Decreased cerebral spinal fluid neurotransmitter levels in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital anomaly syndrome with cognitive impairment and a distinct behavioral phenotype that includes autistic features. SLOS is caused by a defect in 3ß-hydroxysterol Δ(7)-reductase which leads to decreased cholesterol levels and elevated cholesterol precursors, specifically 7- and 8-dehydrocholesterol. However, the pathological processes contributing to the neurological abnormalities in SLOS have not been defined. In view of prior data suggesting defects in SLOS in vesicular release and given the association of altered serotonin metabolism with autism, we were interested in measuring neurotransmitter metabolite levels in SLOS to assess their potential to be used as biomarkers in therapeutic trials. We measured cerebral spinal fluid levels of serotonin and dopamine metabolites, 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) respectively, in 21 SLOS subjects. Results were correlated with the SLOS anatomical severity score, Aberrant Behavior Checklist scores and concurrent sterol biochemistry. Cerebral spinal fluid (CSF) levels of both 5HIAA and HVA were significantly reduced in SLOS subjects. In individual patients, the levels of both 5HIAA and HVA were reduced to a similar degree. CSF neurotransmitter metabolite levels did not correlate with either CSF sterols or behavioral measures. This is the first study demonstrating decreased levels of CSF neurotransmitter metabolites in SLOS. We propose that decreased levels of neurotransmitters in SLOS are caused by a sterol-related defect in synaptic vesicle formation and that CSF 5HIAA and HVA will be useful biomarkers in development of future therapeutic trials.

Repeated cannabinoid administration increases indices of noradrenergic activity in rats.

The present study examined the impact of repeated administration of a synthetic cannabinoid agonist, WIN 55,212-2 on the coeruleo-cortical pathway, a circuit implicated in anxiety. Male Sprague-Dawley rats received repeated systemic injections of WIN 55,212-2 (3.0 mg/kg). A separate group of rats received repeated WIN 55,212-2 injections followed by a period of abstinence. Control animals received vehicle injections. Ninety minutes following the last injection on day 8, anxiety-related behavior was assessed using the elevated plus maze. The abstinent group was tested after another 8 days. Following behavioral testing, brain tissue was extracted from the locus coeruleus (LC) and probed for tyrosine hydroxylase (TH) expression. In a separate group of animals, in vivo microdialysis was used to monitor extracellular norepinephrine efflux in the frontal cortex following repeated WIN 55,212-2 administration and following a period of abstinence. Repeated administration of WIN 55,212-2 evoked an anxiogenic-like response that was accompanied by an increase in TH protein expression in the LC. A similar neurochemical profile was observed using in vivo microdialysis where an augmented increase in cortical norepinephrine efflux was identified in response to a systemic injection of WIN 55,212-2 on day 8. Anxiety-like behavior, catecholamine synthesizing enzyme levels and NE efflux returned to control values after 8 days of abstinence. The present findings indicate that repeated administration of a synthetic cannabinoid receptor agonist induces transient anxiety-like behaviors that correlate with increases in catecholamine synthesizing enzyme expression in the LC and augmented norepinephrine efflux in response to a challenge injection of WIN 55,212-2.

Metabolism and mode of action of cis- and trans-3-pinanones (the active ingredients of hyssop oil).

1. Hyssop oil is an important food additive and herbal medicine and the principal active ingredients are (-)-cis- and (-)-trans-3-pinanones. No information is available on their metabolism or specific mode of action. 2. The metabolites of cis- and trans-3-pinanones were examined from mouse and human liver microsomes and human recombinant P4503A4 with NADPH and on administration to mouse by gas chromatography/chemical ionization mass spectrometry comparison with standards from synthesis. 3. The major metabolite of cis-3-pinanone in each P450 system and in brain of the i.p.-treated mouse in quantitative studies was 2-hydroxy-cis-3-pinanone, and two minor metabolites were hydroxypinanones other than 2-hydroxy-trans-3-pinanone and 4S-hydroxy-cis-3-pinanone. The urine from oral cis-3-pinanone treatment examined on a qualitative basis contained conjugates of metabolites observed in the microsomal systems plus 2,10-dehydro-3-pinanone. 4. Trans-3-pinanone was metabolized more slowly than the cis-isomer in each system to give hydroxy derivatives different than those derived from cis-3-pinanone. 5. Cis- and trans-3-pinanones and hyssop oil act as gamma-aminobutyric acid type A (GABAA) receptor antagonists based on inhibition of 4'-ethynyl-4-n-[2,3-(3)H(2)]propylbicycloorthobenzoate ([(3)H]EBOB) binding in mouse brain membranes (IC(50) of 35-64 microM) and supported by tonic/clonic convulsions in mouse (i.p. LD(50) 175 to >250 mg kg(-1)) alleviated by diazepam. The cis-3-pinanone metabolites 2-hydroxy-cis-3-pinanone and 2,10-dehydro-3-pinanone exhibit reduced toxicity and potency for inhibition of [(3)H]EBOB binding.

Fatty acid amide hydrolase inhibition by neurotoxic organophosphorus pesticides.

Organophosphorus (OP) compound-induced inhibition of acetylcholinesterase (AChE) and neuropathy target esterase explains the rapid onset and delayed neurotoxic effects, respectively, for OP insecticides and related compounds but apparently not a third or intermediate syndrome with delayed onset and reduced limb mobility. This investigation tests the hypothesis that fatty acid amide hydrolase (FAAH), a modulator of endogenous signaling compounds affecting sleep (oleamide) and analgesia (anandamide), is a sensitive target for OP pesticides with possible secondary neurotoxicity. Chlorpyrifos oxon inhibits 50% of the FAAH activity (IC50 at 15 min, 25 degrees C, pH 9.0) in vitro at 40--56 nM for mouse brain and liver, whereas methyl arachidonyl phosphonofluoridate, ethyl octylphosphonofluoridate (EOPF), oleyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (oleyl-BDPO), and dodecyl-BDPO give IC50s of 0.08--1.1 nM. These BDPOs and EOPF inhibit mouse brain FAAH in vitro with > or =200-fold higher potency than for AChE. Five OP pesticides inhibit 50% of the brain FAAH activity (ED50) at <30 mg/kg 4 h after ip administration to mice; while inhibition by chlorpyrifos, diazinon, and methamidophos occurs near acutely toxic levels, profenofos and tribufos are effective at asymptomatic doses. Two BDPOs (dodecyl and phenyl) and EOPF are potent inhibitors of FAAH in vivo (ED50 0.5--6 mg/kg). FAAH inhibition of > or =76% in brain depresses movement of mice administered anandamide at 30 mg/kg ip, often leading to limb recumbency. Thus, OP pesticides and related inhibitors of FAAH potentiate the cannabinoid activity of anandamide in mice. More generally, OP compound-induced FAAH inhibition and the associated anandamide accumulation may lead to reduced limb mobility as a secondary neurotoxic effect.

Phosphoacetylcholinesterase: toxicity of phosphorus oxychloride to mammals and insects that can be attributed to selective phosphorylation of acetylcholinesterase by phosphorodichloridic acid.

Phosphorus oxychloride (POCl(3)) is an intermediate in the synthesis of many organophosphorus insecticides and chemical warfare nerve gases that are toxic to insects and mammals by inhibition of acetylcholinesterase (AChE) activity. It was therefore surprising to observe that POCl(3), which is hydrolytically unstable, also itself gives poisoning signs in ip-treated mice and fumigant-exposed houseflies similar to those produced by the organophosphorus ester insecticides and chemical warfare agents. In mice, POCl(3) inhibits serum butyrylcholinesterase (BuChE) at a sublethal dose and muscle but not brain AChE at a lethal dose. In houseflies, POCl(3)-induced brain AChE inhibition is correlated with poisoning and the probable cause thereof. POCl(3) in vitro is selective for AChE (IC(50) = 12-36 microM) compared with several other serine hydrolases (BuChE, carboxylesterase, elastase, alpha-chymotrypsin, and thrombin) (IC(50) = 88-2000 microM). With electric eel AChE, methylcarbamoylation of the active site with eserine reversibly protects against subsequent irreversible inhibition by POCl(3). Most importantly, POCl(3)-induced electric eel AChE inhibition prevents postlabeling with [(3)H]diisopropyl phosphorofluoridate; i.e., both compounds phosphorylate at Ser-200 in the catalytic triad. Pyridine-2-aldoxime methiodide does not reactivate POCl(3)-inhibited AChE, consistent with an anionic phosphoserine residue at the esteratic site. The actual phosphorylating agent is formed within seconds from POCl(3) in water, has a half-life of approximately 2 min, and is identified as phosphorodichloridic acid [HOP(O)Cl(2)] by (31)P NMR and derivatization with dimethylamine to HOP(O)(NMe(2))(2). POCl(3) on reaction with water and HOP(O)Cl(2) have the same potency for inhibition of AChE from either electric eel or housefly head as well as the same toxicity for mice. In summary, the acute toxicity of POCl(3) is attributable to hydrolytic activation to HOP(O)Cl(2) that phosphorylates AChE at the active site to form enzymatically inactive [O-phosphoserine]AChE.

Chloropicrin dechlorination in relation to toxic action.

Chloropicrin (CCl3NO2) is a widely used soil fumigant with an unknown mechanism of acute toxicity. We investigated the possible involvement of dechlorination in CCl3NO2 toxicity by considering its metabolism, inhibition of pyruvate and succinate dehydrogenases, cytotoxicity in cultured cells, and interaction with hemoproteins. In a newly discovered pathway, CCl3NO2 is metabolized to thiophosgene, which is characterized as the cyclic cysteine adduct (raphanusamic acid) in the urine of mice. CCl3NO2 inhibits porcine heart pyruvate dehydrogenase complex (IC-50 4 microM) and mouse liver succinate dehydrogenase complex (IC-50 13 microM), whereas its dehalogenated metabolites (CHCl2NO2 and CH2ClNO2) are more than 10 times less effective. The inhibitory potency of CCl3NO2 for these dehydrogenase complexes is similar to that of captan, folpet, and dichlone fungicides (IC-50 2-6 microM). CCl3NO2 cytotoxicity with Hepa 1c1c7+ mouse hepatoma cells (IC-50 9 microM) is not correlated with glutathione depletion. Mice treated intraperitoneally with CCl3NO2 at 50 mg/kg but not with an equivalent dose of CHCl2NO2 show increased concentrations of oxyhemoglobin in liver. The acute toxicity of CCl3NO2 in mice is due to the parent compound or metabolites other than CHCl2NO2 or CH2ClNO2 and may be associated with inhibition of the pyruvate dehydrogenase complex and elevated oxyhemoglobin.

Organophosphorus pesticide-induced butyrylcholinesterase inhibition and potentiation of succinylcholine toxicity in mice.

Succinylcholine is the most important rapid-acting depolarizing muscle relaxant during anesthesia. Its desirable short duration of action is controlled by butyrylcholinesterase, the detoxifying enzyme. There are two reported cases of prolonged paralysis from succinylcholine in patients poisoned with the organophosphorus insecticides parathion and chlorpyrifos. The present study examines the possibility that other organophosphorus and methylcarbamate pesticides might also prolong succinylcholine action by inhibiting butyrylcholinesterase using mice treated intraperitoneally as a model and relating inhibition of blood serum hydrolysis of butyrylthiocholine to potentiated toxicity (mouse mortality). The organophosphorus plant defoliant tribufos (4 h pretreatment, 160 mg/kg) and organophosphorus plant growth regulator ethephon (1 h pretreatment, 200 mg/kg) potentiate the toxicity of succinylcholine by seven- and fourfold, respectively. Some other pesticides or analogs are more potent sensitizers for succinylcholine toxicity with threshold levels of 0.5, 1.0, 1.7, 8, 10, and 67 mg/kg for phenyl saligenin cyclic phosphonate, profenofos, methamidophos, tribufos, chlorpyrifos, and ethephon, respectively. Enhanced mortality from succinylcholine is generally observed when serum butyrylcholinesterase is inhibited 55-94%. Mivacurium, a related nondepolarizing muscle relaxant also detoxified by butyrylcholinesterase, is likewise potentiated by at least threefold on 4 hour pretreatment with tribufos (25 mg/kg) or profenofos (10 mg/kg).

Chloropicrin: reactions with biological thiols and metabolism in mice.

Chloropicrin (CCl3NO2) is a preplant soil fumigant as a fungicide, warning agent for other fumigants, and former was gas. Its mode of action is unknown but presumably related to its facile metabolic dechlorination on reaction with biological thiols. These interactions were studied with 14CCl3NO2, 14CHCl2NO2, 14CH2ClNO2, and 14CH3NO2, synthesized for this purpose. Reaction of 14CCl3NO2 with GSH yields the di- and monochloro derivatives, GSSG, and a small amount of nitrite. The initial adduct with GSH is transient, but more stable derivatives are formed at longer times or on catalysis by GSH S-transferase. Human Hb, examined as a model thiol protein, reacts with 1.5 mol equiv of 14CCl3NO2 at pH 7.4 to produce 53-67% 14CHCl2NO2 and 10-11% Hb dducts (radiochemical yields) which, on longer incubation, release 14CHCl2NO2, CHCl2NO2 and CH2ClNO2 react with the same site(s) in Hb but more slowly. The dechlorination reactions are also catalyzed by liver cytosol and microsomes with NADPH. Dechlorination and adduct formation observed in vitro also occur in vivo in ip-treated mice. Thus, 14CCl3NO2 quickly gives 14CHCl2NO2, and some 14CO2 is formed, but the primary urinary metabolites are polar and nonvolatile. The major metabolite from 14CCl3NO2 in urine is less polar than those from 14CHCl2NO2, 14CH2ClNO2, and 14CH3NO2 which, in turn, are different from the 14CH2O and H14CO2Na metabolites. Liver tissue is adducted in vivo by 14CCl3NO2 (43% radiocarbon unextractable with methanol at 1 h), but the total radiocarbon content decreases 4-fold between 1 and 48 h after treatment. Liver effects from a high CCl3NO2 dose are associated with 19% less GSH and 50% more absorbance from soluble hemoproteins than controls. The toxicity of CCl3NO2 is probably due to disruption of multiple targets by its cascade of dechlorination products.

S-methylation as a bioactivation mechanism for mono- and dithiocarbamate pesticides as aldehyde dehydrogenase inhibitors.

S-Methylation is a new bioactivation mechanism for metam and metabolites of methyl isothiocyanate and dazomet in mice. These soil fumigants are converted to S-methyl metam [MeNHC(S)SMe] which reaches peak levels in liver, kidney, brain, and blood 10-20 min after intraperitoneal (ip) treatment. The half-life of S-methyl metam administered ip is 8-12 min in each of these tissues. S-Methyl metam-oxon [MeNHC(O)SMe] is also detected as a metabolite of each of these soil fumigants on analysis by gas chromatography/mass spectrometry with chemical ionization. The conversion of methyl isothiocyanate to S-methyl metam and its oxon probably involves conjugation with glutathione, hydrolysis to S-(N-methylthiocarbamoyl)-cysteine, cleavage by cysteine conjugate beta-lyase to release metam, and finally methylation and oxidative desulfuration. Metam and dazomet are converted to S-methyl metam by mouse liver microsomes on fortification with S-adenosylmethionine. Metam, methyl isothiocyanate, dazomet, and three metabolites (metam-oxon [MeNHC(O)SH], MeNHC(S)SMe, and MeNHC-(O)SMe) administered ip to mice at 40 mg/kg inhibit low-Km liver mitochondrial aldehyde dehydrogenase and elevate ethanol-dependent blood and brain acetaldehyde levels. Several fungicides including the dialkyldithiocarbamates as the disulfide (thiram and the related alcohol-abuse drug disulfiram) and metal salts (ziram) also yield S-methyl thiocarbamate metabolites. Eight S-alkyl and S-(chloroallyl) thiocarbamate herbicides (EPTC, molinate, butylate, vernolate, pebulate, diallate, sulfallate, and triallate), but not their S-chlorobenzyl analog (thiobencarb), undergo sequential liberation of the thiocarbamic acid and then S-methylation, forming the S-methyl thiocarbamates which are new metabolites and potential aldehyde dehydrogenase inhibitors. The S-methyl mono- and dithiocarbamate metabolites of these herbicides and fungicides are easily identified by retention time on gas chromatography and by mass spectrometry giving [MH]+ plus [R1R2NCO]+ or [R1R2NCS]+, respectively, as the two major ions.

Adducts of dienochlor miticide with glutathione, glutathione S-transferases, and hemoglobins.

Dienochlor (Pentac) (C10Cl10) has been used for 30 years as a miticide with little knowledge of its mode of action or metabolic fate except that it is quickly degraded by rats. This study examines the reactions of dienochlor with GSH and proteins as models for its metabolism and interactions with tissues. Dienochlor reacts rapidly with 1.0 mM GSH in phosphate buffer (pH 7.4) at 37 degrees C (t1/2 approximately 11 min) as analyzed by UV/visible spectroscopy and HPLC, yielding a series of more than a dozen adducts. Octachlorofulvalene (C10Cl8), a candidate intermediate, also reacts to give the same apparent products (t1/2 < 0.2 min as above); however, its intermediacy in the dienochlor reaction was not established. Isolation and MS analyses characterized two isomeric C10H2Cl(SG)5 adducts and a C10H2(SG)6 derivative; these products react further in the presence of GSH to yield two even more polar adducts. Cysteine and N-acetylcysteine also react rapidly with dienochlor whereas GSSG and several non-thiol amino acids are much less reactive. Purified GSH S-transferases (GSTs) and hemoglobins, each from six species of mammals including humans, are extensively labeled in vitro by [14C]dienochlor to form adducts separable by gel electrophoresis and HPLC. [14C]Dienochlor readily derivatizes rat liver GSTs even in cytosol and in the presence of high GSH levels. The potency of dienochlor for inhibition of GST activity is maintained or enhanced upon conversion to GSH adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde dehydrogenase of mice inhibited by thiocarbamate herbicides.

The herbicide S-ethyl N,N-dipropylthiocarbamate (EPTC) and three of its candidate metabolites (the sulfoxide, N-depropyl and S-methyl derivatives) inhibit mitochondrial low-Km aldehyde dehydrogenase (ALDH) in liver by 56 to 82% 2 hr after these thiocarbamates are administered intraperitoneally (ip) to mice at 8 mg/kg. They also greatly elevate the acetaldehyde level (determined as the O-benzyloxime ether) in blood (up to 500 microM) and brain (up to 3 ppm) 30 min after two ip treatments, the first with the thiocarbamate at 40 mg/kg and 2 hr later with ethanol at 1000 mg/kg. EPTC at 4 mg/kg inhibits liver ALDH activity by 50% and at 8 and 18 mg/kg gives half of the maximum ethanol-dependent elevation of acetaldehyde levels in blood and brain, respectively. The in vivo effects of other thiocarbamate herbicides at 8 mg/kg on ALDH activity and 40 mg/kg on acetaldehyde levels decrease in the order of thiobencarb, pebulate, vernolate and molinate > butylate and triallate >> cycloate. The percentage inhibition of liver ALDH activity generally correlates with the elevation in blood and brain acetaldehyde under these treatment protocols. B.W. Hart and M.D. Faiman (Biochem. Pharmacol. 43 403-406, 1992) have shown that the alcohol-aversion drug disulfiram is metabolized to S-methyl N,N-diethylthiocarbamate and its sulfoxide as the penultimate and ultimate metabolites inhibiting ALDH. Thus, the thiocarbamate herbicides and their metabolites are similar to the disulfiram metabolites not only in homologous structure but also in their potency range as ALDH inhibitors in vivo. On this basis some of the thiocarbamate herbicides may sensitize agricultural workers to ethanol intoxication.

In vitro susceptibility of bacteria to a ticarcillin-clavulanic acid combination.

In vitro testing of bacterial susceptibility to a combination of ticarcillin and clavulanic acid was done, using 406 aerobic gram-positive and gram-negative isolates (considered to be pathogens) cultured from equine and small animal specimens. A microdilution broth technique of susceptibility testing was performed, using trays with wells containing a range of doubling concentrations of dehydrated ticarcillin (range, 0.50 to 128 micrograms/ml) with fixed concentration of clavulanic acid (4 micrograms/ml). The following isolates of equine origin were (90%) susceptible to concentrations of ticarcillin and clavulanic acid combinations of less than or equal to 16 and 4 micrograms/ml, respectively: Staphylococcus aureus, S intermedius, Klebsiella pneumoniae, Enterobacter aerogenes, Ent agglomerans, Ent cloacae, Escherichia coli, Actinobacillus sp, Corynebacterium pseudotuberculosis, Rhodococcus equi, Proteus vulgaris, and Bordetella bronchiseptica. Isolates of small animal origin (90%) susceptible to less than or equal to 16 and 4 micrograms of ticarcillinclavulanic/ml included S aureus, S intermedius, Ent aerogenes, Ent agglomerans, Pasteurella multocida, B bronchiseptica, Pr mirabilis, and Serratia sp.