The Contribution of Sex Difference on Different Liver Histopathology Between Male and Female Mice After Oral Administration of Caffeine

Background : There are several studies reporting the effect of caffeine on liver histopathology, but it remains controversy. The laboratory animal used in those studies were predominantly male, whereas there is contribution of sex difference on different liver reaction to xenobiotic between male and female subject. Objective : It is necessary to conduct a study to explore the differences between the liver histopathology of male and female mice after oral administration of caffeine. Methods : This study used 36 mice (Mus musculus) that were divided into 4 groups: male & female untreated groups and male & female treated groups which were orally administered with caffeine 0.4 mg / 20 gramBW daily for 30 days. At the end of treatment, mice were euthanized and dissected. Histopathological examination was done to determine the percentage of liver cell death of each group. Results: The percentage of liver cell deathin female treated group was higher than male treated group (p = 0.0001). But there was no significant difference of liver cells death between male control and treated group and between female control


Introduction
Caffeine is one compound which is frequently consumed by people, whether in the form of drink, food or drug. In last period, market of caffeine products also increased, especially caffeinated drink and dietary supplement. 1 Most source of caffeine is found in some plants such as coffee beans, cocoa, tea leaves and cola nut. In addition, caffeine is a psychoactive drug that is most widely used by people in the world. 2 Caffeine is still under discussion whether it has beneficial effects or not in liver. Some studies suggest that caffeine or caffeinated products have effects on liver histopathology. Caffeine and coffee may protect against liver injury induced by diethylnitrosamine (DEN) and thioacetamide (TAA). 3,4 Caffeine alsohas potential effect to protect liver from oxidative stress. 5 However caffeinated drink can damage hepatocytes although the cell damage is reversible. 6 Coffee containing caffeine when orally administered to male rat could cause histological changes on hepatocytes such as parenchymatous degeneration, hydropic degeneration and necrosis. 7 Caffeine when administered 10 mg/kg may exacerbate acute liver injury in female mice by interfering A2A adenosine receptors(A2AR)-mediated-tissueprotecting mechanism. Caffeine in serum are sufficiently high to antagonize adenosine signaling through the A2AR on activated mouse immune cells that caused an increase in the serum protein levels of proinflammatory cytokines such as interferon gamma (IFN-γ). 8 In order to determine the effect of caffeine on liver, the laboratory animal used on previous studies were predominantly male whereas there is contribution of sex differences on different liver reactions to xenobiotics or toxicant. The sex differences in liver toxicity may be produced by physiological differences in the pharmacokinetics and pharmacodynamics phase. Hormonal differences between male and female was also mentioned as a cause of different liver reactions to xenobiotics. 9 Therefore to determine the effect of caffeine on liver histopathology of male and female mice, the research of determining the difference of liver histopathology between male and female mice after oral administration of caffeine is necessary.

Method
Thirty-six mice (Mus musculus) BALB/c strain (male and female) 29.61±3.79 grams were obtained from the Pharmacology laboratory, Faculty of Medicine, Universitas Airlangga. Animals were given food and drink based on laboratory standard. The subjects were randomized and divided equally into 4 groups: male & female untreatedgroups and male & female treated groups which were orally administered with caffeine (CSPC Innovation Pharmaceutical, China) 0.4 mg / 20 gramBWdaily for 30 days. This dose was selected from the conversion of daily dose of caffeine consumtion in human (150-200mg) based on regulation from The National Agency of Drug and Food Control. 10 All solutions were prepared every day to avoid contamination of microorganisms or foreign compounds. Body weight measurement were done during the study to adjust the dose of caffeine administration. At the end of treatment, mice were euthanized and dissected. This study has been approved by the Health Research Ethics Committee in Faculty of Medicine, Universitas Airlangga, Surabaya, based on The The liver of mice weretaken fixed in buffer formalin and made into histopathological slides, Haematoxylin Eosin (H&E) staining, using standard methods in Histotechnic Laboratory, Faculty of Medicine, Universitas Airlangga. Histopathological examination was done to determine the percentage of liver cell death which wascharacterized by pyknotic nucleus by observingon hepatocytes in zone 3 at 6 different central vein that carried out by using a light microscope Olympus CX41 Series with 400xmagnification, OptiLab digital camera and graticule viewed byOptilab ® Viewer Software v2.1 (Fig. 1).Statistical analysis software used in this study was SPSS 16.0. The data were analyzed by t -test for independent samples. Theresults were considered significant if p<0.05.

Result
The percentage of liver cell death in male treated group was the least compared to male control group and female groups (control and treatment), while the percentage of liver cell death in female treated group was the highest compared to the female control group and male groups (control and treatment). Statistic analysis showed a significant difference percentage of liver cell death between male and female treated groups.
Percentage of liver cell death in female treated group was higher than male treated group. However when we compared the control and treated group in each sexes, statistic analysis showed that there is no significant difference of liver cells death between male control and treated group and between female control and treated group, although there was a decreasing percentage of liver cell death in male treated group and an increasing percentage of liver cell death in female treated group (Fig.2).

Discussion
Histopathological liver examination in this study was done by examining on liver cells or hepatocytes around the central vein in zone 3. This zone is far from the hepatic artery perfusion and have an oxygen concentration only 4 -5 % compared tothe zone 1 that have an oxygen concentration as much as 9 -13 %. Therefore, improvements of liver cell necrosis in zone 3 is slower than zone 1. 11 In addition, zone 3 also has higher protein cytochrome P450 than other zones. 12 The effects of caffeine on the liver still remains controversy. There was a tendency of decreasing percentage of liver cell death in male mice and an increase of percentage of liver cell death in female mice. Caffeine can decrease the levels of malondialdehyde (MDA) and increase the levels of nitric oxide (NO) in the liver of male rat. This means that caffeine may have potential effect to protect against oxidative stress in the liver. However, the effect on the liver depends on the dose, route of administration (oral or injection) and the duration of treatment. 5 Caffeine was believed to has hepatoprotective effect because it can prevent liver damage induced by DEN by maintaining the integrity of the plasma membrane. 3 The accumulation of free radicals that trigger oxidative stress and the decreased of defense membrane integrity were indeed one mechanism that can lead to cell damage. 13 However caffeine has a prooxidant effect in the liver of rat. 14 Prooxidant referred in previous study was the effect of depletion or antagonism of some antioxidants such as superoxide dismutase, catalase or vitamin A and E. Antioxidants are associated with reduction of free radicals in the body. Free radicals are produced physiologically in cells during the process of respiration and energy production as well as play a role in cell damage. 13 Caffeine can also trigger intracellular oxidative stress directly. 15 The toxic effect of caffeine is supposed to affect liver cell death based on a study that showed glandular degeneration in the salivary glands after administration of drink containing caffeine. 16 Caffeine significantly worsened the markers of liver cell injury, inflammation, and/or steatosisin nonalcoholic steatohepatitis (NASH) lesions in mice. In the study, serum alanine aminotransferase (ALT) levels, the prevalence of Oil red O-positive areas (reflecting steatosis) and CD45R-positive areas (reflecting lymphocytic infiltration) in the hepatic lobule were significantly higher in the caffeine than in the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) group that was new model of NASH. 17 Moreover caffeine can also induce cell apoptosis through a mechanism of direct modulation of cell cycle or induction of p53. 18 In the osteoblast cells which were administered with caffeine (in vitro) can induce apoptosis through the mitochondrial pathway. 15 Immune mechanisms can explain the liver damage after administration of caffeine in female mice. This is caused by an increase of proinflammatory responses by antagonize of A2AR, a receptor that mediate antiinflamation mechanism. 8 The influence of sex difference on alcoholic liver injury in mice can be observed in somemolecular mechanism. Ethanoltreatment induced more expression of heat shock protein 27 (HSP27) andHSP70, faster hepatocyte proliferation, higher levelof glycogen, and IL-6 signaling pathway activationbut less hepatocyte apoptosis and CYP2E1 expressionin male mice than female mice. 19 Gender dimorphism affect in liver structure and cell composition include liver fibrosis, alcohol injury and post-hepatectomy regeneration. In concept of transplantation, it may be hypothesized that when deprivedwith of estrogenic milieu (inherent to transplantationin a male recipient), the highly hepatocellularfemale liver may start remodeling and the HEP apoptosismay trigger an increased production of pro-inflammatoryinterleukins. This may sentence the femaleorgan to a poorer outcome in the male recipient. 20 Sex difference have also important contribution through the role of fibroblast growth factor 21 (FGF21)in alleviating hepatic injuries associated with a cafeteria diet in young male, but not female mice. 21 The toxicity difference of drugs among sexes is caused by the differences of pharmacokinetics, pharmacodynamics and hormones. 9 Pharmacokinetics of caffeine in liver is associated with biotransformation of caffeine to paraxantin by CYP1A2 which is the main route of metabolism of caffeine. 22 Several studies reported that CYP1A2 is higher in men than women. 11 When caffeine is metabolized rapidly in male, the possibility of liver damage will be less on male mice compared to female mice. Therefore it supports the result of this study that the percentage of liver cell death is higher in female treated group than male treated group. In addition, pharmacokinetic differences can be due to differences in body composition such as variations in body weight, fat composition and plasma volume. 9 Hormonal differences between male and female affect different liver histopathology between male and female mice. This is related to the activity of CYP1A2 in the liver. Estrogen can inhibit the activity of CYP1A2. 22 In addition, oral contraceptives can reduce the activity of CYP1A2. 11 The increasing of sex hormones in women in pregnancy and oral contraceptive users is associated with the decreasing metabolism of caffeine. 23 Therefore the percentage of liver cell death in female treated group is higher than male treated group. Some studies showed that women who are in final stage of pregnancy or in longterm user of oral contraceptives has increased of the half-life of caffeine approximately two times. 2 This is certainly related to the hormonal situation in women that differ from male.
This research suggests that women are more susceptible to liver damage after oral administration of caffeine. Women have more incidence of liver diseases such as acute liver failure, autoimmune hepatitis, benign lesions in the liver, primary biliary cirrhosis and toxinmediated hepatotoxicity. Pathophysiology of sex differences in different incidence and outcome of liver disease is still not understood. Many potential mechanisms could affect the result of this study such as the effect of sex hormones on oxidative and metabolic pathways, different gene transcription in response to an injury and sex differences in regulation of immunity. 24 Human primary hepatocytes in women are more sensitive to hepatotoxic than men. In previous study, there were significant differences in the variables of mitochondria status, endoplasmic reticulum status, nucleus condensation and plasma membrane permeability between male and female. Different liver toxicity between male and female was suspected due to the different sex genome that can determine the behavior of cell when given a toxicant. 9

Conclusion
According to the research the author concludes that there is different liver histopathology between male and female mice after oral administration of caffeine. It is necessary to conduct study to explore about contribution factors on different liver histopathology between male and female that administered with caffeine. Therefore in future study, sex differences were expected to be considered as a contribution factor that affect liver reaction to a substance.