Influence of triazines and lipopolysaccharide coexposure on inflammatory response and histopathological changes in the testis and liver of BalB/c mice.

Background: Triazines are environmental active chemicals that have been reported to alter the inflammatory status of the gonads. We tested the anti-inflammatory effect of the triazines (atrazine; ATZ, simazine; SMZ and cyanazine; CYZ) on the testis and compared it with the more classical liver model that has substantial populations of resident macrophages comparable to the testis. Methods: BalB/c mice were treated with 25 mg/kg ATZ, SMZ and CYZ for 30 days and injected with lipopolysaccharide (0.5 mg/kg i.p.) 6 h before sacrifice. Myeloperoxidase activity and nitric oxide level in the testis and liver homogenates were determined by spectrophotometry whereas tumor necrosis factor-alpha and interleukin-6 concentrations were evaluated by immunoassay. Haematoxylin and eosin stained sections of the tissues were observed using a light microscope. Results: Myeloperoxidase activity, nitric oxide, tumor necrosis factor-alpha, and interleukin-6 levels were decreased in the liver and testis of the triazines co-treated animals. SMZ has the most potent inhibitory effect and ATZ the least effect on inflammatory mediators in both tissues. Microscopic evaluation showed loss of inflammatory cells in the inter-tubular areas of the testis and few patchy masses of infiltrating inflammatory cells around the central vein of the liver. Conclusion: Triazines inhibit the levels of inflammatory mediators in the testis and liver of mice. The anti-inflammatory effect of triazines in a lipopolysaccharide-induced inflammation model was established in this study.

Testosterone restores TM3 and TM4 cell viability, reduces reactive oxygen species generation, and protects against atrazine-induced stereological changes in rat testes.

In this study, we performed the stereological examination of rat testes and evaluated the protective effect of testosterone against atrazine (ATZ) toxicity in TM3 Leydig and TM4 Sertoli cells. Testosterone intake in rats increased the volumetric density of the seminiferous tubules; tubular diameter; germinal epithelial height; number of spermatogonia, primary and secondary spermatocytes, round spermatids, Sertoli cells, and Leydig cells; and Johnsen scores compared with the values after ATZ treatment (p < 0.05). Furthermore, testosterone increased the viability of TM3 cells and reduced reactive oxygen species (ROS) generation in TM4 cells compared to the ATZ-treated group. In conclusion, exogenous testosterone intake maintains testicular morphometry and spermatogenesis in rats, and minimizes cell death and ROS generation in testicular cell lines exposed to ATZ. However, TM4 cells are more responsive to testosterone-mediated regulation of ROS generation induced by ATZ than TM3 cells.

The protective effect of fluted pumpkin seeds against atrazine-induced testicular injury.

The present study evaluated the protective effect of fluted pumpkin seeds (FPS) on atrazine (ATZ)-induced testicular damage. Twenty adult male Wistar rats were divided into four groups of five animals each. The control animals (Group A) received corn oil (2 mL/kg body weight), Group B animals received 50 mg ATZ/kg body weight by gavage, while Groups C and D received 50 mg ATZ/kg body weight once a week in addition to the gavage of 25 mg FPS/kg body weight and 50 mg FPS/kg body weight respectively for 60 days. The results showed that testicular myeloperoxidase activity and nitrite concentration were decreased in all groups compared to the control value. The increase in malondialdehyde and decrease in glutathione concentrations in group B were abrogated in group C (p < 0.05) but not in group D animals. The increase in γ-glutamyl transpeptidase and decrease in lactate dehydrogenase enzyme activities in the group B animals were also normalized to control values in group C but not in group D animals. Interestingly, the testis of the group D animals showed massive depletion of germ cells and the diameter of the seminiferous tubules of these animals were decreased compared to all other groups. However, the number of motile sperms, abnormal sperms and sperm count in Group D animals were similar to the ATZ-treated animals and lower than control values. In conclusion, FPS protected against ATZ-induced testicular damage but can also harm the testis at a higher dose.

The protective effect of rutin against busulfan-induced testicular damage in adult rats.

Here, we studied the protective effect of rutin (RUT) against testicular damage caused by busulfan (BUS) in rats. Adult male Wistar rats were intraperitoneally injected with BUS (4 mg/kg body weight at day 7 and 14), and then treated with RUT (30 mg/kg body weight) by gavage thrice weekly for 60 days. The results showed that BUS-induced increase in 3ß-hydroxysteroid dehydrogenase (3ß-HSD) was significantly decreased by RUT, whereas 17ß-HSD activity and plasma testosterone concentration remained unaffected (p > 0.05). It was also observed that RUT inhibited BUS-induced increase in nitrite concentrations and myeloperoxidase enzyme activities in the plasma and testes (p < 0.05). Similarly, BUS-induced decrease in glutathione and increase in malondialdehyde concentrations in the testes were significantly normalized to control values by RUT. Finally, RUT administration showed some tendency to improve the architecture of the seminiferous epithelium of the rat's testes after BUS treatment. Overall, RUT inhibited BUS-induced oxidative damage and inflammation in the testis of an experimental rat model.

Gallic acid ameliorates busulfan-induced testicular toxicity and damage in mature rats.

Here, we studied the protective effect of gallic acid (GAL) as a potent anti-oxidant and anti-inflammatory agent against damage caused by busulfan (BUS) in the testes of adult rats. The adult Wistar rats were assigned as control, BUS: was intraperitoneally (i.p.) treated with busulfan (15 mg/kg, day 7 and 14), GAL + BUS: was co-treated with busulfan (i.p., 15 mg/kg, day 7 and 14) and orally treated (per os) with gallic acid (60 days, 20 mg/kg) and GAL: was treated with gallic acid (per os, 60 days, 20 mg/kg). The results showed that GAL co-treatment increased the numbers of spermatogonia (Type A and B), spermatocytes (primary and secondary) and round spermatids, along with the tubular diameter, epithelial height and gonado-somatic index. In addition, BUS-induced increase in 3ß-hydroxysteroid dehydrogenase and γ-glutamyl transpeptidase activities were inhibited on GAL co-treatment. Similarly, BUS-induced decrease in gluthathione concentration, catalase and superoxide dismutase activities along with increase in myeloperoxidase activity and malondialdehyde concentration were significantly normalized to control values on GAL co-treatment. Busulfan-induced elimination of tubular germ cells was completely prevented by GAL. Overall, GAL may inhibit BUS-mediated spermatogenesis arrest via decreasing inflammatory-mediated oxidative stress in a rat experimental model.

Antioxidant and anti-inflammatory protective effects of rutin and kolaviron against busulfan-induced testicular injuries in rats.

There are few treatment options, including the use of natural phenolics-based combination therapy for mitigating male infertility conditions associated with chemotherapy. Busulfan is an anti-cancer drug that leads to testicular problems in humans. Here, we studied the effect of co-treatment of rutin and kolaviron against busulfan-induced testis damage. Young adult male Wistar rats were intraperitoneally injected busulfan (4 mg/kg b.w), and then orally administered rutin (30 mg/kg b.w), and kolaviron (50 mg/kg b.w) alone and combined for 60 days. Results revealed that rutin and kolaviron alone or in combination reversed busulfan-induced increase in oxidative stress along with sperm quality of treated animals. However, kolaviron and rutin separately improved the concentrations of MDA and GSH and sperm quality more than when they were combined. Similarly, rutin and kolaviron separately or in combination preserved spermatogenesis and relieved busulfan-induced increase in nitric oxide concentration, myeloperoxidase and 3ß-hydroxysteroid dehydrogenase activities. Co-supplementation with kolaviron but not rutin nor when rutin was combined with kolaviron also improved the testicular level of tumor necrosis-alpha. Finally, the histological features in the testes caused by busulfan were reversed by rutin, whereas treatment with kolaviron alone or in combination with rutin partially protected the testis from busulfan-induced injury as demonstrated by the appearance of few germ cells, damaged tubules, loss of round spermatids and defoliation of the seminiferous epithelium. Thus, the combined treatment regimen of rutin and kolaviron sparingly prevented busulfan-induced testicular injuries in rats.Abbreviations: CAT: Catalase; GSH: Glutathione; 3ß-HSD: 3ß- hydroxysteroid Dehydrogenase; MDA: Malondialdehyde; TNF-α: Tumor necrosis-alpha; BUS: Busulfan; RUT: Rutin; KV: Kolaviron; TBARS: Thiobarbituric Acid Reactive Substances; MPO: Myeloperoxidase; ELISA: Enzyme-Linked Immunoassay; NAD: Nicotinamide Adenine Dinucleotide (oxidized); ROS: Reactive Oxygen Species.

Effect of Anthocleista djalonensis A. Chev Root Extract on Testis of Matured Rats.

OBJECTIVE: To investigate the effects of different extracts of Anthocleista djalonensis on the testis and epididymal sperms of rats. METHODS: Fifty male Wistar rats were randomly divided into 10 groups (n=5 in each group) and orally treated with 50, 100 and 200 mg/kg body weight each of methanol, aqueous ethanol (H-EtOH) and chloroform extracts of A. djalonensis. Corn oil was used as vehicle (2 mL/kg). After 60 days of treatment, testosterone (T) and cholesterol (CHOL) concentrations, catalase (CAT), lactate dehydrogenase (LDH), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and superoxide dismutase (SOD) activities in the testes along with myeloperoxidase (MPO) activities and nitrite concentrations (NO) in the serum and testes as well as sperm quality were measured. RESULTS: T and CHOL concentrations along with 3ß-HSD activity were significantly higher in the animals treated with the low dose than in those treated with the high dose of the chloroform extract (P<0.05). Furthermore, the chloroform extract was more effective than the methanol extract that had the most marginal effect on T level at the high dose and the H-EtOH extract that was only effective at the medium dose. LDH activity was dose-dependently increased by the extracts in all groups. The CAT-SOD antioxidant system was increased in the treated animals at all doses compared to the control values, but the increase in glutathione level reached significant level in those treated with the low dose H-EtOH aqueous ethanol extract (P<0.05). Only the high dose of chloroform extract had significant inhibitory effects on MPO activity (P<0.05). Serum NO concentration was decreased at all doses of the extracts. The inhibitory effects of the extracts on testicular NO concentrations follow this order, chloroform extract > H-EtOH > methanol. Although all extracts at all doses showed excellent stimulatory effects on sperm quality (count, motility and morphology), the methanol extract at the high dose was the most effective on sperm count (P<0.05). CONCLUSION: The chloroform extract of A. djalonensis has better androgen-like and anti-inflammatory effects whereas the methanol extract has the best effect on sperm count.

Protective effects of Anthocleista djalonensis A. Chev root extracts against induced testicular inflammation and impaired spermatogenesis in adult rats.

This study was designed to explore the protective effects of methanol (Meth, 200 mg kg-1 body wt) and aqueous ethanol (Eth-OH, 200 mg kg-1 body wt) extracts of Anthocleista djalonensis roots on testicular inflammation induced by lipopolysaccharide (LPS, 5 mg/kg body wt) and depletion of tubular germ cells induced by busulfan (15 mg/kg body wt) in rats after 60 days of oral administration. As expected, LPS stimulation of the animals significantly increased serum and intra-testicular interleukin-6 and serum nitrite levels which were significantly inhibited in the Eth-OH + LPS and Meth + LPS animals. The increase in testicular and not serum myeloperoxidase activity that was induced by LPS treatment was synergistically increased in the Eth-OH + LPS animals, whereas it was inhibited in the Meth + LPS animals compared to LPS-treated animals. Furthermore, the administration of the Eth-OH or Meth extracts protected against busulfan-induced depletion of tubular germ cells and promotes the re-population of the seminiferous tubules with germ cells (spermatogonia, spermatocytes and round spermatids) at different stages of development. The extracts were found to contain 7'-oxaspiro [cyclopropane-1,4'-tricyclo [3.3.1.0 (6,8)] nonan-2'-one], cis,cis-7,10-hexadecadienal, hexadecanoic acid, methyl ester, hexadecanoic acid, ethyl ester, 9,12-octadecadienoic acid, methyl ester, and 9,12-octadecadienoic acid (Z,Z)-) which may partly explain the observed anti-inflammatory effects. In conclusion, Meth extracts of A. djanonesis have better anti-inflammatory effects than the Eth-OH extract for the management of impaired testicular function due to inflammation. However both extracts exhibited protective effect on the histology of the testis allowing for the recovery of spermatogenesis.

Dietary fluted pumpkin seeds induce reversible oligospermia and androgen insufficiency in adult rats.

Some components of the human diets are believed to be promising male contraceptive agents. The present study examined the antispermatogenic efficacy, reversibility and toxicity of fluted pumpkin seed-supplemented diet (DFPS) in adult male Wistar rats. Adult rats were given DFPS at 2.5, 5 and 10% for 60 days followed by 60 days post-treatment period. The control animals received normal standard rat diet not supplemented with fluted pumpkin seeds. The sperm quality variables, testosterone and follicle-stimulating hormone (FSH), oxidative status of the testis, steroidogenic enzymes and gamma-glutamyl transferase (γ-GT) activities and the histology of the testis were determined to evaluate the anti-fertility activity of fluted pumpkin seeds. Treatment of animals with DFPS at 5% and 10% resulted in decreased serum and intra-testicular testosterone and FSH concentrations. This effect was associated with decreased activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD), increased testicular oxidative stress and poor sperm quality in the 10% diet group. After 60 days DFPS post-treatment, intra-testicular 17ß-HSD and γ-GT activities, FSH and testosterone levels recovered to control values. Furthermore, poor sperm motility, count, morphology and viability as well as severe loss of spermatogonia and other matured epithelial germ cells and Sertoli cells observed especially in the 10% DFPS-treated animals reverted to nearly control values 60 days after withdrawal of treatment. Dietary fluted pumpkin seeds may selectively act on the epithelial germ cells, possibly mediated via Sertoli cells, leading to oligospermia, oxidative damage and androgen insufficiency. The reversibility of these effects to near normal levels after withdrawal of treatment justifies further consideration of DFPS as it may be an effective and readily reversible agent that meets the required criteria of a male contraceptive agent.Abbreviations: GC-MS: gas-chromatography mass spectrophotometry; MPO: myeloperoxidase; NO: nitric oxide; DFPS: dietary fluted pumpkin seeds; DFPS (REV): DFPS post-treatment; MDA: malondialdehyde; SOD: superoxide dismutase; CAT: catalase; GSH: glutathione; 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; 17ß-HSD: 17ß-hydroxysteroid dehydrogenase; NAD: nicotinamide adenine dinucleotide (oxidized); NADH: nicotinamide adenine dinucleotide (reduced); ITT: intra-testicular testosterone; FSH: follicle-stimulating hormone; FPS: fluted pumpkin seeds; NIST: National Institute Standard and Technology; Av: absolute volume; Ac: cross-sectional area; ST: seminiferous tubules; γ-GT: gamma glutamyl transferase.

Effect of Fe and Cd Co-Exposure on Testicular Steroid Metabolism, Morphometry, and Spermatogenesis in Mice.

The mechanism of testicular toxicity of simultaneous multiple exposures to metals is poorly understood. Previous studies reported that the toxic effect of cadmium (Cd) is modified by tissue concentration of iron (Fe). Using the mice (Mus musculus) model in the present study, we demonstrated that combined Cd (25 mg kg-1 bw) and Fe (100 mg kg-1 bw) treatment increased both Cd and Fe testicular concentrations much more than separate exposures to either of the metals. Intratesticular Cd and Fe concentrations were inversely correlated (r = - 0.731, p < 0.05) on administration of Fe but not on combined exposure to both metals when they were positively correlated (versus Cd; r = 0.793, versus Fe; r = 0.779, p < 0.05). Additionally, Cd + Fe treatment increased testicular lipid peroxidation and depleted intratestesticular testosterone, cholesterol and glutathione concentrations much more than their separate treatment. This was also associated with decreased activity of the germ cell marker, testicular lactate dehydrogenase, and increased testicular myeloperoxidase activity. These changes resulted in decreased seminiferous epithelial height, tubular diameter, germ cell (spermatogonia, spermatocytes, and spermatids) numbers, and severe tissue damage. In conclusion, Cd + Fe intake have synergistic toxic effects on testicular steroid formation and spermatogenesis due to the high testicular concentrations of both metals.

Oral administration of cadmium depletes intratesticular and epididymal iron levels and inhibits lipid peroxidation in the testis and epididymis of adult rats.

Cadmium (Cd)-induced tissue injury depends on the accumulated Cd which differentially affects endogenous iron (Fe). To investigate this, adult rats were treated by oral gavage with Cd (50 mg/kg body wt.) once a week for 15, 30 and 60 days and sacrificed a day after last administration. After the 15th and 30th day of treatment, Cd had no effect on thiobarbituric acid reactive substances (TBARS) and endogenous Fe levels but exhibited anti-androgenic effects (p < 0.05) and caused histological damages. At day 60, Cd was accumulated by 156.30% and 364.77% above control values at concentrations that decreased endogenous Fe levels by 46.41% and 50.31% in the testis and epididymis respectively. The histological damages were characterized by decreased tubular diameter, damage to the epithelium leading to loss of tubular germ cells and absent of spermatozoa in the epididymal lumen. Although myeloperoxidase activities were increased, TBARS levels were found to decrease significantly at day 60 in the serum, testis and epididymis suggesting that the histological damages were not caused by lipid peroxidation. Furthermore, TBARS correlated negatively with Cd in the testis (r = -0.251, p < 0.05) and epididymis (r = -0.286, p < 0.05); Fe correlated positively with TBARS in the testis (r = +0.217, p < 0.05) and Cd correlated negatively with Fe in the testis (r = -0.461, p < 0.05) and epididymis (r = -0.109, p < 0.05). The antioxidant enzymes, superoxide dismutase and glutathione peroxidase were also decreased in the gonads after 60 days Cd treatment. Overall, anti-androgenic effects and histo-pathological changes are early indicators of direct effects of Cd and occur before decrease in TBARS which is secondarily related to the modifying of Fe contents.