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 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.

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.

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.

Role of TGF-beta in RA-induced cleft palate in CD-1 mice.

Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. RA exposure on gestational day (GD) 12 in CD-1 mice results in delayed palatal shelf elevation and subsequent clefts in the secondary palate. Given the dynamic and complex nature of palate development, it is not surprising that this system is susceptible to changes in retinoid levels. There is evidence that experimental manipulation of retinoid status during development alters normal transforming growth factor-beta (TGF-beta) status. To study the role of perturbation in TGF-beta levels in RA-induced cleft palate, gravid CD-1 mice were treated with 70 mg/kg RA on GD 12. We examined changes in TGF-beta proteins and the steady-state level of TGF-beta mRNA within the first 24 hr after exposure. The interactions between RA and TGF-beta s were very complex. RA differentially regulated the mRNA and protein levels of TGF-beta 1. Changes in mRNA steady-state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation was evident, because RA treatment resulted in an increase in TGF-beta 1 mRNA steady levels followed by a decrease in the intracellular and extracellular forms of TGF-beta 1 protein. Moreover, the patterns of localization and levels of TGF-beta 2 and TGF-beta 3 proteins were not dramatically affected, although there was an increase in TGF-beta 3 mRNA steady-state levels. The increases in mRNA steady-state levels for TGF-beta 2 and TGF-beta 3, as for TGF-beta 1, were rapid and transient in nature, again arguing for direct mediation by RA. These data provide evidence for interactions between RA and TGF-beta s, and indicate that RA is capable of differentially regulating TGF-beta isoforms through processes involving different stages of TGF-beta synthesis and secretion. Further, changes in TGF-beta isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.

Mesenchymal changes associated with retinoic acid induced cleft palate in CD-1 mice.

Retinoic acid (RA) is teratogenic in many species and is an effective inducer of cleft palate in mice. The pathogenesis of cleft formation varies with the timing of exposure. It has been demonstrated, before formation of the palatal shelves, that RA exposure results in insufficient mesenchymal tissue, and palatal shelves fail to make contact. However, at the palatal shelf outgrowth stage, RA exposure affects shelf elevation and growth in rats, and possibly medial edge epithelium (MEE) differentiation in mice. The objective of this study was to examine the morphologic and functional changes associated with cleft formation in mice following exposure during shelf outgrowth. Particular emphasis was placed on evaluating the timing of palatal shelf elevation in RA exposed embryos and on identifying differentiation events occurring concurrently in the epithelium. On gestational day (GD) 12 (8:00 AM), gravid CD-1 mice were gavaged with 70 mg/kg RA or vehicle. This protocol produced a 100% incidence of cleft palate at term, allowing us to correlate the morphological and/or biochemical changes observed at pre-fusion time points. Embryos were collected at 12 hr intervals through GD 15, beginning 4 hr after exposure. Serial sections of embryos were either stained with H&E, with a battery of lectins [Sambucus nigra (SNA), Arachis hypogaea (PNA), Ricinus communis (RCA-1), Glycine max (SBA), Succinylated Wheat Germ (S-WGA)], or with a probe to hyaluronan. Throughout the period of normal palate development, the shelf mesenchyme showed increasing regional organization and progressive hydration and these changes were correlated with increase Hyaluronan (HA) deposition. RA treatment resulted in lose of regional organization and delayed mesenchyme hydration. In association with these changes there were reductions in HA deposition and extracellular matrix glycoconjugates recognized by PNA in the palate mesenchyme. Further there was a considerable delay in palatal shelf elevation and palate shelf did not make contact at the midline. Our data indicates, in embryos exposed on GD 12 to levels of RA sufficient to induce a 100% incidence of clefting, that cleft formation is a result of palatal shelves failing to make contact. Alterations in mesenchyme development and the subsequent delay in palate shelve elevation are central to RA-induced cleft formation following exposure at the palate shelf out growth stage.

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.

Maternal and developmental toxicity of polychlorinated diphenyl ethers (PCDEs) in Swiss-Webster mice and Sprague-Dawley rats.

Polychlorinated diphenyl ethers (PCDEs) are industrial byproducts found in many ecosystems at low levels. PCDEs are not markedly toxic to adult rodents, but their developmental toxicity has not previously been examined. We evaluated the maternal and perinatal toxicity of nine PCDE congeners to outbred mice when compounds were administered from gestation day (GD) 6 through GD 15. 2,2',4,4',5,6'-hexaCDE and 2,3',4',6-tetraCDE decreased the number of pups born per female mated and the number of pups surviving per litter born. 2,2',4,4',5,5'-hexaCDE and 2,2',4,5,6'-pentaCDE decreased the number of litters born per female mated, without decreasing postnatal survival. The other PCDEs did not decrease survival either pre- or postnatally. None of the PCDEs caused absence of Harderian glands in surviving offspring at the doses administered. Neither induction of cytochromes P450 nor tissue residues of individual congeners correlated well with developmental toxicity. Three PCDEs were also evaluated in outbred (Sprague-Dawley) rats: 2,2',4,5,6'-pentaCDE and 2,3',4',6-tetraCDE, because of their toxicity to mice; 2,2',4,4',5,5'-hexaCDE, because it should exhibit PCB-like toxicity. Each congener was administered at three dose levels from GD 6 through GD 15. 2,2',4,5,6'-pentaCDE decreased the number of litters born at 100 mg/kg/day, and the survival of pups in litters carried to term, at both 50 and 100 mg/kg per day. Postnatal weight gain was also reduced. In contrast to its action in mice, 2,3',4',6-tetraCDE decreased neither the numbers of litters born nor postnatal survival of rat offspring, but did suppress postnatal weight gain at least through PD 5. As in mice, induction of cytochromes P450 was not well correlated with the developmental toxicity of individual congeners.

Effects of maternal exposure to chlorinated diphenyl ethers on thyroid hormone concentrations in maternal and juvenile rats.

Polychlorinated diphenyl ethers (PCDEs) are industrial byproducts and widespread environmental contaminants. Their structural similarity to PCBs suggests that they may exhibit subtle effects on both adult and juvenile mammals. We examined the effects of 3 PCDEs (2,2',4,5,6'-pentachlorodiphenyl ether, 2',3,4,6'-tetrachlorodiphenyl ether, and 2,2',4,4',5,5'-hexachlorodiphenyl ether) on maternal rat thyroid levels shortly after exposure, and on the thyroid levels of 16 day old juvenile rats that had been prenatally exposed. Both 2,2',4,5, 6'-pentachlorodiphenyl ether and 2',3,4,6'-tetrachlorodiphenyl ether depressed thyroxine (T4) levels in the maternal females as well as in both sexes of juvenile rats. 2,2',4,4',5,5'-hexachlorodiphenyl ether did not alter T4 levels in the pregnant females, but depressed juvenile T4 levels. None of the congeners studied significantly altered T3 levels. Effects on thyroid hormones did not correlate with the congeners' induction of cytochrome P450.

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.

Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'-deoxycytidine.

DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5-aza-2'-deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose-related accumulation of cells in the S and G2/M phases was observed at 4 and 8 hr after maternal dosing. S-phase accumulation was the most sensitive indicator of effect; a dose-related increase in the percentage of hindlimb bud cells in S-phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication-associated cytotoxicity, at doses of > or = 0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of > or = 0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of concentration of mixed-function oxidase on concentration of estrogen, rate of egg lay, eggshell thickness, and plasma calcium in laying hens.

The objective of this study was to investigate the effect of induction or depression of mixed-function oxidases (MFO) by xenobiotics on the peripheral concentration of estrogens, plasma Ca, rate of egg lay, and eggshell thickness in laying hens. In Exp. 1, 100 mg of phenobarbital (PB) administered orally each day for 3 or 7 d reduced concentrations of estrone and estradiol (E2) in serum. In Exp. 2, 25, 50, or 100 mg of PB was administered orally to laying hens for 3 or 7 d. Treatment with 100 mg of PB for 3 d or with 25, 50, or 100 mg for 7 d significantly increased liver:body weight ratios. Microsomal protein increased after 7 d of 50 or 100 mg of PB administration. Feeding PB decreased concentration of E2 and total plasma Ca in a dose- and period-dependent manner. Concentration of E2 was reduced to 10% of control, whereas hepatic cytochrome P-450 increased significantly with dose. The correlation between concentration of E2 and P-450 was negative and significant. Total Ca in the plasma was highly, positively correlated with concentration of E2. Eggshells were thinner from hens treated with 100 mg of PB for both 3 or 7 d than those from control hens. Rate of egg lay was reduced by 100 mg of PB for 7 d. In Exp. 3, .5 mL of CCl4 given orally for 1 d decreased P-450 and increased E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Health screening of international adoptees. Evaluation of a hospital based clinic.

OBJECTIVE: To report the findings of health screening of 100 international adoptees. DESIGN: Retrospective case review. SETTING AND PATIENTS: The first 100 children to attend the Outpatient Inter-Country Adoption Clinic at the Royal Children's Hospital, Melbourne. INTERVENTIONS: A medical history was taken and a thorough physical examination and series of screening investigations were performed on all children. RESULTS: The children ranged in age from two months to 16 years (median, five months; mean, 2.8 years). The countries most represented were Korea, with 36 children, and India, with 21. Thirty children fell below the third Australian centile for both height and weight. Abnormalities on physical examination included hepatosplenomegaly (13 children), scabies (six) and severe dental caries (six). Parasitic infestation of the stools was found in 23 children, a positive Mantoux test result in nine (three had active tuberculosis), anaemia in 12, elevated hepatic transaminase levels in nine and positive hepatitis B surface antigen and e antigen in two. One child had acquired syphilis. Screening for human immunodeficiency virus antibody gave negative results in all children. CONCLUSION: International adoptees constitute a special paediatric subgroup requiring assessment and screening investigations as soon as possible after arrival in their adoptive country.

PIP: This retrospective case review sought to report the findings of a health screening of 100 international adoptees. Included were the 1st 100 children who attended the Outpatient Inter-Country Adoption Clinic at the Royal Children's Hospital in Melbourne. A medical history was taken and a thorough physical examination and series of screening investigations were performed on all children. The children range in age from 2 months-16 years (median=5 months; mean=2.8 years). The countries most represented were Korea with 36 children and India with 21. 30 children fell below the 3rd Australian centile for both height and weight. Abnormalities on physical examination included hepatosplenomegaly (13 children), scabies (6), and severe dental caries (6). Parasitic infestation of the stools was found in 23 children, a positive Mantoux test result in 9 (3 with active tuberculosis), anemia in 12, elevated hepatic transaminase levels in 9, and positive hepatitis B surface antigen and e antigen in 2. I child had acquired syphilis. Screening for human immunodeficiency virus antibody gave negative results in all children. These international adoptees constitute a special pediatric subgroup which require assessment and screening as soon as possible after arrival in their adoptive country.

Survey of neonatal meningitis in Australia: 1987-1989.

OBJECTIVE: To identify incidence, bacterial aetiology, outcome after treatment and risk factors for poor outcome of neonatal meningitis. DESIGN: Retrospective survey of neonatal meningitis occurring in Australia between January 1987 and December 1989. Data were obtained from Medical Records and Microbiology Departments of hospitals with neonatal nurseries. SETTING: Neonatal nurseries throughout Australia. PATIENTS: 116 infants under 6 weeks of age with bacterial or fungal meningitis. RESULTS: The minimum incidence was 0.17 per 1000 live births. Traditional neonatal pathogens were responsible for 60% of cases (group B streptococci, 35%; Escherichia coli, 22%), childhood meningeal pathogens for 10% and opportunistic pathogens for 30%. Risk factors for meningitis, including prematurity, were more common among those with meningitis due to E. coli or opportunistic pathogens than among those with infections due to group B streptococci, Listeria monocytogenes or the childhood pathogens (46/60 v. 11/55; P less than 0.0001). Meningitis was more likely to be due to Gram-negative bacteria in premature infants (less than 36 weeks gestation) than in full-term infants (19/30 v. 20/86; P = 0.0002). The mortality overall was 26% but was higher in extremely premature infants (less than 29 weeks) (6/9 v. 24/107; P = 0.009) and among 13 patients who were judged to have had inappropriate initial therapy (7/13 v. 21/97; P = 0.04). Long-term sequelae occurred in at least 23% of survivors, but were more common in those with Gram-negative meningitis (6/10 v. 13/76; P = 0.012). CONCLUSIONS: Initial therapy with penicillin or amoxycillin plus cefotaxime is appropriate for most infants with bacterial meningitis. Since some less common Gram-negative bacteria isolated in this survey were resistant to cefotaxime, an aminoglycoside should be added, initially, in Gram-negative meningitis.

Developmental toxicity of bromoxynil in mice and rats.

The developmental toxicity of the wide-spectrum herbicide bromoxynil (bromoxynil phenol; 3,5-dibromo-4-hydroxyphenyl cyanide) was evaluated in Sprague-Dawley rats and Swiss-Webster mice, and the developmental toxicity of its octanoate ester (2,6-dibromo-4-cyanophenyl octanoate) was evaluated in Sprague-Dawley rats. Animals were treated from Day 6 to Day 15 of gestation [presence of sperm or semen plug = 0 of gestation]. The doses administered were as follows: bromoxynil phenol in the mouse, 342, 114, and 38 mumol/kg/day; bromoxynil phenol and bromoxynil octanoate in the rat, 54, 18, and 6 mumol/kg/day. Some animals were killed on selected days during treatment for measurement of organ weights sensitive to stress. In mice treated with bromoxynil phenol, maternal mortality was noted at 114 and 342 mumol/kg/day, but surviving females gained weight normally. Liver to body weight ratios increased with increasing dose, but no consistent effect was seen on adrenal, thymus, or spleen weights. Fetuses of mice treated with the highest dose of bromoxynil phenol were of lower weight and had a higher incidence of supernumerary ribs than controls. In rats, bromoxynil phenol and its octanoate ester at the highest doses used caused no mortality but resulted in only transient decreases in maternal weight gain and significantly increased the liver to body weight ratio, but did not significantly alter adrenal, thymus, or spleen weight in the dams. No significant maternal effects were seen at lower doses. The highest doses of both compounds increased the incidence of supernumerary ribs in fetuses of treated rats, but did not induce other anomalies.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of supernumerary ribs in rodent developmental toxicity studies: postnatal persistence in rats and mice.

Pregnant Sprague-Dawley rats and Swiss-Webster mice were gavaged with bromoxynil at 15 and 96.4 mg/kg/day, respectively, on Days 6-15 of gestation. The frequency of supernumerary ribs (SNR), which are here defined as any degree of ossification lateral to the first lumbar vertebrae, was determined in fetuses at term and offspring on Postnatal Days 6, 20, and 40. Bromoxynil induced significant increases in the incidence of SNR in fetuses of both species. In rats, SNR occurred in 62% of treated fetuses as compared to 14% in controls; in mice these values were 45% and 11%, respectively. The postnatal incidence and persistence of SNR was species dependent. In the rat, postnatal SNR incidence in treated animals did not differ significantly from controls. In contrast, in mice the bromoxynil-induced elevated incidence of SNR persisted through Day 40 (42.3% in treated vs 0% in controls). Analysis of SNR was also done on the basis of their length (greater or less than 1/2 the length of the 13th rib). In the mouse, the incidence of smaller SNR was much lower on Day 40 as compared to Day 20; in contrast the incidence of larger SNR persisted through Day 40. In the rat, the incidence of larger SNR was too small to draw conclusions as to the postnatal fate of these structures. As in the mouse, however, the incidence of smaller SNR was significantly lower by Day 40. The significance of SNR in developmental toxicity remains problematic. The impact of this anomaly on animals is difficult to assess.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity of desbromoleptophos in chicks: enzyme inhibition, malformations and functional deficits.

The relationship among inhibition of acetylcholinesterase (AChE), inhibition of neuropathy target enzyme (NTE), and developmental toxicity of the organophosphorus ester desbromoleptophos (DBL) was evaluated in chicks exposed on day 3 or day 15 of incubation or 10 days posthatching. DBL induced prolonged inhibition of AChE and NTE when administered either early or late in incubation, structural malformations if administered before organogenesis, posthatching paresis if administered after organogenesis, and delayed deficits of gait if administered after hatching. The posthatching paresis and abnormal gait are not determined solely by either AChE inhibition of NTE inhibition, since they occur in the absence of the latter and are not invariably seen in the presence of the former (Toxicology 49: 253-261; 1988).

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity.

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.

Relative teratogenicity of nitrofen analogs in mice: unchlorinated, monochlorinated, and dichlorinated-phenyl ethers.

To determine the role of number and position of chlorine substituents on the developmental toxicity of diphenyl ether analogs of nitrofen, we have evaluated one unchlorinated, three monochlorinated, and five dichlorinated-phenyl 4'-nitrophenyl ethers with respect to effects on liver weight and on maintenance of pregnancy in females, and with respect to postnatal survival and the occurrence of small or absent Harderian glands in offspring. None of the diphenyl ethers evaluated in these experiments was as active as nitrofen with respect to any parameter evaluated. Both the position and the number of chlorine substituents affected toxicity, but no simple relationship between number or position of chlorine substituents and either maternal or fetal endpoints was established.