Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis

Abstract

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson's disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.

Figure 1. AP site accumulation in mutant LEC vs. wild type LEA rats

(A) Different stages during hepatitis and hepatocellular carcinoma. (B) DNA extracted from the liver tissues of various ages of LEC and LEA rats was used to measure the number of AP sites in DNA using Aldehyde Reactive Probe (ARP) assay as described in “Materials and Methods” section. The data presented are the values (mean ± SD) derived from three independent experiments from three animals per age group of each strain. (* denotes p-value < 0.05.)

Figure 2. APE1 activity in liver extracts from LEC and LEA rat

(A) Typical autoradiograms of denaturing gels showing APE1 activity in liver extracts. The details of the assay procedure are described under “Materials and Methods”. Substrate 50, 50-mer oligonucleotide containing a single THF; Product 26, incised 26-mer oligonucleotide. Loading control is shown as the actin staining of the same protein extracts used for APE1 activity. (B) Quantification of APE1-mediated incision activity. (C) Western blotting of protein expression of APE1 in LEC and LEA rat liver extracts. Image of APE1 Western blotting of liver extracts of two rat strains from different ages. β-actin was used as a loading control and for normalization of APE1 expression. The details of the assay procedure are described under “Materials and Methods.” The data presented are the values (mean ± SD) derived from six independent experiments from three animals per age group of each strain.

Figure 3. APE1 activity in liver extracts from LEC and LEA rats under nonreducing conditions

(A) Typical autoradiograms of denaturing gels showing APE1 activity in liver extracts lacking DTT. The details of the assay procedure are described under “Materials and Methods.” Designations of substrate and product and description of loading control are as described in Figure 2A. (B) Quantification of APE1-mediated incision activity. (C) APE1 protein expression in LEC and LEA rat liver extracts by western blotting, β-actin was used as a loading control and for normalization of APE1 expression. The values (mean ± SD) from the data presented are from six independent experiments from three animals per age group of each strain. (* and ** denote p-value <0.05 and <0.01, respectively).

Figure 4. Effect of α-lipoic acid and DTT on APE1 activity in rat liver tissue extract

Liver tissue extracts from LEC and LEA rats (age 16 weeks) were incubated with various concentrations of α-lipoic acid and DTT. (A) Typical autoradiograms of denaturing gels showing APE1 activity in liver extracts after pre-treatment with various concentrations of α- lipoic acid and DTT. (B) Quantification of APE1-mediated incision activity after pre-treatment of extracts with α-lipoic acid. (C) Quantification of APE1-mediated incision activity after pretreatment of extracts with DTT. The details of the assay procedure are described under “Materials and Methods.” The data presented in panels (B) and (C) are the values (mean ± SD) derived from three independent experiments from three animals of each strain. (* denotes p-value <0.05). The “No extract” control and all LEC and LEA liver extracts samples were resolved on the same gel but were separated from each other by several lanes, as depicted by the division in the gel figure.

Figure 5. Oxidative stress during hepatitis in LEC rat

(A) Levels of oxidized glutathione in liver extracts from LEC and LEA rats. Deproteinated extracts from liver tissues of various ages of LEC and LEA rats were used to measure levels of oxidized and total glutathione utilizing the glutathione assay (B) GAPDH activity in liver extracts from LEC and LEA rats under nonreducing condition. (C) Western blotting of GAPDH protein expression in LEC and LEA rat liver extracts. Western blotting was carried out for the GAPDH protein expression along with the housekeeping protein β-actin as loading control. The details of glutathione assay and the western blotting procedure are described under “Materials and Methods.” The data presented are the values (mean ± SD) derived from three independent experiments from three animals per age group of each strain. (** denotes p-value < 0.01.)