Risk evaluation of occupational exposure of southern Brazilian flower farmers to pesticides potentially leading to cholinesterase inhibition and metals exposure

This work presents a frequency matched observational study comparing flower farmers exposed to pesticides and unexposed individuals as controls. All subjects were interviewed before plasma and urine collection. Manganese and Zinc were measured in theses samples by using dynamic reaction cell inductively coupled mass spectrometry. Cholinesterase activity was analyzed through spectrophotometry by using a modified version of the Ellman method. Seventy-eight percent of subjects reported occupational contact with pesticides, from which 37% reported exposure for over 9 years. Flower farms farmers had increased odds of having headache and irritability, respectively, by factors of 6.2 and 2.4 than the control subjects. While the odds of exposed subjects to have insomnia was smaller than control subjects by a factor of 0.34. Exposure to pesticides had a significant effect regarding the plasmatic plasma and urinary manganese levels and whole blood cholinesterase activity (p < 0.05). High levels of plasma and urinary manganese, as well as cholinesterase inhibition in whole blood, were evident in the flower farmers who participated in the study.     

Gestational exposure to chlorpyrifos: dose response profiles for cholinesterase and carboxylesterase activity

This study investigates the in vivo dose response profiles of the target enzyme cholinesterase (ChE) and the detoxifying enzymes carboxylesterase (CaE) in the fetal and maternal compartments of pregnant rats dosed with chlorpyrifos [(O,O'-diethyl O-3,5,6-trichloro- 2-pyridyl) phosphorothionate], a commonly used organophosphorus insecticide. Pregnant rats were dosed daily (po) with chlorpyrifos in corn oil (0, 3, 5, 7, or 10 mg/kg) on gestational days (GD) 14-18. Animals were sacrificed 5 h after the last chlorpyrifos dose (time of maximum brain cholinesterase inhibition) for analysis of ChE and CaE activity in maternal blood, liver, brain, placenta, and fetal liver and brain. The in vitro sensitivity (i.e., IC50, 30 min, 26 degrees C) of CaE also was determined by assaying the activity remaining after incubation with a range of chlorpyrifos-oxon concentrations. In vivo exposure to 10 mg/kg chlorpyrifos from GD14-18 caused overt maternal toxicity, with dose-related decreases in ChE activity more notable in maternal brain than fetal brain. Dose-related effects were also seen with chlorpyrifos-induced inhibition of fetal liver ChE and maternal brain CaE activities. Gestational exposure caused no inhibition of placental ChE or CaE, fetal brain CaE, or maternal blood CaE. ChE activities in the maternal blood and liver, as well as fetal and maternal liver CaE, however, were maximally inhibited by even the lowest dosage of chlorpyrifos. The in vitro sensitivity profiles of CaE to chlorpyrifos-oxon inhibition were valuable in predicting and verifying the in vivo CaE response profiles. Both the in vivo and in vitro findings indicated that fetal liver CaE inhibition was an extremely sensitive indicator of fetal chlorpyrifos exposure.      

Lack of differential sensitivity to cholinesterase inhibition in fetuses and neonates compared to dams treated perinatally with chlorpyrifos.

Pregnant Sprague-Dawley rats were exposed to chlorpyrifos (CPF; O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) by gavage (in corn oil) from gestation day (GD) 6 to postnatal day (PND) 10. Dosages to the dams were 0 (control), 0.3 (low), 1.0 (middle) or 5.0 mg/kg/day (high). On GD 20 (4 h post gavage), the blood CPF concentration in fetuses was about one half the level found in their dams (high-dose fetuses 46 ng/g; high-dose dams 109 ng/g). CPF-oxon was detected only once; high-dose fetuses had a blood level of about 1 ng/g. Although no blood CPF could be detected (limit of quantitation 0.7 ng/g) in dams given 0.3 mg/kg/ day, these dams had significant inhibition of plasma and red blood cell (RBC) ChE. In contrast, fetuses of dams given 1 mg/kg/day had a blood CPF level of about 1.1 ng/g, but had no inhibition of ChE of any tissue. Thus, based on blood CPF levels, fetuses had less cholinesterase (ChE) inhibition than dams. Inhibition of ChE occurred at all dosage levels in dams, but only at the high-dose level in pups. At the high dosage, ChE inhibition was greater in dams than in pups, and the relative degree of inhibition was RBC approximately plasma > or = heart > brain (least inhibited). Milk CPF concentrations were up to 200 times those in blood, and pup exposure via milk from dams given 5 mg/kg/day was estimated to be 0.12 mg/kg/day. Therefore, the dosage to nursing pups was much reduced compared to the dams exposure. In spite of exposure via milk, the ChE levels of all tissues of high-dosage pups rapidly returned to near control levels by PND 5.     

Relationship between cholinesterase inhibition and thermoregulation following exposure to diisopropyl fluorophosphate in the rat.

This study examined the relationship between inhibition of cholinesterase activity (CA) and thermoregulatory response in the rat following exposure to the organophosphate (OP), diisopropyl fluorophosphate (DFP). Male Long-Evans rats were injected with DFP dissolved in peanut oil in doses ranging from 0 to 1.5 mg/kg (s.c.). Colonic (Tcol) and tail skin temperature (Ttail) were recorded at 0, 1, 2 and 3 h post-injection. At 3 h post-injection the rat was sacrificed and a blood sample was taken by cardiac puncture and analyzed for CA. There was a biphasic dose effect of DFP on Tcol with slight but significant elevation in Tcol in the dose range of 0.01-0.5 mg/kg and a significant depression in Tcol at doses of 1.0 and 1.5 mg/kg. There was a dose-dependent fall in CA with DFP administration in the erythrocyte, plasma, and whole blood fractions. Hypothermia was associated with 80-87% inhibition in CA, whereas the elevation in Tcol was associated with 20-70% inhibition in CA. DFP also elicited significant elevations in Ttail. Overall, the data fail to demonstrate any clear relationship between inhibition of blood CA and thermoregulatory response following exposure to DFP. However, the elevation in Tcol following relatively low doses of DFP may be of relevance to the frequently reported symptom of fever in humans exposed to OP agents.     

Impact of repeated nicotine and alcohol coexposure on in vitro and in vivo chlorpyrifos dosimetry and cholinesterase inhibition

Chlorpyrifos (CPF) is an organophosphorus insecticide, and neurotoxicity results from inhibition of acetylcholinesterase (AChE) by its metabolite, chlorpyrifos-oxon. Routine consumption of alcohol and tobacco modifies metabolic and physiological processes impacting the metabolism and pharmacokinetics of other xenobiotics, including pesticides. This study evaluated the influence of repeated ethanol and nicotine coexposure on in vivo CPF dosimetry and cholinesterase (ChE) response (ChE- includes AChE and/or butyrylcholinesterase (BuChE)). Hepatic microsomes were prepared from groups of naive, ethanol-only (1 g/kg/d, 7 d, po), and ethanol + nicotine (1 mg/kg/d 7 d, sc)-treated rats, and the in vitro metabolism of CPF was evaluated. For in vivo studies, rats were treated with saline or ethanol (1 g/kg/d, po) + nicotine (1 mg/kg/d, sc) in addition to CPF (1 or 5 mg/kg/d, po) for 7 d. The major CPF metabolite, 3,5,6-trichloro-2-pyridinol (TCPy), in blood and urine and the plasma ChE and brain acetylcholinesterase (AChE) activities were measured in rats. There were differences in pharmacokinetics, with higher TCPy peak concentrations and increased blood TCPy AUC in ethanol + nicotine groups compared to CPF only (approximately 1.8- and 3.8-fold at 1 and 5 mg CPF doses, respectively). Brain AChE activities after ethanol + nicotine treatments showed significantly less inhibition following repeated 5 mg CPF/kg dosing compared to CPF only (96 ± 13 and 66 ± 7% of naive at 4 h post last CPF dosing, respectively). Although brain AChE activity was minimal inhibited for the 1-mg CPF/kg/d groups, the ethanol + nicotine pretreatment resulted in a similar trend (i.e., slightly less inhibition). No marked differences were observed in plasma ChE activities due to the alcohol + nicotine treatments. In vitro, CPF metabolism was not markedly affected by repeated ethanol or both ethanol + nicotine exposures. Compared with a previous study of nicotine and CPF exposure, there were no apparent additional exacerbating effects due to ethanol coexposure.     

Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development.

The effects of repeated oral exposures to the organophosphorus insecticide chlorpyrifos (CPS) on brain muscarinic receptor densities, together with cholinesterase (ChE) activity, were studied in early postnatal rats. Initially, the effects on esterases from lactational exposure to CPS were investigated in young rats by administering CPS (100, 150, or 200 mg/kg subcutaneously in corn oil) to dams 1 day postpartum, yielding a significant body burden of CPS in the dams for possible excretion in the milk. Brain ChE inhibition in pups was less severe than in dams, whereas liver carboxylesterase (CbxE) inhibition in pups was at the same level as in dams. Because of the limited brain ChE inhibition obtained following lactation, pups were exposed to CPS directly by gavage, using 3 dosing regimens to yield a dose response. The rats were gavaged with CPS in corn oil on alternate days from postnatal day (PND) 1 through PND 21. Rats in the low-dosage group received 11 treatments at 3 mg/kg, those in the medium-dosage group received 3 treatments at 3 mg/kg and 8 at 6 mg/kg, and those in the high dosage group received 3 treatments at 3 mg/kg, 4 at 6 mg/kg, and 4 at 12 mg/kg. ChE activity in brain homogenates were inhibited significantly by 29% and 63% in the low- and high-dosage groups, respectively, on PND 22 and by 17% in the high dosage group on PND 40. Muscarinic receptor densities in brain synaptosomes were reduced using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB) as ligands, with the effects more prominent from 3H-NMS. Densities of both ligands recovered to the control level several days after terminating treatment. The results indicate that pups are apparently exposed to only limited amounts of chlorpyrifos and/or its oxon through the milk when dams are exposed to extremely high chlorpyrifos levels. In addition, repeated direct oral exposures of early postnatal rats to CPS will result in persistent brain ChE inhibition and will transiently reduce muscarinic receptor density.     

Serum cholinesterase inhibition by omeprazole and lansoprazole

Omeprazole inhibited human and rat serum cholinesterase by approximately 5 to 60% over the 0.5 to 50 mg/L (1.4-140 microM) concentration range. In contrast lansoprazole only produced 20-30% inhibition at the highest concentration of 10 mg/L (29 microM). Thus omeprazole but not lansoprazole is likely to potentiate the effect of succinylcholine at human clinical concentrations by inhibiting its hydrolysis in vivo by serum cholinesterases.     

Comparative effects of oral chlorpyrifos exposure on cholinesterase activity and muscarinic receptor binding in neonatal and adult rat heart

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M(2) muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M(2) receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M(2) receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age-related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac cholinesterase (ChE) activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1x LD(10): neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1x LD(10), relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (approximately 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC(50) values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4h after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity.     

Finding cholinesterase in endotheliocytes: cholinesterase inhibition by organophosphorus compounds leads to endotheliocyte deformation

Toxic action of some organophosphorus cholinesterase inhibitors (including phosphacol) on the state of microvessels was studied in rats. Cholinesterase was found in endotheliocytes and it was established that phosphacol inhibited this enzyme. This was one of the factors responsible for deformation of the luminal relief of the vessels and for violation of the blood microcirculation. The expression of these effects was different for various organophosphorus compounds.     

Tadpole size, cholinesterase activity, and swim speed in four frog species after exposure to sub-lethal concentrations of chlorpyrifos

While physiological biomarkers exist to verify exposure of amphibians in natural populations to agricultural chemicals, the ecological relevance of changes in these parameters is often difficult to determine. We compare the relationship between tadpole cholinesterase (ChE; a common enzymatic biomarker of exposure to OP pesticides) and measures of size and swim speed in four native North American species of anurans (Hyla chrysoscelis, Rana sphenocephala, Acris crepitans, and Gastrophryne olivacea). We used four environmentally realistic levels (1, 10, 100 and 200 μg/l) of a commonly used organophosphate pesticide (OP), chlorpyrifos, and examined tadpole response at the conclusion of 4 days of exposure. We further examined if the presence or absence of pond sediment influenced tadpole responses, and, in two species, we determined how a 12-day exposure influenced responses. We found species-specific differences in response to the pesticide, with H. chrsysoscelis and G. olivacea being most sensitive; however, the levels of inhibition of ChE activity were generally not sufficient to exert an effect on swim speed as we measured it. Generally, tadpole mass was reduced 20–35% in the highest concentration after 4 days of exposure. We found the presence of sediment to influence these responses, although the effects were not consistent among species. Given these differences in ecologically relevant responses, we recommend exercising caution when making generalizations across different anuran amphibian taxa regarding responses to pesticide exposure.     

石杉碱类似物在体外对胆碱酯酶的抑制作用(英文)

用比色法测试表明,14个半合成类似物的抗AChE作用均弱于Hup-A,左旋二氢及四氢类似物的抗BuChE作用稍强于Hup-A,前者属混合型抑制剂,K_i值为0.12μmol·L~(-1)。后者属竞争型抑制剂,K_i值为0.56μmol·L~(-1)。二者不同于异氟磷,与AChE为可逆性结合。     

The effect of chlorpyrifos and chlorpyrifos-oxon on brain cholinesterase, muscarinic receptor binding, and neurotrophin levels in rats following early postnatal exposure.

Chlorpyrifos (CPS) is a widely used diethyl organophosphorus insecticide in agricultural settings. Household and urinary residue analysis has suggested that children in agricultural communities are at risk of exposure to diethyl organophosphorus insecticides. The effects of repeated postnatal exposure to CPS and its metabolite chlorpyrifos-oxon (CPO) on total muscarinic acetylcholine receptor (mAChR) binding, nerve growth factor (NGF) levels, and brain derived neurotrophic factor (BDNF) levels in the forebrain of neonatal rats were investigated. Peak inhibition of brain cholinesterase (ChE) for CPS and CPO was determined after acute exposure to dosages of each compound (a low and a high for each), which produced similar degrees of initial ChE inhibition. Pups were administered CPS (1.5 or 3.0 mg/kg), CPO (0.25 or 0.35 mg/kg), or the corn oil vehicle by daily gavage from postnatal day 1 (PND 1) through PND 6. This exposure paradigm resulted in persistent ChE inhibition by CPS but only transient inhibition by CPO, suggesting that, even though the initial ChE inhibition is similar between compounds, the effects of repeated exposure differ significantly. Forebrain mAChR density, as measured by the binding of 3H-QNB, and NGF levels were significantly reduced on PND 4 and 7 after CPS but not on PND 12. No effects on mAChR density or NGF levels were observed with CPO. No effects on BDNF levels were observed with either compound. The data suggest that the persistent ChE inhibition and decreased mAChR binding may play a role in the decreased NGF levels following CPS exposure.     

Tissue cholinesterase inhibition by propranolol and related drugs

The effect of (+/-)-propranolol and some related drugs have been investigated on the cholinesterase (ChE) enzyme activity of heart and brain tissues of the rat. Brain homogenates hydrolysed more methacholine than benzoylcholine and the reverse was true for the heart tissue. In-vitro, (+/-)-, (+)- and (-)-propranolol, as well as its quaternary analogue, UM-272, all significantly inhibited heart and brain ChE. Timolol and sotalol, however, were less potent. In-vivo, (+/-)-propranolol (30 mumol kg-1) significantly inhibited brain ChE activity in rats when compared with saline controls. It is inferred that propranolol inhibits brain and heart ChE enzyme in a non-stereoselective manner and that this cholinomimetic action could be involved in the mediation of some of its therapeutic effects.     

Stereoselectivity of cholinesterase inhibition by galanthamine and tolerance in humans

Summary The effect of galanthamine (GAL) and its 2 major metabolites on human cholinesterases has been explored. Epigalanthamine, a diastereomer of GAL, was 130-times less potent in vitro in its effect on acetylcholinesterase (AChE) in erythrocytes than the parent compound, and it did not differ significantly from the ketone galanthaminone. In vivo, the maximal 36–55% inhibition of AChE was approached 30 min after oral administration of 10 mg GAL. The duration of the catalytic inhibition corresponded to an elimination half-life of approximately 5–7 h. GAL was well tolerated in 8/8 healthy volunteers, and 3/4 Alzheimer patients tolerated the drug up to a daily dose of 40 mg.     

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62–87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.     

Parasympathetic excitation of sympathetic innervation after cholinesterase inhibition.

1. Orbital parasympathetic innervation normally provides prejunctional muscarinic inhibition of sympathetic neurotransmission without activation of excitatory muscarinic receptors located on the innervated smooth muscle. The present study examines the role of acetylcholinesterase (AChE) in limiting the effects of parasympathetically released acetycholine to prejunctional receptors. 2. Urethane anaesthetized rats were placed in a stereotaxic frame, and parasympathetic activation was achieved by electrical stimulation (20 Hz, < 2.0 V) of the ipsilateral superior salivatory nucleus. Drugs were administered through a femoral venous cannula. Superior tarsal smooth muscle responses were measured by recording eyelid tension. 3. Parasympathetic stimulation alone caused a small decrease in resting tension; previous studies have shown this to be attributable to attention of resting sympathetic tone. Parasympathetic activation following physostigmine administration, however, evoked a large contractile response. Contractions were resistant to atropine but were blocked by gallamine, guanethidine, and phentolamine. 4. We conclude that AChE inhibition results in conversion of orbital parasympathetic nerve function from inhibition of sympathetic neurotransmission to smooth muscle excitation. This occurs as a result of cholinergic activation of excitatory nicotinic receptors on sympathetic varicosities, which elicit the release of noradrenaline.     

Neurochemical effects of repeated gestational exposure to chlorpyrifos in developing rats.

The neurochemical effects in developing rats exposed during gestation to the anticholinesterase organophosphorus insecticide chlorpyrifos (CPS) were determined. Pregnant rats were dosed daily with CPS (0, 3, or 7 mg/kg) in corn oil from gestation days (GD) 6-20. Pups were euthanized on postnatal days (PND) 1, 3, 6, 9, 12, and 30 for the determination of brain cholinesterase (ChE) and choline acetyltransferase (ChAT) activities, along with muscarinic receptor (mAChR) densities, the levels of the high-affinity choline uptake (HACU) system, and the vesicular acetylcholine transporter (VAChT). ChE activities were inhibited about 15 and 30% on PND 1, in the low- and high-dosage groups, respectively, and were not different from control values by PND 6. mAChR densities on PND 1 were reduced in the high-dosage group by about 18, 21, and 17%, using 3H-N-methylscopolamine, 3H-quinuclidinyl benzilate, and 3H-4-DAMP, respectively, as ligands, and were not different from control levels by PND 6. ChAT activity was decreased by approximately 12% in the high-dosage group on PND 9, 12, and 30. HACU levels, using 3H-hemicholinium-3 as the ligand, were reduced by approximately 25% on PND 6 in the low- and high-dosage groups, and by approximately 14 and 21% on PND 12 and 30, only in the high-dosage group. Levels of the VAChT were reduced by a range of 13-31% on PND 3 through 30 in the high-dosage group, using 3H-AH5183 (vesamicol) as the ligand. These data suggest that gestational exposure to 7 mg/kg/day CPS results in long-term alterations of presynaptic cholinergic neurochemistry.     

Color vision and occupational toluene exposure.

We examined the relationship between acquired color vision loss and exposure to toluene and total hydrocarbons among 125 male workers. Seventy-two toluene-exposed printers were compared with 34 workers from the same photogravure plant with ambient background exposure, and with 19 workers from a bookbinding plant located in the same town (nonexposed). Environmental mean toluene exposure level at workstation was estimated from individual 8-h sampling. Historic exposure data from the last 30 years were used to construct two cumulative exposure indices, one for toluene and one for total hydrocarbons. Airborne toluene levels were overall lower than the current Threshold Limit Value (TLV) of 50 ppm. Color vision was assessed by the Lanthony D-15 desaturated panel. Color vision loss was quantitatively established by the Color Confusion Index (CCI) and classified by type of acquired dyschromatopsia according to Verriest's classification. CCI was positively related to current airborne toluene levels, and cumulative exposure indices for toluene and total hydrocarbons (.18< or =r< or =.35). Odds ratios of acquired dyschromatopsia were significant for current airborne toluene, toluene, and total hydrocarbon past exposure (1.27 [1.02-1.58], 1.21 [1.04-1.39], 1.15 [1.02-1.31], respectively). In conclusion, this study suggests that the Lanthony D-15 desaturated panel detects early neurotoxic effects among workers exposed to toluene.