Teratogenic properties of L-2,4-Diaminobutyric acid and polyamine levels in the murine neural tube defect model
Matladi, Dineo Daphney
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L-2,4-Diaminobutyric acid (DABA) is a non-protein amino acid and constituent of numerous plants (i.e. Lathyrus species) and microorganisms. This relatively abundant amino acid is a known neurotoxin, neurolathyrogen and urea cycle inhibitor and commonly occurs in the food chain of vulnerable populations in the rural regions of Africa (i.e. Ethiopia, South Africa) and India. In spite of the fact that DABA is a well-studied toxic agent, it has not yet been reported to be associated with birth defects in man or domestic animals. DABA is a structural homologue of L-ornithine (Orn) and it was subsequently proposed that this potential Orn anti-metabolite may be teratogenic by acting as an inhibitor of ornithine decarboxylase (ODC) and subsequently by modulating polyamine concentrations in experimentally exposed pregnant female mice. The objectives of this investigation were to investigate the purported teratogenic properties of DABA in a murine NTD model, to establish the effect of DABA on hepatic and whole embryo ornithine ODC and to broadly investigate its effect on the general metabolism of pregnant female mice and their embryos. Pregnant Hanover.NMRI female mice were dosed (per os) with DABA (total dose: 450 -1350 mg/kg body mass) on gestation days 7, 8, and 9. Controls animals received a saline solution. The females were kept in metabolic cages - 24-hour urine samples were collected (gestation day 10) and stored (-70 °C). Following decapitation of the pregnant females on (gestation day 18), their livers and embryos were harvested and the livers immediately frozen in at -70 °C. The harvested embryos were stereo-microscopically examined to assess the occurrence, incidence and nature of congenital defects and then stored at -70°C. DABA proved to be highly toxic to mouse foetuses at all administered dose levels and foetal mortality was extremely high. A relatively high proportion of foetuses displayed growth retardation and congenital defects. Neural tube defects (i.e. exencephali) occurred in 5 -17% of DABA-exposed foetuses. These results corroborate the notion that DABA is an embryotoxic and teratogenic agent. Urinary organic acids, acylcarnitines and amino acids were quantified using GC-MS, ES-MS-MS and the Phenomenex EZ:faast® GC-MS procedures respectively. The detrimental effects of DABA on the metabolism of female Hanover.RPMI mice were obvious from the complexity of and profound changes in the urinary metabolic profiles of DABA-treated females, relative to those obtained for control animals. DABA had clearly left an enormous, but non-specific footprint on the general biochemistry of DABA-treated females and a variety of crucial metabolic processes (glycolysis, tricarboxylic acid cycle, urea cycle, etc.) were adversely affected. Enzyme assays were developed and optimised to assess the catalytic activity of ODC in hepatic and whole embryo cytosols and to quantify the total polyamine concentration in tissue (liver, embryo) and urine samples. The catalytic activity of ODC in maternal livers and whole embryos, harvested from DABA-treated pregnant females, were significantly inhibited (-53%; p<0.0001). Total polyamine concentrations in the liver and whole embryo cytosols and maternal urine samples were similarly depressed (-70%; p<0.05). These results unambiguously demonstrated that DABA can act as an Orn anti-metabolite, inhibit the ODC-catalyzed production of Put from Orn and depress the biosynthesis of polyamines (Put, Spd, Spm). The relatively low levels of polyamines during the critical stages of neural tube closure may have adversely affected polyamine-directed regulation of the cell cycle, subsequently causing the dysmorphogenesis (i.e. NTD) observed in some of the embryos of DABA-treated pregnant female mice.