Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling, by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.

Levels of tetrabromobisphenol A, tribromobisphenol A, dibromobisphenol A, monobromobisphenol A, and bisphenol a in Japanese breast milk.

The levels of bisphenol A (BPA) and tetrabromobisphenol A (TeBBPA) were determined in breast milk samples from 19 Japanese mothers. BPA and TeBBPA levels were 36 ng/g lipid (range: 1.4-380 ng/g lipid) and 1.9 ng/g lipid (range: N.D. - 8.7 ng/g lipid), respectively. Tribromobisphenol A (TriBBPA) was similarly detected in all samples (mean: 5.5 ng/g lipid). We investigated the alteration of BPA-related compounds in breast milk over a period of three months. No trend could be observed for time-dependent changes in TeBBPA levels. High levels of TriBBPA were detected in breast milk samples with a high concentration of TeBBPA. We further examined concentration changes in BPA-related compounds in the breast milk of two donors over a period of 24 h. The results suggested that TriBBPA was a debrominated metabolite of TeBBPA, which had been ingested via food consumption and immediately transferred to the breast milk. On the basis of the present results, we estimated and compared the daily intake of BPA, TriBBPA, and TeBBPA from breast milk for infants. The estimated average intake of TriBBPA was 4 times higher than TeBBPA, at 48 and 12 ng/kg/day, respectively. The level of TeBBPA in breast milk was low, suggesting a low risk of causing adverse health effects. In conclusion, the concentration of both TriBBPA and TeBBPA must be determined in breast milk to accurately clarify the exposure of these compounds to infants.

Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk.

Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2',6-tribromobisphenol A (TriBBPA), 2,2'-dibromobisphenol A (2,2'-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPARγ transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary.

In vitro and in vivo induction of cytochrome P450 by coplanar polychlorinated/brominated biphenyls (Co-PXBs) providing high TEQ in mother's milk in Japan.

Coplanar polychlorinated/brominated biphenyls (Co-PXBs) are environmental pollutants previously identified in market fish samples. In this study, we observed that mother's milk in Japan is contaminated with Co-PXBs. Based on assumption that the toxicity of the same congener of PXDDs/DFs and Co-PXBs is nearly equal to that of the corresponding PCDDs/DFs and Co-PCBs, respectively, the toxic equivalent (TEQ) concentration was 10% of the total TEQ concentration (∑PCDDs/DFs, ∑PXDDs/DFs, ∑Co-PCBs and ∑Co-PXBs) in the milk. This observation suggested that humans, and especially infants, are exposed to high levels of Co-PXBs, which might cause adverse effects. However, the toxicity of Co-PXBs has to date not been reported. We assessed the toxic potency of Co-PXBs by studying their effect on the activity of cytochrome P450. Only the mRNA level and activity of CYP1A increased in a dose-dependent manner upon exposure to Co-PXBs. Substitution of bromine for chlorine into Co-PCBs provided higher CYP1A activity in in vitro and in vivo experiments. The expression level of aryl hydrocarbon receptor (AhR) mRNA was not altered, but luciferase activity, an indicator of AhR transcriptional activity, increased following treatment with Co-PXBs. The results suggest that CYP1A induction by Co-PXBs depended on AhR transcriptional activity and not on AhR expression. Although the TEFs of Co-PXBs are not set, if Co-PXBs are included in these calculations because of their higher toxicity compared to Co-PCBs, exposure to Co-PXBs cannot be neglected when assessing human health risks.