John D. Tessari

Pharmacokinetic modeling of disposition and time-course studies with [14C]atrazine.

A physiological pharmacokinetic (PPK) model, with blood, body, and brain compartments, was developed to estimate total plasma chlorotriazine (CI-TRI) time courses (i.e., atrazine [ATRA] and its three chlorinated metabolites) after oral dosing with ATRA. The model, based on disposition data for 14 C-ATRA, tracked two pools of compounds: (1) ATRA and chlorinated metabolites (i.e., the CI-TRIs) and (2) glutathione conjugates. The PPK model developed from total radioactivity was valuable for assessing total plasma CI-TRI concentrations, estimating blood protein binding rates of CI-TRIs, and inferring relationships between tissue exposures of CI-TRIs and administered dose. Absorption of radioactivity into plasma was slow with a rate constant of 0.2 h m 1 . 14 C-disposition data indicated that CI-TRIs react with red blood cells (presumably hemoglobin) and plasma proteins. Second-order rates of reaction of CI-TRIs with hemoglobin and plasma protein were estimated to be 0.008 L/mmol/h and 1.14 2 10 m 7 L/mg/h, respectively. A time-course study, conducted as part of this study, evaluated the absorption, disposition, and elimination characteristics of individual CI-TRIs in plasma after a single oral dose of 90 mg ATRA/kg and indicated (1) that slow uptake into blood reflected both absorption and slow dissolution of the ATRA slurry and (2) that diaminochloro-s-triazine (DACT) was the major, persistent plasma CI-TRI after oral dosing. Optimally, PK model development for pesticide compounds like atrazine should include a combination of radiolabeled studies for residues and speciation studies of important metabolites.

Endotoxin Exposure and Inflammation Markers Among Agricultural Workers in Colorado and Nebraska

The adverse respiratory effects of agricultural dust inhalation are mediated in part by endotoxin, a constituent of gram-negative bacterial cell walls. This study quantified personal work-shift exposures to inhalable dust, endotoxin, and its reactive 3-hydroxy fatty acid (3-OHFA) constituents among workers in grain elevators, cattle feedlots, dairies, and on corn farms. Exposures were compared with post-work-shift nasal lavage fluid inflammation markers and respiratory symptoms. Breathing-zone personal air monitoring was performed over one work shift to quantify inhalable dust (Institute of Medicine samplers), endotoxin (recombinant factor C [rFC] assay), and 3-OHFA (gas chromatography/mass spectrometry). Post-shift nasal lavage fluids were assayed for polymorphonuclear neutrophils (PMN), myeloperoxidase (MPO), interleukin 8 (IL-8), albumin, and eosinophilic cation protein (ECP) concentrations. The geometric mean (GSD) of endotoxin exposure (rFC assay) among the 125 male participants was 888 ± (6.5) EU/m3, and 93% exceeded the proposed exposure limit (50 EU/m3). Mean PMN, MPO, albumin, and ECP levels were two- to threefold higher among workers in the upper quartile of 3‐OHFA exposure compared to the lowest exposure quartile. Even numbered 3-OHFA were most strongly associated with nasal inflammation. Symptom prevalence was not elevated among exposed workers, possibly due to endotoxin tolerance or a healthy worker effect in this population. This is the first study to evaluate the relationship between endotoxins 3-OHFA constituents in agricultural dust and nasal airway inflammation. More research is needed to characterize the extent to which these agents contribute to respiratory disease among agricultural workers.

Exploration of an interaction threshold for the joint toxicity of trichloroethylene and 1,1-dichloroethylene: utilization of a PBPK model

Physiologically based pharmacokinetic (PBPK) modeling and gas uptake experiments were utilized to verify the competitive inhibition mechanism of interaction between trichloroethylene (TCE) and 1,1-dichloroethylene (DCE) and to investigate the presence of an interaction threshold between the two chemicals. Initially, gas uptake experiments were conducted on Fischer 344 rats where the initial concentrations of both DCE and TCE were 2000∶0, 0∶2000, 2000∶2000, 1000∶0, 1000∶1000, and 500∶500 ppm, respectively. When the different modes of inhibition interactions (competitive, uncompetitive and noncompetitive) were employed in the PBPK model, the model description of the competitive inhibition interaction provided the best description of the declining concentrations in the gas uptake chamber. Furthermore, to predict the range at which the interaction threshold would be found, the PBPK model included a mathematical description of the percentage of enzyme sites occupied by either chemical in the presence or the absence of the other. By comparing the percentage of occupied sites by one chemical, in the presence of the other, to those sites occupied in the absence of the latter, the PBPK model predicted a range of concentrations (100 ppm or less) of either chemical where the competitive inhibition interaction would not be observed. Consequently, gas uptake experiments were designed where the initial concentration was selected at 2000 ppm for one chemical while the other chemical was set at 100 in one experiment and 50 ppm in another. Under these conditions, the best simulation to the concentration depletion curves in the gas uptake system of the chemical in the higher concentration was obtained when the PBPK model was run under the assumption of no-interaction. This substantiated the model predictions of the presence of observable interaction only at concentrations higher than 100 ppm.

Field Evaluation of a Portable Photoionization Detector for Assessing Exposure to Solvent Mixtures

To evaluate a portable photoionization detector for assessing personal exposure to solvent mixtures, a set of 26 side-by-side, time-weighted average (TWA) personal breathing zone samples were collected during various construction painting tasks by two different sampling methods: (1) standard charcoal sorbent tubes analyzed by gas chromatography (CST/GC), and (2) a direct-reading photoionization detector coupled with an extended data-logger (PID). The TWA concentrations of the hydrocarbons detected by CST/GC analysis were summed for comparison with the TWA concentration obtained from the direct-reading PID. Based on linear regression between the log TWA concentrations of the two sampling methods, the data were highly correlated (r2 = 0.95). Since the solvents had effects that may be considered additive, threshold limit values (TLVs) for mixtures were developed using American Conference of Governmental Industrial Hygienists formulas to evaluate solvent exposure. The logs of the TLV mixture data from the sampling methods were highly correlated (r2 = 0.94). Based on the linear regression analyses, the response of the portable PID was highly correlated to the CST/GC results for hydrocarbon mixtures encountered during various painting tasks. Due to the short duration of tasks, highly fluctuating exposures, and complexity of the mixtures, the PID may provide the most cost-effective, detailed exposure assessment for solvent mixtures.

Recombinant Factor C (rFC) Assay and Gas Chromatography/Mass Spectrometry (GC/MS) Analysis of Endotoxin Variability in Four Agricultural Dusts

Endotoxin exposure is a significant concern in agricultural environments due to relatively high exposure levels. The goals of this study were to determine patterns of 3-hydroxy fatty acid (3-OHFA) distribution in dusts from four types of agricultural environments (dairy, cattle feedlot, grain elevator, and corn farm) and to evaluate correlations between the results of gas chromatography/mass spectrometry (GC/MS) analysis (total endotoxin) and biological recombinant factor C (rFC) assay (free bioactive endotoxin). An existing GC/MS-MS method (for house dust) was modified to reduce sample handling and optimized for small amount (<1 mg) of agricultural dusts using GC/EI-MS. A total of 134 breathing zone samples using Institute of Occupational Medicine (IOM) inhalable samplers were collected from agricultural workers in Colorado and Nebraska. Livestock dusts contained approximately two times higher concentrations of 3-OHFAs than grain dusts. Patterns of 3-OHFA distribution and proportion of each individual 3-OHFA varied by dust type. The rank order of Pearson correlations between the biological rFC assay and the modified GC/EI-MS results was feedlot (0.72) > dairy (0.53) > corn farm (0.33) > grain elevator (0.11). In livestock environments, both odd- and even-numbered carbon chain length 3-OHFAs correlated with rFC assay response. The GC/EI-MS method should be especially useful for identification of specific 3-OHFAs for endotoxins from various agricultural environments and may provide useful information for evaluating the relationship between bacterial exposure and respiratory disease among agricultural workers.

β‐Benzene hexachloride in breast adipose tissue and risk of breast carcinoma

Epidemiologic studies have recently related benzene hexachloride (BHC) to breast carcinoma risk. Experimental studies have also shown that β‐BHC is weakly estrogenic, hence supporting the alleged association. By directly comparing β‐BHC levels in breast adipose tissue from incident breast carcinoma cases and controls, this study examined the hypothesis that exposure to β‐BHC increases the risk of breast carcinoma in females.

Gas chromatographic determination of catecholestrogens following isolation by solid-phase extraction

A sensitive and specific assay for the determination of the catecholestrogens 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) using gas chromatography with electron-capture detection (GC-ECD) is described. The formation of 2- and 4-OHE2 was assessed following activation of 17beta-estradiol in the microsomal fraction of female rat livers. The analytes were isolated by solid-phase extraction, derivatized to their heptafluorobutyryl esters with heptafluorobutyric acid anhydride, and subjected to solvent exchange prior to analysis; this resulted in minimal chromatographic interference, long column life, and stable derivatized analytes. Derivatized catechols were separated and confirmed with dual column chromatography (DB-5 and DB-608) and quantitated using GC-ECD. The DB-608 column was preferred for quantitation as it provided better 4-OHE2 resolution from interference. Key validation parameters for the assay include sensitivity, intra- and inter-assay precision, and accuracy. Instrument sensitivity and limits of detection (LOD) and quantitation (LOQ) were determined statistically from fortification data approaching expected limits. For 2-OHE2 and 4-OHE2, respective values for these parameters were; instrument sensitivities of 0.4 and 0.7 pg, LODs of 0.8 and 1.3 ng/mg, and LOQs of 2.6 and 4.3 ng/mg.

Evaluation of the Effects of Water Disinfection By-products, Bromochloroacetic and Dibromoacetic Acids, on Frog Embryogenesis

Adverse developmental effects of two haloacetic acids, bromochloroacetic acid (BCA) and dibromoacetic acid (DBA), were determined by using the Frog Embryo Teratogenesis Assay—Xenopus (FETAX). Xenopus embryos (150–400/concentration group) were exposed to 0, 8000, 10,000, 12,000, or 14,000 ppm BCA or 0, 10,000, 12,000, 14,000, or 16,000 ppm DBA for 96 h beginning from stage 8 (mid-blastula) to stage 46 (when primary organogenesis is complete). BCA produced 29, 83, and 100% mortality at 10,000, 12,000 and 14,000 ppm, respectively. Incidence of malformations among surviving embryos at 96 h for 10,000 and 12,000 ppm BCA were 8.4 and 68%. Thus LC50 and EC50 for BCA were between 10,000 and 12,000 ppm. DBA did not produce any significant mortality or malformation at any of the concentrations tested. In summary, BCA affected development of Xenopus embryos only at high concentrations, while DBA did not affect Xenopus development at the concentrations tested.

Two-stage evaluation of exposure to mercury and biomarkers of neurotoxicity at a hazardous waste site.

Communities surrounding the Rocky Mountain Arsenal (RMA), a Superfund site in Colorado, were studied in order to determine whether exposures to mercury were greater among persons who resided there than among residents of a comparison area 12-15 miles distant. From a census-based stratified random sample, 469 persons were interviewed and urine samples were obtained for biomonitoring. Mercury was detected in urine from 32 (6.8%) of the 469 persons sample at a detection limit of 5 ppb. Trace levels of mercury (detectable, but nonquantifiable) were found in 80 (17.1%) of the persons sampled. Neither the frequency of detection, the arithmetic mean, nor the geometric mean value for urine mercury was found to be statistically different when persons living near the site were compared to persons from the more distant comparison area. The risk of mercury exposure associated with demographic variables, residence, occupation, hobbies, dietary habits, water supply, housing, and activity patterns was evaluated. In the second stage of the evaluation, the Neurobehavioral Core Test Battery (NCTB) is being used to assess individual functional deficits and nervous system disorders associated with exposure to mercury and other neurotoxic chemicals.

QUALITY ASSURANCE QUALITY CONTROL PROCEDURES FOR CHLORINATED HYDROCARBONS IN HUMAN BREAST ADIPOSE TISSUE

Extensive literature exists supporting the accumulation of organochlorine pesticides such as DDT [2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane], and polychlorinated biphenyls (PCBs) in human adipose tissue. Debate has surfaced concerning the link between these environmental contaminants and human breast cancer. Accurate residue analysis and proper analytical procedures are critical in determining the extent to which these compounds play a role in human breast cancer. Further, adequate quality assessment/quality control (QA/QC) is critical for reliable residue analysis. The purpose of this research was twofold: (1) to find an appropriate surrogate for human breast adipose tissue for spiking purposes, as human samples are difficult to obtain, and (2) to develop a human breast adipose tissue pool that yields adequate reproducibility with low coefficients of variation (CVs) for each compound of interest. Using a previously validated method developed in the Analytical Laboratory at Colorado State University, rendered ovine adipose tissue was found to be a suitable spiking material, as it was free of interfering compounds and behaved in a manner similar to human breast adipose tissue throughout the analytical method. Further, this analytical method was used to produce data on three control pool preparations: (A) blended human breast adipose tissue (n = 26), (B) blended and partially rendered human breast adipose tissue (n = 12), and (C) fully blended and rendered human breast adipose tissue (n = 15). The CVs between control pools vary up to 20% for a single compound. The most reproducible preparation procedure requires full blending and rendering.