The effect of teratogens on maternal corticosterone levels and cleft incidence in A/J mice.

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-T > HAL > controls. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.     

Maternal toxicity: a possible etiological factor in embryo-fetal deaths and fetal malformations of rodent-rabbit species

Data from animal teratology studies were surveyed to determine whether embryo-fetal mortality and fetal malformations result from a primary action of the agent on the conceptus or if they are secondary to maternal toxicity--a consequence of administration with high dose levels of test chemicals. A fairly strong association between embryo-fetal mortality and maternal toxicity was revealed by analysis of data from hamsters, mice, rats, and rabbits in 234 studies of chemical and physical agents, of which 83 were conducted at both maternotoxic and nonmaternotoxic doses, 94 only at maternotoxic doses, and 49 at nonmaternotoxic doses. In the above studies, only nine chemicals (four each in hamsters and rabbits and one in rats) were reported to induce embryo-fetal deaths at apparently nonmaternotoxic doses. These findings tend to suggest a contributory role for maternal toxicity in the induction of embryo-fetal deaths. The previously reported hypothesis that certain fetal defects in mice may perhaps be caused by maternal toxicity was also found to be true in a review of data on hamsters, rats, and rabbits. Salient maternal toxicity-associated fetal malformations were exencephaly, encephalocele, micro- or anophalmia, and fused ribs in hamsters and defective (fused, missing, or extra) ribs, vertebrae, and sternebrae, ex-, an-, or microphthalmia, and cleft palate in rats and rabbits. These malformations occurred at low frequencies, generally with no readily apparent dose-response relationship. Presumptive evidence indicates that embryo-fetal deaths, and the above-mentioned fetal malformations in experimental animals, which in published literature are presently attributed to chemical induction for a large number of chemicals, may be a consequence of maternal toxicity per se.     

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.     

Dose-response relations of palatal slit, cleft palate, and fetal mortality in mice treated with a glucocorticoid

C57BL/6 (C57BL) and SWV mice were treated subcutaneously with triamcinolone acetonide in a single dose of 1.0-7.0 mg/kg on day 12 of pregnancy, and the palate of their fetuses was examined at term. In C57BL mice palatal slit occurred spontaneously and its frequency increased with increasing doses of triamcinolone. However, this defect was not seen in SWV fetuses, even when dams were treated with the doses that induced cleft palate. The frequency of cleft palate increased in both C57BL and SWV as the dose of triamcinolone increased. Fetal mortality increased in SWV, but not in C57BL, with increasing doses of triamcinolone. Dose-response relations were analyzed by the log-probit transformation method. In C57BL mice, the slope of the dose-response curve of palatal slit was significantly different from that of cleft palate. In contrast, the dose-response curves of cleft palate were similar in both C57BL and SWV; the median effective dose was significantly greater in C57BL than in SWV. The mechanism of induced palatal slit appears to be different from that of induced cleft palate; the mechanism of cleft palate induction may be the same in both C57BL and SWV. The slope of the dose-response curve of fetal mortality in SWV mice was different from that of cleft palate; the mechanisms underlying the resorption and cleft palate responses must be different.     

Maternal cigarette smoking during pregnancy and the risk of having a child with cleft lip/palate

Maternal cigarette smoking during pregnancy as a risk factor for having a child with cleft lip/palate has been suggested by several epidemiologic studies. However, most of these studies contained small sample sizes, and a clear association between these two factors could not be established. The U.S. Natality database from 1996 and a case-control study design were used to investigate the association between maternal smoking during pregnancy and having a child with cleft lip/palate. The records of 3,891,494 live births from the 1996 U.S. Natality database were extracted to obtain cleft lip/palate cases and random controls. The National Center for Health Statistics collects maternal and newborn demographic and medical data from the birth certificates of all 50 states. New York (excluding New York City), California, Indiana, and South Dakota did not collect smoking data, and the data from these states were excluded from the analysis. A total of 2207 live births with cleft lip/palate cases were identified, and 4414 controls (1:2 ratio) were randomly selected (using the SAS program) from live births with no congenital defects. Odds ratios and 95 percent confidence intervals were determined from logistic regression models, adjusting for confounding variables, including maternal demographic and medical risk factors. A significant association was found between any amount of maternal cigarette use during pregnancy and having a child with cleft lip/palate [unadjusted odds ratio 1.55 (1.36, 1.76), p < 0.001]. Univariate analysis showed that maternal education level, age, race, and maternal medical conditions (diabetes and pregnancy-associated hypertension) were potential confounders. After adjusting for these confounders, the odds ratio remained significant [Mantel-Haenszel odds ratio 1.34 (1.16, 1.54), p < 0.001]. To determine the dose response of cigarette smoking during pregnancy, the cigarette consumption per day was divided into four groups: none, 1 to 10, 11 to 20, and 21 or more. A dose-response relationship was found when comparing each smoking category with the no smoking reference group: 1.50 (1.28, 1.76), 1.55 (1.23, 1.95), and 1.78 (1.22, 2.59), respectively. This means that increased cigarette smoking during pregnancy resulted in increased odds of having a child with cleft lip/palate. This is the largest study to date to test the association between maternal cigarette smoking during pregnancy and having a newborn with cleft lip/palate. The significant trend in the dose-response relationship strongly suggests the association of smoking tobacco and this common congenital deformity. These results emphasize the public health risks associated with smoking during pregnancy. To prevent this devastating craniofacial anomaly, educational initiatives should be considered that will alert expectant mothers to the association between smoking during pregnancy and the occurrence of cleft lip/palate.     

Likelihood-Based Modeling and Analysis of Data Underdispersed Relative to the Poisson Distribution

By using a generalization of the Poisson process, distributions can be constructed that show appropriate amounts of underdispersion relative to the Poisson distribution that may be apparent from observed data. These are then used to examine the differences between the distributions of numbers of fetal implants in mice corresponding to different doses of the herbicide 2,4,5-T.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

A prospective evaluation of the prevalence of submucous cleft palate in patients with isolated cleft lip versus controls.

Although there is an established relationship between cleft lip and overt cleft palate, the relationship between isolated cleft lip and submucous cleft palate has not been investigated. To test the hypothesis that patients with isolated cleft lip have a greater association with submucous cleft palate, a double-armed prospective trial was designed. A study group of 25 consecutive children presenting with an isolated cleft lip, with or without extension through the alveolus but not involving the secondary palate, was compared with a control group of 25 children with no known facial clefts. Eligible patients were examined for the presence of physical criteria associated with classic submucous cleft palate, namely, (1) bifid uvula, (2) absence of the posterior nasal spine, and (3) zona pellucida. Nasoendoscopy was subsequently performed just after induction of general anesthesia, and the findings were correlated with digital palpation of the palatal muscles. Patients who did not satisfy all three physical criteria and in whom nasoendoscopy was distinctly abnormal relative to the control group were classified as having occult submucous cleft palate. Classic submucous cleft palate was found in three study group patients (12 percent), all of whom had flattening or a midline depression of the posterior palate and musculus uvulae on nasoendoscopy and palpable diastasis of the palatal muscles under general anesthesia. An additional six study group patients (24 percent) had similar nasoendoscopic criteria and palpable diastasis of the palatal muscles; they were classified as having occult submucous cleft palate. No submucous cleft palate was identified in the control group. Seventeen patients in the study group had an alveolar cleft with a 53 percent (9 of 17) prevalence of submucous cleft palate. In the present study, classic submucous cleft palate in association with isolated cleft lip was 150 to 600 times the reported prevalence in the general population. All children with an isolated cleft lip should undergo peroral examination and speech/resonance assessment no later than the age of 3 years. Any child with an isolated cleft lip with velopharyngeal inadequacy or before an adenoidectomy should be assessed by flexible nasal endoscopy to avoid missing an occult submucous cleft palate.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100.

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test

The chlorinated phenoxyacetic acids 2-methyl-4-chlorophenoxyacetic acid (MCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5-T butoxyethyl ester and the chlorophenols 2,4-dichlorophenol and 2,4,5-trichlorophenol were tested for genotoxicity in the modified Allium test, which is based on exposure to the test chemicals of growing onions. The mean length of growing roots were measured and chromosome damage was recorded. Of the substances tested, MCPA was the most toxic and the chlorophenoxyacetic acids were more toxic than the chlorophenols. The lower threshold values for growth retardation were below 0.1 ppm for the acids, approx. at 0.1 ppm for the ester and less than 5 ppm for the phenols. Though a monocotyledon, Allium cepa was sensitive enough to respond to even low concentrations of these dicotyledon-selecting pesticides.     

Development of the orbicularis oris muscle in normal and cleft lip and palate human fetuses using three-dimensional computer reconstruction

As part of an ongoing study of cleft lip and palate fetal morphology, normal and dysmorphic development of the human fetal orbicularis oris muscle was studied in a cross-sectional sample of 29 human fetuses (20 "normal" and 9 cleft lip and palate) ranging in age from 8 to 21 postmenstrual weeks. The specimens were embedded in celloidin and sectioned at 20 microns, and every tenth section was stained with hematoxylin and eosin. A computer reconstruction technique was applied to produce three-dimensional representations of the orbicularis oris muscle. The orbicularis oris muscle in the normal fetal sample with discernible lip fibers (N = 15) increased symmetrically in both fiber density and complexity from 12 to 21 weeks. Metrically, muscle volume and thickness growth curves were consistent with qualitative observations. In contrast, the unilateral cleft lip and palate fetal specimens with discernible lip fibers (N = 3) exhibited a 3.5-week delay in overall muscle development, asymmetrical fiber distribution, and abnormal fiber insertions. However, quantitatively, no significant (p greater than 0.05) differences were noted in orbicularis oris muscle thickness or volume between the normal and cleft lip and palate fetal specimens through 21 weeks. Findings suggest that orbicularis muscle deficiency, noted clinically in cleft lip and palate neonates, may be a result of perinatal functional dysmorphogenesis rather than congenital mesenchymal reduction or deficiency.     

Birth prevalence of cleft lip and palate in British Columbia between 1952 and 1986: stability of rates.

We examined the birth prevalence of cleft lip with or without cleft palate and of isolated cleft palate in British Columbia between 1952 and 1986 using the data of the BC Health Surveillance Registry. The rates fluctuated over the study period, but linear trend analysis showed no increase or decrease for cleft lip with or without cleft palate; however, there was a significant increase for isolated cleft palate, attributed to improved ascertainment around 1963-66. Given the possible effects of newer agents used in both silviculture and agriculture, as well as the general concern over drugs and other environmental agents, such a long-term monitoring program is important. Furthermore, if significant clustering occurs, good background data are essential for comparison. The general public''s perception is that the rates of birth defects are increasing. Our findings should give some reassurance with respect to orofacial clefts.     

Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives

Objective To estimate the relative risk of recurrence of oral cleft in first degree relatives in relation to cleft morphology.Design Population based cohort study.Setting Data from the medical birth registry of Norway linked with clinical data on virtually all cleft patients treated in Norway over a 35 year period.Participants 2.1 million children born in Norway between 1967 and 2001, 4138 of whom were treated for an oral cleft.Main outcome measure Relative risk of recurrence of isolated clefts from parent to child and between full siblings, for anatomic subgroups of clefts.Results Among first degree relatives, the relative risk of recurrence of cleft was 32 (95% confidence interval 24.6 to 40.3) for any cleft lip and 56 (37.2 to 84.8) for cleft palate only (P difference=0.02). The risk of clefts among children of affected mothers and affected fathers was similar. Risks of recurrence were also similar for parent-offspring and sibling-sibling pairs. The “crossover” risk between any cleft lip and cleft palate only was 3.0 (1.3 to 6.7). The severity of the primary case was unrelated to the risk of recurrence.Conclusions The stronger family recurrence of cleft palate only suggests a larger genetic component for cleft palate only than for any cleft lip. The weaker risk of crossover between the two types of cleft indicates relatively distinct causes. The similarity of mother-offspring, father-offspring, and sibling-sibling risks is consistent with genetic risk that works chiefly through fetal genes. Anatomical severity does not affect the recurrence risk in first degree relatives, which argues against a multifactorial threshold model of causation.     

Involvement of Glucocorticoids in the Development of the Secondary Palate

Genetic differences between various inbred strains of mice in the levels of glucocorticoid receptors embryonic in maxillary mesenchyme cells appear to be reflected in the magnitude of the responses to steroids in these cells. High levels of glucocorticoids cause significant growth inhibition in maxillary mesenchyme cells with subsequent alterations in the production of extracellular matrix components. The presence of higher levels of cytoplasmic glucocorticoid receptor proteins may be one factor which could predispose those strains such as A/J to a greater inhibition of craniofacial growth in vivo by glucocorticoids and therefore increase the frequency of cleft palate production. Furthermore, women with infertility treated with glucocorticoids to support pregnancy give birth to infants with a marked decrease in birth weight [98]. Pharmacologic doses of glucocorticoids can also cause a dramatic reduction in the growth of a number of fetal tissues in mice and humans. In fact, there is evidence that glucocorticoids may be a causative factor in the production of cleft palate in primates [52]. The nature of the molecular elements which determine the biochemical and physiologic responses to glucocorticoids in the palate still remains largely unknown. Although in the mouse there is some evidence to suggest that the major histocompatibility locus (H-2) might be involved, the level(s) at which this control is exerted is unknown. It is possible that this locus may regulate in some manner the level of glucocorticoid receptors and the response to glucocorticoids in the secondary palate. Moreover, there is evidence to suggest that other genes distinct from, but closely linked to the H-2 locus may be important in determining both the strain-dependent differences in susceptibility to glucocorticoid-induced cleft palate and the intracellular levels of cyclic AMP in the secondary palate. It is also apparent that glucocorticoids in conjunction with other hormones or growth factors such as epidermal growth factor and agents which regulate cyclic nucleotide metabolism are essential for the normal development of the secondary palate. Excesses or deficiencies in either the level of these growth regulators and/or in their receptors in specific fetal tissues at defined periods in development are likely to lead to certain fetal malformations. Definition and integration of the genetic, biochemical, and endocrine factors which are involved in the control of cellular growth as influenced by alterations in the composition of cell surface and extracellular matrix components should provide some insights into the events associated with normal palatogenesis.     

Multiplex relative risk and estimation of the number of loci underlying an inherited disease

Knowledge of the number of causative loci is necessary to estimate the power of mapping studies of complex diseases. In the present article, we reexamine a theory developed by Risch and its implications for estimating the number L of causative loci affecting a complex inherited disease. We first show that methods based on Risch's analysis can produce estimates of L that are inconsistent with the observed population prevalence of the disease. We demonstrate this point by showing that the maximum-likelihood estimate for L produced by the method of Farrall and Holder for cleft lip/cleft palate data is not consistent with the prevalence under the multiplicative model. We show how to incorporate disease prevalence and develop a maximum-likelihood method for estimating L that uses the entire distribution of numbers of affected individuals in families containing an affected individual. This method avoids the potential inconsistencies of the Risch method and has greater precision. We apply our method to data on cleft lip/cleft palate and schizophrenia.     

Combined effects of radiation and caffeine on embryonic development in mice

The combined effect of radiation and caffeine has been studied in mouse embryos. Radiation and/or caffeine were administered to ICR mice on Day 11 of gestation. Intrauterine death, gross malformation, and fetal body weight were selected as indicators of effects. Doses of whole-body gamma irradiation were 0.5 to 2.5 Gy and those of caffeine were 100 and 250 mg/kg maternal body wt. Intrauterine mortality increased with increasing radiation dose; this trend was more remarkable in combination with caffeine. Gross malformations such as cleft palate and defects of forelegs and hindlegs appeared frequently in the fetuses treated with both radiation and caffeine. Decreased fetal weight was observed even in mice treated with 0.5 Gy of radiation or 100 mg/kg caffeine. There was a linear relationship between dose and reduction of fetal weight. The fetal weight was a sensitive, precise, and easy-to-handle indicator for the effects of growth retardation. Intrauterine mortality and frequencies of cleft palate and defects of forelegs and hindlegs were higher than the sum of those induced by radiation and by caffeine separately. The results indicated that the combined action of radiation and caffeine on intrauterine death and malformations was synergistic.     

Testing of 2,4,5-T-amino acid conjugates for mutagenic activity in Salmonella typhimurium strains

Since amino acid conjugates are plant metabolites of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 5 amino acid conjugates (aspartic acid, glutamic acid, leucine, methionine and tryptophan) of 2,4,5-T were tested for possible mutagenic activity utilizing 5 strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535 and TA1538) with and without rat-liver microsomal and cytosolic enzymes. These compounds did not cause any significant increase in reversions when compared with controls in the presence or absence of the activating system. Further, linear regression analysis showed no significant (p less than 0.05) dose-response relationships. Thus, it was concluded that the tested amino acid conjugates of 2,4,5-T are not mutagens or promutagens in these assays.     

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate--implications for fetal wound healing

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.