Co-supervisor

Prof. Vinoy K. Shrivastava 

Head of theDepartment of Bioscience

Supervisor

Prof. Arun Raghuwanshi

Faculty of Life Science

Presented By

Neha Jain

M.Phil.Bioscience

“Aspirin induced alterations in the Liver, Kidney and Reproductive

System of female albino rat, Rattus rattus norvegicus”.

2010-2011

CONTENTS

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

SUMMARY

REFERANCES

• What is Aspirin• History of Aspirin• Mechanism of action• Common-side effects• Indications

INTRODUCTION

What is Aspirin

Aspirin belongs to a class of drugs called non-steroidal anti-inflammatory drugs (NSAIDs).

They work by inhibiting the enzymes that make prostaglandins, chemicals that promote inflammation, pain and fever, and are necessary for blood clotting.

Aspirin is an analgesic the brand name Aspirin was coined by the Bayer company of Germany also called acetylsalicylic acid most frequently selled acetylsalicylic acid compound Worldwide

History of aspirin

• in 1897 Felix Hofmann formed acetylsalicylic acid by derivatizing one of the hydroxyl functional groups in

salicylic acid with an acetyl group

• 'Aspirin' was patented in 1899

Felix Hofmann

In the 5 th century people used the bitter powder extracted from a willow bark to relieve pain

salicylic acid was used since 1874 as a drug

Aspirin: Mechanism of Action

Aspirin inhibits an enzyme, called COX which is responsible for the production of prostaglandines there are two forms : COX 1 and COX 2

COX 2• induced ( normally not present in cells)• built only in special cells (A549 lung cells)• used for signaling pain and inflammation• produces prostaglandins for inflammatory response• stimulated only as part of immuneresponse

COX 1• continuously stimulated by the body• its concentration in the body remain stable• creates prostaglandins used for basichouse keeping throughout body• prostaglandins stimulate normal bodyfunctions such as stomach mucousproduction, regulation of gastric acid andkidney water excretion

Membrane Phospholipids

Arachidonic Acid

Cyclooxygenase

Lipooxygenase

PGG2PGH

2

Prostaglandins ThromboxanesLeukotrienes

Aspirin

Aspirin: Mechanism of Action

Platelet Aggregation and Activation

Aspirin: Mechanism of Action

Aspirin: Mechanism of Action

Available Antiplatelet Agents

Indications• Aspirin used as,• Local analgetic effect

• Antipyretic

• Anti-inflammatory

• Antiplatelet• Analgesic &Antipyretic• Rheumatic fever & heumatoid

Arthritis• External applications • Corns & calluses• Colon cancer • Cardiovascular applications• Prophylactically to decrease the

incidence of ischemic attacks & unstable angina

Clinical aspirin resistance

• The inability of aspirin to protect a person from cardiovascular events such as heart attacks and strokes.

AIM

“Aspirin induced alterations in the Liver, Kidney and Reproductive System of female albino rat, Rattus rattus norvegicus”

MATERIALS AND METHODS

Animals Selection

• The present experiment were performed on mature female albino rat, Rattus rattus (n=20). The rats were obtained from the colony of the animal house of Barkatullah University, Bhopal. The rats were 15-16 weeks old, the body weight range 125 ± 5 g and acclimatized up to three weeks. The rats were maintained at suitable temperature of 23 ± 2ºC with an automatically controlled photo-period (12 hours light and 12 hours darkness) and the rats were fed with standard balanced pelleted diet with tap water "ad libitum". The changes in vaginal smear were also measured daily. The above experiment was performed during the month of November-10 to March- 11.

Chemical and Dose Selection

• Aspirin tablets (Acetyl Salicylate, Ecosprin-150) obtained from registered medical shop, Bhopal has been used for present experiment. The experimental animals were administered Aspirin orally at a dose level of 100 mg/Kg b.w./day through gavage.

Experimental design

• Ist Group – The animals of this group were fed with normal diet, were called SET A or Control Group.

• IInd Group – The animals of this group was fed with normal diet and received Aspirin by oral administration for 15 & 30 days, were called Treated Group. This group was divided into 2 sub groups as following.

• Ist Sub Group – these groups of 5 animals was fed with normal diet and received Aspirin by oral administration for 15 days, was called SET B or Treated Group A.

• IInd Sub Group – these groups of 5 animals was fed with normal diet and received Aspirin by oral administration for 30 days, was called SET C or Treated Group B.

For present investigation total 20 female rats were divided into 2 groups (ten each), these different groups and dosing patterns as follows.

Method Design

• All animals were sacrificed after 24 hours of the last dose according to their dosing patterns i.e. 15 days treated animals killed 16th day and 30 days treated animals killed 31st day along with control animals. After sacrificing blood were collected through cardiac puncture and serum were prepared for the different parameters i.e. biochemical parameter etc. At the same time body organs viz. liver, kidney, ovary, oviduct, uterus were quickly removed, washed with physiological saline, dried (by filter paper), weighed (by physical precision balance). Then these organs either kept in Bovines fixative for histopathological study or tissue homogenate were prepared for enzymological study.

Parameter estimated

Body Weight study: From 1-30 days of control and treated animals on different time interval i.e. initial, after 15 days and after 30 days. 

Hematological Study: Hb %, RBCs and WBCs count  

Enzymological Study:1) Acid Phosphatase (ACP): King and Kings Method (1954).2) Alkaline Phosphatase (ALP): King and Kings Method (1954).3) Glutamate Oxalate Transaminase (GOT): Reitman and Frankel Method (1957).4) Glutamate Pyruvate Transaminase (GPT): Reitman and Frankel Method (1957).

Biochemical Study:1) Cholesterol Estimation: By Ferric Chloride and Sulphuric Acid Method (Henly,

1957).2) Protein Estimation: By Follin-Phenol Method (Lowry et al., 1951).3) Glucose Estimation: By Dinitro Salicylic Acid Method (Miller, 1959).4) Bilirubin Estimation: By Malloy and Evelyn Method (1937)5) Calcium Estimation: By Eriochrome Dye Method (Preston Smith et al., 1966).

Histological study: Ovary, Oviduct, Uterus, Liver and Kidney.

Estrus cycle study: From 1-30 days (daily) of control and treated animals.

RESULTS

Body Weight study

Hematological Study

Enzymological Study

Biochemical Study

Histological studyMicrophotograph of Ovary

CL

CL

CL

AF

AF

AF

Histological study : Microphotograph of Uterus (X 40/100/400)

EM

PM

MM

MM

EM

PM

MM

EM

PM

L

L

L

Histological study Microphotograph of Liver

S

S

S

Histological study Microphotograph of Kidney (Cortex Region)

T

T

T

Estrus cycle studyMicrophotograph of Vaginal Smear

S

Proestrus Phase

Diaestrus PhaseMetaestrus Phase

Estrus Phase

Following phases are seen in rat vaginal smear

Epithelial cells

Cornified cells Leucocytes

Х 80Х 80

Cell types in vaginal smears of Rattus rattus•Estrus lasts up to 12 h and is indicated by the presence of large cornified cells in the vaginal smear. •Metestrus lasts 21 h and usually has many neutrophils in the smear and scattered squamous epithelial cells. •Diestrus lasts up to 57 hours and there are abundant neutrophils and a few nucleated non-cornified epithelial cells. •Proestrus lasts 3-12 hours and has abundant nucleated non-cornified epithelial cells.

The regular study of vaginal smears of control and treated groups of rat (Rattus rattus).

DAYS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

GROUPS

CONTROL

D E M P D E M P D E M P D E M P D E M P D E M P D E M P D E

TREATED

E M P D E M P D E M P D E M P D E M P D D D D D D D D D D D

Control Group = This Group contains 5 animals which receive only normal diet from 1 to 30 days.

Treated Group = This Group contains 5 animals which receive normal diet and aspirin (100mg/Kg b.w.) by oral administration from 1 to 30 days.

D, E, M and P = Showing estrus cycle phases D = Diaestrus PhaseE = Estrus PhaseM = Metaestrus PhaseP = Proestrus phase

DISCUSSION

DISCUSSION

• Data of the present study indicate that, body weight of rats (n=5) were not significantly affected by oral administration of Aspirin (100mg/Kg b.w.) for 30 days. One could suggest that oral administration of Aspirin (0.05% w/v) do not affect body weight in rats (Ebuehi et al., 2007; Kwon et al., 1997).

• Our present findings shows that, significant (<0.001) increase in the number of TC WBC in 30 days Aspirin treated rats. The auther suggested that, Aspirin increases the production of blood eosinophils (and possibly blood basophils) (Hirai et al., 2001).

• After the administration of Aspirin (100mg/Kg b.w.) for 30 days do not significant altered TC RBC and Hb %. Accoding to auther, Aspirin had showed no effect or alteration on packed cell volume (Otimenyin, et al).

• In our present study, the serum protein content were not significantly (p<0.01) reduced by oral administration of Aspirin. Ebuehi et al., (2007) suggested that, oral administration of Aspirin may inhibit protein synthesis or many prevent the uptake of amino acids into the tissues (such as in brain tissues).

• The study of Aspirin on the serum Ca level suggest that, the Ca++ concentration significantly (p<0.01) decreased on the dose level 100mg/Kg b.w. of Aspirin. In addition to, Aspirin diet enhances calcium absorption for the improvement in hen’s egg shell percentage and thickness (Abdullah et al., 2010). Low levels of serum Calcium can be caused by low albumin or total protein levels (www.questdiagnostics.com, 2004).

• The administration of Aspirin for 15 and 30 days on rats caused significant reduce the glucose and cholesterol level. Aspirin is known to inhibit glycogenesis and aminotransferase enzymes which may lead to decreased gluconeogenesis. The reduction of glucose and cholesterol levels which accompanied the reduction of corticosterone and T3 levels that consequently may affect its metabolism (Mohammed et al., 2010).

DISCUSSION

• In our present study Aspirin significantly increases ACP and ALP content in Ovarian Utricular tissues and not significantly altered ACP and ALP content in Serum. The present investigation revealed that, Aspirin significantly increased serum GOT and GPT content in 15 days and 30 days treated rats at the dose level 100 mg/Kg (b.w.). As well as those patients, who are treated with NSAIDs, particularly with Aspirin, are at more danger of developing hepatotoxicity than others as well as the liver enzymes (ALT ,ALP, GOT and GPT) started to deteriorate gradually (Kawar et al, 2010).

• In the present data indicates, Aspirin significantly reduces serum bilirubin content in rats compare to control rats. In the treatment of arthritis an anti-inflammatory drug were given (Aspirin level was 200 mg/kg/24hr) for two days found that, the bilirubin level were normal in patients (Kawar et al., 2010).

• After the 15 and 30 days treatment of Aspirin (100mg/Kg b.w.) causes, significantly increase of serum Creatinine. Aspirin treated patients increase of serum Creatinine (Muhammad et al., 2010).

• In the present investigation found that Aspirin induced toxicity on liver and Kidney both along with patho-histological changes at the higher dose level (100mg/Kg b.w./day). The results revealed that Aspirin made changes in antioxidant system even at therapeutic doses, made no changes in hepatic and renal pathology (Balog et al., 2000).

DISCUSSION

• Our present findings show that, the morphological changes found in both ovaries and oviduct and uterus seen in the experimental groups may be due to the vascular properties of Aspirin. long-term administration of Aspirin for 30 days, developed an areas of hemorrhagic spots, at the site of uterine wall vascularization and this may be due to blood vessels weakness induced by the prolonged program of Aspirin-induced treatment (Schafer. et al., 1995).

• Aspirin administration persistently arrest luteolysis (i.e. intact and functional corpora lutea), which has been confirmed by daily vaginal smear, that reflect a cellular picture of diestrous phase. Inhibitors of PGF2α and synthesis (indomethacin and acetylsalicylic acid) as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats (Jezova et al., 1999).

• structural ovarian changes seen: significant increase in the number of corpora lutea and a significant increase in the diameter of the granulose lutein cells, with absence of regressed corpora luteal when compared to the control animals.

• The structural uterine changes by Aspirin namely: the significant decrease in the endometrial lining cell height, endometrial thickness and diameter of endometrial glands in both periods of Aspirin treated rat (15 and 30 days) (Chaffin et al., 2000).

SUMMARY

SUMMARY

This can be achieved through public health education to make people aware of the hazardous effect of these compounds. It is therefore recommended that great precautions and supplements must be taken to minimize the harmful side effects of such chemicals especially to human and animals.

It conclude from present study is that, the long term use of Aspirin at the dose of 100mg/kg b. w. orally is responsible for damage to female reproductive organs, if precautionary measures are not taken while using them. Structural damage to mammalian oviduct may be lead to infertility cause. So in selecting a safe drug, quantities and mode of usage must be strictly monitored to minimize the possibility of injurious complications.

The data of the present study summarized as, the oral administration of Aspirin (100mg/Kg b.w.) do not significantly affected to the body weight, Hb%, TC RBC and protein content. As well as significantly increases TC WBC. In addition, oral administration of Aspirin significantly inhibited the level of Glucose and cholesterol level. Therefore, it could be inferred the level of serum and tissues enzymes such as ACP, ALP, GOT and GPT. And also Aspirin significantly caused alteration in bilirubin and Creatinine concentration. Furthermore information about Aspirin is that it also causes patho-histological changes in liver, kidney and reproductive organ (i.e. ovary, oviduct and uterus).

REFERANCES

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