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Umbelliferone Inhibits Spermatogenic Defects and Testicular Injury in Lead-Intoxicated Rats by Suppressing Oxidative Stress and Inflammation, and Improving Nrf2/HO-1 Signaling

Introduction Lead (Pb) is an environmental toxic metal that threatens human health. Umbelliferone (UMB) is a coumarin with known medicinal and protective properties against cytotoxicity. This study explored the ameliorative effect of UMB against Pb-induced testicular toxicity in rats, focusing on steroidogenesis, oxidative stress and inflammation. Materials and Methods Rats received lead acetate (50 mg/kg) and UMB (25, 50 or 100 mg/kg) via oral gavage for 4 weeks. Results Pb-intoxicated rats exhibited testicular tissue injury and decreased serum levels of LH, FSH and testosterone. The count, viability, motility and normal morphology of the sperms were decreased accompanied with downregulated steroidogenesis markers in Pb-induced group. UMB prevented testicular injury, increased serum levels of LH, FSH and testosterone, upregulated steroidogenesis markers and improved the semen quality. In addition, UMB attenuated oxidative stress and oxidative DNA damage, downregulated the expression of pro-inflammatory mediators and Bax, boosted antioxidant defenses and Bcl-2, and upregulated Nrf2/HO-1 signaling in Pb-intoxicated rats. Conclusion UMB prevents Pb-induced testicular injury by suppressing oxidative damage, inflammation and cell death, and boosting antioxidant defenses, Nrf2/HO-1 signaling and pituitary-gonadal axis. Thus, UMB may represent a protective and cost-effective agent against Pb testicular toxicity, pending further investigations to elucidate other underlying mechanisms.


Introduction
Exposure to lead (Pb) or its compounds may occur occupationally or in a polluted environment and remains one of the concerns of the World Health Organization (WHO). Pb is widely used in industry and can be found in ambient air, dust, foods, drinking water, and in various products such as cosmetics and batteries. 1 However, modern cities are at risk of increasing health problems as exposure to environmental Pb can occur secondary to coal combustion and leaded paints or plumbing as well as mining and quarrying. [2][3][4] Pb is a persistent dangerous toxic heavy metal 5 that can cause a world-wide severe environmental and health problems, such as, neurological, hematological, gastrointestinal, immunological, circulatory, and reproductive disorders. 6,7 Pb is considered among the anticipated human carcinogens 8 and its toxicity is associated with the surplus levels of reactive oxygen species (ROS) and cellular oxidative stress. 7,8 Moreover, Pb-induced ROS generation can suppress activities of the antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), resulting in oxidative injury and cell death. [9][10][11] The toxic effect of Pb can easily damage the male reproductive system and cause male infertility. It has been reported that the toxic effect of Pb on the testis can be direct on testicular Sertoli cells resulting in alteration of sperm production and quality, or indirect on hypothalamic-pituitary-testicular axis resulting in reduced levels of circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. [12][13][14][15] Clinically, the approved treatment against Pb toxicity is by administering chelating agents to form an insoluble complex with Pb and allow its removal from the affected tissues. 16,17 However, most of these chelating agents are unable to completely extract the metal from the intracellular sites and may lead to adverse side effects such as redistribution of the toxic metal to other unaffected tissues or loss of essential metals. 17 Therefore, it is essential to find safe and cost-effective compounds against Pb toxicity.
The nuclear-factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of antioxidant proteins and protect the cells against oxidative stress. Nrf2 exists in the cytoplasm sequestered by Kelch-like ECH-associated protein 1 (Keap1) which mediates ubiquitination of Nrf2 and its subsequent proteasomal degradation through acting as an adaptor molecule for CUL-E3 ligase. 18 Under oxidative stress conditions, Keap1 dissociates from CUL-E3 ligase and Nrf2 accumulates and translocates into the nucleus to elicit the transcription of its target genes. 19 Upregulation of Nrf2 has been reported to protect against Pb hepato-, nephro-and neurotoxicity. [20][21][22] Given the role of oxidative stress in reproductive toxicity, [9][10][11] activation of Nrf2 can protect the testes against the deleterious effects of Pb.
Several studies have demonstrated the protective effects of some natural antioxidant compounds against Pb-induced testicular damage. 9,11,23 For instance, previous studies reported the ability of umbelliferone (UMB), also known as 7-hydroxycoumarin, to scavenge free radicals and increase the activities of antioxidant defensive systems in multiple organs such as the liver, kidneys, heart, and brain. [24][25][26] In addition, we have shown previously that UMB can significantly protect against cellular oxidative stress induced by cyclophosphamide, carbon tetrachloride and ammonium chloride and boost the antioxidant defensive mechanisms. [27][28][29] These findings provide an evidence supporting the ability of UMB to reduce cellular oxidative stress or damage secondary to intoxication by multiple toxic compounds. Therefore, the current study investigated the possible protective effects of UMB on Pb-induced oxidative stress, inflammation and testicular damage in rats, pointing to the role of Nrf2/heme oxygenase 1 (HO-1) signaling. We investigated the effect of UMB on spermatogenesis, steroidogenesis, inflammation, apoptosis and oxidative stress in Pbintoxicated rats.

Experimental Design
Age matched (8-9 weeks) male Wistar rats (150-170 g) were housed in standard cages under standard conditions for 10 days before starting the experiment. The animals were supplied a standard diet and get free access to drinking water. All experiments were performed in line with the guidelines of the National Institutes of Health (NIH publication No. 85-23, revised 2011) and approved by the Animal Care and Use Committee of Beni-Suef University (No. 2019-Z1025).
Lead acetate (Purity ≥99%) and UMB (Purity 99%) (Sigma, USA) were dissolved in distilled water and 0.5% carboxymethyl cellulose (CMC), respectively. Lead acetate and UMB were administered via oral gavage for 4 weeks. The dose of lead acetate was selected based on the findings of previous studies showing that the 50 mg/kg dose induces testicular toxicity in rats. 10,30 Groups I and III received 0.5% CMC via oral gavage for 4 weeks. All treatments were administered once a day. Twenty-four h after the last treatment, rats were sacrificed under thiopental (Eipico, Egypt) anesthesia and blood samples were collected for serum preparation. After coagulation, the blood samples were centrifuged at 3000 rpm for 10 min and serum was collected. The animals were dissected, and the testes were excised and washed in ice-cold phosphate-buffered saline (PBS). Samples from the testes were stored at −80°C and other samples were fixed in Bouin's solution overnight. Other samples from the testes were homogenized (10% w/v) in Tris-HCl buffer (pH 7.4), and the homogenate was centrifuged at 3000 rpm for 20 min, and clear supernatant was separated.

Determination of Sex Hormones and Cytokines
Serum testosterone and gonadotropins (LH and FSH) levels were determined using specific ELISA kits supplied by Cusabio (China) and Novus Biologicals (USA), respectively. Tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-1β were assayed using R&D Systems (USA) ELISA kits.

Sperm Analysis
Cauda epididymis was removed and minced in 5 mL physiological saline, and then incubated for 30 min at 37°C. The number of sperms was determined using Neubauer hemocytometer. Sperm motility was determined as the percentage of motile sperms observed by examining different fields. Samples from the sperm suspension were stained with eosin, smeared on glass slides and examined. Two hundred sperms were counted and those with unstained heads were considered viable. 31 To determine sperm abnormalities, 200 sperms were observed for head and tail defects and expressed as percentage. 32 Determination of ROS, Lipid Peroxidation (LPO), Nitric Oxide (NO) and Antioxidants ROS was assayed in testicular tissue homogenate as previously described. 33 Briefly, testicular homogenate (100 μL) was mixed with 1 mL PBS and 5 μL H 2 DCF-DA (final concentration 10 μM). After incubation for 30 min at 37 ° C, the fluorescence was measured at excitation 490 nm and emission 540 nm. Malondialdehyde (MDA), a marker of LPO, was assayed according to the method of Ohkawa et al, 34 and NO was determined using Griess reagent. 35 Reduced glutathione (GSH), SOD and CAT were determined according to Beutler et al, 36 Marklund and Marklund 37 and Cohen et al, 38 respectively.

Histological Examination
Samples fixed overnight in Bouin's solution were dehydrated and processed for paraffin wax embedding. Five-μm sections were obtained using microtomy and then stained with hematoxylin and eosin (H&E) for microscopic examination.
Determination of HO-1 Activity and DNA Fragmentation HO-1 activity was assayed in testicular tissue homogenate according to the method described by Abraham et al 39 and the absorbance was measured at 464 nm. DNA fragmentation was determined as previously reported by Hickey et al, 40 and absorbance of the color was measured at 600 nm. The obtained results were expressed as a fold change relative to the control.

Gene Expression Analysis
The mRNA expression levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-6, and inducible nitric oxide synthase (iNOS)), steroidogenesis (steroidogenic acute regulatory protein (StAR), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-HSD), apoptosis markers (B-cell lymphoma 2 (BCL-2) and Bcl-2-associated X protein (BAX)), Nrf2, HO-1 and NQO-1 were quantified using qRT-PCR. Briefly, total RNA was extracted from the testicular tissue samples using TRIzol (Invitrogen, USA). After treating the RNA samples with DNase (RNase-free), it was quantified by measuring the absorbance at 260 nm and samples with A260/A280 nm ≥1.8 were selected for cDNA synthesis. The obtained cDNA was amplified using QuantiFast SYBR Green RT-PCR kit (Qiagen, Germany) and primers in supplementary Table 1. The cycling conditions consisted of initial denaturation at 95°C for 2 min followed by 40 cycles of denaturation at 95°C for 5 s, annealing for 10 s and extension at 72°C for 30 s. The data were analyzed using the 2 −ΔΔCt method 41 and normalized to β-actin.

Molecular Docking
Autodock vina 1.5.6 was used to perform molecular docking of UMB and Keap1 protein (PDB ID: 4l7b). The average of the lowest energy of docking was used to show the binding affinity of UMB with Keap1. The best-scored conformation has been chosen and visually analyzed using the PyMoL 1.7.6 software.

Statistical Analysis
The results were represented as mean ± standard error of the mean (SEM). The normality of the data was checked with the Shapiro-Wilk test and the data were normally distributed. The Shapiro-Wilk test is the most powerful test for all types of distribution and sample sizes. 42 All statistical comparisons were determined by one-way ANOVA and Tukey's test using GraphPad Prism 7 (GraphPad Software, USA). A P value less than 0.05 was considered significant.

UMB Alleviated Pituitary-Gonadal Axis Hormones and Prevents Testicular Injury in Pb-Intoxicated Rats
To assess the protective effect of UMB on testicular injury, we determined serum gonadotropins (LH and FSH) and testosterone and conducted a histological investigation. The levels of LH, FSH, and testosterone were significantly decreased in Pb-intoxicated rats as compared with the control (P<0.001; Figure 1). Rats treated with 25 mg/kg UMB showed a significant increase in the levels of LH (P<0.05), FSH (P<0.01) and testosterone (P<0.001). Treatment with either 50 or 100 mg/kg UMB resulted in increased LH (P<0.01), FSH (P<0.001) and testosterone (P<0.001). The 100 mg UMB dose increased serum testosterone significantly when compared with the 25 mg dose (P<0.05). Rats that received UMB alone showed no changes in serum sex hormones.
Histological examination revealed normal structure of the seminiferous tubules and spermatogonia in control rats as depicted in Figure 2A. In contrast, Pb-intoxicated rats exhibited extensive damage of spermatogonia and few number spermatids along with other manifestations, including vacuolations ( Figure 2B-C). Pb-intoxicated rats treated with 25 ( Figure 2D), 50 ( Figure 2E) and 100 mg/kg UMB ( Figure  2F) showed significant improvement of the histological architecture of the seminiferous tubules and abundance of spermatogonia, spermatids and spermatocytes.

UMB Attenuates the Negative Impact of Pb on Spermatogenesis
In addition to the histological examination, we determined different sperm parameters to evaluate the impact of Pb on spermatogenesis and the ameliorative potential of UMB. Rats treated with Pb showed significant decrease in sperm count, motility and viability accompanied with increased percent of sperm abnormalities as represented in Figure 3. Treatment with all doses of UMB remarkably alleviated the decreased count, motility and viability of sperms and attenuated abnormalities in Pb-intoxicated rats. The high dose of UMB did not alter sperm parameters in normal rats.

UMB Alleviates Steroidogenesis in Pb-Intoxicated Rats
The impact of Pb on steroidogenesis and the ameliorative effect of UMB were evaluated via assessment of genes involved in steroid hormone synthesis ( Figure 4). Rats intoxicated with Pb exhibited significant downregulation of testicular StAR ( Figure 4A

UMB Prevents Pb-Induced Testicular Oxidative Stress in Rats
ROS, LPO and NO as well as cellular antioxidants were assayed in testicular tissue to assess the beneficial effect of UMB on Pb-induced oxidative injury. Testicular ROS ( Figure 5A) and MDA ( Figure 5B) were significantly elevated in Pb-intoxicated rats (P<0.001). Similarly, testicular NO was markedly elevated in Pb-intoxicated rats as depicted in Figure 5C. Treatment of the Pb-induced rats with UMB effectively ameliorated testicular ROS, MDA and NO levels (P<0.001) with non-significant differences between effects of the three doses. Testicular GSH ( Figure  5D), SOD ( Figure 5E) and CAT ( Figure 5F) were significantly decreased in Pb-intoxicated rats (P<0.001), an effect that was reversed by all doses of UMB. The high dose of UMB supplemented to normal rats did not induce significant changes in oxidative stress markers.

UMB Upregulates Nrf2/HO-1 Signaling in Pb-Intoxicated Rats
Nrf2, a master regulator of antioxidant and cytodefensive genes, has been reported to be downregulated in different conditions of surplus ROS generation. 28,43,44 Thus, we investigated the effect of Pb on testicular Nrf2 signaling and the ameliorative effect of UMB. Pb-induced rats exhibited a significant downregulation of testicular Nrf2 ( Figure 6A), NQO-1 ( Figure 6B) and HO-1 mRNA ( Figure 6C). The decreased activity of HO-1 in the testicular tissue of Pb-intoxicated rats added support to the negative impact of Pb on Nrf2 signaling ( Figure 6D). Oral supplementation of UMB remarkably upregulated testicular Nrf2, NQO-1 and HO-1 expression as well as HO-1 activity, whereas exerted no effect in normal rats.
Given the central role Keap1 plays in the ubiquitination and degradation of Nrf2, 18 we performed docking of UMB onto Keap1. UMB forms two hydrogen bonds with S363 and N387 at the dimeric interface of Keap1 and at −6.51 kcal/mol ( Figure 6E).

UMB Ameliorates Inflammation in Pb-Intoxicated Rats
To investigate the ameliorative effect of UMB on inflammatory response in Pb-intoxicated rats, the gene expression of pro-inflammatory mediators in the testis as well as their serum levels were determined. Analysis of testicular TNFα, IL-6, IL-1β and iNOS mRNA showed remarkable and significant upregulation in rats which received Pb compared with the control (P<0.001; Figure 7A-D). Treatment of rats  Figure 7E), IL-6 ( Figure 7F) and IL-1β ( Figure 7G). TNF-α, IL-6, and IL-1β were significantly increased in serum of Pb-intoxicated rat (P<0.001). In contrast, rats treated with UMB (25, 50 or 100 mg/kg) exhibited remarkable decrease in the serum levels of these proinflammatory cytokines.

UMB Suppresses Testicular Apoptosis and DNA Fragmentation in Pb-Intoxicated Rats
The results showed a significant downregulation of Bcl-2 ( Figure 8A) accompanied with increased Bax ( Figure 8B) and Bax/Bcl-2 ratio ( Figure 8C) when compared with the control group (P<0.001). Interestingly, all used doses of UMB effectively boosted testicular Bcl-2 while decreased both Bax and Bax/Bcl-2 ratio in Pb-intoxicated rats. The antiapoptotic effect of UMB was further confirmed by its ability to suppress testicular DNA fragmentation ( Figure 8D) which was significantly provoked following Pb administration. The effect of UMB on genes related to inflammation, apoptosis, steroidogenesis and Nrf2 signaling in Pbintoxicated rats are summarized in Figure 9.

Discussion
The environmental and occupational toxicity of Pb remains one of the major health concerns that threaten human reproductive functions, especially in the developing countries. Great attention has been paid since the past decades to deal with men's infertility that arises from exposure to environmental or occupational toxicants. Workers in many industries, including painting, welding, battery repair, glass blowing, and metal smelting are at risk of lead poisoning. Previous reports confirmed the significant harmful effects of Pb poisoning on male reproductive functions, 14,15,45 and several studies have been escalating to find better and effective natural compounds to mitigate Pb-induced testicular damage. 11,23,46 Studies from our lab as well as other investigators have reported the protective effect of UMB against multiple toxicants in different organs, including liver, brain and heart 24-29 and testicular ischemia/reperfusion (I/R) injury in rodents. 47 However, the protective effect of UMB on Pb-induced testicular injury has not been reported yet. Herein, we evaluated the potential role of UMB against Pb-induced testicular damage in rats, emphasizing its modulatory effect on spermatogenesis, steroidogenesis, Nrf2/HO-1 signaling, inflammation and apoptosis. Exposure to Pb caused a hormonal disruption of the pituitary-gonadal axis, including a reduction in serum FSH, LH and testosterone. Our findings are in the line with the findings of other investigators who reported disrupted pituitary-gonadal axis by Pb exposure. 46,48,49 LH is an important stimulator for testosterone secretion from testicular Leydig cells and adequate amount of testosterone is essential for the structural and functional integrity of reproductive organs as well as to maintain the structure and function of male accessory glands. FSH secretion is necessary for the normal spermatogenesis as it stimulates the proliferation, maturation, and function of Sertoli cells to produce signals for the initiation and maintenance of germ cells. 50 The reduced testosterone level in Pb-intoxicated rats could be ascribed to the reduced responsiveness of Leydig cells to LH or insufficient secretion of LH from the pituitary gland. Consequently, we have conducted histological examination and investigated the sperm quality in Pb-intoxicated rats. Our data revealed extensive damage to spermatogonia along with other manifestations, including vacuolations and reduced number of spermatozoa. However, a major limitation of the histological study is not using the Johnsen's scoring system 51 to evaluate spermatogenesis and interpret the histological changes. In addition, Pb-exposed rats exhibited a significant reduction in semen quality (sperm count, motility, viability and normality), which is consistent with the findings of other   11,23 These findings are attributed to the ability of Pb to cross the blood-testis barrier and accumulate in the testicular tissue, thereby affecting the normal spermatogenesis. 23,52 It has been known since decades that Pb-intoxication can markedly delay spermiation, produce immature spermatogenic cells, 53 and disrupt the epididymal functions resulting in asthenospermia, oligospermia, and teratospermia. 54 There is evidence that exposure to Pb decreases the weight and the volume of the testis and increases the chance of male infertility. Previous studies have demonstrated that Pb can significantly decrease the weight of Leydig cells, germ cells, seminiferous tubules, epididymis, seminal vesicles, prostate gland, vas deferens, and the activity of steroidogenic enzymes. 9,11,55 Indeed, the poor semen quality along with the reduced reproductive hormones are suggested to be due to the toxic effect of Pb on endocrine and reproductive systems. 56 Besides its ability to reduce the reproductive hormones, we also found a remarkable downregulation of testicular StAR, 3ß-HSD, CYP17A1 and 17ß-HSD mRNA following Pb intoxication. However, upregulation of these genes is essential for physiologic steroidogenesis and fertility. StAR enhances the movement of cholesterol into the inner mitochondrial membrane. CYP17A1 catalyzes the conversion of pregnenolone to dehydroepiandrosterone which is converted to testosterone via downstream reactions catalyzed by 3ß-HSD and 17ß-HSD. 57 Our findings are in agreement with others who reported downregulation of testicular steroidogenic enzymes by Pb exposure. 23,49,58 The reduced levels of these hormones in conjunction with the decreased expression of steroidogenic genes confirm the toxicity of Pb on endocrine and reproductive organs. 13,48 Interestingly, UMB prevented the negative impact of Pb on the pituitary-gonadal axis evidenced by the elevated serum FSH, LH and testosterone levels. In addition, UMB conferred remarkable protection against the detrimental effects of Pb on testicular tissue integrity, steroidogenesis and spermatogenesis by significantly improving the sperm count, motility and viability, and reducing the number of abnormal sperms. These findings suggest that the improved semen quality as well as the increased reproductive hormones along with the upregulation of steroidogenic genes were attributed to the ability of UMB to work locally within the testes to improve the spermatogenesis or centrally by regulating the pituitarygonadal axis.
Given the well-documented role of oxidative stress in mediating Pb toxicity, we assumed that the antioxidant activity of UMB contributed significantly to its protective role against testicular toxicity. This study showed that Pb toxicity caused testicular oxidative stress determined by the increased testicular ROS, MDA and NO in conjunction with decreased antioxidant defenses. These findings are in consonance with previous studies, 9,11 indicating the ability of Pb to induce testicular oxidative stress. Generation of excess ROS and oxidative stress have been observed in testicular tissue after Pb exposure, 59 which can adversely affect membrane integrity leading to LPO and protein malfunction. Several endogenous defenses such as GSH, SOD, and CAT are essential in protecting the cells against ROS. Nevertheless, Pb is known to cause oxidative stress in different tissues, including the testis, by enhancing the production of ROS, such as superoxide, hydrogen peroxide and hydroperoxide radicals, provoking LPO and reducing antioxidants. 23,59,60 Furthermore, Pb binds to the sulfhydryl groups or metal cofactors in the antioxidant enzymes, thereby reducing their activities. 61 The remarkable elevation in testicular MDA and the reduction in antioxidant enzymes in Pb-intoxicated rats in this study could be attributed to the ability of Pb to bind with the sulfhydryl groups of antioxidant enzymes leading to increased ROS. UMB markedly alleviated testicular oxidative stress induced by Pb as evidenced by the decreased ROS generation, MDA and NO levels, and increased expression of antioxidant enzymes. The antioxidant potential of UMB has been previously reported in our previous studies showing its ability to suppress oxidative stress in the liver of hepatotoxicity and fibrosis rats models 28,29 and brain of hyperammonemic rats. 27 Other investigators have shown suppressed oxidative stress in the heart 25 and kidney 24 of rats challenged with I/R and methotrexate, respectively. However, to determine whether the observed antioxidant properties of UMB were mediated via Nrf2/HO-1 signaling, we analyzed the expression of Nrf2 and its target protective genes NQO-1 and HO-1 in the testis of Pb-intoxicated rats. Nrf2 is a transcription factor that activates the expression of genes encoding detoxifying and antioxidant enzymes, including NQO-1 and HO-1, to protect against cellular oxidative stress, inflammation and apoptosis. 62,63 Therefore, activation of Nrf2 signaling can represent an effective protective strategy against Pb-induced testicular injury. In this study, Pb significantly downregulated the gene expression of testicular Nrf2, NQO-1, and HO-1 which was clearly reversed by UMB treatment. Our findings are consistent with earlier studies reporting remarkable enhancement of Nrf2 or its target protective enzymes by UMB in different organ systems, 24,28 suggesting that the protective effect of UMB against Pb toxicity was partly mediated by the activation of Nrf2 and its target protective genes. Additionally, UMB increased HO-1 activity in the testes of Pb-intoxicated rats. HO-1 catalyzes degradation of heme to biliverdin and bilirubin which are potential antioxidants. 64 Molecular docking simulations revealed the ability of UMB to form two hydrogen bonds with S363 and N387 at the dimeric interface of Keap1. These findings point to the ability of UMB to inhibit Keap1 and activate Nrf2. Therefore, our findings added new information about the involvement of Nrf2 activation in mediating the antioxidant activity of UMB. It is also noteworthy mentioning that UMB itself possesses radical scavenging and metal chelation properties (reviewed in 65 ) which play a role in its antioxidant activity. UMB scavenged hydroxyl radicals in a concentration-dependent manner, 66 inhibited membrane reactive-free hydroxyl radicals on a sitespecific deoxyribose degradation assay, 67 prevented superoxide anion generation 66 and showed a strong chelating effect on ferrous ions. 67 Accordingly, it could be assumed that UMB, through its metal chelation activity, chelated Pb and prevented its accumulation in the testes. Inflammation has been reported to play a key role in Pb toxicity. 68 Recently, we have demonstrated an increase in NF-κB phosphorylation and TNF-α in the liver of rats challenged with 50 mg/kg lead acetate, 60 demonstrating an inflammatory status. NF-κB is a redox-sensitive transcription factor that is activated in conditions of excessive ROS, resulting in the transcription of TNF-α, IL-6, IL-1β, iNOS and other inflammatory mediators. Here, we explored the potential of UMB to mitigate testicular inflammation in Pbintoxicated rats. The expression of TNF-α, IL-6, IL-1β and iNOS genes as well as serum TNF-α, IL-6 and IL-1β were increased in the testicular tissue of Pb-intoxicated rats. TNFα, a potent pro-inflammatory mediator, produced primarily by the macrophages is essential for the initiation and maintenance of inflammatory response. Circulating TNF-α has been increased in a positive correlation with blood Pb levels in male subjects 69 and in Pb-exposed workers. 70 The increased expression of iNOS explained the observed increase in testicular NO levels. In the presence of superoxide radicals, NO can interact producing the versatile oxidant peroxynitrite which exacerbates the inflammatory response 71 and provokes DNA damage as reported in this study.
Inflammation and oxidative stress can work in concert to provoke cell death via apoptosis. In this study, we showed oxidative DNA damage evidenced by the significant increase in DNA fragmentation in the testis of Pb-exposed rats. Our results are in agreement with the previous findings reporting the deleterious effect of Pb on the integrity of DNA in lymphocytes 72 and liver. 60 Given the role of Bcl-2 family proteins in controlling apoptosis, 73 we evaluated the protective effect of UMB against Pb-induced testicular cell death by determining the mRNA expression levels of Bcl-2 and Bax. Apoptosis in the present study was supported by the declined anti-apoptotic factor Bcl-2 and increased Bax expression in Pb-intoxicated rats. Testicular apoptosis is a direct consequence of the excessive release of ROS and pro-inflammatory mediators in Pb-intoxicated rats. Very recently, we have demonstrated the role of glycogen synthase kinase (GSK)-3β in Pb-induced hepatocyte apoptosis in rats. 60 GSK-3 can activate both Bax phosphorylation and mitochondrial translocation. 74 Bax elicits cytochrome c release from the mitochondria and subsequent activation of caspases, provoking cell death. UMB effectively ameliorated Pb-induced inflammation and apoptosis as shown by the decreased expression of testicular pro-inflammatory and pro-apoptotic mediators as well as circulating proinflammatory cytokines. These findings are supported by our previous studies showing that UMB suppressed NF-κB, iNOS and pro-inflammatory cytokines in rat models of hepatotoxicity, liver fibrosis and hyperammonemia. 27-29 UMB protected against oxidative DNA damage in the testicular tissue of Pb-intoxicated rats, adding support to a previous study showing decreased gamma-radiation induced ROS generation and DNA damage in human lymphocytes treated with UMB. 66 The anti-inflammatory and anti-apoptotic effects of UMB could be directly attributed to its antioxidant activity and Nrf2 activation. Nrf2 can suppress inflammation through attenuating the activation of NF-ĸB and release of pro-inflammatory cytokines. 75,76

Conclusions
The present study provided compelling evidence for the negative impact of Pb on steroidogenesis, spermatogenesis and pituitary-gonadal axis, and the substantial protective effects of UMB mediated via its antioxidant, antiinflammatory, and anti-apoptotic properties. Treatment with UMB suppressed Pb-induced testicular injury, and pituitary-gonadal axis, spermatogenesis and steroidogenesis alteration. These beneficial effects of UMB were mediated via its ability to attenuate ROS generation, LPO, inflammation and cell death, and boosting antioxidant defenses and Nrf2/HO-1 signaling (possible mechanisms of action are summarized in Figure 10). Our findings open new therapeutic window by providing protective and cost-effective agent against toxicity in people exposed to Pb. However, further investigations and clinical studies are required to elucidate other mechanisms by which UMB improves the testicular function.