Dose-responsive gene expression changes in juvenile and adult mummichogs (Fundulus heteroclitus) after arsenic exposure.

The present study investigated arsenic's effects on mummichogs (Fundulus heteroclitus), while also examining what role that gender or exposure age might play. Adult male and female mummichogs were exposed to 172 ppb, 575 ppb, or 1720 ppb arsenic as sodium arsenite for 10 days immediately prior to spawning. No differences were noted in the number or viability of eggs between the groups, but there was a significant increase in deformities in 1720 ppb arsenic exposure group. Total RNA from adult livers or 6-week old juveniles was used to probe custom macroarrays for changes in gene expression. In females, 3% of the genes were commonly differentially expressed in the 172 and 575 ppb exposure groups compared to controls. In the males, between 1.1 and 3% of the differentially expressed genes were in common between the exposure groups. Several genes, including apolipoprotein and serum amyloid precursor were commonly expressed in either a dose-responsive manner or were dose-specific, but consistent across genders. These patterns of regulation were confirmed by QPCR. These findings will provide us with a better understanding of the effects of dose, gender, and exposure age on the response to arsenic.

A concentration addition model for the activation of the constitutive androstane receptor by xenobiotic mixtures.

The effects of contaminants are typically studied in individual exposures; however, environmental exposures are rarely from a single contaminant. Therefore, the study of chemical mixtures is important in determining the effects of xenobiotics. The constitutive androstane receptor (CAR) responds to endobiotics and xenobiotics, and in turn induces detoxification enzymes involved in their elimination. First, we compared several androgens as inverse agonists, including androgens allegedly used by Bay Area Laboratory Co-operative to enhance athletic performance. CAR inverse agonists ranked in order of potency were dihydroandrosterone (DHA) > tetrahydrogestrinone (THG) > androstanol > norbolethone. Therefore, we used DHA as an inverse agonist during transactivation assays. Next, we examined the effects of several pesticides, plasticizers, steroids, and bile acids on CAR activation. Our data demonstrates that several pesticides and plasticizers, including diethylhexylphthalate, nonylphenol, cypermethrin, and chlorpyrifos activate CAR. Both full and partial CAR activators were discovered, and EC(50) values and Hillslopes were determined for use in the concentration addition models. Concentration addition models with and without restraint values to account for partial activators were developed. Measured results from transactivation assays with a mixture of two to five chemicals indicate that the concentration addition model without restraints correctly predicts activity unless all of the chemicals in the mixture are partial activators, and then restraint values be considered. Overall, our data indicates that it is important to consider that we are exposed to a milieu of chemicals, and the efficacy of each individual chemical is not the sole factor in determining CAR's activity in mixture modeling.

Using mummichog (Fundulus heteroclitus) arrays to monitor the effectiveness of remediation at a superfund site in Charleston, South Carolina, U.S.A.

We previously developed a cDNA array for mummichogs (Fundulus heteroclitus), an estuarine minnow, that is targeted for identifying differentially expressed genes from exposure to polycyclic aromatic hydrocarbons and several metals, including chromium. A chromium-contaminated Superfund site at Shipyard Creek in Charleston, South Carolina, USA, is undergoing remediation, providing us a unique opportunity to study the utility of arrays for monitoring the effectiveness of site remediation. Mummichogs were captured in Shipyard Creek in Charleston prior to remediation (2000) and after remediation began (2003 and 2005). Simultaneously, mummichogs were collected from a reference site at the Winyah Bay National Estuarine Research Reserve (NERR) in Georgetown, South Carolina, USA. The hepatic gene expression pattern of fish captured at Shipyard Creek in 2000 showed wide differences from the fish captured at NERR in 2000. Interestingly, as remediation progressed the gene expression pattern of mummichogs captured at Shipyard Creek became increasingly similar to those captured at NERR. The arrays acted as multidimensional biomarkers as the number of differentially expressed genes dropped from 22 in 2000 to four in 2003, and the magnitude of differential expression dropped from 3.2-fold in 2000 to no gene demonstrating a difference over 1.5-fold in 2003. Furthermore, the arrays indicated changes in the bioavailability of chromium caused by hydraulic dredging in the summer of 2005. This research is, to our knowledge, the first report using arrays as biomarkers for a weight-of-evidence hazard assessment and demonstrates that arrays can be used as multidimensional biomarkers to monitor site mitigation because the gene expression profile is associated with chromium bioavailability and body burden.

Hexavalent chromium reduces larval growth and alters gene expression in mummichog (Fundulus heteroclitus).

Hexavalent chromium [Cr(VI)] is a common bioavailable metal ion that causes oxidative stress, DNA adducts, and perturbs gene expression. Changes in gene expression are useful biomarkers of toxicant exposure that provide information about an organism's health, adaptability, and toxicant-specific effects. Therefore, we developed a cDNA array for the estuarine sentinel species mummichog (Fundulus heteroclitus). Mummichog larvae were exposed to concentrations ranging from 0 to 24 mg/L (462 microM) of Cr(VI) for 30 d, and growth was measured to determine the no-observable-effect concentration (1.5 mg/L) and the lowest-observable-effect concentration (3 mg/L). Body burdens from Cr(VI)-exposed fish showed a dose-dependent increase and were inversely correlated to body weight. Mummichog larvae exposed to Cr(VI) differentially expressed 16 genes in a dose-dependent manner, including GLUT-2, L-FABP, ATPase synthase 8, type II keratin, TBT-binding protein, and complement component C3-2. Many of these genes are involved in energy metabolism or growth, which is consistent with the reduced growth observed. In subsequent experiments, adults were exposed to Cr(VI) for 7 d at 0, 1.5, or 3 mg/L, because adult mummichog are used in monitoring Superfund sites. Hexavalent chromium altered the expression of 10 genes in adult liver, including HGFA, H-FABP, and complement component C3-2. Many of these genes also are involved in energy metabolism. The mummichog arrays provide a potential mechanism for the effects of Cr(VI) on growth. We anticipate using these arrays and the data they provide to monitor effects at polluted sites, to assess the bioavailability of chromium at these sites, and to investigate the efficacy of remediation in chromium-polluted estuaries.

Alterations in hepatic gene expression by trivalent chromium in Fundulus heteroclitus.

Cr(III) is the dominant toxicant at some Superfund sites within the United States and therefore we are interested in its effects. Cr(III)s mechanisms are not well studied or understood because of its low bioavailability. We have attempted to characterize the effects of Cr(III) on gene expression in Fundulus heteroclitus (mummichog) liver. The NOEC and LOEC were determined at 32 and 64 mg/L, respectively, by measuring growth and mortality after exposing juveniles for 30 days. Secondary adult male exposures were performed at 32 mg/L, livers excised, and RNA extracted. Arrays were probed with cDNA from untreated or Cr(III)-exposed adult fish and gene expression was quantified. Cr(III) at 32 mg/L altered the expression of five genes, including GSTtau, GSTalpha, and ALDH4. Ultimately, we anticipate using this gene expression information to ascertain whether Cr(III) is bioavailable at potentially adverse concentrations in contaminated sites.

Physiological changes and differential gene expression in mummichogs (Fundulus heteroclitus) exposed to arsenic.

Arsenic has been detected as a contaminant in water bodies around the world. Although a number of studies have shown toxicity to adult fish, little is known about its effects on the offspring. However, human epidemiological studies have shown that arsenic increases the number of stillbirths and prematurely born infants. We examined changes in the morphology and gene expression in juvenile mummichogs (Fundulus heteroclitus) whose parents were exposed to 230 ppb arsenic for 10 days immediately prior to spawning. The hatchlings of exposed fish had a 2.8-fold increased incidence of curved or stunted tails. Total RNA from 6-week-old hatchlings, reared in clean water, was used to construct a cDNA subtractive hybridization library. Using this library, we found 13 genes whose expression was altered in the hatchlings as a result of arsenic exposure. We confirmed differential expression by real-time PCR and found significant up-regulation of myosin light chain 2 (4.2-fold), type II keratin (1.5-fold), tropomyosin (3.1-fold) and parvalbumin (3.5-fold) in the hatchlings whose parents were exposed to arsenic. These genes are important during embryogenesis and their differential expression may be linked to the morphological changes observed in the hatchlings.

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder (Pleuronectes americanus): comparison to the effects of Saint John's Wort.

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6beta- and 16beta-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder (Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6beta-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16beta-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8 x), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.

Construction of a subtractive library from hexavalent chromium treated winter flounder (Pseudopleuronectes americanus) reveals alterations in non-selenium glutathione peroxidases.

Chromium is released during several industrial processes and has accumulated in some estuarine areas. Its effects on mammals have been widely studied, but relatively little information is available on its effects on fish. Gene expression changes are useful biomarkers that can provide information about toxicant exposure and effects, as well as the health of an organism and its ability to adapt to its surroundings. Therefore, we investigated the effects of Cr(VI) on gene expression in the sediment dwelling fish, winter flounder (Pseudopleuronectes americanus). Winter flounder ranging from 300 to 360 g were injected i.p. with Cr(VI) as chromium oxide at 25 microg/kg chromium in 0.15N KCl. Twenty-four hours following injections, winter flounder were euthanized with MS-222 and the livers were excised. Half of the livers were used to make cytosol and the other half were used to isolate mRNA for subtractive hybridization. Subtractive clones obtained were spotted onto nylon filters, which revealed several genes with potentially altered expression due to Cr(VI), including an alpha class GST, 1-Cys peroxiredoxin (a non-selenium glutathione peroxidase), a P-450 2X subfamily member, two elongation factors (EF-1 gamma and EF-2), and complement component C3. Semi-quantitative RT-PCR was performed and confirmed that Cr(VI) down-regulated complement component C3, an EST, and two potential glutathione peroxidases, GSTA3 and 1-Cys peroxiredoxin. In addition, cytosolic GSH peroxidase activity was reduced, and silver stained SDS-PAGE gels from glutathione-affinity purified cytosol demonstrated that a 27.1 kDa GSH-binding protein was down-regulated greater than 50%. Taken together, Cr(VI) significantly altered the expression of several genes including two potential glutathione peroxidases in winter flounder.

Differential gene expression in mummichogs (Fundulus heteroclitus) following treatment with pyrene: comparison to a creosote contaminated site.

Mummichogs, Fundulus heteroclitus, an estuarine fish with a relatively small home range found along the eastern coast of the United States are well-suited to monitoring contaminant effects, including those of polycyclic aromatic hydrocarbons (PAHs). One of the common PAHs in estuaries is pyrene. We report here on efforts to develop multiple biomarkers of pyrene exposure in this species. Adult male mummichogs were exposed in the laboratory to the weak aryl hydrocarbon receptor (AhR) agonist pyrene at 0, 30, or 50 microg/L in 7-day static renewal exposures. The RNA was extracted from livers and alterations in mRNA expression were assessed by subtractive hybridization and differential display in order to produce multiple biomarkers of pyrene exposure. Genes demonstrating differential expression were confirmed by quantitative-PCR (Q-PCR) and include cytochrome P-450 1A (CYP1A), a putative hepatocyte growth factor activator, a X-ray inducible retrotransposon, and several expressed sequenced tags (ESTs). Some of these genes represent new biomarkers of pyrene exposure and potential biomarkers of PAH exposure. Therefore, similar changes were investigated at a Superfund site in Charleston, SC. Mummichogs from a creosote contaminated site and from a reference site (North Inlet National Estuarine Research Reserve near Georgetown, SC) were trapped, RNA extracted from the livers, and Q-PCR performed. Many of the genes differentially expressed following pyrene exposure were not altered at the creosote contaminated site in comparison to the reference site. However, CYP1A and an EST were induced. CYP1A induction at Diesel Creek indicates that this population of fish does not demonstrate refractory CYP1A phenotypes observed at several sites with high levels of AhR agonists. Ultimately, we anticipate that the use of multiple biomarkers of PAH exposure will provide useful information on the potential effects of toxicants.