose-dependent decrease in the level of serum T3 and T4 following intoxication of male rats with PENZ. A direct toxic effect of PENZ on the thyroid gland was histologically confirmed in the current study (data not shown). It has been reported that pesticides, including triazoles, can devastate the reproductive system by changing the hormonal balance [37].
Accordingly, the disturbance in serum androgen-estrogen balance and fertility hormones level in the present study is a clear evidence for the endocrine disrupting potential of PENZ. Generally, triazoles increase serum testosterone level and lead to hepatomegaly and fertility reduction [38,39].
Triazoles had been reported to disrupt testosterone homeostasis in rats via downregulating the expression of hepatic genes involved in testosterone metabolism, along with upregulating testicular expression of genes involved in testosterone synthesis [40]. As compared to the respective positive control groups, the slight significant reduction in serum estradiol level of PENZ-intoxicated We previously demonstrated that PENZ induced oxidative damage in the testis of adult male rats, as evidenced by the enhancement of lipid peroxidation and suppression of antioxidant enzymes (catalase and superoxide dismutase), and that administration of SSO prior to PENZ intoxication protected the testes from the induced oxidative stress by lowering the testicular lipid peroxidation level and inducing antioxidant enzymes activity [42]. Antioxidant vitamins, including A, C and E, were reported to reduce chlorpyrifos-induced lipid peroxidation in different organs, by inhibiting the DNA damage induced by reactive oxygen metabolites, preventing therefore ultimate genetic changes [43].
Vitamin C is present in the spermatogenic chamber and Leydig cells and protects spermatogenesis, increases testosterone levels, prevents sperm agglutination and plays a major role in semen integrity and fertility [44]. Also, vitamins A and E act as antioxidants and stabilize testicular cell membranes by reducing lipid peroxidation [45]. The depletion of the aforementioned vitamins in the testis of PENZ-treated rats resulted from their utilization in antioxidant reactions with radicals generated by PENZ. By contrast, administration of SSO prior to PENZ intoxication normalized testicular vitamin A level and significantly increased vitamin C and E levels, compared to PENZ-treated rats. Sesame seed oil is well known for its oxidative stability, which is attributed to its vitamin C and tocopherol contents. Hemalatha & Ghafoorunissa [46]reported that sesame lignans increase tissue tocopherol levels by inhibition of CYP450 3A-dependent n-hydroxylase pathways of tocopherol catabolism, enhancing therefore the quality and morphology of the produced sperms, which ultimately protected the testis from PENZ-induced damage in the current study.
Therefore, SSO supplementation decreased the generation of ROS, prevented PENZ-induced derangements in the antioxidant system and improved the redox status in testicular tissues.

Conclusion
The current study demonstrated an endocrine disrupting potential and a testicular toxicity for PENZ in adult male rats. Oral administration of SSO was effective in minimizing the endocrine disruption and counterbalancing the toxic effects of PENZ on the male reproductive system. It is important to consider the possibility of SSO as a food supplement for men in agriculture practice to minimize the toxic effects on male fertility and to limit the toxicity against pesticides-exposure on the reproductive system.