Expression of stem cell markers as useful complementary factors in the early detection of urinary bladder carcinogens by immunohistochemistry for γ-H2AX.

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.