Frank Welsch

Brain cholinergic, behavioral, and morphological development in rats exposed in utero to methylparathion☆

The purpose of this study was to determine the effects of subchronic administration of the organophosphate methylparathion (MPTH) during gestation on behavior and development of brain cholinergic neurons in the offspring. Pregnant rats received daily po doses of MPTH from Day 6 through Day 20 of gestation at doses causing no (1.0 mg/kg) or minimal (1.5 mg/kg) visible signs of maternal toxicity. Acetylcholinesterase (AChE) and choline acetyltransferase (CAT) activities, and [3H]quinuclidinyl benzilate (QNB) binding to muscarinic receptors, were determined in several brain regions at 1, 7, 14, 21, and 28 days postnatal age and in maternal brain at Day 19 of gestation. Prenatal exposure to 1.5 mg MPTH/kg reduced AChE and increased CAT activity in all brain regions at each developmental period and in maternal brain. Similar exposure to 1.0 mg MPTH/kg caused a significant but smaller and less persistent reduction in AChE activity but no change in brain CAT activity of the offspring. Both doses of MPTH decreased the Bmax of 3H-QNB binding in maternal frontal cortex but did not alter the postnatal pattern of 3H-QNB binding. In parallel studies, prenatal exposure to MPTH did not affect a variety of behaviors. However, cage emergence, accommodated locomotor activity, and operant behavior in a mixed paradigm were impaired in rats exposed to 1.0 but not to 1.5 mg/kg MPTH. No morphological changes were observed in hippocampal or cerebellar tissue. Thus, subchronic prenatal exposure to MPTH altered postnatal development of cholinergic neurons and caused subtle alterations in selected behaviors of the offspring.

Studies on accumulation and metabolic fate of (N-Me3h)choline in human term placenta fragments.

Abstract Fragments from human term placenta accumulated [N-Me3H]choline against a concentration gradient. Uptake was linearly related to incubation time and temperature. Analysis of the kinetics of choline accumulation revealed the concurrent existence of a diffusional component occuring both at low temperature as well as high (50 mM) choline concentration and a carrier-mediated transport which had characteristics predicted by the Michaelis-Menten equation showing a Km = 3.46 × 10−4 M and a Vmax of 75 nmoles/ml intracellular water × min−1. Net concentration ratios, corrected for diffusion and extracellular water, were larger than 1.0 within 5 min and about 4.0 after 15 min. Hemicholinium-3 was a competitive inhibitor of choline uptake with a Ki of 0.45 mM. [3H]Choline accumulation was decreased by conditions known to lower intracellular ATP levels. Thus, 2,4-dimitrophenol (1 mM), sodium cyanide (5 mM) and anaerobic incubation reduced [3H]choline accumulation 36, 54 and 33 per cent respectively. Ouabain (0.1 mM) also decreased the concentration ratios by 50 per cent. Modification of the ionic environment led to an increase of 36 per cent in the amount of tritium in intracellular water when Na+ was reduced to one half of the usual 145 mM or 150 per cent when it was completely omitted and replaced by an osmotically equivalent amount of sucrose. Li+ was without effect, while high K+ (> 25mM), Rb+ and Cs+ (145 mM) depressed [3H]choline accumulation. The metabolic fate of [3H]choline was studied. Following a 5-min incubation with 5 μM [3H]choline 95 per cent of the radioactivity was acid-soluble and 5 per cent remained in the acid-insoluble fraction. After 30 min the distribution was 88 and 12 per cent, respectively. Paper high voltage electrophoretic analysis of the acid-soluble material showed that after 5 min 55 per cent of the 3H had a mobility equal to authentic choline, 35 per cent equal to acetylcholine, 6 per cent to phosphorylcholine and 1 per cent to betaine. After 20 min it was 25 per cent in choline, 60 per cent in acetylcholine, 10 per cent in phosphorylcholine and 2 per cent in betaine. A chloroform-methanol extract from the acid-insoluble residue revealed a linear increase of 3H-content suggesting incorporation of [3H]choline into phospholipids.

Effect of subchronic administration of methyl parathion on in vivo protein synthesis in pregnant rats and their conceptuses

Pregnant rats received daily po doses of the organophosphate methyl parathion (MPTH) from Day 6 through Day 15 or 19 of gestation at doses causing no (1.0 mg/kg) or minimal (1.5 mg/kg) signs of maternal toxicity. Following the dose of MPTH on Day 15 or 19, in vivo protein synthesis was measured 0.5, 1.0, and 2.0 hr after sc injection of L-[1-14C]valine at a dose of 5 microCi/mmol/100 g body wt. The specific activity of [14C]valine in the free amino acid pool and protein bound pool was significantly reduced in various regions of maternal brain and in maternal viscera, placenta, and whole embryos (Day 15), and in fetal brain and viscera (Day 19). The inhibitory effect of MPTH on net protein synthesis was dose dependent, greater on Day 19 than 15 of gestation and more pronounced in fetal than in maternal tissues.

Choline metabolism in human term placenta--studies on de novo synthesis and the effects of some drugs on the metabolic fate of [N-methyl 3H]choline.

Abstract Fragments from human term placentae were incubated with [3H]methyl-Iabeled methionine or S-adenosyl methionine as methyl donors and ethanolamine or phosphatidyldimethylethanolamine as methyl acceptors. Analysis of acid-soluble and lipid-soluble extracts by high voltage electrophoresis or thin-layer Chromatography, respectively, gave no indication for the synthesis of products which would be expected to contain [3H]label in the choline (Ch) moiety. These findings led to the conclusion that the placenta could not perform the methylation steps required for de novo synthesis of choline. Short incubations (1, 2.5, 5 and 10 min) with [3H]Ch (5 μM, 0.8 μCi/ml) and paraoxon (10 μM) as an inhibitor of cholinesterases revealed that [3H]Ch was quite rapidly incorporated into [3HJacetylcholine (ACh) and that 18 per cent of the acid-soluble radioactivity was associated with ACh after 1 min. In the lipid-soluble fraction the major labeled product was identified as phosphatidylcholine while a minor amount of [3H](

Choline acetyltransferase in aneural tissue: Evidence for the presence of the enzyme in the placenta of the guinea pig and other species

Abstract Homogenates from placentas of guinea pig, dog, cat, mouse, horse, and cow were incubated in a medium containing 1- 14 C-acetylcoenzyme A and choline to determine whether they contained the enzyme choline acetyltransferase (ChAc). Human placenta, known as extraordinarily rich in ChAc, and human fetal membranes were examined for comparative purposes. Any 1- 14 C-acetylcholine (ACh) formed was isolated by anion-exchange chromatography, paper chromatography, and/or periodide precipitation and high-voltage paper electrophoresis. Migration rates of the synthesized labeled material were compared to those of authentic ACh. Sensitivtiy to hydrolysis by acetylcholinesterase was used as an additional criterion to establish the identity of the 14 C-labeled product with ACh. The results indicated that placentas of all species examined contained an enzyme capable of synthesizing a 14 C-labeled product which behaved indistinguishably from ACh. However, except for human placenta and human fetal membranes, this metabolite was neither the only or the major 14 C-labeled component. The observations suggested that placental ChAc is not unique to man and higher primates as previously assumed.

Acetylcholine in human placenta

SummaryExtracts from human term placentae were analyzed by pyrolysis gas chromatography (GC) combined with mass spectrometry. The only choline ester of carbonic acid whose presence could be unequivocally established was acetylcholine (ACh). The mass spectrum of its demethylated tertiary derivative dimethylaminoethyl acetate (nor-ACh) agreed entirely with that of authentic pyrolyzed ACh.The concentrations of ACh were determined in placentae obtained after vaginal or Caesarean delivery to investigate, the claim that the latter resulted in tissue with higher concentrations of ACh-like activity presumably because ACh was not expended during labor and delivery. No differences in ACh content were found with GC analysis. The ACh concentrations were 102±17 (n=8) nmoles/g fresh tissue (vaginal delivery) vs. 105±16 (n=6) nmoles/g (Caesarean section). The fetal membranes (amnion and chorion) and the umbilical cord contained no ACh under the analytical conditions with a limit of sensitivity of 200 pmoles ACh.

The cholinergic system in tissues without innervation: Choline acetyltransferase, choline and acetylcholine in the placenta of the rhesus monkey (Macaca mulatta)

Abstract Six near-term placentae from rhesus monkeys ( Macaca mulatta ) were analyzed for their contents of choline acetyltransferase (ChAc), acetylcholine (ACh) and choline (Ch). ChAc was measured by a radiometric assay in the presence of [1- 14 C]acetylcoenzyme A and choline and distinguished from other acetyltransferases by a differential assay involving acetylcholinesterase or selective ion pair extraction of [1− 14 C]ACh with tetraphenylboron. At 150 days of gestation the rhesus placenta synthesized 4.067 ± 0.737 μmoles ACh/g wet weight/hr. Ch and ACh were determined with a radiochemical method based on the phosphorylation of free Ch by choline kinase in the presence of [ 32 P]ATP. ACh was first isolated by ion pair extraction and high voltage electrophoresis. Tissue levels of Ch ranged from 737 to 3892 and ACh from 8.8 to 29.0 (nmoles/g wet weight). The rhesus monkey appears to be a suitable animal model to study the physiological significance of ACh in the placenta in vivo .

Uptake of acetylcholine by human placenta fragments and slices from guinea pig and rat placenta

Abstract Fragments of human term placenta took up [ 3 H]acetylcholine (ACh) into the intracellular water fraction against a concentration gradient when incubated in a medium which contained 10 μM paraoxon as an inhibitor of cholinesterases. Intracellular concentrations rose to levels 3- to 4-fold over the concentration in the medium in a time- and temperature-dependent manner. The ACh uptake was saturable and followed the Michaelis-Menten equation. It required metabolic energy and was markedly reduced by drugs and conditions which decreased cell levels of ATP. Concentrative uptake was inhibited by alterations in the ionic environment as introduced by high K + , Li + , Rb + and Cs + . Slices from guinea pig placenta reached intracellular concentrations which were only equal to the concentration in the incubation medium, suggesting equilibration by diffusion, while rat placenta slices concentrated ACh more slowly than human placenta and reached ratios of 2.0. These species variations further confirm known differences of the cholinergic system in placenta and stress the need for caution in extrapolating findings from the placenta of one species to another.

Ein gelber Farbstoff aus Formaldehyd und Kynurenin bei hexaminbehandelten Ratten

ZusammenfassungKynurenin wurde als ein Bestandteil von Rattenhaaren identifiziert, der mit Formaldehyd allein oder zusammen mit Ammoniak einen Stoff bildet, der für die Gelbfärbung des Felles von Albinoratten verantwortlich ist, die nach oraler oder parenteraler Gabe von Hexamethylentetramin oder von formaldehydhaltigem Futter auftritt. Haare von anderen Tierarten werden bei der Einwirkung von Formaldehyd und Ammoniak nicht gelb.Am lebenden Tier wird die Gelbfärbung verstärkt und beschleunigt, wenn die Aktivität der Tryptophanpyrrolase durch Tryptophan- oder Cortisoninjektionen gesteigert ist, so daß mehr Kynurenin entsteht.Die Gelbfärbung ist zum großen Teil durch Kontamination der Haare mit hexaminhaltigem Harn oder formaldehydhaltigem Futter bedingt. Ob der Farbstoff auch innerhalb des Körpers entsteht, konnte noch nicht geklärt werden. Die chemische Struktur des gelben Farbstoffes ist noch nicht ermittelt.Die Bedeutung der Untersuchungen für die Beurteilung der Duldbarkeit von Hexamin oder Formaldehyd als Konservierungsstoffe in Lebensmitteln wird besprochen.SummaryThe coat of albino rats changes to yellow after the administration of methenamine or formaldehyde by different routes. This effect is identified as caused by a reaction between formaldehyde, ammonia and kynurenine which is a normal constituent of the hair in rats but obviously not in other species.The chemical structure of the yellow compound has not yet been determined although there are some physico-chemical data established.The yellow color develops mainly by contamination of the coat with urine which contains methenamine or with feed which contains formaldehyde. Experimentally it has not been excluded that kynurenine and formaldehyde react also within the body to form the yellow compound.The significance of these investigations for the acceptability of methenamine and formaldehyde as food additives is discussed.

Effect of chloramphenicol pretreatment on malathion‐induced acute toxicity in the rat

Pretreatment of rats with chloramphenicol (CAP) (100 mg/kg, ip) 30 min prior to a single oral LD50 dose of malathion (MTH) at 340 mg/kg completely protected against MTH-induced signs of cholinergic toxicity. Pretreatment with CAP also decreased the extent and duration of MTH-induced inhibition of cholinesterase (ChE). It was previously established that CAP inhibits (1) the cytochrome-P-450-catalyzed oxidative desulfuration of methylparathion to the much more toxic oxygen analog methylparaoxon (MOX) and (2) the carboxyesterase in rat liver. Since carboxyesterases account for 60% or more of the catabolism of MTH in the rat, the present results were surprising. Thus it appears that the inhibition of MTH toxicity by CAP pretreatment is attributable to inhibition by CAP of the metabolic activation of MTH to MOX.