Shirley J. Gee

In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens : Receptor-Based Bioassay Screens

Background Concerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects. Objectives In this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor–responsive and calcium signaling bioassays. Materials and methods We determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor–responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1). Results Some carbanilide compounds, including TCC (1–10 μM), enhanced estradiol (E2)-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1–10 μM) significantly enhanced the binding of [3H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca2+] in primary skeletal myotubes, but carbanilides had no effect. Conclusions Carbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca2+ mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS.

Immunochemical techniques for environmental analysis II. Antibody production and immunoassay development

Abstract Antibody production is the key step of any immunochemical technique. The antibody determines to a large extent the specificity and sensitivity of the resulting immunochemical technique. These features may be modulated by judicious design of the immunogen and by rational immunoassay development. Criteria for hapten design and the steps involved in the obtention of antibodies and the development of competitive assays are described.

Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity

The synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (amino carboxylic acid) attached at the pesticide thiophosphate group was simplified to a large extent. While the previous synthetic approach for this type of haptens requires seven steps, the present process involves only two steps. Using this process, five haptens of fenthion differing in spacer arm length (4–8 atoms) were synthesized and they were conjugated to bovine serum albumin and keyhole limpet hemocyanin to be used as immunogens. Rabbits were immunized with these hapten–protein conjugates for production of polyclonal antibodies against fenthion. The five haptens were conjugated to ovalbumin to be used as plate-coating antigens and twenty polyclonal antisera to the haptens were screened against each of the five coating antigens using noncompetitive and competitive indirect enzyme-linked immunosorbent assay (ELISA). The titer difference between the homologous and heterologous combinations was small, suggesting that heterology in spacer arm length is not important for the antigen recognition by antibodies. While the heterology in spacer length of the coating antigen had no significant effect on the sensitivity of ELISA, heterology in spacer structure of the coating antigen produced a remarkable improvement in the sensitivity of ELISA.

Magnetic/luminescent core/shell particles synthesized by spray pyrolysis and their application in immunoassays with internal standard

Many types of fluorescent nanoparticles have been investigated as alternatives to conventional organic dyes in biochemistry; magnetic beads also have a long history of biological applications. In this work we apply flame spray pyrolysis in order to engineer a novel type of nanoparticle that has both luminescent and magnetic properties. The particles have magnetic cores of iron oxide doped with cobalt and neodymium and luminescent shells of europium-doped gadolinium oxide (Eu:Gd(2)O(3)). Measurements by vibrating sample magnetometry showed an overall paramagnetic response of these composite particles. Luminescence spectroscopy showed spectra typical of the Eu ion in a Gd(2)O(3) host-a narrow emission peak centred near 615 nm. Our synthesis method offers a low-cost, high-rate synthesis route that enables a wide range of biological applications of magnetic/luminescent core/shell particles. Using these particles we demonstrate a novel immunoassay format with internal luminescent calibration for more precise measurements.

Immunochemical techniques for environmental analysis I. Immunosensors

Abstract Immunoassays are analytical techniques based on the avidity and specificity of the antigen-antibody reaction. However, new immunochemical techniques have emerged recently as a consequence of the incorporation of scientific advances and knowledge from other areas such as molecular biology, microelectronics and chemistry. The principles of these techniques and their application to environmental analysis are presented.

Use of a 96-well microplate reader for measuring routine enzyme activities

A method is described for the routine determination of the rate of colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. This approach is illustrated by monitoring esterase activity using three common products: release of thiol, release of ethanol, and release of p-nitrophenylate ion. Examples include monitorings of the rate of hydrolysis of acetylthiocholine iodide by eel acetylcholinesterase and the rate of hydrolysis of malathion and nonconventional esters such as O-methyl, O-ethyl, and O-isobutyl carbonates of p-nitrophenol by commercial porcine liver carboxylesterase. Data obtained from the plate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. Absorbance measurements made in both machines on the same solution, as well as absorbance changes measured over time, were similar. The use of the 96-well plate format tremendously increased the number of enzyme assays carried out per person and the interface with a personal computer allowed rapid manipulation of the absorbance values to calculate the desired rate data. This approach should be generally applicable to many routine colorimetric assays in the research laboratory.

Investigation of human exposure to triclocarban after showering, and preliminary evaluation of its biological effects

The antibacterial soap additive triclocarban (TCC) is widely used in personal care products. TCC has a high environmental persistence. We developed and validated a sensitive online solid-phase extraction-LC-MS/MS method to rapidly analyze TCC and its major metabolites in urine and other biological samples to assess human exposure. We measured human urine concentrations 0-72 h after showering with a commercial bar soap containing 0.6% TCC. The major route of renal elimination was excretion as N-glucuronides. The absorption was estimated at 0.6% of the 70±15 mg of TCC in the soap used. The TCC-N-glucuronide urine concentration varied widely among the subjects, and continuous daily use of the soap led to steady state levels of excretion. In order to assess potential biological effects arising from this exposure, we screened TCC for the inhibition of human enzymes in vitro. We demonstrate that TCC is a potent inhibitor of the enzyme soluble epoxide hydrolase (sEH), whereas TCCs major metabolites lack strong inhibitory activity. Topical administration of TCC at similar levels to rats in a preliminary in vivo study, however, failed to alter plasma biomarkers of sEH activity. Overall the analytical strategy described here revealed that use of TCC soap causes exposure levels that warrant further evaluation.

Development of toxicity identification evaluation procedures for pyrethroid detection using esterase activity

Recent agrochemical usage patterns suggest that the use of organophosphate (OP) pesticides will decrease, resulting in a concomitant increase in pyrethroid usage. Pyrethroids are known for their potential toxicity to aquatic invertebrates and many fish species. Current toxicity identification evaluation (TIE) techniques are able to detect OPs, but have not been optimized for pyrethroids. Organophosphate identification methods depend upon the use of piperonyl butoxide (PBO) to identify OP-induced toxicity. However, the use of PBO in TIE assays will be confounded by the co-occurrence of OPs and pyrethroids in receiving waters. It is necessary, therefore, to develop new TIE procedures for pyrethroids. This study evaluated the use of a pyrethroid-specific antibody, PBO, and carboxylesterase activity to identify pyrethroid toxicity in aquatic toxicity testing with Ceriodaphnia dubia. The antibody caused significant mortality to the C. dubia. Piperonyl butoxide synergized pyrethroid-associated toxicity, but this effect may be difficult to interpret in the presence of OPs and pyrethroids. Carboxylesterase activity removed pyrethroid-associated toxicity in a dose-dependent manner and did not compromise OP toxicity, suggesting that carboxylesterase treatment will not interfere with TIE OP detection methods. These results indicate that the addition of carboxylesterase to TIE procedures can be used to detect pyrethroids in aquatic samples.

Evaluation of an enzyme‐linked immunosorbent assay (ELISA) for the direct analysis of molinate (Ordram®) in rice field water

A direct ELISA for the thiocarbamate herbicide molinate was used to study distribution and dissipation of the compound in a treated rice field. No sample preparation other than buffering and dilution was required for the analysis of field water samples. Analyses were performed in 96‐well microplates and required less than 0–5 man‐hour per sample (three dilutions per sample, four replicate wells per dilution). Spiked samples and selected field samples were split for analysis by ELISA and gas chromatography. Two control samples of 92 and 93 ppb (after dilution) had between run coefficients of variation of 13.8 and 13.9% for 37 ELISA runs. A nested ANOVA analysis revealed that the largest source of error for the ELISA was due to within replicate variability, partly attributable to interwell variability of the 96‐well plates. Practical aspects of reducing assay error and handling ELISA data are discussed. Quality control data showed that reliability of the direct ELISA is comparable to the gas chromatography ...

Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.