Mapping of a new locus for autosomal recessive non-syndromic mental retardation in the chromosomal region 19p13.12-p13.2: further genetic heterogeneity.

OBJECTIVE: To identify and clinically evaluate four consanguineous families of Israeli Arab origin with non-syndromic mental retardation (NSMR), comprising a total of 10 affected and 24 unaffected individuals. PARTICIPANTS AND METHODS: All the families originated from the same small village and had the same family name. Association of the condition in these families with the two known autosomal recessive NSMR loci on chromosomes 3p25-pter and 4q24 (neurotrypsin gene) was excluded. RESULTS: Linkage of the disease gene to chromosome 19p13.12-p13.2(Zmax = 7.06 at theta = 0.00) for the marker D19S840 was established. All the affected individuals were found to be homozygous for a common haplotype for the markers cen-RFX1-D19S840-D19S558-D19S221-tel. CONCLUSIONS: The results suggest that the disease is caused by a single mutation derived from a single ancestral founder in all the families. Recombination events and a common disease bearing haplotype defined a critical region of 2.4 Mb, between the loci D19S547 proximally and D19S1165 distally.

Oxidative bioactivation of methamidophos insecticide: synthesis of N-hydroxymethamidophos (a candidate metabolite) and its proposed alternative reactions involving N-->O rearrangement or fragmentation through a metaphosphate analogue.

The systemic insecticide methamidophos, MeO(MeS)P(O)NH2, is a very weak inhibitor of acetylcholinesterase (AChE) in vitro relative to in vivo suggesting bioactivation. This hypothesis is supported by finding that brain AChE inhibition and poisoning signs from methamidophos are greatly delayed in mice and houseflies pretreated with oxidase inhibitors in an order for effectiveness of methimazole > N-benzylimidazole >> piperonyl butoxide. In contrast, the order for delaying parathion-induced AChE inhibition and toxicity is N-benzylimidazole >> piperonyl butoxide or methimazole, suggesting that different oxidases are involved in methamidophos and parathion activation. N-Hydroxylation is examined here as an alternative to the controversial S-oxidation proposed earlier for methamidophos activation. N-Hydroxymethamidophos [MeO(MeS)P(O)NHOH], synthesized by coupling MeO(MeS)P(O)Cl and Me3SiNHOSiMe3 followed by desilylation, is unstable at pH 7.4 (t1/2 = 10 min at 37 degrees C) with decomposition by two distinct and novel mechanisms. The first mechanism (A) is N-->O rearrangement to MeO(MeS)P(O)ONH2 and then hydrolysis to MeO(MeS)P(O)OH, a sequence also established in the analogous series of (MeO)2P(O)NHOH-->(MeO)2P(O)ONH2-->(MeO)2P(O)OH. The second mechanism (B) is proposed to involve tautomerism to the phosphimino form [MeO(MeS)P(OH)=NOH] that eliminates MeSH forming a metaphosphate analogue [MeOP(O)=NOH] trapped by water to give MeO(HO)P(O)NHOH that undergoes the N-->O rearrangement as above and hydrolysis to MeOP(O)(OH)2. As a metaphosphate analogue, the metaphosphorimidate generated from MeO(MeS)P(O)NHOH in aqueous ethanol yields MeOP(O)(OH)2 and MeO(EtO)P(O)OH in the same ratio as the solvents on a molar basis. Reactions of the N- and O-methyl derivatives of MeO(MeS)P(O)NHOH and (MeO)2P(O)NHOH are consistent with proposed mechanisms A and B. N-Hydroxymethamidophos is less potent than methamidophos as an AChE inhibitor and toxicant possibly associated with its rapid hydrolysis. Bioactivation of methamidophos via a metaphosphate analogue would directly yield a phosphorylated and aged AChE resistant to reactivating agents, an intriguing hypothesis worthy of further consideration.

Acephate insecticide toxicity: safety conferred by inhibition of the bioactivating carboxyamidase by the metabolite methamidophos.

Acephate is an important systemic organophosphorus insecticide with toxicity attributed to bioactivation on metabolic conversion to methamidophos (or an oxidized metabolite thereof) which acts as an acetylcholinesterase (AChE) inhibitor. The selective toxicity of acephate is considered to be due to facile conversion to methamidophos in insects but not mammals. We show in the present investigation that a carboxyamidase activates acephate in mice and in turn undergoes inhibition by the hydrolysis product, i.e., methamidophos; thus, the bioactivation is started but immediately turned off. These relationships are established by finding that 4 h pretreatment of mice with methamidophos i.p. at 5 mg/kg has the following effects on acephate action: reduces methamidophos and acephate levels in liver by 30-60% in the first 2 h after i.p. acephate dosage; inhibits the liver carboxyamidase cleaving [14CH3S]acephate to [14CH3S]methamidiphos with 50% block at approximately 1 mg/kg; strongly inhibits 14CO2 liberation from [CH3(14)C(O)]acephate in vivo; markedly alters the pattern of urinary metabolites of acephate by increasing O- and S-demethylation products retaining the carboxyamide moiety; greatly reduces the brain AChE inhibition following acephate treatment; doubles the LD50 of i.p.-administered acephate from 540 to 1140 mg/kg. Methamidophos pretreatment in rats also markedly alters the metabolism of dimethoate (another systemic insecticide) from principally carboxyamide hydrolysis to mainly other pathways. In contrast, methamidophos pretreatment of houseflies does not alter the acephate-induced toxicity and brain AChE inhibition. The safety of acephate in mammals therefore appears to be due to conversion in small part to methamidophos which, acting directly or as a metabolite, is a potent carboxyamidase inhibitor, thereby blocking further activation.

S-methylation of O,O-dialkyl phosphorodithioic acids: O,O,S-trimethyl phosphorodithioate and phosphorothiolate as metabolites of dimethoate in mice.

O,O,S-Trimethyl phosphorodithioate and phosphorothiolate [(MeO)2P(S)SMe and (MeO)2P-(O)SMe, respectively are known from earlier studies to be impurities, delayed toxicants, and detoxication inhibitors in several major O,O-dimethyl phosphorodithioate insecticides. Our recent studies show extensive S-methylation of mono- and dithiocarbamic acids in mice, suggesting the possibility that phosphorodithioic acids such as (MeO)2P(S)SH might also undergo S-methylation. This possibility was examined in ip-treated mice with emphasis on the metabolites of dimethoate [(MeO)2P(S)SCH2C(O)NHMe], one of the most important organophosphorus insecticides. The urinary metabolites of dimethoate, which contains no P-SMe substituent, were found to include four compounds with P-SMe moieties identified by 31P NMR spectroscopy as MeO(HS)P(O)SMe, MeO(HO)P(O)SMe, (MeO)2P(S)SMe, and (MeO)2P-(O)SMe; the latter two compounds are also established by GC-MS as dimethoate metabolites in mouse urine, liver, kidney, and lung. Several approaches verified unequivocally that the previously unknown P-SMe metabolites in urine and tissues are due to in vivo S-methylation rather than to impurities. Studies with other O,O-dimethyl and O,O-diethyl phosphorodithioate insecticides established the analogous S-methylation pathway for ethion, malathion, phenthoate, phosalone, and phosmet in mice. Thus, metabolism of O,O-dialkyl phosphorodithioate insecticides in mammals is shown here for the first time to yield S-methyl phosphorodithioates and phosphorothiolates from in vivo S-methylation of the intermediate O,O-dialkyl phosphorodithioic acids.

Retro-Diels-Alder reaction: possible involvement in the metabolic activation of 7-oxabicyclo[2.2.1]hepta-2(3),5(6)-diene-2,3-dicarboxylates and a phosphonate analog.

A discontinuous structure-activity relationship signaled a change in mode of action and led to the discovery of a possible novel metabolic activation mechanism. The toxicity of the herbicide endothal (exo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid) to mice (ip LD50 = 14 mg/kg) is attributed to the inhibition of protein phosphatase 2A (PP2A) at the cantharidin binding site. The potency is reduced by the introduction of a 2,3- or 5,6-double bond. Surprisingly, high toxicity (ip LD50's = 15-50 mg/kg) is restored in oxabicyclohepta-2(3),5(6)-dienes substituted in the 2- and 3-positions with bis(methyl carboxylate), bis(ethyl carboxylate), and diethyl phosphonate/ethyl carboxylate, whereas the dicarboxylic acid, bis(tert-butyl carboxylate), and bis(dimethyl phosphonate) are inactive. The diene adducts do not inhibit the cantharidin binding site of PP2A. Two observations provided an alternative working hypothesis that the active but not the inactive diene adducts are protoxicants: GC analyses revealed that selected bicyclic dienes readily undergo thermal dissociation by retro-Diels-Alder reactions to liberate disubstituted acetylenes; the liberated acetylenes have mouse ip LD50's of 8-25 mg/kg. Apparent exceptions to this hypothesis are that bicyclic dienes with bis(tert-butyl carboxylate) and bis(dimethyl phosphonate) substituents are not toxic, yet their corresponding acetylenes are quite toxic. These apparent anomalies are resolved by finding that only the toxic bicyclic dienes readily react with albumin and 4-nitrobenzenethiol and that their low-toxicity analogs are much less reactive. Albumin can be replaced by hemoglobin but not by myoglobin or chymotrypsin in reaction with a bicyclic diene indicating the importance of the free thiol group. Diethyl oxabicycloheptadienedicarboxylate readily reacts with GSH to give two products, which are also formed from the corresponding acetylene, identified as the cis and trans isomers of the GSH-acetylene conjugate. This is the first proposal, to our knowledge, that a retro-Diels-Alder-type reaction is involved in the metabolic activation of a toxicant.