The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC50) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity.

Zielsetzung: Wir verglichen die prognostische Bedeutung von CD8 und CD4 positiven tumorinfiltrierenden Lymphozyten sowie Immunglobulin Kappa C (IGKC) positiven tumorinfiltrierenden Plasmazellen bei unterschiedlichen molekularen Subtypen.

Hintergrund: Infiltration durch zytotoxische T Lymphozyten (CTL) ist ein häufig auftretendes Phänomen beim Mammakarzinom. Wir untersuchten die prognostische Bedeutung von CD8 positiven CTL bei nodal-negativen Mammakarzinomen

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Fragestellung: Immunologische Mechanismen gewinnen beim Mammakarzinom an Bedeutung. Wir haben die prognostische und prädiktive Bedeutung von Immunglobulin Kappa C (IGKC) beim Mammakarzinom untersucht.

Abstract Background: Infiltration of cytotoxic T lymphocytes (CTL) is a common feature in breast cancer. We examined the prognostic impact of CD8 positive CTL in formalin-fixed-paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) in node-negative breast cancer and validated our findings in previously published cohorts using RNA expression. Methods: CD8 was evaluated in FFPE tissue of 339 medically untreated node-negative breast cancer patients utilizing IHC. Results were validated utilizing microarray based gene-expression data of four cohorts of medically untreated node-negative breast cancer patients (n=824). Impact of CD8 on metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression. Meta-analysis of previously published cohorts was performed using a random effects model. Prognostic significance was examined in the whole cohort and in different molecular subtypes (ER+/HER2−, ER−/HER2−, HER2). Correlation between RNA expression and IHC was analyzed according to Spearman. Results: Immunohistochemical detection of CD8+ CTL was associated with MFS in univariate (hazard ratio [HR] 0.76, 95% confidence interval [95% CI] 0.59−0.98, P=0.037) as well as in multivatiate analysis (HR 0.61, 95% CI 0.46−0.81, P=0.001). Protein levels of CD8 had a good correlation with RNA expression (r=0.488). Higher RNA expression of CD8 was related to better MFS in a meta-analysis of the whole cohort (HR 0.74, 95% CI 0.60−0.90, P=0.0023). However, prognostic significance was confined to ER+/HER2− (HR 0.71, 95% CI 0.51−0.99, P=0.0201) and HER2+ (HR 0.60, 95% CI 0.38−0.97, P=0.037) but not to ER−/HER2− molecular subtypes (HR 0.77, 95% CI 0.52−1.14, P=0.1965). Conclusion: CD8 positive CTL have independent prognostic significance in node-negative breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-13.