Variability of Microcystin-LR Standards Available from Seven Commercial Vendors.

Microcystins (MCs) are a large group of heptapeptide cyanobacterial toxins commonly produced in harmful algal blooms (HABs) and associated with adverse health effects in wildlife, livestock, pets, and humans. MC chemical standards are extracted from cyanobacteria biomass rather than produced synthetically and are used in water assessment methods and toxicological studies. MC standards are generally supplied in less than 1 mg quantities, and verification of the mass can only be accomplished by analytical chemistry methods using a certified reference of the specific MC for comparison. Analytical quantification of MCs in environmental samples and toxicology studies using accurate doses of test chemicals administered to experimental animals rely on the availability and accuracy of chemical standards. To check the accuracy and purity of available standards, seven individual microcystin-LR (MCLR) standards were purchased from separate commercial vendors and analyzed to determine the actual mass supplied and identify the presence of potential contaminants. To determine the effect of varying toxin mass in toxicological studies, each MCLR standard was administered to CD-1 mice in doses based on mass purchased, by a single 40 µg/kg intraperitoneal injection. The measured mass purchased varied from the vendor label mass by more than 35% for two of the seven MCLR standards. Contaminants, including trifluoroacetic acid (TFA), were identified in four of the seven samples. Comparative in vivo hepatotoxicity between vendor samples closely reflected the actual amount of MCLR present in each standard and demonstrated the toxicological impact of varying cyanotoxin mass.

A rapid assessment bioaccumulation screening (RABS) study design for emerging per-and polyfluoroalkyl substances in mice exposed to industrially impacted surface water.

The shift away from PFOS and PFOA production in the past 20 years towards shorter chain and replacement PFAS has led to the environmental release of complex mixtures of emerging PFAS for which bioaccumulation potential and toxicology are largely unknown. The rate at which emerging PFAS can be prioritized for research in these complex mixtures is often limited by the lack of available chemical standards. We developed a study design that rapidly assesses which emerging PFAS in an environmentally derived mixture have the potential for mammalian bioaccumulation and thus prioritize these emerging chemicals for standard synthesis and toxicity testing. Surface water was collected at an impacted site downstream of an industrial fluorochemical manufacturing outfall and concentrated 100-fold via weak anion exchange, solid-phase extraction. The concentrated extract contained 13 previously identified emerging PFAS, including hexafluoropropylene oxide-dimer acid (HFPO-DA). BALB/c mice were orally dosed with surface water concentrate once a day for seven days. Twenty-four hours after the last dose, liver, serum, urine, and feces were collected and the emerging PFAS were semi-quantified based on peak area counts. Of the 13 emerging PFAS, Nafion byproduct-2 (Nafion BP2), Hydro-EVE, PFO4DA, and PFO5DoA had the largest increases in percent composition when comparing serum and liver to the dosing solution, suggesting that these PFAS may have the highest bioaccumulation potential. This finding supports other studies that detected bioaccumulation of the same four PFAS in human serum collected from communities with contaminated drinking water. In the future, the Rapid Assessment Bioaccumulation Screening (RABS) study design can be extended to other complex industrial chemical mixtures impacting surface water in order to better inform chemical prioritization for acquisition and in vitro/in vivo toxicity testing of the potential pollutants.

Dose-Response Study of Microcystin Congeners MCLA, MCLR, MCLY, MCRR, and MCYR Administered Orally to Mice.

Microcystins are common freshwater cyanobacterial toxins that affect liver function. The toxicities of five microcystin congeners (microcystin-LA (MCLA), MCLR, MCLY, MCRR, and MCYR) commonly observed in harmful algal blooms (HABs) were evaluated in BALB/c mice after a single oral administration of doses ranging from those that were no observed adverse effect levels (NOAELs) to lowest observed adverse effect levels (LOAELs). Animals were monitored for changes in behavior and appearance, and euthanized 24 h after dosing. Test endpoints included clinical changes, necropsy observations, and serum indicators of hepatic toxicity and general homeostasis. Doses were 0.5-7 mg/kg MCLA, 0.5-11 mg/kg MCLR, 1-7 mg/kg MCLY, 7-22 mg/kg MCRR, and 3-11 mg/kg MCYR. MCLA at 3 mg/kg elevated liver/body weight ratio and liver score, ALT, AST, and GLDH, indicating hepatic toxicity, reduced serum glucose and highly elevated total serum bilirubin. MCLR and MCLY induced similar effects with LOAELs of 5 mg/kg, although a greater extent and severity of effects were observed in MCLR animals. MCRR exposure at 22 mg/kg was associated with reduced serum glucose. MCYR induced scattered liver effects at 7 mg/kg and reduced serum glucose levels at 5 mg/kg. The results indicate significant differences in congener-induced toxicity after microcystin exposure.

Toxicity of Balb-c mice exposed to recently identified 1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2).

1,1,2,2-tetrafluoro-2-[1,1,1,2,3,3-hexafluoro-3-(1,1,2,2-tetrafluoroethoxy)propan-2-yl]oxyethane-1-sulfonic acid (PFESA-BP2) was first detected in 2012 in the Cape Fear River downstream of an industrial manufacturing facility. It was later detected in the finished drinking water of municipalities using the Cape Fear River for their water supply. No toxicology data exist for this contaminant despite known human exposure. To address this data gap, mice were dosed with PFESA-BP2 at 0, 0.04, 0.4, 3, and 6 mg/kg-day for 7 days by oral gavage. As an investigative study, the final dose groups evolved from an original dose of 3 mg/kg which produced liver enlargement and elevated liver enzymes. The dose range was extended to explore a no effect level. PFESA-BP2 was detected in the sera and liver of all treated mice. Treatment with PFESA-BP2 significantly increased the size of the liver for all mice at 3 and 6 mg/kg-day. At the 6 mg/kg-day dose, the liver more than doubled in size compared to the control group. Male mice treated with 3 and 6 mg/kg-day and females treated with 6 mg/kg-day demonstrated significantly elevated serum markers of liver injury including alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and liver/body weight percent. The percent of PFESA-BP2 in serum relative to the amount administered was similar in male and female mice, ranged from 9 to 13 %, and was not related to dose. The percent accumulation in the liver of the mice varied by sex (higher in males), ranged from 30 to 65 %, and correlated positively with increasing dose level.

The Comparative Toxicity of 10 Microcystin Congeners Administered Orally to Mice: Clinical Effects and Organ Toxicity.

Microcystins (MCs) are common cyanobacterial toxins that occur in freshwaters worldwide. Only two of the >200 MC variants have been tested for potential toxicity after oral exposure. This paper reports on the toxicity of 10 different MC congeners identified in algal blooms, microcystin-LR (MCLR), MCLA, MCLF, MCLW, MCLY, MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, MCWR, and MCYR after single administrations to BALB/c mice. In a preliminary MCLR dose-response study of 3 to 9 mg/kg doses, ≥5 mg/kg induced clinical changes, increased serum levels of ALT, AST, and GLDH, liver congestion, increased liver/body weight ratios, and reduced serum glucose and total protein. Based on the extent of these effects, the 10 congeners were administered as single 7 mg/kg oral doses and toxicity evaluated. The greatest toxicity was observed with MCLA and MCLR including a high percentage of moribundity. In addition to eliciting effects similar to those listed above for MCLR, MCLA also induced serum alterations indicative of jaundice. MCLY, and MCYR induced changes like those noted with MCLR, but to lesser extents. MCLW and MCLF exhibited some serum and morphological changes associated with hepatic toxicity, while there were few indications of toxicity after exposures to MCRR, [Asp3]MCRR, [Asp3,Dhb7]MCRR, or MCWR. These data illustrate a wide spectrum of hepatic effects and different potencies of these MC congeners.

Pathology in Ecological Research With Implications for One Health: Session Summary.

This session explored the effects of pollutants on One Health at the ecosystem level that included microbes, insects, fish, and humans. The concept of One Health seeks to synergize medical, veterinary, and other health science disciplines to more effectively advance human and animal health. Presentations explored the interactions of pesticides, pathogens, phytochemicals, and xenobiotic biotransformation in bee colony losses critical for food security (bees have been recently listed under the 2017 US Food and Drug Administration (FDA) veterinary feed directive); the role of pathology in identifying the effects of pollutants on fish as sentinels for human health; the effects in rats of per- and polyfluoroalkyl substances (PFAS) that can persist in the environment and contaminate drinking water; harmful algal blooms and toxin production leading to animal and human disease; and the processing of environmental carcinogens by intestinal microbiota.

The analysis of underivatized ß-Methylamino-L-alanine (BMAA), BAMA, AEG & 2,4-DAB in Pteropus mariannus mariannus specimens using HILIC-LC-MS/MS.

ß-Methylamino-L-alanine (BMAA) has been identified as the potential cause of the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) observed in the Chamorro people of Guam. The principal hypothesis for BMAA exposure and intoxication relies on the biomagnification of BMAA in flying fox specimens ingested by the Chamorro people. Although high levels of BMAA were quantitated in flying fox specimens utilizing liquid chromatography-fluorescence (LC-FL), there have not been any confirmatory analyses conducted to date. Therefore, a method for the tissue homogenization, extraction and direct analysis of BMAA (including BAMA, 2,4-DAB and AEG) was utilized. The approach was applied to mammalian dried skin and hair from various rodent species (negative controls) and archived flying fox (Pteropus mariannus mariannus) specimens. A positive control sample of homogenized mussel (Mytelius edulis) with native BMAA was used to verify the method. It was determined that the direct analysis using HILIC MS/MS required additional quality control in order to allow for the confident identification of BMAA due to the near co-elution of BAMA. BMAA was not present above 0.2 µg g-1 (free fraction) or 2.8 µg g-1 (total fraction) in the flying fox specimens. While analysis did not result in BMAA detection in flying fox or negative control samples, the positive control sample and spiked samples were successfully detected.

pH dependent octanol-water partitioning coefficients of microcystin congeners.

Hazardous algal blooms can generate toxic compounds with significant health impacts for exposed communities. The ubiquitous class of algal toxins known as microcystins exhibits significant heterogeneity in its peptide structure, which has been minimally studied, given the significant impact this has on hydrophobicity, acid/base character and related environmental fate and health effects. Octanol-water partition coefficients for the microcystin congeners MCLR, MCRR, MCLY, MCLF, and MCLA were calculated over an environmentally and physiologically relevant pH range. Microcystin-LR log(Kow) partition coefficient values were found to be consistent with previously established literature values, 1.67 to -1.41 between pH 1 and 8. Microcystin RR was found to be pH insensitive with a log(Kow) of -0.7. The remaining congeners exhibit similar pH dependence as MCLR, with systematic increases in hydrophobicity driven by the introduction of more hydrophobic residues to their variable amino acid region. The variation in pH dependent hydrophobicity suggests increased propensity for bioaccumulation and alternate environmental fates for differing microcystin forms, requiring further investigation.

A review on cylindrospermopsin: the global occurrence, detection, toxicity and degradation of a potent cyanotoxin.

Cylindrospermopsin is an important cyanobacterial toxin found in water bodies worldwide. The ever-increasing and global occurrence of massive and prolonged blooms of cylindrospermopsin-producing cyanobacteria poses a potential threat to both human and ecosystem health. Its toxicity is associated with metabolic activation and may involve mechanisms that adversely affect a wide variety of targets in an organism. Cylindrospermopsin has been shown to be cytotoxic, dermatotoxic, genotoxic, hepatotoxic in vivo, developmentally toxic, and may be carcinogenic. Human exposure may occur through drinking water, during recreational activities and by consuming foods in which the toxin may have bioaccumulated. Drinking water shortages of sufficient quality coupled with growing human pressures and climate variability and change necessitate an integrated and sustainable water management program. This review presents an overview of the importance of cylindrospermopsin, its detection, toxicity, worldwide distribution, and lastly, its chemical and biological degradation and removal by natural processes and drinking water treatment processes.

ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its impact on study design and data interpretation.

Workshops on maternal toxicity were held at the annual Society of Toxicology, Teratology Society, and European Teratology Society meetings in 2009. Speakers presented background information prior to a general discussion on this topic. The following recommendations/options are based on the outcome of the discussions at the workshops: 1. A comprehensive evaluation of all available data from general toxicity studies, range-finding Developmental and Reproductive Toxicology (DART) studies, class effects, structure-activity relationships, exposure studies, etc. is essential for appropriate dose selection for definitive DART studies. The intent is to avoid marked maternal toxicity leading to mortality or decreased body weight gains of greater than 20% for prolonged periods. (a) Evaluate alternative endpoints for dose selection and data interpretation (e.g., target tissue effects and pharmacology) for biotherapeutics. (B) Evaluate additional maternal parameters based on effects and/or target organs observed in short-term (e.g., 2- or 4-week) general toxicity studies. 2. Evaluate all available data to determine a cause-effect relationship for developmental toxicity. (a) Conduct a pair-feeding/pair-watering study as a follow-up. (b) Evaluate individual data demonstrating maternal toxicity in the mother with adverse embryo-fetal outcomes in the litter associated with the affected mother. (c) Conduct single-dose studies at increasing doses as a complement to conventional embryo-fetal toxicity studies for certain classes of compounds that affect the hERG channel. 3. Support statements that embryo-fetal effects are caused by maternal toxicity and/or exaggerated pharmacology, especially for malformations. (a) Provide mechanistic or other supporting data. (b) Establish the relevance of the DART findings in animals for human exposures. Birth Defects Res (Part B) 92:36-51, 2010. © 2011 Wiley-Liss, Inc.

Hypoxia and the Edema Syndrome: elucidation of a mechanism of teratogenesis.

The elucidation of mechanisms and pathogenesis of birth defects is exceedingly complex. Consequently, there are few examples where the etiology of birth defects caused by a specific agent has been well described. One such example is the "Edema Syndrome" first described by Casimer Grabowski in the 1960s as a mechanism of hypoxia-induced malformations in the chick embryo. The Edema Syndrome comprised a series of events in the embryo starting with osmotic imbalances followed by edema, distention, blisters, hematomas, and hemorrhage in or near developing structures. Malformation or deformation of structures resulted from mechanical disruption or loss of blood supply. A similar etiology has since been described by others in a variety of laboratory mammals following treatment with drugs including epinephrine, hydroxyurea, cocaine, phenytoin, and potassium channel-blocking drugs. Free radical excess following transient hypoxia may be a common factor in all of these insults. Vascular disruption is also associated with a number of birth defects in humans, including limb and digit reduction defects and urogenital defects.

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.

Short-term exposures to dihaloacetic acids produce dysmorphogenesis in mouse conceptuses in vitro.

The haloacetic acids (HAAs) are a family of xenobiotics found in tap water as a result of drinking water disinfection. Administration of HAAs to rats produces a variety of adverse effects, including developmental toxicity. The dysmorphogenic potencies of all nine bromo/chloro-acetic acids have been determined in rodent whole embryo culture using standard 26-h exposure. Since the half-lives of the HAAs in vivo are typically <8 h, the developmental effects of short-term exposures to dihaloacetates were evaluated. Gestation day 8 (3-6 somite pairs) CD-1 mouse conceptuses were exposed to 11,000 microM dichloroacetic acid (DCA), 300 microM dibromoacetic acid (DBA) or 300 microM bromochloroacetic acid (BCA) for culture periods of 1, 3, 6 or 26 h. Following 1, 3 or 6 h of exposure to HAAs, conceptuses were transferred to control medium to complete a 26-h culture period. The amounts of HAAs present in embryos after 1, 3 and 6h of exposure were determined. Increased incidences of dysmorphic embryos were produced by 6 or 26-h exposures to DCA; a 26-h exposure to DBA; or 3, 6 or 26-h exposures to BCA. The dysmorphology produced was dependent upon the length of exposure and chemical. The embryonic concentration of each HAA (104.5, 2.5 and 2.6 pmol/microg protein for DCA, DBA and BCA, respectively) was reached by 1h of exposure and did not change at the subsequent time points examined. The current studies demonstrate that BCA is more potent than DBA or DCA at disrupting embryogenesis since shorter exposures alter morphogenesis. Since the embryonic HAA concentrations were the same at the three time points measured, the time-dependence in dysmorphogenesis does not appear to be a simple function of increasing embryonic concentration of these chemicals. These studies demonstrate that for these dihaloacetic acids relatively high concentrations and long exposures are needed to alter rodent development in vitro.

Bromochloro-haloacetic acids: effects on mouse embryos in vitro and QSAR considerations.

The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection by-products. We previously reported that haloacetic acids, including several bromo- and chloro-HAAs, alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. Craniofacial dysmorphogenesis was observed in exposed embryos and a quantitative structure activity relationship (QSAR) for induction of cranial neural tube dysmorphogenesis was established for a series of 10 HAAs, which also included fluoro- and iodo-HAA representatives. In the current study, we evaluate the effects of exposing neurulation staged (3-6 somite pairs) CD-1 mouse conceptuses to bromochloro- (BCA), dibromochloro- (DBCA) and bromodichloro-acetic (BDCA) acids in whole embryo culture at concentrations ranging from 50 to 2500 microM. Morphological development was assessed after a 26 h exposure period. Exposure of conceptuses to these HAAs produced dysmorphogenesis, including prosencephalic and pharyngeal arch hypoplasia as well as eye and heart tube abnormalities. Benchmark concentrations for induction of neural tube dysmorphogenesis were 63, 500 and 536 microM for BCA, DBCA and BDCA, respectively. Our previously developed HAA QSAR accurately predicted placement of these three chemicals in the larger context of the previously tested di- and tri-HAAs, also correctly predicting that BCA would be more potent than DBCA and BDCA, and that the latter two HAAs would be near equi-potent. This study describes the concentration-dependent induction of dysmorphogenesis in whole embryo culture by three mixed chloro/bromo-HAAs and demonstrates the ability of the HAA QSAR to predict relative potencies within this family of xenobiotics.

Evaluation and interpretation of maternal toxicity in Segment II studies: issues, some answers, and data needs.

Biologically rational regulatory policies with regards to developmental toxicity are often based on the extrapolation of standard laboratory rodent bioassay results to the human population. Significantly contributing to the difficulty of this task is the possibility that general toxic effects on the maternal organism may affect the developing conceptus. This review examines maternal factors which may bear directly or indirectly upon developmental outcome, with emphasis on those of greatest relevance to the hazard assessment process. Standard teratology testing protocols call for top dosage levels that induce overt maternal toxicity, and the developmental effects of this toxicity (both alone, and with concurrent embryo/fetal insult) continue to present regulators with considerable interpretive difficulties. In response to these problems, there have been both research and literature review efforts dealing with the relationship of maternal and developmental toxicity. Maternally mediated developmental toxicity occurs with a number of agents, and toxicant-induced alterations in maternal physiology may affect the conceptus at dosages not causing overt maternal toxicity. Relevant studies are reviewed here, and suggestions for avenues of future research are offered including the identification of any syndromes of developmental effects occurring at maternally toxic levels irrespective of the causative agent, and experimental approaches for the characterization of maternal toxicity.

Supernumerary ribs in developmental toxicity bioassays and in human populations: incidence and biological significance.

Supernumerary or accessory ribs (SNR), either lumbar (LR) or cervical (CR), are a common finding in standard developmental toxicology bioassays. The biological significance of these anomalies within the regulatory arena has been problematic and the subject of some debate. In rodents, the spontaneous incidence of SNR is species and strain related and ranges from <1% to >30%. Compound-induced LR are induced by a wide variety of chemical and physical agents when pregnant animals are exposed during specific gestational periods. A significant portion of the agent-induced LR may be due to maternal factors, as it has been shown that stress alone will induce LR in rodents. SNR are not isolated phenomena and signify basic alterations in the architecture of the axial skeleton. LR are associated with longer ribs, increased numbers of vertebrosternal ribs, and the presence of extra presacral vertebrae ("anteriorization"). CR are associated with reduced numbers of vertebrosternal ribs (posteriorization). It is evident that SNR are not a single anomaly, but consist of two unrelated structures: an extra rib that has a cartilaginous segment at the distal end, and an ossification site that lacks cartilage. These have a bimodal size distribution, with the population of extra ribs being significantly longer than the ossification sites, and 0.6 mm can be used as an approximate length for distinguishing the two populations in mice. Extra ribs are permanent structures in contrast to ossification sites that disappear postnatally, probably becoming part of the lateral transverse vertebral processes. SNR are also found in humans although, in contrast to laboratory species, CR are more commonly noted. SNR are associated with adverse heath effects, and CR with inducing thoracic outlet disease characterized by diminished blood flow and altered position of the ganglia and nerve roots in the area of the C7-T1 vertebrae. LR are associated with lower back pain and L4-5 degeneration. The incidence of CR is greatly reduced in adult humans as compared to fetuses, and it has been hypothesized that fetal "SNR" may be largely composed of ossification sites that disappear postnatally. The mechanisms involved in the formation of extra ribs are not understood at this time, although the fact that the early sensitive periods for their initiation during embryogenesis is coupled with the associated changes in the axial skeleton argues for their induction being due to fundamental changes in gene expression. The sum of the experimental evidence supports the idea of SNR being composed of two different structures: extra ribs that are permanent dysmorphological structures that may be induced by xenobiotics and/or maternal stress, and ossification sites that may be transient variations in the formation of the lateral processes of the vertebrae.

Chemically induced supernumerary lumbar ribs in CD-1 mice: size distribution and dose response.

Supernumerary ribs (SNR) of differing sizes are commonly observed in rodent developmental toxicity studies, and the significance of treatment-related increases in SNR in standard studies has been contentious. We induced dose-related increases in SNR in fetal CD-1 mice by treating on gestation days 7-8 with benomyl (BEN; 0, 75, 150 mg/kg/d), dinoseb (DIN; 0, 30, 50 mg/kg/d); 2-methoxyethanol (2-ME; 0, 75, 150 mg/kg/d), or valproic acid (VPA; 0, 125, 250 mg/kg/d). Incidences of SNR were 9.3-27.6% in controls and 19.3-84.4% in the high dosage groups. SNR length showed a bimodal distribution with peaks at 0.3-0.4 mm and 0.9-1.1 mm in both treated and control groups. Based on length distributions, we used an actual length of 0.6 mm to separate short (rudimentary) from long (extra) SNR. DIN, 2-ME, and VPA induced a dose-related increase of extra ribs, while the incidence of rudimentary ribs remained at control levels. There was no apparent correlation of the presence of either type of SNR in a fetus and the occurrence of other anomalies. These data support the idea that extra and rudimentary SNR may reflect separate developmental phenomena, and should be considered and reported separately in developmental toxicity studies for risk assessment.

Periods of vertebral column sensitivity to boric acid treatment in CD-1 mice in utero.

Boric acid (BA) has many uses as an industrial compound and is widely distributed in the environment. BA has been shown to produce rib agenesis, a rare effect in laboratory animals. This study was conducted to determine if there is a period of sensitivity to this unusual effect. BA (500 or 750 mg/kg) was administered p.o. to pregnant CD-1 mice once daily on gestational days (GDs) 6-10. A reduction of 13th rib length occurred at both dose levels. BA 400mg/kg was also administered twice daily on GD 6, 7, 8, 9, or 10 or on GDs 6-8. A significant decrease in average fetal weight was observed in all treatment groups. Significant increases in the incidence of cervical ribs/ossifications resulted from treatments on GD 7 and GDs 6-8. Rib agenesis occurred with treatment on GD 8 and GDs 6-8. Reduced rib length, a decreased incidence of supernumerary ribs (SNR), and an increased incidence of fused and/or branched ribs occurred when dams were treated GDs 6-8. Doses of 750 mg/kg given twice on day 8 produced significant increases in several thoracic skeletal anomalies. Further studies of pathogenesis are necessary to determine the earliest perturbations and the processes that are affected. The sensitivity of embryos to treatment on GD 8 to rib agenesis suggests that BA is affecting early processes such as gastrulation and presomitic mesoderm formation and patterning in this area.

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse.

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.