Study of The effect of L-Riboce-D-Cystein on X-Ray-Induced Testiculur Damage in Adult Wistar Rats

Dare Babatunde Joseph¹, Olayemi Olamide Samuel², Luqman Adepoju Hassan²,

Oladele Oluwabukola Margeret², Adetoro Kehinde Emmanuela³, Olaniyi Oluwafemi Samuel², Oletuegbe Oghenekome Gift⁴

¹Department of Anatomy, Osun State University, Osogbo, Nigeria.

²Department of Anatomy, University of Ilesa, Ilesa, Osun State, Nigeria.

³Department of Physiology, University of Ilesa, Ilesa, Osun State, Nigeria.

⁴Department of Anatomy, University of Lagos, Lagos, Nigeria.

*Corresponding Author E-mail: luqman_hassan@unilesa.edu.ng

ABSTRACT:

This research investigated the effect of D-Riboce-L-cysteine on X–ray induced testicular injury in adult Wistar rat. Twenty male rats were split into five (5) groups of four (4) animals at random. Group 1 consisted of control animals that were given only regular food and drink. Only X-rays were used to induce Group 2, these were administered at approximately 95 kV, 12.5 mA/s, and 50 focal field distances (FFD) per animal, primarily in the pelvic and perineum regions. Group 3 underwent treatment with 30 mg/kg body weight with D-Riboce-L-cysteine, followed by an X-ray treatment at 95 kV, 12.5 mA.s, and 50 FFD each animal. Before receiving 30 mg/kg body weight of D-ribose-L-cysteine, Group 4 was treated with 95 kv, 12.5 milliampere-seconds (mA.s), and 50 FFD of X-ray each animal. Only 30 mg/kg body weight of D-ribose-L-cysteine was administered to Group 5. Following a 21-day course of treatment, the animals were sacrificed, the testes were removed and preserved in Bouins fluids for histological examination, and the right testis was homogenized in 5% sucrose solution to measure tissue malonialdehyde (MDA) and the activity of glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione peroxidase (GSH-P). The caudal portion of the epididymis was immersed in a standard saline solution to assess sperm motility, count, and morphology. The animals receiving no treatments showed significantly greater sperm count, sperm motility and sperm morphology when compared with the treated groups (Group 2,3 and 4) at (p>0.05) and minor differences found when compared with group 5 (D-Riboce-L-cysteine alone) at (p>0.05). The control group exhibited a statistically significant increase in G-6-PDH activity in relation to all other groups (p>0.05). The control group had lower MDA levels than all other groups, with the exception of group 5 (antioxidant alone), which had marginally higher MDA levels (p>0.05). In contrast, both the control group and the animals receiving D-Ribocine-L-cysteine group. The testicular lobules, seminiferous epithelium, and its constituent cells are all visible in the photomicrograph of the testis from the animals in group 1 (Control), which displays normal testicular histology. The animals in group 2 have a wider interstitial gap, a loss of basal laminal tissue, and spermatogonia production following vaculation in the photomicrograph (X-ray only). There was also evidence of seminiferous tubule germinal epithelium loss. The photomicrograph of the testis of the animals in groups 3 and 4 (D-Riboce-L-cysteine+X-ray) revealed aberrant interstitial space expansion, decreased leydig cell, and spermatogonial cell degeneration, all of which indicate vaculation. In Conclusion, D-ribose-L-cysteine seems to has a significant impact on preventing testicular damage caused by X-rays. This suggests that D-Riboce-L-cysteine has the potential to enhance fertility.

KEYWORDS: D-Riboce-L-cysteine, X–ray, Leydig, Spermatogenesis, Testis.

1. INTRODUCTION:

Humans have grown more vulnerable than ever to the harmful effects of using ionizing radiation in several facets of modern life due to the advent of nuclear technology¹. Ionizing radiation causes oxidative stress, which can lead to extensive damage or destruction of a variety of biomolecules. This is how it causes its harmful effects. Male infertility is caused by enobiotic-induced testicular dysfunctions, which are associated with oxidative stress processes². Under normal conditions, the testis is afforded with antioxidant protection as an elaborate array of antioxidant enzymes, free radical scavengers, and low oxygen tension in order to support the well as leydig cells steroidogenic function². However, a wide variety of endogenous and exogenous factors are known compromise male fertility by generating free radicals in testes³. In order to prevent and relieve the hazard to human reproductive health induced by ionizing radiation exposure, avoid the toxicity and the side effects and for the labialization of anti- radiation drugs⁴; It is right that radioprotectors made from traditional foods and medicinal plant sources be given careful thought and attention. When oxygen interacts with specific molecules, free radicals—atoms or groups of atoms with an odd (unpaired) number of electrons—can be created⁵. Whereby, when these extremely reactive radicals are created, start a domino effect-like chain reaction. Important biological components like DNA and the cell membrane are reacted with by radiation, changing the functioning of the cell and ultimately causing it to die⁶. Antioxidants are therefore part of the body's defense mechanism against free radical damage. Free radicals are known to be crucial in a number of disorders⁷. The emission or transmission of energy via space or a material medium in the form of waves or particles is known as radiation. Depending on the energy of the particles that emit radiation, radiation is frequently classified as either ionizing or non-ionizing REF. More energy than 10 eV is carried by ionizing radiation, which is sufficient to ionize atoms and molecules and disrupt chemical bonds. Radiography are electromagnetic waves that have wavelengths more than 310 17 Hz and 1,240 eV, but less than roughly 10 −9m.

Chemically reactive chemical compounds that contain oxygen are known as reactive oxygen species (ROS). Nonetheless, ROS levels can rise sharply in response to environmental stressors like heat or UV exposure⁸. Cell structures may sustain serious harm as a result of this. This is collectively referred to as oxidative stress. Increased oxidizing species generation or a marked decline in the efficacy of antioxidant defense, including glutathione, are linked to oxidative stress⁹. The testes feature a number of defense procedures that reduce the potentially harmful effects of these reactive oxygen species¹⁰ to guarantee that oxidative stress does not affect the organ's twin processes, spermatogenic and steroidogenic functions. One of the most crucial elements of good fertility that both men and women should concentrate on is D-ribose-L-cysteine¹¹. Antioxidants are a collection of vitamins, minerals and other elements that protect the body from the harm produced by free radicals¹². Antioxidants neutralize free radicals by acting as a kind of defensive mechanism. They function as the body's equivalent of the police. Free radicals are "quenched" by them, and they stop the ROS from spreading and harming our cells¹³. It has been demonstrated that radiation has carcinogenic impact on both experimental animals and people¹⁴. One of the main mechanisms via which the carcinogenic effect is mediated has been proposed to be the formation of reactive oxygen species (ROS) and its interaction with the cellular antioxidant system (Smits RM et al., 2019). D-ribose-L-cysteine has an exceptionally strong antioxidant capacity because of its high natural vitamin C concentration (Falana, 2020). The current investigation examines how D-ribose-L-cysteine affects testicular damage caused by radiation in adult wistar rats.

2. MATERIALS AND METHOD:

2.1 Animal source and handling:

Bolamid farm in Iwo, Osun State provided twenty (20) adult male wistar albino rats, each weighing between fifty and eighty grams. The rats were housed in Osun State University's animal control department and allowed to acclimate for two weeks, or until they reached a weight of 100–150g.The rat were fed on starter mash (Vital Feeds Grand Cereals Ltd. Ibadan); water was given ad libitum and maintained under standard circumstances. The animal room had 12 hours of photoperiodic day/night ventilation and a temperature range of 25–27.

2.2 Animal Grouping and Treatment:

Twenty (20) Wistar rats weighing between 100 and 150 g were randomly divided into 5 groups with n = 4.

2.3 Calculation of the drug:

1 capsule of D-Riboce-L-cysteine = 125mg

10 capsules of D-Riboce-L-cysteine = 1250mg

125ml of distilled were used to dissolved 1250mg (10 capsules of D-Riboce-L-cysteine)

Then

30mg/kg body weights of animals were used according to the average weight of animal in each group.

Group 3:

Average body weight of animals = 133.20

Average drug received each = 4mg

1ml of the solution = 10mg of D-Riboce-L-cysteine

Therefore each animal in group 3 received 0.4ml of the solution

Group 4:

Average body weight of animals = 139.96

Average drug received each = 4.2mg

1ml of the solution = 10mg of D-Riboce-L-cysteine

Therefore each animal in group 4 received 0.42ml of the solution

Group 5:

Average body weight of animals = 146.65

Average drug received each = 4.4mg

1ml of the solution = 10mg of D-Riboce-L-cysteine

Therefore each animal in group 5 received 0.44ml of the solution

2.4 Administration:

D-Riboce-L-cysteine was procured from Max International, dissolved in distilled water, and administered orally using oral gavage.

Group A (control group) received water and starter mash only.

Group B (radiation only) received water, starter mash and 95kv, 12.5milliampere-seconds (mA.s), 50 focal field distance (FFD) of X-ray per animal.

Group C received water, starter mash and 30mg/kg body weight of D-Riboce-L-cysteine before receiving 95kv, 12.5milliampere-seconds (mA.s), 50 focal field distance (FFD) of X-ray per animal.

Group D received water, starter mash and 95kv, 12.5milliampere-seconds (mA.s), 50 focal field distance (FFD) of X-ray per animal before receiving 30mg/kg body weight of D-Riboce-L-cysteine.

Group E received water, starter mash and 30mg/kg body weight of D-Riboce-L-cysteine only.

2.5 Animal Sacrifice:

After the last dose, the animals were sacrificed 24hours later. The heart was harvested for its blood. The animals were sacrificed using ethyl ether, and when the abdomen was cut open, the testes were removed and preserved in 10% formal-saline for histological examination. The epididymis was preserved in regular saline to assess sperm motility, count, and shape. For the enzyme testing, the testes were homogenized in a 5% sucrose solution.

2.6 Determination of sperm motility, sperm count, and sperm morphology:

The WHO technique was used to measure sperm motility¹⁷. A prepared drop of epididymal fluid was placed on a glass slide, coated with a coverslip (measuring 22 x 22mm), and immediately viewed under an Olympia light microscope (Germany). Spermatozoa motility was carefully scanned, and the results were rated as progressive, non-progressive, and dead. Using the subjective method, at least ten high power fields were viewed at a magnification of X400. The relative percentage of spermatozoa in each category was calculated and recorded to the closest 5%¹⁸. The conventional hemocytometer method was used to determine the epididymal sperm counts. After using a vortex to completely mix the epididymal fluid with 10 mL of normal saline, around 10μL of this diluted specimen was placed on slides in the Bio-Rad counting chambers and counted using an automated cell counter. Every specimen was counted on both sides of the counting chamber, and the average was recorded to the closest millions/milliliter¹⁹. Using eosin-nigrosine staining on a glass slide with dry, smeared, diluted epididymal fluid, the morphology of the sperm was examined under a 400X light microscope (Olympia, Germany). A percentage was calculated based on the number of normal spermatozoa, abnormal heads, abnormal tails, and abnormal midpiece spermatozoa.

2.6 Enzyme Histo- Chemistry:

Glucose -6- phosphate Dehydrogenase (G-6-PDH) assay:

The Lohr and Walker method was used to measure the activity of G6PD in homogenate. Reagent R2 (Buffered)-1.0ml. Reagent R2 (NAOP)-30µl. 15µl of homogenate, 5 minutes at 37^◦C incubation R3: 15µl of glucose 6 phosphate. Read initial absorbance at 365nm, and start timer concurrently (against air). Read absorbance again after 1, 2 and 3 minutes. The homogenate activity of G6PDH is equal to 60571 mu/ml rate of change in absorbance per minute. G6PDH in units of homogenate (u/l) = 6057 absorbance change per minute (m/min).

Malondialdehyde assay:

The modified method of Buege and Aust (25) was used to measure the thiobarbituric acid reactive substances present in the blood serum in order to determine the MDA concentration. One milliliter of the serum was added to two milliliters of a reagent consisting of trichloroacetic acid, thiobarbituric acid, and hydrochloric acid (TCA/TBA/HCl) in a 1:1:1 ratio. It was thoroughly shaken, then boiled for 15 minutes, allowed to cool on ice, centrifuged for 10 minutes at 3000rpm, and the absorbance was measured at 532nm in comparison to a blank.

Glutathione Peroxidase assay:

The homogenate's glutathione peroxidase activity was measured using the Paglia and Valentine method with a reagent kit from Rando Lab, Ardmore, Diamond Road, Crumin Co., UKBT294QY. It was decided to let the homogenate come to room temperature. A 5 minute centrifugation at 5000g was performed on an aliquot of the homogenate, and 25 microliters of the resulting sample were pipetted into 0.5milliliters of glutathione peroidase dilution agent.

2.7 Statistical Analysis:

All calculations were done using the ANOVA statistical software package for analysis of the data. The data were presented as mean standard error of mean (SEM), and statistical analysis carried Fout using the student’s t-test. Differences were considered to be of statistical significance at an error probability of less than 0.05 (p>0.05).

3. RESULT:

Table 1: Shown standard error of mean and P-value results for Sperm analysis (microscopic sperm count {cell/ml}, sperm morphology {Normal/Abnormal %} and sperm motility {Motile/Non-motile %}.

Sperm characteristcs from table 1: showed that sperm characteristics were significantly altered in the animals exposed to X-ray radiation; sperm count was significantly reduced in the animals that were exposed only to X-ray radiation, morphology observation showed a higher significant increase in control group (group one) and antioxidant group (group five) as compared to radiation only group (group two), motility from table 1: showed that there was a higher significant differences in control group and all other groups except group five (antioxidant only) with higher value of motility although with no significant. Increased sperm count, morphological and motility integrity was maintained in the control animals, but significant increase in sperm count, motility and morphology was noticed in antioxidant group as shown in table-1.

The enzyme results obtained were analyzed at 0.05 level of significance. Changes in tissue levels of Glutathione peroxidase (GP) and lipid peroxidations and the activity of the enzymes glucose -6- phosphate dehydrogenase (G-6-PDH) are presented in the table 2.

Table 1: Semen analysis

Parameter		Group One (Control) Mean±SEM	Group Two (Radiati on Only Mean±SEM	Group Three (Antioidant+ Radiation) Mean±SEM	Group Four (Rad Iation+an Tioidant) Mean±SEM	Group Five (Antio Idant Only) Mean±SEM
Sperm Count (10^6cell/ml)		690.0±10.00	320.0±20.00 **0.0036	470.0 ± 10.00 **0.0041 ˢ	500.0±20.00 *0.0136 ˢ	710.0±10.00 ˢˢ0.0033
Sperm Morphology grading (%)	Normal	87.50 ±2.500	47.50 ±2.500 **0.0077	62.50 ± 2.500 *0.0194	72.50 ± 2.500 ˢ0.0194	89.00 ± 1.000 ˢˢ0.0042
	Abnormal	12.50 ±2.500	52.50 ±2.500 **0.0077	37.50 ± 2.500 *0.0194	27.50 ± 2.500 ˢ0.0194	11.00 ± 1.000 ˢˢ0.0042
Sperm Motility grading (%)	Motile	82.50 ±2.500	51.50 ±6.500 *0.0111	37.50 ± 2.500 **0.0061	47.50 ± 2.500 *0.0101	86.00 ±1.000 ˢˢ0.0082
	Non- motile	17.50 ±2.500	70.00 ±5.000 *0.0111	62.50 ± 2.500 **0.0061	52.50 ± 2.500 *0.0101	14.00 ± 1.000 ˢˢ0.0082

*Significantly different from group one at p < 0.05

SEM = Standard error of mean

Table 2: serum level of enzymes of carbon hydrates metabolism (G-6-PDH), lipid peroxidations (MDA) and antioxidant enzymes (GPx) activ ities

Groups	MDA (µmol/L) Mean ± SEM	GP (IU/L) Mean ± SEM	G6PDH (IU/L) Mean±SEM
Group one (Control)	19.50 ± 0.5000	3259 ± 104.5	3916 ± 26.00
Group Two  (X-ray only)	32.00 ± 1.000 ** 0.0079	2237 ± 157.0 *	2112 ± 104.0 ** 0.0035
Group Three (D-Riboce-L-Cysteine +X-ray)	29.50 ± 0.5000 ** 0.0050	2484 ± 366.0	2668 ± 208.0 *0.0271
Group Four (X-ray +D-Riboce-L-Cysteine)	28.00 ± 1.000 * 0.0169	2514 ± 38.00 *	3023 ± 61.00 ** 0.0055
Group Five (D-Riboce-L-Cysteine)	17.50 ± 0.5000 **  0.0059	3409 ± 82.50	4002 ± 16.50 **0.0031

*Significantly different from group one at p < 0.05

SEM = Standard error of mean

MDA= Malondialdehyde

GPx= Glutathione peroxidase

G6PDH activity was significantly higher in the control group compared to all other groups. The enzyme activity was also higher in control group compared to group two (radiation only) although the difference was significant. The enzyme activity was also significantly lower in group two (radiation only) compared to group four (radiation + antioxidant) and group five (antioxidant only) with a slight significant. MDA level was lower in the control group compared to all other groups except group five (antioxidant only) which was higher although it was insignificant; other differences were only significant to group two, three and four. MDA level was higher in group two (radiation only) compared to all other group, although with no significant however only significant to group five (antioxidant only). GP level was higher in the control group compared to all other groups except group five which was higher although with no significantly different.

Histological Observations:

The photomicrograph of the testis of control animals in group one showed basic histology arrangement of the testis with the testicular lobules, seminiferous epithelium and its constituent cells. Moreso, the testis of the animals in group two (X-ray only) showed widening of the intersial space, loss basal laminal, degeneration in spermatogia with vacuolation observed. Losess of germinal epithelium of seminiferous tubules were also observed in X-ray group, disrupt spermatogenesis by destroying the spermtids and spermtogonia.

The photomicrograph of the testis of the animal in group three (D-Ribose-L-cysteine + X-ray) also showed abnormal widening of the interstitial spaces, reduced leydig cells and degeneration of the spermatogia cells showing vacuolation.

Animal in group four (X-ray + D-Ribose-L-cysteine) also showed abnormal widening of the intersial space, reduced leydig cells and degeneration of the spermatogia cells.

The photomicrograph of the testis of the animal in group three (D-Ribose-L-cysteine) also showed an intact testicular integrity maintained with interstitial spaces, spermatogonia at different stages well expressed; an indication of fertility enhancing ability of D-Ribose-L-cysteine.

Figure 1: Testicular section of rats in control group stain with H/E X400, showing the spermatogonia in the basal laminar, spermatogonia A and B are well expressed. The interstitial space was with intact leydig cell

Figure 2: Testicular section of rats expose to radiation only, stain H/E X 400, widening of the interstitial space, loss of the basal laminal, generation in spermatogonia with vacuolation were observed

Figure 3: Testicular section of rats treated with D-Riboce-L-cysteine + X ray stain with H/E X400; abnormal widening of the interstitial spaces, reduced leydig cells and degeneration of the spermatogonial cell showing vaculation

Figure 4: Testicular section of rats treated with X ray + D-Riboce-L-cysteine stain with H/E X400; abnormal widening of the interstitial spaces, reduced leydig cell and degeneration of the spermatogonial cell showing vacuolation

Figure 5: A and B Testicular section of rats treated D-Riboce-L-cysteine stain with H/E X400 Intact testicular integrity was maintain with interstitial space, spermatogonia at different stages were well expressed.

Figure 6: Testicular section of rats in control group stain with PAS X400, showing the spermatogonia in the basal laminal, spermatogonia A and B are well expressed. The interstitial space was with intact leydig cell

Figure 7: Testicular section of rats expose to radiation only, stain PAS X 400, widening of the interstitial space, loss of the basal laminal, generation in spermatogonia with vaculation were observed

Figure 8: Testicular section of rats treated with D-Riboce-L-cysteine + X ray stain with PAS X400; abnormal widening of the interstitial spaces, reduced leydig cell and degeneration of the spermatogonial cell showing vaculation

Figure 9: Testicular section of rats treated with D-Riboce-L-cysteine + X ray stain with PAS X400; abnormal widening of the interstitial spaces, reduced leydig cell and degeneration of the spermatogonial cell showing vaculation

Figure 10 Testicular section of rats treated D-Riboce-L-cysteine stain with PAS X400 Intact testicular integrity was maintain with interstitial space, spermatogonia at different stages were well expressed.

4. DISCUSSION:

The development of radioprotective agents is important for protecting patients from the side- effects of radiotherapy, as well as occupational workers in nuclear and radiation plants¹⁵. Natural compounds have been evaluated as radioprotectors and seem that they exert their effect through antioxidants content and immunostimulant activities¹⁶. Testicular weight decreased following radiation exposure in the current investigation. If alterations in the interstitial tissue or Sertoli cells do not correspond with this drop, it can be the result of the actual loss of germinal epithelial cells¹⁸.

When animals exposed to X-ray only (group 2) were compared to control group members, the analysis of carbohydrate metabolic enzymes revealed a highly significant decrease in glucose-6-phosphate dehydrogenase (G-6-PDH) activity. This reduction could be explained by the consumption of G-6-PDH enzymes as a compensatory response to oxidative stress, where the enzymes were used to maintain sufficient levels of NADPH in response to the oxidative stress²⁰. But in contrast to D-ribose-L-cysteine, there was also a notable drop in the activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the X-ray only group. sole group, as well as a marginally significant difference between ray +D-Riboce-L-cysteine or D-Riboce-L-cysteine+ ray was seen.

An indicator of oxidative damage to cellular structures is the estimation of lipid peroxidation end products such malondialdehyde (MDA)²³. The X-ray group's significantly higher MDA level than the control group's indicated that the generation of free radicals, which results in damage and the loss of functional characteristics, is the cause. Tissue levels of glutathione peroxidase (GSH-P) and malondialdehyde (MDA) are reliable markers of oxidative stress brought on by lipid peroxidation²⁴. The oxidative stress-induced tissue damage in the X-ray group may be the cause of the decrease in sperm parameters when compared to the control group. Conversely, the antioxidant action of D-Riboce-L-cysteine may be the reason for the notable rise in sperm parameters found in the D-Riboce-L-cysteine group. Certain vitamins are thought to aid increase male fertility²⁵.

The decrease in sperm parameters in X-ray group compared to control group may be due to damage caused by oxidative stress in the tissue and also a significant increase in sperm parameters observed in D-Riboce-L-cysteine group may be as a result of antioxidant effect of D-Riboce-L-cysteine. It is believed that taking certain vitamins may help improve male fertility²⁶.

The exposure of rats to X-ray induces a biological and histological effects in the testes which can be attributed to increase oxidative stress resulting from radiation intoxication²⁷. D-Riboce-L-cysteine produce a regenerative effect against radiation damage and increase in G-6-PDH level as well as normal architecture of the testis; particularly in relation to the leydig cells and germinal epithelium of the testes of the rats treated with D-Riboce-L-cysteine. This study showed that histological and histochemical parameters changed when Wistar rats with X-ray-induced testicular injury were given D-ribose-L-cysteine.

5. CONCLUSION:

Rats exposed to X-rays experience biochemical and histological changes in their testes, which are related to elevated oxidative stress brought on by radiation poisoning. When applied to the testes of rats treated with D-ribose-L-cysteine, it has a regenerative impact against radiation damage, increases G6PDH levels, and preserves the testes' natural architecture. This is especially true in respect to the leydig cells and germinal epithelium.

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Received on 05.12.2024      Revised on 30.12.2024

Accepted on 16.01.2025 Published on 28.02.2025

Available online from March 03, 2025

Asian J. Pharm. Res. 2025; 15(1):29-35.

DOI: 10.52711/2231-5691.2025.00006

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