Apigenin Ameliorates Lead Acetate induced Hyperlipidemia, Hypothyroidism and Hypogonadism in Male Rats

Lead (Pb) is an environmental pollutant and has detrimental effects on human health. Apigenin (APG) is a avonoid that have antioxidant, anti-inammatory, antiallergic, and cardioprotective effects and is presented as treatment of some diseases. The aim of the present study was to evaluate the probable protective effect of APG against Pb-induced toxicity in rats. Adult male rats were given either Pb (as lead acetate; 20 mg/kg) alone or in combination with APG (20 mg/kg) daily for 4 weeks by intraperitoneally injection (i.p). At the end of the experimental period, Pb accumulation, lipid prole, thyroid, and testicular function alterations were assessed. In addition, histopathological changes in the testis were assessed.Results revealed that Pb treatment signicantly increased Pb concentrations in blood, thyroid gland, and in testis of rats. Further, the blood levels of hormones related to thyroid gland and testis were altered in Pb-treated rats. In parallel, low sperm count and sperm motility, increased sperm abnormalities, and marked pathological changes in testis were observed. On the contrary, the treatment with both Pb and APG recorded amelioration of the deleterious effects of Pb, involving attenuation of changes in lipid prole, thyroidal and testicular hormonal levels, sperm parameters and pathological changes in Pb treated rat’s testis. In conclusion, it appears that dietary APG can ameliorate lead acetate induced hyperlipidemia, hypothyroidism and hypogonadism in male rats. peroxidation, Superoxide dismutase, Catalase and glutathione peroxidase activities were determined using commercial kits (catalog # MD2528, SD2520, CA2516, GP2524 respectively) obtained from Diagnostic, Giza, Egypt. TNF α was determined using Human ELISA Kit (catalog # EA100365) OriGene Technologies Inc., MD, IL-6 was determined using Rat Interleukin 6 (IL-6) ELISA Kit (catalog # MBS726707), IL-4 was determined using Rat Interleukin 4 (IL-4) ELISA Kit (catalog #MBS162452) , Interleukin 10 (IL-10) was determined using Interleukin 10 (IL-10) ELISA Kit ELISA Kit (catalog #MBS764911) MyBioSource, Inc., San Diego, USA. Determination of caspase-3 was done using Biotechne kit (catalog # BF3100) (USA). T4 ELISA kit (Cat. No. 60863) was purchased from Kamiya Biomedical Company, WA, USA. T3 solid-phase competitive ELISA kit (Cat. No. T3043T-100) was obtained from CalbioTech Inc., Spring Valley, Canada. TSH ELISA kit (Cat. No. CSBE05115r) was obtained from CUSABIO Biotech Co., LTD, Wuhan, China. Kit for determination of T was purchased from K-assay, WA, USA. LH and FSH kits were obtained from Biovender, Tokyo, Japan. Determination of caspase-3 was done using Biotechne kit (catalog # BF3100) (USA). 1 drop of sperm suspension was placed on a glass slide to analyze 200 motile sperm in 4 different elds. The motility of the sperm was evaluated microscopically within 2– 4 min of their isolation from the testis, and data were expressed as percentage motility (Morrissey et al. 1988). The sperms were counted using a hemocytometer following the method of Freud and Carol (1964). The technique of Evans and Maxwell (1987) was adopted for sperm abnormality study. Briey, smears were made by placing a drop from the sperm suspension and one or two drops of the previously warmed (37 °C) eosin–nigrosin stain. The smears were allowed to dry in the air and then examined under the microscope using a high power (100×) oil immersion objective. The normal sperm cells were counted and the percentage was calculated. induced by Pb. This nding is in agreement with that of (El-Barky et al., 2019) in diabetic rats and (Khudiar et al., 2017) in cadmium chloride treated rats. This may be due to the ability of APG to reduce the level of blood fat by promoting cholesterol absorption and conversion, and accelerating reverse cholesterol transport (Zhang et al., 2016). the was in tests and of with indicate that had the ability induce apoptosis in these These results in agreement with studies on Pb apoptic effect in the cells


Introduction
Hyperlipidemiaoccured when serum cholesterol or triglycerides levels are elevated and reach levels linked with an increased risk of is chemic heart disease (Walker and Edwards, 2004). Several studies suggested that exposure to lead (Pb) may induce hypercholesterolemia, hypertriglyceridemia and hyperphosphatemia (Vgbaja et al., 2013).
Thyroid gland is an endocrine gland that regulates many body functions by secreting triiodothyronine (T3) and thyroxine (T4) and calcitonin hormones (Tortora and Derrickson, 2017). Previous studies have found that lead has the ability to induce hypothyroidism (Krieg, 2019).
Infertility is a health problem that affects about 15% of couples of reproductive ages (Lukac et al., 2009). Pb is a heavy metal with many industrial uses such as manufacturer of lead acid batteries, coloring agents, paints, smelters, and printing presses (Zhang et al., 2020). It causes environmental pollution and health problems (Okereafor et al., 2020). Pb is a major factor affecting male fertility (Al-Chalabi et al. Therefore, the present study was carried out to investigate the protective role of APG against the alteration in lipid pro le and toxicity of Pb in testis and thyroid gland of treated rats. This was done using biochemical and structural assessments.

Animals
Twenty-four adult male rats (Rattus norvegicus) weighing 180-200gm were purchased from the animal house of Faculty of Science king Faisal University, Saudi Arabia. All experimental procedures were done according to the research ethics at King Faisal University. The rats were housed in plastic cages (6 per cage), oored with soft a wood shaving that was changed three times per week. The animals were acclimatized for 2 weeks prior the study and were maintained under a 12 h light/dark cycle at (25 °C ± 2 °C), with free access to water and rat chow.

Experimental design:
Animals were randomly divided into four groups of six animals each as follows: Group I: served as the control group. Rats were daily injected (i.p.) with normal saline (0.9% Na cl) as vehicle.
Group II: Served as lead acetate -treated group. Rats were daily injected (i.p) with lead acetate dissolved in normal saline at a dose of 20mg/kg b.w.
Group III: Served as lead acetate and Apigenin-treated group. Rats were daily injected (i.p) with lead acetate dissolved in normal saline at a dose of 20mg/kg (b.w.) followed by injection (i.p) with Apigenin dissolved in normal saline at dose of 20mg/kg b.w.
Group IV: Served as Apigenin-treated group. Rats were daily injected (i.p) with Apigenin dissolved in normal saline at dose of 20mg/kg b.w.
At the end of the 4-week experimental period, the animals were sacri ced and samples of blood and testis tissues were collected for analysis.
Estimation of studied parameters: a) Evaluation of lead level: The lead concertation was determined in blood and cerebellum tissue using Atomic Absorption Spectroscopy. II-Oxidative stress markers -Activities of catalase, superoxide dismutase, glutathione peroxidase were determined in chosen tissues using commercial kits. The enzymes activities were expressed as U/mg protein. The level of MDA, as an index of lipid peroxidation, was determined through its reaction with thiobarbituric acid (TBA) using the lipid peroxidate kit according to the instruction of the supplier. TBARS values were calculated as nmol MDA per g tissue.

III-In ammatory markers
The sera TNF-µ , IL-6, IL-4 and IL-10 levels were measured using standard kits according to the manufacturer's protocol.

IV-Apoptosis marker
The levels of caspase-3 enzyme in cerebellum tissue were measured according to the manufacturer's protocol.

V-Estimation of serum hormonal concentrations
Thyroid hormones were determined by measuring the serum levels of thyroxine (T4), triiodothyronine (T3) and thyroid-stimulating hormone (TSH). T4 was determined following the protocol described in T4

VI-Assessment of sperm concentration, motility, and abnormality
The left testis of each rat was harvested, then minced in pre-warmed saline (37 °C), and the resulted suspension was used in semen analysis. To analyze sperm motility, 1 drop of sperm suspension was placed on a glass slide to analyze 200 motile sperm in 4 different elds. The motility of the sperm was evaluated microscopically within 2-4 min of their isolation from the testis, and data were expressed as percentage motility (Morrissey et al. 1988). The sperms were counted using a hemocytometer following the method of Freud and Carol (1964). The technique of Evans and Maxwell (1987) was adopted for sperm abnormality study. Brie y, smears were made by placing a drop from the sperm suspension and one or two drops of the previously warmed (37 °C) eosin-nigrosin stain. The smears were allowed to dry in the air and then examined under the microscope using a high power (100×) oil immersion objective. The normal sperm cells were counted and the percentage was calculated.

C. Microscopic examination
For preparation of samples used in the examination by light microscope, specimens of testis were collected from all experimental groups and xed in 10% neutral buffered formalin and routinely processed for stained with hematoxylin and eosin stain (H& E) (Bancroft and Camble, 2002), then sections were examined using light microscope.

D-Statistical analysis
All variables were compared using one-way analysis of variance (ANOVA) followed by LSD multiple range test. Differences at P<0.05 were considered as statically signi cant.

Results
Effect of Apigenin on lead levels in the testis and thyroid gland of rats treated with lead acetate In this study, there was a signi cant increase in lead level in the serum, thyroid gland and testis tissues of rats treated with lead acetate (GII) relative to control-group (GI). Treatment with APG caused decrease in lead level in serum and the tissues of rats intoxicated with lead acetate ( Table 1). The order of bioaccumulation of lead in selected organs was as follow: Serum> thyroid gland> testis.

Effect of Apigenin on lipid pro le in sera of rats treated with lead actate
Effect of APG on lipid pro le in rats treated with lead acetate is shown in Table 2. It is obvious that exposure of rats to lead acetate produced signi cant increase in the values of TC, TGs and LDL-C, relative to the control group. Also, the results showed that HDL-C level was signi cantly decreased relative to that obtained for control group. On the contrary, APG treatment attenuated these changes in lipid pro le.
Effect of APG on oxidative stress markers in testis and thyroid gland tissues of rats treated with lead acetate In the present study, as shown in Table 3, rats treated with lead acetate (GII) showed signi cant decrease in studied antioxidant enzymes activities in thyroid gland and testis tissues as compared to the control group (GI). Also, there was a signi cant increase in the MDA levels in these tissues as compared to the control group (GI). These changes were markedly attenuated by the treatment with APG.
Effect of APG on in ammation response in testis and thyroid gland tissues of rats treated with lead acetate Exposure of rats to lead acetate caused increase in levels of proin ammatory cytokines (IL-6 and TNF-α) and decrease in levels of antiin ammatory cytokines (IL-4 and IL-10) response in testis and thyroid gland tissues compared to the control group. On the other hand, rats treated with APG showed a signi cant decrease in the levels of pro-in ammatory cytokines and a signi cant increase in the levels of antiin ammatory cytokines in comparison to the lead acetate treated group ( Table 4).

Effect of Apeginin on caspase 3 in testis and thyroid gland tissues of rats treated with lead acetate
In this study, there was a signi cant increase in caspace-3 activities (Table 5) in testis and thyroid gland tissues of rats treated with lead acetate (GII) relative to control-group (GI). These changes were markedly ameliorated by the treatment with APG.
Effect of Apigenin on testicular and thyroid gland hormones in sera of rats treated with lead acetate In the present study, it was observed a decrease in T, LH and FSH in sera of rats treated with Pb. Furthermore, there was a reduction in levels of T3 and T4 and an increase in TSH level. on the contrary, APG increased the levels of altered hormones in the sera of rats treated with Pb ( Table 6).

Effect of APG on sperm parameters in rats intoxicated with lead acetate
The result of this study showed that Lead treatment induced a signi cant decrease in sperm count and sperm motility and increase in sperm abnormalities. However, the alterations in these sperm parameters were ameliorated in animals that received Pb plus APG when compared to rats treated with Pb alone (Table 7).

Control group and Apigenin treated -group:
In para n cross sections of Control & Apigenin rat's testes xed in formaldehyde and stained with H&E, it was noticed well preserved seminiferous tubules separated by the interstitial tissue (Fig.1,7). Leydig cells occupied the interstitial tissue and each seminiferous tubule was lined with multiple layer of spermatogenic epithelium. The lumina of seminiferous tubules were occupied by the spermatozoa (Fig. 2,8).
Lead acetate-treated group: In this group, light microscopically examination of H&E testicular sections revealed extremely severe and widespread degenerative changes appeared as deformities of seminiferous tubules lined with wavy outline and surrounded by irregular basement membrane. Also, distinctive appearance of genotoxicity with degenerated interstitial tissue led to large spaces between the seminiferous tubules (Fig.3). Tubules exhibit focal or diffuse extensive necrosis with a few viable cells. The cells were identi ed in some tubules mostly composed of immature form of germ cells which appeared as small acidophilic cells have darkly stained nuclei. The irregular basement membranes of seminiferous tubules were lined with one or two layers of cells (Fig.4).
Lead acetate and Apigenin treated group: In contrast, prior Apigenin treatment reduced the degree and the number of necrotic seminiferous tubules. There were relatively well preserved interestitium between seminiferous tubules. The interstitial spaces were narrow and most of the seminiferous tubules regained their normal appearance with more spermatozoa (Figs.6,7). Less improvement was recorded in few seminiferous tubules with depletion of germ cells but with healthy Leydig and spermatogenic cells (Fig.8).

Effect of APG on oxidative stress markers in testis and thyroid gland of rats treated with lead acetate
The data of the present study showed that Pb can induced oxidative stress in tissues of testes and thyroid gland of treated rats as shown by the observed decrease in the activities of studied antioxidant enzymes and increase in level of MDA in these tissues. This result is in agreement with that reported by Ezejiofor and Orisakwe, (2019) .On the contrary, APG reduced this observed oxidative stress in testis and thyroid gland. These results agree with the results of Dang et al. (2017) in case of testis of acrylamide treated rats and Panda and Kar (2007) in the thyroid gland of diabetic mice.
The ability of APG treatment to reduce the oxidative stress induced by Pb in testis and thyroid glands may be due to the antioxidant activity of APG which is mainly determined by three hydroxyl groups at its 4', 5, 7 position and double bond at C2 and C3 (Pan et al., 2020).

Effect of APG on in ammation response in testis and thyroid gland of rats treated with lead acetate
The result of the present study showed that Pb treatment caused increase in proin ammatory cytokines levels (TNF-α and I L-6) and decrease in anti-in ammatory cytokines levels (IL-4 and IL-10) in both testis and thyroid gland. These results can be supported by previous nding of The decrease in T level by Pb may be due to its ability to produce reactive oxygen species which promote Leydig cells aging and apoptosis. It is well known that Leydig cells are responsible for T secretion (Adekola (2015). The observed alterations in testicular and thyroid gland hormones indicated impairment in function of testis and hypothyroidism.
The observed hypogonadism may be a consequence of hypothyroidism where it is well known that hypothyroidism is associated with a decrease in male sex hormones, abnormal spermatogenesis, and retardation of Sertoli cell differentiation (Mohamed and Gawad, 2017). T3 hormone has the ability to stimulate the basal testosterone biosynthesis by increasing LH receptors and steroidogenesis of Leydig cells The observed thyroid dysfunction may be related to structural damage of thyroid follicular cells due to accumulation of Pb in the thyroid gland (Baoliei et al., 2009). The observed increase in serum TSH level is likely a response to decreases serum T4 and T3 level.
On the contrary, APG increased the levels of altered hormones in the sera of rats treated with Pb. This effect can be supported by the nding of Akilah et al. (2018) in testes of rats treated with chloroquinone and Panda and Karin in thyroid gland of diabetic mice.

Effect of APG on sperm parameters in rats intoxicated with lead acetate
In the present study, exposure of rats to Pb showed altered sperm parameter where there were a decrease in sperm count and motility and an increase in sperm abnormality. This result can be supported by that of Oyeyemi et al. (2019).
It is well known that sperm plasma membranes are rich in polyunsaturated fatty acids, containing double bonds vulnerable to free radical attack and initiation of lipid peroxidation which causes impaired membrane structure and functions (Tvrda et al., 2011, Asadpour et al., 2013).
The observed decrease in sperm count may be due to the ability of Pb to cause oxidative stress and consequently increased death and decreased number of sperms (Tvrda et al., 2011).
Pb may interact with sulfhydral groups on the proteins of the outer dense bers and brous sheath, which are cytoskeletal components of the agellum, causing its detachment from the plasma membranes and this may affect the sperm motility (Gomes et al., 2015).
On the other hand, APG treatment ameliorated the alteration in sperm parameters. This may be due to the ability of APG to reduce oxidative stress as demonstrated in this study. Similar result was obtained by (Akilah et al.,2018) in testes of rats treated with chloroquinone.

Effect of Apigenin on testis histology
In the present study, lead acetate led to degenerative changes in the testis of treated rats. This result can be con rmed by

Conclusion
In summary, it can be concluded that 20mg/kg b.w of lead acetate for 4 weeks can induce toxicity in testis and thyroid gland of rats. On the contrary, treatment with APG had (20mg/kg b.w) can attenuate change in lipid pro le and had bene cial effect for prophylaxis of Pb-induced testicular and thyroid gland damages.
Results of the current study shows that APG treatment may exhibit these prophylactic effects through anti-hyperlipidemic, antioxidant, antiin ammatory and anti-apoptic activities of APG.

Ethical approval
The experimental protocol of this investigation was approved by Institutional Animal Care and Use Committee (IACUC) at the King Faisal University.

Funding
No funding agency support this research.

Declaration of Competing Interest
The authors report no declarations of interest.

Data and materials Availability
We have no con ict to publish our data Author Contributions • Abdulmohsen I. Algefare conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared gures and/or tables, authored or reviewed drafts of the paper, approved the nal draft.
•Azza Sedky conceived and designed the experiments, performed the experiments, analyzed the data, prepared gures and/or tables, authored or reviewed drafts of the paper, approved the nal draft.
• Manal Alfwuaires analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the nal draft.
. • Omar Mahmoud analyzed the data, prepared gures and/or tables, authored or reviewed drafts of the paper, approved the nal draft.

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Consent to Publish
The authors report no con ict to publish  The in ammatory cytokines are expressed as pg/mg protein. Effect of Apigenin on some testicular and thyroid gland hormones in sera of rats treated with lead acetate Figure 1 Please See the Supplemental Files section for the complete gure caption