Double Staining of Skeleton Using Microwave Irradiation

The fetal skeleton double staining method is used to reveal developmental abnormalities in the skeletal system. We used alizarin red S and al-cian blue successfully with microwave irradiation for skeletal double staining. The fixation time was reduced from 4-7 days to 2-2.5 min and the staining time was reduced from 4 days to 23 min.     

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale.     

High-throughput staining for the evaluation of fetal skeletal development in rats and rabbits

Typical developmental toxicity studies require the assessment of fetal skeletal development. Regulatory guidelines require the assessment of bone ossification and indicate preferences for an assessment of both ossified bone as well as cartilaginous elements. Current manual methods to process fetuses for skeletal examination, whether single or double staining, are laborious and time consuming, and ultimately extend the time before study interpretations. There is a definite need for a quick and efficient, yet reliable, procedure to generate stained fetal skeletons for analysis. A non-automated high-throughput method for single and double staining rat and rabbit fetuses for skeletal evaluations is described, which results in excellent quality specimens ready for evaluations in approximately 3 days for rats and 7 days for rabbits.     

An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon PathcentreTM, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.     

Sensitive Windows of Skeletal Development in Rabbits Determined by Hydroxyurea Exposure at Different Times throughout Gestation

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo–fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day,GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio‐facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.     

二噁英致大鼠先天骨骼发育畸形中软骨细胞的病理学改变

目的探讨大鼠骨骼发育过程中环境类致畸因子对大鼠骨骼发育的先天性致畸作用。方法应用不同剂量的环境类致畸因子-二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD）构建先天性Wistar大鼠骨骼发育畸形动物模型;茜素红染色法制作并观察透明骨骼标本;采用光镜和透射电镜观察胎鼠趾骨骨化中心的软骨细胞病理学变化及细胞超微结构的改变。结果TCDD在5～15μg／kg浓度下诱导了大鼠骨骼发育畸形,畸形包括：内翻足、脊柱裂、腭裂、无尾畸形等,并存在剂量依赖性生物学效应;光镜下可见在畸形胎鼠的肢端骨化中心软骨发生带缩小,软骨细胞变性。透射扫描电镜下见软骨细胞核内粗面内质网扩张,核基质降解,线粒体嵴不规则。结论在高雌激素水平下,TCDD可以诱导大鼠骨骼发育畸形;TCDD可能通过干扰软骨细胞的形态和功能代谢,引起原发性骨化中心的结构紊乱而发挥骨骼致畸效应。     

The Developmental Basis of Skeletal Cell Differentiation and the Molecular Basis of Major Skeletal Defects

Vertebrate skeletal differentiation retains elements from simpler phyla, and reflects the differentiation of supporting tissues programmed by primary embryonic development. This developmental scheme is driven by homeotic genes expressed in sequence, with subdivision of skeletal primordia driven by a combination of seven transmembrane‐pass receptors responding to Wnt‐family signals, and by bone morphogenetic family signals that define borders of individual bones. In sea‐dwelling vertebrates, an essentially complete form of the skeleton adapted by the land‐living vertebrates develops in cartilage, based on type II collagen and hydrophilic proteoglycans. In bony fishes, this skeleton is mineralized to form a solid bony skeleton. In the land‐living vertebrates, most of the skeleton is replaced by an advanced vascular mineralized skeleton based on type I collagen, which reduces skeletal mass while facilitating use of skeletal mineral for metabolic homeostasis. Regulation of the mammalian skeleton, in this context, reflects practical adaptations to the needs for life on land that are related to ancestral developmental signals. This regulation includes central nervous system regulation that integrates bone turnover with overall metabolism. Recent work on skeletal development, in addition, demonstrates molecular mechanisms that cause developmental bone diseases.     

Developmental toxic effects of antifungal flusilazole administered by gavage to mice

BACKGROUND: The developmental toxicity of flusilazole was studied in CD-1 mice after oral administration. METHODS: Pregnant mice were given flusilazole at doses of 0 (corn oil), 10, 20, and 40 mg/kg/day, by gavage, on gestational days (GD) 6-15. RESULTS: Maternal toxicity, as evidenced by reduction in body weight gain and signs of toxicity, was observed at the middle- and high-dose groups. No significant incidence of resorptions or death was observed in any of dose groups. There was a pronounced reduction in fetal weight, which was significantly lower than control from 20 and 40 mg/kg/day. There was no significant increase in the incidence of fetuses with external or visceral malformations in any of dose groups, but there was a significant increase in the incidence of skeletal malformations was observed at 20 and 40 mg/kg/day. CONCLUSIONS: The results of this study reported marked maternal toxicity, growth retardation, and skeletal abnormalities in the mid- and high-dose groups. It seems likely that marked maternal toxicity contributed to the observed alterations in fetal growth retardation and skeletal development. The no-observed-effect level in the present study for maternal and developmental toxicity was 10 mg/kg/day.     

The role of maternal toxicity in lovastatin-induced developmental toxicity

The role of maternal toxicity in lovastatin-induced developmental toxicity in rats was examined in a series of studies. The first study administered lovastatin at 100, 200, 400, or 800 mg/kg/day (mkd) orally to mated rats from Gestation Day (GD) 6 through 20. Maternal toxicity was observed as transient dose-related body weight losses at the initiation of dosing; there were also deaths and/or morbidity at 400 and 800 mkd. These toxicities occurred in conjunction with forestomach lesions. Mean fetal weights were decreased in all groups (-5 to -16%), and the incidence of skeletal malformations, variations, and incomplete ossifications was increased. The 2 highest doses produced the most severe maternal and developmental effects. Using the same dosages, the second study avoided gestational maternal weight losses and morbidity by starting treatment 14 days before mating with dosing continued to GD 20. There were transient dose-related body weight losses after the start of dosing and deaths in the 400- and 800-mkd groups; however, there was no evidence of maternal toxicity during gestation. Developmental toxicity was evident only as slight, but generally significant (p< or =0.05) decreases in mean fetal weights in groups given > or =200 mkd (-2 to -5%). Significantly, no skeletal abnormalities were observed. A third study administered the pharmacologically active metabolite of lovastatin subcutaneously at dose levels that matched oral maternal drug exposures. In the high-dose group, maternal weight gain and mean fetal weight were slightly decreased but there were no treatment-related skeletal abnormalities. Finally, a series of toxicokinetic studies assessed whether the 2 different developmental toxicity profiles were due to differences in drug exposure between the developmentally toxic and non-toxic dosing regimes. The data showed that groups with no skeletal abnormalities had maternal and embryonic/fetal drug concentrations similar to or even greater than the groups with fetal abnormalities. These results indicate that fetal skeletal abnormalities observed at lovastatin dose levels > or =100 mkd are not due to a direct teratogenic effect, but are the result of excessive maternal toxicity, which most likely involves a nutritional deficiency associated with forestomach lesions and reduced maternal food intake.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Altered axial skeletal development

The axial skeleton is routinely examined in standard developmental toxicity bioassays and has proven to be sensitive to a wide variety of chemical agents. Dysmorphogenesis in the skull, vertebral column and ribs has been described in both human populations and in laboratory animals used to assess potential adverse developmental effects. This article emphasizes vertebrae and rib anomalies both spontaneous and agent induced. Topics discussed include the morphology of the more common effects; incidences in both human and experimental animal populations; the types of anomalies induced in the axial skeleton by methanol, boric acid, valproic acid and others; the postnatal persistence of common skeletal anomalies; and the genetic control of the development of the axial skeleton. Tables of the spontaneous incidence of axial anomalies in both humans and animals are provided.     

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs

Delayed (or incomplete) ossification of developing fetal bones and wavy ribs are some of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. Although they tend to be regarded as minor effects, they can be quite sensitive and consequently may influence the study lowest-observed-adverse-effect levels (LOAELs), and thus, impact classification, labeling, and risk assessment. In this study, we review the underlying mechanisms of these skeletal variations, evaluate different scenarios in which they have been observed, offer guidance for their interpretation, and comment on their use for risk assessment. Both minor delays in ossification and wavy ribs seem to be readily repairable via postnatal skeletal remodeling, are not mechanistically linked to malformation, and often are seen in the presence of maternal or fetal toxicity. As such, these minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal findings, information available on normal skeletogenesis patterns, mode of action of the test agent, and historical control incidence using a weight of evidence approach.     

Dose–Response Effects of Diphenylhydantoin on Pregnant Dams and Embryo‐Fetal Development in Rats

Despite the widespread use of diphenylhydantoin (DPH), there is a lack of reliable information on the teratogenic effects, correlation with maternal and developmental toxicity, and dose–response relationship of DPH. This study investigated the dose–response effects of DPH on pregnant dams and embryo‐fetal development as well as the relationship between maternal and developmental toxicity. DPHwas orally administered to pregnant rats from gestational days 6 through 15 at 0, 50, 150, and 300 mg/kg/day. At 300 mg/kg, maternal toxicity including increased clinical signs, suppressed body weight, decreased food intake, and increased weights of adrenal glands, liver, kidneys, and brain were observed in dams. Developmental toxicity, including a decrease in fetal and placental weights, increased incidence of morphological alterations, and a delay in fetal ossification delay also occurred. At 150 mg/kg, maternal toxicity manifested as an increased incidence of clinical signs, reduced body weight gain and food intake, and increased weights of adrenal glands and brain. Only minimal developmental toxicity, including decreased placental weight and an increased incidence of visceral and skeletal variations, was observed. No treatment‐related maternal or developmental effects were observed at 50 mg/kg. These results show that DPH is minimally embryotoxic at a minimal maternotoxic dose (150 mg/kg/day) but is embryotoxic and teratogenic at an overt maternotoxic dose (300 mg/kg/day). Under these experimental conditions, the no‐observed‐adverse‐effect level of DPH for pregnant dams and embryo‐fetal development is considered to be 50 mg/kg/day. These data indicate that DPH is not a selective developmental toxicant in the rat.     

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.     

Effects of tert-butyl acetate on maternal toxicity and embryo-fetal development in Sprague-Dawley rats

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.     

Developmental toxicity evaluation of orthovanadate in the mouse

Sodium orthovanadate in deionized water was administered once daily by gavage on gestational days 6-15 to mice at doses of 0, 7.5, 15, 30, and 60 mg/kg. Dams were killed on day 18 of pregnancy, and fetuses were examined for external, visceral, and skeletal defects. Maternal toxicity was observed at the highest doses of sodium orthovanadate, as evidenced by a significant number of deaths (60 and 30 mg/kg/d) and reduced weight gain and food consumption (30 and 15 mg/kg/d). Embryolethality and teratogenicity were not observed at maternally toxic doses and below, but fetal toxicity was evidenced by a significant delay in the ossification process of some skeletal districts at 30 mg/kg/d. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was 7.5 mg/kg/d, and 15 mg/kg/d represented a NOAEL for developmental toxicity in mice under the conditions of this study.     

Sequential changes in weight of the skeleton and in length of long limb bones of Macaca mulatta.

In a collection of 274 monkeys (Macaca mulatta) the relative weight of the dry, fat-free skeleton, expressed as a proportion of total body weight, increases significantly throughout the gestational period to approximately 6% with only random variation after birth. The weight of the fetal skeleton increases exponentially with age. In the postnatal period the skeletal weight increases asymptotically to adulthood, which is considered to be 6.5 years of age. Equations for estimating skeletal weight are presented. Of four subdivisions of the skeleton, the skul contributes the greatest proportion of total skeletal weight in the fetal stage with the proportion decreasing to adulthood. The contributions of the other subdivisions, postcranial axial, superior limb, and inferior limb, and inferior limb, are nearly equal in the fetal stage, with that of only the inferior limb increasing to adulthood, when it makes up the greatest proportion of total skeletal weight. Until the last third of the gestational period, the humerus is longer than the femur and the radius longer than the tibia. Thereafter, the inferior limbs grow at a faster rate than the superior limbs, resulting in an intermembral index of approximately 95% by birth and less than 90% by adulthood.     

Commentary on the Role of Maternal Toxicity on Developmental Toxicity

Maternal mammalian toxicity impacts prenatal development, with general systemic maternal toxicity, from reduced weight gain to morbidity, causative for reduced fetal weights/litter and increased fetal variations (especially skeletal)/litter, but not, in the author's opinion, for increased fetal malformations, reduced litter sizes or full litter losses. Increased fetal malformations are likely due to exposure to specific chemicals which alter specific maternal functions at critical point(s) in pregnancy, typically exaggerated effects from higher doses by drugs under development with known, desired pharmacological effects. Malformations can also be from genetic/epigenetic alterations, specific altered proteins, molecular pathways, etc. Full litter losses are triggered by the mother and are rare in rats. Information to inform maternal (and developmental) toxicity includes ovarian corpora lutea counts, uterine implantation profile, degree of litter reduction (if present), timing and extent of maternal toxicity relative to those of adverse embryofetal effects, etc. The view of maternal toxicity as confounding results in in vivo developmental toxicity studies, worldwide concerns about increased research animal usage, increasing time, labor, costs, and new software and hardware sophistication all drive the interest in development, validation, and performance of in vitro/in silico assays. These assays are fast, inexpensive, responsive to animal use concerns and amenable to mechanistic questions. The strength of these in vitro/in silico assays is considered by many to be the absence of the maternal organism/placenta. These assays inform mechanism and hazard, but NOT risk. The Environmental Protection Agency currently estimates that these new assays are approximately 70% accurate versus the whole animal tests.