Abstract

Introduction: Tamoxifen is a chemotherapy drug used in cancer treatment. In addition to its anticancer effect, tamoxifen also has hepatotoxic effects. This study aims to evaluate the induction effect of tamoxifen in the development of fatty liver in New Zealand white rabbits by employing ultrasonography.

Methods: Twenty female, adult New Zealand rabbits with a weight range of 1.5–2.5 kg and ages of 8–14 months were obtained from the Pasteur Institute in Tehran. After the adaptation period, the rabbits were randomly divided into four groups with 5 rabbits each. The first group, as a control group, received distilled water during the study period, and the other three groups received tamoxifen orally with daily doses of 1, 2, and 3 mg/kg body weight for 7 days. After 7 days of drug administration, a blood sample was taken from the middle ear artery of the rabbits to examine the serum levels of liver enzymes and assess several blood parameters. For liver imaging, a Mindray Z5 ultrasound machine with 5 MHz frequency and a linear probe was used to check the brightness of the liver before drug administration and 7 days after the final administration (i.e., day 14).

Results: The results show that tamoxifen dose-dependent dose-dependently caused a significant (p < 0.05) increase in alanine aminotransferase, aspartate aminotransferase, triglycerides, and low-density lipoprotein enzymes, and the amounts of high-density lipoprotein and alkaline phosphatase decreased significantly (p < 0.05). It should be noted that cholesterol changes were not significant (p > 0.05). From ultrasound imaging, tamoxifen changes the appearance of the liver from hyperechoic to hypoechoic.

Conclusions: The results show that oral administration of tamoxifen citrate at doses of 1, 2, and 3 mg/kg for 7 days causes liver damage and increases the leakage of liver enzymes and the levels of several blood parameters in New Zealand adult female rabbits, which can be evaluated by ultrasound imaging.

Introduction

Fatty liver is recognized as the most common cause of cirrhosis and liver failure, as well as one of the causes of liver cancer1. In addition, people with fatty liver are at increased risk of cardiovascular and cerebrovascular complications, hypertension, high blood lipids, and premature death2, 3, 4. Fatty liver disease also has more serious consequences in younger individuals and children. Thus, fatty liver disease should be considered a serious threat in national prevention and treatment programs5. Notably, laboratory methods are not sufficient for diagnosing fatty liver disease6. In most cases, fatty liver disease is asymptomatic and is accidentally discovered by observing elevated liver enzymes in blood tests performed for routine health checks or abdominal ultrasound performed for other reasons, although patients sometimes complain of vague upper right abdominal pain or early fatigue7.

The disease has two main causes, namely drugs or toxins and metabolic disorders. The metabolic syndrome involves a combination of high blood pressure, increased blood lipids, obesity, and diabetes, and recent studies indicate that as the number of diseases that make up this syndrome increases, the severity of fatty liver disease also increases8. Currently, it is believed that the progression of fatty liver disease toward cirrhosis is determined based on the intensity of chemical mediators produced during inflammation and the inflammatory process in liver cells; however, numerous chemical mediators may play a role in this pathway, but they have not been identified. Among them, leptin, angiotensin, and norepinephrine, which stimulate the proliferation of fibroblast-producing cells, require further investigation. These chemical mediators act unlike adiponectin, and by increasing fibrotic tissue, they cause rapid progression of the disease toward chronic and irreversible liver disease or cirrhosis9, 10. Clinical criteria, such as obesity, diabetes, hypertension, and being over 50 years old, are indicative of the severity of fatty liver disease. An increase in liver enzymes more than twice the normal limit and a level of triglycerides (TG) in the blood greater than 250 mg/dl are also laboratory criteria for the severity of fatty liver disease11.

Liver enzymes, such as aspartate aminotransferase and alanine aminotransferase, are present in liver cells and enter the serum upon destruction of the liver cells. These liver enzymes are the most strongly correlated with the increase in fatty liver disease12. Moreover, ultrasonography of the liver is the most common method for diagnosing fatty liver and is non-invasive, relatively inexpensive, and accessible, but does not have a high degree of correlation with histological changes. Therefore, imaging methods do not indicate the severity of fatty liver disease and cannot replace liver biopsy samples to determine the severity and prognosis of the disease13. Liver biopsy sampling is the most accurate method for evaluating the extent and severity of liver damage in individuals with a history or clinical suspicion of fatty liver disease14.

Tamoxifen is a non-steroidal anti-estrogen drug that has long been used to treat breast cancer, in addition to liver, brain, and pancreatic cancers15. However, it also has numerous side effects, including hot flashes and sweating, nausea and vomiting, loss of appetite, weight gain, vaginal discharge, irregular menstruation, excessive fatigue, headache, vaginal dryness, itching in the area surrounding the vagina, and more serious side effects such as liver disease, blood clots, depression, vision problems, and uterine or endometrial cancer16. Although the exact mechanism of action of the drug remains unclear, it likely works by blocking estrogen receptors in tumor cells that require estrogen for growth. The tamoxifen-estrogen receptor complex may enter the nucleus of tumor cells and inhibit DNA synthesis there17.

The present study focused on the evaluation of fatty liver disease induced by tamoxifen in New Zealand white rabbits, using ultrasound for the assessment. Fatty liver disease occurs because of the destruction of liver cells, and if not diagnosed early and properly treated, it can lead to an advanced and irreversible liver disease called cirrhosis. Fatty liver disease includes a spectrum of mild liver diseases in the form of fat accumulation in liver cells, which in its course may create inflammation of liver cells and result in the further destruction of liver cells. The coexistence of hypertension, increased blood fat, obesity, and diabetes, all of which are components of the metabolic syndrome18.

Results

To conduct this research, 20 female, adult New Zealand rabbits with a weight range of 1.5–2.5 kg and an age range of 8 to 14 months were obtained from the Pasteur Institute of Iran. Water and food were cut off for 6 hours before transporting the rabbits to the laboratory environment (animal house of the Faculty of Veterinary Medicine, University of Science and Research), and all rabbits were kept in specially designed transport cages and cared for following international ethical and health guidelines for animal welfare. To examine their health after transferring them to the laboratory, all animals were examined for physical health, and animals with suspicious symptoms were not included in the study. To allow the animals to adapt to the environment and become accustomed to the researcher, they were kept under 12-hour light-dark cycles, fed based on 5% of their body weight, provided free access to water, maintained at a temperature of approximately 22 °C and relative humidity of 55%, and prevented from undergoing any prior experiments or stress. In the next stage, the rabbits were randomly and equally divided into four groups of 5:

Group 1 (Control): Received placebo orally

Group 2 (Treatment): Received tamoxifen orally at a dose of 1 mg/kg for 7 days

Group 3 (Treatment): Received tamoxifen orally at a dose of 2 mg/kg for 7 days

Group 4 (Treatment): Received tamoxifen orally at a dose of 3 mg/kg for 7 days

Sampling was performed in two stages; once before the start of the experiment to determine the normal range, and again 7 days after the final daily administration of tamoxifen (i.e., on the 14th day). On the day of sampling, all animals underwent a 12-hour fast and only had access to water. The central artery of the ear was used to obtain blood samples. First, the hair in the desired area was shaved, and then the area was disinfected with alcohol and betadine. Next, using an insulin syringe and a suitable angiocatheter, 3 cc of fresh blood was collected into yellow-capped citrate tubes and transferred to the laboratory under standard conditions while maintaining the cold chain in less than an hour. Using a Prestige 24i AutoAnalyzer, the TG was measured based on GPO-PAP, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured based on IFCC without pyridoxal phosphate, and low-density lipoprotein (LDL) was measured by enzymatic determination.

To perform the ultrasound, the hair in the abdominal area of the animal was shaved using a modified Moser TiG40 shaver, and then the animal was placed on a special table in a supine position. Ultrasound was performed by an experienced radiologist using a Mindray Z5 machine and a linear probe before drug administration and 14 days after beginning administration, and the brightness level of the liver was evaluated. Based on the echogenicity of the liver and its comparison with adjacent kidneys, as well as the comparison of the spleen and the appearance of the diaphragm and the margin of intrahepatic vessels, the animals were classified into four groups: healthy, mild fatty liver (GI), moderate fatty liver (GII), and severe fatty liver (GIII). After performing the sonography and obtaining blood samples, the animals were removed from the study, for which a decapitator was used under anesthesia, and all ethical principles and animal rights were observed.

SPSS statistical software (version 21) was used for data analysis. The sample size was estimated to be 10 New Zealand rabbits based on the Cochran formula at a 0.05 level of error, and the Pearson correlation test, dependent t-test, and Kolmogorov–Smirnov test were used to determine the normality of the data distribution.

× Figure 1 . Ultrasound images of the liver during the treatment with on 1e mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows that the liver is hypoacute. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. Figure 1 . Ultrasound images of the liver during the treatment with on 1e mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows that the liver is hypoacute. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. × Figure 2 . Ultrasound images of the liver during the treatment with one mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows hypocauterization and reduction of liver granulosity. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. Figure 2 . Ultrasound images of the liver during the treatment with one mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows hypocauterization and reduction of liver granulosity. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. × Figure 3 . Liver ultrasound images during the treatment with 2 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows hypoechoic and complete loss of liver granulosity. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. Figure 3 . Liver ultrasound images during the treatment with 2 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows hypoechoic and complete loss of liver granulosity. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. × Figure 4 . Liver ultrasound images during the treatment with 2 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows the liver becoming hyperechoic and some crossing. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. Figure 4 . Liver ultrasound images during the treatment with 2 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows the liver becoming hyperechoic and some crossing. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. × Figure 5 . Liver ultrasound images during the treatment with 3 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows that the liver has become hyperechoic and softer. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C) Liver after drug administration in day of 14. Figure 5 . Liver ultrasound images during the treatment with 3 mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows that the liver has become hyperechoic and softer. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C) Liver after drug administration in day of 14. × Figure 6 . Liver ultrasound images during the treatment with threemg/kg tamoxifen . The comparison of liver changes before and after drugadministration shows tat the liver has become hyperechoic and softer. A ) Liver before drugadministration. B ) Liver after drug administration in day of 14. Figure 6 . Liver ultrasound images during the treatment with threemg/kg tamoxifen . The comparison of liver changes before and after drugadministration shows tat the liver has become hyperechoic and softer. A ) Liver before drugadministration. B ) Liver after drug administration in day of 14. × Figure 7 . Liver ultrasound images during the treatment with three mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows complete hyperechoicization and crossing of the liver. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14. Figure 7 . Liver ultrasound images during the treatment with three mg/kg tamoxifen . The comparison of liver changes before and after drug administration shows complete hyperechoicization and crossing of the liver. A ) Liver before drug administration. B ) Liver after drug administration in day of 14. C ) Liver after drug administration in day of 14.

Table 1.

The statistical value obtained for the tamoxifen group with a dose of 1 mg/kg

Variable Time Mean Standard deviation Alanine transaminase enzyme (international unit per liter) 1 st day 50 11.59 21 st day 65 10.26 Aspartate transaminase enzyme (international unit per liter) 1 st day 25.66 0.33 21 st day 36.66 1.85 Alkaline phosphatase enzyme (international unit per liter) 1 st day 293.67 17.57 21 st day 230.33 38.8 Blood cholesterol (mg/dL) 1 st day 126.33 40.10 21 st day 149.33 58.26 Blood triglycerides (mg/dL) 1 st day