Repeated dose toxicity and developmental toxicity of diisopropanolamine to rats.

The repeated dose oral and dermal toxicity of diisopropanolamine (DIPA) was evaluated in rats and compared to the reported toxicity of the related secondary alcohol amine, diethanolamine (DEA). Fischer 344/DuCrl rats were given up to 750 mg/kg/day by dermal application, 5 days/week, for 4 weeks; or up to 1,000 mg DIPA/kg/day by drinking water for 13 weeks to evaluate potential toxic effects. Time-mated female CRL:CD(SD) rats were given up to 1,000 mg/kg/day by gavage on gestation days (GD) 6-20 for evaluation of maternal and fetal effects. In the dermal toxicity study, no adverse treatment-related in-life effects other than mild irritation at the site of dermal application at >or= 500 mg/kg/day were observed. There were no systemic effects in rats given up to 750 mg/kg/day. In the subchronic oral toxicity study, the most significant effects were an increase in absolute and relative kidney weights, unaccompanied by histopathologic changes, at >or= 500 mg/kg/day DIPA. The latter effect was ameliorated following a 4-week recovery period. In the developmental toxicity study, there were no maternal or developmental effects at any dose level evaluated. The toxicity of DIPA contrasts with that of DEA which has been shown to affect a number of organ systems when repeatedly administered orally or dermally at similar or lower dosages.

Developmental toxicity evaluation of triclopyr butoxyethyl ester and triclopyr triethylamine salt in the CD rat.

Triclopyr (3,5,6-trichloro-2-pyridyloxyacetic acid) is an herbicide used extensively in the control of woody plants and broadleaf weeds, and is often formulated as a triethylamine salt (T-TEA) or butoxyethyl ester (T-BEE). This study evaluated the developmental toxicity of T-TEA or T-BEE in time-mated CD rats gavaged on gestation days 6-15 with 0, 30, 100 or 300 mg/kg body weight(bw)/day. The doses of each compound were equimolar and equivalent to 22, 76, 216 mg/kg bw/day of triclopyr, based on prior studies indicating rapid cleavage of the salt or ester and equivalent pharmacokinetics for the active ingredient. T-TEA caused maternal toxicity, evidenced by the death of one high-dose dam, reduced body weight gain, increased relative liver and kidney weights (300 mg/kg bw/day), reduced feed consumption, and increased water consumption (100 and 300 mg/kg bw/day). Developmental effects were limited to slightly decreased fetal body weight and reduced skeletal ossification at the high dose level. T-BEE caused similar, albeit slightly more severe, maternal toxicity, with three maternal deaths at 300 mg/kg bw/day, and slight maternal effects at 30 mg/kg bw/day. Due to an equivocal increase in malformations, which were mainly clustered in litters from three high dose dams with marked maternal toxicity, the T-BEE study was repeated using 30 dams/group, investigator-blind fetal evaluations, and an additional dose group (5 mg/kg bw/day). In the repeat study, the only reproducible fetal effect was an increased incidence of 14th thoracolumbar rib at 300 mg/kg bw/day. Overall analysis of the two T-BEE studies suggested that the fetal malformations unique to the initial study likely reflected a combination of spontaneous events, exacerbated by marked maternal toxicity. The combined weight of evidence from these developmental toxicity studies, coupled with their known pharmacokinetic equivalence, indicates that T-BEE and T-TEA are not selectively toxic to the fetus. The respective maternal toxicity no-observed effect levels (NOEL) for T-BEE and T-TEA were 5 and 30 mg/kg bw/day, while the NOEL for developmental toxicity was 100 mg/kg bw/day for both compounds.

Developmental toxicity studies in Crl:CD (SD) rats following inhalation exposure to trichloroethylene and perchloroethylene.

The potential for trichloroethylene (TCE) and perchloroethylene (PERC) to induce developmental toxicity was investigated in Crl:CD (SD) rats whole-body exposed to target concentrations of 0, 50, 150 or 600 ppm TCE or 0, 75, 250 or 600 ppm PERC for six hours/day, seven days/week on gestation day (GD) 6-20 and 6-19, respectively. Actual chamber concentrations were essentially identical to target with the exception of the low PERC exposure level, which was 65 ppm. The highest exposure levels exceeded the limit concentration (2 mg/L) specified in the applicable test guidelines. Maternal necropsies were performed the day following the last exposure. Dams exposed to 600 ppm TCE exhibited maternal toxicity, as evidenced by decreased body weight gain (22% less than control) during GD 6-9. There were no maternal effects at 50 or 150 ppm TCE and no indications of developmental toxicity (including heart defects or other terata) at any exposure level tested. Therefore, the TCE NOEC for maternal toxicity was 150 ppm, whereas the embryo/fetal NOEC was 600 ppm. Maternal responses to PERC were limited to slight, but statistically significant reductions in body weight gain and feed consumption during the first 3 days of exposure to 600 ppm, resulting in a maternal NOEC of 250 ppm. Developmental effects at 600 ppm consisted of reduced gravid uterus, placental and fetal body weights, and decreased ossification of thoracic vertebral centra. Developmental effects at 250 ppm were of minimal toxicological significance, being limited to minor decreases in fetal and placental weight. There were no developmental effects at 65 ppm.

Sex ratio of the offspring of Sprague-Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study.

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.

A unifying concept for assessing toxicological interactions: changes in slope.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.

Development of a physiologically based pharmacokinetic model for ethylene glycol and its metabolite, glycolic Acid, in rats and humans.

An extensive database on the toxicity and modes of action of ethylene glycol (EG) has been developed over the past several decades. Although renal toxicity has long been recognized as a potential outcome, in recent years developmental toxicity, an effect observed only in rats and mice, has become the subject of extensive research and regulatory reviews to establish guidelines for human exposures. The developmental toxicity of EG has been attributed to the intermediate metabolite, glycolic acid (GA), which can become a major metabolite when EG is administered to rats and mice at high doses and dose rates. Therefore, a physiologically based pharmacokinetic (PBPK) model was developed to integrate the extensive mode of action and pharmacokinetic data on EG and GA for use in developmental risk assessments. The resulting PBPK model includes inhalation, oral, dermal, intravenous, and subcutaneous routes of administration. Metabolism of EG and GA were described in the liver with elimination via the kidneys. Metabolic rate constants and partition coefficients for EG and GA were estimated from in vitro studies. Other biochemical constants were optimized from appropriate in vivo pharmacokinetic studies. Several controlled rat and human metabolism studies were used to validate the resulting PBPK model. When internal dose surrogates were compared in rats and humans over a broad range of exposures, it was concluded that humans are unlikely to achieve blood levels of GA that have been associated with developmental toxicity in rats following occupational or environmental exposures.

The effects of feed restriction during in utero and postnatal development in rats.

This study determined the effects of feed restriction (FR) during in utero and postnatal life on standard reproductive toxicity and developmental immunotoxicity end points. Groups of 26 time-mated CD rats were fed various amounts of Purina 5002 diet from gestation day 7 through lactation. Control rats were fed once per day in amounts based on historical control feed consumption data, while the amounts fed to the FR groups were reduced by 10% (10% FR), 30% (30% FR), or 50% (50% FR) relative to controls. Selected F1 weanlings were necropsied on postnatal day (PND) 22, assessed for immunotoxicity end points between PND 22 and 27 or PND 52 and 56, or maintained on FR through PND 70. Thereafter, half the remaining F1 rats in each group were fed ad lib (recovery subgroup), while the rest continued on FR. Both subgroups were necropsied at 21 weeks of age. In the 10% FR group, slight decreases in maternal body weight had no effect on F1 offspring body weights, but did decrease F1 liver weights. FR at the 30% level reduced maternal body weights by 10-20%, reduced F1 offspring body weights by as much as 21%, caused changes in numerous weanling organ weights, but did not affect reproductive or immune system function. Dams in the 50% FR group were 17-32% lighter than controls, resulting in F1 body weights that were 12-47% lower than controls. F1 estrous cycle length was increased, puberty was delayed by 6 days (males and females), and anogenital distance, epididymal sperm counts, and all organ weights were decreased in this group. Antibody responses were unaffected despite decreased spleen and thymus weights. Essentially all effects of feed restriction showed evidence of reversibility.

Significance of 2-methoxypropionic acid formed from beta-propylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits.

Commercial grade propylene glycol monomethyl ether (PGME), which is composed of > 99.5% alpha-isomer and < 0.5% beta-isomer, has been shown in several studies to have a low potential for developmental toxicity. Nonetheless, questions have been raised about potential human developmental toxicity due to beta-PGME, because it can be metabolized to 2-methoxypropionic acid (MPA), a compound bearing structural similarity to the teratogen, methoxyacetic acid (MAA). Accordingly, a series of in vivo developmental toxicity, whole embryo culture, and in vivo pharmacokinetic experiments were conducted in New Zealand White rabbits (highly sensitive to these compounds) to better understand the developmental toxicity potential of MPA and the kinetics of its formation from beta-PGME. For the in vivo developmental toxicity studies, groups of 20 inseminated rabbits were gavaged with 0, 10, 26, or 78 mg/kg/day of MPA on gestation day (GD) 7-19, followed by fetal evaluation on GD 28. Results with MPA were compared with those of rabbits similarly dosed with 0, 2.5, 7.5, or 15 mg/kg/day of MAA. Developmental toxicity no-observable-effect levels (NOEL) were approximately 10-fold higher for MPA (26 mg/kg/day) than for MAA (2.5 mg/kg/day). Also, the severity of effects caused by MPA was less than that of MAA, and unlike MAA, MPA was not selectively toxic to the fetus. This differential toxicity was also seen in whole embryo cultures of GD 9 rabbit embryos, in which there were no adverse effects of MPA (1.0, 5.0 mM) or its parent compound, beta-PGME (0.5, 2.0 mM), but severe dysmorphogenesis in 100% of embryos cultured in 5.0 mM MAA. The pharmacokinetics study showed rapid and complete conversion of beta-PGME to MPA, with a relatively long elimination half-life (33-44 h) for MPA. However, peak and AUC concentrations of MPA in blood associated with the MPA LOEL dose of 78 mg/kg/day were 1.3 mM and 52.9 mM-h/l, respectively, suggesting a relatively high threshold based on internal dosimetry. Taken together, these data indicate a negligible risk of developmental toxicity due to MPA formation from the small amounts of beta-isomer present in commercial PGME.

Evaluation of spinosad in a two-generation dietary reproduction study using Sprague-Dawley rats.

Spinosad, an insecticide derived from a naturally occurring bacterium via fermentation, represents a new class of insecticides acting by a novel mode of action. A dietary study was conducted in Sprague-Dawley rats in which groups of 30 rats/sex/dosage level were given diets that provided 0, 3, 10, or 100 mg spinosad/kg body weight/day, 7 days/week, for 2 successive generations. Following 10 weeks of dietary exposure, the P1 generation was mated twice to produce F1a and F1b litters. After weaning, groups of 30 rats/sex/dosage level were selected from the F1a litters, given diets containing spinosad for 12 weeks, and mated to produce the F2 generation. Dietary administration of spinosad to rats at a dosage of 100 mg/kg/day over 2 generations produced parental toxicity and effects on the offspring. Among adult males, body weights and weight gains were decreased 2-9% relative to controls, with P1 males more affected than P2. Absolute and relative liver, kidney, heart, spleen, and thyroid weights were increased by from 12% to as much as 240% of control values. Histologic changes consistent with cationic amphiphilic compounds were noted in the kidneys, lungs, mesenteric lymph nodes, spleen, and thyroid of P1 and P2 males and females. In females given 100 mg/kg/day, though premating body weights were not affected, weight gains during the F1a and F1b gestation periods were depressed 15-16%. Increased incidences of dystocia, and vaginal bleeding and mortality occurred during parturition and lactation at 100 mg/kg/day. Effects on the offspring (decreased litter size and survival through day 4 of lactation) were limited to the high-dosage group. Signs indicative of poor maternal care noted in the pups (stomachs void of milk, cold, thin, etc.) were observed at 100 mg/kg/day. Early postnatal effects on the offspring were considered likely secondary to the effects in maternal animals around the time of parturition. At 100 mg/kg/day, weight gain in pups was depressed throughout lactation, with statistically significantly decreased weights noted toward the latter half of the lactation period. There were no treatment-related effects on adults or their offspring at 3 or 10 mg/kg/day in either generation. Based on these results, spinosad is not considered a selective reproductive toxicant, (i.e., no effects on reproductive parameters were noted below a level that produced toxicity in the adults) and the no observed effect level (NOEL) for both parental and reproductive/perinatal toxicity was 10 mg/kg/day.

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents.

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 microM) or DEM (5-25 microM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.

Dose-dependent nonlinear pharmacokinetics of ethylene glycol metabolites in pregnant (GD 10) and nonpregnant Sprague-Dawley rats following oral administration of ethylene glycol.

The kinetics of orally administered ethylene glycol (EG) and its major metabolites, glycolic acid (GA) and oxalic acid (OX), in pregnant (P; gestation day 10 at dosing, GD 10) rats were compared across doses, and between pregnant and nonpregnant (NP) rats. Groups of 4 jugular vein-cannulated female rats were administered 10 (P and NP), 150 (P), 500 (P), 1000 (P), or 2500 (P and NP) mg (13)C-labelled EG/kg body weight. Serial blood samples and urine were collected over 24-hr postdosing, and analyzed for EG, GA, and OX using GC/MS techniques. Pharmacokinetic parameters including Cmax, Tmax, AUC, and betat((1/2)) were determined for EG and GA. Pregnancy status (GD 10-11) had no impact on the pharmacokinetic parameters investigated. Blood levels of GA were roughly dose-proportional from 10 to 150 mg EG/kg, but increased disproportionately from 500 to 1000 mg EG/kg. EG and GA exhibited dose-dependent urinary elimination at doses > or = 500 mg EG/kg, probably due to saturation of metabolic conversion of EG to GA, and of GA to downstream metabolites. The shift to nonlinear kinetics encompassed the NOEL (500 mg EG/kg) and LOEL (1000 mg EG/kg) for developmental toxicity of EG in rats, providing additional evidence for the role of GA in EG developmental toxicity. The peak maternal blood concentration of GA associated with the LOEL for developmental toxicity in the rat was quite high (363 microg/g or 4.8 mM blood). OX was a very minor metabolite in both blood and urine at all dose levels, suggesting that OX is not important for EG developmental toxicity.

Differential antioxidant enzyme activities and glutathione content between rat and rabbit conceptuses.

Redox status regulates numerous cellular processes like transcription factor activation and binding, protein folding, and calcium sequestration. Because the most abundant reducing equivalent in the cell is glutathione (GSH), it could play a role for teratogens that cause oxidative stress and disrupt pathways involved in differentiation and proliferation. Investigation of the redox status of two species that have demonstrated differential sensitivity to teratogens represents a novel approach for determining the role of redox alteration in teratogenesis. Furthermore, examining specific regions of the embryo may also help to explain why certain tissues are uniquely sensitive, while others are resistant to oxidative insult. In the presented study, New Zealand White rabbit (GD 12) and Sprague Dawley rat embryos (GD 13) were removed from the uterus on days of similar development. Each embryo was dissected into three portions-the limbs, the head, and the trunk. Samples were placed in the appropriate buffers for the measurement of both direct and indirect redox status contributors-GSH, cysteine, thioredoxin, glutathione disulfide, protein-glutathione mixed disulfides, superoxide dismutase, glutathione peroxidase, and glutathione disulfide reductase. Species comparison of whole embryos indicated that the rabbit embryo possesses a higher redox potential (more oxidative) than the rat embryo. Findings, in general, show that the rabbit may be more sensitive to redox-altering teratogens because it is inherently more pro-oxidizing and may be more easily perturbed resulting in misregulation of cellular processes. Differences were most apparent in the limb as compared to the embryonic head and trunk, where the rabbit limb has a significantly more pro-oxidizing redox environment than the rat limb. Species comparisons like these may help in the understanding of how redox shifts affect cellular processes and would contribute to regulation of biochemical and molecular events that may be associated with mechanisms of teratogenesis. These may contribute to a more complete rationale for choosing a species for study and provide a better correlation with human developmental toxicants.

Evaluation of the male pubertal onset assay to detect testosterone and steroid biosynthesis inhibitors in CD rats.

The male pubertal onset assay has been recommended by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) as an alternate Tier I screening assay to detect potential endocrine-active chemicals (EACs). Recently, this assay was evaluated by several laboratories using a variety of dosing schemes. This study used a 30-day dosing period to confirm and extend previous work on the assay's ability to detect steroid biosynthesis inhibitors. Weanling male rats were dosed by gavage from 21 to 50 days of age with vehicle (0.5% methocel) or chemicals from the following EAC classes: an androgen (testosterone propionate [TP], 0.1 or 0.4 mg/kg/day), a broad-spectrum steroid biosynthesis inhibitor (ketoconazole [KETO], 24 mg/kg/day), a 5alpha-reductase inhibitor (finasteride [FIN], 20 or 80 mg/kg/day), a moderately specific aromatase inhibitor (testolactone [TL], 220 mg/kg/day), or a highly specific aromatase inhibitor (fadrozole [FAD], 0.6 or 6.0 mg/kg/day). None of these treatments altered relative thyroid weights. However, TL, KETO, and FIN were positive for endocrine activity based on decreases in one or more reproductive or accessory sex gland organ weights. Of these three inhibitors, only TL significantly increased the age at PPS, indicating that PPS was less sensitive for detecting these EACs. Based on its profile of effects, TL may have been detected as an antiandrogen. TP and FAD were negative in this assay, even at doses that caused effects in other studies. With TP, oral administration limited assay sensitivity such that higher TP doses would be needed for detection. FAD decreased body weight gains, but did not significantly alter any other assay end points; thus, the capacity of this assay to detect aromatase inhibitors remains in question.

Evaluation of the male pubertal assay's ability to detect thyroid inhibitors and dopaminergic agents.

The male pubertal onset assay is under consideration as an alternate Tier I screening assay to detect potential endocrine active chemicals (EACs) acting through a variety of steroid hormone and thyroid hormone receptor-mediated and non-receptor-mediated mechanisms. This study focused on the assay's ability to detect several non-receptor-mediated EACs. Weanling male CD rats (21 days old) were dosed for 30 d by gavage with vehicle (0.5% METHOCEL) or the following EAC classes (mg/kg/d): a potent thyroid agent (6-propylthiouracil, PTU, 240), a weak thyroid agent (phenobarbital, PB, 50 or 100), a dopamine antagonist (haloperidol, HALO, 2 or 4), or a dopamine agonist (bromocryptine, BRC, 10 or 50). In vehicle-treated males, preputial separation (PPS) occurred at 44.4 +/- 2.0 days of age. Age at PPS was delayed with PTU and 50 BRC, treatments that also delayed growth. Absolute testes and/or epididymal weights were decreased by PTU and 100 PB. BRC (50) and PB (100) decreased absolute prostate and seminal vesicle weights. Relative thyroid weights were altered by HALO, PTU, and PB, agents that significantly decreased serum T(4) levels. PTU increased serum thyroid-stimulating hormone (TSH) by 8.5 times and markedly altered thyroid histology, whereas HALO and PB did not significantly increase TSH and had marginal effects on thyroid histology. Thus, this assay detected both strong (PTU) and weak (PB) thyroid agents as well as the dopamine agonist BRC; however, its ability to detect dopamine antagonists remains unproven. These results confirm that thyroid weight measurements, although not required in the current male pubertal assay protocol, may add valuable information for interpretation of thyroid effects. Due to the apical nature of the male pubertal assay end points, additional work will be required to establish definitive criteria for a positive result in this assay.

The rabbit as a model for reproductive and developmental toxicity studies.

The rabbit has many advantages as a nonrodent and second model for assessing the effects of toxic agents on semen quality, fertility, developmental toxicity, and teratology. The male and female reproductive systems of the rabbit are described, and data on growth, sexual development and reproduction are compared with mice, rats, and humans. Techniques for semen collection and evaluation in the male, and artificial insemination, superovulation, embryo culture, and embryo transfer in the female are included as useful procedures in toxicity testing. Examples of the use of rabbits and experimental replication for toxicity testing are given. Special features of the visceral yolk sac and development of the chorioallantoic placenta of the rabbit are compared with rodents. The rabbit extraembryonic membranes more closely resemble the human than do the rodents, in some respects. The use of the rabbit in developmental toxicity and teratology studies is discussed.

Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay.

A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.

Incorporation of S-9 activation into an ER-alpha transactivation assay.

We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-alpha transactivation assay. 17beta-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded consistently in the assay with average EC(50) values of 9.6 x 10(-11) M and 1.2 x 10(-5) M, respectively. In the presence of a 0.1% S-9 fraction, the EC(50) for E2 was increased to 1.4 x 10(-9) M and that for MXC decreased to 4.9 x 10(-7) M, with both compounds demonstrating increased secondary metabolite formation as evidenced by HPLC analysis. Consistent with these data, metabolites of E2 and MXC exhibited decreased and increased potencies, respectively, in the assay system relative to the parent molecules. S-9 was compatible with the MCF-7 reporter assay and has the potential to enhance detection of proestrogenic materials.

Developmental toxicity studies in rats and rabbits with 3,5,6-trichloro-2-pyridinol, the major metabolite of chlorpyrifos.

3,5,6-Trichloro-2-pyridinol (TCP), the primary metabolite of chlorpyrifos and chlorpyrifos-methyl, was evaluated for potential developmental toxicity. Groups of 32-34 bred female Fischer 344 rats were given 0, 50, 100, or 150 mg TCP/kg/day by gavage on gestation days 6-15; the fetuses were evaluated on gestation day 21. Similarly, groups of 16 inseminated female New Zealand White rabbits were given 0, 25, 100, or 250 mg TCP/kg/day by gavage on gestation days 7-19, and fetuses were evaluated on gestation day 28. No clinical signs of toxicity attributed to TCP were noted in either species. In rats, at 150 mg/kg/day, maternal effects included slight decreases in feed consumption, significantly depressed body weight gain (25% relative to controls) resulting in significantly lower maternal terminal body weights, and increased relative liver weight. At 100 mg/kg/day, maternal body weight gain in rats was depressed approximately 22%. Among rabbits, maternal effects were limited to the group given 250 mg/kg/day, which lost an average of approximately 70 g during the treatment period (vs. 140 g in the controls). There were no effects on fetal weight or viability, nor were there significant increases in any fetal alteration in either species. A slightly higher (not statistically significant) than usual incidence of central nervous system anomalies occurred in rabbits, but these anomalies were found in both treated and control groups in this study as well as contemporaneous studies of unrelated compounds. This, and the fact that these anomalies were not seen with the parent compound, chlorpyrifos, suggest that their origin was spontaneous. Thus, TCP was not considered fetotoxic or teratogenic in either rats or rabbits, even at dose levels that produced maternal toxicity.

Optimization of an estrogen receptor-alpha transcriptional activation assay for testing a diverse spectrum of chemicals.

Estrogen receptor (ER) transactivation assays were initially designed to study endogenous mechanisms of steroid hormone action, but more recently have been used to assess industrial chemicals for potential estrogenic activity. Given the diverse spectrum of physicochemical properties of these chemicals, we examined the effects of pH (a weak organic and strong inorganic acid and base), hyperosmolality (NaCl, mannitol) and two different vehicles (DMSO, Triton X-100) on responses to estradiol-17beta (E2) in an ER transactivation assay. MCF-7 human breast cancer cells were transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc), treated with E2 under various test conditions, and then assessed for ER-mediated luciferase activity. Maximal E2-induced reporter activity was observed at pH 7.8 (pre-incubation), but was markedly reduced at pH < or =7.5, or > or =8.0 (P < 0.001), even though there was no evidence of cytotoxicity. Hyperosmolality induced by addition of mannitol (> or =25 mM) resulted in significant decreases in overall assay responsiveness, whereas NaCl (> or =80 mM) decreased the sensitivity of the assay by increasing the no-observed-effect level for E2 compared to control cultures (330 mOsm). The maximal DMSO concentration that resulted in consistently high E(2)-induced reporter activity was 0.1%, whereas concentrations of Triton X-100 above 1 ppm inhibited E2-induced reporter responses and were cytotoxic above 10 ppm. These results indicate that various physicochemical factors have the potential to confound assay data if not kept within predefined operational limits.

Differential alteration by thalidomide of the glutathione content of rat vs. rabbit conceptuses in vitro.

Thalidomide has been shown to cause limb reduction defects in rabbits with much greater potency than in rats, possibly due to inherent biochemical differences between the two species. Whole embryo culture was used to make direct comparisons between thalidomide-sensitive New Zealand White rabbits and thalidomide-resistant Sprague-Dawley rats, focusing on the possible roles of glutathione (GSH) and cysteine in mechanisms of thalidomide teratogenicity. Conceptuses were treated by adding thalidomide (0, 5, 15, and 30 microM) directly to the culture media containing conceptuses of similar gestational stages. Embryos and visceral yolk sacs (VYS) were measured for changes in GSH and cysteine content using HPLC after 24 h of exposure in vitro. Thalidomide-induced (15 and 30 microM) depletion of VYS GSH occurred only in the rabbit, where GSH concentrations (pmol/microg protein) fell significantly to about 50% of control. Rat VYS did not show a significant GSH depletion at any thalidomide concentration tested. Comparison between species showed that the control rabbit VYS contained 35% less GSH than the control rat VYS. Control rat embryos and control rabbit embryos contained similar concentrations of GSH, but thalidomide treatment preferentially depleted GSH in the rabbit at lower thalidomide concentrations (5 micro/M). Cysteine concentrations were not significantly altered from control in the embryo or VYS of either species when treated with thalidomide. However, although control cysteine concentrations did not differ significantly between rat and rabbit VYS, control cysteine levels in rabbit embryos were 65% lower than those in control rat embryos. Rabbit conceptuses displayed lower species-specific GSH and cysteine levels and a greater propensity for thalidomide-induced GSH depletion than in rat conceptuses, consistent with the greater sensitivity of the rabbit to thalidomide teratogenicity. These thalidomide-induced and inherent species differences implicate a possible role for GSH and redox status in the mechanisms of thalidomide teratogenicity.