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 nongenotoxic 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 BEnegative 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. (Cancer Sci 2010; 101: 1093-1098 P eroxisome proliferators (PP), including clofibrate (CF), induce hepatomegaly, proliferation of peroxisomes, and expression of several peroxisomal enzymes in rodent livers, such as acyl-CoA oxidase and L-bifunctional enzyme (BE), which participate in the b-oxidation of fatty acids and result in the production of hydrogen peroxide.(1-3) Prolonged administration of PP to rats is associated with the development of hepatic preneoplastic and neoplastic lesions.(4,5) As PP do not cause mutagenic effects in short-term in vitro assays, (6,7) administration of PP to rats for 60-100 weeks is required to evaluate their carcinogenic potential. PP include a broad spectrum of compounds of industrial, pharmaceutical, and agricultural importance, such as phthalate ester plasticizers, lipid-lowering drugs, and herbicides.(8) Thus, methods for early detection of the carcinogenic potential of PP are required. PP are non-genotoxic rodent carcinogens and their carcinogenic processes are suggested to differ from the processes of genotoxic carcinogens. (6,7) Gene expression profiles differ between hepatic lesions induced by PP and those by genotoxic agents.(9,10) The former lesions do not express GST-P, (11) a relia...

Glutathione Transferases☆

“…Immunohistochemistry revealed that GSTP1-1 was localized in preneoplastic foci and neoplastic tissues. GSTA4 was found to be strongly expressed in this case, and may be used as a positive marker for such lesions (Shimizu et al, 2010). Such GSTP1-positive single cells may be 'initiated cells', the DNA of which is modified with reactive metabolites derived from carcinogens in the first step of carcinogenesis (Satoh et al, 1989).…”

Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR

“…However, organic solvents, including acetone, has also been employed to fix tissues for some cases, in which milder fixation was suitable for downstream analyses, such as immunohistochemistry with specific antibodies [ 16 , 17 ]. Indeed, we have used acetone fixation for the immunohistochemistry of rat tissues [ 18 ]. Thus, we first examined whether DNA extracted from AFPE tissue specimens could be used for ORNi-PCR.…”

“…Rat livers were removed following the animal faculty guidelines. The livers were immediately stored at −80 °C or subjected to AFPE treatment as described previously [ 18 ]. DNA was extracted from frozen rat liver tissues (ca.…”

Decrease of hepatic stellate cells in rats with enhanced sensitivity to clofibrate‐induced hepatocarcinogenesis

“…This study was performed at 8 weeks of CF administration, because our previous study revealed that preneoplastic foci lacking BE expression developed in sensitive KS‐type rats at this time‐point. ( 12,22 ) α‐SMA, CD68 and CD34 were used as markers for the HSC, Kupffer cell and sinusoidal endothelial cell, respectively, ( 21 ) and the numbers of cells positive for the respective marker were compared between the KS‐ and NC‐type rat livers. Individual staining results are presented in Figure 1.…”