1. Mechanism of protection by diallyl disulfide against cyclophosphamide- induced testicular toxicity and oxidative stress in rats

3. Introduction

4. Cancer Cancer is the first leading cause of death in Korea and in many other nations in the world. Cancer chemotherapy is typically associated with severe side effects.

5. Introduction Cyclophosphamide (CP) CP was introduced in 1958. Endoxan ®, Cytoxan ® Alkylaing agent solid tumors, Hodgkin’s disease, non-neoplastic conditions, and transplant rejection combatant drug (West, 1997) Pharmacological efficacy of CP

6. Introduction Limitation of CP chemotherapy injury to normal tissue Muti-organ toxicity Testicular toxicity (Rezvanfar et al., 2008) CP causes several adverse effects including testicular toxicity in human and experimental animals. (Qureshi et al., 1972; Elangovan et al., 2006; Rezvanfar et al., 2008) CP causes several adverse effects including testicular toxicity in human and experimental animals. (Qureshi et al., 1972; Elangovan et al., 2006; Rezvanfar et al., 2008)

7. Introduction Testicular toxicity of CP Therefore, a potential therapeutic approach to protect or reverse CP-induced testicular toxicity would have very important clinical consequences.

8. CP metabolism-liver Toxic metabolite

9. The concomitant use of CP with other drugs that inhibit or induce the CYP2B, CYP2C, or CYP3A enzymes can lead to drug-drug interactions (Chang et al., 1997; Rae et al., 2002; Yu et al., 1999). Introduction CP CP is a prodrug, which requires hepatic biotransformation to exert its testicular toxic effect. Rate and pattern of CP metabolism Altering of hepatic CYP

10. CP Acrolein ROS production Oxidative damage ROS production Oxidative damage Introduction Adult male patients: oligospermia or aspermia – male infertiltiy Male rat: oligospermia or aspermaia, biochemical and structural changes in the testis and epididymis (Mirkes et al., 1984; Matalon et al., 2004) CP is cytotoxic to rapidly dividing cells - Testis: good target CP is cytotoxic to rapidly dividing cells - Testis: good target rich in polyunsaturated fatty acids low antioxidant capacity rich in polyunsaturated fatty acids low antioxidant capacity LPO of sperm membrance impair energy metabolism and motility (Aitken et al., 1993; Alvarez and Storey, 1995) Spermatotoxicity

11. Introduction CP To avoid these toxic side effects, CP is typically used in combination with various detoxifying and protective agents to reduce or eliminate its adverse toxic effects. Antioxidant agents have protective action against CP- induced testicular toxicity. Taurine (Abd-Allah et al., 2005) Flavonoids (Ozcan et al., 2005) Melatonin (Tripathi and Jena, 2010) Trigonella foenum-graecum L. (Bhatia et al., 2006) Thus, a combination of the drug delivered together with a potent antioxidant may be appropriate to reduce the testicular toxic effects of CP.

12. Potent antioxidant Potent antioxidant Testicular toxicity

13. Introduction Diallyl disulfide (DADS) Garlic (Allium sativum L.) contains more than 20 organosulfur compounds. Experimental animal studies have shown inhibition of chemically induced carcinogenesis in different organs by certain sulfur-containing compounds. (Sparnins et al., 1988; Wattenberg et al., 1989)

14. Introduction 4.7% 21.9% 41.5% Diallyl disulfide (DADS) A major component of the secondary metabolites derived from garlic A potent compound to prevent cancer, genotoxicity, nephrotoxicity, urotoxicity, and hepatotoxicity (Nakagawa et al., 2001; Guyonnet et al., 2002; Pedraza-Chaverrí et al., 2003; Fukao et al., 2004; Kim et al., 2014)

15. Introduction Diallyl disulfide (DADS) phase I enzymes, such as hepatic CYP phase II enzymes : GSTs antioxidant-system capacity (Pan et al., 1993; Singh et al., 1998; Wu et al., 2001; Guyonnet et al., 2002; Pedraza- Chaverrí et al., 2003; Fukao et al., 2004) Phase I Phase II

16. Introduction 0 0 Despite the favorable pharmacological properties of DADS, its protective capacity against testicular toxicity caused by CP has not been explored previously. Therefore, the aim of the present study was to evaluate the protective effects of DADS on CP-induced testicular toxicity. To study the protective mechanism of DADS, potential effects of DADS on the expression of hepatic CYP involved in the metabolism of CP, oxidative stress, and apoptotic changes in spermatogenic germ cells were also assessed. Despite the favorable pharmacological properties of DADS, its protective capacity against testicular toxicity caused by CP has not been explored previously. Therefore, the aim of the present study was to evaluate the protective effects of DADS on CP-induced testicular toxicity. To study the protective mechanism of DADS, potential effects of DADS on the expression of hepatic CYP involved in the metabolism of CP, oxidative stress, and apoptotic changes in spermatogenic germ cells were also assessed. The Aim of Present Study…

17. Materials and methods

18. Animals: Sprague-Dawley male rats aged 9 weeks Experimental groups: Total 24 rats were assigned into four experimental group. Each group consisted of 6 rats. Test substance and treatment: DADS was gavaged to rats once daily for 10 days at 50 mg/kg/day. (Guyonnet et al., 1999; Wu et al., 2002) On the first 2 days, CP (150 mg/kg/day) was injected intraperitoneally to rats 1 h after the DADS treatment. (Matsui et al., 1995; Senthilkumar et al., 2006) All animals were sacrificed 11 days after DADS administration. GroupsControlCPCP&DADSDADS Treatment (mg/kg/day): CP/DADS 0/0150/0150/500/50

19. Materials and methods Body weight & food consumption: days 1, 3, 7, and 11(10) Reproductive organ weight: prostates, seminal vesicles, testes, and epididymides Sperm examination: epididymal sperm head count, epididymal motility, and sperm morphology Histopathologic examinations (H&E) -Testis Quantitative morphometry of spermatogenic epithelia -Stages II, V, VII, and XII -Spermatogonia, primary spermatocytes, secondary spermatocyte, spermatid. Apoptosis -Caspase-3 IHC, TUNEL assay

20. Materials and methods Oxidative stree analysis: MDA, GSH, CAT, GR, and GST (testis) Preparation of hepatic microsomes: (Jeong and Yun, 1995) – CYP analysis Western blot: β-actin, CYP2B1/2, CYP2C11, and CYP3A1 Statistics: One-way analysis of variance followed by Tukey’s multiple comparison test on GraphPad InStat Software.

21. Results

22. Table 1. Body weight changes and food consumption in male rats treated CP and/or DADS ** P < 0.01 vs Control group; †† P < 0.01 vs CP group Items Group ControlCPCP&DADSDADS No. of rats6666 Body weight Day 1280.8±12.86278.0±11.46276.2±10.60276.0±15.61 Day 3299.7±10.72263.6±11.08 ** 267.5±13.99 ** 291.6±16.91 Day 7320.3±9.16236.5±9.81 ** 248.9±9.20 ** 318.0±20.93 Day 11334.3±11.02219.9±37.02 ** 272.3±5.56 **,†† 329.1±24.40 Food consumption Day 120.7±2.86 a 10.0±5.67 ** 13.0±3.73 ** 19.4±0.73 Day 323.9±2.269.9±5.49 ** 14.3±2.64 ** 23.6±1.84 Day 721.6±0.743.7±4.04 ** 15.2±1.44 **,†† 22.4±1.70 Day 1025.7±2.298.5±7.01 ** 17.8±0.24 **,†† 25.2±1.18

23. Table 2. Absolute and relative reproductive organ weights in male rats treated with CP and/or DADS *, ** P < 0.05, P < 0.01 vs Control group; † P < 0.05 vs CP group Items Group ControlCPCP&DADSDADS No. of rats6666 Prostates (g)0.38±0.060.19±0.03 ** 0.24±0.05 ** 0.38±0.06 per body weight (%)0.11±0.020.09±0.02 0.11±0.02 Seminal vesicles (g)1.27±0.140.69±0.18 ** 0.97±0.10 **,† 1.25±0.13 per body weight (%)0.38±0.040.32±0.090.36±0.030.38±0.04 Testes (g)3.45±0.373.15±0.333.33±0.353.27±0.27 per body weight (%)1.03±0.111.45±0.15 ** 1.22±0.10 *,† 1.00±0.08 Epididymides (g)0.75±0.080.64±0.080.72±0.080.71±0.05 per body weight (%)0.22±0.030.29±0.03 ** 0.27±0.03 * 0.22±0.02

24. Table 3. Sperm analysis of male rats treated with CP and/or DADS Items Group ControlCPCP&DADSDADS No. of rats6666 Sperm count (×10 6 /cauda epididymis) 141.3±13.16146.8±18.35155.1±21.27157.7 ±18.31 Sperm motility (%)79.8±3.7048.7±7.37 ** 81.8±5.59 †† 74.8±8.40 Sperm abnormalities (%)6.6±1.677.5±2.437.0±3.327.5±1.52 Small head0.0±0.00 Amorphous head0.0±0.000.3±0.520.6±0.890.2±0.41 Two heads/tails0.0±0.00 Excessive hook0.2±0.450.2±0.410.2±0.450.2±0.41 Straight hook3.2±1.302.8±2.042.8±1.301.8±2.23 Folded tail0.8±0.841.8±1.940.8±1.101.3±1.97 Short tail0.6±0.890.7±0.820.0±0.001.3±0.82 No tail1.8±1.301.7±1.372.6±1.522.7±2.50 *, ** P < 0.05, P < 0.01 vs Control group; †, †† P < 0.05, P < 0.01 vs CP group

25. Figure 1. Representative photographs of testis sections treated with CP and/or DADS. desquamation in all types of cells (black arrow), vacuolization (white arrow), degeneration of spermatocytes (black arrow head), and decreased number of spermatocytes/spermatogonia (white arrow head). VC CP CP&DADS

26. Table 4. The number of spermatogenic cells in seminiferous tubules of male rats treated CP and/or DADS *, ** P < 0.05, P < 0.01 vs Control group; †, †† P < 0.05, P < 0.01 vs CP group Items Group ControlCPCP&DADSDADS Stage II Spermatogonia18.7±1.63 a 5.3±3.08 ** 15.2±3.06 †† 18.7±1.21 Pachytene spermatocytes48.5±3.8330.0±9.49 ** 42.2±6.43 † 48.0±4.05 Round spermatids155.0±8.49152.0±10.45160.5±13.26157.0±8.20 Elongated spermatids151.0±12.00156.0±8.49158.2±13.01150.7±9.50 Sertoli cells15.8±2.6419.5±3.2717.5±2.5916.8±3.06 Stage V Spermatogonia33.5±4.518.2±5.31 ** 25.2±7.52 †† 34.0±3.95 Pachytene spermatocytes50.8±5.0437.3±5.53 ** 42.3±5.1651.2±5.38 Round spermatids150.8±10.94152.0±10.45151.8±10.36150.2±10.28 Elongated spermatids159.3±8.96156.0±8.49165.2±8.04161.7±6.19 Sertoli cells16.2±2.4818.0±2.2817.2±1.8316.7±2.73 Stage VII Spermatogonia1.5±1.641.5±1.051.8±1.472.2±1.17 Preleptotene spermatocytes37.0±5.2217.7±5.47 ** 33.2±7.41 †† 37.2±3.66 Pachytene spermatocytes53.5±6.8655.2±5.6752.0±7.6756.0±4.77 Round spermatids151.2±8.98151.8±15.05152.3±8.78150.7±8.38 Elongated spermatids154.2±9.89150.7±8.62149.3±10.88152.5±10.03 Sertoli cells17.7±1.3717.2±1.4717.8±1.3317.7±1.63 Stage XII Spermatogonia4.0±1.411.3±2.03 * 3.7±1.373.5±1.52 Zygotene spermatocytes46.8±4.4024.7±5.85 ** 38.7±4.93 *,†† 45.2±3.71 Pachytene spermatocytes59.0±5.5158.0±5.0261.0±6.8157.7±6.65 Elongated spermatids164.8±5.67163.2±11.48159.0±11.90164.5±5.36 Sertoli cells17.8±1.7218.5±1.3819.3±2.8017.7±2.07

27. Figure 2. Representative photographs of TUNEL analysis in testis sections treated CP and/or DADS VC CP DADS CP&DADS ** P < 0.01 vs Control group; †† P < 0.01 vs CP group ** P < 0.01 vs Control group; †† P < 0.01 vs CP group

28. Figure 3. Representative photographs of immunohistochemical analysis of caspase-3 in testis sections treated CP and/or DADS VC CP DADS CP&DADS ** P < 0.01 vs Control group; †† P < 0.01 vs CP group ** P < 0.01 vs Control group; †† P < 0.01 vs CP group

29. Figure 4. Western blot analysis of hepatic microsomal CYP2B1/2, CYP2C11, and CYP3A1 expressions in male rats treated with CP and/or DADS. *, ** P < 0.05, P < 0.01 vs Control group; †† P < 0.01 vs CP group

30. Discussion

31. Cellular damage Sperm damage, histopathologic lesions, spermatogenic cell damage, apoptosis Testicular toxicity

32. Discussion `

33. Conclusion  DADS had protective effects against CP-induced testicular toxicity in rats.  These findings suggest that DADS, which is a naturally occurring antioxidant from commonly consuming plants of allium spices, may be a useful protective agent against various testicular toxicities induced by oxidative stress.

34. Conclusion CP ROS Production & Oxidative Damage ROS Production & Oxidative Damage Testicular toxicity Phase I CYPs Acrolein DADS Toxic metabolite

35. Induction of cytochrome P450 3A1 expression by diallyl disulfide: Protective effects against cyclophosphamide-induced embryo-fetal developmental toxicity

36. Developmental toxicity

37. Introduction Effects of pregnancy on CYPs (Maternal liver) Non pregnantMidpregnantLate pregnant (He et al., 2005)

38. Introduction Effects of pregnancy on CYPs (Placenta) The bands positive for CYP1A1, 2B1, 2C6, 2C12, 2D1, 2D4, 2E1 and 4A1 were not detected through pregnancy. CYP3A1 in the placenta is mainly detected in the cytoplasm of giant cells in the trophoblastic region, which is thought to be important in exchanging many substrates between the maternal and fetal circulation (Okajima et al., 1993). These results suggest that CYP3A1 may be a major component of CYP system in the rat placenta. (Ejiri et al., 2001) GD9 GD11 GD13 GD16 GD19 Positive CYP3A1

40. Conclusion Our results show that DADS has protective effects against CP-induced embryo-fetal developmental toxicity in rats, and that the protective effects of DADS may be due to a reduction in oxidative stress and its ability to promote detoxification of CP by inducing CYP3A1 in the maternal liver and placenta.