Annarita Meneguz

CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes

The bioactivation of azinphos-methyl (AZIN), chlorpyrifos (CPF), diazinon (DIA), and parathion (PAR), four widely used organophosphorothioate (OPT) pesticides has been investigated in human liver microsomes (HLM). In addition, the role of human cytochrome P450 (CYPs) in OPT desulfuration at pesticide levels representative of human exposure have been defined by means of correlation and immunoinhibition studies. CYP-mediated oxon formation from the four OPTs is efficiently catalyzed by HLM, although showing a high variability (>40-fold) among samples. Two distinct phases were involved in the desulfuration of AZIN, DIA, and PAR, characterized by different affinity constants (K(mapp1) = 0.13-9 microM and K(mapp2) = 5- 269 microM). Within the range of CPF concentrations tested, only the high-affinity component was evidenced (K(mapp1) = 0.27-0.94 microM). Oxon formation in phenotyped individual HLM showed a significant correlation with CYP1A2-, 3A4-, and 2B6-related activities, at different levels depending on the OPT concentration. Anti-human CYP1A2, 2B6, and 3A4 antibodies significantly inhibited oxon formation, showing the same OPT concentration dependence. Our data indicated that CYP1A2 is mainly involved in OPT desulfuration at low pesticide concentrations, while the role of CYP3A4 is more significant to the low-affinity component of OPT bioactivation. The contribution of CYP2B6 to total hepatic oxon formation was relevant in a wide range of pesticide concentrations, being a very efficient catalyst of both the high- and low-affinity phase. These results suggest CYP1A2 and 2B6 as possible metabolic biomarkers of susceptibility to OPT toxic effect at the actual human exposure levels.

Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice

Neonatal mice were treated daily on postnatal days (pnds) 1 through 4 or 11 through 14 with the organophosphate pesticide chlorpyrifos (CPF), at doses (1 or 3 mg/kg) that do not evoke systemic toxicity. Brain acetylcholinesterase (AChE) activity was evaluated within 24 h from termination of treatments. Pups treated on pnds 1-4 underwent ultrasonic vocalization tests (pnds 5, 8, and 11) and a homing test (orientation to home nest material, pnd 10). Pups in both treatment schedules were then assessed for locomotor activity (pnd 25), novelty-seeking response (pnd 35), social interactions with an unfamiliar conspecific (pnd 45), and passive avoidance learning (pnd 60). AChE activity was reduced by 25% after CPF 1-4 but not after CPF 11-14 treatment. CPF selectively affected only the G(4) (tetramer) molecular isoform of AChE. Behavioral analysis showed that early CPF treatment failed to affect neonatal behaviors. Locomotor activity on pnd 25 was increased in 11-14 CPF-treated mice at both doses, and CPF-treated animals in both treatment schedules were more active when exposed to environmental novelty in the novelty-seeking test. All CPF-treated mice displayed more agonistic responses, and such effect was more marked in male mice exposed to the low CPF dose on pnds 11-14. Passive avoidance learning was not affected by CPF. These data indicate that developmental exposure to CPF induces long-term behavioral alterations in the mouse species and support the involvement of neural systems in addition to the cholinergic system in the delayed behavioral toxicity of CPF.

Kinetic parameters of OPT pesticide desulfuration by c-DNA expressed human CYPs

The role of different cytochrome P450 isoforms (CYPs) in the desulfuration of four organophosphorothionate pesticides (OPTs), namely diazinon (DIA), azinphos-methyl (AZ), chlorpyrifos (CPF) and parathion (PARA), at OPT levels representative of actual human exposure has been investigated. For this purpose c-DNA expressed human CYPs and a method, based on acetylcholinesterase (AChE) inhibition, able to detect nM levels of oxon have been used. Our results indicate that the four tested OPTs at low concentration were mainly desulfurated by CYP2B6, 2C19 and 1A2, showing K(m) values in the range 0.8-5 μM and the highest efficiency (intrinsic clearance (ICL)) values. CYP3A4 was generally endowed with high K(m) and resulted linear up to 25-100 μM OPT, concentrations saturating the most efficient CYPs. The tentative extrapolation of the relative contribution of single CYPs, taking into account the average content of different isoforms in the human liver, indicate that CYP1A2 is the major responsible for oxon formation. Indeed this CYP catalyses the 50-90% of desulfuration reaction, depending on the OPT. As CYP3A4 activity is not completely saturated up to 100 μM OPT, and due to the high hepatic content, its contribution to oxon formation may result relevant in poisoning episodes, when individuals are exposed at high doses of OPTs.

Influence of urethane and ketamine on rat hepatic cytochrome P450 in vivo

The aim of this study was to identify the effects of widely used laboratory anaesthetics on cytochrome (CYP) activity in male Sprague Dawley rats in vivo. The anaesthetics used were urethane and ketamine. 7-Ethoxyresorufin (EROD), 7-pentoxyresorufin (PROD), aniline and ethylmorphine were used as substrates for CYP 1A, CYP 2B, CYP 2E1 and CYP 3A, respectively. Urethane increased EROD (CYP 1A) activity by 40 % (p < 0.01), and hydroxylation of aniline (CYP 2E1) by 14 % in the early phase of anaesthesia and by 60 % (p < 0.01) in the later one. Urethane also reduced the demethylation of ethylmorphine by 37 % (p < 0.01), but did not affect CYP 2B activity significantly. Ketamine did not significantly affect CYP 1A, 2B or 2E1. However, it reduced the demethylation of ethylmorphine (i.e. CYP 3A) by 32 % (p < 0.01). From these data, we concluded that a single dose of urethane inhibits CYP 3A but increases CYP 2E1 and CYP 1A, and that a single dose of ketamine inhibits the activity of CYP 3A.

Synthesis and cholinesterase activity of phenylcarbamates related to Rivastigmine, a therapeutic agent for Alzheimer's disease.

In order to develop new cholinesterase agents effective against Alzheimers disease (AD) we synthesized some phenylcarbamates structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine.

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.

Mechanisms of Recovery of Brain Acetylcholinesterase in Rats During Chronic Intoxication by Isoflurophate

During chronic intoxication by diisopropyl fluorophosphate (Isoflurophate, DFP; s.c. treatment on alternate days - first dose of 1.1 mg/kg, subsequent doses of 0.7 mg/kg each until the 23rd day) a partial recovery of enzymatic activity was found at 24 h after each DFP administration. Relative to maximal AChE depression at 90 min, these rises were more pronounced in the soluble portion of the enzyme than in total enzyme preparation, i.e., that containing mainly membrane-bound AChE. Moreover, from the 2nd DFP administration on, there was a persistent increase of medium-molecular-weight forms both in soluble and in total AChE. The results suggest an important role of the soluble portion of AChE and of medium forms in the process of recovery of enzymatic activity.

Change in the distribution of acetylcholinesterase molecular forms in frontoparietal cortex of the rat following nucleus basalis lesions with kainic acid.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G4/G1 ratio fell from 5.4 +/- 0.8 in contralateral to 3.0 +/- 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimers disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

Alterations in the distribution of cholinesterase molecular forms in maternal and fetal brain following diisopropyl fluorophosphate treatment of pregnant rats.

Previous work in this laboratory showed that during intoxication of rats with diisopropyl fluorophosphate at day 20 of pregnancy the recovery of ChE activity was faster in fetal than in maternal brain. In the present study the differences between recovery rates in dam and fetus brain were evaluated in terms of molecular forms and spontaneous reactivation. Using ultracentrifugation on sucrose gradient two molecular forms of ChE, namely 10S (tetrameric globular G4 form) and 4S (monomeric G1 form) were detected both in maternal and fetal brain of untreated rats. The ratios 10S/4S were about 5.0 and 0.75 for dams and 20-day fetuses, respectively. DFP administration (1.1 mg/kg sc) inducing at 90 min an about 80% inhibition of ChE in maternal brain caused a shift in its 10S/4S ratio to 1.63, and to 0.53 in fetal brain (in which overall inhibition was about 70%). This means that 10S forms were preferentially inhibited by DFP both in maternal and fetal brain. After 24 and 48 hr there was a negligible recovery of overall ChE in maternal brain with no shift in the ratio. On the other hand, complete recovery of ChE in fetal brain within 48 hr was accompanied by almost total normalization of the 10S/4S ratio. Rapid recovery of fetal ChE appeared not to depend on hydrolysis of DFP-inhibited ChE. In fact, maternal and fetal DFP-inhibited enzyme preparations following the addition of oximes (pralidoxime or obidoxime) in vitro showed similar rates of reactivation. The overall data indicate considerable differences in recovery rate of molecular forms between dams and fetuses, but not in reactivation by dephosphorylation.

Developmental Factors Affecting Brain Acetylcholinesterase Inhibition and Recovery in DFP-Treated Rats

The effect of a single dose of diisopropyl fluorophosphate (DFP; Isoflurophate, 1.1 mg/kg s.c.) administered to rats during pregnancy was evaluated by measuring postpartum maternal and newborn brain-soluble and total acetylcholinesterases (AChE) and their molecular forms at intervals of 1, 2, 3, 4 and 10 days between treatment and sacrifice. Subsequently, the effects of DFP were studied in 18-day-pregnant rats, fetuses and placentae at 90 min and 24 h after treatment. The inhibition of postpartum maternal enzymatic activity did not differ from that previously found in adult males, while inhibition was considerably less pronounced in newborns at all time intervals, with a nearly complete recovery already at 48 h after treatment. An even faster recovery of brain enzyme was observed in 18-day fetuses from DFP-treated mothers (24-hour interval between treatment and sacrifice). In this experiment, a comparable inhibition was observed at 90 min after treatment in the adult and the developing brain, excluding a major influence of disposition factors in the differential recovery phenomena. An experiment on weanling rats yielded intermediate results between those of newborn and those of adult animals. Finally, most data confirmed previous findings that the soluble portion of brain AChE and medium molecular weight enzyme forms may have special significance in the initial phases of recovery.