Title: Butyrylcholinesterase genotype and enzyme activity in relation to Gulf War illness: preliminary evidence of gene-exposure interaction from a case-control study of 1991 Gulf War veterans Steele L, Lockridge O, Gerkovich MM, Cook MR, Sastre A Ref: Environ Health, 14:4, 2015 : PubMed
BACKGROUND: Epidemiologic studies have implicated wartime exposures to acetylcholinesterase (AChE)-inhibiting chemicals as etiologic factors in Gulf War illness (GWI), the multisymptom condition linked to military service in the 1991 Gulf War. It is unclear, however, why some veterans developed GWI while others with similar exposures did not. Genetic variants of the enzyme butyrylcholinesterase (BChE) differ in their capacity for metabolizing AChE-inhibiting chemicals, and may confer differences in biological responses to these compounds. The current study assessed BChE enzyme activity and BChE genotype in 1991 Gulf War veterans to evaluate possible association of this enzyme with GWI. METHODS: This case-control study evaluated a population-based sample of 304 Gulf War veterans (144 GWI cases, meeting Kansas GWI criteria, and 160 controls). BChE enzyme activity levels and genotype were compared, overall, in GWI cases and controls. Potential differences in risk associated with cholinergic-related exposures in theater were explored using stratified analyses to compare associations between GWI and exposures in BChE genetic and enzyme activity subgroups. RESULTS: Overall, GWI cases and controls did not differ by mean BChE enzyme activity level or by BChE genotype. However, for the subgroup of Gulf War veterans with less common, generally less active, BChE genotypes (K/K, U/AK, U/A, A/F, AK/F), the association of wartime use of pyridostigmine bromide (PB) with GWI (OR = 40.00, p = 0.0005) was significantly greater than for veterans with the more common U/U and U/K genotypes (OR = 2.68, p = 0.0001). CONCLUSIONS: Study results provide preliminary evidence that military personnel with certain BChE genotypes who used PB during the 1991 Gulf War may have been at particularly high risk for developing GWI. Genetic differences in response to wartime exposures are potentially important factors in GWI etiology and should be further evaluated in conjunction with exposure effects.
Title: Complex factors in the etiology of Gulf War illness: wartime exposures and risk factors in veteran subgroups Steele L, Sastre A, Gerkovich MM, Cook MR Ref: Environmental Health Perspectives, 120:112, 2012 : PubMed
BACKGROUND: At least one-fourth of U.S. veterans who served in the 1990-1991 Gulf War are affected by the chronic symptomatic illness known as Gulf War illness (GWI). Clear determination of the causes of GWI has been hindered by many factors, including limitations in how epidemiologic studies have assessed the impact of the complex deployment environment on veterans' health. OBJECTIVE: We sought to address GWI etiologic questions by evaluating the association of symptomatic illness with characteristics of veterans' deployment. METHODS: We compared veteran-reported wartime experiences in a population-based sample of 304 Gulf War veterans: 144 cases who met preestablished criteria for GWI and 160 controls. Veteran subgroups and confounding among deployment variables were considered in the analyses. RESULTS: Deployment experiences and the prevalence of GWI differed significantly by veterans' location in theater. Among personnel who were in Iraq or Kuwait, where all battles took place, GWI was most strongly associated with using pyridostigmine bromide pills [odds ratio (OR) = 3.5; 95% confidence interval (CI): 1.7, 7.4] and being within 1 mile of an exploding SCUD missile (OR = 3.1; 95% CI: 1.5, 6.1). For veterans who remained in support areas, GWI was significantly associated only with personal pesticide use, with increased prevalence (OR = 12.7; 95% CI: 2.6, 61.5) in the relatively small subgroup that wore pesticide-treated uniforms, nearly all of whom also used skin pesticides. Combat service was not significantly associated with GWI. CONCLUSIONS: Findings support a role for a limited number of wartime exposures in the etiology of GWI, which differed in importance with the deployment milieu in which veterans served.
Title: Naturally occurring mutation, Asp70his, in human butyrylcholinesterase Boeck AT, Fry DL, Sastre A, Lockridge O Ref: Annals of Clinical Biochemistry, 39:154, 2002 : PubMed
BACKGROUND: People with genetic variants of butyrylcholinesterase can have hours of prolonged apnoea after a normal dose of succinylcholine or mivacurium. METHODS: Serum samples from 308 persons living in mid-USA were phenotyped to identify the atypical and fluoride variants. 308 samples were analysed for the K variant by DNA amplification, digestion with Mae III and gel electrophoresis. Amplified DNA from 16 samples was sequenced to identify the D70G, T243M and D70H mutations. Values for kcat and Km were determined for the D70H mutant BChE expressed in 293T cells. RESULTS: A new mutation, Asp70His, was identified. This mutation is located in the peripheral anionic site of butyrylcholinesterase, where it causes a 10-fold decrease in binding affinity for positively charged substrates. CONCLUSION: People homozygous for the Asp70His mutation are expected to have prolonged apnoea in response to succinylcholine or mivacurium, similar to people with the Asp70Gly mutation.
Title: Physiological and performance effects of pyridostigmine bromide in healthy volunteers: a dose-response study Cook MR, Graham C, Sastre A, Gerkovich MM Ref: Psychopharmacology (Berl), 162:186, 2002 : PubMed
RATIONALE: Questions have been raised about the role pyridostigmine bromide (PB) plays in the etiology of Gulf War veterans' illnesses. There is a need to understand better the physiological and behavioral effects of this drug, particularly at the 30-mg/8-h regimen recommended by the US Military. OBJECTIVE. To perform a double-blind, cross-over, dose-response study of PB in 67 healthy, young volunteers (31 women, 36 men). METHODS: Volunteers were initially trained on a standardized test battery. Supervised administration of placebo (PL) and PB (every 8 h/5 days) occurred in each of two dosing weeks, separated by a non-dosing week. One group received 30 mg PB and PL, and the other 60 mg PB and PL. In each dosing week, the battery was performed after the first pill and again when steady-state plasma PB levels were achieved. RESULTS: PB was associated with an overall improvement in reaction time on tests of memory and attention, and with a reduction in RMS error on a tracking task. PB slowed heart rate and decreased the high frequency component of heart rate variability (HF HRV). Dose-response effects were found only for HF HRV, and RMS error. The extent of cholinesterase inhibition was directly related to the magnitude of the HF HRV decrease, and was predicted by the weight-normalized PB dose. Cholinesterase inhibition was not related to the extent or severity of reported drug side effects. CONCLUSIONS: PB does not appear to have detrimental physiological or performance consequences at the recommended 30-mg dose, or at twice that dose, when evaluated under non-stressful laboratory conditions.
Title: Side effects of low-dose pyridostigmine bromide are not related to cholinesterase inhibition Cook MR, Gerkovich MM, Sastre A, Graham C Ref: Aviat Space Environ Med, 72:1102, 2001 : PubMed
BACKGROUND Pretreatment with pyridostigmine bromide PB has become part of standard military procedures for protection against the effects of possible chemical warfare attack The purpose of the work reported here was to quantify the type intensity and frequency of side effects of low-dose PB and to examine factors that predict the intensity and frequency of side effects METHOD A double-blind cross-over placebo PL)-controlled design was used Of the 67 subjects 33 received 30 mg PB every 8 h for 13 doses and 34 received 60 mg on the same schedule Order of PB and PL administration was counterbalanced RESULTS Overall side effects were mild even at the 60-mg dose level More side effects were reported when volunteers were taking PB than when they were taking placebo Women reported more symptoms than men Neither cholinesterase inhibition nor plasma levels of PB predicted side effect scores during the PB week the best predictor of side effect scores during the PB week was side effect scores during the PL week CONCLUSION PB is well tolerated by healthy young people even when twice the recommended military dose is administered
Title: Cholinergic receptors and contraction of smooth muscle in canine portal vein Milnor WR, Sastre A Ref: Journal of Pharmacology & Experimental Therapeutics, 245:244, 1988 : PubMed
The properties of cholinergic receptors in homogenates of canine portal vein were determined with the radioligand [3H]quinuclinidyl benzilate [( 3H]QNB), and correlated with the functional responses of that vessel to carbamylcholine (CCh) in vitro. [3H]QNB bound to a single population of sites in the homogenates, with a dissociation constant (Kd) of 125 pM (+/- 19), and a total receptor capacity of 24.4 fmol/mg of protein, which corresponded to 224 (+/- 67.3) fmol/g wet weight of the initial vascular wall sample. Competition of CCh with [3H]QNB in eight experiments revealed two binding sites of different affinity, present in about equal numbers with Kd = 0.48 microM and 31 microM (in the absence of 5'-guanylylimidodiphosphate), and Kd = 1.5 microM and 42 microM (in the presence of 100 microM 5'-guanylylimidodiphosphate). Vascular strips in vitro contracted in response to CCh, with a threshold of approximately 0.3 microM, ED50 of 1.87 microM and maximum response of 344 g/cm2 (65% of the maximum response to l-norepinephrine). Strips precontracted by l-norepinephrine or by high potassium concentrations were slightly (4%) relaxed by 0.1 microM CCh, but further contracted by higher concentrations. The contractile responses were not altered by removal of the endothelium, and were blocked by atropine but not hexamethonium. The results demonstrate the existence in this vein of muscarinic cholinergic receptors that mediate smooth muscle contraction, and the receptor properties resemble those reported in cardiac muscle and brain.
Title: Obligatory role of a Tris/choline allosteric site in guanine nucleotide regulation of [3H]-L-QNB binding to muscarinic acetylcholine receptors Murphy KM, Sastre A Ref: Biochemical & Biophysical Research Communications, 113:280, 1983 : PubMed
Tris and choline reduce the maximal binding capacity (RT) of the muscarinic cholinergic antagonist [3H]-L-quinuclidinyl benzilate ([3H]-L-QNB) to atrial membranes, when compared to control values in physiological salt solution (PBS) or NaPi buffer. Addition of guanine nucleotides (GN) to incubations containing choline or Tris reverses the effect of choline and Tris on RT and restores it to levels determined in NaPi or PBS alone. GN addition fails to alter RT or KD values determined in NaPi or PBS in the absence of choline and Tris. This GN effect follows a nucleotide specificity similar to that of the GN regulatory proteins coupled to adenylate cyclase. Tris or choline are required for the expression of GN regulation of [3H]-L-QNB binding to muscarinic acetylcholine receptors (mAChR). An allosteric site recognizing choline and Tris appears involved in the interaction between the guanine nucleotide regulatory protein and antagonist binding to mAChR.
Title: Myocardial muscarinic acetylcholine receptor: choline and tris unmask heterogeneity of antagonist binding sites Sastre A, Murphy KM, Rusher MM Ref: Biochemical & Biophysical Research Communications, 104:383, 1982 : PubMed
Title: Cholinergic and adrenergic receptors on mouse cardiocytes in vitro Lane MA, Sastre A, Law M, Salpeter MM Ref: Developmental Biology, 57:254, 1977 : PubMed
Title: Innervation of heart cells in culture by an endogenous source of cholinergic neurons Lane MA, Sastre A, Salpeter MM Ref: Proc Natl Acad Sci U S A, 73:4506, 1976 : PubMed
Hearts of embryonic mice 9 days in utero were found to have an endogenous source of cholinergic neurons which can survive in dispersed cell cultures. These neurons are electrically excitable, have ultrastructural characteristics of cholinergic embryonic neurons, and functionally innervate heart cells in culture. The nature of the innervation described is muscarinic cholinergic.
