Polybrominated Diphenylether (DE-71) Exposure Skews Phenotypic Sex Ratio, and Alters Steroid Hormone Levels and Steroidogenic Enzyme Activities in Juvenile Silurana tropicalis.

The impact of the brominated flame-retardant mixture, DE-71, on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined. A partial lifecycle study exposing S. tropicalis to varying concentrations of DE-71 (0.0, 0.65, 1.3, 2.5, and 5.0 µg/l [nominal]) was conducted from early gastrula-stage embryo to 150 days postmetamorphosis (dpm). Exposure of S. tropicalis to DE-71 induced liver necrosis and induced abnormal ovary development characterized by previtellogenic oocyte necrosis and arrested development of vitellogenic oocytes in females in a concentration-dependent manner. Decreased mean plasma dihydrotestosterone (DHT) and T, gonad T, and increased mean plasma E2 levels were found in 150 dpm DE-71-treated male S. tropicalis compared to controls. Plasma E2 levels in females were not significantly altered compared to control S. tropicalis, although lower plasma and gonad T were detected. Mean gonadal CYP 19 aromatase activity in both male and female S. tropicalis exposed to DE-71 was not appreciably affected. Decreased mean male 5α-reductase and CYP17 activities in both male and females were observed compared to control frogs. Overall, these studies suggested that PBDE exposure induced liver necrosis and abnormal ovary development; and reduced circulating and gonadal androgens resulting in a phenotypic skew in sex ratio toward the female sex in S. tropicalis.

Effect of perfluorooctanesulfonate exposure on steroid hormone levels and steroidogenic enzyme activities in juvenile Silurana tropicalis.

The impact of the perfluoro-chemical, perfluorooctanesulfonate (PFOS), on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined because of its ubiquity in the environment, bioaccumulative nature and potential to disturb endocrine activity. A partial life cycle study exposing S. tropicalis to varying concentrations of PFOS 0.06, 0.13, 0.25, 0.50 and 1.0 mg PFOS/L [nominal]) was conducted. Gonad and plasma samples were collected from juvenile control specimens and organisms exposed to PFOS from early embryo through 150 days post-metamorphosis. Gonad CYP17, aromatase and 5α-reductase activities were measured. Plasma estradiol, testosterone, dihydrotestosterone (DHT) and gonadal testosterone were measured in both males and females. Increased plasma DHT and gonadal testosterone were found in PFOS-treated juvenile male S. tropicalis compared to controls. Decreased plasma estradiol, but not testosterone, was detected in PFOS-treated female S. tropicalis compared to controls. Plasma DHT was not detected and an increase in gonadal testosterone was detected in PFOS-treated female frogs. Female S. tropicalis exposed to PFOS exhibited a concentration-related decrease in the mean aromatase activity, but not 5α-reductase. PFOS exposure in male frogs induced a concentration-related increase in 5α-reductase activity, but did not alter aromatase activity compared to control frogs. A concentration-related increase in CYP 17,20-lyase activity, but not 17-hydroxylase activity, was found in both female and male S. tropicalis exposed to PFOS.

Evaluation of the developmental toxicity of perfluorooctanesulfonate in the Anuran, Silurana tropicalis.

A 150-day post-metamorphosis (dpm) partial lifecycle study exposing Silurana tropicalis to <0.03 (control), 0.06, 0.13 0.25, 0.5 and 1.0 mg/L perfluorooctanesulfonate (PFOS) was conducted. A subset of specimens from the control and each treatment were evaluated at metamorphic completion. A significant increase in the median metamorphosis time was observed in the 1.0 mg/L PFOS treatment relative to the control. A modest increase in the occurrence, but not severity, of mild follicular hypertrophy was found in thyroid glands from organisms exposed to the 0.62 and 1.1 mg/L PFOS treatments. At 150 dpm, a concentration-dependent increase in whole body PFOS residues was measured ranging from 29.6 to 163.5 mg/kg in the 0.05 and 1.1 mg/L PFOS treatments. Decreased body weight and snout-vent length were noted in specimens exposed to 1.1 mg PFOS/L at the completion of metamorphosis. Body weight was reduced in the 1.1 mg/L PFOS concentration; however, snout-vent length was not affected by PFOS exposure at 150 dpm. An increased proportion of phenotypic males were noted in the 0.62 and 1.1 mg/L PFOS treatments. Abnormal ovary development characterized by size asymmetry, necrosis and formation of excessive fibrous connective tissue was identified in females exposed to 0.29 and 1.1 mg PFOS/L. Asymmetrically misshaped testes were found at 1.1 mg/L PFOS. Results suggested that PFOS is capable of interfering with S. tropicalis growth before metamorphic completion and growth and gonad development during juvenile development.

Inhibition of germinal vesicle breakdown in Xenopus oocytes in vitro by a series of substituted glycol ethers.

A 24 hour in vitro Xenopus oocyte maturation (germinal vesicle breakdown [GVBD]) assay developed by Pickford and Morris (Environmental Health Perspectives, 1999, 107, 285-292) was used to screen a series of substituted glycol ethers (GEs). Substituted GEs included: ethylene glycol monomethyl ether (EGME); EG monoethyl ether (EGEE); EG monopropyl ether (EGPE); EG monobutyl ether (EGBE); EG monohexyl ether (EGHE); diethylene glycol monomethyl ether (DGME); triethylene glycol monomethyl ether (TGME); ethylene glycol monophenyl ether (EGPhE); EG monobenzyl ether (EGBeE); EG diphenyl ether (EGDPhE); and propylene glycol monophenyl ether (PGPhE). The GEs inhibited progesterone- or androstenedione-induced GVBD with the following relative potency: EGPhE > PGPhE > EGME >> EGEE ≥ EGBeE > EGPE >> EGBE >EGHE > EGDPhE >> DGME ≥ TGME, or EGPhE >> PGPhE >> EGBeE > EGDPhE > EGEE > EGME > EGPE > EGBE, EGHE, DGME and TGME, respectively. Further, [3 H]progesterone or [3 H]androstenedione binding affinities to the oocyte plasma membrane progesterone receptor (OMPR) or classical androgen receptor (AR) were: EGME > EGPhE ≥ PGPhE ≥ EGEE > EGBeE >> EGPE >> EGBE ≥ EGHE > EGDPhE, TGME, and DGME, or EGPhE > PGPhE >> EGBeE > EGDPhE >> EGEE ≥ EGME >> EGPE, EGBE, and EGHE > DGME and TGME, respectively. Binary joint mixture studies with the GVBD model using flutamide (AR antagonist) and EGPhE indicated that flutamide/EGPhE mixture acted in a concentration additive manner. The effects of substituted GE series, however, may be mediated through the OMPR; the potency of EGPhE may be the result of bimodal inhibition of both the OMPR and AR pathways.

Assessment of sensitization risk of a laundry pre-spotter containing protease.

When proteolytic enzymes were first introduced to common laundry detergents in the 1960s, their ability to cause hypersensitivity due to exposure by inhalation was soon recognized as a problem, especially for production workers. Subsequently, formulations and manufacturing methods were developed to minimize exposure to enzymes via inhaled dust particles. Although detergents containing proteases are now considered safe for consumers, the experience with laundry pre-spotter products is not as extensive. Two studies were undertaken to examine the risk of sensitization to protease (i.e. Savinase(®)) used in a trigger-spray laundry pre-spotter product. The first was a laboratory study simulating a very heavy-use scenario in a controlled environment cubical chamber (14.5 m(3)). The product was applied to a series of fabric targets held vertically over a standard washing machine. Eight replicates of the experiment were done, using 30 sprays for each replicate. Airborne particle distributions in the breathing zone were characterized using a TSI particle analyzer. Enzyme concentrations in air were measured using PTFE membrane filters that were frozen until analyzed by an enzyme linked immunosorbent assay (ELISA). Results indicated that aerosol concentrations returned to baseline within 10 min, during which the average enzyme concentration in air was 17 ± 1.6 and 12 ± 0.92 ng/m(3) using low- and high-volume samplers, respectively. The corresponding amount of enzyme that could be inhaled was significantly less than allowed in occupational situations. The second study was a 6-month, controlled-use study involving approximately 100 subjects with confirmed atopic status by skin prick testing with common aeroallergens. The study involved daily exaggerated use of the pre-spotter product for 6 months, with prick testing for the protease carried out at baseline, 3 and 6 months. Results from the clinical study indicated that none of the subjects exhibited reactions that would indicate sensitization to the protease by inhalation. The principal limitations of the study were the relatively small number of subjects and the limited duration (96 completed the entire 6-month exposure program).

Effect of methoxychlor on various life stages of Xenopus laevis.

The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at /=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the frequency of amplexus, fertilization, and embryo viability was also affected by adult female methoxychlor exposure, and to a lesser extent by male exposure. Overall, these results suggested that sensitivity to methoxychlor is most dramatic during the reproductive and metamorphic phases of the life cycle and least sensitive during early embryo-larval development.

Evaluation of the developmental and reproductive toxicity of methoxychlor using an anuran (Xenopus tropicalis) chronic exposure model.

The chronic toxicity of methoxychlor to the South African clawed frog, Xenopus (Silurana) tropicalis, was evaluated using a life cycle approach. The chronic exposure period ranged from mid-cell blastula stage [NF (Nieuwkoop and Faber, 1994) stage 8] to 90 days of exposure, during which time the organisms generally completed metamorphosis and emerged as juvenile frogs. Methoxychlor concentrations ranged from 1 to 100 micrograms/l. Methoxychlor concentrations >10 micrograms/l caused delayed development. Organisms exposed to 10 micrograms/l methoxychlor for 30 days showed enlarged thyroid glands with follicular hyperplasia. No increase in mortality or external malformation was observed at any of the test concentrations during early embryo-larval development (NF stage 8 to NF stage 46; ca. 2 days exposure). A concentration-dependent increase in external malformations and internal abnormalities of the liver and gonads were noted after 90 days of exposure, however. Skewing of the sex ratio toward the female gender decreased ovary weight and number of oocytes, and increased oocyte immaturity and necrosis were noted at methoxychlor concentrations of 100 micrograms/l. Reductions in testis weight and sperm cell count were also detected at 100 micrograms/l methoxychlor. Results from these studies suggested that methoxychlor was capable of altering the rate of larval development, but did not adversely affect early embryo-larval development (2 days of exposure) as manifested in external malformations. Internal malformations, increases in the ratio of phenotypic females, were induced by chronic methoxychlor exposure. In addition, reproductive endpoints, most notably in the female specimens, were adversely affected by methoxychlor exposure. These studies add to the standardization and validation of a useful amphibian test methods capable of evaluating both reproductive and developmental effects of potential endocrine disrupting chemicals over a life cycle exposure.