Keith R. Cooper

Embryonic exposure to tetrabromobisphenol A and its metabolites, bisphenol A and tetrabromobisphenol A dimethyl ether disrupts normal zebrafish (Danio rerio) development and matrix metalloproteinase expression.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant that is persistent in the environment and detected in human serum and breast milk. TBBPA is microbiologically transformed in anaerobic environments to bisphenol A (BPA) and in aerobic environments to TBBPA dimethyl ether (TBBPA DME). Despite the detection of TBBPA DME in the environment, the resulting toxicity is not known. The relative toxicity of TBBPA, BPA and TBBPA DME was determined using embryonic exposure of zebrafish, with BPA and TBBPA DME exhibiting lower potency than TBBPA. TBBPA exposure resulted in 100% mortality at 3 (1.6mg/L) and 1.5μM (0.8mg/L), whereas BPA and TBBPA DME did not result in significant embryonic mortality in comparison to controls. While all three caused edema and hemorrhage, only TBBPA specifically caused decreased heart rate, edema of the trunk, and tail malformations. Matrix metalloproteinase (MMP) expression was measured due to the role of these enzymes in the remodeling of the extracellular matrix during tissue morphogenesis, wound healing and cell migration. MMP-2, -9 and -13 expression increased (2-8-fold) after TBBPA exposure followed by an increase in the degradation of collagen I and gelatin. TBBPA DME exposure resulted in only a slight increase (less than 2-fold) in MMP expression and did not significantly increase enzymatic activity. These data suggest that TBBPA is more potent than BPA or TBBPA DME and indicate that the trunk and tail phenotypes seen after TBBPA exposure could be due in part to alteration of proper MMP expression and activity.

Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos

Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

A proposed multigeneration protocol for Japanese medaka (Oryzias latipes) to evaluate effects of endocrine disruptors

Definitive data on reproductive impairment of chronically exposed populations may be required to assess the appropriateness of the existing test methods for hazard identification and prioritization of endocrine modulators. Multigeneration toxicity testing protocols for wildlife receptors are lacking. To help address this gap we describe a multigeneration fish assay using the freshwater fish, Japanese medake (Oryzias latipes). This test species has been used for the evaluation of carcinogenic, teratogenic and reproductive effects and is sensitive to estrogen exposure producing ovo-testis, altered biochemical parameters and phenotypic characteristics. Due to the short life cycle, a multigeneration test with medaka can be conducted in 1 year. Endpoints evaluated include: survival, growth, sex ratio, fecundity, embryonic lesion occurrence, embryonic stage development, gonadal and hepatic somatic indices, histopathology and biochemical parameters. As new endpoints are developed they can be incorporated into the protocol. Results of a positive control (17 beta-estradiol) study are presented to give an indication of the baseline associated with various test endpoints and to highlight the importance of nutrition in the experimental design. 17 beta-Estradiol treatment induced vitellogenin production in male and female medaka, feminized males, and disrupted egg production. The proposed protocol provides researchers with an effective multigeneration fish test that can be used to examine potential effects of stressors at the population, individual, cellular and subcellular level.

Glucocorticoids Alter Craniofacial Development and Increase Expression and Activity of Matrix Metalloproteinases in Developing Zebrafish (Danio rerio)

Teratogenic effects are observed following long-term administration of glucocorticoids, although short-term glucocorticoid therapy is still utilized to reduce fetal mortality, respiratory distress syndrome, and intraventricular hemorrhage in preterm infants. However, the mechanism of glucocorticoid-induced teratogenicity is unknown. We hypothesize that glucocorticoid-induced teratogenesis is mediated through the glucocorticoid receptor (GR) and results from altering the expression and activity of the matrix metalloproteinases (MMPs). During embryogenesis, degradation of the extracellular matrix to allow for proper cellular migration and tissue organization is a tightly regulated process requiring appropriate temporal and spatial expression and activity of the MMPs. Studies have demonstrated that MMP gene expression can be either inhibited or induced by glucocorticoids in a variety of model systems. Using the zebrafish (Danio rerio) as a model of development, the data presented here demonstrate that embryonic exposure to the glucocorticoids dexamethasone or hydrocortisone increased expression of two gelatinases, MMP-2 ( approximately 1.5-fold) and MMP-9 (7.6- to 9.0-fold), at 72 h postfertilization (hpf). Further, gelatinase activity was increased approximately threefold at 72 hpf following glucocorticoid treatment, and changes in craniofacial morphogenesis were also observed. Cotreatment of zebrafish embryos with each glucocorticoid and the GR antagonist RU486 resulted in attenuation of glucocorticoid-induced increases in MMP expression (52-84% decrease) and activity (41-94% decrease). Furthermore, the abnormal craniofacial phenotype observed following glucocorticoid exposure was less severe following RU486 cotreatment. These studies demonstrate that in the embryonic zebrafish, dexamethasone, and hydrocortisone alter expression and activity of MMP-2 and -9, and suggest that these increases may be mediated through the GR.

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/PCDF) in food samples collected in southern Mississippi, USA

In 1994, we analyzed 43 foodstuff samples from local supermarkets in southern Mississippi, USA, for PCDD/PCDF. 2,3,7,8-Cl4DD could be quantified in 31 of these samples. On a lipid basis, levels in meat (0.53-1.10 pg I-TEQ/g) and dairy products (0.42-1.10 pg I-TEQ/g) were slightly lower than those reported from other industrialized countries. While levels in dairy samples from the United States and Europe are comparable, there is a difference in the contribution of individual congeners to the I-TEQ: for example, in milk samples from Germany approximately 40% of the I-TEQ is due to the presence of 2,3, 4,7,8-Cl5DF while in the Mississippi samples this congener only contributes 16%. The highest concentrations of PCDD/PCDF in our study were detected in the farm-raised catfish (10.2-27.8 pg I-TEQ/g). A unique finding was that in addition to the 2,3,7,8-substituted PCDD/PCDF the catfish samples contained many non-2,3,7,8-substituted congeners. This is unusual because vertebrate animals selectively eliminate or metabolize the non-2,3,7,8-substituted congeners.

Comparison of the toxicity of several polychlorinated dibenzo-p-dioxins and 2,3,7,8-tetrachlorodibenzofuran in embryos of the Japanese medaka (Oryzias latipes)

Nine polychlorinated dibenzo-p-dioxins (PCDDs) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were tested in the Japanese medaka embryo bioassay. Individual embryos were exposed to varying nominal concentrations of a PCDD or 2,3,7,8-TCDF under static conditions. The LC50 with 95% confidence intervals for survival to 3 days post-hatch were as follows: 2,3,7,8-TCDD, 13 (10–17) nanograms (ng)/liter (L) (parts per trillion); 2,3,7,8-TCDF, 16 (12–22) ng/L; 1,2,3,7,8-PeCDD, 27 (23–31) ng/L and 1,2,3,4,7,8-HCDD, 2900 (718–11717) ng/L. Both 1,2,7,8-TCDD and 2,3,7-TriCDD caused a concentration dependent increase in visible lesions from 50 to 50000 ng/L. In the 1,2,7,8-TCDD exposed embryos, lethality was observed only at the highest concentration tested (50000 ng/L). For 2,3,7-TriCDD, there was no concentration related increase in lethality from 50 to 50000 ng/L. At 50000 ng/L, 1,3,6,8-TCDD, 2,8-DCDD and 2,3-DCDD did not cause any visible toxicity. The octachlorodibenzo-p-dioxin congener did not cause any visible toxicity at 10000 ng/L. The toxic PCDDs and 2,3,7,8-TCDF caused the same sequela of lesions in the developing Japanese medaka, suggesting that they may act through a common mechanism. These results demonstrate that 2,3,7,8-TCDF and PCDDs with the four lateral positions chlorine substituted and at least one other position non-substituted are toxic to the Japanese medaka embryo. The structure activity relationship observed here is similar to that observed in guinea pigs, mice and trout eleutheroembryos.

PCDD and PCDF contamination in catfish feed from Arkansas, USA

One combined catfish feed sample from Arkansas, USA, and its eight ingredients were analyzed for PCDDs and PCDFs. One of the ingredients, soybean meal, was highly contaminated by PCDDs, especially the toxic 2,3,7,8-substituted congeners, e.g., 7.3 pg/g dry weight or 370 pg/g lipid for the 2,3,7,8-tetra CDD. The I-TEQ value for the soybean meal was 11.4 pg/g dry weight or 576 pg/g fat. The corresponding values for the combined catfish feed concentrations were approximately 3 times lower. The congener pattern, the congener profile and the ratio sigma PCDDs/sigma PCDFs for the soybean meal were quite unique. We are not aware of any environmental sample or technical product with similar characteristics. As a result, natural formation of the PCDDs found in the soybean meal cannot be ruled out.

Exposure assessment for heavy metal ingestion from a sport fish in Puerto Rico : estimating risk for local fishermen

Sport or subsistence fishermen sometimes underestimate the risks from eating local fish and distrust authorities regarding potential hazards from fish. We report on lead, cadmium, selenium, and mercury levels in snook, locally called robalo (Centropomus spp.), consumed by local fishermen in Puerto Rico, who ate up to 23 fish/wk. Median levels (in ppb, dry weight) for muscle, adipose, and liver ranged from 683 to 780 for lead, 37 to 600 for cadmium, 50 to 146 for mercury, and 361 to 3986 for selenium. Lead levels were similar in all tissues, and selenium and cadmium levels were highest in the liver. Mercury was higher in muscle than in liver. Computing exposure via ingestion for the average fisherman indicated that if one were to consume robalo throughout the year one would be exposed in excess of the EPA Reference Dose (RfD) for mercury. Using a widely accepted maximum consumption of 200 g/d for fishermen yielded hazard indices (daily consumption/RfD) below 1 for all four metals, with a value approaching 1 for mercury in adults, and over 1 for 16-kg children. The possibility that in certain localities and cultures sport or subsistence fishermen may consume fish at levels substantially above 200 g/d should be investigated in performing risk assessments, particularly outside the continental United States.

Impaired reproductive health of killifish (Fundulus heteroclitus) inhabiting Newark Bay, NJ, a chronically contaminated estuary.

A battery of biomarkers were used to evaluate the reproductive health and contaminant exposure of Atlantic killifish (Fundulus heteroclitus) inhabiting the heavily industrialized Newark Bay and a reference population from Great Bay, Tuckerton, NJ. The biomarkers investigated included classical endpoints (gonad and liver histopathology, body and tissue morphometrics), hepatic mRNA expression (CYP1A and vitellogenin I), hepatic protein levels (CYP1A and vitellogenin), gonadal aromatase mRNA expression, and chemical exposure analyses (bile PAHs). Our data showed no significant differences between populations for body size and body weight. However, Newark Bay killifish exhibited molecular and morphological changes indicative of impaired reproductive health and endocrine disruption compared to the reference population. Newark Bay males had decreased gonad weight, altered testis development and decreased gonadal aromatase mRNA expression. Newark Bay females had decreased gonad weight, inhibited gonadal development, decreased hepatic vitellogenin production (mRNA and protein) and increased mRNA expression of gonadal aromatase. In addition, Newark Bay females had a significant increase in the percent of pre-vitellogenic follicles (43% at Tuckerton, 64% at Newark Bay) and a significantly decreased percent of follicles at the mid-vitellogenic and mature stages (25% mature at Tuckerton and 3% at Newark Bay). In addition to reproductive endpoints, killifish at Newark Bay exhibited high basal levels of CYP1A mRNA and protein expression which indicated exposure to aryl hydrocarbon receptor (AhR) agonists. An inverse relationship between hepatic CYP1A protein and hepatic vitellogenin mRNA expression was established suggesting a possible link between AhR agonist exposure and vitellogenesis. Killifish in the NY-NJ Harbor Estuary are exposed to a number of chemicals that can interact with the AhR pathway and stimulate enzymatic activity along with chemicals that can modify reproductive success in this indigenous species. Similar effects on the reproductive development in less resilient species may limit their ability to repopulate the NY-NJ Harbor Estuary and similarly contaminated water systems.

PCDDs and PCDFs in soil and river sediment samples from a rural area in the United States of America

In 1994, we collected 36 soil samples and 61 sediment samples from southern Mississippi, USA. Each sample was analyzed for PCDDs and PCDFs by HRMS using either a polar or a non-polar HRGC column. Although most Cl4  Cl8 PCDDs and PCDFs were detected in each sample, PCDD levels (particularly Cl7  Cl8) were higher in sediment than soil samples. The PCDF pattern in two sediment samples indicated a possible PCB source. The concentrations of 2,3,7,8-tetra CDD and 2,3,7,8-tetra CDF were somewhat higher upstream of a pulp mill than downstream from the mill.