Investigation of SAMD1 ablation in mice.

SAM domain-containing protein 1 (SAMD1) has been implicated in atherosclerosis, as well as in chromatin and transcriptional regulation, suggesting a versatile and complex biological function. However, its role at an organismal level is currently unknown. Here, we generated SAMD1-/- and SAMD1+/- mice to explore the role of SAMD1 during mouse embryogenesis. Homozygous loss of SAMD1 was embryonic lethal, with no living animals seen after embryonic day 18.5. At embryonic day 14.5, organs were degrading and/or incompletely developed, and no functional blood vessels were observed, suggesting failed blood vessel maturation. Sparse red blood cells were scattered and pooled, primarily near the embryo surface. Some embryos had malformed heads and brains at embryonic day 15.5. In vitro, SAMD1 absence impaired neuronal differentiation processes. Heterozygous SAMD1 knockout mice underwent normal embryogenesis and were born alive. Postnatal genotyping showed a reduced ability of these mice to thrive, possibly due to altered steroidogenesis. In summary, the characterization of SAMD1 knockout mice suggests a critical role of SAMD1 during developmental processes in multiple organs and tissues.

Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of tissue microsomes or recombinant human cytochrome P-450 oxidoreductase: implications for possible roles in gasotransmission.

Carbon monoxide (CO), an endogenously produced gasotransmitter, regulates inflammation and vascular tone, suggesting that delivery of CO may be therapeutically useful for pathologies like preeclampsia where CO insufficiency is implicated. Our strategy is to identify chemicals that increase the activity of endogenous CO-producing enzymes, including cytochrome P-450 oxidoreductase (CPR). Realizing that both riboflavin and pyrroloquinoline quinone (PQQ) are relatively nontoxic, even at high doses, and that they share chemical properties with toxic CO activators that we previously identified, our goal was to determine whether riboflavin or PQQ could stimulate CO production. Riboflavin and PQQ were incubated in sealed vessels with rat and human tissue extracts and CO generation was measured with headspace-gas chromatography. Riboflavin and PQQ increased CO production ∼60% in rat spleen microsomes. In rat brain microsomes, riboflavin and PQQ increased respective CO production approximately fourfold and twofold compared to baseline. CO production by human placenta microsomes increased fourfold with riboflavin and fivefold with PQQ. In the presence of recombinant human CPR, CO production was threefold greater with PQQ than with riboflavin. These observations demonstrate for the first time that riboflavin and PQQ facilitate tissue-specific CO production with significant contributions from CPR. We propose a novel biochemical role for these nutrients in gastransmission.

Rabbit Whole Embryo Culture.

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48 h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.

Regulation and Control of AP-1 Binding Activity in Embryotoxicity.

The electrophoretic mobility shift assay (EMSA) is a sensitive and relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined, and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the 32P-labelled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "super-shift", is also presented. The important points are underscored, and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.

The Application of High-Resolution Ultrasound for Assessment of Cardiac Structure and Function Associated with Developmental Toxicity.

Congenital heart defects (CHD) are the most prevalent anomaly both clinically and in laboratory animal species. Historically, it was difficult to assess the longitudinal progression or repair of such anomalies because assessment methodologies were too invasive (gross exams and/or histology). Recently, technological advances in the field of diagnostic imaging have led to the manufacture of high-resolution ultrasound (HRUS), capable of characterizing both embryonic and maternal cardiovascular structure and function in small animals (rat and mouse). HRUS is relatively noninvasive, facilitating the longitudinal assessment of heart development throughout gestation and postnatally, providing a comprehensive evaluation of changes in cardiovascular performance following toxicant exposure.Described herein is a brief overview of important theoretical and practical considerations when applying HRUS to understand the impact of perturbations on the fetal heart. Examples are given from our own work to help the reader interpret their own HRUS images and more readily identify anomalies in utero. In addition to embryonic assessment, maternal pathologies may adversely affect the cardiovascular performance of the conceptus indirectly. Umbilical blood flow is particularly vulnerable to such effects and procedures to assess this endpoint are described. Neonatal rats, born with CHD, may respond pathologically to cardiovascular challenges as they mature, and we outline the use of HRUS to evaluate cardiac performance over the lifetime of the animal. Some of the caveats related to HRUS are discussed, particularly with the emphasis on how this may impact experimental design.

Tacrolimus in the prevention of adverse pregnancy outcomes and diabetes-associated embryopathies in obese and diabetic mice.

BACKGROUND: T2DM is a high-risk pregnancy with adverse fetal and maternal outcomes including repeated miscarriages and fetal malformations. Despite the established association between placental insufficiency and poor maternal Th1-adaptability to the development of pregnancy complications in T2DM, there have been no established data to assess benefits of pre-pregnancy immunosuppression relative to gestational outcomes in T2DM. We hypothesized that pre-pregnancy macrolide immune suppression can re-establish normal placental development and uterine vascular adaptation in a mouse model of obesity-associated T2DM. METHODS: Fetal live birth rate, postnatal variability, mid-gestational uterine and umbilical flow dynamics and certain morphological features of spiral artery modification were examined in the New Zealand Obese (NONcNZO10/Ltj) female mice (n = 56) weaned to ages of 32 weeks on a 60% calories/g high-fat diet (also referred to as HFD-dNONcNZO), and which received either tacrolimus (0.1 mg/kg s.c. q2d) , its vehicle (castor oil and ethanol) or metformin (in drinking water 200 mg/dL p.o. ad libitum). HFD-BALBc-Rag2/IL2-gc female mice (n = 24) were used as HFD-immunodeficient controls. RESULTS: Treatment of the HFD-dNONcNZO female mice with tacrolimus improved live birth rates and postnatal viability scores (p < 0.01), normalized OGTT (p < 0.001), inhibited fetal malformation rates, restored morphology of spiral arterial modification; and improved uterine arterial and umbilical blood flow (p < 0.01). Placental production of TNFαand IL16 in the tacrolimus-treated HFD-dNONcNZO dams were restored to non-diabetic levels and the treatment resulted in the inhibition of aberrant monocyte/macrophage activation during pregnancy in the HFD-dNONcNZO dams. CONCLUSIONS: Our present data suggest a casual association between chronic maternal overnutrition and aberrancy in the maternal Th1-immune maladaptation to pregnancy and defective spiral artery modification, placental insufficiency and adverse fetal outcomes in the T2DM subjects. Further safety studies into the use of tacrolimus in the pre-pregnancy glycemic control may be beneficial.

Moderate Exercise Attenuates Lipopolysaccharide-Induced Inflammation and Associated Maternal and Fetal Morbidities in Pregnant Rats.

Fetal growth restriction (FGR) and coagulopathies are often associated with aberrant maternal inflammation. Moderate-intensity exercise during pregnancy has been shown to increase utero-placental blood flow and to enhance fetal nutrition as well as fetal and placental growth. Furthermore, exercise is known to reduce inflammation. To evaluate the effect of moderate-intensity exercise on inflammation associated with the development of maternal coagulopathies and FGR, Wistar rats were subjected to an exercise regime before and during pregnancy. To model inflammation-induced FGR, pregnant rats were administered daily intraperitoneal injections of E. coli lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 and sacrificed at GD 17.5. Control rats were injected with saline. Maternal hemostasis was assessed by thromboelastography. Moderate-intensity exercise prevented LPS-mediated increases in white blood cell counts measured on GD 17.5 and improved maternal hemostasis profiles. Importantly, our data reveal that exercise prevented LPS-induced FGR. Moderate-intensity exercise initiated before and maintained during pregnancy may decrease the severity of maternal and perinatal complications associated with abnormal maternal inflammation.

Dimethadione embryotoxicity in the rat is neither correlated with maternal systemic drug concentrations nor embryonic tissue levels.

Pregnant rats treated with dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, produce offspring having a 74% incidence of congenital heart defects (CHD); however, the incidence of CHD has high inter-litter variability (40-100%) that presents a challenge when studying the initiating events prior to the presentation of an abnormal phenotype. We hypothesized that the variability in CHD incidence was the result of differences in maternal systemic concentrations or embryonic tissue concentrations of DMO. To test this hypothesis, dams were administered 300 mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses). Maternal serum levels of DMO were assessed on GD 11, 12, 13, 14, 15, 18 and 21. Embryonic tissue concentrations of DMO were assessed on GD 11, 12, 13 and 14. In a separate cohort of GD 12 embryos, DMO concentrations and parameters of growth and development were assessed to determine if tissue levels of DMO were correlated with these endpoints. Embryos were exposed directly to different concentrations of DMO with whole embryo culture (WEC) and their growth and development assessed. Key findings were that neither maternal systemic concentrations nor tissue concentrations of DMO identified embryos that were sensitive or resistant to DMO in vivo. Direct exposure of embryos to DMO via WEC also failed to show correlations between embryonic concentrations of DMO with developmental outcomes in vitro. We conclude that neither maternal serum nor embryonic tissue concentrations of DMO predict embryonic outcome.

In Utero Exposure to a Cardiac Teratogen Causes Reversible Deficits in Postnatal Cardiovascular Function, But Altered Adaptation to the Burden of Pregnancy.

Congenital heart defects (CHD) are the most common birth anomaly and while many resolve spontaneously by 1 year of age, the lifelong burden on survivors is poorly understood. Using a rat model of chemically induced CHD that resolve postnatally, we sought to characterize the postnatal changes in cardiac function, and to investigate whether resolved CHD affects the ability to adapt to the increased the cardiovascular (CV) burden of pregnancy. To generate rats with resolved CHD, pregnant rats were administered distilled water or dimethadione (DMO) [300 mg/kg b.i.d. on gestation day (gd) 9 and 10] and pups delivered naturally. To characterize structural and functional changes in the heart, treated and control offspring were scanned by echocardiography on postnatal day 4, 21, and 10-12 weeks. Radiotelemeters were implanted for continuous monitoring of hemodynamics. Females were mated and scanned by echocardiography on gd12 and gd18 during pregnancy. On gd18, maternal hearts were collected for structural and molecular assessment. Postnatal echocardiography revealed numerous structural and functional differences in treated offspring compared with control; however, these resolved by 10-12 weeks of age. The CV demand of pregnancy revealed differences between treated and control offspring with respect to mean arterial pressure, CV function, cardiac strain, and left ventricular gene expression. In utero exposure to DMO also affected the subsequent generation. Gd18 fetal and placental weights were increased in treated F2 offspring. This study demonstrates that in utero chemical exposure may permanently alter the capacity of the postnatal heart to adapt to pregnancy and this may have transgenerational effects.

In utero dimethadione exposure causes postnatal disruption in cardiac structure and function in the rat.

In utero exposure of rat embryos to dimethadione (DMO), the N-demethylated teratogenic metabolite of the anticonvulsant trimethadione, induces a high incidence of cardiac heart defects including ventricular septal defects (VSDs). The same exposure regimen also leads to in utero cardiac functional deficits, including bradycardia, dysrhythmia, and a reduction in cardiac output (CO) and ejection fraction that persist until parturition (10 days after the final dose). Despite a high rate of spontaneous postnatal VSD closure, we hypothesize that functional sequelae will persist into adulthood. Pregnant Sprague Dawley rats were administered six 300 mg/kg doses of DMO, one every 12 h in mid-pregnancy beginning on the evening of gestation day 8. Postnatal cardiac function was assessed in control (CTL) and DMO-exposed offspring using radiotelemetry and ultrasound at 3 and 11 months of age, respectively. Adult rats exposed to DMO in utero had an increased incidence of arrhythmia, elevated blood pressure and CO, greater left ventricular volume and elevated locomotor activity versus CTL. The mean arterial pressure of DMO-exposed rats was more sensitive to changes in dietary salt load compared with CTL. Importantly, most treated rats had functional deficits in the absence of a persistent structural defect. It was concluded that in utero DMO exposure causes cardiovascular deficits that persist into postnatal life in the rat, despite absence of visible structural anomalies. We speculate this is not unique to DMO, suggesting possible health implications for infants with unrecognized gestational chemical exposures.

Regulation and control of AP-1 binding activity in embryotoxicity.

The electrophoretic mobility shift assay (EMSA) is a sensitive relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the ³²P-labeled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "supershift," is also presented. The important points are underscored and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.

Noninvasive high-resolution ultrasound reveals structural and functional deficits in dimethadione-exposed fetal rat hearts in utero.

BACKGROUND: We previously showed dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant trimethadione, induces ventricular septation defects (VSD) and other heart anomalies in rat (Weston et al., 2011). Because of the relationship between cardiac structure and function, we hypothesized that DMO-induced structural defects of the heart are associated with in utero functional deficits. To test the hypothesis, the goals were (1) define the parameters for ultrasound in the rat conceptus, and; (2) use ultrasound to identify structural and functional deficits following DMO treatment. METHODS: Different ultrasound modes (B-mode, M-mode, and Pulse-wave Doppler) using four high-resolution ultrasound transducer heads of varying frequency (25-40 MHz) were tested on gestational day (GD) 14, 15, 16, 17, and 21. Having identified the optimal conditions, pregnant Sprague-Dawley rats were administered six 300 mg/kg doses of DMO every 12 hr beginning at 19:00 hr on GD 8 to generate conceptuses with a high incidence of VSD. RESULTS: The three ultrasound modalities were used to identify VSD and several novel and rare structural heart anomalies (cardiac effusions and bifurcated septum) in live rat fetuses. DMO-treated hearts had an array of functional deficits including a decrease in mean heart rate, ejection fraction, and cardiac output and increased incidence of bradycardia and dysrhythmia. CONCLUSIONS: The ultrasound biomicroscope is an effective tool for the real-time characterization of the structure and function of embryo/fetal rat hearts. DMO causes significant deficits to in utero heart function for up to ten days (GD 21) following its final administration, suggesting long-term or possible permanent changes cardiac function.

Co-variation in frequency and severity of cardiovascular and skeletal defects in Sprague-Dawley rats after maternal administration of dimethadione, the N-demethylated metabolite of trimethadione.

BACKGROUND: The anticonvulsant trimethadione is a potent inducer of ventricular septation defects, both clinically and in rodents. Teratogenicity requires its N-demethylation to dimethadione, the proximate teratogen. It was previously demonstrated trimethadione only induced membranous ventricular septation defects in rat (Fleeman et al., 2004), and our present goal is to determine whether direct administration of dimethadione increases the incidence and severity of septation defects. METHODS: Pregnant Sprague-Dawley rats were divided into five groups and administered either distilled water (control) or four different regimens of dimethadione. The core treatment was 300 mg/kg dimethadione b.i.d. on gestation day 9, 10 with additional groups given one additional dose of dimethadione 12 hr earlier, 12 hr later or two additional doses 12 hr earlier and later. Caesarian sections occurred on gestation day 21 and fetuses were examined for standard developmental toxicity endpoints. RESULTS: The broadest dosing regimen yielded the highest incidence and the most severe heart and axioskeletal findings with a decrease in mean fetal body weight. The overall incidence of ventricular septation defects was 74%, of which 68% were membranous and 9% muscular. Outflow tract anomalies (17%) were also observed, as were malformations of the axioskeleton (97%), but not of the long bones, and of particular interest was the high incidence of sternoschesis. CONCLUSIONS: Unlike trimethadione, dimethadione induces more serious muscular septation defects that are believed to be more clinically relevant. This, when taken together with the high incidence of total septation anomalies suggests dimethadione is useful for the study of chemically induced ventricular septation defects.

Not a walk in the park: the ECVAM whole embryo culture model challenged with pharmaceuticals and attempted improvements with random forest design.

BACKGROUND: The European Committee for the Validation of Alternative Methods (ECVAM) supported the development of a linear discriminant embryotoxicity prediction model founded on rat whole embryo culture (Piersma et al. (2004). Altern Lab Anim 32:275­307). Our goals were to (1) assess the accuracy of this model with pharmaceuticals, and (2) to use the data to develop a more accurate prediction model. METHODS: Sixty-one chemicals of known in vivo activity were tested. They were part of the ECVAM validation set (N513), commercially available pharmaceuticals (N531), and Pfizer chemicals that did not reach the market, but for which developmental toxicity data were available (N517). They were tested according to the ECVAM procedures. Fifty-seven of these chemicals were used for Random Forest modeling to develop an alternate model with the goal of using surrogate endpoints for simplified assessments and to improve the predictivity of the model. RESULTS: Using part of the ECVAM chemical test set, the ECVAM prediction model was 77% accurate. This approximated what was reported in the validation study (80%; Piersma et al. (2004). Altern Lab Anim 32:275­307). However, when confronted with novel chemicals, the accuracy of the linear discriminant model dropped to 56%. In an attempt to improve this performance, we used a Random Forest model that provided rankings and confidence estimates. Although the model used simpler endpoints, its performance was no better than the ECVAM linear discriminant model. CONCLUSIONS: This study confirms previous concerns about the applicability of the ECVAM prediction model to a more diverse chemical set, and underscores the challenges associated with developing embryotoxicity prediction models.

Cyclophosphamide and the Teratology Society: an awkward marriage.

Cyclophosphamide (CPA) is a potent and highly effective chemotherapeutic and immunosuppressive agent that has been marketed for about 50 years. Reports of its teratogenicity emerged just after the Teratology Society was established, and from that time forth CPA has been inextricably linked to the Society's goal of understanding and preventing birth defects. CPA teratogenesis was previously reviewed (Mirkes, 1985), and since that time the pathways leading to teratogenesis have become more complicated, with many contradictions. By causing DNA strand breaks, crosslinks, and adducts, CPA is highly effective at disrupting the integrity of the genome. This was the focus of CPA teratogenesis research for many years. However, it is now clear that CPA disrupts the embryonic epigenome and the functionality of the proteome, and that these perturbations are related to teratogenesis. CPA also induces cell-cycle arrest and apoptosis in the embryo but there is conflicting data as to whether these changes are embryoprotective or teratogenic. In addition, CPA has made a number of diverse contributions to the field of developmental toxicology. For example, the concept of male-mediated teratogenesis, in the absence of compromised fertility parameters, was established using CPA. Antivivisectionist sentiment has produced a resurgent interest in in vitro developmental toxicity screens, and with it the need to identify proteratogens that typically are false negatives in such systems due to the relative dearth of P-450 activity in early embryonic tissues. The requirement of P-450 for CPA-mediated embryotoxicity has made CPA an excellent tool with which to probe the metabolic competence of adjunct P-450 supplements in these in vitro systems. Recently, it was noted that in utero exposure to CPA disrupts the immunofunction markers at parturition, suggesting CPA may be a future model for developmental immunotoxicology.

Defects in embryonic development of EGLN1/PHD2 knockdown transgenic mice are associated with induction of Igfbp in the placenta.

The HIF (hypoxia inducible factor) hydroxylases EGNL1/PHD2 has been implicated in embryonic development. Here we knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. The partial inhibition of EGLN1 in utero is embryonic lethal in some, but not all mice on gestation day 14, and is associated with defects in placental and heart development, similar to those noted in the EGLN1 knockout mouse. Importantly, the in utero inhibition of EGNL1 varied greatly between the embryo proper and the placenta. Using this as a tool we show that the embryopathic effects are associated with knockdown of EGNL1 and the associated induction of Igfbp1 (insulin-like growth factor binding protein-1) mRNA in the placenta, but not the embryo.

Analysis of the role of the HIF hydroxylase family members in erythropoiesis.

HIF (hypoxia-inducible factor) hydroxylases have been implicated in EPO (erythropoietin) production and erythropoiesis. Here we examined the role of each of the three EGLN family members and the HIF asparaginyl hydroxylase FIH (factor inhibiting HIF) in EPO production. We examined the effect of inhibiting individual as well as combinations of HIF hydroxylases with RNAi. We found that inhibition of EGLN1 (egl nine homolog 1) as well as other members of the EGLN family (EGLN2 and EGLN3) led to accumulative EPO production in vitro. We then knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. Among all the in vitro and in vivo studies, no or minimal VEGF (vascular endothelial growth factor) mRNA or protein stimulation resulted from inhibition of EGLN1.

Refinement of a morphological scoring system for postimplantation rabbit conceptuses.

BACKGROUND: The rabbit is used extensively in developmental toxicity testing, yet basic information on rabbit embryo development is lacking. The goals of this study were to refine a rabbit embryo morphology scoring system, and use it to evaluate rabbit whole embryo cultures (WEC). METHODS: A total of 265 conceptuses were harvested between GD 8.0 and 12.0 (coitus = GD 0) at 6-hr intervals and examined in detail. Discreet developmental landmarks were then established for 18 morphological features and assigned scores ranging from 0 up to 6. The scoring system was then validated on a subset of randomly selected in vivo conceptuses, and was used to evaluate conceptuses grown for 12, 24, 36, or 48 hr in WEC beginning from GD 9.0 or 10.0. A few embryos also were examined using microscopic computed tomography (microCT)-based virtual histologytrade mark to assess the utility of this technology. RESULTS: Morphology scores of in vivo developed conceptuses increased linearly (r2 = 0.98) with advancing gestational age, from means of 0.0 on GD 8.0 to 67.9 on GD 12.0. Application of the scoring system, supplemented with evidence from Virtual histologytrade mark, indicated that the WEC system supported normal morphological development of rabbit conceptuses. However, when explanted at GD 9, the rate of development was about 20% slower than in vivo, whereas the rate of development in WEC from GD 10 was indistinguishable from in vivo. CONCLUSIONS: This work enhances the evaluation tools available to study mechanisms of normal and abnormal development in this widely used animal testing species.

Thoracic skeletal defects and cardiac malformations: a common epigenetic link?

Congenital heart defects (CHDs) are the most common birth defects in humans. In addition, cardiac malformations represent the most frequently identified anomaly in teratogenicity experiments with laboratory animals. To explore the mechanisms of these drug-induced defects, we developed a model in which pregnant rats are treated with dimethadione, resulting in a high incidence of heart malformations. Interestingly, these heart defects were accompanied by thoracic skeletal malformations (cleft sternum, fused ribs, extra or missing ribs, and/or wavy ribs), which are characteristic of anterior-posterior (A/P) homeotic transformations and/or disruptions at one or more stages in somite development. A review of other teratogenicity studies suggests that the co-occurrence of these two disparate malformations is not unique to dimethadione, rather it may be a more general phenomenon caused by various structurally unrelated agents. The coexistence of cardiac and thoracic skeletal malformations has also presented clinically, suggesting a mechanistic link between cardiogenesis and skeletal development. Evidence from genetically modified mice reveals that several genes are common to heart development and to formation of the axial skeleton. Some of these genes are important in regulating chromatin architecture, while others are tightly controlled by chromatin-modifying proteins. This review focuses on the role of these epigenetic factors in development of the heart and axial skeleton, and examines the hypothesis that posttranslational modifications of core histones may be altered by some developmental toxicants.

Rat embryos express transcripts for cyclooxygenase-1 and carbonic anhydrase-4, but not for cyclooxygenase-2, during organogenesis.

BACKGROUND: Acetylsalicylic acid (ASA) is a rat teratogen, and exposures on gestational days (GDs) 9 and 10 induce diaphragm, cardiac, and midline defects. ASA inhibits members of the cyclooxygenase (COX) family and potentially members of the carbonic anhydrase (CA) family. The objective of this study was to determine whether the mRNA developmental expression pattern for any COX or CA isoform was consistent with a model in which ASA teratogenicity is mediated through direct interaction with one of these enzymes within embryos or within the adjacent ectoplacental cone (EPC) or yolk sac. METHODS: Staged embryos, over a range (GD 9.5-12) that included ASA-sensitive and ASA-insensitive stages of organogenesis, were assayed for COX and CA mRNA levels by three techniques: microarrays; in situ hybridization quantitated by a micro-imager; and quantitative reverse transcription polymerase chain reaction. ASA- and vehicle-treated embryos also were compared to determine whether inhibition led to upregulated COX or CA mRNA expression. RESULTS: COX-2 mRNA was undetectable in embryos throughout organogenesis by any assay (although it was abundant in EPC). In contrast, COX-1 mRNA was moderately abundant in embryos throughout organogenesis. One CA isoform, CA-4, demonstrated developmentally regulated embryonic mRNA expression that coincided with ASA sensitivity ASA exposure failed to induce upregulation of any of these mRNAs. CONCLUSIONS: Although ASA may affect the embryo indirectly through interaction with COX-2 within EPC, failure to detect embryonic COX-2 mRNA argues against COX-2 functioning as a direct mediator of ASA teratogenic activity in induction of cardiac, diaphragm, and midline defects. Correlation of COX-1 and CA-4 expression with ASA sensitivity suggested that embryonic COX-1 and possibly CA4 are much more likely candidates for mediators of ASA developmental toxicity.