Effect of isotretinoin (Netlook) on the testis of adult male albino rats and the role of omega 3 supplementation: A histological and biochemical study

Abstract Isotretinoin is an oral retinoid which used across the world in the treatment of patients especially adolescents complaining of acne. In spite of the prevalent clinical use of isotretinoin, the generation of oxidative stress with the affection of several organs leads to the limitation and restriction of its use. Omega‐3 (N‐3) is an essential polyunsaturated fatty acid (PUFAs) with powerful antioxidant properties. The aim of this study was to investigate the histological and biochemical changes occurring in the rat testis following isotretinoin intake and to evaluate the role of omega 3 supplementation in ameliorating testicular damage. Thirty adult male albino rats were divided equally into three groups. Group I is the control group, group II received isotretinoin (1.0 mg/kg/day) dissolved in distilled water and group III received isotretinoin (1.0 mg/kg/day) and omega 3 (400 mg/kg/day). Testis samples were collected and processed for light and electron microscopic examination. The blood samples were collected for biochemical assessments. Results indicated that isotretinoin caused histological changes in all stages of spermatogenesis and alterations of the hormonal assay. These changes in the rat testis which were corrected by omega 3 use.

docosahexaenoic acid (DHA). They are found naturally in fish oil, flaxseed and some nuts and have anti-inflammatory effects. 5 Omega3 supplementation have protective roles in the liver, kidney, cardiovascular system, immune system disorders, psychological status, maternal and offspring health. 6 N-3 PUFAs are important ingredients of cell membranes and play a role in the integrity of various membrane channels and receptors. 7 In this study, omega 3 was used as a protection against the effect of isotretinoin testicular damage which may lead to reproductive dysfunction. To our knowledge, this is the first work to assess the protective effects of omega-3 against isotretinoin testicular damage.

| Animals
Thirty adult male albino rats weighting 140-170 g were used in this study. All animal procedures were performed in accordance with the guide for the care and use of laboratory animals and approved by the Animal Ethical Committee at The Faculty of Medicine for Girls, Al-Azhar University. The principles of laboratory animal care were followed, as well as the specific national laws, when applicable. The examined rats were divided equally into 3 groups: G I: Control group was kept without medications.
G II: Received isotretinoin at a single oral daily dose of (1.0 mg/ kg/day) for 21 successive days according to 8 then sacrificed. This administrated dose was equivalent to the average human therapeutic dose.
G III: Received isotretinoin (1.0 mg/kg/day) concomitant with omega 3 (400 mg/kg/day) orally for 21 successive days 9 then sacrificed. This administrated dose was adjusted by using the formula of Paget and Barnes. 10 By using this formula, the rat dose = the human dose × 18/1000. Isotretinoin tablet containing 20 mg was dissolved in 20 ml distilled water to produce a suspension containing (1 mg of isotretinoin /ml). Rat dose = The human dose (1 mg) × 18/1000 = 0.018 mg. Each rat received 0.018 ml which contained 0.018 mg isotretinoin.
Each omega 3 soft gelatinous capsule contained 1000 mg omega-3 fish oil. The capsule was carefully evacuated by 1 ml syringe; then, the content was dissolved in in 9 ml corn oil. So, the 10 ml of oily solvent will contain 1000 mg omega-3 fish oil. Rat dose = The human dose (400 mg) × 18/1000 = 7.2 mg. The equivalent dose 7.2 mg of omega 3 was administered in 0.72 ml of the oily solvent.

| Drugs
Isotretinoin was purchased as a tablet containing 20

| Biochemical technique
At the end of the experiment, blood samples were collected from 12 to 14 h fasting rats, from retro-orbital sinus by capillary tubes under light ether anaesthesia. The collected blood samples were centrifuged then sera were separated and stored at −20°C until the time of use. Serum testosterone was assayed by rat testosterone ELISA kit of DRG International, Inc., USA, as previous publication. 11 Luteinizing hormone (LH) was assayed by rat LH ELISA kit of DRG International, Inc., USA, as mentioned. 12 Malondialdehyde (MDA) production following the method of 13 superoxide dismutase (SOD) activity was determined by ELISA as described previously. 14

| Histological techniques
All studied animals were sacrificed by ether inhalation with excision of the two testes. One testis was fixed in Bouin's solution and processed for light microscopic examination 15 to be stained by Haematoxylin and eosin stain (H&E) and Periodic acid Schiff's (PAS).
In addition, proliferating cell nuclear antigen (PCNA) immunohistochemical techniques were used for detection of active cell proliferation in testicular tissue. 16 The other testis was fixed in glutaraldehyde and processed for electron microscopic (E M) examination. 17

| Morphometrical measurements
It was carried out using a computerized image analysis system (Software Leica Quin 500). The basement membrane thickness, epithelial height, seminiferous tubular diameter and the mean area % of PCNA-positive cells were measured in 10 non-overlapping fields per group using a magnification (400×).

| Statistical analysis
Statistical Analysis was conducted using (SPSS) program version 22, data expressed as mean ± SD for statistical analysis, using analysis of variance [anova] test to compare between the different studied groups. The value of significance was taken at (p-value ≤0.05).

| A: Biochemical results
Administration of isotretinoin to rats (G II) caused significant decrease in serum testosterone and SOD and significant increase in LH and MDA if compared to control group (G 1). Co-administration of isotretinoin and omega 3 to rats (G III) caused a significant increase in serum testosterone and SOD and a significant decrease in LH and MDA if compared to G II, but these parameters still significantly changed if compared to G I (Table 1). 2. Administration of isotretinoin to (G II) caused a significant decrease in tubular diameter and epithelial height compared to the control group (G I). In (G III), co-administration of isotretinoin and omega 3 caused a significant increase in epithelial height and tubular diameter compared to G II (Table 3 and histogram 3).

| DISCUSS ION
Testing medicines to determine the possible harmful health effects and exposure levels that can be considered safe is of utmost importance. Most adverse effects involving the use of retinoids, including isotretinoin, are related to the skin and mucous membranes, nervous system, skeletal muscle, haematopoietic, lymphatic, gastrointestinal, cardiorespiratory and genitourinary systems. 18  and Leydig cells, which might affect the spermatogenesis. 19 These histological changes in the current study were confirmed by significant decrease in serum testosterone and significant increase in LH levels. The same results were reported with a significant decrease in serum testosterone and a significant increase in serum LH level with isotretinoin treatment. 20 The increase in LH level might be explained by the negative feedback effect from reduced testosterone level. 21 This reduction might be mediated via the gonads which contained abundant retinoid receptors. 22 Reduction in androgen, testosterone and rostenedione levels were reported after 12 weeks from isotretinoin use. 23 These effects were mediated via presence of retinoid receptors in tests. 24 Reduced in retinoic acid level might cause alterations in the differentiation of spermatogonia either at the beginning of meiosis or at the beginning of the cycle of the seminiferous epithelium. 27 Retinoic acid was a potent endocrinologic regulator of the testicular development and germ cell proliferation. 22 Vitamin A is an essential factor during spermatogenesis. 19 Vitamin A deficiency could result in disturbance of testosterone secretion, meiotic germ cells degeneration and cessation of spermatogenesis. 24 However, hypervitaminosis A could lead to disturbance of spermatogenesis rhythm, and retinoid deficiency or excess may be harmful to spermatogenesis and steroidogenesis. 19 In contrary to these results, systemic isotretinoin caused no change in total testosterone or LH levels with positive effects on all the parameters of the spermiogram. 28 Furthermore, the positive effect of retinoic acid was dosedependent, and with higher doses, signs of hypervitaminosis and degeneration of the spermatocytes were noticed. So isotretinoin effect on sex hormones is time and dose dependent. 29 In this study, a notable elevation of MDA (representing massive lipid peroxidation) and significant decrease in SOD level with isotretinoin use may reflect increased cellular oxidative stress, which may be another factor in isotretinoin testicular damage. This finding was supported by mitochondrial changes observed by EM. Accordingly, mitochondrial reductive stress might be a possible source of free radicals with the accumulation of reducing equivalents in the mitochondrial electron transport chain, which enhance the production of reactive oxygen species (ROS). 30,31 In addition, mitochondria may become an oxidative target resulting in lipid peroxidation, DNA cleavage and impaired ATP production. 31,32 This factor was explained by other studies which clarified that isotretinoin treatment resulted in loss of biological membrane fluidity, neutrophil activation and lipid peroxidation with production of ROS. 33,34 Regarding level of SOD; which is an indicator of antioxidant activity as it directed the superoxide anion to form hydrogen peroxide. 35 In this study, the decreased SOD activity may reflect accumulation of superoxide radicals and hydrogen peroxide resulting in a state of oxidative stress. As mentioned before the steroidogenic capacity and proliferation of Leydig cells were deteriorated and apoptosis occur with exposure to oxidants. 36 Furthermore, ROS induced lipid peroxidation which resulted in changes in testicular functions. 37 In this study, co-administration of omega 3 and isotretinoin caused improvement of histological changes caused by isotretinoin nearly to be similar to the control group G 1. These findings could be explained by the high amounts of PUFA in the composition of the male germ cell membranes, which are mainly susceptible to lipid peroxidation. 38 Due to the high content of DHA in omega-3, it is capable to restore fertility through the protection of the germ cells, spermatozoa and spermatids membranes architecture. 39 These findings were similar to other studies which found that fish oil in obese male mice could improve sexual hormones synthesis by increasing the number of Leydig cells and diminishing apoptosis then stimulating the testosterone biosynthesis through the adenyl cyclase, cyclic AMP, protein kinase A and splicing factor-1 pathways. 40 Our data showed that compared to isotretinoin alone, the treatment of rats with omega 3 and isotretinoin increases levels of antioxidants enzymes (SOD) and reduced (MDA). The administration of omega 3 to male rats caused a significant increase in glutathione level and a significant decrease in MDA level. 41 Therefore, PUFAs oil improves antioxidant activity and decreased the oxidative stress, so diet rich in omega-3 can raise the SOD level. 42 Diet enriched with omega-3 from fish oil and linseed oil decreases serum cholesterol, triglycerides level and enhances antioxidative status. 43 In addition, incorporation of PUFAs in cell membrane may reduce the susceptibility of cells to lipid peroxidation, change the fluidity of cell membrane, elevate enzymes activity, enhance receptor function and influence the production of lipid mediator. 44 This may be associated with the reaction between free radical and PUFAs resulting in avoiding free radical conjugating with fatty acids of the membrane. 45 ROS had been concerned with reduced fertility and high levels of nitric oxide, which is related to ROS generation. 46

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available