Susanne M. Brander

The in vivo estrogenic and in vitro anti‐estrogenic activity of permethrin and bifenthrin

Pyrethroids are highly toxic to fish at parts per billion or parts per trillion concentrations. Their intended mechanism is prolonged sodium channel opening, but recent studies reveal that pyrethroids such as permethrin and bifenthrin also have endocrine activity. Additionally, metabolites may have greater endocrine activity than parent compounds. The authors evaluated the in vivo concentration-dependent ability of bifenthrin and permethrin to induce choriogenin (an estrogen-responsive protein) in Menidia beryllina, a fish species known to reside in pyrethroid-contaminated aquatic habitats. The authors then compared the in vivo response with an in vitro assay--chemical activated luciferase gene expression (CALUX). Juvenile M. beryllina exposed to bifenthrin (1, 10, 100 ng/L), permethrin (0.1, 1, 10 µg/L), and ethinylestradiol (1, 10, 50 ng/L) had significantly higher ng/mL choriogenin (Chg) measured in whole body homogenate than controls. Though Chg expression in fish exposed to ethinylestradiol (EE2) exhibited a traditional sigmoidal concentration response, curves fit to Chg expressed in fish exposed to pyrethroids suggest a unimodal response, decreasing slightly as concentration increases. Whereas the in vivo response indicated that bifenthrin and permethrin or their metabolites act as estrogen agonists, the CALUX assay demonstrated estrogen antagonism by the pyrethroids. The results, supported by evidence from previous studies, suggest that bifenthrin and permethrin, or their metabolites, appear to act as estrogen receptor (ER) agonists in vivo, and that the unmetabolized pyrethroids, particularly bifenthrin, act as an ER antagonists in cultured mammalian cells.

Toxicity of a dissolved pyrethroid mixture to Hyalella azteca at environmentally relevant concentrations.

Use of pyrethroid pesticides, which are highly toxic to aquatic organisms, has increased substantially over the past decade. In 2006, the pyrethroid pesticides cyfluthrin and permethrin were measured in Sacramento-San Joaquin (SSJ) Delta (CA, USA) water at 5 and 24 ng/L (pptr), respectively. To elucidate any interactions between the two pyrethroids, a 10-d laboratory exposure was performed with 7- to 14-d-old amphipods (Hyalella azteca). Cyfluthrin and permethrin were tested singly and in combination at detected levels and also at half and twice the detected levels, both with and without the addition of 25 ppb of piperonyl butoxide (PBO). Mortality in all treatments was significantly higher than in controls, with the median lethal concentration (LC50) for permethrin with PBO (13.9 ng/L) and the LC50s with and without PBO for cyfluthrin (5.7 and 2.9 ng/L, respectively) at or below levels measured in SSJ Delta water samples. The LC50 for permethrin alone was estimated to be 48.9 ng/L. To evaluate combined toxicity, logistic regression models containing terms for concentrations of cyfluthrin, permethrin, and PBO, as well as models containing all possible combinations of these terms and interactions, were run and compared using Akaikes information criterion. The most parsimonious set of models indicated slight antagonism between cyfluthrin and permethrin. Results indicate that a dissolved mixture of cyfluthrin and permethrin is toxic at environmentally relevant concentrations in the water column.

From ‘Omics to Otoliths: Responses of an Estuarine Fish to Endocrine Disrupting Compounds across Biological Scales

Endocrine disrupting chemicals (EDCs) cause physiological abnormalities and population decline in fishes. However, few studies have linked environmental EDC exposures with responses at multiple tiers of the biological hierarchy, including population-level effects. To this end, we undertook a four-tiered investigation in the impacted San Francisco Bay estuary with the Mississippi silverside (Menidia audens), a small pelagic fish. This approach demonstrated links between different EDC sources and fish responses at different levels of biological organization. First we determined that water from a study site primarily impacted by ranch run-off had only estrogenic activity in vitro, while water sampled from a site receiving a combination of urban, limited ranch run-off, and treated wastewater effluent had both estrogenic and androgenic activity. Secondly, at the molecular level we found that fish had higher mRNA levels for estrogen-responsive genes at the site where only estrogenic activity was detected but relatively lower expression levels where both estrogenic and androgenic EDCs were detected. Thirdly, at the organism level, males at the site exposed to both estrogens and androgens had significantly lower mean gonadal somatic indices, significantly higher incidence of severe testicular necrosis and altered somatic growth relative to the site where only estrogens were detected. Finally, at the population level, the sex ratio was significantly skewed towards males at the site with measured androgenic and estrogenic activity. Our results suggest that mixtures of androgenic and estrogenic EDCs have antagonistic and potentially additive effects depending on the biological scale being assessed, and that mixtures containing androgens and estrogens may produce unexpected effects. In summary, evaluating EDC response at multiple tiers is necessary to determine the source of disruption (lowest scale, i.e. cell line) and what the ecological impact will be (largest scale, i.e. sex ratio).

Pyrethroid Pesticides as Endocrine Disruptors: Molecular Mechanisms in Vertebrates with a Focus on Fishes

Pyrethroids are now the fourth most used group of insecticides worldwide. Employed in agriculture and in urban areas, they are detected in waterways at concentrations that are lethally and sublethally toxic to aquatic organisms. Highly lipophilic, pyrethroids accumulate in sediments and bioaccumulate in fishes. Additionally, these compounds are demonstrated to act as endocrine disrupting compounds (or EDCs) in mammals and fishes, and therefore interfere with endocrine signaling by blocking, mimicking, or synergizing endogenous hormones through direct receptor interactions, and indirectly via upstream signaling pathways. Pyrethroid metabolites have greater endocrine activity than their parent structures, and this activity is dependent on the enantiomer present, as some pyrethroids are chiral. Many EDCs studied thus far in fish have known estrogenic or antiestrogenic effects, and as such cause the inappropriate or altered expression of genes or proteins (i.e., Vtg-vitellogenin, Chg-choriogenin), often leading to physiological or reproductive effects. Additionally, these compounds can also interfere with other endocrine pathways and immune response. This review highlights studies that focus on the mechanisms of pyrethroid biotransformation and endocrine toxicity to fishes across a broad range of different pyrethroid types, and integrates literature on the in vitro and mammalian responses that inform these mechanisms.

Thinking Outside the Box: Assessing Endocrine Disruption in Aquatic Life

Exposures to environmental concentrations of endocrine disrupting compounds (EDCs) are now a known threat to both human and ecological health. A large body of work has established that EDCs can agonize, antagonize or synergize the effects of endogenous hormones, resulting in physiological and behavioral abnormalities in aquatic organisms. Examples of disruption in fishes include altered secondary sexual characteristics and male production of female reproductive proteins. The universe of potential EDCs is expanding as new pesticides and pharmaceuticals constantly enter the marketplace, and the monumental tasks of prioritizing the backlog of compounds to be assessed and reducing their release into the environment remains. In the recent past, the majority of EDC research has focused on reproductive impacts, particularly those caused by estrogenic compounds, or to a lesser extent, androgenic compounds. Attention is now being directed toward impacts inflicted via novel mechanisms and toward impacts on other aspects of the endocrine system. Examples of lesser-known impacts of EDCs on fish include changes in somatic growth and modulation of the immune system. EDCs are known to disrupt pathways mediated by thyroid hormone, glucocorticoids, progestogens, and prostaglandins via receptor-binding, to interfere with cellular signaling cascades, or alter steroidogenesis. The challenge for ecotoxicologists is to determine which end points should be measured in fish in order to most accurately predict impacts at the population and even at the ecosystem level. Furthermore, in addition to assessing risk at multiple biological scales, the effects of complex environmental mixtures, differences in species sensitivity, adaptation to pollution, and the potential for epigenetic change must also be integrated into determinations of “safe” EDC concentrations. Considering the propensity of EDCs to exert effects at low doses and to exhibit nonmonotonic responses, this is a task that will require increased collaboration and ingenuity amongst researchers in the field.

The role of P450 metabolism in the estrogenic activity of bifenthrin in fish.

Bifenthrin, a pyrethroid pesticide, is estrogenic in vivo in fishes. However, bifenthrin is documented to be anti-estrogenic in vitro, in the ER-CALUX (estrogen receptor) cell line. We investigated whether metabolite formation is the reason for this incongruity. We exposed Menidia beryllina (inland silversides) to 10ng/l bifenthrin, 10ng/l 4-hydroxy bifenthrin, and 10ng/l bifenthrin with 25μg/l piperonyl butoxide (PBO) - a P450 inhibitor. Metabolite-exposed juveniles had significantly higher estrogen-mediated protein levels (choriogenin) than bifenthrin/PBO-exposed, while bifenthrin alone was intermediate (not significantly different from either). This suggests that metabolites are the main contributors to bifenthrins in vivo estrogenicity.

Transcriptomic changes underlie altered egg protein production and reduced fecundity in an estuarine model fish exposed to bifenthrin

Pyrethroid pesticides are a class of insecticides found to have endocrine disrupting properties in vertebrates such as fishes and in human cell lines. Endocrine disrupting chemicals (EDCs) are environmental contaminants that mimic or alter the process of hormone signaling. In particular, EDCs that alter estrogen and androgen signaling pathways are of major concern for fishes because these EDCs may alter reproductive physiology, behavior, and ultimately sex ratio. Bifenthrin, a pyrethroid with escalating usage, is confirmed to disrupt estrogen signaling in several species of fish, including Menidia beryllina (inland silverside), an Atherinid recently established as a euryhaline model. Our main objective was to broadly assess the molecular and physiological responses of M. beryllina to the ng/L concentrations of bifenthrin typically found in the environment, with a focus on endocrine-related effects, and to discern links between different tiers of the biological hierarchy. As such, we evaluated the response of juvenile Menidia to bifenthrin using a Menidia-specific microarray, quantitative real-time polymerase chain reaction (qPCR) on specific endocrine-related genes of interest, and a Menidia-specific ELISA to the egg-coat protein choriogenin, to evaluate a multitude of molecular-level responses that would inform mechanisms of toxicity and any underlying causes of change at higher biological levels of organization. The sublethal nominal concentrations tested (0.5, 5 and 50ng/L) were chosen to represent the range of concentrations observed in the environment and to provide coverage of a variety of potential responses. We then employed a 21-day reproductive assay to evaluate reproductive responses to bifenthrin (at 0.5ng/L) in a separate group of adult M. beryllina. The microarray analysis indicated that bifenthrin influences a diverse suite of molecular pathways, from baseline metabolic processes to carcinogenesis. A more targeted examination of gene expression via qPCR demonstrated that bifenthrin downregulates a number of estrogen-related transcripts, particularly at the lowest exposure level. Choriogenin protein also decreased with exposure to increasing concentrations of bifenthrin, and adult M. beryllina exposed to 0.5ng/L had significantly reduced reproductive output (fertilized eggs per female). This reduction in fecundity is consistent with observed changes in endocrine-related gene expression and choriogenin production. Taken together, our results demonstrate that environmental concentrations of bifenthrin have potential to interfere with metabolic processes, endocrine signaling, and to decrease reproductive output.

The Role of Epigenomics in Aquatic Toxicology

Over the past decade, the field of molecular biology has rapidly incorporated epigenetic studies to evaluate organism-environment interactions that can result in chronic effects. Such responses arise from early life stage stress, the utilization of genetic information over an individuals life time, and transgenerational inheritance. Knowledge of epigenetic mechanisms provides the potential for a comprehensive evaluation of multigenerational and heritable effects from environmental stressors, such as contaminants. Focused studies have provided a greater understanding of how many responses to environmental stressors are driven by epigenetic modifiers. We discuss the promise of epigenetics and suggest future research directions within the field of aquatic toxicology, with a particular focus on the potential for identifying key heritable marks with consequential impacts at the organism and population levels. Environ Toxicol Chem 2017;36:2565-2573.

The effects of fipronil and the photodegradation product fipronil desulfinyl on growth and gene expression in juvenile blue crabs, Callinectes sapidus , at different salinities

Endocrine disrupting compounds (EDCs) are now widely established to be present in the environment at concentrations capable of affecting wild organisms. Although many studies have been conducted in fish, less is known about effects in invertebrates such as decapod crustaceans. Decapods are exposed to low concentrations of EDCs that may cause infertility, decreased growth, and developmental abnormalities. The objective herein was to evaluate effects of fipronil and its photodegradation product fipronil desulfinyl. Fipronil desulfinyl was detected in the eggs of the decapod Callinectes sapidus sampled off the coast of South Carolina. As such, to examine specific effects on C. sapidus exposed in early life, we exposed laboratory-reared juveniles to fipronil and fipronil desulfinyl for 96h at three nominal concentrations (0.01, 0.1, 0.5μg/l) and two different salinities (10, 30ppt). The size of individual crabs (weight, carapace width) and the expression of several genes critical to growth and reproduction were evaluated. Exposure to fipronil and fipronil desulfinyl resulted in significant size increases in all treatments compared to controls. Levels of expression for vitellogenin (Vtg), an egg yolk precursor, and the ecdysone receptor (EcR), which binds to ecdysteroids that control molting, were inversely correlated with increasing fipronil and fipronil desulfinyl concentrations. Effects on overall growth and on the expression of EcR and Vtg differ depending on the exposure salinity. The solubility of fipronil is demonstrated to decrease considerably at higher salinities. This suggests that fipronil and its photodegradation products may be more bioavailable to benthic organisms as salinity increases, as more chemical would partition to tissues. Our findings suggest that endocrine disruption is occurring through alterations to gene expression in C. sapidus populations exposed to environmental levels of fipronil, and that effects may be dependent upon the salinity at which exposure occurs.

The Ecotoxicology of Plastic Marine Debris

ABSTRACT The accumulation of plastic in the oceans is an ever-growing environmental concern. Plastic debris is a choking and entanglement hazard for wildlife; plastics also leach toxic compounds into organisms and ecosystems. Educating students about the marine debris problem introduces fundamental concepts in toxicology, ecology, and oceanography. Students will learn about the toxicity of plastics, collect and analyze data on plastic debris, and put their new knowledge to work by writing a congressional bill that addresses the problem of marine debris.