Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis

Healing Right Way: study protocol for a stepped wedge cluster randomised controlled trial to enhance rehabilitation services and improve quality of life in Aboriginal Australians after brain injury.

IntroductionDespite higher incidence of brain injury among Aboriginal compared with non-Aboriginal Australians, suboptimal engagement exists between rehabilitation services and Aboriginal brain injury survivors. Aboriginal patients often feel culturally insecure in hospital and navigation of services post discharge is complex. Health professionals report feeling ill-equipped working with Aboriginal patients. This study will test the impact of a research-informed culturally secure intervention model for Aboriginal people with brain injury. METHODS AND ANALYSIS: Design: Stepped wedge cluster randomised control trial design; intervention sequentially introduced at four pairs of healthcare sites across Western Australia at 26-week intervals.Recruitment: Aboriginal participants aged ≥18 years within 4 weeks of an acute stroke or traumatic brain injury.Intervention: (1) Cultural security training for hospital staff and (2) local, trial-specific, Aboriginal Brain Injury Coordinators supporting participants.Primary outcome: Quality-of-life using EuroQOL-5D-3L (European Quality of Life scale, five dimensions, three severity levels) Visual Analogue Scale score at 26 weeks post injury. Recruitment of 312 participants is estimated to detect a difference of 15 points with 80% power at the 5% significance level. A linear mixed model will be used to assess the between-condition difference.Secondary outcome measures: Modified Rankin Scale, Functional Independence Measure, Modified Caregiver Strain Index, Hospital Anxiety and Depression Scale at 12 and 26 weeks post injury, rehabilitation occasions of service received, hospital compliance with minimum care processes by 26 weeks post injury, acceptability of Intervention Package, feasibility of Aboriginal Brain Injury Coordinator role.Evaluations: An economic evaluation will determine the potential cost-effectiveness of the intervention. Process evaluation will document fidelity to study processes and capture changing contexts including barriers to intervention implementation and acceptability/feasibility of the intervention through participant questionnaires at 12 and 26 weeks.Ethics and disseminationThe study has approvals from Aboriginal, university and health services human research ethics committees. Findings will be disseminated through stakeholder reports, participant workshops, peer-reviewed journal articles and conference papers.Trial registration numberACTRN12618000139279

Synthesis, structural characterization, antimicrobial and cytotoxic effects of aziridine, 2-aminoethylaziridine and azirine complexes of copper(II) and palladium(II).

The synthesis, spectroscopic and X-ray structural characterization of copper(II) and palladium(II) complexes with aziridine ligands as 2-dimethylaziridine HNCH2CMe2 (a), the bidentate N-(2-aminoethyl)aziridines C2H4NC2H4NH2 (b) or CH2CMe2NCH2CMe2NH2 (c) as well as the unsaturated azirine NCH2CPh (d) are reported. Cleavage of the cyclometallated Pd(II) dimer [μ-Cl(C6H4CHMeNMe2-C,N)Pd]2 with ligand a yielded compound [Cl(NHCH2CMe2)(C6H4CHMe2NMe2-C,N)Pd] (1a). The reaction of the aziridine complex trans-[Cl2Pd(HNC2H4)2] with an excess of aziridine in the presence of AgOTf gave the ionic chelate complex trans-[(C2H4NC2H4NH2-N,N′)2Pd](OTf)2 (2b) which contains the new ligand b formed by an unexpected insertion and ring opening reaction of two aziridines (“aziridine dimerization”). CuCl2 reacted in pure HNC2H4 or HNCH2CMe2 (b) again by “dimerization” to give the tris-chelated ionic complex [Cu(C2H4NC2H4NH2-N,N′)3]Cl2 (3b) or the bis-chelated complex [CuCl(C2H2Me2NC2H2Me2NH2-N,N′)2]Cl (4c). By addition of 2H-3-phenylazirine (d) to PdCl2, trans-[Cl2Pd(NCH2CPh)2] (5d) was formed. All new compounds were characterized by NMR, IR and mass spectra and also by X-ray structure analyses (except 3b). Additionally the cytotoxic effects of these complexes were examined on HL-60 and NALM-6 human leukemia cells and melanoma WM-115 cells. The antimicrobial activity was also determined. The growth of Gram-positive bacterial strains (S. aureus, S. epidermidis, E. faecalis) was inhibited by almost all tested complexes at the concentrations of 37.5–300.0 μg mL−1. However, MIC values of complexes obtained for Gram-negative E. coli and P. aeruginosa, as well as for C. albicans yeast, mostly exceeded 300 μg mL−1. The highest antibacterial activity was achieved by complexes 1a and 2b. Complex 2b also inhibited the growth of Gram-negative bacteria. Graphical abstract: Synthesis, structural characterization, antimicrobial and cytotoxic effects of aziridine, 2-aminoethylaziridine and azirine complexes of copper(ii) and palladium(ii

Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions

Estrogen receptor alpha (ERalpha) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERalpha is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERalpha (C451A-ERalpha) to obtain membrane-specific loss of function of ERalpha. The abrogation of membrane localization of ERalpha in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERalpha mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERalpha mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERalpha (ERalpha-AF2(0)) provided selective loss of function of nuclear ERalpha actions. In ERalpha-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERalpha-AF2(0), whereas in C451A-ERalpha it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles

Line-shape transition of collision broadened lines

Using the newly developed technique of THz time-domain spectroscopy, we have measured the far-wing absorption line profile of the ensemble of collision broadened ground state rotational lines of methylchloride vapor out to more than 200 linewidths from resonance, corresponding to frequency offsets as much as 5X the resonant frequency. On these far wings the measured absorption is approximately an order of magnitude less than that predicted by the van Vleck-Weisskopf theory. Our observations show that at higher frequencies a transition occurs from the regime of the van Vleck- Weisskopf theory to the regime of the Lorentz theory.Peer reviewedElectrical and Computer Engineerin

Predicting Diabetic Nephropathy Using a Multifactorial Genetic Model

AIMS: The tendency to develop diabetic nephropathy is, in part, genetically determined, however this genetic risk is largely undefined. In this proof-of-concept study, we tested the hypothesis that combined analysis of multiple genetic variants can improve prediction. METHODS: Based on previous reports, we selected 27 SNPs in 15 genes from metabolic pathways involved in the pathogenesis of diabetic nephropathy and genotyped them in 1274 Ashkenazi or Sephardic Jewish patients with Type 1 or Type 2 diabetes of >10 years duration. A logistic regression model was built using a backward selection algorithm and SNPs nominally associated with nephropathy in our population. The model was validated by using random "training" (75%) and "test" (25%) subgroups of the original population and by applying the model to an independent dataset of 848 Ashkenazi patients. RESULTS: The logistic model based on 5 SNPs in 5 genes (HSPG2, NOS3, ADIPOR2, AGER, and CCL5) and 5 conventional variables (age, sex, ethnicity, diabetes type and duration), and allowing for all possible two-way interactions, predicted nephropathy in our initial population (C-statistic = 0.672) better than a model based on conventional variables only (C = 0.569). In the independent replication dataset, although the C-statistic of the genetic model decreased (0.576), it remained highly associated with diabetic nephropathy (χ(2) = 17.79, p<0.0001). In the replication dataset, the model based on conventional variables only was not associated with nephropathy (χ(2) = 3.2673, p = 0.07). CONCLUSION: In this proof-of-concept study, we developed and validated a genetic model in the Ashkenazi/Sephardic population predicting nephropathy more effectively than a similarly constructed non-genetic model. Further testing is required to determine if this modeling approach, using an optimally selected panel of genetic markers, can provide clinically useful prediction and if generic models can be developed for use across multiple ethnic groups or if population-specific models are required

Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefit from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carcinoma

INTRODUCTION: Little is known of the function and clinical significance of intratumoral dysregulation of xenobiotic-metabolizing enzyme expression in breast cancer. One molecular mechanism proposed to explain tamoxifen resistance is altered tamoxifen metabolism and bioavailability. METHODS: To test this hypothesis, we used real-time quantitative RT-PCR to quantify the mRNA expression of a large panel of genes coding for the major xenobiotic-metabolizing enzymes (12 phase I enzymes, 12 phase II enzymes and three members of the ABC transporter family) in a small series of normal breast (and liver) tissues, and in estrogen receptor alpha (ERα)-negative and ERα-positive breast tumors. Relevant genes were further investigated in a well-defined cohort of 97 ERα-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. RESULTS: Seven of the 27 genes showed very weak or undetectable expression in both normal and tumoral breast tissues. Among the 20 remaining genes, seven genes (CYP2A6, CYP2B6, FMO5, NAT1, SULT2B1, GSTM3 and ABCC11) showed significantly higher mRNA levels in ERα-positive breast tumors than in normal breast tissue, or showed higher mRNA levels in ERα-positive breast tumors than in ERα-negative breast tumors. In the 97 ERα-positive breast tumor series, most alterations of these seven genes corresponded to upregulations as compared with normal breast tissue, with an incidence ranging from 25% (CYP2A6) to 79% (NAT1). Downregulation was rare. CYP2A6, CYP2B6, FMO5 and NAT1 emerged as new putative ERα-responsive genes in human breast cancer. Relapse-free survival was longer among patients with FMO5-overexpressing tumors or NAT1-overexpressing tumors (P = 0.0066 and P = 0.000052, respectively), but only NAT1 status retained prognostic significance in Cox multivariate regression analysis (P = 0.0013). CONCLUSIONS: Taken together, these data point to a role of genes coding for xenobiotic-metabolizing enzymes in breast tumorigenesis, NAT1 being an attractive candidate molecular predictor of antiestrogen responsiveness

Genetic variants of CYP3A5, CYP2D6, SULT1A1, UGT2B15 and tamoxifen response in postmenopausal patients with breast cancer

INTRODUCTION: Tamoxifen therapy reduces the risk of recurrence and prolongs the survival of oestrogen-receptor-positive patients with breast cancer. Even if most patients benefit from tamoxifen, many breast tumours either fail to respond or become resistant. Because tamoxifen is extensively metabolised by polymorphic enzymes, one proposed mechanism underlying the resistance is altered metabolism. In the present study we investigated the prognostic and/or predictive value of functional polymorphisms in cytochrome P450 3A5 CYP3A5 (*3), CYP2D6 (*4), sulphotransferase 1A1 (SULT1A1; *2) and UDP-glucuronosyltransferase 2B15 (UGT2B15; *2) in tamoxifen-treated patients with breast cancer. METHODS: In all, 677 tamoxifen-treated postmenopausal patients with breast cancer, of whom 238 were randomised to either 2 or 5 years of tamoxifen, were genotyped by using PCR with restriction fragment length polymorphism or PCR with denaturing high-performance liquid chromatography. RESULTS: The prognostic evaluation performed in the total population revealed a significantly better disease-free survival in patients homozygous for CYP2D6*4. For CYP3A5, SULT1A1 and UGT2B15 no prognostic significance was observed. In the randomised group we found that for CYP3A5, homozygous carriers of the *3 allele tended to have an increased risk of recurrence when treated for 2 years with tamoxifen, although this was not statistically significant (hazard ratio (HR) = 2.84, 95% confidence interval (CI) = 0.68 to 11.99, P = 0.15). In the group randomised to 5 years' tamoxifen the survival pattern shifted towards a significantly improved recurrence-free survival (RFS) among CYP3A5*3-homozygous patients (HR = 0.20, 95% CI = 0.07 to 0.55, P = 0.002). No reliable differences could be seen between treatment duration and the genotypes of CYP2D6, SULT1A1 or UGT2B15. The significantly improved RFS with prolonged tamoxifen treatment in CYP3A5*3 homozygotes was also seen in a multivariate Cox model (HR = 0.13, CI = 0.02 to 0.86, P = 0.03), whereas no differences could be seen for CYP2D6, SULT1A1 and UGT2B15. CONCLUSION: The metabolism of tamoxifen is complex and the mechanisms responsible for the resistance are unlikely to be explained by a single polymorphism; instead it is a combination of several mechanisms. However, the present data suggest that genetic variation in CYP3A5 may predict response to tamoxifen therapy