Perturbation of critical metabolic processes associated with 3-hydroxynorvaline induced teratogenesis
Neural tube defects (NTD) are a group of folate-responsive congenital defects that occur relatively frequently in humans. NTD display a multi-factorial aetiology, resulting from a complex interplay of genetic and environmental factors (i.e. dietary folate and/or vitamin B12 deficiency, teratogenic xenobiotics, etc.). β-Hydroxynorvaline (HNV) is a proven toxic, non-protein amino acid (xenobiotic agent), structurally related to L-threonine and L-serine and able to substitute L-threonine in the primary structure of proteins. The main objectives of this study were to investigate the teratogenic potential of HNV in the chicken embryo and Hanover NMRl mouse embryo models and to elucidate some of the molecular mechanisms involved in the aetiology of NTD. HNV was dosed to chicken embryos (in ovo), 24 h post incubation (p.i.) at 37.8 °C ± 0.5 °C. Controls received a sterile saline solution. Chicken embryos were removed 12 days p.i., weighed, fixed in Allen's solution and investigated stereo-microscopically to assess the incidence and nature of dysmorphogenic events (i.e. NTD). Body, toe and beak lengths of the chicken embryos were measured. Chicken embryo fibroblasts were cultured and used to measure the effect of HNV on the biosynthesis of DNA in fibroblasts. Pregnant Hannover NMRl female mice were dosed with HNV or a saline solution (per os) on days 7-9 post coitus (p.c.). Following the last dose of HNV on day 9, the pregnant mice were placed in metabolic cages for 24 h to collect urine samples. Urinary organic acids (GC-MS), acylcarnitines and amino acids (ESI-MSMS) were quantitatively and qualitatively determined to assess the catabolic breakdown of H NV and its effects on vital metabolic processes, such as amino acid catabolism and the P-oxidation of fatty acids. Control and HNV exposed mouse embryos were removed on days 10 or 18 post coitus (p.c.). Embryos, removed from each individual mother on day 10 were pooled and either immediately used to assess the catalytic activity of the glycine cleavage system (GCS), or stored at -75 °C until the catalytic activities of cytosolic (cSHMT), mitochondria1 serine hydroxymethyltransferase (mSHMT) and citrate synthase (CS) could be assayed. Mouse embryos removed on day 18 p.c., weighed and stereo-microscopically investigated to assess the incidence and nature of dysmorphogenic events. Bio-indicators of the effect of HNV on the flow of one-carbon units through the folate and remethylation cycles (i.e. [3H]-thyrnidine incorporation, DNA methylation and synthesis, polyamine synthesis, carnitine synthesis, etc.) were determined in the liver tissues of pregnant females and in pooled batches of whole embryos. HNV proved to be embryotoxic and displayed the capacity to induce a variety of congenital defects, including NTD, in both the chicken and mouse embryo models. The incidence of NTD in both models proved to be dose-dependent. Selected stereoisomers of HNV were rapidly catabolised and the main HNV derived metabolite in the urines of HNV treated pregnant mice, was identified as 2,3- dihydroxypentanoic acid (DHPA; GC-MS). The structure of DHPA was confirmed by chemical synthesis and subsequent GC-MS, NMR (13c-NMR, 1H-NMR, HETCOR and COSY) spectroscopy and IR spectrometry. HNV altered the flow of one-carbon units through the folate and remethylation cycles, causing a decrease in DNA synthesis, DNA methylation, polyarnine biosynthesis, carnitine and trimethyllysine synthesis. Free carnitine stores in HNV treated pregnant mice appeared to be depleted, probably due to a combined effect of the detoxification of vast amounts of accumulated metabolites, generated as a result of HNV toxicosis and decreased carnitine biosynthesis. HNV also appeared to have altered serinelglycine interconversion, due to an inhibition of cSHMT and to a lesser degree the inhibition of GCS. Organic acid profiles of urine samples, collected from HNV treated pregnant mice, suggested that HNV had induced a general ketothiolase defect in pregnant females by inhibiting the P-oxidation of fatty acids, isoleucine catabolism and ketone body utilisation. HNV affected the hornocysteine to cysteine transulffuration by acting as a substrate for CBS, culminating in the biosynthesis of Sethylcysteine (GC-MS). The presence of 3-ethylcysteine in the urines of HNV treated pregnant mice was confirmed by GC-MS. following its in vitro synthesis, employing a reaction system containing mouse liver hornogenate, homocysteine, HNV and pyridoxal-5- phosphate. In conclusion, HNV can apparently cause multiple metabolic perturbations in pregnant mice and their developing embryos. One-carbon flux, energy metabolism and a number of other vital biochemical processes can be adversely affected, resulting in a disturbance of normal embryonic development (i.e. proper closure of the neural tube) and subsequent dysmorphogenesis in developing embryos.