Serine-enhanced restoration of 2-methoxyethanol-induced dysmorphogenesis in the rat embryo and near-term fetus.

Effects of serine on restorative growth were characterized by comparing embryo/fetal responses after maternal exposure to 2-methoxyethanol (ME) and ME + serine by gavage on gestation day (gd) 13, a day of heightened limb sensitivity. Paws (gd 20) and limb buds (gd 15) were examined after ME alone at 50, 100, and 250 mg/kg, and after ME (either 100 or 250 mg ME/kg) + serine (1734 mg serine/kg) administered within minutes (0 hr) to 24 hr after ME. Paw development was not altered after ME at 100 mg/kg, but was highly sensitive to 250 mg ME/kg with all fetuses and litters exhibiting defects (ectrodactyly, syndactyly, and short digit) in the preaxial region. In contrast, the limb bud displayed dose-related incidences of abnormalities after maternal treatment with the low and high levels of ME. The condensing (precartilaginous, pentadactyl pattern) and noncondensing (undifferentiated mesenchymal cells) regions exhibited changes in their size, number, and location. Serine administration after 250 mg ME/kg was effective in reducing the occurrence of paw dysmorphogenesis with its protection potency inversely related to its delay of administration (i.e., 0% paw defect incidence after 0-hr delay, 25% after 4-hr delay, 41-45% after 8- and 12-hr delays, and 76% after 24-hr delay). The occurrences of limb bud pattern disturbances produced by ME were also markedly decreased by serine cotreatment. Higher incidences of embryonic defects versus those of fetal defects demonstrate that restorative growth followed ME exposure. Serine attenuation of ME teratogenicity appears to emanate from enhanced restorative growth so that tissue damage, which otherwise would be expressed as a defect at parturition, is repaired and replaced to resume development of the limb toward its normal structure.

Developmental phase specificity and dose-response effects of 2-methoxyethanol in rats.

Methoxyethanol (ME) produces embryotoxic effects in rodents, rabbits, and nonhuman primates. Mechanistic evaluations of ME dysmorphogenesis have focused mainly on developmental insults and chemical disposition in the mouse. These assessments in mice were based on developmental phase specificity (DPS) and dose-response relationship (DRR) of ME. DPS and DRR indicated treatments for selectively inducing defects to study ME disposition and expressed dysmorphogenesis. This study was conducted to establish DPS and DRR of ME in the rat. DPS was determined by injecting 500 mg ME/kg (6.6 mmol/kg) into the tail vein on Gestational Day (gd; sperm-positive day = gd 0) 10, 11, 12, 13, 14, or 15 (n = 6 dams/gd; saline controls on gd 12). On gd 20, embryolethality incidence was 100% after gd 10 dosing; at gd 11 through 15, it was 50, 32, 15, 2, and 5% respectively (control, 2%). Incidences of external defects in live fetuses exposed on gd 11-15 were 97, 98, 100, 44, and 0% and those of viscera were 100, 62, 44, 10, and 0%, respectively. The predominant anomalies observed were ectrodacyly and renal agenesis. DRR was determined on gd 13, when live embryos/litter and external malformations (ectro- and syndactyly, micromelia) were maximal. Dams (n = 8/dose group) were injected intravenously with 0, 100, 250, 350, or 500 mg ME/kg. On gd 20, fetal defect rates were 0, 0, 82.5, 83.0, and 100% at these concentrations, respectively. Based on these studies, appropriate ME doses, times of maternal exposure, and critical phases of development in the rat model are available for reproducing selective defects to investigate biochemical and pharmacokinetic determinants underlying their expression.

Developmental toxicity evaluation of diethyl and dimethyl phthalate in rats.

Diethyl phthalate (DEP) and dimethyl phthalate (DMP), phthalic acid ester (PAE) plasticizers, were evaluated for developmental toxicity because of reports in the literature that some PAE were embryotoxic and teratogenic. A previous study (Singh et al., '72) suggested that an increased incidence of skeletal defects in rats might result from gestational exposure to DEP (0.6-1.9 g/kg) or DMP (0.4-1.3 g/kg), ip, on gestational days (gd) 5, 10, and 15. In the current study DEP (0, 0.25, 2.5, and 5%) or DMP (0, 0.25, 1, and 5%) in feed (approximately 0.2-4.0 g/kg/day) were supplied to timed-mated rats from gd 6 to 15. Treatment with 5% DMP resulted in increased relative maternal liver weight. Also, animals exhibited reduced body weight gain during treatment (5% DEP or DMP) and during gestation (5% DEP). Weight gain corrected for gravid uterine weight was also reduced in animals fed 5% DEP. However, high-dose treatment with either DEP or DMP resulted in changes in food and water consumption paralleling the body weight reductions, suggesting that apparent toxic effects on maternal body weight may reflect PAE/feed unpalatability. Treatment with 2.5% DEP resulted in only transient changes in body weight during early treatment. The only maternal effects at 0.25 or 1% DMP were minor changes in food and/or water consumption, and there were no effects at 0.25% DEP. Thus, the NOAELs for maternal toxicity were 1% DMP and 0.25% DEP. In contrast to the observed maternal toxicity, there was no effect of DEP or DMP treatment on any parameter of embryo/fetal development, except an increased incidence of supernumerary ribs (a variation) in the 5% DEP group. These results do not support the conclusion of other investigators that DEP and DMP are potent developmental toxicants. Rather, they suggest that the short-chain PAE are less developmentally toxic than PAE with more complex substitution groups, e.g., di(2-ethylhexyl) phthalate, mono(2-ethylhexyl) phthalate, and butyl benzyl phthalate.

Codeine: developmental toxicity in hamsters and mice.

Timed-pregnant LVG Syrian hamsters and Swiss CD-1 mice were dosed orally twice daily (b.i.d.) with codeine in water on Gestational Days (gd) 5-13 (0, 10, 50, or 150 mg/kg, b.i.d.--hamsters) or 6-15 (0, 37.5, 75, 150, or 300 mg/kg, b.i.d.--mice). Dams were necropsied on gd 14 (hamsters) or 17 (mice), and fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations. No maternal deaths were observed in hamsters, while 19% of the pregnant mice in the high-dose group died. Maternal weight gain (gestational and treatment periods) and gravid uterine weights were significantly depressed in hamsters (150 mg/kg, b.i.d.) and in mice (300 mg/kg, b.i.d.). However, the corrected weight gain for both species, although decreased, was not significantly different from that of the controls. In both species, maternal liver weights (relative) were significantly increased in the high-dose groups. There were increases in the percentage resorptions per pregnant dam and in the proportion of litters with 100% resorptions in the high-dose groups of both species. Considering only live litters, the number of live fetuses per litter and the sex ratio were unaffected in both species. Mean fetal body weights were also significantly decreased in the 50 and 150 mg/kg, b.i.d. (hamsters), and the 150 and 300 mg/kg, b.i.d. (mice), groups. The no-observed-adverse-effect levels (NOAELs) for developmental toxicity were 10 (hamsters) and 75 (mice) mg/kg, b.i.d., whereas the NOAELs for maternal toxicity were 50 (hamsters) and 150 (mice) mg/kg, b.i.d. The predominant structural malformation in hamsters was meningoencephalocele (high-dose group only), affecting 3% of fetuses and 19% of litters (neither statistically significant). Codeine did not induce any increase in structural malformations in mice. Thus, codeine produced developmental toxicity (as indicated by decreased fetal body weight) at doses below those producing maternal toxicity in both hamsters and mice. In the hamster, the more sensitive species to codeine developmental toxicity, effects were observed at a total daily dose of 100 mg/kg, which is only 11 times the maximum human therapeutic oral dose.

Developmental toxicity evaluation of acrylamide in rats and mice.

Acrylamide (ACRL), a widely used industrial chemical with neurotoxic effects, was evaluated for developmental toxicity. ACRL in distilled water was administered once daily by gavage on gestational days (gd) 6-17 to mice (0, 3, 15, or 45 mg/kg) and on gd 6-20 to rats (0, 2.5, 7.5, or 15 mg/kg). Following termination (gd 17, mice; gd 20, rats) fetuses were examined for external, visceral, and skeletal malformations. Maternal toxicity during treatment was observed at the highest dose as reduced body weight gain in both species and hindlimb splaying in treated mice only. Weight gain corrected for gravid uterine weight was also reduced in rats at 7.5 and 15 mg/kg/day. Embryo/fetal toxicity was not observed in rats, but fetal weight was reduced in mice administered 45 mg/kg/day. No increase in the incidence of malformations was observed in either species; however, the incidence of variations (predominately extra rib) increased with dose. In summary, administration of ACRL during organogenesis produced maternal and developmental toxicity at 45 mg/kg/day in mice and maternal, but not developmental, toxicity at doses greater than or equal to 7.5 mg/kg/day in rats.

The relationship of embryotoxicity to disposition of 2-methoxyethanol in mice.

Paw development of CD-1 mice is uniquely sensitive to 2-methoxyethanol (ME) given by gavage (po) on gestation day (gd) 11 (copulation plug day = gd 0). The relation between induction of paw dysmorphogenesis and disposition of po ME (3.3 or 4.6 mmol/kg) in the maternal and conceptus compartments was investigated. The expression of digit malformations depends on metabolism of ME to methoxyacetic acid (MAA). ME and MAA were equipotent in causing teratogenicity. Alcohol dehydrogenase (ADH) catalyzes the initial rate-limiting oxidation that leads to embryotoxicity. The ADH inhibitor 4-methylpyrazole (0.12 or 1.2 mmol/kg) or ethanol (43.3 mmol/kg, single dose concomitant with ME or additional ethanol 5 and 10 hr later) reduced the incidence of malformations 60-100%, depending on the dosing regimen. Elimination of 14C from 1,2-14C-ME occurred predominantly via urine where 80% of a teratogenic dose was excreted and 6% appeared in CO2. Oxidation of ME to MAA was nearly complete after 1 hr when approximately 90% of 14C in maternal plasma and conceptus coeluted with authentic 14C-MAA upon HPLC. 14C-MAA levels in embryos were 1.2 X those in plasma 1 and 6 hr after dosing, although by 6 hr concentrations had declined to approximately 50% of 1-hr values. Concomitant ethanol did not affect 14C kinetics as measured in maternal blood after oral 14C-ME, but retarded ME conversion to MAA by about 2 hr. Furthermore, embryo 14C-MAA levels then reached only 50% of the peak in embryos from dams dosed with ME alone, an effect that coincided with less 14C incorporation into macromolecules synthesized by the embryo within 6 hr. These data imply that the attenuation of digit malformations by concomitant ethanol may be explained by changes in MAA disposition. However, delayed ethanol (5 and 10 hr after 3.3 mmol ME/kg) reduced teratogenicity by 25%, although MAA was present in the embryo up to 5 hr. Dams given 14C-MAA by iv injection had higher 14C blood levels than after MAA po but their offspring had fewer digit malformations. Peak and steady-state plasma levels of MAA as well as embryo concentrations of the chemical do not appear to determine the embryotoxic outcome whereas further metabolism of MAA does.

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo.

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.

Attenuation of 2-methoxyethanol and methoxyacetic acid-induced digit malformations in mice by simple physiological compounds: implications for the role of further metabolism of methoxyacetic acid in developmental toxicity.

The ethylene glycol ether 2-methoxyethanol (ME) and its oxidation product methoxyacetic acid (MAA) are selective embryotoxins and equipotent as inducers of digit malformations when given by gavage to pregnant Crl:CD-1 ICR BR mice on gestation day 11. Earlier observations showed that the teratogenic effects were attenuated by delayed administrations of ethanol given at a time when all ME is already converted to MAA. That outcome suggested that acetate from ethanol catabolism might compete with methoxy-acetate in biosynthetic reactions relevant to MAA-induced malformations. Furthermore, 14C derived from [1,2-14C]-ME or [1-14C]-MAA is incorporated into all macromolecular fractions of the embryo, and 14C is exhaled by the dam in 14CO2. Those data indicate that 14C derived from 14C-ME catabolism enters into many metabolic reactions. The present study examined acetate and other simple physiological compounds with close relationships to carbon and one-carbon moiety metabolic pathways for their ability to attenuate digit malformations upon concomitant dosing with ME. All of the agents examined reduced the teratogenic effect significantly with a potency rank order of formate much greater than acetate = glycine much greater than D-glucose. The common link for their efficacy may be the one-carbon moiety oxidation pathway that involves tetrahydrofolic acid as a catalyst of one-carbon transfer into purines and thymidylate. Carbon from all of the attenuators administered is incorporated into those bases and then into DNA. It appears as if methoxyacetate enters into biochemical reactions analogous to those of acetate. This speculation is supported by the metabolic fate of 14C from 14C-ME in dam and embryo. Based on the indirect evidence obtained with all of the simple compounds that attenuate the ME-induced digit malformations, we postulate that abnormal macromolecules are generated by anabolic reactions and that those products disrupt normal paw development.

Localization of radioactivity from 2-methoxy[1,2-14C]ethanol in maternal and conceptus compartments of CD-1 mice.

2-Methoxyethanol (ME) induces paw malformations in CD-1 mice when given by gavage on gestation day (gd) 11 (vaginal plug + day = gd 0). The distribution of radioactivity originating from 2-methoxy[1,2-14C]ethanol ([14C]ME) was examined by liquid scintillation spectrophotometry and whole body autoradiography in pregnant (gd 11) CD-1 mice from 5 min to 48 hr after oral administration. Each dam received either a trace dose of [14C]ME (0.92 mumol; 13 muCi) combined with an unlabeled teratogenic dose (187 mumol). By 5 min after the trace dose was administered, 14C had distributed throughout the maternal and conceptus compartments. Radioactivity in the maternal compartment was most concentrated in the liver, blood and gastrointestinal tract. Conceptus 14C was associated with the placenta, yolk sac, and embryonal structures such as limb buds, somites, and neuroepithelium. The concentration of blood 14C plateaued within 30 min after administration of the trace or combined trace/teratogenic dose. It remained stable for 1.5 hr and then gradually declined, reaching 2 to 10% of the maximal concentration by 48 hr. 14C content in the maternal liver, conceptuses, and embryos per se was always greater than that of the blood and was inversely related to ME dose at 6 hr but not 48 hr. At 6 hr after administration of the trace dose, 69% of total liver and 33% of embryonal 14C were acid insoluble. Tissue-specific interaction with [14C]ME was demonstrated by the distribution of acid insoluble radioactivity among various cellular components of the maternal liver and embryo. The findings indicate that the embryo is readily susceptible to blood borne ME and/or its metabolites. In addition, the chemical characteristics of the labeled molecule(s) apparently favored label incorporation into macromolecules by the liver and embryo.

Developmental phase-specific and dose-related teratogenic effects of ethylene glycol monomethyl ether in CD-1 mice.

Several animal species have shown a teratogenic response to inhaled or ingested ethylene glycol monomethyl ether (EGME). The present study examined the developmental phase specificity and dose-response characteristics of EGME-induced embryotoxicity. Pregnant CD-1 mice (vaginal plug positive day = gestation Day [gd]0) received multiple or single doses of EGME by gavage between gd 7 and 14. Fetuses were examined on gd 18 for external and skeletal malformations. EGME was not maternally toxic after multiple doses of 250 mg/kg or a single administration of up to 500 mg/kg. EGME induced embryotoxicity as manifested by reduced gd 18 fetal weights and increased resorptions. The observed malformations were specifically related to the developmental stage at the time of exposure. Exencephaly resulted after EGME exposure between gd 7 to 10 whereas paw anomalies (syndactyly, oligodactyly, and stunted digit No. 1) predominated during later stages of development. Paw anomalies were maximal after administration on gd 11, and forepaws exhibited greater susceptibility than hindpaws. The no observed effect dose for the induction of digit malformations after a single administration of EGME on gd 11 was 100 mg/kg. At 175 mg EGME/kg digit anomalies were induced without any concurrent reduction in fetal body weight while at 250 mg/kg and above, digit anomalies occurred concurrently with reduced fetal body weight.

Homogeneous populations of Artemia nauplii and their potential use for in vitro testing in developmental toxicology.

Efforts have begun to establish test subjects other than the intact pregnant mammal to serve as models for rapid teratology screens. Artemia nauplii transcending instar I to later instars were examined to determine their potential for indicating chemicals as potential developmental hazards and thus prioritizing them for more extensive in vivo testing. Several criteria selected for assessing the system's potential for screening were the ability to: collect homogeneous populations of instar I nauplii; characterize intermediate development by technically simple measurements; and demonstrate development-related differentials in naupliar vulnerability. Homogeneous populations of nauplii were harvested from a flow-through hatching and cold storage system. Nauplii accumulated in the system are stored at 4 degrees C in a quiescent state with little physical (body length, body water volume) and biochemical (DNA and protein levels) change and thus are maintained at instar I. Intermediate development of nauplii transcending instar I to IV was characterized after the onset of 25 degrees C incubation by measuring changes in drinking activity, body length, body water volume, and DNA and protein levels. During the first day of incubation, development was greatest between 6 and 24 hours of incubation. Development-related differentials in naupliar vulnerability were shown by comparing median lethal concentrations (LC50) estimated for cadmium sulfate (CdSO4), mercuric chloride (HgCl2), and sodium azide (NaN3) at various times during incubation. With cadmium and mercury, LC50s decreased as nauplii aged and developed; whereas, with azide, LC50s did not vary. Developing nauplii were differentially vulnerable to cadmium and inorganic mercury. Artemia nauplii transcending instar I to IV appear useful for indicating chemicals that preferentially interact with developmental events.

Improved methods for harvesting and counting synchronous populations of Artemia nauplii for use in developmental toxicology.

Artemia nauplii have, within recent years, gained popularity as a test organism for short-term toxicity testing. Because nauplii exhibit rapid development and growth within 48 hr after hatch, their potential as a model organism for teratology screening has been considered. To do this, synchronous populations of nauplii at different developmental intervals must be available. A dual-chamber hatching vessel which enables multiple harvesting of synchronous populations of nauplii from one sample of prepared cysts has been developed. An accumulation period of 2 hr defines the synchronous population and produces around 1000 nauplii among five hatching vessels. The body lengths of nauplii harvested 2 to 9 hr after the initial harvest were equivalent to and lower than that of animals incubated at the hatching temperature (31 +/- 1 degree C); e.g., the body length of freshly harvested nauplii at 9 hr was 473 +/- 6.2 micron (+/- SE) and for nauplii incubated for 8 to 10 hr, the body lengths were 643 +/- 10.7 and 702 +/- 10.9 micron, respectively. A counting method was developed that enabled precise counting up to 400 nauplii in suspension and distinguishing dead from live nauplii. Measurements of body length, body water volume, and whole animal DNA and protein of nauplii harvested sequentially from the same sample of prepared cysts indicated that in addition to temporal differences in hatching, the nauplii may differ both physically and chemically.

Plasmid-determined ability of a Salmonella tennessee strain to ferment lactose and sucrose.

The ability of a Salmonella tennessee strain to ferment both lactose and sucrose was attributed to a conjugally transmissible plasmid, deoxyribonucleic acid molecular weight 164 x 10(6), bearing the genetic determinants of both fermentation characters.