CONTEXTUALES DE LA INVESTIGACIÓN
MOVILIDAD EN LA FORMACIÓN DEL
6.1 Introduction
The importance of folic acid supplementation in the prevention of human NTD is well documented (Section 1.3.3). However there has been little progress since the preventive action of vitamin supplementation was described (Smithells et a l 1981), towards
understanding the molecular mechanism by which folate acts to normalise embryonic development. One reason for this is the practical and ethical difficulty of studying embryonic processes in humans. An animal model of folate-preventable NTD would be advantageous since it would provide a tool to study the specific role of folate in the development of NTD and permit experimental studies to analyse the underlying developmental and molecular mechanisms.
6.1.1 Detection of disturbed folate metabolism in NTD
Recent studies of NTD in humans have been concerned with finding a link between deranged folate metabolism and the incidence of NTD.
6.1.1.1 Folate status in the mother and embryo
Initial studies of the folate status of women with pregnancies affected by NTD have proven inconclusive. Measurements of serum folate levels from blood samples taken at birth (Giles, 1966; Emery et a l 1969; Pritchard et a l 1969; Scott et a l 1970) do not provide an accurate assessment of the folate status of the mother at the time that the embryo was undergoing neurulation. Rather more pertinent studies have involved the retrospective analysis of folate status in women with pregnancies affected by NTD, as assessed from blood taken at the first antenatal visit (Hall, 1972; Molloy et a l 1985; Mills et a l 1992). These studies have failed to find an association between maternal serum folate levels and offspring affected by NTD and support the hypothesis that the
preventive role of folate may be to overcome a metabolic block within the embryo rather than to correct a deficiency in the mother. However, subsequent and more detailed studies on folate status, measuring both serum and red blood cell (RBC) folate levels, have shown a reduced RBC folate status associated with pregnancies affected by NTD whereas there is no significant difference between serum folate in controls and affected groups (Smithells et a l 1976; Yates et a l 1987). These findings suggest that the lack of association in early studies may have been due to a lack of sensitivity in detection.
6.1.2 Folate antagonists and NTD
The mode of action of many drugs used in anti-cancer chemotherapy is to block cell proliferation. Folate antagonists are widely used in such treatments to prevent DNA synthesis by blocking specific enzymes in the folate cycle. Retrospective studies have been carried out on births affected by NTD, associating the developmental defect with administration of a folate antagonist at the time of neurulation. These studies have shown that methotrexate (Van den Hof et a l 1990), aminopterin (Feldkamp and Carey,
1993) and 5-fluorouracil (Stephens et a l 1980) increase the risk of spontaneous abortion and are associated with the occurrence of NTD in offspring of mothers undergoing treatment in early pregnancy (Feldkamp and Carey, 1993). More commonly, however, these drugs are associated with an increased incidence of such defects as cleft lip and palate and limb deformities such as syndactyly and polydactyly (Feldkamp and Carey, 1993). These craniofacial and limb defects are associated with drug administration later in the gestation period, after the completion of neurulation, suggesting that the defects are specific to the stage of organogenesis occurring at the time of administration and that the use of antagonists in early gestation is more likely to result in embryonic loss.
The defects seen in human foetuses exposed to folate antagonists mirror those seen in teratology studies in rodents. Administration of 5-fluorouracil to pregnant female mice from ElO onwards results in polydactyly and some craniofacial abnormalities (Dagg et a l 1966a)and at E14 produces hindlimb defects and cleft palate (Dagg et a l 1966b). Administration of the same agent to organ cultured embryonic tissue results in
abnormalities of palate and hind limb development (Shuey et a l 1994). The similarities between human and rodent embryos exposed to folate antagonists gives support to the hypothesis that folate plays a role within the same developmental pathways in both species.
6.1.3 The deoxyuridine (dU) suppression test
The dU suppression test was first described by Killmann in 1964 as a method of measuring vitamin B12 and folic acid disorders in the bone marrow of patients with megaloblastic anaemia. The principle of the test is as follows. There are two sources of deoxythymidine monophosphate (dTMP) for dividing cells: thymidine can be recycled via the salvage pathway or synthesised de novo. Deoxyuridine monophosphate (dUMP) is the main source of dTMP in de novo synthesis, but this is dependant on efficient folate metabolism. In the case of vitamin B 12 or folic acid deficiency, the folate cycle is perturbed and de novo synthesis declines so that the salvage pathway becomes the dominant source of thymidine. In the dU suppression test, radioactively labelled thymidine is provided in the culture medium and its incorporation into DNA is
measured. Prior incubation with dUMP suppresses the uptake of labelled thymidine in normal cells, since de novo synthesis can be stimulated. In contrast, cells with folate metabolic defects cannot respond to a dUMP challenge by increasing their de novo
synthesis, owing to a shortage of 5, 10 methylene tetrahydrofolate, and so incorporation of radioactive thymidine fails to be suppressed. The test is summarised in Figure 6.1.
Haematological studies have shown that an abnormal dU suppression test can be corrected when patients are treated with vitamin B12, where there is a known B 12 deficiency (Metz et a l 1968), and that both folinic acid (Ganeshaguru and Hoffbrand,
1978) and 5-methyl tetrahydrofolate (Taheri et a l 1982) are corrective in cases of both B12 and folic acid deficiency. Thus, the test is sensitive to both underlying metabolic defects, and preventive therapies.
6.1.4 Application of the dU suppression test to whole embryo culture
At present, there seems to be no adequate model for folate-sensitive NTD (reviewed in Section 1.5). The dU suppression test was therefore adapted for use in the whole embryo culture system and applied to mutant mouse models of NTD in an attempt to find a more suitable model of the human condition. The use of the in vitro system is highly significant as the folate status of individual embryos can be assessed in isolation from the maternal environment, which is complicated not only by nutritional variations, but also by the diurnal cycle of folate metabolism within the adult (Wegner and Nau,
1991). The culture system also allows the selection of embryos at the correct stage of development (i.e. prior to and during the period of neural tube closure). This specific timing would be impossible to achieve in vivo as even timed matings produce litters of markedly varied developmental stages, in addition to the developmental variation within individual litters. The development and use£he dU suppression test is described in the present Chapter.