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Dose-responses in rat hepatic protein modification and expression following exposure to the rat hepatocarcinogen
F C Richardson1, D M Horn, N L Anderson
1Toxicology Research Laboratories, Lilly Research Laboratories, Greenfield, IN.
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.
Insights
Methapyrilene (MP) causes dose-dependent mitochondrial protein modifications in rats, detectable even at low doses. These molecular changes occur without cellular toxicity, suggesting their use as early biological markers.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Methapyrilene (MP) is a chemical agent with known toxicological effects.
- Understanding dose-response relationships at a molecular level is crucial for risk assessment.
- Two-dimensional gel electrophoresis (2-D PAGE) is a powerful tool for analyzing protein expression and modification.
Purpose of the Study:
- To investigate the dose-related effects of methapyrilene (MP) on protein modification and expression in rat liver.
- To determine if molecular changes occur at sub-toxic doses of MP.
- To assess the utility of 2-D PAGE in identifying molecular dosimeters.
Main Methods:
- Male F-344 rats were administered varying doses of MP (0-1000 p.p.m.) for 12 weeks.
- Liver proteins were separated and analyzed using two-dimensional gel electrophoresis (2-D PAGE) and ISO-DALT technology.
- Changes in protein abundance and covalent modifications were quantified using Kepler software and a charge modification index.
- Dose-response relationships were statistically analyzed using Tukey's trend test.
Main Results:
- Covalent modification of three specific mitochondrial proteins showed a linear relationship with MP dose.
- Dose-dependent effects on protein modification were observed for 2 out of 3 proteins, even at low doses.
- Two patterns of protein expression changes were identified: dose-dependent and those occurring only at the highest toxic dose.
- Molecular effects were detected at doses below those causing cellular toxicity or increased cell replication.
Conclusions:
- Mitochondrial protein modifications induced by MP serve as molecular dosimeters, detectable at sub-toxic levels.
- These modifications are likely early indicators of MP exposure but may not be direct drivers of carcinogenesis.
- 2-D PAGE is a valuable technique for delineating dose-dependent effects on protein expression and modification.