There was no significant difference in food consumption between phytol-fed mice and control mice (data not shown). Body weights at the end of the study were significantly lower in male mice on 1% phytol but not 0.5% phytol (). These effects were even more prominent in female mice fed 1% phytol or even 0.5% phytol (). On DEXA scans, the phytol-induced weight loss was a result of reduction of both FTM and LTM (data not shown). Liver weight was significantly increased in most groups of phytol-fed mice, but there was no obvious sex difference (). Peroxisome proliferation was evident in phytol-fed mice, especially females, as determined by quantitative analysis of Western blots for catalase expression () and light microscopic evaluation of sections stained with DAB for catalase (data not shown).
Figure 1 Body weight and liver weight. (A, B) Male mice fed 1% phytol and female mice fed 0.5% or 1% phytol have lower body weights than mice fed a control diet. (C, D) Phytol-fed mice have increased liver weights compared with control mice. Data represent means (more ...)
Figure 2 Catalase expression in liver determined by Western blot analysis. Phytol-fed mice exhibit increased expression of catalase compared with control mice. Data represent means ± SEM for three to eight mice per group. * p < 0.05 vs. control; (more ...)
Grossly, some livers from female mice fed 0.5% phytol and male and female mice fed 1% phytol in the diet were enlarged, pale, and friable, with an accentuated lobular pattern. Microscopically, livers from male mice on 0.5% phytol were usually normal in appearance or exhibited subtle hepatocellular hypertrophy (). In contrast, all female mice on 0.5% phytol and all male and female mice on 1% phytol exhibited necrosis and loss of hepatocytes (), which tended to be most severe in female mice fed 1% phytol (). The necrosis was predominantly midzonal in distribution. In some mice, the necrosis extended to involve centrilobular hepatocytes, though there was a tendency to preserve at least the last one or two rows of hepatocytes surrounding the central veins. Based on morphology, cell death appeared to be occurring by both apoptosis (, inset) and non-apoptotic mechanisms (). An inflammatory reaction, consisting mainly of macrophages, with fewer neutrophils in places, was associated with the necrosis in some but not all affected livers. The severity of the inflammation tended to correlate with the severity of the necrosis. In some areas, midzonal apoptosis was occurring with no or minimal associated inflammation. In the most severely affected livers, occasional multinucleated giant cells and foci of mineralization were observed. Additional changes, more pronounced in male and female mice on 1% phytol, included hepatocellular hypertrophy (mainly centrilobular, but occasionally also periportal), and periportal hepatocellular fatty vacuolation. The midzonal necrosis tended to occur at the edge of the zone of periportal hepatocytes undergoing fatty vacuolation (). Increased numbers of mitotic figures and immunohistochemical staining for PCNA indicated a higher rate of hepatocellular proliferation in phytol-fed mice (). Minimal to mild hyperplasia of bile ductule epithelial cells was observed in a minority of phytol-fed mice, but there was no cholestasis, necrosis, or inflammation of the biliary epithelium to suggest injury to the biliary system.
Figure 3 Representative liver sections stained with hematoxylin and eosin. Examples of portal triads (PT) and central veins (CV) are marked. (A) Male mice fed a control diet. (B) Female mice fed a control diet. (C) Liver from a male mouse fed 0.5% phytol exhibits (more ...)
Figure 4 Severity grading of hepatocellular necrosis. Male mice fed 1% phytol and female mice fed 0.5% or 1% phytol exhibited necrosis of hepatocytes, which tended to be most severe in female mice fed 1% phytol. Data represent means ± SEM for three to (more ...)
Representative liver sections stained immunohistochemically for PCNA. Note increased number of positively stained nuclei in phytol-fed mice (B) compared with control mice (A), indicating increased hepatocellular proliferation. Bar = 75 µm.
Liver levels of the phytol metabolites phytanic acid and pristanic acid were significantly elevated in female mice on 0.5% phytol and male and female mice on 1% phytol (). In female mice, these changes appeared to be time and dose dependent.
Figure 6 Phytanic acid and pristanic acid levels in liver. Male mice fed 1% phytol and female mice fed 0.5% or 1% phytol exhibit elevated levels of phytanic acid (A, B) and pristanic acid (C, D) in liver compared with control fed mice. Data represent means ± (more ...)
Hepatic expression of SCP-x, determined by quantitative analysis of Western blots, was five-to twelve-fold lower in control female mice than in control male mice (). Expression of SCP-x was significantly increased in male mice fed 0.5% phytol and in female mice fed 0.5% phytol and 1% phytol compared with control mice.
Figure 7 Sterol carrier protein-x (SCP-x) expression in liver determined by Western blot analysis. Female mice fed a control diet have significantly lower levels of SCP-x than male mice fed a control diet. Male mice fed 0.5% phytol and female mice fed 0.5% phytol (more ...)