3. Introduction

4. Introduction
• 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 CPPharmacological efficacy of CP

6. Introduction
Limitation of CP chemotherapyLimitation 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
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).
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 CYPAltering of hepatic CYP

10. CPCP AcroleinAcrolein 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
LPO of sperm
membrance
impair energy
metabolism and motility
impair energy
metabolism and motility
(Aitken et al., 1993; Alvarez and Storey,
1995)
(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.
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 IPhase I Phase IIPhase II

16. Introduction
00
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…The Aim of Present Study…

17. Materials and methods

18. Materials and methods
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.
Groups Control CP CP&DADS DADS
Treatment (mg/kg/day):
CP/DADS
0/0 150/0 150/50 0/50

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**
P < 0.01 vs Control group; ††
P < 0.01 vs CP group
Items
Group
Control CP CP&DADS DADS
No. of rats 6 6 6 6
Body weight
Day 1 280.8±12.86 278.0±11.46 276.2±10.60 276.0±15.61
Day 3 299.7±10.72 263.6±11.08**
267.5±13.99**
291.6±16.91
Day 7 320.3±9.16 236.5±9.81**
248.9±9.20**
318.0±20.93
Day 11 334.3±11.02 219.9±37.02**
272.3±5.56**,††
329.1±24.40
Food consumption
Day 1 20.7±2.86a
10.0±5.67**
13.0±3.73**
19.4±0.73
Day 3 23.9±2.26 9.9±5.49**
14.3±2.64**
23.6±1.84
Day 7 21.6±0.74 3.7±4.04**
15.2±1.44**,††
22.4±1.70
Day 10 25.7±2.29 8.5±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*, **
P < 0.05, P < 0.01 vs Control group; †
P < 0.05 vs CP group
Items
Group
Control CP CP&DADS DADS
No. of rats 6 6 6 6
Prostates (g) 0.38±0.06 0.19±0.03**
0.24±0.05**
0.38±0.06
per body weight (%) 0.11±0.02 0.09±0.02 0.09±0.02 0.11±0.02
Seminal vesicles (g) 1.27±0.14 0.69±0.18**
0.97±0.10**,†
1.25±0.13
per body weight (%) 0.38±0.04 0.32±0.09 0.36±0.03 0.38±0.04
Testes (g) 3.45±0.37 3.15±0.33 3.33±0.35 3.27±0.27
per body weight (%) 1.03±0.11 1.45±0.15**
1.22±0.10*,†
1.00±0.08
Epididymides (g) 0.75±0.08 0.64±0.08 0.72±0.08 0.71±0.05
per body weight (%) 0.22±0.03 0.29±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
Control CP CP&DADS DADS
No. of rats 6 6 6 6
Sperm count
(×106
/cauda epididymis)
141.3±13.16 146.8±18.35 155.1±21.27 157.7 ±18.31
Sperm motility (%) 79.8±3.70 48.7±7.37**
81.8±5.59††
74.8±8.40
Sperm abnormalities (%) 6.6±1.67 7.5±2.43 7.0±3.32 7.5±1.52
Small head 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00
Amorphous head 0.0±0.00 0.3±0.52 0.6±0.89 0.2±0.41
Two heads/tails 0.0±0.00 0.0±0.00 0.0±0.00 0.0±0.00
Excessive hook 0.2±0.45 0.2±0.41 0.2±0.45 0.2±0.41
Straight hook 3.2±1.30 2.8±2.04 2.8±1.30 1.8±2.23
Folded tail 0.8±0.84 1.8±1.94 0.8±1.10 1.3±1.97
Short tail 0.6±0.89 0.7±0.82 0.0±0.00 1.3±0.82
No tail 1.8±1.30 1.7±1.37 2.6±1.52 2.7±2.50
*, **
P < 0.05, P < 0.01 vs Control group; †, ††
P < 0.05, P < 0.01 vs CP group*, **
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).
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 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*, **
P < 0.05, P < 0.01 vs Control group; †, ††
P < 0.05, P < 0.01 vs CP group
Items
Group
Control CP CP&DADS DADS
Stage
II
Spermatogonia 18.7±1.63a
5.3±3.08**
15.2±3.06††
18.7±1.21
Pachytene spermatocytes 48.5±3.83 30.0±9.49**
42.2±6.43†
48.0±4.05
Round spermatids 155.0±8.49 152.0±10.45 160.5±13.26 157.0±8.20
Elongated spermatids 151.0±12.00 156.0±8.49 158.2±13.01 150.7±9.50
Sertoli cells 15.8±2.64 19.5±3.27 17.5±2.59 16.8±3.06
Stage
V
Spermatogonia 33.5±4.51 8.2±5.31**
25.2±7.52††
34.0±3.95
Pachytene spermatocytes 50.8±5.04 37.3±5.53**
42.3±5.16 51.2±5.38
Round spermatids 150.8±10.94 152.0±10.45 151.8±10.36 150.2±10.28
Elongated spermatids 159.3±8.96 156.0±8.49 165.2±8.04 161.7±6.19
Sertoli cells 16.2±2.48 18.0±2.28 17.2±1.83 16.7±2.73
Stage
VII
Spermatogonia 1.5±1.64 1.5±1.05 1.8±1.47 2.2±1.17
Preleptotene spermatocytes 37.0±5.22 17.7±5.47**
33.2±7.41††
37.2±3.66
Pachytene spermatocytes 53.5±6.86 55.2±5.67 52.0±7.67 56.0±4.77
Round spermatids 151.2±8.98 151.8±15.05 152.3±8.78 150.7±8.38
Elongated spermatids 154.2±9.89 150.7±8.62 149.3±10.88 152.5±10.03
Sertoli cells 17.7±1.37 17.2±1.47 17.8±1.33 17.7±1.63
Stage
XII
Spermatogonia 4.0±1.41 1.3±2.03*
3.7±1.37 3.5±1.52
Zygotene spermatocytes 46.8±4.40 24.7±5.85**
38.7±4.93*,††
45.2±3.71
Pachytene spermatocytes 59.0±5.51 58.0±5.02 61.0±6.81 57.7±6.65
Elongated spermatids 164.8±5.67 163.2±11.48 159.0±11.90 164.5±5.36
Sertoli cells 17.8±1.72 18.5±1.38 19.3±2.80 17.7±2.07

27. Figure 2. Representative photographs of TUNEL analysis in testis sections
treated CP and/or DADS
VC CP
DADSCP&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
DADSCP&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*, **
P < 0.05, P < 0.01 vs Control group; ††
P < 0.01 vs CP group

30. Discussion

31. Cellular damageSperm 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 IPhase I
CYPs Acrolein
DADSDADS
Toxic
metabolite
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 pregnant Midpregnant Late pregnant
(He et al., 2005)

38. Introduction
• Effects of pregnancy on CYPs (Placenta)
• Thebandspositivefor CYP1A1, 2B1, 2C6, 2C12, 2D1, 2D4, 2E1and4A1were notdetectedthroughpregnancy.
• CYP3A1intheplacenta ismainlydetectedinthecytoplasmofgiantcellsinthe trophoblasticregion, whichisthoughttobeimportant in exchanging
manysubstratesbetweenthematernalandfetalcirculation (Okajima etal.,1993).
• TheseresultssuggestthatCYP3A1may be amajorcomponentof CYPsystemintheratplacenta.
(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.