Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA).

We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.

Vitellogenin induction in painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol.

Ponds within cattle farms often support turtle and fish populations and are impacted by manure runoff. Cattle excrete metabolized (glucuronide-conjugated) hormones in feces and urine into these ponds, and bacteria cleave the glucuronide metabolites to active steroids, which can be stable for several weeks in wastewater. The objectives of this study were to (1) assess levels of xenoestrogens found in ponds near livestock pastures; and (2) assess whether these levels of xenoestrogens induce vitellogenin (VTG) in painted turtles in the laboratory and field. We collected water twice, 6 weeks apart, and placed turtle traps weekly into two ponds, which receive runoff from beef cattle pastures, and into one pond with no cattle farm effluents. Water E(2) levels were analyzed using C(18) solid phase extraction disks and detected in a radioimmunoassay (RIA). Plasma was collected from painted turtles (Chrysemys picta) captured from these ponds and VTG levels were measured via enzyme linked immunosorbent assay (ELISA). Nine additional turtles were collected from a pond at the South Carolina Botanical Gardens, which receives no farm runoff, and were exposed in the laboratory to nominal concentrations of 0.15, 1.5, and 15 ng/l estradiol (static renewal) over a 28-day period, followed by 14 days in clean water. Plasma samples were taken weekly for VTG measurement via ELISA. Levels of free estradiol in the water column of farm ponds range from 0.05 to 1.80 ng/l, as measured by RIA, and up to 7.4 ng/l as measured by ER-beta binding affinity. This is similar to what has been reported in streams receiving sewage treatment works (STW) effluents. In the laboratory, plasma VTG in male painted turtles could not be induced even at the high E(2) dose (9.45 ng/l) after 28 days. In the field, VTG levels were induced only in females when compared with animals from the SC Botanical Gardens. Adult male turtles need to be primed with high doses of E(2) prior to being able to respond to exogenous E(2). Given that males would not typically be sensitized in the wild, environmentally relevant levels of E(2) may not be sufficient to affect them. However, higher VTG levels in females could potentially change their reproductive fitness by altering egg size or by shifting energy allocations away from other survival needs. Long-term studies are needed to study potential impacts of VTG induction on female turtle reproductive success.

Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones.

Many plant compounds are able to modulate growth and reproduction of herbivores by directly interacting with steroid hormone systems. In insects, several classes of phytochemicals, including the phytoestrogens, interfere with molting and reproduction. We investigated whether the anti-ecdysone activity may be due to interaction with the ecdysone receptor (EcR) using a reporter-gene assay and a cell differentiation assay of an ecdysone-responsive cell line, Cl.8+. We tested rutin (delays molt in insects); four flavones: luteolin and quercetin (metabolites of rutin), and apigenin and chrysin; and three non-flavones, coumestrol and genistein (both estrogenic) and tomatine (alters molt in insects). None of the phytochemicals tested were ecdysone agonists in the reporter-gene assay, but the flavones were able to significantly inhibit EcR-dependent gene transcription. In the Cl.8+ cells, quercetin and coumestrol were mixed agonists/antagonists, while genistein, tomatine and apigenin showed a synergistic effect with ecdysteroid in the reduction of cell growth. We suggest that the rutin effects on molting in insects are most likely due to the metabolites, luteolin or quercetin, while tomatine acts via a non-EcR pathway. Flavones not only interact with EcR and estrogen receptor (ER), but also signal nitrogen-fixing bacteria to form root nodules. The NodD protein which regulates this symbiosis has two ligand-binding domains similar to human ERalpha. The evolutionary significance of these findings are discussed.

Gender benders at the beach: endocrine disruption in marine and estuarine organisms.

Several consensus definitions of the term endocrine disruptor have appeared recently, but all definitions include the important, though frequently implicit, stipulation that the animal is not distressed or in obvious discomfort. Instead, a superficially healthy animal is experiencing alterations in hormone synthesis, transport, receptor interaction, metabolism, excretion, or feedback regulation. In addition, hormone disruption may occur during sex differentiation, and its effects may not be manifested until after sexual maturation. Many cases of chemically induced reproductive impairment have been reported for both freshwater and marine species. However, reproductive impairment may not necessarily result from hormone disruption and should be considered suggestive, but not conclusive, evidence of endocrine disruption. A suite of in vivo and in vitro assays will more adequately assess whether a compound is truly endocrine disrupting. This review will cover basic endocrinology of marine and estuarine invertebrates and vertebrates, methods for detecting endocrine disruption, and examples of endocrine disruption in various species.

Peptide hormones versus steroid hormones: case studies from snail and turtle populations.

The proposed mechanisms of masculinization in gastropod mollusks (imposex) and feminization in oviparous vertebrates has focused primarily on the interaction of xenobiotics with the steroid hormone system. The evidence does not support the proposed mechanism of imposex induction, inhibition of CYP 19 (aromatase), since the changes in steroid titers occur only in the later stages of imposex. In addition, the role of vertebrate sex steroid hormones is limited in gastropods. Recent evidence suggests that peptide hormones play a key role in masculinization in snails and may be a more plausible mechanism of imposex induction. Steroid hormones play a role in feminization of oviparous vertebrates. Vitellogenin (Vtg, egg yolk) induction has been used extensively as a biomarker of feminization. However, in some species of turtles there is evidence that high levels of estradiol are necessary to initially induce Vtg. Once this imprinting occurs, lower levels of estradiol can then induce high levels of Vtg. It was found that environmental levels of estradiol do not always induce Vtg in male and juvenile turtles. However, these same levels of estradiol significantly elevate female Vtg levels. Elevated female Vtg may lead to larger, but fewer, eggs, and therefore fewer offspring. However, these offspring may have better survivorship. Alternatively, elevated Vtg may lead to a larger number of smaller eggs, which have been shown to produce smaller hatchlings with reduced survival. The effects of elevate female Vtg on fitness are difficult to assess in such long-lived species.

Purification of vitellin from grass shrimp Palaemonetes pugio, generation of monoclonal antibodies, and validation for the detection of lipovitellin in Crustacea.

Much effort has been put into developing vitellogenin antibodies against a wide variety of aquatic vertebrate species to study potential estrogen or anti-estrogen endocrine disrupters. Little work has been done on endocrine disruption in aquatic invertebrates. Although some antibodies have been produced against blue crab and penaeid shrimp lipovitellin, they have only poor cross-reactivity with the important estuarine grass shrimp, Palaemonetes pugio. Vitellin was purified from eggs, monoclonal antibodies were produced using standard techniques, and hybridoma supernatants were screened by ELISA. Western blots were done using extracts from male and female grass shrimp to verify specificity of the monoclonal antibodies. Two low molecular mass bands in the range of 68-85 kD and two high molecular mass bands in the range of 190-221 kD were found. In addition to grass shrimp, several other crustacean species were screened and cross-reactivity found, including blue crab (Callinectes sapidus), mud crab (Rhithropanopeus harrisii), red swamp crayfish (Procambarus clarkii ) and Daphnia magna. To further investigate the use of the antibody, we performed a chronic 6-week pyrene exposure study. We found that vitellin was upregulated in females after 6 weeks and that this may be a protective measure against lipophilic xenobiotics.

The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta.

We investigated whether neuropeptides which control sexual differentiation in mollusks can induce imposex-a condition where female snails grow male accessory sex organs after exposure to tributyltin (TBT). Mud snails, Ilyanassa obsoleta, were dosed with one of four neuropeptides: APGWamide, conopressin, LSSFVRIamide, or FMRFamide for seven or fourteen days. TBT and testosterone (T) were used as positive controls and induced imposex as expected. APGWamide significantly induced imposex, with a threshold dose near 10(-)(16) moles. The other neuropeptides had no effect on imposex induction. We propose that TBT could act as a neurotoxin to induce imposex via abnormal release of APGWamide.

Long-term pyrene exposure of grass shrimp, Palaemonetes pugio, affects molting and reproduction of exposed males and offspring of exposed females.

The objective of this study was to investigate the impact of long-term pyrene exposure on molting and reproduction in the model estuarine invertebrate, the grass shrimp (Palaemonetes pugio). Grass shrimp were exposed to measured concentrations of 5.1, 15.0, and 63. 4 ppb (microg/L) pyrene for 6 weeks, during which time we determined molting and survivorship. At the end of the exposure, we immediately sacrificed some of the shrimp for biomarker (CYP1A and vitellin) analyses. The remaining shrimp were used to analyze fecundity and embryo survivorship during an additional 6 weeks after termination of pyrene exposure. Male shrimp at the highest pyrene dose (63 ppb) experienced a significant delay in molting and in time until reproduction, and showed elevated ethoxycoumarin o-deethylase (ECOD) activity immediately after the 6-week exposure period. In contrast, 63 ppb pyrene did not affect these parameters in female shrimp. Females produced the same number of eggs per body weight, with high egg viability (98-100%) at all exposure levels, but with decreased survival for the offspring of the 63-ppb pyrene-exposed females. In addition, vitellin levels were elevated only in females at 63 ppb pyrene after the 6-week exposure. We hypothesize that the elevated vitellin binds pyrene and keeps it biologically unavailable to adult females, resulting in maternal transfer of pyrene to the embryos. This would account for the lack of effect of pyrene exposure on ECOD activity, molting, and reproduction in the adult females, and for reduced survival of their offspring.

Benthic community structure and biomarker induction in grass shrimp in an estuarine system

The impact of polycyclic aromatic hydrocarbons (PAHs) and lead contamination on benthic community structure and grass shrimp (Palaeomonetes sp.) biochemical markers were investigated in a bayou that has been heavily contaminated by PAHs and heavy metals. The benthic community had decreased species richness as well as decreased numbers of individuals along a contamination gradient. Grass shrimp collected in the field showed a contaminant-gradient increase in heat shock protein 63 and cytochrome P450 1A (as measured by ECOD metabolism). Grass shrimp had elevated ECOD metabolism when exposed in the laboratory to sediments from the most contaminated site. However, individual variation was too great for statistically significant changes. In addition, heat shock protein levels were not significantly elevated in laboratory exposed shrimp. Benthic community structure and wild-caught grass shrimp are clearly impacted in this bayou.http://link.springer-ny. com/link/service/journals/00244/bibs/37n4p512.html

Interaction of PAHs and PCBs with ecdysone-dependent gene expression and cell proliferation.

This study was done to determine whether PAHs and PCBs can interact with the arthropod steroid hormone system. Ecdysteroid molting hormones control growth, molting, and reproduction in arthropods. A spike in 20-OH ecdysone (20 HE) triggers the molt cycle in crustaceans, and earlier studies have shown that PAHs can affect this molt cycle in several crab species. However, the mechanism of this molt cycle interaction is unknown. Both PAHs and PCBs interact with other nuclear receptors; however, nothing is known about their ability to interact with the invertebrate ecdysone receptor (EcR). Four PAHs, benzo[a]pyrene, benzo[b]fluoranthene, pyrene, and chrysene, and the commercial PCB mixture, Aroclor 1254, were used to determine the ability of these classes of compounds to induce ecdysone-dependent reporter gene expression and to modify the proliferation and differentiation response of the ecdysteroid-responsive Cl.8+ cell line. The four PAHs were each able to enhance the ecdysteroid response in both the reporter gene and the cell proliferation assays only when given in conjunction with ecdysteroids. Aroclor 1254 had no effect in either system, either alone or in conjunction with ecdysteroids. These studies show that although the PAHs alone do not activate ecdysteroid-dependent gene expression or cell differentiation, they are able to enhance the effect of ecdysteroids, presumably through a non-receptor-mediated process. This mechanism may explain the effects on molting which have been reported after low-level crude oil exposures in crustaceans.

Environmental signaling: a biological context for endocrine disruption.

Endogenous and exogenous chemical signals have evolved as a means for organisms to respond to physical or biological stimuli in the environment. Sensitivity to these signals can make organisms vulnerable to inadvertent signals from xenobiotics. In this review we discuss how various chemicals can interact with steroid-like signaling pathways, especially estrogen. Numerous compounds have estrogenic activity, including steroids, phytoestrogens, and synthetic chemicals. We compare bioavailability, metabolism, interaction with receptors, and interaction with cell-signaling pathways among these three structurally diverse groups in order to understand how these chemicals influence physiological responses. Based on their mechanisms of action, chemical steroid mimics could plausibly be associated with recent adverse health trends in humans and animals.

Alteration of [14C]-testosterone metabolism after chronic exposure of Daphnia magna to tributyltin.

Tributyltin (TBT) is a marine biocide that has been shown to alter the activity of cytochrome P450 monooxygenases and elicit toxicity indicative of androgenization in some species. The present study was conducted to determine whether TBT altered P450-, reductase-, and transferase-mediated testosterone metabolic processes in Daphnia magna at sublethal exposure concentrations. Two generations of daphnids were continuously exposed for 21 days to nominal TBT concentrations ranging from 0.31 to 2.5 microg/L TBT. The highest TBT concentration (2.5 microg/L) was lethal to 60% of the exposed organisms. Lower TBT concentrations elicited no adverse effects on molting or reproduction of the daphnids. No differences were observed in the response of the first- and second-generation daphnids to the toxicity of TBT. The ability of daphnids to metabolize [14C]-testosterone in vivo was assessed following exposure of each generation to TBT. Production of hydroxylated, reduced/dehydrogenated, and glucose-conjugated metabolites of testosterone were all elevated following exposure of both generations to 1.25 microg/L TBT. These findings indicate that, under these conditions, TBT elicits no discernible effects on molting and reproduction of daphnids at sublethal concentrations, and testosterone metabolism is enhanced at concentrations approaching those that are lethal to organisms. Alterations of steroid metabolism by xenobiotics can be used as a more sensitive indicator of sublethal exposure in daphnids than reproductive endpoints.