Chemical genomics profiling of environmental chemical modulation of human nuclear receptors

Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays.Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program.Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure–activity relationships.Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality.Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.     

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts. II. Synthesis and processing of RNA.

Incorporation of tritiated uridine into cellular RNA is decreased in senescent human fibroblast cultures when measured per cellular RNA content. Since early and late passage cells demonstrate similar kinetics of RNA precursor pool labeling and saturation, this decreased tritiated uridine incorporation reflects decreased RNA synthesis. However, cellular RNA contents are markedly elevated in senescent cell cultures. When data are measured per cell number or DNA content, net RNA synthesis is slightly increased in senescent cultures. Separation of labeled RNAs by polyacrylamide gel electrophoresis revealed the same diminished specific activities (CPM/RNA) and increased net synthesis (CPM/cell number) of major ribosomal and transfer RNAs. However, this slight increase in RNA synthesis is probably not of sufficient magnitude to completely explain the observed increase in cellular RNA content in senescent cells.     

Implantable microencapsulated dopamine (DA): a new approach for slow-release DA delivery into brain tissue

Biodegradable controlled-release systems constitute an exciting new technology for drug delivery to the central nervous system (CNS). The present study describes functional and histochemical observations, indicating that implantation of DA microcapsules into striatal tissue assures a prolonged release of the transmitter in situ. This technology has considerable potential for basic and possibly also clinical research.     

Responses of transgenic mouse lines p53(+/-) and Tg.AC to agents tested in conventional carcinogenicity bioassays.

The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.     

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K⁺) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC₅₀ potencies ranging from 0.26 to 22 μM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC₅₀ value of 260 nM in the thallium influx assay and 80 nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.     

Quantitative high-throughput screening for chemical toxicity in a population-based in vitro model

A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d'Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM-46.0μM) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of genetic diversity are now feasible through the use of human lymphoblast cell lines.     

Risk Factors for Community-associated Methicillin-resistant Staphylococcus Aureus Cellulitis - and the Value of Recognition

Objectives

To identify the risk factors for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) cellulitis.

Methods

A review of risk factors for CA-MRSA skin and soft tissue infection in previously published literature was first performed. A retrospective cohort study was then conducted in a teaching ambulatory-care clinic of a tertiary medical center in Honolulu, Hawai‘i.

Results

Of 137 cases with cellulitis diagnosed from January 2005 to December 2007, MRSA was recovered from 85 (62%) of patients who presented with either abscesses or skin ulcers. The recovery of MRSA was significantly associated with obesity (p=0.01), presence of abscesses (p=0.01), and lesions involving the head and neck (p=0.04). Independent risk factors by multivariate logistic regression analysis included the presence of abscesses [adjusted odds ratio (aOR) 2.72; 95% confidence interval (CI) 1.27–5.83; p=0.01] and obesity (aOR 2.33; 95% CI 1.10–4.97; p =0.03). Patients with CA-MRSA were less likely to receive an appropriate antibiotic (p=0.04) and were more likely to require antibiotic change at evaluation in one week (p=0.04) compared with patients infected with non-MRSA bacteria.

Conclusions

The presence of abscesses and obesity were signifi cantly associated with CA-MRSA cellulitis. Empiric therapy with antibiotics active against MRSA should be guided by these risk factors.     

Quantitation of Staphylococcus aureus in Seawater Using CHROMagar? SA

A microbiological algorithm has been developed to analyze beach water samples for the determination of viable colony forming units (CFU) of Staphylococcus aureus (S. aureus). Membrane filtration enumeration of S. aureus from recreational beach waters using the chromogenic media CHROMagar™SA alone yields a positive predictive value (PPV) of 70%. Presumptive CHROMagar™SA colonies were confirmed as S. aureus by 24-hour tube coagulase test. Combined, these two tests yield a PPV of 100%. This algorithm enables accurate quantitation of S. aureus in seawater in 72 hours and could support risk-prediction processes for recreational waters. A more rapid protocol, utilizing a 4-hour tube coagulase confirmatory test, enables a 48-hour turnaround time with a modest false negative rate of less than 10%.