Carbamate protection of AChE against inhibition by agricultural chemicals.

The carbamate pyridostigmine bromide has been used as a pretreatment to protect individuals from the nerve agent soman. Previous research showed that pyridostigmine significantly protected human muscle acetylcholinesterase in vitro from soman and bovine red blood cell acetylcholinesterase from some organophosphorous pesticides. Research presented here demonstrates that pretreatment with other carbamates also protects acetylcholinesterase from inhibition by the pesticides chlorpyrifos-oxon and diazinon-oxon, but not from malaoxon.

Chronic hydrocarbon exposure of harlequin ducks in areas affected by the Selendang Ayu oil spill at Unalaska Island, Alaska.

We evaluated chronic exposure of harlequin ducks (Histrionicus histrionicus) to hydrocarbons associated with the 2004 M/V Selendang Ayu oil spill at Unalaska Island, Alaska. We measured levels of hepatic 7-ethoxyresorufin-O-deethylase activity (EROD) in liver biopsy samples as an indicator of hydrocarbon exposure in three oiled bays and one reference bay in 2005, 2006, and 2008. Median EROD activity in ducks from oiled bays was significantly higher than in the reference bay in seven of nine pairwise comparisons. These results indicated that harlequin ducks were exposed to lingering hydrocarbons more than three years after the spill.

Pyridostigmine bromide protection against acetylcholinesterase inhibition by pesticides.

Pyridostigmine bromide (PB) has been used to protect soldiers from the toxic effects of soman, a chemical warfare agent. Recent research shows that pyridostigmine bromide protects a significant percentage of acetylcholinesterase in isolated human intercostal muscle. Findings presented here indicate that red blood cell acetylcholinesterase is similarly protected by pyridostigmine bromide from the action of diisopropyl fluorophosphate and several organophosphate pesticides including chlorpyrifos-oxon, diazinon-oxon, and paraoxon, but not malaoxon, using the bovine red blood cell as a subject. These findings suggest that pretreatment with PB may protect growers, farmworkers, first responders, and the public, in general, from the effects of selected pesticides.

Protection of human muscle acetylcholinesterase from soman by pyridostigmine bromide.

INTRODUCTION: Pretreatment with pyridostigmine bromide (PB) of human intercostal muscle fibers exposed to the irreversible acetylcholinesterase (AChE) inhibitor soman was investigated. METHODS: Muscles were pretreated with 3 × 10(-6) M PB or saline for 20 minutes, then exposed to 10(-7) M soman for 10 minutes. RESULTS: AChE of muscles treated with soman alone was inhibited >95%. In contrast, PB pretreatment of soman-exposed bundles protected 20% of AChE activity. AChE of bundles exposed to PB alone recovered after 4 hours, but bundles exposed to both PB and soman did not. Soman-induced reduction of resting membrane potentials and increment of amplitudes and decay times of miniature endplate potentials (MEPPs) were partially corrected by PB pretreatment. CONCLUSIONS: In vitro pretreatment of human muscles with PB protected up to 20% of muscle AChE and ameliorated some deleterious effects on endplate physiology induced by soman.

Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil.

We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow's goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (≥ 2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow's goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.

Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill.

Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed.

Selenium accumulation in coturnix quail fed seleno-L-methionine.

The accumulation of selenium in Coturnix quail, a sensitive species to selenium toxicity, was studied. Quail were fed diets containing 0.7 µg/g (control), 12 µg/g (low-Se), and 22 µg/g (high-Se) as seleno-L-methionine for four weeks. At the end of the four weeks, blood selenium in the treatment groups had a positive significant relationship to diet, and selenium in blood was approximately two times, and in livers was approximately three times, the dietary concentration. Quail in the low-Se group accumulated selenium to higher levels over the same time period than American kestrels and mallards fed selenomethionine at similar concentrations in other studies. Food consumption was similar among the groups throughout the study. Body mass, as a percent of initial body mass, was similar in the control and low-Se groups (102%) but lower in the high-Se group (97%). Liver mass, as a percent of initial body mass, showed a positive dose-dependent response to dietary selenium, and liver mass was significantly higher in quail fed 22 µg Se/g than in control quail. Unlike other species studied, accumulation of selenium in blood did not plateau but continued to increase until the study ended.

Normal range of human red blood cell acetylcholinesterase activity.

The normal range of human erythrocyte acetylcholinesterase (RBC-AChE) activity is important when monitoring exposure to pesticides and chemical warfare agents. A modification of Michel's method measured RBC-AChE activities from 991 individuals (818 males and 173 females) presumably unexposed to nerve agents. Median age was 42 (range, 18-76) years. RBC-AChE (mean +/- SD) was 0.74 +/- 0.06 delta pH units/hour. Multivariate linear regression showed an association with age (slope +0.0008 delta pH units/hour for each year; P < 0.001) unlikely to be clinically significant. The findings represent the largest study of human RBC-AChE to date providing measures of central tendency and variation.

Blood cholinesterases from Washington State orchard workers.

Court-ordered monitoring of blood cholinesterases (ChEs) from orchard workers in Washington State is underway. In 2008, the mean red blood cell acetylcholinesterase (AChE, EC 3.1.1.7) activity was 9.65 +/- 1.11 micromoles/min/ml (n = 1,793) and the mean serum (BChE, 3.1.1.6) activity was 5.19 +/- 0.90 micromoles/min/ml (n = 1,811). Determinations were made using the Ellman assay and automated equipment of Pathology Associates Medical Laboratories (PAML), Spokane, Washington.

Polycyclic aromatic hydrocarbon exposure in Steller's eiders (Polysticta stelleri) and Harlequin ducks (Histronicus histronicus) in the eastern Aleutian Islands, Alaska, USA.

Seaducks may be affected by harmful levels of polycyclic aromatic hydrocarbons (PAHs) at seaports near the Arctic. As an indicator of exposure to PAHs, we measured hepatic enzyme 7-ethoxyresorufin-O-deethylase activity (EROD) to determine cytochrome P4501A induction in Steller's eiders (Polysticta stelleri) and Harlequin ducks (Histronicus histronicus) from Unalaska, Popof, and Unga Islands (AK, USA) in 2002 and 2003. We measured PAHs and organic contaminants in seaduck prey samples and polychlorinated biphenyl congeners in seaduck blood plasma to determine any relationship to EROD. Using Akaike's information criterion, species and site differences best explained EROD patterns: Activity was higher in Harlequin ducks than in Steller's eiders and higher at industrial than at nonindustrial sites. Site-specific concentrations of PAHs in blue mussels ([Mytilus trossilus] seaduck prey; PAH concentrations higher at Dutch Harbor, Unalaska, than at other sites) also was important in defining EROD patterns. Organochlorine compounds rarely were detected in prey samples. No relationship was found between polychlorinated biphenyl congeners in avian blood and EROD, which further supported inferences derived from Akaike's information criterion. Congeners were highest in seaducks from a nonindustrial or reference site, contrary to PAH patterns. To assist in interpreting the field study, 15 captive Steller's eiders were dosed with a PAH known to induce cytochrome P4501A. Dosed, captive Steller's eiders had definitive induction, but results indicated that wild Steller's eiders were exposed to PAHs or other inducing compounds at levels greater than those used in laboratory studies. Concentrations of PAHs in blue mussels at or near Dutch Harbor (approximately 1,180-5,980 ng/g) approached those found at highly contaminated sites (approximately 4,100-7,500 ng/g).

Determination of cholinesterase in blood and tissue.

The widespread use of organophosphate and organocarbamate pesticides and the dangers of related chemical warfare agents dramatize the importance of rapid, accurate, and sensitive assays for blood and tissue cholinesterases (ChEs), important targets for neurotoxic chemicals. Two ChE enzymes used as biomarkers of exposure are the specific acetylcholinesterases (AChE, EC 3.1.1.7) and the nonspecific plasma cholinesterases (BChE, EC 3.1.1.8). This unit contains two protocols for measuring ChE activity: (1) a colorimetric kinetic method and (2) a radiometric endpoint assay and selective inhibitors that are used to distinguish between the two classes of enzymes.

Monitoring cholinesterases to detect pesticide exposure.

Progress toward a standard blood cholinesterase assay to assess pesticide exposures in the agricultural workplace and to identify possible victims of chemical warfare agents is discussed. Examples given are drawn from collaborations with clinical laboratories in California and the Department of Defense Cholinesterase Reference Laboratory (CRL).

Cholinesterases in aquatic biomonitoring: assay optimization and species-specific characterization for a California native fish.

Cholinesterase (ChE) enzyme activity measurements are widely applied in aquatic organisms for water quality monitoring, especially for pesticide contamination in agricultural watersheds. These biomarkers are amenable to measurement in a variety of species, and are therefore useful for examining effects in model organisms relevant to the ecosystem of interest. However, extensive variation in ChE biochemistry exists among tissues and species. This variation is rarely characterized and may lead to biases in the interpretation of activity determinations. We optimized ChE activity measurement parameters and characterized ChE biochemistry in Sacramento sucker (Catostomus occidentalis), a widely distributed fish native to watersheds of the Central Valley of California. Acetylcholinesterase (AChE) was the predominant ChE present in C. occidentalis brain and muscle, and muscle AChE was most sensitive to diazinon inhibition. Field caging experiments indicated that exposures to ChE-inhibiting pesticides were insufficient to induce neurotoxic effects. However, pesticide usage in the Central Valley is highly variable among years, and long-term monitoring of in-stream effects would be necessary to evaluate trends in pesticide contamination. Recent changes to the State Water Code require agricultural landowners to participate in a regional water quality monitoring plan. As with most regional monitoring plans, measurements of in-stream effects, and effects in resident species, are not scheduled to be included. We suggest that inclusion of biomarker measures would lend important information to the monitoring process, and propose these procedures as a template for adapting ChE activity measurements into region-specific monitoring programs to assess in-stream effects of pesticide contamination on native species.

Influence of water temperature on acetylcholinesterase activity in the Pacific tree frog (Hyla regilla).

This investigation evaluated whether acetylcholinesterase (AChE) in Pacific tree frogs (Hyla regilla) from different geographical locations was influenced by different temperatures during early aquatic life stages, independent of pesticide exposure. Tadpoles were collected from both a California coastal pond and a Sierra Nevada mountain range pond, USA. Groups of frogs from each location were raised in temperatures representative of either the Sierra Nevada (8 degrees C) or the coastal (19 degrees C) location. Metamorphs from both locations raised as tadpoles at 19 degrees C had AChE activities of 42.3 and 38.7 nm/min/mg protein, while those raised as tadpoles at 8 degrees C had activities of 26.9 and 28.2 nm/min/mg protein. A two-way analysis of variance revealed temperature to be the significant factor in determining AChE activity (F = 22.3, p < 0.001), although origin was not important (F = 0.09, p = 0.75). Interpretations regarding the influence of pesticides upon AChE activity in Pacific tree frogs must consider the influence of environmental temperature to enable cross-population comparisons.

Toxicity of storm-water runoff after dormant spray application in a french prune orchard, Glenn County, California, USA: temporal patterns and the effect of ground covers.

Organophosphorous (OP) insecticides, especially diazinon, have been detected routinely in surface waters of the Sacramento and San Joaquin River watersheds, coincident with rainfall events following their application to dormant orchards during the winter months. Preventive best management practices (BMP) aim at reducing off-site movement of pesticides into surface waters. Two proposed BMPs are: The use of more hydrophobic pyrethroid insecticides believed to adsorb strongly to organic matter and soil and the use of various types of ground cover vegetation to increase the soil's capacity for water infiltration. To measure the effectiveness of these BMPs, storm water runoff was collected in a California prune orchard (Glenn County, CA, USA) during several rainstorms in the winter of 2001, after the organophosphate diazinon and the pyrethroid esfenvalerate were applied to different orchard sections. We tested and compared acute toxicity of orchard runoff from diazinon- and esfenvalerate-sprayed sections to two species of fish (Pimephales promelas, Onchorhynchus mykiss) and three aquatic invertebrates (Ceriodaphnia dubia, Simocephalus vetelus, Chironomus riparius), and determined the mitigating effect of three ground cover crops on toxicity and insecticide loading in diazinon-sprayed orchard rows. Runoff from the esfenvalerate-sprayed orchard section was less toxic to waterflea than runoff from the diazinon-sprayed section. However, runoff from the orchard section sprayed with esfenvalerate was highly toxic to fish larvae. Samples collected from both sections one month later were not toxic to fish, but remained highly toxic to invertebrates. The ground cover crops reduced total pesticide loading in runoff by approximately 50%. No differences were found between the types of vegetation used as ground covers.

Molecular and cellular biomarker responses to pesticide exposure in juvenile chinook salmon (Oncorhynchus tshawytscha).

We determined the effects of two pesticides, chlorpyrifos and esfenvalerate in juvenile Chinook salmon. Four to five month old salmon were exposed to a range of pesticide concentrations, and tissue samples of surviving fish were analyzed for stress protein expression, cytokine transcription, and acetylcholinesterase (AChE) activity. At the highest concentrations, both pesticides led to complete mortality, whereas medium and low concentrations resulted in high survival rates. Significant differences in stress protein expression, cytokine transcription and AChE activity were found between control and surviving chlorpyrifos-exposed fish. Elevated stress protein expression was the only detectable response to esfenvalerate.

Meeting requirements of the California cholinesterase monitoring program.

California (CA) has a long-standing formal blood cholinesterase (ChE) monitoring program for mixers, loaders, and applicators of pesticides. When the authors found commercial clinical kits were not optimal for assaying blood ChEs, CA regulations were revised to specify use of the Ellman ChE assay or to demonstrate a conversion factor with a correlation (r(2)) of 0.9 or better. The authors were enlisted to work with the clinical laboratories. Only two of seven participating laboratories generated an acceptable correlation for red blood cells (RBCs), whereas four of five laboratories had an acceptable correlation for plasma ChE. Subsequently, the CA Department of Pesticide Regulation (DPR) restated the need to meet this requirement and the authors worked with several of the clinical laboratories using a bovine ghost RBC ChE as a reference. Unfortunately, only 3 of 10 laboratories had acceptable correlations. Next, the authors provided all interested laboratories with human blood and plasma samples to perform the comparison study outlined in the regulation (Section 6728f). Fourteen laboratories participated; 9 met the ChE criteria for whole blood, 14 for plasma, and 6 for RBCs. Based on such data, on July 8, 2003, DPR notified the CA Agricultural Commissioners that nine of the participating laboratories were approved for ChE testing. Later work resulted in acceptable RBC values for two of the laboratories and their approval. The authors continue to work with laboratories interested in being on the approved list. The current list may be seen at www.cdpr.ca.gov/docs/whs/lablist.htm.

Standardization of clinical cholinesterase measurements.

Previous studies showed that commonly used kits for measuring cholinesterases were not optimal for determining acetylcholinesterase (AChE) activity. Clinical use of different kits and methodologies resulted in AChE levels being reported in different units and activities that were not reproducible among laboratories. Findings such as these led to a revision in California regulations (covering AChE measurements for pesticide worker safety) calling for clinical laboratories to standardize their findings. The laboratories were contacted and invited to participate in a split-sample study of human blood AChE and nonspecific cholinesterase (BChE) assays. Participating laboratories measured erythrocyte (RBC) AChE and/or plasma BChE from undiluted and 50% diluted blood, according to their practices. Aliquots of blood samples were shipped to University of California Davis for measurement, using an optimized semiautomated plate reader version of the method of Ellman. Nine of 25 laboratories sent samples for comparison. Two others performed their own comparisons and submitted data to the state. Best correlations were obtained with BChE activity. Correlations (r(2)) were .88 or above for four of five laboratories for BChE, and above .9 for two of seven laboratories for AChE. Reasons for poor correlations may include difficulties in pipetting RBCs, storage, and processing. A bovine AChE RBC ghost "standard" was devised and tested. Activity of the preparation was maintained at -70 degrees C for approximately 11 months. A test with an East coast laboratory resulted in a high correlation, demonstrating the reliability of the RBC ghost standard and that one laboratory can replicate the AChE findings of another. The overall poor correlation of interlaboratory cholinesterase results points to the need to further standardize sample handling and assay methods.

Cholinesterase measurements with an automated kit.

BACKGROUND: The Test-mate kit determines acetylcholinesterase (AChE, EC 3.1.1.7) and hemoglobin content of a drop of blood, displaying enzyme activities normalized to 25 degrees C. Previous models produced inconsistent results at different temperatures. This report focuses on the current model, ChE 400, and two instruments of a previous OP model. METHODS: AChE activities were determined by the Ellman assay, using the three kits and a 96-well microplate reader. Temperatures ranged from 10 to 37 degrees C. Fetal bovine serum was the source of AChE. RESULTS: Normalized activities decreased below 20 degrees C in the ChE model and below 25 degrees C in the OP models. Activities of the same serum sample differed between the three Test-mate kits, ranging from 1.03 to 1.49 micromoles/min/ml. Percent errors were greater than with the microplate reader at all temperatures. CONCLUSIONS: Neither we nor the manufacturer recommend the current Test-mate model for fieldwork. Nevertheless, there have been field measurements with Test-Mate kits, and we recommend that an enzyme activity standard be run in parallel with their use.

Actions of pyridostigmine and organophosphate agents on chick cells, mice, and chickens.

Gulf War veterans were given pyridostigmine bromide (PB) tablets to enhance the therapeutic effect of antidotes to nerve agents in the event of exposure. The goal of this research is to examine whether combined exposure to PB and sarin (agent GB) is more neurotoxic to sensitive surrogate animals, mice and chickens, than if given separately. Scoping trials were performed to establish appropriate dose-response ranges for sarin and control chemicals. IC50 values were determined in chickens and mice for in vitro inhibition of acetylcholinesterase (AChE) and neuropathy target esterase (NTE). The results indicated PB neither inhibits NTE nor does it spare sarin's inhibition of AChE. Chick embryo nerve cells in vitro showed more inhibition of AChE activity and no faster recovery when PB treatment was followed by DFP treatment than the other way around. Experiments on chickens also indicated that PB treatment did not inhibit NTE and that it crossed the blood brain barrier inhibiting brain AChE although to a lesser extent than it inhibited blood cholinesterases. Other experiments determined multiple dose levels in chickens for sarin and DFP that inhibited > 80% of NTE, considered a threshold for triggering organophosphate-induced delayed neuropathy.