New aspects in deriving health-based guidance values for bromate in swimming pool water.

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1-5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460-1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose-response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587-594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73-98%) by swallowing water, followed by the dermal route (2-27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Genome-wide expression changes induced by bisphenol A, F and S in human stem cell derived hepatocyte-like cells.

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.

Subtype-specific prognostic impact of different immune signatures in node-negative breast cancer.

BACKGROUND: The role of different subtypes of immune cells is still a matter of debate. METHODS: We compared the prognostic relevance for metastasis-free survival (MFS) of a B-cell signature (BS), a T-cell signature (TS), and an immune checkpoint signature (CPS) in node-negative breast cancer (BC) using mRNA expression. Microarray-based gene-expression data were analyzed in six previously published cohorts of node-negative breast cancer patients not treated with adjuvant therapy (n = 824). The prognostic relevance of the individual immune markers was assessed using univariate analysis. The amount of independent prognostic information provided by each immune signature was then compared using a likelihood ratio statistic in the whole cohort as well as in different molecular subtypes. RESULTS: Univariate Cox regression in the whole cohort revealed prognostic significance of CD4 (HR 0.66, CI 0.50-0.87, p = 0.004), CXCL13 (HR 0.86, CI 0.81-0.92, p < 0.001), CD20 (HR 0.76, CI 0.64-0.89, p = 0.001), IgκC (HR 0.81, CI 0.75-0.88, p < 0.001), and CTLA-4 (HR 0.67, CI 0.46-0.97, p = 0.032). Multivariate analyses of the immune signatures showed that both TS (p < 0.001) and BS (p < 0.001) showed a significant prognostic information in the whole cohort. After accounting for clinical-pathological variables, TS (p < 0.001), BS (p < 0.05), and CPS (p < 0.05) had an independent effect for MFS. In subgroup analyses, the prognostic effect of immune cells was most pronounced in HER2+ BC: BS as well as TS showed a strong association with MFS when included first in the model (p < 0.001). CONCLUSION: Immune signatures provide subtype-specific additional prognostic information over clinical-pathological variables in node-negative breast cancer.

Prognostic significance of interferon regulating factor 4 (IRF4) in node-negative breast cancer.

PURPOSE: The transcription factor IRF4 regulates immunoglobulin class switch recombination as well as plasma cell differentiation. We examined the prognostic significance of IRF4 expression in node-negative breast cancer (BC). METHODS: IRF4 expression was evaluated by immunostaining in a cohort of 197 node-negative BC patients not treated in adjuvant setting, referred to as Mainz cohort. The prognostic significance of immunohistochemically determined IRF4 expression for metastasis-free survival (MFS) was examined by Kaplan-Meier survival analysis as well as univariate and multivariate Cox analysis adjusted for age, pT stage, histological grade, ER, and HER2 status. For verification of immunohistochemical results, IRF4 mRNA expression was evaluated using microarray-based gene expression profiling in four previously published cohorts (Mainz, Rotterdam, Transbig, Yu) consisting of 824 node-negative breast cancer patients in total, who were not treated with adjuvant therapy. The prognostic significance of IRF4 mRNA expression on metastasis-free survival (MFS) was examined by univariate and multivariate Cox analysis in the Mainz cohort and by a meta-analysis of all node-negative BC patients and different molecular subtypes. IRF4 mRNA levels were compared to immunohistochemically determined IRF4 expression in 140 patients of the Mainz cohort using Spearman correlation. RESULTS: Immunohistochemically determined high IRF4 expression was associated with higher MFS in univariate Cox regression (HR 0.178, 95% CI 0.070-0.453, p < 0.001). IRF4 maintained its significance independently of established clinical factors for MFS (HR 0.088, 95% CI 0.033-0.232, p < 0.001). Immunohistochemically, determined IRF4 correlated moderately with IRF4 mRNA expression (ρ = 0.589). Higher expression of IRF4 was associated with better MFS in a meta-analysis of the total cohort (HR 0.438, 95% CI 0.307-0.623, p < 0.001). Prognostic significance was more pronounced in the HER2+ molecular subtype (HR 0.215, 95% CI 0.090-0.515, p = 0.001) as compared to the luminal A (HR 0.549, 95% CI 0.248-1.215, p = 0.139), luminal B (HR 0.444, 95% CI 0.215-0.916, p = 0.028), and basal-like subtypes (HR 0.487, 95% CI 0.269-0.883, p = 0.018). Further, IRF4 expression showed independent prognostic significance in a multivariate analysis of the Mainz cohort (HR 0.236, 95% CI 0.105-0.527, p < 0.001). CONCLUSIONS: IRF4 had independent prognostic significance in node-negative BC. Higher expression of IRF4 was associated with improved outcome. The prognostic impact differed between diverse molecular subtypes and was most pronounced in HER2+ breast cancer.

Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery.

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-ß and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay.

In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens.

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.

[Bladder cancer at an early age in father and son]. / Harnblasenkarzinom in jungen Jahren bei Vater und Sohn.

Bladder cancer may be caused by external factors like tobacco smoking, but may also be familial. We report on a father and son who developed this tumour at the ages of 45 and 35. Testing various genetic markers including the mismatch repair proteins MLH1, MSH2 and MSH6, whose loss is associated with a higher risk for hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), did not point to a familial disease. Thus the heavy smoking habits of the two patients must be considered as causal.

Critical evaluation of key evidence on the human health hazards of exposure to bisphenol A.

Despite the fact that more than 5000 safety-related studies have been published on bisphenol A (BPA), there seems to be no resolution of the apparently deadlocked controversy as to whether exposure of the general population to BPA causes adverse effects due to its estrogenicity. Therefore, the Advisory Committee of the German Society of Toxicology reviewed the background and cutting-edge topics of this BPA controversy. The current tolerable daily intake value (TDI) of 0.05 mg/kg body weight [bw]/day, derived by the European Food Safety Authority (EFSA), is mainly based on body weight changes in two- and three-generation studies in mice and rats. Recently, these studies and the derivation of the TDI have been criticized. After having carefully considered all arguments, the Committee had to conclude that the criticism was scientifically not justified; moreover, recently published additional data further support the reliability of the two- and three-generation studies demonstrating a lack of estrogen-dependent effects at and below doses on which the current TDI is based. A frequently discussed topic is whether doses below 5 mg/kg bw/day may cause adverse health effects in laboratory animals. Meanwhile, it has become clear that positive results from some explorative studies have not been confirmed in subsequent studies with higher numbers of animals or a priori defined hypotheses. Particularly relevant are some recent studies with negative outcomes that addressed effects of BPA on the brain, behavior, and the prostate in rodents for extrapolation to the human situation. The Committee came to the conclusion that rodent data can well be used as a basis for human risk evaluation. Currently published conjectures that rats are insensitive to estrogens compared to humans can be refuted. Data from toxicokinetics studies show that the half-life of BPA in adult human subjects is less than 2 hours and BPA is completely recovered in urine as BPA-conjugates. Tissue deconjugation of BPA-glucuronide and -sulfate may occur. Because of the extremely low quantities, it is only of minor relevance for BPA toxicity. Biomonitoring studies have been used to estimate human BPA exposure and show that the daily intake of BPA is far below the TDI for the general population. Further topics addressed in this article include reasons why some studies on BPA are not reproducible; the relevance of oral versus non-oral exposure routes; the degree to which newborns are at higher systemic BPA exposure; increased BPA exposure by infusions in intensive care units; mechanisms of action other than estrogen receptor activation; and the current regulatory status in Europe, as well as in the USA, Canada, Japan, New Zealand, and Australia. Overall, the Committee concluded that the current TDI for BPA is adequately justified and that the available evidence indicates that BPA exposure represents no noteworthy risk to the health of the human population, including newborns and babies.