Invisible endocrine disruption and its mechanisms: A current review.

The research on impacts of environmental chemicals on crustacean molting dates back to the 1970s when ground-breaking studies investigated the disruption of molting in Crustacea by organochlorines. With the emergence of a new scientific inquiry, termed environmental endocrine disruption, in the early 1990s, increasing attention has been attracted to the possibility that environmental chemicals capable of wreaking havoc on sex steroid-regulated processes in vertebrates can also adversely affect ecdysteroid-mediated processes, e.g. molting, in crustaceans. Given the fact that many molting-disrupting chemicals accumulate in crustacean tissues and that the effect on molting is not readily visible in the field, the disruption of molting by environmental chemicals has been dubbed the invisible endocrine disruption. In recent years, much advancement has been made in both the documentation of the phenomenon of molting disruption and the search for mechanisms, by which molting disruption occurs. This review provides an overview of the current status of the field of invisible endocrine disruption, and perspectives on future directions are also presented.

Exogenous 20-hydroxyecdysone induces epidermal carbonic anhydrase but inhibits exoskeletal calcification in the post-ecdysial blue crab, Callinectes sapidus.

The crustacean exoskeleton is composed primarily of chitin, proteins and various inorganic compounds. It is the inorganic compounds, such as calcium and magnesium, that underlie the exoskeletal mineralization process following ecdysis. Little is known about the hormonal mechanism for this process in crustaceans. Carbonic anhydrase (CA) in the epidermis has been suggested to aid in deposition of calcium and magnesium carbonates to the exoskeleton in crustaceans by generating bicarbonate ions, resulting in mineralization. Given a similar pattern of fluctuation in prevalence between epidermal CA and ecdysteroids during the crab molting cycle, it has been proposed in a previous study that post-ecdysial epidermal CA and subsequent metal deposition to the exoskeleton are controlled by the ecdysteroid molting hormones in the blue crab, Callinectes sapidus. This study sought to acquire evidence to support such a proposition. Early postmolt and early intermolt blue crabs were used to quantify epidermal CA mRNA expression when exposed in vitro to three physiologically relevant concentrations of 10 nM, 100 nM and 1 µM 20-hydroxyecdysone (20-HE), using the epidermis-with-exoskeleton (EWE) tissue assay method. It was found that 100 nM 20-HE significantly induced the mRNA of CasCAg, a CA isoenzyme, in epidermal tissues of early intermolt crabs and that injection of 20-HE at a dose of 15 ng/g significantly elevated epidermal CA activity in vivo in early intermolt crabs. These two lines of evidence clearly show that post-ecdysial epidermal CA is influenced by the molting hormone. Interestingly, exoskeletal calcium content was significantly lower in the 20-HE treated crabs than in the control, whereas magnesium content was unchanged. The inhibition of calcification in the post-ecdysial exoskeleton by the exogenous molting hormone may implicate that sclerotization and mineralization of the new shell must be coordinated and that enhanced deposition of carbonate salts as a result of increased epidermal CA activity following the administration of exogenous molting hormone would be inhibited to avoid the formation of a structurally disrupted exoskeleton.

Effects of cadmium alone and in combination with low molecular weight chitosan on metallothionein, glutathione-S-transferase, acid phosphatase, and ATPase of freshwater crab Sinopotamon yangtsekiense.

Cadmium (Cd) is an environmental contaminant showing a variety of deleterious effects, including the potential threat for the ecological environment and human health via food chains. Low molecular weight chitosan (LMWC) has been demonstrated to be an effective antioxidant. Metallothionein (MT) mRNA levels and activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), acid phosphatase (ACP), Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as malondialdehyde (MDA) contents in the gills of the freshwater crab Sinopotamon yangtsekiense were analyzed in vivo in order to determine the injury of Cd exposure on the gill tissues as well as the protective effect of LMWC against this injury. The results showed that there was an apparent accumulation of Cd in the gills, which was lessened by the presence of LMWC. Moreover, Cd(2+) significantly increased the gill MT mRNA levels, ACP activity and MDA content while decreasing the activities of SOD, GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in the crabs relative to the control. Cotreatment with LMWC reduced the levels of MT mRNA and ACP but raised the activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in gill tissues compared with the crabs exposed to Cd(2+) alone. These results suggest that LMWC may exert its protective effect through chelating Cd(2+) to form LMWC-Cd(2+) complex, elevating the antioxidative activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as alleviating the stress pressure on MT and ACP, consequently protecting the cell from the adverse effects of Cd.

Zinc is incorporated into the exoskeleton during post-ecdysial mineralization and inhibits exoskeletal calcification in the blue crab, Callinectes sapidus.

Zinc is an essential but toxic metal with both natural and anthropogenic sources. Zinc has been reported to be present in crustacean exoskeleton, but it has remained unknown as to when zinc is incorporated into the shell during the molting cycle and the effects zinc has on exoskeleton properties. This study was conducted to identify a period during the molting cycle, in which zinc is incorporated into the exoskeleton of the blue crab, Callinectes sapidus, and to identify deleterious effects of zinc incorporation on the exoskeleton. It was hypothesized that zinc would be incorporated into the shell during the mineralization phase using calcium transporters, which would inhibit exoskeletal calcification. Post-ecdysial blue crabs were given two injections of zinc in the form of zinc chloride dissolved in Pantin's crustacean saline at the arthrodial membrane at three treatment levels: 0.0, 1.0 and 5.0 µg Zn/g wet weight. Exoskeletal and hemolymph samples were then analyzed for zinc, calcium, and magnesium content. Gill, muscle, and hepatopancreas samples were analyzed for zinc only. Epidermis samples were analyzed for carbonic anhydrase activity. The results showed that the injection dose of 1.0 µg Zn/g wet weight resulted in significant accumulation of zinc in the exoskeleton. There was no significant accumulation of exoskeletal zinc following 5.0 µg Zn/g wet weight injections. A significant reduction in exoskeletal calcium content in crabs treated with 1.0 or 5.0 µg Zn/g wet weight was also observed. The hypothetical model explaining zinc's incorporation into the exoskeleton and inhibition of exoskeletal calcification is proposed. Additionally, for the soft tissues examined, significant zinc accumulation was only observed in the hepatopancreas following zinc treatment. Our data points to the existence of crustacean zinc transporter. This study is the first to present evidence that zinc is deposited to the exoskeleton during post-ecdysial mineralization and inhibits exoskeletal calcification in a crustacean.

Polybrominated diphenyl ethers disrupt molting in neonatal Daphnia magna.

Polybrominated diphenyl ethers (PBDEs) are flame-retardants which can bioaccumulate and biomagnify and are found worldwide despite their banned usage in some countries. In recent years, the possibility that PBDEs may disrupt endocrine functions in vertebrates has been well investigated, but little attention has been paid to the endocrine disrupting potential in aquatic invertebrates. The current study aimed to investigate whether PBDEs affect molting in neonatal Daphnia magna. Prior to molting studies, 48 h LC50 values were tested for several environmentally prevalent PBDEs: PBDEs-28, -47, -99, -100 and -209. The 48 h LC50s determined were 110.7, 7.9, 2.6, and 11.1 µg/L for PBDEs-28, -47, -99, and -100, respectively, but the highest concentration of PBDEs-209 tested (2.5 mg/L) did not affect survival at 48 h. Sublethal concentrations of these were used to investigate their potential effects on molting, assessed by the time taken to reach 4 molts. Molting studies found that PBDE-28 at 12 µg/L significantly increased the time it took to complete 4 molts. PBDE-47 at 20 µg/L inhibited daphnid molting initially but such an inhibitory effect disappeared with the prolongation of exposure due to the death of sensitive individuals. No other PBDEs affected molting at the concentrations tested, while still maintaining relatively high survival rates. In conclusion, this study found that PBDEs-28 and -47 can delay molting at µg/L concentrations, which raises concern for disrupted molting in crustaceans exposed to PBDEs.

Lead Inhibits Postecdysial Exoskeletal Calcification in the Blue Crab (Callinectes sapidus).

Postecdysial mineralization in crustaceans involves the deposition of carbonate salts, such as calcium carbonate, to the organic matrix. Because of the resemblance between Pb2+ and Ca2+ , the present study was carried out to investigate whether Pb is incorporated into the new shell during postecdysial mineralization using the blue crab (Callinectes sapidus) as the model crustacean. It was hypothesized that injected Pb would be deposited in the shell via calcium transporters in the epidermis during the mineralization process. Postecdysial blue crabs were injected with two doses of 5 µg Pb/g wet weight each in lead acetate, and then Pb, Ca, and Mg contents were analyzed in the exoskeleton, while only Pb bioaccumulation was quantified for the hepatopancreas, gills, muscles, and hemolymph. The results showed a statistically nonsignificant increase in exoskeletal Pb content in Pb-treated crabs compared to control, suggesting that exoskeletal Pb may not be a sensitive proxy for aquatic Pb pollution. There was a significant decrease in Ca content in Pb-treated crabs, suggesting that Pb hindered the deposition of Ca to crab exoskeleton, thereby obstructing calcification. A trend of a decrease in exoskeletal Mg was also observed in Pb-treated crabs. There was a significant increase in Pb content found in the gills, hepatopancreas, muscle, and hemolymph in Pb-treated crabs. The rank of the Pb level among three soft tissues in a decreasing order is hepatopancreas > gill > muscle. This is the first study to present evidence that Pb disrupts postecdysial exoskeletal calcification in a crustacean. Environ Toxicol Chem 2022;41:474-482. © 2021 SETAC.

How Does Carbon Dioxide-Induced Acidification Affect Postecdysial Exoskeletal Mineralization in the Blue Crab (Callinectes sapidus)?

Carbon dioxide (CO2 ) enrichment in seawater because of increased use of fossil fuels can possibly cause detrimental effects on the physiological processes of marine life, especially shell builders, due to CO2 -induced ocean acidification. We investigated, for the first time, specifically the effect of CO2 enrichment on postecdysial shell mineralization in Crustacea using the blue crab, Callinectes sapidus, as the model crustacean. It was hypothesized that CO2 enrichment of seawater would adversely affect exoskeletal mineralization in the blue crab. We used two groups of postecdysial crabs, with one group exposed to seawater at a pH of 8.20 and the other group treated with CO2 -acidified seawater with a pH of 7.80-7.90. After a period of 7 days, samples of exoskeleton and hemolymph were collected from the survivors. Enrichment was found to significantly increase exoskeletal magnesium content by 104% relative to control, whereas a statistically nonsignificant elevation of 31% in exoskeletal calcium was registered. Because CO2 treatment did not change the content of magnesium and calcium in the hemolymph, we postulate that increased exoskeletal mineralization in postecdysial blue crabs must stem from an increased influx of bicarbonate ions from the medium through the gill, to the hemolymph, and across the epidermis. In addition, the observed significant increase in the mass of exoskeleton following CO2 treatment must be at least partly accounted for by enhanced postmolt carbonate salt deposition to the shell. Environ Toxicol Chem 2022;41:2950-2954. © 2022 SETAC.

Cadmium is deposited to the exoskeleton during post-ecdysial mineralization in the blue crab, Callinectes sapidus.

Despite the fact that cadmium has been reported to be present in crustacean exoskeletons, no study has previously been done to determine when cadmium is deposited to the exoskeleton and what effects cadmium has on the shell-hardening process. This project sought to address these scientific questions using the blue crab, Callinectes sapidus, as the model crustacean. It was hypothesized that cadmium would be incorporated into the exoskeleton during post-ecdysial mineralization through ionic mimicry because of the resemblance between cadmium and calcium ions. To test this, soft shell blue crabs were injected with cadmium chloride, and cadmium content in the exoskeleton was subsequently quantified using ICP-OES. Carbonic anhydrase catalyzes the carbon dioxide hydration reaction, which generates bicarbonate ions essential for calcium carbonate formation in the shell. The effect of cadmium injection on epidermal carbonic anhydrase activity as well as exoskeletal calcification was also investigated. It was found that cadmium injection into post-ecdysial Callinectes sapidus significantly increased cadmium content in the exoskeleton, suggesting that cadmium is deposited to the new exoskeleton during post-ecdysial mineralization. Cadmium administration had no effect on epidermal carbonic anhydrase activity or exoskeletal calcification. Interestingly, magnesium content in the exoskeleton was significantly elevated following cadmium treatment. This is most likely a "pseudo" effect stemming from the cadmium-induced reduction in exoskeleton weight. The fact that cadmium had no effect on exoskeletal calcium and that cadmium decreased the weight of the exoskeleton suggests that cadmium has a detrimental effect on the formation of the organic matrix of the exoskeleton. The presence of cadmium in control crabs and exuviae and the amplification of cadmium content in cadmium-treated crabs clearly show that crab shell is a cadmium repository and can be used as a biomarker for aquatic cadmium pollution.

Effects of hypoxia and sedimentary naphthalene on the activity of N-acetyl-beta-glucosaminidase in the epidermis of the brown shrimp, Penaeus aztecus.

The brown shrimp, Penaeus aztecus, is subject to dual stresses of environmental hypoxia and contamination of polycyclic aromatic hydrocarbons (PAHs) in the northern Gulf of Mexico. The effects of hypoxia and sedimentary naphthalene, administered alone and in combination, on epidermal activity of N-acetyl-beta-glucosaminidase (NAG), a biomarker for molt-interfering effects in P. aztecus, were investigated. It was found that hypoxia and sedimentary naphthalene, when given simultaneously, significantly inhibited epidermal NAG activity, suggesting that these two environmental stressors together can have adverse effects on molting of the brown shrimp. The results of this study also show that sedimentary naphthalene potentiates hypoxia effects on epidermal NAG activity.

A molecular biomarker for disruption of crustacean molting: the N-acetyl-beta-glucosaminidase mRNA in the epidermis of the fiddler crab.

Several environmentally persistent chemicals have been found to be capable of disrupting crustacean molting. Considering the importance of molting in the life of crustaceans, there is a need to develop a molecular biomarker that can reflect the disrupting effects of contaminants on ecdysteroid signaling in crustaceans. N-acetyl-beta-glucosaminidase (NAG) is a chitinolytic enzyme found in crustacean epidermis. The results of the present investigation show that the transcription of NAG gene in the epidermis of the fiddler crab, Uca pugilator, is inducible by the molting hormone 20-hydroxyecdysone, which validates the use of NAG mRNA as a biomarker for molt-disrupting effects of xenobiotics.

Hypoxia does not promote naphthalene bioaccumulation in the brown shrimp, Penaeus aztecus.

Since increased ventilation is known to be a common strategy used by aquatic animals to cope with hypoxia, we tested in present study the hypothesis that hypoxia can promote the bioaccumulation of naphthalene, a representative polycyclic aromatic hydrocarbon (PAH), in Penaeus aztecus, a penaeid shrimp subject to hypoxia and PAH contamination in the northern Gulf of Mexico. For each of the two naphthalene concentrations, five groups of shrimps were, respectively, subjected to five different conditions, namely, clean seawater under normoxia, seawater containing acetone under normoxia and hypoxia, and seawater containing 10 or 250 microg/L naphthalene under normoxia and hypoxia. Our results suggest that hypoxia does not significantly alter naphthalene bioaccumulation in either the gills or the hepatopancreas of P. aztecus. The absence of a promoting effect of hypoxia on naphthalene bioaccumulation is attributed to the increased disposition of naphthalene when the shrimps are subjected to hypoxia.

Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro.

Polybrominated diphenyl ethers (PBDEs) are environmentally pervasive flame retardants that have been linked with endocrine disruption in a variety of organisms. BDE-47, one of the most prevalent congeners found in aquatic environments, has recently been shown to inhibit crustacean molting, but little is known about the specific mechanism through which molt-inhibition occurs. This study examined whether the inhibitory effect on molting arises from the disruption of hormone signaling in the epidermis using the blue crab, Callinectes sapidus, as the model crustacean. First, we partially sequenced cDNA of N-acetyl-ß-glucosaminidase (NAG) from the epidermis, a terminal enzyme in the molting hormone-signaling cascades that is commonly used as the biomarker for ecdysteroid signaling. This partial cDNA sequence was then used to create primers for quantification of NAG gene expression. Then, a new tissue culture technique was developed and dubbed the epidermis-with-exoskeleton (EWE) method, wherein epidermal tissue, along with the overlying exoskeleton, is immersed in a medium of physiologically relevant osmolarity. Using this EWE tissue culture method, we assessed the inducibility of NAG mRNA by 20-hydroxyecdysone (20-HE) in vitro. Exposures to 1µM 20-HE were found to induce NAG mRNA at a significantly higher level than the control. Using NAG expression as a biomarker for ecdysteroid signaling, the effects of BDE-47 were measured. BDE-47 alone at 100nM and a combination of 1µM BDE-47 and 1µM 20-HE were found to significantly increase NAG mRNA. A trend of increasing NAG gene expression in the binary BDE-47 exposure as compared to 1µM BDE-47 and 1µM 20-HE alone is suggestive of a synergistic effect of these two chemicals on ecdysteroid signaling in the cultured epidermis. Discussion on the mechanism for inhibition of crustacean molting by BDE-47 is presented.

Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus.

During the crustacean molting cycle, the exoskeleton is first mineralized in postmolt and intermolt and then presumably demineralized in premolt in order for epidermal retraction to occur. The mineralization process calls for divalent metal ions, such as Ca(2+) and Mg(2+) , and bicarbonate ions whereas protons are necessary for dissolution of carbonate salts. Carbonic anhydrase (CA) has been suggested to be involved in exoskeletal mineralization by providing bicarbonate ions through catalyzing the reaction of carbon dioxide hydration. However, results of earlier studies on the role of epidermal CA in metal incorporation in crustacean exoskeleton are not consistent. This study was aimed to provide further evidence to support the notion that epidermal CA is involved in exoskeletal mineralization using the blue crab, Callinectes sapidus (Rathbun 1896), as the model crustacean. Significant increases first in calcium and magnesium then in manganese post-ecdysis indicate significant metal deposition during postmolt and intermolt. Significant positive correlation between calcium or magnesium content and epidermal CA activity in postmolt and intermolt constitutes evidence that CA is involved in the mineralization of the crustacean exoskeleton. Additionally, we proposed a hypothetical model to describe the role of epidermal CA in both mineralization and demineralization of the exoskeleton based on the results of epidermal CA activity and exoskeletal metal content during the molting cycle. Furthermore, we found that the pattern of epidermal CA activity during the molting cycle of C. sapidus is similar to that of ecdysteroids reported for the same species, suggesting that epidermal CA activity may be under control of the molting hormones.

Cadmium inhibits molting of the freshwater crab Sinopotamon henanense by reducing the hemolymph ecdysteroid content and the activities of chitinase and N-acetyl-ß-glucosaminidase in the epidermis.

Molting is an essential process during the growth of crustaceans, which is coordinated by ecdysteroids secreted by the Y-organ, molting inhibiting hormone secreted by the X-organ sinus-gland complex, as well as chitinase and N-acetyl-ß-glucosaminidase synthesized by the epidermis. Cadmium is one of the toxic metals in the aquatic environment. However, the endocrine effects of cadmium on the molting of freshwater crabs and the underlying mechanisms are unknown. To investigate these, freshwater crabs (Sinopotamon henanense) were acutely exposed to 0, 7.25, 14.5 and 29 mg/l Cd for 3, 4, 5 days or in some experiments for 4 days after eyestalk-ablation. The concentration of hemolymph ecdysone and the activities of the molting enzymes chitinase and NAG were measured. Histological changes in the epidermal tissues were documented. Our results showed that eyestalk ablation increased the ecdysteroid content as well as the activities of chitinase and NAG, which were inhibited by cadmium in a concentration-dependent manner; histological examinations demonstrated that eyestalk ablation produced storage particles in the epidermal tissues, which was also reduced by cadmium in a concentration-dependent manner. Our data suggest that cadmium disrupts endocrine function through inhibiting the secretion of ecdysteroids by the Y-organ and altering with the regulation of chitinase and NAG activity in the epidermis. This work provides new insights into the mechanisms underlying the molting inhibition effect of cadmium on the crabs.

Long-term exposure to beta-hexachlorocyclohexane (beta-HCH) promotes transformation and invasiveness of MCF-7 human breast cancer cells.

Due to its lipophilicity and persistence, an organochlorine compound, beta-hexachlorocyclohexane (beta-HCH), is known to frequently accumulate in human adipose and breast tissues. An epidemiological study has indicated that exposure to beta-HCH could be one of the significant environmental risk factors for the development of human breast cancers. Additionally, beta-HCH has recently been identified as an environmental estrogen capable of activating estrogen receptor (ER) through a ligand-independent pathway. In the present investigation, we examined the impact of long-term in vitro exposure to beta-HCH on cell transformation and the metastatic potentials of MCF-7 cells. We found that continuous exposure of MCF-7 cells to beta-HCH at 100 nM and 1 microM or to 17beta-estradiol (E(2)) at 1 nM for up to 13 months (33 passages) not only enhanced their transformation tendencies but also promoted their invasiveness. Western blot analysis revealed that beta-HCH induced transformation-related biochemical changes in MCF-7 cells, such as a decline in the levels of ERalpha and p44/42 MAP kinase and a significant increase in expression of c-ErbB2 and MMP-9 levels. In contrast, long-term E(2) treatment resulted in the downregulation of ERalpha and p44/42 MAP kinase and upregulation of MMP-9 only, but no changes in c-ErbB2. Together, these results indicate that these biochemical changes induced by beta-HCH are consistent with the events taking place in these cells to promote the phenotypical expression of transformed cells. Our results provide the in vitro mechanistic basis supporting the hypothesis that beta-HCH is one of the epigenetic risk factors assisting the progression of breast cancer cells to an advanced state of malignancy.

Foci formation of MCF7 cells as an in vitro screening method for estrogenic chemicals.

Previously we reported a novel phenomenon that some organochlorine compounds mainly act through activation of c-Neu tyrosine kinase without being strong agonists for the estrogen receptor. In this study we tested the possibility of developing an assay system to identify estrogenic compounds acting through this c-Neu-mediated mechanism. We describe herein an assay that utilizes foci formation of MCF7 cells as an endpoint, antibody 9G6 to neutralize the c-Neu-mediated pathway and 4-hydroxytamoxifen to block the ER. Aroclors 1242 and 1248, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2'-dichlorobiphenyl (PCB), cis- and trans- permethrins, and chlorothalonil were found to render estrogenic effects through this c-Neu-mediated mechanism, while α and ß- endosulfans appeared to act through a pathway independent of the c-Neu-mediated one. Pentachloronitrobenzene was found to be capable of antagonizing the 17ß-estradiol effect, which has never been reported previously.

Estrogenic action of ß-HCH through activation of c-Neu in MCF-7 breast carcinoma cells.

ß-HCH is known to be a poor agonist for the estrogen receptor (ER), and yet it has been shown to act like an estrogen in stimulating foci formation in MCF-7 cells. We investigated the reason for such an action of ß-HCH, using a rat prolactin-luciferase reporter system transfected to MCF-7 cells. We found that the presence of c-Neu (erbB2), ER and ERE is needed for ß-HCH to act estrogenic at the transcription activation level in this cell line. We compared the action of ß-HCH to that of EGF which is known to act estrogenic without being an agonist for ER in this cell and found that their action patterns are quite similar, the only difference being that the former action is blocked by an antibody against c-Neu and the latter by both c-Neu and EGF receptor antibody. We concluded that ß-HCH's estrogenic action in this cell model is mediated through "ligand-independent activation of ER pathway".

Does crustacean ethoxyresorufin O-deethylase activity vary during the molting cycle?

The authors examined fluctuation in microsomal ethoxyresorufin O-deethylase (EROD) activity in the hepatopancreas during the molting cycle of the fiddler crab, Uca pugilator. Results showed that microsomal EROD activity fluctuates significantly during the molting cycle, with the lowest enzymatic activity occurring in the late premolt stage. These results clearly show that molting physiology influences crustacean EROD activity, suggesting that when using crustacean EROD assays in evaluating pollution, only individuals from the same molt stage should be used. The authors propose that the high level of EROD activity in postmolt and intermolt stages is an additional mechanism crustaceans use to prevent any untimely rise in ecdysteroid levels.

Impacts of molt-inhibiting organochlorine compounds on epidermal ecdysteroid signaling in the fiddler crab, Uca pugilator, in vitro.

Because of their chemical stability and lipophilicity, many organochlorine compounds (OCs) can readily accumulate in fatty tissues of crustaceans. Several OCs have been reported to inhibit crustacean molting. To determine whether the disruption of crustacean molting by these OCs involves interference with ecdysteroid signaling in the epidermis, the impacts of five molt-inhibiting OCs on the level of N-acetyl-beta-glucosaminidase (NAG, EC 3.2.1.30) mRNA in cultured epidermal tissues from the fiddler crab, Uca pugilator, were investigated using quantitative real-time PCR. The NAG mRNA was found to be inducible by 20-hydroxyecdysone (20-HE) in cultured epidermal tissues. The inducibility of NAG mRNA in cultured epidermal tissues by 20-HE is not only further direct evidence that epidermal expression of NAG gene in U. pugilator is controlled by the molting hormone but also validates the use of the NAG mRNA as a biomarker for epidermal ecdysteroid signaling. When Aroclor 1242, 2,4,5-trichlorobiphenyl (PCB29), endosulfan or kepone was administered alone, the expression of NAG gene in cultured epidermal tissues was upregulated, while heptachlor had no effects. Under binary exposure to both 20-HE and an OC, a condition similar to the natural hormonal milieu of epidermal tissues of animals impacted by OCs, both Aroclor 1242 and endosulfan were found to be capable of antagonizing ecdysteroid signaling in cultured epidermal tissues. This antagonizing effect on epidermal ecdysteroid signaling can at least partly explain the inhibitory effects of these two agents on crustacean molting. PCB29, when given together with 20-HE, produced an additive effect on epidermal ecdysteroid signaling but such an additive effect was not observed when kepone was combined with 20-HE.

Activity of glutathione S-transferase in the hepatopancreas is not influenced by the molting cycle in the fiddler crab, Uca pugilator.

Glutathione S-transferase (GST) in the hepatopancreas of crustaceans has been suggested as a biomarker for organic pollution. However, much of crustacean physiology is known to exhibit a cyclic characteristic because of the periodic shedding of the confining exoskeleton. The goal of this study was to determine whether hepatopancreatic GST activity varies during the molting cycle using the fiddler crab, Uca pugilator, as the model. Neither the molting cycle nor 20-hydroxyecdysone injection had a significant effect on hepatopancreatic GST activity, suggesting GST activity is not under control of the molting hormone in Uca pugilator.