Glutathione S-transferase A4 is a positive marker for rat hepatic foci induced by clofibrate and genotoxic carcinogens

Abstract

Peroxisome proliferators (PP), including clofibrate (CF), are non-genotoxic rodent carcinogens, and oxidative DNA damages are suggested as a causative event for carcinogenesis. Gene expression profiles differ between hepatic lesions induced by PP and genotoxic carcinogens. Our previous study revealed that expression of L-bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE) was repressed in preneoplastic lesions induced by PP, whereas it was enhanced in the surrounding tissues. In the present study, we immunohistochemically examined expression of the specific glutathione S-transferase (GST) form, GST-A4, which detoxifies 4-hydroxy-alkenal, the end-product of lipid peroxides, and nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor for many genes encoding drug-metabolizing enzymes and defending enzymes against oxidative stress, during rat hepatocarcinogenesis induced by CF and genotoxic carcinogens. GST-A4 and Nrf2 were not expressed in BE-negative foci at 8 weeks of CF administration, but were expressed in the foci at 60 weeks. GST-A4-positive foci appeared at later stages than BE-negative foci, but its localization was coincidental with that of the latter foci. The areas of GST-A4-positive foci were larger than those of BE-negative foci without GST-A4 expression. Most GST-A4-positive foci were also positive for Nrf2. In rat livers induced by genotoxic carcinogens, GST-P-negative foci as well as GST-P-positive foci were demonstrated. GST-A4 and Nrf2 were expressed in GST-P-negative foci, whereas they were not expressed in most GST-P-positive foci. Thus, GST-A4-positive foci developed in rat livers by CF and genotoxic carcinogen administration, indicating that the enzyme is a positive marker for hepatic foci induced by these different carcinogens.

Figure 1

Immunohistochemical staining of serial sections of rat hepatic foci induced by clofibrate (CF) administration with anti‐L‐bifunctional enzyme (BE) antibody (a), anti‐glutathione S‐transferase (GST) A4 antibody (b), and non‐immune γ‐globulin (c). The section is also stained with hematoxylin–eosin (d). The liver samples were obtained from SD rats administered CF for 30 weeks. Original magnification, ×100.

Figure 2

Changes in the numbers (a) and areas (b) of L‐bifunctional enzyme (BE)‐negative foci (closed and open triangles) and glutathione S‐transferase (GST) A4‐positive foci (closed and open circles) in the livers of SD rats administered clofibrate (CF) (closed triangles and closed circles) for up to 60 weeks or control rats without CF administration (open triangles and open circles). Five rats were analyzed at each time point and SDs are indicated by bars. The numbers of foci are expressed as values per unit area (cm²) of liver sections. Asterisks represent statistical significance at P < 0.05 (*) and P < 0.01 (**) between numbers of BE‐negative foci (closed triangles) and those of GST‐A4‐positive foci (closed circles) in CF‐administered rats at the respective time points.

Figure 3

Immunohistochemical staining of rat hepatic foci induced by clofibrate (CF) administration with anti‐glutathione S‐transferase (GST)‐A4 antibody (a), and anti‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) antibody (b), and anti‐L‐bifunctional enzyme (BE) antibody (c). The liver samples were obtained from SD rats administered CF for 60 weeks. Original magnification, ×100.

Figure 4

Immunohistochemical staining of rat hepatic foci induced by the Solt–Farber protocol by anti‐glutathione S‐transferase (GST)‐P antibody (a), anti‐GST‐A4 antibody (b), and anti‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) antibody (c). The liver samples were obtained at 2 weeks after partial hepatectomy. A GST‐P‐negative and GST‐A4‐positive focus is encircled by blue broken line; a GST‐P‐positive and GST‐A4‐negative focus by red line; a GST‐P‐positve and GST‐A4‐positve focus by black line; Original magnification, ×100.