Barry W. Wilson

MAJOR PHYSIOLOGIC AND HISTOCHEMICAL CHARACTERISTICS OF INHERITED DYSTROPHY OF THE CHICKEN

Inherited muscular dystrophy of the chicken was first described more than 20 years ago.’ Since then. several genetic lines have been developed, some have even become extinct, and many studies have been undertaken of the morphologic, physiologic, and biochemical properties of muscles of birds with the disorder. Early research on the abnormality has been reviewed by Julian and Asmundson2 and Asmundson et ~ 1 . ~ This paper discusses some of the major characteristics of the dystrophic chicken that occur in the several lines developed and maintained at the University of California, at Davis, and describes how these properties have been investigated in our laboratory.

Production and release of acetylcholinesterase by cultured chick embryo muscle.

Abstract The acetylcholinesterase (AChE) activity of cultures from 11-day-old chick embryo muscle cells was studied for up to 4 weeks in vitro. AChE activity was found in mononucleated cells and multinucleated myotubes. The activity increased greatly after fusion. Maximum AChE levels were reached after 7–10 days of incubation and tended to decline thereafter. Multiple forms of AChE found in embryo muscle in situ were present in cultures before and after fusion. Selective inhibitors and substrates were used to show that AChE was released by the cells into their medium. Within a 2-day period the AChE that accumulated in the medium averaged over 6 times that remaining in the cells. Release of AChE from the cells was inhibited by cycloheximide, and AChE levels in cells and medium were much reduced when differentiation was inhibited by bromodeoxyuridine. Little AChE was present in subcultures of fibroblasts from muscle cultures. Acetyl-β-methylcholine and, to a lesser degree, choline itself, prevented the decrease in AChE levels of 2- to 3-week-old muscle cultures.

Tissue acetylcholinesterase in plasma of chick embryos and dystrophic chickens

Abstract The plasma cholinesterases of chickens (line 304) with inherited muscular dystrophy and 2 normal lines were examined from 12 days of incubation to 14 weeks after hatching. Acetylcholinesterase (AChE) and non-specific cholinesterase (BChE) activities were distinguished by spectrophotometric, electrophoretic and titrimetric analyses using acetylcholine, butyrylcholine, and their thioester analogs acetylthiocholine and butyrylthiocholine as substrates and 284C51 and iso-OMPA as selective inhibitors. Plasma from normal and dystrophic embryos and from dystrophic chicks had high AChE activity. For example, by 12 weeks of age, 37% of plasma acetylthiocholine hydrolysis was inhibited by the anti-AChE agent 284C51. The results support the view that embryo plasma contains AChE and BChE forms. After hatching the AChE forms decrease greatly in normal plasma and a shift in BChE forms occurs. Later, the AChE forms return in dystrophic line plasma. AChE activity in plasma was circumstantially associated with multiple molecular forms of AChE in the sarcoplasm of embryo and dystrophic muscles and it is likely that these muscles are sources of the plasma AChE activity. The results also confirmed that acetyl-β-methylcholine, unlike the situation which exists in mammals, is hydrolyzed by both AChE and BChE forms in the chicken, and cannot be used to distinguish these cholinesterases.

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.

Factors in standardizing automated cholinesterase assays.

A scientific panel assembled by the U.S. Environmental Protection Agency (EPA) determined that variability in cholinesterase (ChE) activities in the agencys pesticide/animal study database likely was due to a lack of accepted guidelines for ChE methodology. A series of trials was held in which participating laboratories measured ChE activity in blood and brain samples from untreated and pesticide-treated rats using a colorimetric assay method. The degree of inhibition of ChE activity in plasma and brain samples compared to controls was consistent among most of the laboratories. The ChE activity in erythrocyte samples differed more between laboratories due to a high blank, low erythrocyte AChE activity and hemoglobin absorption at the wavelength of the assay. Strategies are suggested for minimizing the variability of ChE activity in hemoglobin-rich samples.

Neuropathology of organophosphate-induced delayed neuropathy (OPIDN) in young chicks.

To examine the phenomenon of apparent age resistance of young chicks to organophosphate-induced delayed neuropathy (OPIDN), groups of either 2- or 10-week-old chicks were exposed subcutaneously daily for 4 days to the neuropathic organophosphate (OP), di-isopropylfluorophosphate (DFP, 1 mg/kg), the non-neuropathic OP, paraoxon (PO, 0.25 mg/kg) or atropine (20 mg/kg). Subsequently, all birds were examined at post-exposure intervals (calculated from the last day of exposure) for up to 56 days for neurological deficits and morphological lesions in the central and peripheral nervous systems (CNS, PNS). Clinically, none of the birds in the 2-week-old groups, or in the 10-week-old PO or atropine exposed groups had neurological deficits. However, all birds in the 10-week-old DFP exposed group developed ataxia by 7 days post-exposure (DPE) and then progressive paralysis. Therefore, all birds in the 10-week-old groups were killed at 14 DPE. Pathologically, the 2-week-old DFP exposed chicks had increasingly severe lesions of Wallerian-like degeneration predominantly in the spinal cord from 7 DPE and subsequently. In the 10-week-old DFP exposed chicks, the degenerative lesions of OPIDN were first detected in the CNS at 3 DPE and then with equally increasing severity in the CNS and PNS up to 14 DPE. A higher incidence of neuronal necrosis and chromatolysis in ventral motor horn neurons of spinal cord grey matter and in dorsal root ganglia occurred in both the DFP exposed age groups compared with those lesions in other groups. These results demonstrate that after neuropathic DFP exposure, 2-week-old chicks develop pathological lesions in the spinal cord without neurological deficits. In both age groups, onset of degenerative lesions in the spinal cord preceeded those in the PNS. The claim of apparent age resistance of chicks to OPIDN needs to be re-evaluated.

Assessment of azinphosmethyl exposure in California peach harvest workers

We compared measurements of urinary alkylphosphate metabolites and oxime-induced reactivation of plasma cholinesterase (P-ChE) and erythrocyte acetylcholinesterase (RBC-AChE) with measurements of foliar residues, skin and clothing contamination, and P-ChE and RBC-AChE activities among 20 Northern California peach orchard workers exposed to the organophosphate agent azinphosmethyl (Guthion). Subjects entered orchards treated 30 d previously with azinphosmethyl and worked 21 d in treated fields during the ensuing 6 wk. Dislodgeable foliar residues ranged from 0.32-0.96 micrograms/cm2. Median reduction in RBC-AChE activity was 7% (p < .001) over the initial 3-d period of exposure and 19% (p < .01) over the 6-wk season. Urinary metabolites were the most sensitive indicator of recent exposure and correlated moderately with dermal and clothing levels (rs = +0.31-(+)0.55); urinary metabolites correlated well with RBC-AChE drawn 3 d after exposure began (rs = -0.77). No significant oxime-induced reactivation was found.

Blood esterase determinations as markers of exposure.

The bases of using blood enzyme activity measurements [e.g. AChE, non-specific cholinesterase (BChE), carboxylesterase] as markers of organophosphate ester (OP) exposure are inhibition of activity by the binding of OPs to serine active sites in the enzymes, and the accessibility of the enzymes in RBCs and serum. The methods used to determine esterases in the blood of humans, experimental animals, and wildlife are outlined with emphasis on the acetylcholinesterase (AChE) of the red blood cell. Adaptations of an acetylthiocholine ester assay of Ellman et al. (1961) are common, but other colorimetric procedures, radiometric assays, and pH methods are also in use. Optimized, standardized methods are needed to assess exposures and provide a solid basis for risk assessment analyses. Useful adjuncts to ChE measurements are oxime reactivation tests and assay of neuropathy target esterase, an enzyme associated with organophosphate-induced delayed neuropathy. Determination of urinary metabolites compliments, but does not substitute for, the information obtained from blood ChE studies. Future assays are likely to involve antibodies to OP-protein complexes. Improvements in techniques permit the detection of small decreases in ChE activities. Whether or not such small decreases in ChE activities can, by themselves, constitute an adverse effect for input into risk assessment analyses is a controversial matter.

Cholinesterase Activity and Inherited Muscular Dystrophy of the Chicken

Summary The cholinesterase activities of pectoral, biceps, and adductor muscles from selected lines of normal and genetically dystrophic chickens were investigated using spectrophotometric assays of acetylthiocholine and butyrylthiocholine hydrolysis and acryl-amide disk gel electrophoresis. It was found that predominately white-fibered muscles such as the pectoral or biceps of dystrophic chickens has 30-40 times the acetylcholinesterase activity of their normal counterparts when the data were expressed on a per protein basis. In contrast, the cholinesterase activity of the predominately red-fibered adductor muscle was little different in samples from dystrophic and normal chickens. Acryl-amide gel electrophoresis revealed that the high acetylcholinesterase activity of dystrophic pectoral and biceps muscles was associated with isoenzyme bands of cholinesterase activity characteristic of embryonic skeletal muscle. In the normal these bands disappeared from the gels before the chicks were 2 weeks of age; in dystrophic lines many of the embryonic isoenzyme bands were retained until the chickens were at least 1 year of age. The possible significance of these findings with respect to the mechanism of action of the gene causing muscular dystrophy of the chicken was discussed.

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 soils 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.