1. Thymoquinone attenuates cisplatin induced toxicity and oxidative damage in rat kidney
By: Zeba Farooqui
Department of Biochemistry
Aligarh Muslim University, India
Supervisor
Dr. Farah Khan

2. Overview
Cisplatin (cis-diamminedichloroplatinum II, CP) is one of the most effective platinum-based chemotherapeutic agent with a broad range of antitumor activities.
Therapeutic utility of CP is limited due to its associated side effects including acute and chronic nephrotoxicity, hepatotoxicity and ototoxicity.
Multifactorial mechanisms might be involved in CP induced nephrotoxicity. However, one of the well known mechanism involved is CP induced reactive oxygen species (ROS) generation.
Considering the effectiveness of CP, it is necessary to prevent dose limiting side effects that inhibit its use at tumoricidal doses.
Since oxidative stress has been implicated in the etiology of CP induced toxicity, renewed interest has been centred on the role of natural antioxidants having free radical scavenging and/or antioxidant properties to counteract CP toxicity.
Thymoquinone, a phytochemical isolated from Nigella sativa seeds, has been tested for its therapeutic effects in many diseases.

3. INTRODUCTION

4. Cisplatin Inorganic complex of platinum, synthesized by Michael
Peyrone in 1845.
Effectiveness as anticancer drug was discovered by
Rosenberg and co workers in 1969.
Approved by Food and Drug Administration (FDA) in 1978.
Used for the treatment of various human solid tumors including those of head, neck, ovary, testes and breast.
Health risks: nephropathy, liver damage, hearing loss, myelosuppression, nausea and vomiting.

5. Pathophysiology of cisplatin in kidney
Adverse morphological changes in the S3 subsegment of renal proximal tubule
Loss of brush border membrane
Alters membrane permeability
Depletes intracellular glutathione and interact with enzyme/protein sulphydryl groups
Perturbs antioxidant defense system
Tubular necrosis

6. Protection and/or prevention of cisplatin induced toxicity
Strategies to
ameliorate cisplatin induced
toxicity
Antioxidants: Flaxseed oil, Fish oil, Nigella sativa oil, melatonin, selenium, curcumin, silymarin, rutin

7. Thymoquinone Thymoquinone (2-Isopropyl-5-methyl-1,4-benzoquinone,
TQ) is a monoterpene isolated from essential oil of Nigella
sativa (NS) seeds.
TQ exhibit promising therapeutic potential against many
diseases such as diabetes, artherosclerosis and cancer.
TQ possesses an antioxidant action to scavenge free radicals and also up regulate the expression and/or activities of antioxidant enzymes.
The strong antioxidant properties of TQ is related to the redox properties of its quinone structure and its ability to cross the morpho-physiological barriers and hence easy access to sub-cellular compartments that facilitates its free radical scavenging activity.

8. HYPOTHESIS
Thymoquinone would be able to prevent/reduce cisplatin induced adverse effects on kidney
Anti- tumor
Cisplatin
Nephrotoxicity
Thymoquinone

9. Experimental design
C : control; CP: cisplatin treated; TQ: Thymoquinone administered; CPTQ: thymoquinone+cisplatin treated; i.p : intraperitoneal injection ; cisplatin injection ( ) ; normal saline injection ( )

10. Experiments were conducted to study the effect of CP alone and in combination with TQ on
various biochemical parameters in serum
enzymes of brush border membrane (BBM) in cortical and medullary homogenates and in cortical BBM vesicles (BBMV)
various oxidative stress parameters in cortical and medullary homogenates
histopathological examination of rat kidney

11. RESULTS

12. Table 1. Effect of TQ administration with and without CP treatment on serum parameters
Groups
Creatinine (mg/dl)
BUN (mg/dl)
Cholesterol (mg/dl)
Phospholipid (mg/dl)
Phosphate (μmols/ml)
Glucose (mg/dl)
Control
0.93 ± 0.03
12.83 ± 1.265
54.39 ± 2.64
± 5.72
2.50 ± 0.10
± 0.45 CP
1.85 ± 0.29* (+98.92%)
24.21 ± 1.773*
(+88.69%)
70.06 ± 3.61*
(+28.81%)
± 6.28*
(+82.42%)
1.31 ± 0.18*
(-47.6%)
74.29 ± 1.10*
(-26.02%) TQ
0.99 ± 0.064
(+6.45%)
13.11 ± 1.65
(+2.18%)
49.92 ± 3.15
(-2.74%)
125.9 ± 6.71
(+8.34%)
2.22 ± 0.21
(-11.2%)
± 6.69
(+16.76%)
CPTQ
1.13 ± 0.05†
(+21.50%)
15.24 ± 1.20†
(+18.78%)
62.16 ± 2.99
(+14.28%)
± 7.73*†
(+49.44%)
1.91 ± 0.38
(-23.6%)
92.48 ± 7.43†
(-7.91%)
CP: cisplatin treated; TQ: Thymoquinone administered; CPTQ: Thymoquinone administered + cisplatin treated; BUN: blood urea nitrogen.
Results are mean ± SEM for three different preparations.
*Significantly different from control.
† Significantly different from CP at p<0.05 by one way ANOVA.
Values in parenthesis represent percent change from control

13. † † *
Figure 1. Effect of TQ administration with and without CP treatment on ALP activity in (A) cortical homogenate (B) medullary homogenate and (C) cortical BBMV

14. A
Figure 2. Effect of TQ administration with and without CP treatment on GGTase and LAP activities in (A) cortical homogenate (B) medullary homogenate and (C) cortical BBMV

15. A B
Figure 3. Effect of TQ administration with and without CP treatment on non enzymatic antioxidant parameters in the homogenates of (A) cortex and (B) medulla

16. Figure 4. Effect of TQ administration with and without CP treatment on enzymatic antioxidant parameters in the homogenates of (A) cortex and (B) medulla

17. Histopathology of rat kidney
A. Control group with normal corpuscle and tubular epithelium; B. CP treated group, reveals extensive damage of both components. Glomerulus appear congested and tubules appear edematous with obvious interstitial bleeding (all along the lower field); C. TQ alone group, shows histoarchitecture of both renal corpuscle and renal tubules very similar to control group ; D. CPTQ group, shows reasonably good preservation of renal corpuscle and tubules. Though, the glomerulus appears mildly congested, tubules are not edematous and there is no obvious interstitial bleeding.
Figure 5. Histopathology of rat kidney showing glomerular capillary tuft ( ) in the renal corpuscle and renal tubule ( ).

18. Cisplatin + Thymoquinone
CONCLUSION
CP elicited deleterious nephrotoxic and other adverse effects as indicated by significant increase in serum creatinine and blood urea nitrogen (BUN), decrease in the activities of various BBM enzymes and suppression of antioxidant defense system.
TQ administration markedly ameliorated CP induced nephrotoxicity.
TQ might have ameliorated the damage caused by CP in the following ways:
TQ administration increased the levels of antioxidant enzymes in the renal tissues resulting in enhanced antioxidant defense against CP generated ROS.
Owing to its antioxidant/free radical scavenging property, TQ might have reduced the CP induced generation of free radicals and ROS.
Cisplatin treatment
Damaged proximal tubular membrane
Caused oxidative stress
Increased lipid peroxidation
Decreased antioxidant enzyme activities
Renal tissue
Cisplatin + Thymoquinone
co treatment
Reduced proximal tubular
damage
Diminished oxidative stress

19. THANK YOU