Jesus A. Reyes

Evidence for thyroid endocrine disruption in wild fish in San Francisco Bay, California, USA. Relationships to contaminant exposures

It is well documented that many coastal and estuarine environments adjacent to developed and industrialized urban centers, such as the San Francisco Bay Area, are significantly contaminated by anthropogenic chemicals. However, it is not well understood to what extent existing contaminants, many with continuing inflows into the environment, may impact exposed wildlife. This study provided an initial characterization of thyroid endocrine-related effects and their relationship to accumulated contaminants in two indigenous fish species sampled from different San Franicsco Bay Area study sites. Plasma concentrations of thyroxine (T4) were significantly reduced in fish sampled from highly impacted locations such as Oakland Inner Harbor and San Leandro Bay as compared with fish from other locations representing relatively lower human impact, including Bodega Bay, Redwood City and a remote site on Santa Catalina Island. Triiodothyronine (T3) levels also varied significantly by location, with differing T3/T4 ratios in fish from some locations suggestive of altered peripheral deiodinase activity. The changes in thyroid endocrine parameters were significantly correlated with hepatic concentrations of certain environmental contaminants. A large number of polychlorinated biphenyl (PCB) congeners, both co-planar (dioxin-like) and non-co-planar, exhibited significant inverse correlations with T4 levels in the fish, while in contrast, T3 and T3/T4 ratio were positively correlated with PCB exposures. The positive correlation between T3/T4 ratio and PCBs supports the hypothesis that environmental PCBs may alter T4 deiodination or turnover, actions of PCBs reported in laboratory experiments. Some relationships between chlorinated pesticides including DDT and chlordanes, but fewer relationships with PAHs, were also observed. Together, these findings indicate that the thyroid endocrine system is exhibiting alterations associated with different aquatic environments in the San Francisco Bay Area, which are significantly related to current-day exposures of the fish to contaminant chemicals such as PCBs.

Analysis of Endocrine Disruption in Southern California Coastal Fish Using an Aquatic Multispecies Microarray

Background Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. Objectives We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. Methods We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. Results Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. Conclusions The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Genomic and phenotypic response of hornyhead turbot exposed to municipal wastewater effluents

Laboratory tests with marine flatfish were conducted to investigate associations among gene expression, higher biological responses and wastewater effluent exposure. In the present study, male hornyhead turbot (Pleuronichthys verticalis) were exposed to environmentally realistic (0.5%) and higher (5%) concentrations of chemically enhanced advanced-primary (PL) and full-secondary treated (HTP) effluents from two southern California wastewater treatment plants (WWTP). Hepatic gene expression was examined using a custom low-density microarray. Alterations in gene expression (vs. controls) were observed in fish exposed to both effluent types. Fish exposed to 0.5% PL effluent showed changes in genes involved in the metabolism of xenobiotics, steroids, and lipids, among other processes. Fish exposed to 5% PL effluent showed expression changes in genes involved in carbohydrate metabolism, stress responses, xenobiotic metabolism, and steroid synthesis, among others. Exposure to 5% HTP effluent changed the expression of genes involved in lipid, glutathione and xenobiotic metabolism, as well as immune responses. Although no concentration-dependent patterns of response to effluent exposure were found, significant Spearman correlations were observed between the expression of 22 genes and molecular and/or higher biological responses. These results indicate that microarray gene expression data correspond to higher biological responses and should be incorporated in studies assessing fish health after exposure to complex environmental mixtures.

Evaluation of reproductive endocrine status in hornyhead turbot sampled from Southern California's urbanized coastal environments.

As part of a regionwide collaboration to determine the occurrence of contaminants and biological effects in coastal ecosystems offshore of urban southern California, the present study characterized the reproductive endocrinology of an indigenous flatfish, the hornyhead turbot (Pleuronichthys verticalis), and compared groups sampled from different study sites representing varying degrees of pollution to screen for potential endocrine disruptive effects. Turbot were sampled from locations near the coastal discharge sites of four large municipal wastewater treatment plants (WWTPs) located between Los Angeles and San Diego, California, USA, and were compared with fish sampled from three far-field reference locations in the region. Despite environmental presence of both legacy contaminants and contaminants of emerging concern and evidence for fish exposure to several classes of contaminants, both males and females generally exhibited coordinated seasonal reproductive cycles at all study sites. Patterns observed included peaks in sex steroids (17β-estradiol, testosterone, 11-ketotestosterone) in the spring and low levels in the fall, changes corresponding to similarly timed gonadal changes and plasma vitellogenin concentrations in females. Comparisons between fish captured at the different study sites demonstrated some regional differences in plasma levels of estrogens and androgens, indicative of location-associated effects on the endocrine system. The observed differences, however, could not be linked to the ocean discharge locations of four of the largest WWTPs in the world.

Biological responses of marine flatfish exposed to municipal wastewater effluent.

There is increasing concern over the presence of pharmaceutical compounds, personal care products, and other chemicals collectively known as contaminants of emerging concern (CECs) in municipal effluents, yet knowledge of potential environmental impacts related to these compounds is still limited. The present study used laboratory exposures to examine estrogenic, androgenic, and thyroid-related endocrine responses in marine hornyhead turbot (Pleuronichthys verticalis) exposed to CECs from municipal effluents with 2 degrees of treatment. Fish were exposed for 14 d to environmentally realistic concentrations of effluent (0.5%) and to a higher concentration (5%) to investigate dose responses. Plasma concentrations of estradiol (E2), vitellogenin (VTG), 11-keto testosterone, and thyroxine were measured to assess endocrine responses. Contaminants of emerging concern were analyzed to characterize the effluents. Diverse types of effluent CECs were detected. Statistically significant responses were not observed in fish exposed to environmentally realistic concentrations of effluent. Elevated plasma E2 concentrations were observed in males exposed to ammonia concentrations similar to those found in effluents. However, exposure to ammonia did not induce VTG production in male fish. The results of the present study highlight the importance of conducting research with sentinel organisms in laboratory studies to understand the environmental significance of the presence of CECs in aquatic systems.

Application of a targeted endocrine q-PCR panel to monitor the effects of pollution in southern California flatfish

Current environmental monitoring programs focus primarily on legacy contaminants identified in the past as priority pollutants. Many new chemicals including so-called contaminants of emerging concern (CECs) are being widely dispersed into the environment and the development of sensitive tools that can screen endocrine responses and monitor the harmful effects of pollutants has become a priority. We developed a focused quantitative polymerase chain reaction (q-PCR) panel containing key endocrine gene targets for investigation of the effects of contaminants on transcriptional patterns in the demersal marine flatfish, hornyhead turbot (Pleuronichthys verticalis). An additional objective was to develop this tool so that it had broad applicability for monitoring endocrine disruption in other sentinel species, including California flatfish. This endocrine q-PCR panel was validated by studying transcriptional perturbations in 2 important sentinel species, hornyhead turbot and English sole (Parophrys vetulus), both sampled from sewage outfall and reference coastal waters in Southern California. In addition, the cross species utility of the endocrine q-PCR panel was examined by its application to the study of gene expression changes in laboratory male zebrafish (Danio rerio) following exposure to 17-β estradiol and 4-nonylphenol.