Introduction

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.

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

IntroductionLimitation of CP chemotherapyLimitation of CP chemotherapy

injury to normal tissue

Muti-organ toxicityTesticular 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)

Introduction

Testicular toxicity of CP

Therefore, a potential therapeutic approach to protect or reverse CP-induced testicular toxicity would have very

important clinical consequences.

CP metabolism-liver

Toxic metabolite

Toxic metabolite

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

CPCP AcroleinAcrolein ROS production Oxidative damageROS 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 acidslow antioxidant capacity

rich in polyunsaturated fatty acidslow 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

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.

Potentantioxidant

Potentantioxidant

Testicular toxicity

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)

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)

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

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…

Materials and methods

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

Materials and methodsBody 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

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.

Results

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

ItemsGroup

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

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

ItemsGroup

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

Table 3. Sperm analysis of male rats treated with CP and/or DADS

ItemsGroup

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

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

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

ItemsGroup

Control CP CP&DADS DADS

StageII

Spermatogonia 18.7±1.63a 5.3±3.08** 15.2±3.06†† 18.7±1.21Pachytene spermatocytes 48.5±3.83 30.0±9.49** 42.2±6.43† 48.0±4.05Round spermatids 155.0±8.49 152.0±10.45 160.5±13.26 157.0±8.20Elongated spermatids 151.0±12.00 156.0±8.49 158.2±13.01 150.7±9.50Sertoli cells 15.8±2.64 19.5±3.27 17.5±2.59 16.8±3.06

StageV

Spermatogonia 33.5±4.51 8.2±5.31** 25.2±7.52†† 34.0±3.95Pachytene spermatocytes 50.8±5.04 37.3±5.53** 42.3±5.16 51.2±5.38Round spermatids 150.8±10.94 152.0±10.45 151.8±10.36 150.2±10.28Elongated spermatids 159.3±8.96 156.0±8.49 165.2±8.04 161.7±6.19Sertoli cells 16.2±2.48 18.0±2.28 17.2±1.83 16.7±2.73

StageVII

Spermatogonia 1.5±1.64 1.5±1.05 1.8±1.47 2.2±1.17Preleptotene spermatocytes 37.0±5.22 17.7±5.47** 33.2±7.41†† 37.2±3.66Pachytene spermatocytes 53.5±6.86 55.2±5.67 52.0±7.67 56.0±4.77Round spermatids 151.2±8.98 151.8±15.05 152.3±8.78 150.7±8.38Elongated spermatids 154.2±9.89 150.7±8.62 149.3±10.88 152.5±10.03Sertoli cells 17.7±1.37 17.2±1.47 17.8±1.33 17.7±1.63

StageXII

Spermatogonia 4.0±1.41 1.3±2.03* 3.7±1.37 3.5±1.52Zygotene spermatocytes 46.8±4.40 24.7±5.85** 38.7±4.93*,†† 45.2±3.71Pachytene spermatocytes 59.0±5.51 58.0±5.02 61.0±6.81 57.7±6.65Elongated spermatids 164.8±5.67 163.2±11.48 159.0±11.90 164.5±5.36Sertoli cells 17.8±1.72 18.5±1.38 19.3±2.80 17.7±2.07

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

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

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

Discussion

Cellular damageSperm damage, histopathologic lesions, spermatogenic cell damage, apoptosis

Testicular toxicity

Discussion

`

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.

Conclusion

CP

ROSProduction

&OxidativeDamage

ROSProduction

&OxidativeDamage Testicular

toxicity

Phase IPhase I

CYPs Acrolein

DADSDADS

Toxic metabolite

Toxic metabolite

Induction of cytochrome P450 3A1 expression by diallyl disulfide: Protective effects against cyclophosphamide-induced

embryo-fetal developmental toxicity

Developmental toxicity

Introduction

• Effects of pregnancy on CYPs (Maternal liver)

Non pregnant Midpregnant Late pregnant

(He et al., 2005)

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

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.