Neuroactive steroids modulate crustacean locomotor activity.

We examined the effects of neuroactive steroids known to modulate gamma-aminobutyric acid(A) (GABA)(A) receptor activity, on locomotor activity in a submerged circular open-field apparatus. Juvenile male lobsters, Homarus americanus, were treated with a single administration of an agonist, 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-TH PROG), an antagonist, pregnenolone sulfate (PREGS), or vehicle alone. 3alpha,5alpha-TH PROG treatment (125 and 250 microg) significantly reduced while PREGS significantly elevated locomotor activity in a dose-dependent manner similar to diazepam. PREGS increased locomotor activity at 30 and 60 microg, while diminishing such activity and altering locomotor patterns at 120 microg. These results suggest that neuroactive steroids may affect crustacean GABA receptors in a fashion similar to the GABA(A) type found in the vertebrates, and that they may be involved in the regulation of locomotor behavior.

Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure.

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. One of the primary functions of stress proteins (HSPs) is to aid in the recovery of damaged proteins by chaperoning their refolding. These and other biomarkers of xenobiotic exposure and resulting effects have not been studied in crustacean larvae. This information is of potential importance for environmental management and risk assessment. In this work, we have given Homarus americanus larvae single 24 h exposures to the cyclodiene pesticide heptachlor, a known environmental endocrine disruptor (EDC) on different days of the 1st larval instar. We followed these larvae during the first larval stage for effects on timing of ecdysis to 2nd stage, ecdysteroid molting hormone titers, and alterations in the levels of cytochrome P450 CYP45 and HSP70 proteins. Delays in ecdysis were correlated with alterations in ecdysteroid levels. This result provides clues that this pesticide may function as an environmental endocrine disruptor in crustaceans. CYP45 and HSP70 levels were significantly elevated for several days following heptachlor exposure. The elevation in HSP70 was prolonged depending on the day of pesticide exposure and this was directly related to the increase in mortality. These results demonstrate the utility of these measurements as potential biomarkers in crustacean larval developmental toxicology and EDC effects research.

Identification of a new cytochrome P450 family, CYP45, from the lobster, Homarus americanus, and expression following hormone and xenobiotic exposures.

A new cytochrome P450, the first member of the CYP45 family, was identified from the hepatopancreas of the American lobster, Homarus americanus. The lobster CYP45 shares significant sequence homologies to the vertebrate CYP3 and the invertebrate CYP6, CYP9, CYP28, and CYP30 families, perhaps indicating a common ancestor of these P450s. Of seven tissues examined, CYP45 was expressed only in the hepatopancreas, the crustacean equivalent of the vertebrate liver, pancreas, and intestine. Over the course of the lobster molt cycle, CYP45 expression mirrored the hemolymph titer of ecdysteroids, suggesting its potential involvement in molting hormone dynamics. This idea was strengthened further by ecdysteroid treatment of intermolt-stage lobsters during the lowest hemolymph titers and CYP45 expression levels. Significant elevations in hepatopancreas CYP45 mRNA levels were elicited by such injections over a 2- to 4-day interval. Similar experiments were performed by intubating juvenile lobsters with various xenobiotics. Induction of CYP45 expression occurred following phenobarbital and heptachlor administration, but not by beta-naphthoflavone. Hormonal and xenobiotic modulation of lobster CYP45 expression provides a potential pathway for endocrine disruption in lobsters.

Role of the midgut gland in metabolism and excretion of ecdysteroids by lobsters, Homarus americanus.

The chromatographic profile of ecdysteroids (Ecds) from the midgut gland (MG) of juvenile female lobsters, Homarus americanus, was examined using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) over four stages of the molt cycle. Upon initial examination, highly polar Ecd conjugates appeared to be the principal metabolites found in all molt stages. HPLC fractions containing apolar Ecds initially exhibited low RIA activity. Upon hydrolysis with a Helix pomatia enzyme preparation and reanalysis, significant amounts of other Ecds were released. Amounts of apolar Ecd conjugates were estimated, at their highest levels, to be at least 50% of the total Ecds in MGs of molt stage D3 lobsters. Only the MG formed significant amounts of apolar Ecds upon in vitro culture with [3H]ecdysone ([3H]E). Epidermis and antennal gland significantly increased their rates of [3H]E metabolism in vitro between molt stages C4 and D1. This result further supports the idea that regulation of ecdysteroid metabolism, at least in selected tissues, may be important in the molt cycle regulation of hormone titers. Using gel filtration column chromatography and sucrose density gradient centrifugation analyses, evidence was found for association of apolar Ecds with a protein(s) from MG cytosol. The protein was estimated to have a molecular weight of 180,000-200,000 and specifically bound apolar Ecds.

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. II. excretion of metabolites.

Ecdysteroid (Ecd) excretion patterns were followed during the molt cycle of adult male and female lobsters. Homarus americanus. Urine was the major route of Ecd elimination, amounting to greater than or equal to 96% of the excreted radioimmunoassay activity for all molt stages. The other identified route of Ecd elimination from the hemolymph was the feces, which accounted for the remaining 4% of the total Ecd excretion. High polarity metabolites (HP), including 20,26-dihydroxyecdysone (2026E) and 20-hydroxyecdysonoic acid (20EA), were the major types of Ecds found in the urine. Other urinary Ecd components included 20-hydroxyecdysone (20E), ecdysone (E), and ponasterone A (P). The major portion of urinary HP was composed of conjugates of 2026E, 20E, E, P, and other unidentified metabolites. The fecal Ecds were predominately HP and apolar metabolites. Apolar fecal Ecds were hydrolyzable to release 20EA, 2026E, 20E, E, P, and other metabolites. By means of intubation, [3H]E was placed directly into the cardiac stomach of lobsters. The gut pathway formed an apolar conjugate of [3H]E which was found exclusively in the feces. Lobsters are therefore capable of excreting ingested Ecds without absorption.

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. I. Hemolymph titers and metabolites.

Hemolymph ecdysteroid (Ecd) titers were measured using radioimmunoassay (RIA) during the molt cycle of the American lobster, Homarus americanus. Individual animals showed small, transitory rises of Ecds which increased in magnitude with the onset of premolt and culminated in a large premolt peak at morphological stages D2(2)-D3(1). Male lobsters had significant postmolt peaks and late premolt titers that remained high until ecdysis. In females, postmolt peaks were absent and late premolt titers reached basal levels before ecdysis. At least seven different Ecd metabolites were identified by high-performance liquid chromatography-RIA analyses. High polarity products (HP) were the most abundant metabolites in virtually every molt stage. Titers of HP were significantly higher in males during late postmolt-early intermolt and in late premolt. Levels of 20-hydroxyecdysone (20E) were equivalent in both sexes and correlated with the morphological changes associated with premolt. Evidence was also obtained for the presence of ecdysone, ponasterone A, and other as yet unidentified metabolites. The pattern of Ecd metabolites in the hemolymph supports other data indicative of 20E as the major molting hormone. Metabolism of 20E is primarily toward more polar compounds, including conjugates.

Imipenem therapy of Pseudomonas aeruginosa bacteraemia in neutropenic rats.

Rats were made neutropenic by intraperitoneal (ip) injection of cyclophosphamide. Those neutropenic (mean white blood cell count of 470/mm3) rats were challenged intraperitoneally with Pseudomonas aeruginosa to assess the efficacy of single agent therapy with either imipenem, latamoxef (moxalactam) or amikacin, or combination therapy with imipenem-amikacin or latamoxef (moxalactam)--amikacin. Pharmacokinetic studies were performed in rats to assure that therapy was equivalent during therapeutic trials. Three levels of bacterial challenge (4 LD50, 13 LD50 and 250 LD50) were examined. At all challenge levels, single agent therapy with latamoxef (moxalactam) failed to significantly protect rats from fatal bacteraemia. Single-agent therapy with amikacin did significantly protect rats from fatal bacteraemia at the lower challenge levels, but not at the 250 LD50 challenge. Single agent therapy with imipenem significantly protected rats at all challenge. Single agent therapy with imipenem significantly protected rats at all challenge levels. In-vitro studies established a synergistic effect when combination antibiotics were used. This correlated with in-vivo findings that combination therapy resulted in improved rat survival and recovery of fewer Ps. aeruginosa isolates. The latamoxef (moxalactam)-amikacin combination was more effective than either agent alone, but was not more effective than imipenem alone. The imipenem-amikacin combination was the most effective therapeutic regimen tested. These results suggest that imipenem alone, and particularly when combined with an aminoglycoside, is effective in treating serious Ps. aeruginosa infections in neutropenic rats. Clinical studies in infected immunocompromised patients may be warranted.