Phytol-induced hepatotoxicity in mice

Abstract

Phytanic acid is a branched-chain, saturated fatty acid present in high concentrations in dairy products and ruminant fat. Some other dietary fats contain lower levels of phytol, which is readily converted to phytanic acid after absorption. Phytanic acid is a peroxisome proliferator binding the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) to induce expression of genes encoding enzymes of fatty acid oxidation in peroxisomes and mitochondria. Administration of dietary phytol (0.5% or 1%) to normal mice for twelve to eighteen days caused consistent PPARalpha-mediated responses, such as lower body weights, higher liver weights, peroxisome proliferation, increased catalase expression, and hepatocellular hypertrophy and hyperplasia. Female mice fed 0.5% phytol and male and female mice fed 1% phytol exhibited midzonal hepatocellular necrosis, periportal hepatocellular fatty vacuolation, and corresponding increases in liver levels of the phytol metabolites phytanic acid and pristanic acid. Hepatic expression of sterol carrier protein-x (SCP-x) was five- to twelve-fold lower in female mice than in male mice. These results suggest that phytol may cause selective midzonal hepatocellular necrosis in mice, an uncommon pattern of hepatotoxic injury, and that the greater susceptibility of female mice may reflect a lower capacity to oxidize phytanic acid because of their intrinsically lower hepatic expression of SCP-x.

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 ± SEM for three to eight mice per group. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

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; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

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 subtle hepatocellular hypertrophy but is otherwise essentially normal. Livers from female mice fed 0.5% phytol (D), male mice fed 1% phytol (E) and female mice fed 1% phytol (F) exhibit necrosis and loss of mid-zonal hepatocytes (arrows), accompanied by inflammation, mild to moderate fatty vacuolation, and hypertrophy of hepatocytes around central veins. (D, inset) Note apoptotic hepatocytes in phytol-fed mice. Bar = 75 µm.

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 eight mice per group. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control.

Figure 5

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.

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 ± SEM for three to eight mice per group. * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control. ND, not detected.

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 or 1% phytol exhibit increased expression of SCP-x compared with control mice. Data represent means + SEM for three to eight mice per group. ** p < .01 vs. control; *** p < .001 vs. control; ## p < .01 vs. the respective male control group; ### p < .001 vs. the respective male control group.