Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

Abstract

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.

Figure 1

Liver/body weight ratios, hepatocellualr proliferation and surface area. Mice were fed control diet or a diet containing Wy-14,643 (100 ppm), rotenone (600 ppm), or a mixture of both for seven days. Animal body and liver weights were recorded, and ratios were calculated (A). Bromodeoxyuridine incorporation into hepatocyte DNA (B), and hepatocyte surface areas (C) were determined as detailed under "Materials and Methods". Data are means ± SEM from 4–6 mice per group. * P < 0.05 compared to control values. **p < 0.05 compared to rotenone values, and ***p < 0.05 compared to Wy-14,643 values.

Figure 2

Perturbations of serum thyroid levels by rotenone and Wy-14,643. Animals were treated, and sera were collected, and analyzed, as detailed under "Materials and Methods", for T3 (A), and T4 (B). Free Thyroxine Index (C) was calculated [(T3% Uptake/100) × T4 μg/dl]. Data are means ± SEM from 4–6 mice per group. * P < 0.05 compared to control values. **p < 0.05 compared to rotenone values, and ***p < 0.05 compared to Wy-14,643 values.

Figure 3

Increase in serum corticosterone levels by rotenone. Corticosterone was quantified in sera from various animal groups as described under "Materials and Methods". Data are means ± SEM from 4–6 mice per group. * P < 0.05 compared to control values. **p < 0.05 compared to rotenone values, and ***p < 0.05 compared to Wy-14,643 values.

Figure 4

Correlation between serum hormone levels and hepatocellular changes. Relationship between serum thyroid hormone and corticosterone levels and either hepatocye BrDU labeling (A,C), or hepatocyte surface area (B,D) were evaluated. Regression coefficient analyses were performed using the Prism Software (GraphPad Software, Inc., San Diego, CA).