The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M(1) subtype and cell-specific markers for developing neurons (beta-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin-associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M(1) mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. beta-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat.

Although the use of organophosphate (OP) insecticides has been restricted, sufficient exposure can occur to induce detrimental neurobehavioral effects. In this study, we measured physical and reflex development and spatial learning and memory in rats repeatedly exposed to incremental doses of chlorpyrifos (CPS) and methyl parathion (MPS) from postnatal day (PND) 1 to PND21. Other than decreased body weight in the higher dosage groups, no effects on physical or reflex development were observed. Significant hippocampal cholinesterase inhibition was induced in all treatment groups for up to 19 days following exposure. Beginning on PND36, working and reference memory was tested using a 12-arm radial maze, with subject animals trained and tested 4 days a week for 4 weeks. In males, working memory was decreased with the medium and high dosage of MPS but only the high dosage of CPS; while in females, no deficits were observed. For reference memory, errors were significantly increased in males exposed to the high dosage of CPS and all dosages of MPS. In females, enhanced performance was observed within the medium and high dosages of CPS but not with MPS. These data show that repeated developmental exposure to OP insecticides can induce sex-selective alterations and long-lasting changes in spatial learning and memory formation when measured using a radial arm maze and that MPS and CPS induce different neurobehavioral outcomes.

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.

A possible link between Parkinson's disease and pesticide exposure has been suggested, and recently it was shown that the herbicide atrazine (ATR) modulates catecholamine metabolism in PC12 cells and affects basal ganglia function in vivo. Hence, the objectives of this study were to: (i) determine if ATR is capable of modulating dopamine (DA) metabolism in striatal tissue slices in vitro and (ii) to explore possible mechanisms of its effects. Striatal tissues from adult male Sprague Dawley rats were incubated with up to 500 μM ATR in a metabolic shaker bath at 37 °C and an atmosphere of 95% O 2 and 5% CO 2 for 4 h. At the end of incubation, samples were collected for both tissue and media levels of DA and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC and homovanillic acid, HVA), which were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). To gain some mechanistic insight in to the way ATR affects DA metabolism, several pharmacological manipulations were performed. Striata exposed to ATR at concentrations of 100 μM and greater had a dose-dependent decrease of tissue levels of DA. At doses of ATR 50 μM and greater, the DOPAC+HVA/DA ratio was dose-dependently increased. Tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) protein levels and activity were not affected by ATR treatment. However, high potassium induced DA release into the medium was decreased, whereas the increase in media DA observed in the presence of the DA uptake inhibitor nomifensine was increased even further by ATR in a dose-dependent manner. All of these effects of ATR were observed at levels that were not toxic to the tissue, as LDH release into the medium (lactate dehydrogenase, an index of non-specific cytotoxicity) was not affected by ATR. Taken together, results from this study suggest that ATR decreases tissue DA levels not by affecting TH activity, but possibly by interfering with the vesicular storage and/or cellular uptake of DA.

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Exposure to chlorpyrifos (CPF) during the late preweanling period in rats inhibits the endocannabinoid metabolizing enzymes fatty acid hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resulting in accumulation of their respective substrates anandamide (AEA) and 2-arachidonylglycerol (2-AG). This occurs at 1.0 mg/kg, but at a lower dosage (0.5 mg/kg) only FAAH and AEA are affected with no measurable inhibition of either cholinesterase (ChE) or MAGL. The endocannabinoid system plays a vital role in nervous system development and may be an important developmental target for CPF. The endocannabinoid system plays an important role in the regulation of anxiety and, at higher dosages, developmental exposure to CPF alters anxiety-like behavior. However, it is not clear whether exposure to low dosages of CPF that do not inhibit ChE will cause any persistent effects on anxiety-like behavior. To determine if this occurs, 10-day old rat pups were exposed daily for 7 days to either corn oil or 0.5, 0.75, or 1.0 mg/kg CPF by oral gavage. At 12 h following the last CPF administration, 1.0 mg/kg resulted in significant inhibition of FAAH, MAGL, and ChE, whereas 0.5 and 0.75 mg/kg resulted in significant inhibition of only FAAH. AEA levels were significantly elevated in all three treatment groups as were palmitoylethanolamide and oleoylethanolamide, which are also substrates for FAAH. 2-AG levels were significantly elevated by 0.75 and 1.0 mg/kg but not 0.5 mg/kg. On day 25, the latency to emerge from a dark container into a highly illuminated novel open field was measured as an indicator of anxiety. All three CPF treatment groups spent significantly less time in the dark container prior to emerging as compared to the control group, suggesting a decreased level of anxiety. This demonstrates that repeated preweanling exposure to dosages of CPF that do not inhibit brain ChE can induce a decline in the level of anxiety that is detectable during the early postweanling period.