Dose-responses in rat hepatic protein modification and expression following exposure to the rat hepatocarcinogen methapyrilene

Abstract

Dose-related effects of methapyrilene (MP) on protein modification and expression were examined using two-dimensional gel electrophoresis (2-D PAGE) coupled with computer analysis. Methapyrilene was administered ad libitum at doses of 0, 62.5, 125, 250 and 1000 p.p.m. to male F-344 rats for 12 weeks beginning at 8 weeks of age. Following treatment, livers were removed and frozen for 2-D PAGE analysis. Separation of hepatic proteins was conducted using ISO-DALT technology. Changes in abundance and modification of hepatic proteins were determined using the Kepler software package. Covalent modifications of three specific mitochondrial proteins were quantified using a charge modification index. Dose-response relationships were analyzed using Tukey's trend test. Results demonstrated that covalent modification of the three mitochondrial proteins was linearly related to dose and that a dose effect could be found at all dose levels in 2 out of 3 proteins. Two forms of change in protein expression were observed: (i) a dose-dependent change with effects at all doses and (ii) a change only at the toxic dose of 1000 p.p.m. MP. These results demonstrate a molecular effect of MP at doses that do not produce cellular responses including toxicity or increases in cell replication suggesting that these specific mitochondrial modifications are molecular dosimeters but are probably not direct factors and/or sufficient factors in carcinogenesis. This study also demonstrates the potential use of 2-D PAGE electrophoresis to delineate the effect of dose on expression of specific proteins.

A Metabolomics Investigation of Non-genotoxic Carcinogenicity in the Rat

“…It has been suggested that it causes liver mitochondrial proliferation in the rat and has high binding affinity for mitochondrial proteins. 54 In our study, the carnitine profile remained the same and did not indicate changes in mitochondrial biogenesis or increased mitochondrial fatty acid β-oxidation. Increased relative concentrations of TAGs and decreased concentrations of PCs also suggest against any increase in β-oxidation and mitochondrial proliferation.…”

“…MP HCl causes liver toxicity and regenerative hyperplasia. It has been suggested that it causes liver mitochondrial proliferation in the rat and has high binding affinity for mitochondrial proteins 52 . In our study, the carnitine profile remained the same and did not indicate changes in mitochondrial biogenesis or increased mitochondrial fatty acid β- oxidation.…”

Methapyrilene Toxicity: Anchorage of Pathologic Observations to Gene Expression Alterations

“…Treatment of rats with MP has been shown to form reactive metabolites that covalently bind specifically to mitochondrial proteins (50) and damage to the inner mitochondrial membrane has been proposed to play a significant role in MP toxicity (51,60). Co-incubation of MP-treated hepatocytes with phenylalkylamine Ca ++ channel blocker verapamil, but not treating the cells in a nominally Ca ++ -free medium, abrogated cell death and afforded approximately a 100-fold protection against MP effects suggesting that intracellular Ca ++ is involved in MP-induced cell death (44).…”

Systems Toxicology: Integrated Genomic, Proteomic and Metabonomic Analysis of Methapyrilene Induced Hepatotoxicity in the Rat

“…19 Two glucuronide conjugates of methapyrilene were also found in bile, however it is unclear how, if at all, they relate to the formation of a reactive metabolite. In rats methapyrilene induces liver mitochondrial proliferation 20 and promotes covalent modification of proteins within the organelle 21 which might be associated with the drug or a metabolite. 22 In isolated rat hepatocytes, inhibition of mitochondrial transition permeability pore opening and prevention of intracellular Ca 2+ mobilization reduced methapyrilene toxicity, strongly implicates loss of mitochondrial Ca 2+ homeostasis in the toxic mechanism.…”