Captan exposure and evaluation of a pesticide exposure algorithm among orchard pesticide applicators in the Agricultural Health Study.

Pesticide exposure assessment in the Agricultural Health Study (AHS) has relied upon two exposure metrics: lifetime exposure days and intensity-weighted lifetime exposure days, the latter incorporating an intensity score computed from a questionnaire-based algorithm. We evaluated this algorithm using actual fungicide exposure measurements from AHS private orchard applicators. Captan was selected as a marker of fungicide exposure. Seventy-four applicators from North Carolina and Iowa growing apples and/or peaches were sampled on 2 days they applied captan in 2002 and 2003. Personal air, hand rinse, 10 dermal patches, a pre-application first-morning urine and a subsequent 24-h urine sample were collected from each applicator per day. Environmental samples were analyzed for captan, and urine samples were analyzed for cis-1,2,3,6-tetrahydrophthalimide (THPI). Task and personal protective equipment information needed to compute an individual's algorithm score was also collected. Differences in analyte detection frequency were tested in a repeated logistic regression model. Mixed-effects models using maximum-likelihood estimation were employed to estimate geometric mean exposures and to evaluate the measured exposure data against the algorithm. In general, captan and THPI were detected significantly more frequently in environmental and urine samples collected from applicators who used air blast sprayers as compared to those who hand sprayed. The AHS pesticide exposure intensity algorithm, while significantly or marginally predictive of thigh and forearm captan exposure, respectively, did not predict air, hand rinse or urinary THPI exposures. The algorithm's lack of fit with some exposure measures among orchard fungicide applicators may be due in part to the assignment of equal exposure weights to air blast and hand spray application methods in the current algorithm. Some modification of the algorithm is suggested by these results.

Identification of human urinary metabolites of acetochlor in exposed herbicide applicators by high-performance liquid chromatography-tandem mass spectrometry.

Acetochlor is a preemergent chloroacetanilide herbicide used to control annual grasses and small-seeded broadleaf weeds. It is the second most abundantly applied herbicide on corn crops in the United States; however, human metabolites associated with known exposure to acetochlor have not been positively identified and confirmed. We positively identified acetochlor mercapturate (ACM) as a metabolite of acetochlor in urine samples collected during a 24-h period from custom (commercial) applicators who had applied acetochlor on either the day of or the day before urine collection. Concentrations in applicator urine samples ranged from 0.5 to 449 microg/l (0.3-121 microg/g creatinine). We found that ACM accounted for as much as 42% of the total acetochlor-derived metabolites; however, as the exposure level decreased (based on total acetochlor metabolite level), ACM became a less abundant metabolite of acetochlor (<17%). Unmetabolized acetochlor was also measured in the urine samples analyzed. At high exposures (classified as >100 microg/l), acetochlor accounted for about 0.8% of the total excreted acetochlor metabolites (approximately 2% of the ACM concentrations). At lower exposures (classified as ACM<10 microg/l), ACM and acetochlor concentrations were similar. Additionally, we tentatively identified another acetochlor metabolite that appeared to be important at low levels of exposure.

Rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin g in serum and whole blood.

Evidence from animals suggests that anti-anthrax protective antigen (PA) immunoglobulin G (IgG) from vaccination with anthrax vaccine adsorbed (AVA) is protective against Bacillus anthracis infection. Measurement of anti-PA IgG in human sera can be performed using either enzyme-linked immunosorbent assay or fluorescent covalent microsphere immunoassay (ELISA) (R. E. Biagini, D. L. Sammons, J. P. Smith, B. A. MacKenzie, C. A. Striley, V. Semenova, E. Steward-Clark, K. Stamey, A. E. Freeman, C. P. Quinn, and J. E. Snawder, Clin. Diagn. Lab. Immunol. 11:50-55, 2004). Both these methods are laboratory based. We describe the development of a rapid lateral-flow immunochromatographic assay (LFIA) test kit for the measurement of anti-PA IgG in serum or whole-blood samples (30-microl samples) using colloidal gold nanoparticles as the detection reagent and an internal control. Using sera from 19 anthrax AVA vaccinees (anti-PA IgG range, 2.4 to 340 microg/ml) and 10 controls and PA-supplemented whole-blood samples, we demonstrated that the LFIA had a sensitivity of approximately 3 microg/ml anti-PA IgG in serum and approximately 14 microg/ml anti-PA IgG in whole blood. Preabsorption of sera with PA yielded negative anti-PA LFIAs. The diagnostic sensitivity and specificity of the assay were 100% using ELISA-measured anti-PA IgG as the standard. This kit has utility in determining anti-PA antibody reactivity in the sera of individuals vaccinated with AVA or individuals with clinical anthrax.

Mixed-effect models for evaluating multiple measures of atrazine exposure among custom applicators.

The exposure of custom (or commercial) applicators to the herbicide atrazine was measured in environmental (hand wash and dermal patch) and biological (urine and saliva) samples. Surrogate exposure data, such as amount of atrazine sprayed, were also collected. A systematic sampling design was used that included both spray and nonspray days. Fifteen applicators were sampled 5 to 7 days each during a 6-week spring spray season for a total of 89 sampled days. Mixed-effect regression modeling was used to examine the relationship among the surrogate, environmental, and biological atrazine exposure measures. Surrogate measures of atrazine application (either kg of atrazine sprayed or spray atrazine [yes/no]) were significantly associated with increased levels of atrazine or atrazine equivalents (eq) in hand wash, thigh patch, 4-6 p.m. saliva, and 24-hour urine samples. Two days of spraying information (day of sampling and day before sampling) were needed to optimally estimate atrazine biomarkers in the biological samples, whereas only 1 day of spraying information (day of sampling) was needed to estimate atrazine levels in the environmental samples. Thigh and hand atrazine exposures were significantly associated with increased atrazine and atrazine eq. levels in the 4-6 p.m. saliva and 24-hour urine samples, respectively. Levels of 4-6 p.m. salivary atrazine were also significantly associated with increased levels of 24-hour urinary atrazine eq. Atrazine levels in the 4-6 p.m. saliva samples tracked most closely with evening and next morning urinary atrazine eq. Number of days into the study at the time of sample collection predicted urinary and salivary atrazine levels independent of other fixed effects. These results indicate that either surrogate, environmental, or biological exposure measures can be used in appropriately specified models to estimate urinary and salivary atrazine biomarker levels.

Dibenzotetraaza crown ethers: a new family of crown ethers based on o-phenylenediamine.

Dibenzotetraaza (DBTA) crown ethers possess two o-phenylenediamine moieties. They are homologues of dibenzo crown ether phase-transfer catalysts and were prepared from the condensation of benzimidazoles with oligo(ethyleneglycol) dichlorides and oligo(ethyleneglycol) ditosylates. Compounds with ring sizes ranging from 18-crown-6 to 42-crown-14 were prepared. In addition, various altered benzimidizoles were used to produce DBTA crown ethers with modified substituents and ether bridges, as well as benzimidazolidine crown ethers. The synthetic approach presented here proved to be a convenient route to a new family of crown ethers with overall yields of up to 48% based on the benzimidazole. Yields for the ring-closing step were generally high, ranging from 51% to 94%, without the need for high-dilution conditions. Reaction of the DBTA crown ethers with alkyl and benzyl halides was found to be a facile way to obtain the corresponding tetra(N-organyl) compounds. Picrate extraction studies were carried out to determine phase-transfer catalytic capabilities. Extraction efficiencies for alkali-metal ions were lower than those for dibenzo-18-crown-6. Efficiencies were higher for other metal ions, with some selectivity for Pb(2+). Tetra(N-methyl) DBTA-18-crown-6 generally exhibited higher extraction efficiencies than its N-H analogue, but the selectivity was lower.

Evaluation of the prevalence of antiwheat-, anti-flour dust, and anti-alpha-amylase specific IgE antibodies in US blood donors.

BACKGROUND: Asthma in bakery workers is one of the most frequently occurring forms of occupational asthma in the world. Experience from other countries has shown the prevalence of sensitization (IgE) to bakery-associated allergens (BAAs) (wheat [W], flour dust [FD], alpha-amylase [AA]) in bakery workers to be 5% to 53%, whereas the prevalence in nonoccupationally exposed individuals was estimated to be 1.2% to 6.4%. OBJECTIVE: To estimate the prevalence of BAA sensitization by measuring BAA specific IgE in the residual serum tubes of volunteer blood donors. METHODS: Serum samples from 534 volunteer blood donors were measured for anti-W, anti-FD, and anti-AA specific IgE antibodies (in duplicate) using the AlaSTAT microplate assay. Samples with BAA IgE concentrations of 0.35 kU/L or greater were considered positive. RESULTS: Nineteen of 530 serum samples (3.6%; 95% confidence interval [CI], 3.3%-3.9%) were positive for W (range, 0.38-3.61 kU/L), whereas 31 of 534 (5.8%; 95% CI, 5.3%-6.3%) were positive for FD (range, 0.35-2.34 kU/L) and 5 of 529 (1.0%; 95% CI, 0.9%-1.1%) were positive for AA (range, 0.38-1.59 kU/L). Thirteen serum samples were positive for both W and FD; 1 sample each was positive for W and AA and FD and AA. CONCLUSIONS: The prevalence of IgE sensitization in serum samples from a relatively large unselected population of volunteer blood donors is 1.0% for AA, 3.6% for W, and 5.8% for FD, which agrees well with data from other countries for sensitization prevalence rates for nonoccupationally exposed individuals.

Comparison of a multiplexed fluorescent covalent microsphere immunoassay and an enzyme-linked immunosorbent assay for measurement of human immunoglobulin G antibodies to anthrax toxins.

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 microgram/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 micro g of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 microgram/ml, while the dynamic range was 0.06 to 1.7 microgram/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 microgram of anti-PA IgG per ml, the RDL was 0.016 microgram/ml, and the whole-serum equivalent MDC was 1.5 micrograms/ml. The dynamic range was 0.006 to 6.8 microgram/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

Method for simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides.

We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.

Biological monitoring for selected herbicide biomarkers in the urine of exposed custom applicators: application of mixed-effect models.

Metabolites and/or parent compounds of the herbicides atrazine, alachlor, metolachlor, cyanazine and the 2-ethylhexyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D) were measured in the urine of 15 custom applicators who each provided from five to seven 24 h urine samples during a 6 week period (n = 87). Each applicator provided a pre-season urine sample and a reference population (n = 46) provided first-morning urine samples. Urinary biomarkers were measured by either immunoassay or gas chromatography. During the spraying season, the geometric mean amount of alachlor mercapturate equivalents (eq.), atrazine eq., 2,4-D and metolachlor mercapturate eq. excreted in 24 h was 17, 19, 110 and 22 nmol, respectively. Mixed-effect models were used to determine predictors of the amount of atrazine eq. and 2,4-D excreted in 24 h. The specific days of herbicide spraying associated with increased biomarker excretion varied for the two analytes, and included one or more days prior to urine collection. This confirms the importance of collecting covariate information on day(s) most relevant to the biomarker of interest. The within-worker variance component, expressed as a geometric standard deviation ((W)GSD range: 2.5-2.9), was substantially larger than the between-worker component ((B)GSD range: 1.3-1.5) for the modeled biomarkers. Alachlor mercapturate eq. and metolachlor mercapturate eq. were detected in more than half of the applicator pre-season urine samples. All biomarkers were detected infrequently in the reference population. Evaluation of non-spray exposure determinants was limited by inclusion of prior day spraying, adjustment for time and the small sample size.

Variance of Microsomal Protein and Cytochrome P450 2E1 and 3A Forms in Adult Human Liver.

Differences in the pharmacokinetics of xenobiotics among humans makes them differentially susceptible to risk. Differences in enzyme content can mediate pharmacokinetic differences. Microsomal protein is often isolated from liver to characterize enzyme content and activity, but no measures exist to extrapolate these data to the intact liver. Measures were developed from up to 60 samples of adult human liver to characterize the content of microsomal protein and cytochrome P450 (CYP) enzymes. Statistical evaluations are necessary to estimate values far from the mean value. Adult human liver contains 52.9 +/- 1.476 mg microsomal protein per g; 2587 +/- 1.84 pmoles CYP2E1 per g; and 5237 +/- 2.214 pmols CYP3A per g (geometric mean +/- geometric standard deviation). These values are useful for identifying and testing susceptibility as a function of enzyme content when used to extrapolate in vitro rates of chemical metabolism for input to physiologically based pharmacokinetic models which can then be exercised to quantify the effect of variance in enzyme expression on risk-relevant pharmacokinetic outcomes.

Susceptibility to the ototoxic properties of toluene is species specific.

Toluene is the most widely used industrial solvent. It has been shown to be ototoxic in mice and rats, and to increase permanent threshold shift in conjunction with exposure to noise. Chinchillas are widely used for studying noise effects on the cochlea. The present study was initiated to study toluene and noise interaction in chinchillas. Thirty-three chinchillas were exposed to a 95 dBA 500 Hz octave band noise plus 2000 ppm toluene, 8 or 12 h per day for 10 days. Auditory function was estimated using the auditory brainstem response (ABR) to tones between 500 Hz and 16 kHz. There was no effect on the ABR of toluene alone. Noise alone produced a threshold shift. There was no interaction of noise and toluene on the ear. The present study suggests that chinchillas are markedly less susceptible to the ototoxic effect of toluene than mice and rats. A working hypothesis as to the species differences was that chinchilla liver was able to detoxify the toluene. Hepatic microsomes from chinchillas, rats and humans were tested for their ability to convert toluene to the more water-soluble compound - benzyl alcohol. Chinchilla livers were found to contain more of the P450 enzymes CYP2E1 and CYP2B than rats or humans. In addition, the data show that the P450 enzymes are more active in chinchillas than in rats and humans. In conclusion, the results suggest that rats and mice are a more appropriate model for human toluene ototoxicity. However, chinchillas may provide a valuable model for investigating how ototoxic agents can be detoxified to less damaging compounds.