Effects of carbon monoxide and hypoxia on cleft lip in A/J mice.

Epidemiologic evidence indicates an increase in cleft lip with or without cleft palate [CL(P)] in infants of mothers who smoke cigarettes. It appears that the principle mechanism is through carbon monoxide (CO) decreasing the oxygen (O2) available to the embryo. Previous studies have shown that maternal respiratory hypoxia can increase the incidence of CL(P) in mice. The present investigation was designed to analyze the effects of altered levels of CO and O2 in respiratory gases on the incidence of CL(P) in genetically susceptible A/J mice. Results from blood gas analysis, after a 24-hour exposure of pregnant mice during the time of primary palate development, showed that CO levels of 180 ppm in air decrease oxyhemoglobin (%O2Hb) and increased carboxyhemoglobin (%COHb) to slightly above the high end of the range found in human studies of cigarette smokers. Interestingly, the control COHb levels were higher in our CL(P) sensitive mouse strain compared with those of the range of increases found in human smokers, versus nonsmoker studies, and that the increase for treated mice (3x) was at the low end of the range for smokers. Decreasing O2 levels to 10% from 21% (normal percentage in air) more severely decreased %O2Hb and moderately decreased %COHb. At 24 hours of exposure, the incidence of CL(P) and resorption was approximately the same for both the CO and the control groups, but there were significant increases in the incidence of resorptions in the hypoxia group and of CL(P) in relation to the CO group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal craniofacial development: new insights on normal and abnormal mechanisms.

Technical advances are radically altering our concepts of normal prenatal craniofacial development. These include concepts of germ layer formation, the establishment of the initial head plan in the neural plate, and the manner in which head segmentation is controlled by regulatory (homeobox) gene activity in neuromeres and their derived neural crest cells. There is also a much better appreciation of ways in which new cell associations are established. For example, the associations are achieved by neural crest cells primarily through cell migration and subsequent cell interactions that regulate induction, growth, programmed cell death, etc. These interactions are mediated primarily by two groups of regulatory molecules: "growth factors" (e.g., FGF and TGFalpha) and the so-called steroid/thyroid/retinoic acid superfamily. Considerable advances have been made with respect to our understanding of mechanisms involved in primary and secondary palate formation, such as growth, morphogenetic movements, and the fusion/merging phenomenon. Much progress has been made on the mechanisms involved in the final differentiation of skeletal tissues. Molecular genetics and animal models for human malformations are providing many insights into abnormal development. A mouse model for the fetal alcohol syndrome(FAS), a mild form of holoprosencephaly, demonstrates a mid-line anterior neural plate deficiency which leads to olfactory placodes being positioned too close to the mid-line, and other secondary changes. Work on animal models for the retinoic acid syndrome (RAS) shows that there is major involvement of neural crest cells. There is also major crest cell involvement in similar syndromes, apparently including hemifacial microsomia. Later administration of retinoic acid prematurely and excessively kills ganglionic placodal cells and leads to a malformation complex virtually identical to the Treacher Collins syndrome. Most clefts of the lip and/or palate appear to have a multifactorial etiology. Genetic variations in TGF alpha s, RAR alpha s, NADH dehydrogenase, an enzyme involved in oxidative metabolism, and cytochrome P-450, a detoxifying enzyme, have been implicated as contributing genetic factors. Cigarette smoking, with the attendant hypoxia, is a probable contributing environmental factor. It seems likely that few clefts involve single major genes. In most cases, the pathogenesis appears to involve inadequate contact and/or fusion of the facial prominences or palatal shelves. Specific mutations in genes for different FGF receptor molecules have been identified for achondroplasia and Crouzon's syndrome, and in a regulatory gene (Msx2) for one type of craniosynostosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Prenatal craniofacial development: new insights on normal and abnormal mechanisms.

Technical advances are radically altering our concepts of normal prenatal craniofacial development. These include concepts of germ layer formation, the establishment of the initial head plan in the neural plate, and the manner in which head segmentation is controlled by regulatory (homeobox) gene activity in neuromeres and their derived neural crest cells. There is also a much better appreciation of ways in which new cell associations are established. For example, the associations are achieved by neural crest cells primarily through cell migration and subsequent cell interactions that regulate induction, growth, programmed cell death, etc. These interactions are mediated primarily by two groups of regulatory molecules: "growth factors" (e.g., FGF and TGF alpha) and the so-called steroid/thyroid/retinoic acid superfamily. Considerable advances have been made with respect to our understanding of the mechanisms involved in primary and secondary palate formation, such as growth, morphogenetic movements, and the fusion/merging phenomenon. Much progress has been made on the mechanisms involved in the final differentiation of skeletal tissues. Molecular genetics and animal models for human malformations are providing many insights into abnormal development. A mouse model for the fetal alcohol syndrome (FAS), a mild form of holoprosencephaly, demonstrates a mid-line anterior neural plate deficiency which leads to olfactory placodes being positioned too close to the mid-line, and other secondary changes. Work on animal models for the retinoic acid syndrome (RAS) shows that there is major involvement of neural crest cells. There is also major crest cell involvement in similar syndromes, apparently including hemifacial microsomia. Later administration of retinoic acid prematurely and excessively kills ganglionic placodal cells and leads to a malformation complex virtually identical to the Treacher Collins syndrome. Most clefts of the lip and/or palate appear to have a multifactorial etiology. Genetic variations in TGF alpha s, RAR alpha s, NADH dehydrogenase, an enzyme involved in oxidative metabolism, and cytochrome P-450, a detoxifying enzyme, have been implicated as contributing genetic factors. Cigarette smoking, with the attendant hypoxia, is a probable contributing environmental factor. It seems likely that few clefts involve single major genes. In most cases, the pathogenesis appears to involve inadequate contact and/or fusion of the facial prominences or palatal shelves. Specific mutations in genes for different FGF receptor molecules have been identified for achondroplasia and Crouzon's syndrome, and in a regulatory gene (Msx2) for one type of craniosynostosis. Poorly co-ordinated control of form and size of structures, or groups of structures (e.g., teeth and jaws), by regulatory genes should do much to explain the very frequent "mismatches" found in malocclusions and other dentofacial "deformities". Future directions for research, including possibilities for prevention, are discussed.

Foodhandling practices of Dunedin caterers: a cause for concern.

AIM: Foodhandling practices in the Dunedin catering industry were assessed to determine if they were correct, preventing the transmission of foodborne disease to the public. METHOD: A random sample of 300 foodservice outlets was selected. Information was collected during personal interviews with managers using a prepared questionnaire. Potentially dangerous practices were further analysed and summed to identify "at risk" operations. RESULTS: Response rate was 71%. Thirty-six percent of restaurants, 27% of takeaway bars and 25% of deli-bars and butchers could have been considered at risk operations. Few managers had any formal training in preparing and serving food, and less than 20% of managers and personnel had any food hygiene training. CONCLUSIONS: Current legislation and its enforcement is unable to adequately address incorrect food handling practices. It is recommended that revisions of the law emphasise the importance of correct practices and their enforcement. Food hygiene training should become compulsory for all who work in the catering industry.

Animal models for human craniofacial malformations.

Holoprosencephaly malformations, of which the fetal alcohol syndrome appears to be a mild form, can result from medial anterior neural plate deficiencies as demonstrated in an ethanol treated animal model. These malformations are associated with more medial positioning of the nasal placodes and resulting underdevelopment or absence of the medial nasal prominences (MNPs) and their derivatives. Malformations seen in the human retinoic acid syndrome (RAS) can be produced by administration of the drug 13-cis-retinoic acid in animals. Primary effects on neural crest cells account for most of these RAS malformations. Many of the malformations seen in the RAS are similar to those of hemifacial microsomia, suggesting similar neural crest involvement. Excessive cell death, apparently limited to trigeminal ganglion neuroblasts of placodal origin, follows 13-cis retinoic acid administration at the time of ganglion formation and leads to malformations virtually identical to those of the Treacher Collins syndrome (TCS). Secondary effects on neural crest cells in the area of the ganglion appear to be responsible for the TCS malformations. Malformations of the DiGeorge Syndrome are similar to those of the RAS and can be produced in mice by ethanol administration or by "knocking out" a homeobox gene (box 1.5). Human and animal studies indicate that cleft lips of multifactorial etiology may be generically susceptible because of small MNP)s or other MNP developmental alterations, such as those found in A/J mice, that make prominence contact more difficult. Experimental maternal hypoxia in mice indicates that cigarette smoking may increase the incidence of cleft lip by interfering with morphogenetic movements. Other human cleft lips may result from the action of a single major gene coding for TGF-alpha variants. A study with mouse palatal shelves in culture and other information suggest that a fusion problem may be involved.

Congenital malformations of the external, middle, and inner ear produced by isotretinoin exposure in mouse embryos.

Isotretinoin (Accutane), a widely used dermatologic drug, produces severe congenital malformations when used during pregnancy. The isotretinoin teratogen syndrome consists of multiple cardiovascular and craniofacial anomalies, most commonly involving the external ear. This study examined the pathogenesis of isotretinoin teratogenicity in a mouse model, using microdissection and histologic examination of fetal mouse ears after treatment with the drug at various stages of embryonic development. In this study, earlier treatment times frequently produced microtia similar to that seen in affected infants, as well as recognizable patterns of temporal bone and ossicular abnormalities; exposure at a later developmental stage resulted in facial tags with less severely affected ears. Possible teratogenic mechanisms of isotretinoin are discussed. Suitability of the mouse model for studying human congenital craniofacial malformations, such as Goldenhar's and Treacher Collins Syndrome, is also explored.

Pathogenesis of cleft palate in Treacher Collins, Nager, and Miller syndromes.

Abnormalities of the secondary palate were studied in an animal model in which features of Treacher Collins syndrome (TCS) and Nager or Miller syndromes (both of which are facially similar to Treacher Collins, but include limb malformations) were induced by acute maternal exposure to 13-cis-retinoic acid (13-cis-RA, isotretinoin, Accutane). Previous work in our laboratory has illustrated that excessive cell death in the proximal aspect of the maxillary and mandibular prominences of the first visceral arch and in the apical ectodermal ridge of the limb bud probably accounts for the characteristic craniofacial and limb abnormalities observed (Sulik et al, 1987; Sulik and Dehart, 1988). The current study shows that maternal treatment with 400 mg per kilogram 13-cis-RA at 8 days 14 hours (8d14hr) or 9d6hr post fertilization results in abnormalities of the secondary palate that vary in incidence and severity. Following the earlier treatment time, 82 percent (68 of 74) of the 18d fetuses were affected, with, severely hypoplastic, unfused palatal shelves present in 34 percent (25 of 74). The less severely affected fetuses had malformations that involved primarily the posterior aspect of the palatal shelves. This malformation (foreshortening of the posterior portion of the palate) constituted the major developmental alteration that resulted from treatment at the later time, at which time a 52 percent (26 of 50) malformation incidence was seen. The change in pattern of malformations with treatment time is consistent with the changing pattern of programmed cell death, which was observed to occur in the first visceral arch.

Mandibulofacial dysostosis (Treacher Collins syndrome): a new proposal for its pathogenesis.

Acute exposure to 400 mg/kg 13-cis retinoic acid (13-cis RA, isotretinoin, Accutane) on the ninth day postfertilization in mice (a time that corresponds to the fourth week postfertilization in humans) results in malformations that characterize mandibulofacial dysostosis (MFD, Treacher Collins syndrome). Deficiencies in the infraorbital region and in the mandibular ramus and condyle, abnormalities of the secondary palate, and external ear malformations were observed. Light and scanning electron microscopic analyses of affected embryos illustrate that within 12 hours of maternal 13-cis RA treatment, markedly excessive (possibly premature) cell death occurs in regions where some of the cells are normally destined to undergo programmed cell death. Previous studies with retinoids have shown that they labilize lysosomal membranes and expand and strengthen regions of programmed cell death. Of particular interest for this study was cell death occurring in the dorsal (proximal) aspects of the maxillary and mandibular prominences of the first visceral arch, the second visceral arch, and the first visceral cleft, areas that correspond to the locations of the first and second arch ectodermal ("ganglionic") placodes and first closing membrane, respectively. The derivatives of this region are those that are severely affected in MFD. As described in previous reports from this laboratory, 13-cis RA is known to interfere with neural crest cells, resulting in major craniofacial malformations. However, the exposure times involved were earlier than those described herein. It is hypothesized that effects on the first and second arch ectodermal placodal cells at a time following the release from the neural folds of neural crest cells into the developing cranial region are of great significance in the pathogenesis of MFD. This is in contrast to the prevailing hypothesis that these malformations are the direct result of a primary interference with neural crest cells.

Abnormal heart and great vessel development following acute ethanol exposure in mice.

Two maternal intraperitoneal doses of ethanol (2.9 g/kg) administered on gestational day (GD) 8, 12 hours (8 d, 12 h) and 8 d, 16 h result in abnormal heart and great vessel development in C57B1/6J mice. Heparinized hearts from GD 9 to 18 conceptuses were dissected to expose the forming or already completed ventricular septum and great vessels and then routinely processed for scanning electron microscopy. As early as GD 9 (12 hours post-treatment), size deficiency and abnormal external contour of the cardiac tube were notable. By GD 12, deficiencies in the size of the conal and atrioventricular (A-V) endocardial cushions, as well as abnormal positioning of the A-V canal, were demonstrable. On GD 13, when the ventricular septum should be complete, a range of deficiencies in the conal tissue was observed. Deficiencies observed were a lack of closure of the ventricular septum in the region of the membranous septum, and lack of septation of the conal portion of the developing heart. These deficiencies persist and have been documented through GD 18. Other abnormalities noted on GD 18 include double-outlet right ventricle, as well as distal defects of the great vessels including interrupted aortic arch, right aortic arch, and a vascular ring. While these defects are comparable to those seen in the fetal alcohol syndrome, they also overlap considerably with cardiac defects that are characteristic of those in the DiGeorge Syndrome as well as in the CHARGE Association. Recent work by others as well as the fact that acute ethanol exposure in this animal model corresponds to a time of neural crest cell migration has led to the speculation that this cell population is involved in the cardiovascular pathogenesis described.

Isotretinoin embryopathy and the cranial neural crest: an in vivo and in vitro study.

Severe congenital malformations have been associated with the inadvertant use in early pregnancy of a new dermatological drug, isotretinoin. We present proposals for the pathogenesis of this embryopathy based on the study of animal models. The characteristic malformations of the face, thymus, and great vessels were induced in mice by prenatal exposure to the drug during the early somite stages of development. From histological examination of mouse embryos it was shown that the drug directly interferes with the development of cranial neural crest cells. Subsequent deficiency of crest cell-derived mesenchyme adequately explains most of the observed malformations. Rat embryo culture studies showed that, when used at concentrations of 500 ng/ml, both isotretinoin and its main metabolite in the human, 4-oxo-isotretinoin, induce malformations similar to those seen in vivo. Since during normal repetitive dosing in the human the mean trough blood concentration of isotretinoin ranges from 132 to 196 ng/ml, while 4-oxo-isotretinoin ranges from 610 to 791 ng/ml, it is likely that the metabolite plays a major role in the induction of the isotretinoin embryopathy.

Morphogenesis of hypoxia-induced cleft lip in CL/Fr mice.

Cleft lip with or without associated cleft palate [CL(P)], one of the most common human malformations, is in most cases, believed to be caused by a combination of genetic and environmental factors. Previous studies have shown that maternal respiratory hypoxia (10% O2) increases the incidence of CL(P) from the spontaneous level of 36% to 89% in CL/Fr mice. The current investigation was designed to study, morphologically, the developmental alterations of the primary palate primordia in CL/Fr embryos, following a reduction in maternal respiratory oxygen levels. Scanning electron microscopy was utilized to compare the development of 35-43 somite hypoxia and control (normoxia) embryos. Hypoxia increased the incidence of resorptions and increased the incidence of CL(P) in viable embryos, compared to normoxia. Debris, most of which was limited to the deeper aspects of the invaginating nasal placode, was present in hypoxia embryos at stages prior to primary palate fusion and was absent in comparably staged normoxia embryos. It is believed that this debris is cellular in nature and that associated retardation of placodal invagination is primarily responsible for the increased incidence of CL(P). Other effects on morphogenesis and/or growth retardation may also be contributing factors.

Fetal alcohol syndrome and DiGeorge anomaly: critical ethanol exposure periods for craniofacial malformations as illustrated in an animal model.

Acute maternal ethanl (alcohol) administration induces different craniofacial anomalies in the offspring of experimental animals, depending on the gestational day of teratogen exposure. Previous studies in our laboratories have illustrated the sequence of developmental changes leading to the "typical" fetal alcohol syndrome (FAS) craniofacial phenotype which results from teratogen exposure during gastrulation. These facial features are accompanied by deficiencies in median forebrain derivatives. Ethanol teratogenesis at this time apparently results in a loss of midline territory of the embryonic disc with little effects on neural crest-dependent laterally derived structures including the visceral arches. Acute ethanol exposure in mice 1 1/2 days later, at a time when neural crest cells are populating the frontonasal prominence and the visceral arches, results in a craniofacial phenotype that is similar to that noted in the DiGeorge anomaly or sequence. Sequential scanning electron microscopic analysis in our laboratory of embryos exposed on day 8 1/2 have illustrated deficiencies in the developing facial prominences and the visceral arches. The developing forebrain and midbrain appear hypoplastic. We have also observed heart, great vessel, and thymus abnormalities in these fetuses. Histologic analyses indicate that a common pathogenetic basis for the above-mentioned (day 8 1/2-induced) fetal alcohol effects appears to be an interference with the integrity of the cranial (including occipital) neural crest. Other discrete cell populations may also be involved since we have observed abnormalities in other regions, including placodal and closing membrane tissues. This animal model provides evidence linking maternal ethanol abuse during the 3rd or 4th weeks of human gestation to the development in the conceptus of FAS or DiGeorge anomally craniofacial characteristics, respectively. As the DiGeorge anomaly has been noted in the offspring of alcoholic women, this animal model indicates that ethanol and/or its metabolites is, in these cases, the causative agent.

Normal and abnormal development of the lip and palate.

Largely because fusion of the lip and the palate are developmental weak points, common facial clefts may arise in a great many ways, both experimentally and in man. However, we believe that the vast majority of human clefts have similar origins with minor variations. One must always question the appropriateness of animal models. Apparently appropriate animal models are giving us considerable insight into the manner in which genetic and environmental factors alter development and how they interact with one another in the developing embryo. These studies help us understand the nature of the multifactorial threshold concept as it applies to cleft lip and cleft palate, and they emphasize the potential importance of even "minor" environmental factors in determining on which side of the threshold for clefting an individual embryo may fall.

An implant labeling technique employing sable hair probes as carriers for 3H-thymidine: applications to the study of facial morphogenesis.

An implant labeling technique is described that utilizes sable hair probes as carriers for a tritiated thymidine marker. The protocol that was developed produced localized labeling of specific embryonic cell populations. This procedure was applied to the analysis of facial process development in chick embryos. Evaluation of the technique demonstrated that the probe preparation procedure was consistently successful in producing labeled probes. Using labeled probes, the procedure was reliable in producing acceptable levels of labeling in chick embryonic tissues and labeling of localized cell populations was possible using the implant labeling technique. Surrounding the center of labeling, a gradient in intensity of labeling was often observed. This pattern presumably reflects declining availability of labeled thymidine as distance from the probe increased. The technique allowed excellent survival rates to be achieved provided that aseptic procedures were followed. Additionally, careful analysis of older embryos failed to reveal any malformations induced by the implant labeling procedure. The localized labeling patterns that were demonstrated during this investigation suggest that the implant labeling technique would provide a useful tool for following cell migration during facial process formation.

Craniofacial development: the possible mechanisms for some malformations.

Normal craniofacial development is reviewed and some mechanisms in the fusion of mammalian facial 'processes' are discussed. The manipulation of environmental factors and effects of teratogens are related to genetic susceptibility of animal models, and extrapolated to craniofacial malformations in man.

Maternal phenytoin administration affects DNA and protein synthesis in embryonic primary palates.

Recent studies have shown that phenytoin (Dilantin) administration to pregnant A/J mice on day 10 causes reduced growth in embryonic primary palates. The current investigation concentrates on biochemical and autoradiographic changes toward the end of primary palate formation (gestational day 11), which coincides with the developmental period used for the previously conducted morphological studies. On gestational day 10, one group of pregnant A/J mice was injected intraperitoneally (IP) with 60 mg/kg phenytoin and the other group with vehicle. Twenty-three hours after phenytoin administration, all animals were injected (IP) with either [3H]-thymidine or [3H]-leucine. After one hour of incorporation, animals were sacrificed, embryos removed and placed in ice-cold Eagle's minimum essential medium containing 0.02% NaN3 for biochemical assay or fixed immediately in Bouin's solution for autoradiography. For biochemical analyses, palates and limb buds were removed, homogenized, TCA precipitated, lyophilized, and acid hydrolyzed. Examination of the data revealed that DNA synthesis in control palates was 3.8-fold greater than in primary palates from embryos of phenytoin-treated mothers. Results were similar for limb buds from control embryos and from embryos of phenytoin-treated mothers. Experiments utilizing [3H]-leucine indicated that protein synthesis was 2.6-fold greater in primary palates from phenytoin-treated mothers than in control primary palates. Similar results were obtained for protein synthesis in limb-bud tissue from controls and embryos of phenytoin-treated mothers. Autoradiographic data supported the biochemical findings. DNA synthesis in primary palates from embryos of phenytoin-treated mothers decreased 3-fold; protein synthesis increased 2.2-fold compared with control primary palates.(ABSTRACT TRUNCATED AT 250 WORDS)