Cylindrospermopsin toxicity in mice following a 90-d oral exposure.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

A critical review of the postulated role of the non-essential amino acid, ß-N-methylamino-L-alanine, in neurodegenerative disease in humans.

The compound BMAA (ß-N-methylamino-L-alanine) has been postulated to play a significant role in four serious neurological human diseases: Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) found on Guam, and ALS, Parkinsonism, and dementia that occur globally. ALS/PDC with symptoms of all three diseases first came to the attention of the scientific community during and after World War II. It was initially associated with cycad flour used for food because BMAA is a product of symbiotic cycad root-dwelling cyanobacteria. Human consumption of flying foxes that fed on cycad seeds was later suggested as a source of BMAA on Guam and a cause of ALS/PDC. Subsequently, the hypothesis was expanded to include a causative role for BMAA in other neurodegenerative diseases including Alzheimer's disease (AD) through exposures attributed to proximity to freshwaters and/or consumption of seafood due to its purported production by most species of cyanobacteria. The hypothesis that BMAA is the critical factor in the genesis of these neurodegenerative diseases received considerable attention in the medical, scientific, and public arenas. This review examines the history of ALS/PDC and the BMAA-human disease hypotheses; similarities and differences between ALS/PDC and the other diseases with similar symptomologies; the relationship of ALS/PDC to other similar diseases, studies of BMAA-mediated effects in lab animals, inconsistencies and data gaps in the hypothesis; and other compounds and agents that were suggested as the cause of ALS/PDC on Guam. The review concludes that the hypothesis of a causal BMAA neurodegenerative disease relationship is not supported by existing data.

The course of toxicity in the pregnant mouse after exposure to the cyanobacterial toxin cylindrospermopsin: clinical effects, serum chemistries, hematology, and histopathology.

Cylindrospermopsin (CYN) is a toxin produced by a variety of fresh-water cyanobacterial species worldwide and induces significant adverse effects in both livestock and humans. This study investigated the course of CYN-induced toxicity in pregnant mice exposed daily during either the period of major organogenesis (gestation days [GD] 8-12) or fetal growth (GD13-17). Endpoints include clinical signs of toxicity, serum analyses to evaluate hepatic and renal function, histopathology of liver and kidney, and hematology. Study animals were administered 50 µg/kg CYN once daily by ip route and euthanized 24 h after 1, 2, 3, 4, or 5 consecutive doses, or 6 or 13 d after the dosing period. The course of the CYN-induced effects was determined at all euthanasia times for the endpoints just outlined. Results indicated that CYN is a toxin, producing lethality in dams during the early part of gestation, significant weight loss, and bleeding in the gastrointestinal tract, tail tip, and peri-orbital tissues. Effects also included alterations in serum markers for liver function, histopathological changes in liver and kidney tissues, electrolyte abnormalities, leukocytosis, and posttreatment thrombocytopenia and reticulocytosis. The onset of symptoms was rapid, producing reductions in weight gain in GD8-12 animals, bleeding in the vaginal area in GD13-17 animals, and significant increases in sorbitol dehydrogenase (SDH) in both groups after a single dose. Although the GD8-12 dams displayed a 50% lethality, in GD13-17 animals only a single death occurred. Alterations seen in hepatic and renal function or histopathology do not appear to be of sufficient severity to produce death. Evidence indicates that bleeding may play a critical role in the onset of symptoms and eventually, in the observed lethality.

Toxicity and recovery in the pregnant mouse after gestational exposure to the cyanobacterial toxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post-dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8-12 induced significantly more lethality than GD13-17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8-12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13-17 group. Gene expression changes persisted up to 2 weeks post-dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter-animal variability within the treated groups.

Moderate developmental undernutrition: Impact on growth and cognitive function in youth and old age.

Low weight at birth is a common adverse developmental effect reported in human populations and animal toxicity studies. Epidemiological evidence links low birth weight to a syndrome of metabolic changes that increase later risk for obesity, type 2 diabetes, hypertension, and cardiovascular disease. The present study used a four-treatment cross-over experimental design to evaluate the selective impact of early nutritional deficiency on metabolism and brain function across the lifespan of male Sprague Dawley rats. Undernutrition was induced prenatally by restricting maternal food intake to 50% of ad lib from GD3 to birth. Postnatal undernutrition was induced by fostering pups at birth to naïve dams in large (n=16) litters as opposed to small (n=8) control litters. Body weights were monitored in the early neonatal period, in early adulthood beginning at 5months and through to senescence at 21months of age. In contrast to recent reports, no increase in the prevalence of obesity was seen in animals born to food restricted dams and reared under ad lib feeding conditions. Behavioral tests of locomotion, learning and memory were performed in young, middle-aged, and aged animals. No effects of pre or postnatal nutritional history were detected. Age-dependent reductions in locomotor activity were detected, as well as deficits in spatial learning as measured in the Morris water maze and in context fear conditioning. These findings indicate that moderate fetal undernutrition followed by neonatal adequate nutrition does not appear to lead to obesity or neurological dysfunction in young adulthood or old age.

Reproductive effects of maternal and pre-weaning undernutrition in rat offspring: age at puberty, onset of female reproductive senescence and intergenerational pup growth and viability.

Maternal and/or postnatal undernutrition are widespread in human populations and are components of many experimental developmental and reproductive toxicology bio-assays. This study investigated in utero and/or pre-weaning undernutrition effects on reproductive maturation and senescence in the Sprague-Dawley rat as well as potential intergenerational effects. Pregnant rats were given food ad libitum or at 50% of normal dietary intake throughout pregnancy. Their offspring (control or IUGR) were cross-fostered to control dams with litter sizes of 8 or 16 pups (control and undernourished). Offspring body weights were reduced and onset of male puberty slightly delayed in animals from large postnatal litters. Similar body weight effects were observed in females but there was no difference in the age of vaginal opening. Female reproductive senescence as measured by onset of estrus acyclicity occurred at a younger age in IUGR-8-pup and Control-16-pup groups compared to Control-8-pup or IUGR-16-pup groups. Females were bred to control males and no evidence of adverse reproductive effects was found in any F2 groups. The offspring of the F1 generation did not show an intergenerational effect as documented in humans.

The relationship of maternal and fetal toxicity in developmental toxicology bioassays with notes on the biological significance of the "no observed adverse effect level".

Standard developmental toxicology bioassays are designed to identify agents with the potential to induce adverse effects and include dose levels that induce maternal toxicity. The work reported here was undertaken to evaluate the relationship of maternal and fetal toxicity. It constitutes an analysis of 125 developmental toxicity bioassays in the mouse, rat, and rabbit conducted by the National Toxicology Program. Although varying by species, general findings include: (1) most lowest observable adverse effect levels (LOAELs) were determined by reduced maternal gestational weight gain or fetal weight at term. (2) Maternal weight reductions are associated with reduced food intake for a variety of dissimilar test agents. (3) Lower fetal weights were associated with reduced maternal weight gains late in gestation. (4) The degree of fetal weight reduction is correlated with the extent of the maternal weight loss. In a substantial number of the studies, reduced fetal weights at term may, therefore, be due to maternal undernutrition caused by general toxicity rather than direct developmental insult. Consequently, such test agents may be erroneously classified as primary developmental toxicants. Experimental approaches to test the hypothesis that maternal undernutrition in standard developmental toxicology bioassays may be responsible for significant term fetal weight decrements are discussed.

The cyanobacterial toxin, cylindrospermopsin, induces fetal toxicity in the mouse after exposure late in gestation.

Cylindrospermopsin (cyn) is a cyanobacterial toxin implicated in human and wildlife poisonings. We have completed studies investigating the potential of purified cyn to induce developmental toxicity in mammals. The teratology study involved intraperitoneal injections (8.0-128 microg kg(-1)) on gestational days (GD) 8-12 with subsequent examination of term fetuses for viability, weight and morphological anomalies. Cyn was lethal to a significant portion of the dams receiving > or = 32 microg kg(-1). Surviving pregnant females were killed and fetuses removed for examination. Analysis indicates no adverse effects on litter size, fetal weight, or incidence of anomalies. Subsequently, 50 microg kg(-1) cyn was administered on GD 8-12 or 13-17. Animals were allowed to give birth and litters monitored for growth and viability. A reduction in litter size occurred in treated groups. Avg. pup wt. was only affected in the GD 13-17 group. GD 13-17 dams did not exhibit the toxicity noted in the GD 8-12 group but gave birth significantly earlier than controls. There was a significant number of dead GD 13-17 pups and incidences of blood in the gastrointestinal tract and hematomas in the tips of the tails in survivors. Pups were cross-fostered to control mothers in litters of 10. On postnatal days (PND) 5-6 there were no significant differences in weight gain or viability in GD 8-12 litters, while GD 13-17 litters had significantly reduced weight gain and viability. GD 13-17 exposed male pups still weighed significantly less than the controls after 15 months.

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.

Nicotine effects on the activity of mice exposed prenatally to the nicotinic agonist anatoxin-a.

Anatoxin-a is a nicotinic agonist produced by several genera of cyanobacteria, and has caused numerous deaths of wildlife, livestock and domestic animals world-wide. Several studies in the literature have shown that exposure of mice and rats to nicotine early in development alters its effects when the rodents are subsequently challenged with nicotine. We therefore determined the effect of nicotine on the motor activity of adult mice that had been exposed prenatally to anatoxin-a. Pregnant CD-1 mice received either saline vehicle or one of two doses of (+/-) anatoxin-a (125, 200 microg/kg), i.p., on GD13-17. As adults (8 months), control mice of both genders were used to determine the effect of nicotine (0, 0.1, 0.3, 1.0 or 3.0 mg/kg, s.c.) on motor activity measured for 30-min in a photocell device. Under these conditions, nicotine produced dose-related decreases in both horizontal and vertical activity, with an ED50 estimated to be 0.65 mg/kg. Next, additional control mice and mice exposed prenatally to anatoxin-a received the nicotine ED50 and saline vehicle, in a counterbalanced fashion, with one week separating treatments. Nicotine decreased both horizontal and vertical activity in all mice, regardless of prenatal anatoxin-a treatment. Thus, no enduring effects of prenatal anatoxin-a were obtained in adult mice following nicotine challenge.

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.

Axial skeletal and Hox expression domain alterations induced by retinoic acid, valproic acid, and bromoxynil during murine development.

Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.

Lack of teratogenicity of microcystin-LR in the mouse and toad.

Microcystin-LR (MC-LR) is a cyanobacterial toxin generated by the organism Microcystis aeruginosa. Although the hepatotoxicity of this chemical has been characterized, the potential developmental toxicity in vertebrates has not been well studied. The purpose of this study was to elucidate the effects of this toxin on the in vivo and in vitro development of mammals and the development of an Anuran (toad). Initial acute toxicity experiments with female CD-1 mice were accomplished with MC-LR administered i.p. in saline. Lethality occurred at 128 and 160 microg kg (-1) and histopathology revealed massive hepatic necrosis with diffuse hemorrhage. Developmental toxicity studies were done with MC-LR administered i.p. for 2-day periods: gestation days 7-8, 9-10 or 11-12. Doses used ranged from 2 to 128 microg kg(-1). On gestation day 17, fetuses were weighed and analyzed for gross morphological and skeletal defects. No treatment-related differences were seen in litter size, viability, weight or the incidence of anomalies. Groups of dams dosed with 32-128 microg kg(-1) on gestation days 7-8, 9-10 or 11-12 were allowed to give birth and the growth and development of their pups were followed postnatally. There were no significant effects noted in the offspring of the treated dams. Neurulation-staged CD-1 mouse conceptuses were exposed to 50-1000 nM MC-LR in whole embryo culture for 24 h. No significant increase in abnormalities or developmental delays was observed. Finally, exposure of the developing toad. Bufo arenarum was done from stage 17 (tail bud) for 10 days at concentrations of 1-20 mg l(-1). No effect on morphological development or survival was noted in any exposed groups. These data indicate that microcystin does not appear to affect development adversely in the mouse (in vivo or in vitro) or the toad at the doses and exposure parameters used.

5-AZA-2'-deoxycytidine-induced dysmorphogenesis in the rat.

5-aza-2'-deoxycytidine (d-AZA) causes temporally related defects in the developing mouse. Treatment of 1.0 mg/kg on gestation day (GD) 8 results in axial skeletal defects; on GD9, cleft palate and vertebral defects; on GD10, hindlimb phocomelia; and on GD11, digital defects. An unusual aspect of d-AZA teratogenicity in mice is that the phocomelia appears to be specific to the hindlimb, and the forelimb is not similarly affected regardless of treatment day. The current study was initiated to evaluate the embryonic response of another species, the rat, to this unique teratogen. Pregnant Sprague Dawley (CD) rats were treated with d-AZA or vehicle control. The compound was administered i.p. on GD9, 10, 11, or 12 to parallel developmental staging of the mouse. The highest dose (1.0 mg/kg) elicited effects indicating increased sensitivity to the compound in the rat as compared to the mouse. GD9 treatment was characterized by massive resorptions; GD10, by a predominance of axial skeletal defects and cleft palate; GD11, by a predominance of forelimb phocomelia and missing ribs; and GD12 by hindlimb phocomelia and forelimb digit defects. These data indicate significant differences in the developmental responses to d-AZA of the mouse and the rat. This may reflect interspecies differences in the temporal expression of genes involved in morphogenesis and/or the methylation patterns of such genes. Molecular data generated in the mouse will be compared to that of the rat to further characterize the developmental dynamics responsible for the interspecies differences. Teratogenesis Carcinog. Mutagen. 19:329-338, 1999.

Maternal and developmental toxicity of halogenated 4'-nitrodiphenyl ethers in mice.

In an ongoing effort to delineate structure-activity relationships in the developmental toxicity of diphenyl ethers, we evaluated the maternal and developmental toxicity of 10 diphenyl ethers related to the herbicide nitrofen. All possible trichlorophenyl 4'-nitrophenyl ethers were evaluated, as were the 2,4-difluorophenyl and 2,4-dibromophenyl 4'-nitrophenyl ethers. We also evaluated bifenox and chlomethoxyfen, which are 2,4-dichlorophenyl congeners with meta-substituents on the 4'-nitrophenyl ring. Nitrofen (2,4-dichlorophenyl 4'-nitrophenyl ether) was included for comparison. Identity of the halogen affected the postnatal (but not prenatal) mortality induced by 2,4-dihalogenated 4'-nitrophenyl ethers. The presence of 3'-substituents on the 4'-nitrophenyl ring reduced both pre- and postnatal toxicity of 2,4-dichlorinated congeners. Among chlorinated 4'-nitrophenyl congeners without meta-substituents on the nitrophenyl ring, the position of chlorine substituents strongly affected the congener's potential for inducing prenatal vs. postnatal syndromes. All congeners increased liver to body weight ratios in unmated females, but such increases were not well-correlated with either prenatal or postnatal embryotoxicity.

Differentially expressed genes associated with 5-Aza-2'-deoxycytidine-induced hindlimb defects in the Swiss Webster mouse.

5-Aza-2'-deoxycytidine (d-AZA) inhibits methylation of DNA, a process that serves as an epigenetic regulator of gene expression. We have shown that d-AZA causes temporally related defects in mice. Gestational day (GD) 10 treatment induced severe long-bone defects of the hindlimb but not the forelimb. Exposure of younger embryos (GD 8 or 9) does not induce similar defects in forelimbs. This limb-dependent response suggests that methylation alterations in genes specific for fore- or hindlimbs may contribute to the observed pattern of defects. Subtraction hybridization (SH) studies were conducted to identify differential expression of DNA subsequent to the administration of d-AZA to mice on GD 10. Hindlimb buds collected from both treated and untreated embryos at 4, 12, and 24 hours post-treatment were used. A clone isolated from the untreated sample (down-regulation in treated tissue) was identified as a member of the murine B1 family of repetitive sequences. The two other clones isolated from the treated tissue (up-regulation) were homologous to avian myogenic regulatory protein mRNA and activin receptor type II gene. Both species are active during embryogenesis. These findings suggest that the isolated clones may have roles in abnormal embryonic development when inappropriately expressed.

Infectious dermatitis in a ball python (Python regius) colony.

Seven wild-caught ball pythons (Python regius), including six gravid females and one male, were obtained from Africa and were housed in a government animal facility in Research Triangle Park, North Carolina. Upon arrival, the snakes were found to be infested with ticks (Aponomma latus), which were manually removed. Four weeks following arrival, vesicular skin lesions began to appear on the snakes. Despite treatment of all affected female snakes with amikacin (5 mg/kg i.m., every 3 days) and cefotaxime (25 mg/kg i.m., every 3 days), the condition progressed and five of the female snakes died 7 wk after arrival. The remaining male and one female improved after an increase in environmental temperature, with ecdysis followed by healing. Physiologic stress, ectoparasites, and shipping may have predisposed the snakes to sepsis.

Teratogenic effects of the demethylating agent 5-aza-2'-deoxycytidine in the Swiss Webster mouse.

5-Aza-2'-deoxycytidine (d-AZA) replaces cytidine in DNA thereby altering gene expression by passively removing methyl groups. This study determined the temporal patterns of morphological defects induced by d-AZA in mice. The dosages (0, 0.3, or 1.0 mg/kg) were administered by a single i.p. injection on gestational days (GD) 8, 9, 10, or 11. Mice were killed on GD 17 and fetal skeletons examined. The 1.0 mg/kg dose elicited characteristic defects for each treatment day: GD 8, supernumerary ribs, (significantly above background), fused vertebrae and ribs; GD 9, cleft palate and vertebral variations; GD 10, hind limb defects (especially phocomelia); GD 11, digital defects of fore and hindlimbs. The known demethylating ability of d-AZA coupled with the induction of longbone defects only in the hindlimbs suggests that d-AZA may act by disrupting specific hindlimb gene function through DNA hypomethylation.

Supernumerary lumbar rib: manifestation of basic alteration in embryonic development of ribs.

Supernumerary ribs (SNR) are a common variant in some strains of mice used in standard teratology bioassays. We have previously demonstrated that increased incidence of SNR may be induced by a wide variety of xenobiotics and/or general maternal stress. The significance of this defect in cross-species extrapolations has been problematic and recent studies, including this one, have shown that this anomaly is more complex than previously thought. The SNR in mice have a bimodal distribution composed of 'rudimentary ribs' (RR) with a mode of 0.3-0.4 mm and 'extra ribs' (ER) with a mode of 0.9-1.1 mm. The studies reported here examine the relationship between the presence of SNR and the 13th rib length and the gross morphological development of the anomaly. Supernumerary ribs were induced in CD-1 mice by surgical stress (subcutaneous micropump implanted on gestational day (GD), restraint stress (GD8), food and water deprivation (GD8) or maternal administration of the pesticide dinoseb (50 mg kg-1 on GD7 and GD8). Fetuses from untreated litters were also examined. Dinoseb-treated mice were killed on GD14, 15, 16 or 17. All other groups were killed on GD17. The lengths of the 13th and 14th ribs were measured and other anomalies were recorded. Femur length was used as an indicator of fetal size. The SNR frequency was higher in all treatment groups compared to controls. We found that ER and RR were morphologically distinct. The ER were flat ended and distally joined by a cartilaginous portion, while RR were usually rounded distally and were without cartilaginous extensions. The 13th ribs were significantly longer in fetuses having SNR than in those not having SNR, whether treated or untreated. This relationship was present in all fetal ages examined and with both ER and RR groups. These results suggest that SNR are indicative of basic alterations in the development of the axial skeleton.

Teratogenicity of low doses of all-trans retinoic acid in presomite mouse embryos.

This study was designed to examine the developmental dose response for all-trans retinoic acid (TRA) administered at presomite stages in mouse embryos. Previous studies using hamsters [Shenefelt (1972) Teratology 5:103-118] have shown that developmental stages corresponding to those present early on gestational day (GD) 7 in mice are most sensitive to retinoid-induced teratogenesis. Our preliminary studies showed that at this treatment time, gavage dosages of 7.5 mg/kg maternal body weight administered to C57B1/6N mice, an inbred strain, resulted in severe craniofacial malformations representing the holoprosencephaly, aprosencephaly spectrum. Additionally, in an outbred mouse strain, CD-1, exencephaly was induced by dosages of 2.5 mg/kg TRA and above. Readily detectable abnormalities of the eyes, including anophthalmia and severe microphthalmia and iridial colobomata, were induced by even lower doses cf TRA in the C57B1/6N strain. Incidences of micro/anophthalamia were 6.7%, 8.1%, 12.9%, and 32.4% at 0, 0.313, 0.625, and 1.25 mg/kg, respectively. The dosages required to induce significant incidences of exencephaly (2.5 mg/kg) and severe ocular abnormalities (1.25 mg/kg) on GD 7 in mice are approximately 50-100-fold less than those that are commonly used to examine the teratogenicity of this compound at later developmental stages in this species. The trend toward an increase in the incidence of severe ocular malformations at the lowest dose examined and the fact that subtle ocular malformations were not taken into account for this study suggest that even lower dosages may be effective.(ABSTRACT TRUNCATED AT 250 WORDS)