Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.

Evidence for multifactorial origin of diabetes-induced embryopathies.

Serum factors characteristically altered in the diabetic state, e.g., glucose, ketone bodies (beta-hydroxybutyrate), and somatomedin inhibitors, induce dysmorphogenesis with or without growth retardation in rodent embryos in whole-embryo culture. Furthermore, serum from diabetic animals, which contains combinations of altered factors, is teratogenic, thereby implying that the diabetic embryopathy is of a multifactorial origin. However, a detailed investigation with various combinations of factors at known concentrations to test this hypothesis has not been conducted. Therefore, we employed combinations of hyperglycemia (300 and 600 mg/dl; 16.6 and 33.3 mM), hyperketonemia (8 and 16 mM D-beta-hydroxybutyrate), and an 800- to 1000-Mr somatomedin-inhibitor serum fraction (at an amount equal to that found in 0.05 and 0.1 ml of serum from a diabetic rat per deciliter culture medium), which represented doses with low teratogenic potential, to determine if interactions of the factors could occur that would increase the risk of malformations in mouse embryos exposed in whole-embryo culture during gastrulation and neurulation. The results demonstrate that glucose and D-beta-hydroxybutyrate can act synergistically to produce growth retardation and additively to induce malformations. The addition of the somatomedin inhibitor exacerbates the induction of malformations produced by the ketone body and glucose. Thus, the origin of the diabetic embryopathy may be multifactorial, involving several altered maternal factors.

Transient modulation of gene expression in the neurulation staged mouse embryo.

Transient modulation of gene expression in the embryo during early organogenesis will allow studies to be conducted that determine tissue- and stage-specific function(s) of genes. To achieve this goal, viral vectors and antisense oligodeoxynucleotides have been used to produce gain-of-function and loss-of-function models. Adenoviral transduction of whole embryos, embryonic heart and vasculature, and primary neural crest cell culture has been reported. The morphological consequences of overexpression or decreasing expression of selected genes have been evaluated using these tools. Gene-teratogen interaction studies have also been performed. The viral vectors appear to be important tools for modulating gene expression and hold great promise for future research.

Effects of altered maternal metabolism during gastrulation and neurulation stages of embryogenesis.

In summary, many congenital malformations are produced during gastrulation and neurulation stages of embryogenesis at a time when no definitive chorioallantoic placenta has been established. In rodents, altered maternal metabolism may have a direct impact on the embryo or an indirect impact via disruption of the nutritive function of the visceral yolk sac. If similar mechanisms operate in human embryos, these factors probably alter functions of the trophoblastic shell. In any case, it is crucial to remember that the metabolic status of the embryo is rapidly changing and during early stages of organogenesis may respond to alterations in nutrients quite differently during the first four weeks of gestation than at later stages of organogenesis and the fetal period.

Metabolism of D- and DL-beta-hydroxybutyrate by mouse embryos in vitro.

Rates of 14CO2 production by mouse conceptuses in vitro from D- and DL-[3-14C]-beta-hydroxybutyrate (beta OHB) were determined during the period of organogenesis (days 9 through 12 of gestation) in the presence of 4 to 32 mmol/L DL-beta OHB. During this time period the rates 14CO2 production from D-beta OHB metabolism are concentration-dependent, increase on each day of gestation, and the site of metabolism appears to shift from the visceral yolk sac placenta to the embryo proper. In contrast to fetal and neonatal tissues, the rates of 14CO2 production from DL-beta OHB oxidation is significantly greater than from D-beta OHB suggesting that the utilization of the L-isomer may be equal to or greater than that of the D-form.

Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

Haloacetic acids (HAs) are embryotoxic contaminants commonly found in drinking water. The mechanism of HA embryotoxicity has not been defined, but may be mediated in part by protein kinase C (PKC) inhibition. This study was conducted to evaluate the pathogenesis of HA embryotoxicity, and to compare these data with those from specific (Bis I) and non-specific (staurosporine) inhibitors of PKC. Embryos were incubated for varying times with several HAs, Bis I, staurosporine, or Bis V (a negative control). Cell cycle analysis was performed by flow cytometry following nuclear staining with propidium iodide; apoptosis was evaluated by fluorescence microscopy following LysoTracker staining. At concentrations producing 100% embryotoxicity with no embryolethality, only staurosporine perturbed the cell cycle. However, flow cytometry revealed accumulation of sub-G1 events (an apoptotic indicator) across time with bromochloroacetic acid, dichloroacetic acid, and staurosporine, but not dibromoacetic acid, Bis I, or Bis V. Sub-G1 events were particularly prominent in the head region, and remained at control levels in the heart. LysoTracker staining confirmed a similar pattern of apoptosis in the intact embryo; BCA and DCA produced intense staining in the prosencephalon, with virtually no staining in the heart. These data indicate that while cell-cycle perturbation may not mediate the pathogenesis of HA embryotoxicity, these agents do induce embryonic apoptosis. In addition, the lack of Bis I-induced apoptosis indicates that PKC inhibition is unlikely to be the sole mediator of HA embryotoxicity.

Dysmorphogenic effects of a specific protein kinase C inhibitor during neurulation.

Protein kinase C (PKC) plays a key role in signal transduction and is an important mediator of events throughout development. However, no information exists regarding the effect of a specific PKC inhibitor on mammalian embryogenesis during neurulation. This investigation was undertaken to examine the effects of a specific inhibitor of PKC, as well as inhibitors of other important kinases, on cultured mouse embryos. CD-1 mouse embryos (3 to 6 somite stage) were exposed to bisindolylmaleimide I (a specific PKC inhibitor) as well as specific inhibitors of PKA, PKG, and MAP kinase kinase for 24 h. The PKC inhibitor was a potent embryotoxicant and elicited malformations at concentrations as low as 0.01 microM. Inhibitors of other kinases also produced malformations but at much higher concentrations than those required to produce similar defects with the PKC inhibitor. These data suggest that PKC plays an important role in mammalian neurulation. Further research is required to clarify the mechanism by which PKC inhibition at this developmental stage produces malformations and the potential effects of environmental toxicants with PKC inhibitory properties on this signal transduction pathway.

Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture.

The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic acid are developmentally toxic in mouse whole embryo culture. Human exposure to these contaminants in drinking water would involve simultaneous exposure to all three HAs. This study explores the question of developmental toxicity interactions between these compounds. Gestational day (GD) 9.5 rat embryos were exposed to various concentrations of the three HAs (singly or in combination) for 48 h and then evaluated for dysmorphology. The embryonic effects from exposure to the single compounds and mixtures were evaluated using developmental score (DEVSC) as the parameter of comparison. Concentrations of individual compounds and mixtures were chosen (based on a dose-additivity model) which were predicted to produce scores 10 or 20% lower than control levels. Evaluations were performed on all possible combinations of the three HAs. The HAs were dysmorphogenic and resulted in primarily rotation and heart defects and to a lesser extent prosencephalic, visceral arch, and eye defects. The percent anomalies in the rat were comparable to those previously published for the mouse at comparable toxicant concentration. There was a low incidence of neural tube defects in the rat following exposure to the HAs. The rat neural tube appeared less sensitive to the HAs than did the mouse resulting in a higher rate of neural tube dysmorphology in the mouse. Following exposures to BCA and DBA, alone and in combination, there was a significant incidence of delayed embryonic caudal development with apparent normal development anterior to the second visceral arch. The developmental scores for embryos exposed to combinations of the three compounds, when compared to scores for embryos exposed to the single compounds, indicated that the dose-additivity model adequately predicted the observed toxicity and that the developmental toxicity of these water disinfection by-products appears to be additive in whole embryo culture (WEC).

Lack of evidence for intergenerational reproductive effects due to prenatal and postnatal undernutrition in the female CD-1 mouse.

The impacts of adverse environments during the prenatal and/or early postnatal periods may be manifested as functional deficits that occur later in life. Epidemiological studies have shown an association of sub-optimal pregnancy outcomes in one generation with similar events in the following one, a phenomenon termed the "intergenerational effect". Data indicate that the incidence of adverse pregnancy outcomes and/or low birth weight infants is more closely correlated with the mother's perinatal environment than with that during her pregnancy. However, epidemiological studies are inherently limited given the variability of lifestyles, ethnicity, nutritional status, and exposures to environmental factors. An appropriate animal model would permit control of parameters that may be impossible to evaluate in human populations. The current studies investigated the mouse as a possible animal model. Pregnant CD-1 mice were placed on an ad libitum or food-restricted diet (50% normal) throughout gestation to generate control (CON) and intrauterine growth retarded (IUGR) litters. At birth (postnatal day (PD) 1) pups (F1) were cross-fostered to control dams in litters of either 8 (CON) or 16 (postnatal food restriction (FR)). The experimental groups thus generated represented adequate nutrition (CON-CON) and undernutrition during the prenatal (IUGR-CON), or postnatal periods (CON-FR), or both (IUGR-FR). Pups of dams on a restricted diet during gestation had significant IUGR (P<0.001) as compared to controls (birth weights of 1.32 g versus 1.63 g). At weaning, the average weight of the pups was dependent on postnatal litter size and the difference in birth weights between IUGR and CON animals was not a significant factor. CON-CON pup weight was 24.1g and IUGR-CON was 22.2 g as compared to the CON-FR (17.0 g) and IUGR-FR (17.3 g) groups. The difference in weaning pup weights between the FR and CON groups was significant (P<0.01). The F1 FR females did not reach CON female weights at any time point through 11 months after weaning. At PD60, a single breeding period for all groups of females with CON males began and continued for 75 days with 17 opportunities for breeding. Animals that became pregnant during this time were removed and allowed to litter. No significant differences were noted in average F2 litter size or average pup weight at birth: (CON-CON 12.2/1.62 g; IUGR-CON 11.9/1.6 2 g; CON-FR 10.9/1.70 g; IUGR-FR 11.3/1.61 g). We conclude that body weight at birth in the CD-1 mouse is not correlated with growth through the period of weaning (PD28). We did not find any evidence for an intergenerational reproductive effect after developmental undernutrition.

Embryonic metabolism of foetal fuels in whole-embryo culture.

The whole-embryo culture technique has been extensively used to evaluate embryonic growth and development using morphological criteria. However, the system also provides an opportunity to assess the status of embryonic metabolism to determine biochemical maturation and alterations induced by xenobiotics or changes in substrate availability. For example, using (14)C-labelled substrates the rates of product formation may be determined for a variety of foetal fuels such as glucose and the ketone body, beta-hydroxybutyrate. During organogenesis glucose metabolism is characterized by high rates of glycolytic metabolism at the early-somite stage and an increased dependence on Krebs cycle concomitant with the establishment of the chorioallantoic placenta (c. 30-somite stage) to initiate an increasingly 'foetal' type of pattern. The dependence of the neurulating embryo on glucose as a fuel is further demonstrated by the embryotoxic effects of insulin-induced hypoglycaemia. Although decreased glycolytic metabolism is the earliest effect of this treatment, both the Krebs cycle and oxidative pentose phosphate pathway are also inhibited. Therefore, the induction of malformations by hypoglycaemia is the result of a multifactorial biochemical alteration. In addition to glucose, mouse conceptuses have the capacity to metabolize alternative foetal fuels such as the ketone bodies d- and dl-BOHB at rates that are concentration dependent. However, the energy production from these substrates is limited by the low rates of Krebs-cycle metabolism at the early-somite stage.

Effects of cocaine and cocaine metabolites on mouse developmentin vitro.

The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 mum or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 mum. EME also induced defects at concentrations of 400 mum or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 mum or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 mum, exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDsin vitro is higher than the teratogenic concentrationin vivo. Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reportedin vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.

Effects of short-term exposure to ethanol on mouse embryos in vitro.

The adverse developmental effects of ethanol consumption have been documented in humans and in animal models. In animal models, the organ system affected by ethanol administration is dependent on the point in gestation at which the xenobiotic is administered. Previous studies have shown that an exposure of 24-48 hr beginning at the early somite stage in rodent conceptuses alters neural tube closure in vitro. However, the concentration and time dependency of this effect have not been fully defined. Whole embryo culture was therefore used to expose 3-6-somite mouse conceptuses (ICR strain) to ethanol at 300, 450, 600 and 800 mg/dl. The higher concentrations were selected to approximate the peak serum ethanol concentrations that have been shown to be teratogenic in vivo. A 24-hr exposure produced a concentration-dependent increase in neural tube defects (NTDs) and concomitant growth retardation. When shorter exposure periods were used (8, 10, 12 or 20 hr) the incidence of NTDs was dependent on the ethanol concentration and exposure period. At the 600 and 800 mg/dl concentrations an exposure of 8 hr or more produced NTDs, but shorter periods (4 and 6 hr) did not affect neural tube closure when evaluated at the end of a 24-hr culture period. At the 450 mg/dl concentration a 20-hr exposure induced NTDs, but a 12-hr exposure to this level did not. Exposure of conceptuses to ethanol for periods similar to their half-life in vivo did not induce NTDs and the highest concentration produced only a trend towards a reduction in protein content. When the incidence of NTDs was plotted against the area under the time and concentration curve (AUC) the correlation coefficient was 0.5779. An analysis of covariance indicated that the relationships between NTDs and AUC were similar at the 300 and 450 mg/dl concentrations and also at the 600 and 800 mg/dl concentrations. In contrast, the relationships between embryonic protein content and AUC did not differ at the 300, 450 and 600 mg/dl concentrations, but all differed from that at the 800 mg/dl level. These results indicate that ethanol-induced NTDs do not appear to be due solely to embryonic growth retardation. Additionally, ethanol-induced neural tube defects are a function of duration of exposure as well as of peak serum concentration.

Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin.

Some 2000 species of cyanobacteria (blue-green algae) occur globally in aquatic habitats. They are able to survive under a wide range of environmental conditions and some produce potent toxins. Toxin production is correlated with periods of rapid growth (blooms) and 25%-70% of blooms may be toxic. Anatoxin-a is an alkaloid neurotoxin that acts as a potent neuro-muscular blocking agent at the nicotinic receptor. Acute toxicity, following consumption of contaminated water, is characterized by rapid onset of paralysis, tremors, convulsions and death. Human exposures may occur from recreational water activities and dietary supplements, but are primarily through drinking water. The current studies were conducted to examine the effect of in utero exposure on postnatal viability, growth and neurodevelopment, to evaluate the potential of in vitro embryotoxicity, and to explore the synergistic relationship between anatoxin-a and the algal toxin microcystin-LR by the oral route. The results of preliminary studies on amphibian toxicity are also reported. Time-pregnant mice received 125 or 200 microg kg(-1) anatoxin-a by intraperitoneal injection on gestation days (GD) 8-12 or 13-17. Pup viability and weight were monitored over a 6-day period. Maternal toxicity (decreased motor activity) was observed at 200 microg kg(-1) in both treatment periods. There were no significant treatment-related effects on pup viability or weight on postnatal day (PND) 1 or 6. The GD 13-17 pups were evaluated on PND 6, 12 and 20 for standard markers of neurodevelopmental maturation (righting reflex, negative geotaxis and hanging grip time). No significant postnatal neurotoxicity was observed. In vitro developmental toxicity was evaluated in GD 8 mouse embryos exposed to 0.1-25 microm anatoxin-a for 26-28 h. Perturbations in mouse yolk sac vasculature were noted from the 1.0 microm concentration in the absence of significant embryonic dysmorphology. Potential algal toxin synergism was tested in mice receiving either 0, 500 or 1,000 microg kg(-1) microcystin-LR by gavage and approximately 50 min later receiving either 0, 500, 1,000 or 2,500 microg kg(-1) anatoxin-a by the same route. No deaths occurred at any dose and no definitive signs of intoxication were observed. Stages 17 and 25 toad embryos (Bufo arenarum) were exposed to 0.03-30.0 mg l(-1) of anatoxin-a for 10 days. Adverse effects included a dose-dependent transient narcosis, edema and loss of equilibrium. Most notable was the occurrence of 100% mortality at the high dose in both groups 6-13 days post-exposure. The observed delay between initial exposure and death is highly unusual for anatoxin-a.

Inhibitors of glycolytic metabolism affect neurulation-staged mouse conceptuses in vitro.

In order to evaluate the apparent discordance between altered glucose metabolism and embryonic energy production, the effects of inhibitors of glucose utilization on morphological development and biochemical changes in mouse embryos in culture were evaluated. Day 9 ICR mouse conceptuses having 3-6 pairs of somites were prepared for culture as previously described. 2-Deoxyglucose (2DG) produced a concentration-dependent effect on development. A 25 microM 2DG concentration did not induce neural tube closure defects (NTDs) but 100 microM, 100% of embryos exhibited this defect. A 17% reduction in the rate of lactate production by the conceptus was produced by a 24-hr exposure period to 100 microM 2DG. Iodoacetate, which inhibits glyceraldehyde-3-phosphate dehydrogenase in adult tissues, produced high rates of NTDs at concentrations > or = 2.5 microM. Following a 24 hour exposure to iodoacetate, lactate production was inhibited at 10 and 25 microM. The effects of 2DG on embryonic ATP content were assessed to test the hypothesis that effects on glucose utilization would effect embryonic ATP content. Despite using 2DG concentrations that alter development and inhibit glycolysis, there were no effects on whole embryo or visceral yolk sac (VYS) ATP content. However, when the embryo was divided into regions, there was a specific reduction in ATP content in the head following a 24-hr exposure period. No effect of 2DG on head ATP content was produced after 12 hr of exposure. To determine if there were region specific differences in 2DG uptake and distribution that could account for the differential effects of 2DG on ATP content, 14C-2DG accumulation in different regions of the embryo and VYS was determined over the 24-hr culture period. The uptake of 2DG was dependent on the medium 2DG concentration and suggested a higher accumulation in regions with decreased ATP. However, when the uptake was monitored for a 1-hr period after a 24-hr exposure, there was no region specific differences in 2DG uptake. These studies further document the adverse developmental effects of inhibitors of glucose utilization during the early stage of neurulation. The biochemical mechanism for induction of these defects is unclear, but an effect on ATP content does not appear to be solely responsible for the dysmorphogenesis.

Hypoglycemia: how little is too much for the embryo?

The effects of hypoglycemia on mammalian embryos undergoing neurulation (third to fourth week of human development) were investigated. Mouse embryos were maintained for 28 hours in whole embryo culture in serum collected from rats that had received 50 units of 100 United States Pharmacopeia insulin units per milliliter. Glucose concentrations used were 40, 60, 80, and 147 mg/dl (normal blood glucose in the pregnant mouse is 125 mg/dl). After the culture period embryos were evaluated for malformations and growth and compared with those maintained under euglycemic conditions. The results demonstrate that glucose concentrations approximately 50% of normal maternal levels were teratogenic but not growth inhibitory, whereas concentrations in the range of 30% to 40% of maternal levels were lethal to the embryo. Furthermore, a 14-hour exposure to reduced blood sugar in either the first or second half of the culture period produced malformations.

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro.

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.

Quantitative structure-activity relationships for the developmental toxicity of haloacetic acids in mammalian whole embryo culture.

Developmental toxicity in mouse whole embryo culture assay has been reported for acetic acid (AA) and a series of ten haloacetic acids, including mono-, di-, tri-fluoro (MFA, DFA, TFA), chloro (MCA, DCA, TCA), bromo (MBA, DBA, TBA), and monoiodo (MIA) acetic acids. Benchmark concentrations (BCm), calculated as the lower 95% confidence limit of molar acid concentration producing a 5% increase in embryos with neural tube defects, provided potency estimates for development of quantitative structure-activity relationships (QSARs). The best overall regression was obtained for the ten halo-acids (excluding AA) and related log (1/BCm) to the energy of the lowest unoccupied molecular orbital (Elumo) and acid dissociation constant (pKa) with a correlation coefficient of r = 0.97, and a sample size-adjusted r2 = 0.92. This QSAR suggested a common basis for the mechanism of HA activity, which would imply additivity for mixtures of these acids. Examination of QSARs for subsets of the total data set (e.g., monohaloacids) highlighted parameter relationships embedded in the total QSAR, helping to unravel the separate contributions of Elumo and pKa to the overall potency. The relevance of these parameters is discussed in terms of postulated mechanisms of developmental toxicity involving changes in intercellular pH and redox metabolism. The whole embryo assay results pertain to direct embryo exposure and toxicity without the confounding influence of maternal factors. The resulting QSAR model offers possible insight into the mechanism of embryo toxicity that will hopefully contribute to understanding of the more complex, in vivo teratogenicity problem.