INTRODUCTION:
The alkylating agent cyclophosphamide (CPA) belongs to the oxazaphosphorine class. An alkylating agent is a type of drug that binds to the cell's a nucleophilic DNA and prevents it from replicating. It can be taken oral or intravenous. The active ingredient is inactive in vitro and is replaced by metabolites, the most important of which is phosphoramide mustard, which the P450 system of hepatic microsomal enzymes is responsible for the production of this substance. Even though more than 150 substances have been recognized to date, their pharmacokinetics and potential harm are unknown1,2.
CPA is primarily used as a chemotherapy drug that was approved in USA in 1959 for the treatment of breast cancer, Hodgkin's acute myeloid leukemia, non-lymphoma, acute lymphoblastic leukemia, and chronic myelogenous leukemia among other indications3. After three days of CPA treatment, bone marrow suppression was observed, with hemoglobin, RBC, and WBC levels significantly reduced. It also affects the immune system, preventing both cell-mediated and humoral immunity4. It is commonly used in auto-immune illnesses such as organ transplant and Nephrotic syndrome treatment protocols to avoid refusal of organs because it also has immune-suppressive properties. CPA prevents cellular/humoral refusal and preserves immune suppression without any need for corticosteroid supplementation5. The suggested CPA dosage for treatment varies depending on the period of the syndrome, patient's weight, and the route of administration6. It has a wide range of toxic effects, despite it is being very effective at inhibiting tumor growth and it is playing a crucial role in immune suppression. In Wisteria mice, oral dosing of 10mg/kg CPA for thirty days resulted in a reduction in the weight of body and a delayed hypersensitivity. The cellularity of the thymus and spleen was significantly reduced histopathological changes. The liver injury caused by CPA is primarily dose dependent. It appears to be the result of impaired CPA metabolism, particularly of its metabolite, acrolein7. Continued exposure to CPA metabolites causes hepatotoxicity, which manifests as symptoms of liver dysfunction associated with sinusoidal obstruction syndrome, including elevated aminotransferase and bilirubin levels8-10. Mild and transitory elevations in serum aminotransferase levels were noticed in most cancer cases treated with cyclophosphamide. The increase in enzyme levels is more noticeable in case where in elevated doses of CP are directed directly to bloodstream via i/v administration11. It's unusual for regular cyclophosphamide doses to cause clinically noticeable liver and kidney damage12. The onset occurs in the first weeks of CPA treatment, and there is an initial increase in serum enzymes. In certain cases, the destruction is self-limited, and it resolves when the drug is stopped, with a recurrence upon re-exposure13. Numerous experimental animal models with CPA have been reported in the literature. It demonstrates CPA's negative effects on tissues. A study of the histopathology of Swiss albino mice's livers revealed signs of necrosis inside liver tissue14.
MATERIALS AND METHODS:
The study was conducted on 20 healthy male albino mice weighing an average of 213g. Fresh water and food were freely available to the animals. They were divided into two group, 10 mice for each. The first group (CPA group) was treated with single dose of cyclophosphamide at the dose of 200mg per kg body weight intraperitoneally, while the second group was given the Phosphate Buffered Saline (PBS) solution and considered as control. On day 25, mice from each group were sacrificed. An incision was introduced into midline of the abdomen using scalpel, liver and kidney were recognized and separated out. The tissues were secure in Bouin’s solution and then kept in 70% of alcohol for histopathological study. The stored tissues were processed via dehydration in 90% alcohol, and finally immersed in xylol. The liver and kidney tissues were embedded in paraffin wax before being cut into blocks and labeled. A rotatory microtome was used to cut sections of 5m thickness. Hematoxylin and Eosin stains were used for staining of the sections. Slides were examined under 100X amplification. Three factors were assessed using a semi quantitative method: The level of lymphocytic inflammatory, inflammation in the portal area, infiltrations, and hepatocellular necrosis. Similarly, the cortex, medulla, and tubule structures of kidney sections were examined. Samples of blood were collected from the heart and centrifuged at 1500 rpm for 20 minutes to extract serum, which was then tested for markers of renal and liver function15.
Analytical Statistics:
Student T-test used to detect the Statistical differences between control and CPA-treated group. Results represented Mean and standard deviation for each experiment. P value was (P<0.05) and (P<0.01) levels.
RESULTS AND DISCUSSION:
Histopathological results of CPA-treated group showed a dilatation with congestion of blood vessels, massive necrosis of hepatic parenchyma and an increased in apoptosis level (Fig.1). in addition, other sections showed severe hemorrhage with huge infiltration of neutrophils, focal aggregation of neutrophils and lymphocytes in hepatic parenchyma (Fig.2).
The portal blood vessels and sinusoids contained an accumulated of serum protein as compared with control. Livers of mice in the control group showed normal histological structure (Fig. 3). While, the kidney of treated group tissue sections showed a cloudy swelling, vacuolar degeneration, and necrosis of the lining epithelium of the renal tubules, as well as glomerular tuft atrophy (Fig.4).
Moreover, there were a congestion of blood vessels with perivascular lymphocytic cuffings, with complete destruction of renal tissue with severe hemorrhage (Fig.5). Hyperplasia of lymphoid tissue showed in the perirenal adipose tissue compared with control group (Fig. 6).
The current study's histological results were like previous studies conducted by various authors10,11. The metabolites of CPA damaged the lining endothelial cells of the liver sinusoids in vitro, resulting in sinusoidal obstruction syndrome also known as veno occlusive disease16. The histological findings in current study were including the enlargement of portal vein, congestion of sinusoid, and degeneration of hepatocyte, veno occlusive disease point because of increased oxidative stress.
Researchers claimed that CPA has no negative effects on the kidneys. This is because acrolein, a by-product, is urotoxic rather than nephrotoxic17-19. Pathological changes were observed in our study such as acute inflammatory cells infiltration in the cortex, edema of tubular cells, loss of brush margin, and finally pyknotic cells20.
Our data also reflected a clear congestion and dilation of the blood vessels of the kidney, vacuolations of renal tubules epithelial lining, and of glomerular tuft atrophy after in CPA-treated group. These alterations were attributed to CPA-prompted oxidative stress, as evidence by an increase in oxidative biomarkers in f kidney tissue 20-23.
Fig (1) Histopathological section of liver mouse treated with CPA showed dilatation and congestion of blood vessels, massive necrosis of hepatic parenchyma and an increased in apoptosis (H&E)(100x)
Fig (2) Histopathological section of liver mice of CPA group showed a severe hemorrhage with infiltration of large numbers of neutrophils and lymphocytes in hepatic parenchyma and sinusoids. (H&E)(100x)
Fig (3): Histopathological section of liver of mouse of (control group) showed a normal histological structure. (H&E) (100x)
Fig (4): Histopathological section of mouse kidney
treated group showed a cloudy swelling, vacuolar degeneration and necrosis of
the lining epithelium of the renal tubules ( 
) with glomerular tuft atrophy ()
(H&E)(100x)
Fig(5): Histopathological section of mouse kidney treated group showed a congestion of blood vessels with perivascular lymphocytic cuffing, with complete destruction of renal tissue with severe hemorrhage () (H&E)(100x).
Fig(6) Histopathological structure of kidney of control group showed the normal histological structure (H&E)(100x)
kidney weights (0.90±0.03), serum urea (97.13±4.99) and creatinine (7.79±0.30) levels were increased significantly (P≤0.01) in comparison to control (Table 1). The serum levels of urea and creatinine were elevated in mice treated with CPA. Our findings are agreed with findings of previous study24. The elevated levels of these parameters in CPA-treated mice indicated a renal dysfunction. This might be due to reduced creatinine and urea excretion, as well as a lower rate of glomerular filtration in mice treated with CPA25-27. Serum albumin is a biochemical index used to assess kidney function, particularly in malnourished condition 28.
Table 1: Effects of cyclophosphamide on some kidney function parameters and its weight of albino mice.
|
Groups |
Body weight at the beginning (g) |
Body weight at the end (g) |
Weight of kidney (g) |
Level of urea (mg/dL) |
Level of creatinine (mg/dL) |
|
CPA group |
216.5± 13.8 |
222.1±13.6 |
0.90± 0.03 |
97.13± 4.99a |
7.79± 0.30a |
|
Control |
210.4± 11.1 |
226.2±10.8 |
0.78± 0.07 |
23.4± 2.23b |
1.98± 0.10b |
CPA; cyclophosphamide, (P≤0.01).
Table (2): Effects of cyclophosphamide on albino mice's serum liver function and weight.
|
Groups |
Weight of liver (g) |
AST (U/L) |
ALT (U/L) |
Albumin (g/dL) |
TB (U/L) |
|
CPA group |
8.56± 0.29 |
343.1± 12.4a |
242.8±10.9a |
1.87± 0.09b |
41.3± 4.99a |
|
Control |
8.46± 0.15 |
62.0± 4.99b |
65.1± 3.24b |
5.55± 0.20a |
7.80± 0.42b |
CPA; cyclophosphamide, (P≤0.05).
In comparison to the control group, the administration of CPA had no significant effects on liver and body weights (Table 2). When compared to control mice, AST, ALT serum levels and TB were statistically significant (P<0.05) higher in mice administered CPA. In comparison to control, albumin levels were significantly decreased (P<0.05). The enzymes were leaching from liver hepatocytes, which results in increased systemic activities and circulation, is one of the hepatic injury consequences caused by CPA29-31. In the recurrent study, mice administered CPA had statistically significant increases in ALT and AST levels in the liver and serum. This finding is due to cellular damage in the liver and changes in membrane function32. Bilirubin is an endogenous anion formed when hemoglobin in red blood cells is degraded. It is bound to albumin and transported to the liver. Hepatic excretory function is impaired when plasma conjugated bilirubin levels are high. Higher bilirubin levels in the blood are directly related to the point of injury of hepatocyte and the duration of the disease in hepatitis33. Total bilirubin levels were found to be higher in mice exposed to CPA in this study. The increased total bilirubin levels seen in mice intoxicated with CPA are toxicity sign, and they agree with previous results34,35. This finding could be explained by CPA-induced bilirubin overproduction, conjugation, or elimination of conjugated or unconjugated bilirubin into bile ducts from hepatocytes.
CONCLUSION:
Our data revealed there were a toxic effect of CAP in albino mice, which characterized by pathological changes in the histology of the liver and kidney tissues, as well as a clear effects on some biochemical parameters of these organs.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank the department of medical laboratories technique, technical institute-Kut / Middle technical university/Iraq for their kind support during hematological and all other lab studies.
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Received on 17.11.2021 Modified on 11.01.2022
Accepted on 24.02.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2022; 15(10):4655-4659.
DOI: 10.52711/0974-360X.2022.00781