Chlorella Vulgaris alleviates Lead-induced testicular toxicity better than Zingiber Officinale: An Ultrastructural

study

XXIV International Symposium on Morphological Sciences

Sep. 3, 2015

Introduction

Material and Methods

Results

Discussion

Conclusion

References

Introduction

Introduction

• The toxicity of lead is related to oxidative stress through the creation of reactive oxygen species (ROS).

• The deterioration of reproductive ability in males is the main manifestation of occupational exposure to lead.

• Chlorella vulgaris is a green unicellular algae contains metal–ion binding site affinity in the chloroplasts within the cell wall that have sulfhydryl which chelates metals.

• Zingiber officinale contains phytochemicals such as shogaols and gingerols which recognized to have antioxidant properties.

Introduction con’t

Aim of the current work

Aim of the current work

is to compare the chelating ability of Chlorella vulgaris and the antioxidant ability of Zingiber officinale in the alleviation of adverse consequences of lead exposure.

Material and Methods

Material and Methods

• Ethical Approval. by the Medical Research Ethics Committee of Faculty of Medicine, King Abdulaziz University (Reference No 1167-13).

• Sixty adult male Wistar rats were distributed into four groups. Group 1 was considered control, group 2 received 200 mg/l PbAc, group 3 received 50 mg/kg/rat of C. vulgaris extract and 200 mg/l PbAc, and group 4 received 100 mg/kg/rat of Z. officinale and 200 mg/l PbAc for 90 days.

Material and Methods con’t

• Chlorella vulgaris Extract (CVE). CV Beijerinck (strain 072), a strain of unicellular green algae was obtained from Medical supplier (Kuala Lumpur, Malaysia).

• Z. officinale root was obtained from the local market, cut into tiny parts, and dried for 6 days. The dried root was ground in a grinder and powder was obtained. Fifty grams of powder were extracted in 250 ml of ethanol for 18 h in a Soxhlet extractor, and then the extract was dried at low pressure and stored at 4ᴼC.

Material and Methods con’t

• Testicular Histology. with hematoxylin and eosin (H&E) and examined using Olympus BX53 microscope equipped with a DP73 camera.

Material and Methods con’t

• Morphometry. At least 30 tubular profiles of each animal. The mean seminiferous tubule diameter (MSTD) was obtained by measuring across the minor and major axes. The seminiferous epithelium height was measured for the same tubules. The measurements were taken at different magnifications using Image-Pro Plus v6.0 (Media Cybernetics, Maryland, USA) and NIHImageJ (v1.50) (http://rsb.info.nih.gov/ij/).

Material and Methods con’t

• The Ultrastructural Picture. with a Philips 201 transmission electron microscope at 60–80 kV in the Transmission Electron Microscope Unit (Philips Industries, Eindhoven, Netherlands).

• Statistical Analysis. All data are presented as the mean ±standard deviation (SD) of different parameters between treated groups. Data evaluation was carried out using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using SPSS software v22.

Results

Histological Picture

Fig. (1A): Control. Fig. (1B): group 2 (Lead) showed spermatogenic cells with shrunken pyknotic nuclei (arrow), hyaline material (H), and numerous vacuoles (V). Fig. (1C): group 3 showed rebuilding of the spermatogenic layers. Fig. (1D): group 4 showed restoration of the spermatogenic series (H&E. Scale bar, 20 µm).

Results con’t

Morphometric Evaluation

Mean Seminiferous Tubular Diameter m (Morphometry)

Germinal Epithelial Height m (Morphometry)

Results con’t

Semithin Picture

Fig. (2A): Control. Fig. (2B): group 2 shows depletion and separation of the spermatogenic cells with vacuoles (V) and empty spaces between and inside the spermatogenic cells. Fig. (2C): group 3 shows healthy spermatogenic series. Fig. (2D): group 4 shows restoration of the spermatogenic cells.(Toluidine Blue. Scale bar, 5 µm).

Fig. (3A): Control. Fig. (3B): group 2 shows wide interstitial spaces with many interstitial cells of Leydig (L), a lot of vacuolated cytoplasm (V). Note the large congested blood vessel (C). Fig. (3C): group 3 showed almost healthy architecture. Fig. (3D): group 4 shows restoration of interstitial cells of Leydig (L) with minimal vacuoles. (Toluidine Blue. Scale bar, 5 µm.)

Results con’t

Ultrastructure Picture

Fig. (4A): group 2 shows a Sertoli cell (St), basal lamina (BL), primary spermatocytes (Sc), mitochondria (M), vacuoles (V), zone of tight junctions (arrowhead) and smooth endoplasmic reticulum (star). Fig. (4B):Rounded spermatids (Rsd) with rounded or oval nuclei that have disturbed chromatin; peripherally arranged mitochondria with lost cristae (M) and the characteristic acrosomal cap (Ac) can be observed. Note the myelin-like figure (F) and numerous empty spaces (V) all around. Fig. (4C): Cross-sections of mid-pieces (MP) of the sperm, with distortion of the central axoneme (Ax), mitochondrial sheath (M), and fibrous sheath (FS). Fig. (4D): Leydig cells near the basal lamina (BL) with irregular euchromatic nucleus (N) and peripheral heterochromatin. Electron-dense bodies of variable sizes (D), mitochondria (M), dilated sER (star), and lipid droplets (L) are noticed. Fig. 4A, 4B, and 4D, the scale bar is 2 µm; for Fig. 4C, it is 500 nm.

Fig. (5A). group 3 that showed a Sertoli cell (St) with a euchromatic-indented nucleus and apparent nucleolus (N) and the spermatogonia (Sg) resting on the thickened basal lamina (BL) with an apparent myoid cell (My) and collagen (C). Some mitochondria are affected, while others are healthy (M). Fig. (B): Rounded spermatids (Rsd) with peripherally arranged unhealthy mitochondria (M) and the characteristic acrosomal cap (Ac) enwrapped by Golgi saccules (G). Note that there are few empty spaces (V) in the cytoplasm. Fig. (C): Cross-sections in the mid-pieces (MP) of the sperms that have a central axoneme (Ax) surrounded by a mitochondrial sheath (M), a fibrous sheath (FS), and a peripheral cell membrane (P). Fig. (D): Leydig cells with irregular euchromatic nucleus (N) and peripheral heterochromatin. Electron-dense bodies of variable sizes (D), mitochondria (M), and lipid droplets (L) are present in the cytoplasm. Fig. 5A, 5B, and 5D, scale bar is 2 µm; for Fig.

5C is 500 nm.

Fig. (6A): An electron micrograph of a testis from the group 4 shows a Sertoli cell (St) with a euchromatic-indented nucleus (arrow) resting on the thickened basal lamina (BL) with an apparent myoid cell (My) and collagen (C). Note the intact zone of tight junctions (Z) and numerous unhealthy mitochondria with lost cristae (M). Fig. (6B):Rounded spermatids (Rsd) with the characteristic acrosomal cap (Ac) and peripherally arranged unhealthy mitochondria (M) can be observed. Note some empty spaces (V) all around. Fig. (6C):Cross-sections of mid-pieces (MP) of the sperms with minimal distortion of the mitochondrial sheaths (M) and fibrous sheath (FS). Note that the end-pieces (EP) have central axoneme (Ax) surrounded by outer cell membranes (P). Fig. (6D): Leydig cells with irregular euchromatic nuclei (N) and external heterochromatin. Variable-sized electron-dense bodies (D), mitochondria (M), and lymph (Ly) are observed in the cytoplasm. Fig. 6A, 6B, and 6D, the scale bar is 2 µm; for

Fig. 6C, it is 500 nm.

Discussion

• C. vulgaris provides remarkable defensive mechanisms against PbAc-induced injury; this consists of the elimination of heavy metal through the digestive function, either affecting absorption or excretion. When dietary C. vulgaris is given to animals with heavy metal, this stimulates toxicant excretion in the urine and feces.

Discussion con’t

• Previous research noted that the administration of C. vulgaris extract (CVE) reduced the blood level of lead in PbAc-treated groups, which is attributable to the chelating influence of the sulfhydryl-containing elements in CVE.

• As an additional factor, the mineral elements of CVE includes iron, zinc, calcium, phosphorus, and magnesium may prevent lead absorption from the alimentary tract, probably through competition for shared absorptive receptors in the intestinal mucosa.

Discussion con’t

• The defensive action of Z. officinale is due to its content of volatile oils, which revealed immunomodulatory and anti-inflammatory outcomes that can guard against testicular damage caused by the inflammatory process. These volatile oils are able to suppress T-lymphocyte-dependent immune reactions.

• Furthermore, the anti-inflammatory action of Z. officinale is due to its components as gingerol analogs (shogaols and paradols). Previous studies have noted that these ingredients’ capable to inhibit prostaglandin and leukotriene synthesis immediately.

Discussion con’t

• Results of presented experiments clarify the chelating effects and the antioxidant effects of Chlorella vulgaris extract on the ultrastructural changes of lead induced toxicity in rat testes.

• The chelating effect of CVE gave better results as regards diameter of seminiferous tubules and germinal epithelium thickness, and the sperm structure.

• While the antioxidant effects of Zingibar officinale extract was less pronounced.”

Conclusion

• Chlorella vulgaris and Z. officinale can be recommended as an adjunct therapeutic strategy to combat lead-induced testicular damage.

• C.vulgaris showed superior potentials through its antioxidant ability and chelating properties of heavy metals.

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