Impact of Two Doses of Vitamin K2 (Menaquinone-7) on Doxorubicin- Induced Hepatotoxicity in Rats

The objective of this study was to evaluate the impact two doses of Menaquinones-7 on hepatotoxicity induced by doxorubicin in rats. Sixty adult rats of both sexes were used in this study; the animals were randomly enrolled into six groups of 10 animals each. Group I: negative control (rats administered distilled water); Group II: Menaquinones-7 at a dose of 16 μg/kg; Group III: Menaquinones7 at a dose of 48 μg/kg; Group IV: positive control (Doxorubicin 15 mg/kg); Group V: Menaquinones-7 at a dose of 16 μg/kg administered prior to a single dose of Doxorubicin 15 mg/kg; Group VI: Menaquinones7 at a dose of 48 μg/kg administered prior to a single dose of Doxorubicin 15 mg/kg. On day twelve of the study, blood was collected for serum preparation for the estimation of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin (TB). The liver of each animal was excised for histological examination. High dose of MK-7 significantly (P<0.05) decreased serum ALT, ALP, and TB and there was an improvement in the histopathological lesions of the liver in group V and group VI compared to group IV. In conclusion, MK-7 may have protective effect against Dox-induced hepatotoxicity in rats.


Introduction
In the complicated world of malignancy treatment, the administration of chemotherapeutic agents deliberately considered to be cytotoxic and causes negative consequences.
The liver is the primary site of metabolism for many drugs, and this liver-drug interaction must be accounted while dosing chemotherapy (1) . Doxorubicin (Dox) was approved in 1974 by Food and Drug Administration (FDA); it is an anthracycline still widely used in modern cancer treatments for different type of malignancy (2 , 3) despite the advent of targeted therapy (4) . However, its beneficial effect was limited by its adverse effects on heart (5) , kidney (6) , liver (7) and other organs (8,9) with its main toxicity on heart and liver. Dox has adverse effects on the liver. One reason may be that the liver is the main organ contributed to metabolism of exogenous and endogenous toxins including Dox, leading to toxic metabolites formation. Hence, resulting in hepatotoxicity (10) ; where, the hepatic damage in this respect was reported to be irreversible in a dose-dependent manner as reported by investigators (11,12) . Another reason is that Dox may drastically increase lipid oxidation and mitochondrial reactive oxygen species (ROS) content, and decrease liver antioxidant enzymes and mitochondrial function (13) .
Fat-soluble vitamin K is an essential micronutrient (14) for which there is two forms: phylloquinone (vitamin K1) and the menaquinones (vitamin K2; MK-n). Menaquinones vary in the length and in the degree of saturation at the aliphatic side chains. Menaquinone-7 (MK-7) has long half-life and good bioavailability (15) ; it is produced by Bacillus subtilis natto (16) . Several authors demonstrated the beneficial protective role of long chain vitamin k against cardiovascular and bone diseases (17)(18)(19) .
The aim of this study is to evaluate the impact of two doses of MK-7 on Dox-induced hepatotoxicity in rats.

Experimental animals
Sixty adult albino rats of both sexes, three months old, weighing 160-250gm were used in this study; they were obtained from and maintained in the Animal House of the College of Pharmacy, Baghdad University under conditions of controlled temperature. The animals were fed commercial pellets and tap water ad libitum throughout the experiment period. The study was approved by the Scientific-and the Ethical-Committees of the College of Pharmacy/ University of Baghdad.

Experimental protocol
Rats were randomly allocated into six groups, each containing 10 rats (5 males and 5 females) as follow: Group I: Rats were received 0.5 ml of distilled water (DW) as intraperitoneal (IP) dose. This group served as a negative control. Group II: Rats were received MK-7 (16 µg/kg body weight/day) orally by oral gavage for 11 consecutive days. Group III: Rats were administered MK-7 (48 µg/kg body weight/day) orally by oral gavage for 11 consecutive days. Group IV: Rats were intraperitoneally injected with single dose of Dox (15 mg/kg body weight). This group served as a positive control. Group V: Rats were orally administered MK-7 at a dose of 16 µg/kg body weight/day prior to 15 mg/kg of Dox. Group VI: Rats were orally administered 48 µg/kg body weight/day prior to 15mg/kg of Dox.
In groups (V and VI) animals, each dose of MK-7 was administered once daily for 11 consecutive days; and at day 11, they received single dose of Dox (15 mg/kg body weight) by IP injection. Twenty-four hour after the end of the treatment duration (i.e. at day 12), the animals were euthanized by diethyl ether and blood was collected for serum preparation.

Estimation of serum biochemical parameters:
Serum samples were used for the estimation of the enzymes activities of alanine aminotransferase (ALT), and alkaline phosphatase (ALP), in addition to total bilirubin (TB) level by Automated Biochemistry analyzer (KENZA, Biolabo, France).

Histological examination
After necropsy, liver of each rat was removed and used for a histopathological examination according to the routine method (20) utilizing paraffin sections technique; the fragments were fixed in 10% formaldehyde solution, embedded in paraffin, segmented, and then stained with haematoxyline/eosin. Morphological examination of the samples was studied using light microscopy.

Statistical analysis
Data were expressed as mean±standard error of the mean (SEM). Unpaired Student t-test was used for testing the significant difference between two groups. The statistical significance of the differences among various groups was determined by one-way analysis of variance (ANОVA) and Least Significant Difference (LSD) analysis by IBM SPSS (statistical package for social sciences) version 23. Differences were considered statistically significant for P-value less than 0.05. Table 1 showed that group II rats, which received MK-7 (16 µg/kg body weight/day) orally by oral gavage for 11 consecutive days and group III rats, which administered MK-7 (48 µg/kg body weight/day) orally by oral gavage for 11 consecutive days, produced non-significant (P>0.05) differences in the serum ALT, ALP enzymes activities and TB with respect to negative control group. Besides, single intraperitoneal dose of Dox significantly (P<0.05) increased the serum activities of ALT, ALP, and TB in comparison with negative control group. Furthermore, in group V rats, which received MK-7 at a dose of 16 µg/kg body weight/day orally prior to 15 mg/kg of Dox, there were non-significant (P>0.05) differences in the intended enzymes compared to positive control group; however, in group VI rats received MK-7 at a dose of 48 µg/kg body weight/day orally prior to 15 mg/kg of Dox exhibited significant (P<0.05) decrease in serum ALT, ALP enzymes activities, and TB compared to positive control group.

Group
Serum ALT levels (IU/L)

The histopathological examination of rats' liver tissue
Histological examinations of liver sections of the negative control group, group II (rats orally received MK-7 16 µg/kg for 11 consecutive days), and group III (rats orally received MK-7 48 µg/kg for 11 consecutive days) showed normal liver section (

Discussion
Toxicity is the major factor hindering Dox treatment. The mechanisms of Dox mediated cell death include oxidative stress, apoptosis, intracellular calcium dysregulation, topoisomerase II poisoning, DNA adduct formation, and ceramide overproduction (21,22) . Additionally, Dox initiates inflammation via markedly increase of inflammatory-related proteins including TNF-a, IL-1 , and IL-6 in the liver (23) .
The current study revealed that Doxinduced hepatotoxicity, which was evident by significant (P<0.05) elevation in serum activities of ALT and ALP with respect to negative control group. The results are in agreement with studies of others (24)(25)(26) . The elevation of the serum activities of the intended enzymes may be attributed to the recognized hepatotoxic effect of doxorubicin that result in cellular damage and leakage of such enzymes to the extracellular space. Furthermore, there was a significant (P<0.05) increase in serum TB level in animals received single intraperitoneal dose (15 mg/kg body weight) of Dox compared with the negative control group. These results are in line with those of Salman (2013) (27) . Since the liver is responsible for clearing the blood from bilirubin thus, increasing serum TB level indicated a reduction in the excretory capability of the liver as a consequence of liver injury (28) .
In the present study, MK-7 administered at a dose of 48 µg/kg prior to single IP dose of Dox 15 mg/kg body weight (group VI), significantly (P<0.05) lowered serum level of ALT, ALP, and TB compared to positive control group. Moreover, there were improvement of the histopathological lesions of the liver in -group V rats administered MK-7 at a dose of 16 µg/kg prior to single IP dose of Dox 15 mg/kg body weight and -group VI rats that received 48 µg/kg MK-7 prior to single IP dose of Dox 15 mg/kg body weight compared to positive control group. This hepatoprotective effect of MK-7 could be attributed to potent antioxidant capacities of MK-7 when reduced to KH2 (dihydroquinone) during vitamin k cycle (29) , that in turn may result in attenuation of oxidative stress induced by Dox.
Several beneficial effects of MK-7 on bone and cardiovascular system were elucidated by authors (18,19) . Moreover, it has been reported that, many of the positive impacts of MK-7 could be accredited to menaquinones-4 (MK-4) where, all vitamin K homologues can be converted to MK-4 in vivo (30) and many studies reported the antioxidant and anti-inflammatory influence of vitamin K analogues in vivo and in vitro; Furthermore, Vervoort et al. (1997) showed that vitamin K2 was an inhibitor of microsomal lipid peroxidation in rat liver microsomes (31) . Additionally, Vitamin k1 and MK-7 had the capability to block activation of 12-lipooxygenase (12-LOX) and to inhibit ROS generation in pre-oligodendrocytes, hence, prevent oxidative cell death (32) .
In conclusion, MK-7, in a high dose, may have protective effect against Dox-induced hepatotoxicity in rats as demonstrated by liver function evaluation and histological examination. To the best of our knowledge, this is the first study that examines the effects of MK-7 at two doses (16 µg/kg and 48 µg/kg) each prior to doxorubicin on the liver. Therefore, we did not have a thorough chance to compare the results of this study with other reports.