Comparison of High Fat and Low Fat Diets on Histopathological Changes in Liver in a Mouse Model of 4NQO‐induced Oral Cancer

Abstract

IntroductionWorldwide, liver cancer remains among the top ten most common cancers. Risk factors for liver cancer include tobacco usage, alcohol consumption, obesity, and sex. Administration of 4‐nitroquinoline 1‐oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. The purpose of this study was to compare the effects of high and low in saturated fat diets (HF and LF, respectively), and sex, on liver histopathology in 4NQO‐treated mice and controls. We hypothesized that 4NQO’s carcinogenic effects would cause histopathological changes in the liver. We also hypothesized that a HF diet would increase inflammation, steatosis, and fibrosis when compared to the LF diet.Materials and methodsMale and female C57Bl/6 mice (Jackson Labs, 36 each gender), 5 weeks old, were divided into a low fat (10 kcal% fat; LF) or HF (60 kcal% fat) diet. Within each dietary group, mice were randomly assigned to one of 3 water treatment groups for 17 weeks: water alone (control); propylene glycol in water (1.25%; PG‐H2O); or 4NQO in PG‐H2O (50 mg/ml; 4NQO). After 17 weeks, all mice were given water alone for 6 weeks prior to euthanasia. Livers were harvested, portions were frozen or formalin fixed, sectioned, and stained with hematoxylin and eosin (H&E). Histopathology criteria used for tissue assessment included steatosis, fibrosis, and lymphoid cell infiltration. Frozen liver samples were hydrolyzed and a colorimetric test was used to measure triglyceride content. Trichrome staining of collagen was performed, and slides were graded visually for fibrosis using an adapted Metavir scale. T‐cells (CD3+ cells), macrophages (CD68+ cells), and neutrophils (Ly6+ cells) were detected by immunohistochemistry, counted, and normalized to tissue area. Statistical analysis was done using GraphPad Prism 7.0. All animal work was in compliance with the regulations of IACUC at Midwestern University, Downers Grove, IL.ResultsCompared to water controls, 4NQO‐treatment significantly increased hepatic inflammatory cell infiltration by neutrophils (11± 1 vs. 31± 3 cells/mm2), T cells (76 ± 7 vs. 99 ± 6 cells/mm2), and macrophages (468 ± 40 vs. 562 ± 25 cells/mm2). Infiltration with inflammatory cells was accompanied by liver fibrosis. Interestingly, HF diet caused hepatic histopathologic changes with severe fatty changes in males, and only moderate fatty changes in female control mice. Thus, triglyceride content was significantly higher in HF males when compared to all other control groups (HF male 14.0 ± 3.2 %, LF male 2.9 ± 0.8 %, HF female 3.2 ± 0.4 %, and LF female 2.3 ± 0.5 %). Fatty change was significantly reduced by 4NQO treatment, possibly as a result of cachexia.ConclusionThis mouse model of oral cancer, initially established to mimic the effects of smoking, may be extended to serve as a model for the effects of tobacco and diet on liver as well.Support or Funding InformationWe recognize the Midwestern University Deans of the College of Dental Medicine and the College of Graduate studies for their support of this research. Student research was supported by the Office of Research and Sponsored Programs.