Background: Treatment with the chemotherapy drug doxorubicin (DOX) may lead to toxicities that affect non-cancer cells including the liver. Supplementing the diet with creatine (Cr) has been suggested as a potential intervention to minimize DOX-induced side effects, but its effect in alleviating DOX-induced hepatoxicity is currently unknown. Therefore, we aimed to examine the effects of Cr supplementation on DOX-induced liver damage. Methods: Male Sprague-Dawley rats were fed a diet supplemented with 2% Cr for four weeks, 4% Cr for one week followed by 2% Cr for three more weeks, or control diet for four weeks. Animals then received either a bolus i.p. injection of DOX (15 mg/kg) or saline as a placebo. Animals were then sacrificed five days-post injection and markers of hepatoxicity were analyzed using the liver-to-body weight ratio, aspartate transaminase (AST)-to- alanine aminotransferase (ALT) ratio, alkaline phosphatase (ALP), lipemia, and T-Bilirubin. In addition, hematoxylin and eosin (H&E) staining, Picro-Sirius Red staining, and immunofluorescence staining for CD45, 8-OHdG, and β-galactosidase were performed to evaluate liver morphology, fibrosis, inflammation, oxidative stress, and cellular senescence, respectively. The mRNA levels for biomarkers of liver fibrosis, inflammation, oxidative stress, and senescence-related genes were measured in liver tissues. Chromosomal stability was evaluated using global DNA methylation ELISA. Results: The ALT/AST ratio and liver to body weight ratio tended to increase in the DOX group, and Cr supplementation tended to attenuate this increase. Furthermore, elevated levels of liver fibrosis, inflammation, oxidative stress, and senescence were observed with DOX treatment, and Cr supplementation prior to DOX treatment ameliorated this hepatoxicity. Moreover, DOX treatment resulted in chromosomal instability (i.e., altered DNA methylation profile), and Cr supplementation showed a tendency to restore chromosomal stability with DOX treatment. Conclusion: The data suggest that Cr protected against DOX-induced hepatotoxicity by attenuating fibrosis, inflammation, oxidative stress, and senescence.

Non-Alcoholic Steatohepatitis (NASH) is the progressive form of Non-Alcoholic Fatty Liver Disease (NAFLD). NASH is distinguished by severe hepatic fibrosis and inflammation. The plant-derived, non-psychotropic compound cannabigerol (CBG) has potential anti-inflammatory effects similar to other cannabinoids. However, the impact of CBG on NASH pathology is still unknown. This study demonstrated the therapeutic potential of CBG in reducing hepatic steatosis, fibrosis, and inflammation. Methods: 8-week-old C57BL/6 male mice were fed with methionine/choline deficient (MCD) diet or control (CTR) diets for five weeks. At the beginning of week 4, mice were divided into three sub-groups and injected with either a vehicle, a low or high dose of CBG for two weeks. Overall health of the mice, Hepatic steatosis, fibrosis, and inflammation were evaluated. Results: Increased liver-to-body weight ratio was observed in mice fed with MCD diet, while a low dose of CBG treatment rescued the liver-to-body weight ratio. Hepatic ballooning and leukocyte infiltration were decreased in MCD mice with a low dose of CBG treatment, whereas the CBG treatment did not change the hepatic steatosis. The high dose CBG administration increased inflammation and fibrosis. Similarly, the expression of cannabinoid receptor (CB)1 and CB2 showed decreased expression with the low CBG dose but not with the high CBG dose intervention in the MCD group and were co-localized with mast cells. Additionally, the decreased mast cells were accompanied by decreased expression of transforming growth factor (TGF)-β1. Conclusions: Collectively, the low dose of CBG alleviated hepatic fibrosis and inflammation in MCD-induced NASH, however, the high dose of CBG treatment showed enhanced liver damage when compared to MCD only group. These results will provide pre-clinical data to guide future intervention studies in humans addressing the potential uses of CBG for inflammatory liver pathologies, as well as open the door for further investigation into systemic inflammatory pathologies.

Background and aim Doxorubicin is a chemotherapeutic agent that is widely used for several malignancies. It is known to have severe side effects such as hepatotoxicity, cardiotoxicity and increase in nitric oxide concentrations in skeletal muscles. On the other hand, creatine monohydrate is a supplementation used as a therapeutic agent and plays a significant role as regenerative agent. So far, there is not enough information about the effect of doxorubicin on the overall liver functionality and how creatine monohydrate helps in easing the hepatotoxicity; therefore, our aim is to investigate the role of creatine monohydrate supplementation in alleviating the hepatotoxicity caused by doxorubicin. Methods Sprague‐Dawley rats were fed rodent chow, 2% creatine, 4% followed by 2% creatine supplementation for our weeks; and 15 mg/kg doxorubicin was given once the day before they were sacrificed. Peripheral blood and liver were harvested and snap frozen. Serum chemistry was obtained to evaluate the liver function by looking at the levels of Lipemia, T‐Bilirubin, AST, ALP and ALT. Liver damage was evaluated by using Hematoxylin and Eosin staining; whereas liver fibrosis was evaluated by Sirius Red staining. RT‐PCR was performed to assess the gene expression of fibrotic genes, proliferative genes, oxidative stress and apoptosis‐related genes at the transcription level, and western blotting was performed to measure the gene expression at the protein level. Results There was no significant difference found in the serum chemistry in the treatment groups when compared to the control group. However, the histology showed an increase in inflammation and fibrosis in the doxorubicin group when compared to the control, where there was a decrease observed in the group treated with 4% creatine followed by 2% creatine. A significant increase in the gene expression of Bax, CK‐19 and Col1‐a1 has been observed in the 2% creatine + doxorubicin. Further, there was a decrease in the protein expression of CASP3 in the 2% creatine + doxorubicin and 4%/2% creatine + doxorubicin when compared to the doxorubicin group. Conclusion Based on the collected data and observed behavior of groups, we conclude that the gradual dose of creatine monohydrate supplementation does alleviate the hepatotoxicity caused by doxorubicin. Support or Funding Information Graduate Student Association at the University of Northern Colorado

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Background and aim Obesity is becoming a major global health concern, as it is associated with many diseases, such as Non‐Alcoholic Fatty Liver Disease (NAFLD). NAFLD is associated with diabetes, metabolic syndrome and, and is known to be multifactorial. Meaning that it is affected by multiple external and environmental factors. The progression of NAFLD to Non‐alcoholic Steatohepatitis (NASH), liver carcinoma and further to liver failure, makes it very crucial that we pay attention to the disease at its earlier stages. Further, NAFLD is affected by the circadian rhythm, which is a 24‐hour cycle that controls biological activities of all cells. In the liver, this rhythmic cycle is called liver clock and is very sensitive to nutritional intake, physical activity and sleeping patterns, and it is controlled by Clock genes. The expression of clock genes can be controlled by DNA methylation, which is a modification that acts as a switch to turn genes on and off. Therefore, this study aimed to study the methylation status of clock genes and clock‐controlled genes at the promoter region. Methods C57BL/6 mice were treated with high‐fat diet (HFD) or control (CTR) for 8 wk. Mouse livers were harvested for further analysis. Liver function was evaluated by serum chemistry, fibrosis was measured by IHC and PCR and lipid deposition were evaluated by Oil red O staining. Genomic DNA and total RNA were obtained for measuring the DNA methylation of core clock genes and RNA expression as described above. Methylation‐Specific PCR (MSP) and Sanger sequencing were used to evaluate the change in the gene expression of genes of interest; CLOCK, BMAL‐1 and PPAR‐a in a high‐fat diet group and control‐diet group. Results We found increased steatosis and hepatic fibrosis in mice fed a high‐fat diet compared to control. Expression of fibrotic markers (a‐SMA, FN1 and collagen I) was upregulated in mice fed high‐fat diet compared to control. Differential DNA methylation status in promoter regions was observed in different core clock genes in high‐fat‐fed mice. Conclusion DNA methylation of core clock genes and clock‐controlled genes may lead to dysregulation of the circadian rhythm during consumption of a high fat diet, thus leading to increased steatosis and liver fibrosis.