Threshold For Cholinesterase Inhibition Research Articles

The Role of Matricaria recutita L. and Asparagus officinalis L. against the Neurotoxicity of Diazinon in Rats

Diazinon (DZN) is an organophosphate insecticide used mainly in agriculture and in sheep dips, and is designed as an irreversible acetylcholine esterase inhibitor [1,2]. It is classified as moderately hazardous class-II organophosphate insecticide [3]. Many systems could be affected by organophosphate intoxication are the immune system, urinary system, reproductive system, pancreas and haematological and biochemical changes [4-8]. In addition, showed that neonatal DZN exposure, at doses below the threshold for cholinesterase inhibition has been lasting effects on emotional responses, with preferential effects on males [9,10]. The microsomal enzymes in the liver oxidize DZN are generating more potent acetylcholinesterase inhibitors, such as diazoxon, hydroxydiazoxon and hydroxydiazinon [11]. Diazinon affects mitochondrial membrane transportation in rat liver [12]. Moreover, it interrupts cytochrome P450 system in human liver [13]. Oxidative stress can also be induced by pesticides, either by the overproduction of free radicals or by alteration in antioxidant defence mechanisms, including detoxification and scavenging enzymes [14]. Oxidative stress has been reported to play an important role in the toxicity of various pesticides, including organochlorines, carbamates and pyrethrods [15,16]. The higher oxidative stress in pesticide sprayers is evidenced by increased concentration of plasma and red blood cell thiobarbituric acid reactive substances (TBARS), changes in antioxidant status, and altered activities of cellular enzymes [17]. Treatment of rats with DZN significantly enhances renal lipid peroxidation, which is accompanied by a decrease in the activities of renal antioxidant enzymes (e.g. catalase (CAT), glutathione peroxidise, glutathione reductase (GSH-R), glucose- 6-phosphate dehydrogenase, glutathione-s-transferase (GST) and depletion in the level of glutathione reduced (GSH). In blood, normal erythrocyte function depends on the intactness of cell membrane, which is the target for many toxic factors, including pesticides. Erythrocyte GSH together with glutathione peroxidase (GSH-Px), GSH-R, GST, gamma-glutamyl transferase (γ-GT), superoxide dismutase (SOD) and CAT efficiently scavenge toxic free radicals and are partly responsible for protecting against lipid peroxidation due to acute/chronic pesticide exposure [18]. There is a relationship between pesticide exposure and the decrease of antioxidant enzymes [19]. Oxidative stress and genotoxic effects of DZN were documented through the changes in total antioxidant capacity (TAC), reduced GSH and oxidative DNA damage [20]. Exposure to low-level of pesticides is known to produce a variety of biochemical and molecular changes, some of which may be responsible for the adverse biological effects reported in human and experimental studies. Many studies have reported that DZN can induce molecular changes and alter gene expression. Jamshidi et al. have found that administration of DZN to rats at doses of 60 mg/ kg significantly decreased expression of glutamate dehydrogenase, the key enzyme of Langerhans islet for the secretion of insulin, gene 18- hour post-administration [21]. Other studies showed that DZN had affected the expression of neurotrophic factors that coordinate neuronal cell differentiation and brain assembly [22]. In addition, Timofeeva et al. found that persisting effect of developmental DZN cholinergic and serotonergic neurotransmitter systems and gene expression as well as behavioural function [9]. Matricaria recutita L. (family Asteraceae, commonly known as German chamomile) is one of the most widely used and welldocumented medicinal plants in the world [23]. Chamomile is also

Reply to Juberg: Using the biologically based dose response to compare exposures across species

In his Letter to the Editor (1), Juberg conflates two sequential statements in our paper (2). The references cited in his letter pertain to the sentence preceding the one in question and relate to the “direct neurotoxic effects of early CPF exposure in animals” (ref. 2, p. 7874). The sentence in question, “The exposures at which these mechanisms become manifest in animal models are comparable to exposure levels in our own population,” (ref. 2, p. 7874) was not referenced, because this concept was presented with citations earlier in the paper, in the section detailing the inadequacy of cholinesterase as a predictor of developmental neurotoxicity. Juberg’s contention violates a longstanding principle of toxicology in that he treats external dose (milligrams per kilogram) between species as the sole determinant of “exposure,” when it is the internal dose or, even better, the biologic effect that should be compared across species when establishing equivalent exposures. This concept, now universally accepted, is inherent in the adoption of “physiologically based pharmacokinetics” as the basis for internal dose comparisons and “biologically based dose response” for the biologic effects across species. In this particular instance, cholinesterase inhibition provides a benchmark for establishing biological exposure equivalence between rats and humans. The animal studies cited in this paper involved nonsymptomatic exposures that are at or below the threshold for any detectable cholinesterase inhibition and therefore did guide us appropriately to look for similar effects at the even lower human exposures in our work. Our study found structural defects at human exposures well below the safety limits currently in place, and these specific defects were predicted from animal studies at exposure levels below the threshold for cholinesterase inhibition.

Developmental neurotoxicity of organophosphates targets cell cycle and apoptosis, revealed by transcriptional profiles in vivo and in vitro

Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1–4, in doses straddling the threshold for barely-detectable cholinesterase inhibition, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20–25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60–70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme.

Developmental diazinon neurotoxicity in rats: Later effects on emotional response

Developmental exposure to the organophosphorus pesticides chlorpyrifos and diazinon (DZN) alters serotonergic synaptic function at doses below the threshold for cholinesterase inhibition, however there are some indications that the two agents may differ in several important attributes. Previously, we found that low-dose chlorpyrifos exposure in neonatal rats causes lasting changes in emotional response and in the current study we did a comparable evaluation for DZN. Male and female Sprague–Dawley rat pups ( N = 10–12 of each sex per treatment group) were given 0, 0.5 or 2 mg/(kg day) of DZN s.c. daily on postnatal days (PND) 1–4. These doses bracket the threshold for barely-detectable cholinesterase inhibition. Starting on PND 52, these rats began a battery of tests to assess emotional reactivity. In the elevated plus maze, there was a slight decrease in the time spent in the open arms for DZN-exposed males, while DZN-exposed females were not different from control females. In the novelty-suppressed feeding test, DZN-exposed males had significantly shorter latencies to begin eating than did control males, reducing the values to those normally seen in females. DZN-exposed rats of either sex showed reduced preference for chocolate milk in the anhedonia test that compared the consumption of chocolate milk to water. These findings show that neonatal exposures to DZN at a dose range below the threshold for cholinesterase inhibition nevertheless evokes specific, later alterations in emotional behaviors, particularly in males. The effects show not only some similarities to those of chlorpyrifos but also some differences, in keeping with neurochemical findings comparing the two agents.

Developmental neurotoxicity of chlorpyrifos: what is the vulnerable period?

Previously, we found that exposure of neonatal rats to chlorpyrifos (CPF) produced brain cell damage and loss, with resultant abnormalities of synaptic development. We used the same biomarkers to examine prenatal CPF treatment so as to define the critical period of vulnerability. One group of pregnant rats received CPF (subcutaneous injections in dimethyl sulfoxide vehicle) on gestational days (GD) 17-20, a peak period of neurogenesis; a second group was treated on GD9-12, the period of neural tube formation. In the GD17-20 group, the threshold for a reduction in maternal weight gain was 5 mg/kg/day; at or below that dose, there was no evidence (GD21) of general fetotoxicity as assessed by the number of fetuses or fetal body and tissue weights. Above the threshold, there was brain sparing (reduced body weight with an increase in brain/body weight ratio) and a targeting of the liver (reduced liver/body weight). Indices of cell packing density (DNA per gram of tissue) and cell number (DNA content) similarly showed effects only on the liver; however, there were significant changes in the protein/DNA ratio, an index of cell size, in fetal brain regions at doses as low as 1 mg/kg, below the threshold for inhibition of fetal brain cholinesterase (2 mg/kg). Indices of cholinergic synaptic development showed significant CPF-induced defects but only at doses above the threshold for cholinesterase inhibition. With earlier CPF treatment (GD9-12), there was no evidence of general fetotoxicity or alterations of brain cell development at doses up to the threshold for maternal toxicity (5 mg/kg), assessed on GD17 and GD21; however, augmentation of cholinergic synaptic markers was detected at doses as low as 1 mg/kg. Compared with previous work on postnatal CPF exposure, the effects seen here required doses closer to the threshold for fetal weight loss; this implies a lower vulnerability in the fetal compared with the neonatal brain. Although delayed neurotoxic effects of prenatal CPF may emerge subsequently in development, our results are consistent with the preferential targeting of late developmental events such as gliogenesis, axonogenesis, and synaptogenesis.

Estimating a worker entry interval for the carbamate pesticide furadan 4F insecticide.

Existing methods for the calculation of worker entry intervals by the use of kinetic data have focused on the anticholinesteratic organic phosphates. As carbamate pesticides share with these materials a common site for inhibition, the kinetic method was applied in estimating an entry interval for the carbamate, carbofuran, applied to citrus and grapes as Furadan 4F. No allowance was made for the more transient carbamylation of cholinesterase attributed to N-methyl carbamates, compared with the phosphorylation of the enzyme by organic phosphates. Thus, a measure of safety was built into the calculations which can then be used to establish safe reentry intervals to insure residue levels below the physiological threshold for cholinesterase inhibition.