Reproductive alterations in adult grass shrimp, Palaemonetes pugio, following sublethal, chronic endosulfan exposure.

Grass shrimp (Palaemonetes pugio) populations exposed to anthropogenic contaminant sources in South Carolina (SC) have reduced densities when compared with populations at SC-reference sites. This laboratory study examined the effects of a commonly used agricultural insecticide, endosulfan, on grass shrimp reproduction. Reproductively active grass shrimp were chronically exposed to sublethal concentrations of endosulfan (200 or 400 ng/l) for 43 days. The cumulative number of females that became gravid and the rate at which they became gravid were measured. Endosulfan exposure reduced the cumulative number of gravid females by 31% in the 200 ng/l exposure and 39% in the 400 ng/l exposure. The first appearance of gravid females in the population was significantly delayed in treated populations compared with the control treatment in a dose dependent manner. Clutch size in these gravid females was not significantly different among the treatments. Additionally, there was no difference in the onset of reproduction in the treated populations. These results implicate a population reduction due to a decrease in the overall number of females becoming gravid in a population over time, not a reduction in clutch size per individual. While the mechanisms of action have yet to be defined, these results indicate that sublethal endosulfan concentrations may have a negative effect on grass shrimp reproductive biology.

Toxicity and bioconcentration potential of the agricultural pesticide endosulfan in phytoplankton and zooplankton.

Agricultural pesticide runoff in southeastern coastal regions of the United States is a critical issue. Bioconcentration of pesticides by phytoplankton and zooplankton at the base of the aquatic food web may increase the persistence of pesticides in aquatic ecosystems and cause effects at higher trophic levels. This study examined the toxicity of a widely used agricultural pesticide, endosulfan, to Pseudokirchneriella subcapitatum (freshwater green alga) and Daphnia magna (freshwater cladoceran). We then investigated the potential of both plankton species to sequester endosulfan from their surrounding media. We also assessed the degree to which endosulfan is accumulated by D. magna via food (endosulfan-contaminated P. subcapitatum). A 96-h growth rate EC50 of 427.80 microg/L endosulfan was determined for P. subcapitatum, whereas a 24-h immobilization EC50 of 366.33 microg/L endosulfan was determined for D. magna. The 5-h EC50s for filtration and ingestion in D. magna were 165.57 microg/L and 166.44 microg/L, respectively. An average bioconcentration factor (BCF) of 2,682 was determined for P. subcapitatum exposed to 100 microg/L endosulfan for 16 h. An average BCF of 3,278 was determined for D. magna in a 100 microg/L endosulfan water-only exposure. There was negligible uptake of endosulfan by D. magna feeding on contaminated algae in clean water (BCF approximately 0). Different proportions of parent isomers (endosulfan I and II) and the primary degradation product (endosulfan sulfate) were detected among treatments. Endosulfan was rapidly accumulated and concentrated from water by P. subcapitatum and D. magna neonates. Endosulfan contained in phytoplankton, however, was not bioaccumulated by zooplankton. These findings may prove useful in assessing ecosystem risk, because uptake from the water column appears to be the dominant route for bioconcentration of endosulfan by zooplankton.

Determination of acute mortality in adults and sublethal embryo responses of Palaemonetes pugio to endosulfan and methoprene exposure.

Adult grass shrimp (Palaemonetes pugio) were exposed to endosulfan or methoprene for 96 h and LC(50) values were calculated. Male and female P. pugio cohorts were also exposed to endosulfan for 96 h in an attempt to determine potential differences in sensitivity between the sexes. Results from the methoprene exposure indicated that this pesticide was not acutely toxic to adult grass shrimp at 1 mg l(-1). Due to the lack of sensitivity, sex specific tests with methoprene were not performed. The calculated LC(50) for a population of grass shrimp, including both males and females exposed to endosulfan, was 0.62 microg l(-1). The LC(50) determinations for the sex specific tests were 0.92 microg l(-1) for males and 1.99 microg l(-1) for females. Following these acute exposures, reproductively active grass shrimp were chronically exposed to 200 ng l(-1) endosulfan or 1 mg l(-1) methoprene and were allowed to produce embryos. The resulting embryos were assessed for potential sublethal toxicity. There were no observed differences in the percent successfully hatching or larval mortality 3-days post hatch among the treatments. However, endosulfan treated embryos had a significantly increased hatching time (9.76 days compared to 8.72 days in controls). Methoprene treated embryos also took longer to hatch (9.55 days), but this delay was not significantly different from controls. These findings suggest that endosulfan may preferentially affect male grass shrimp and exposed female grass shrimp may produce embryos with delayed hatching times.

Prostaglandin-independent anovulatory mechanism of indomethacin action: inhibition of tumor necrosis factor alpha-induced sheep ovarian cell apoptosis.

Indomethacin, a nonsteroidal anti-inflammatory agent, is a potent inhibitor of ovulation in vertebrates. The presumptive obligate anovulatory mode of indomethacin action is via suppression of ovarian prostaglandin production. We report that a very high systemic dose of indomethacin (800 mg i.m.) is required to block ovulation in gonadotropin-treated anestrous ewes. A lower dose of indomethacin (200 mg), which negated the preovulatory rise in follicular prostaglandin (PGF(2alpha)) biosynthesis, did not prevent ovulation. Endothelial secretion of tumor necrosis factor (TNF)-alpha within the apical follicular wall (prospective site of rupture) was not altered by indomethacin; notwithstanding, the apoptosis (DNA-fragmentation)-inducing effect of TNF-alpha (a determinant of ovulatory stigma formation) was attenuated by 800 (but not 200) mg indomethacin. A suprapharmacological concentration of indomethacin also was necessary to protect ovarian surface epithelial cells from a (prostaglandin-independent) cytotoxic effect of TNF-alpha in vitro. It is concluded that indomethacin inhibits ovulation by anti-apoptotic mechanisms that can be dissociated from the paradigm of prostanoid down-regulation.

Mitogenic and antioxidant mechanisms of estradiol action in preovulatory ovine follicles: relevance to luteal function.

The objectives of this investigation were to determine the intrafollicular mechanisms and physiological consequences of estradiol actions in preovulatory ovine follicles. Acute suppression of estradiol production in proestrous ewes by an aromatase inhibitor (Arimidex) was associated with follicular lipid peroxidation, testosterone accumulation, and a granulosa cell deficiency (decreased proliferation/increased apoptosis). Estradiol-17beta stimulated granulosa proliferating cell nuclear antigen (PCNA) and protected cells from oxidative (H(2)O(2)) stress-induced apoptosis in vitro; the PCNA, but not the antiapoptotic response, was negated by the transcriptional inhibitor actinomycin D. Thus, it appears that genomic/mitotic and cytoprotective (oxygen-scavenging) modes of estradiol action operate in preovulatory follicles. Luteal (large steroidogenic cell) function was diminished following ovulation induction of estradiol-deficient follicles. It is suggested that inadequate exposure of the preovulatory follicle to estradiol caused the granulosa lutein insufficiency.

Culture and characterization of sinusoidal endothelial cells isolated from human liver.

Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Comparison of fluoxetine, bupropion, and placebo in the treatment of premenstrual dysphoric disorder.

Serotonergic antidepressants have been shown to be effective treatments for premenstrual dysphoric disorder (PMDD). The efficacy of nonserotonergic antidepressants is less well studied. This study was a two-center, parallel design, placebo-controlled, randomized trial of fluoxetine, bupropion, and placebo in women with PMDD. Thirty-four women with PMDD completed 1 month of single-blind placebo and 2 months of fluoxetine 20 mg/day (N = 10), bupropion 100 mg three times daily (N = 12), or placebo (N = 12). Clinical Global Impressions (CGI) Scale, an expanded form of the Hamilton Rating Scale for Depression (HAM-D), and Global Assessment Scale (GAS) ratings were obtained premenstrually in each of the three treatment cycles. The three treatment groups differed significantly in efficacy by CGI ratings. Fluoxetine was superior to both bupropion and placebo. Comparison of posttreatment to pretreatment HAM and GAS scores demonstrated significant superior efficacy of fluoxetine compared with placebo. Posttreatment HAM and GAS scores for bupropion were intermediate between but not significantly different from fluoxetine or placebo. In summary, fluoxetine was significantly superior to bupropion and placebo as an effective treatment for PMDD. Although some improvement with bupropion was noted, and both medications were well tolerated, patient satisfaction was far greater with fluoxetine.

Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression.

Prostacyclins have long been shown to have anti-metastatic activity. One hypothesis is their modulation of cell adhesion molecule (CAM) expression by target organ endothelial cells. We have postulated that prostacyclin, its analogs, and mechanistic mimics decrease colon carcinoma adhesion to cytokine-stimulated endothelial cells by blocking endothelial expression of the adhesion molecule E-selectin, but not the vascular cell adhesion molecule-1 (VCAM-1). Cultured human microvascular endothelial cells (HDMEC) were pre-incubated with prostacyclin (PGI2), dibutyrl-cAMP (dbcAMP), forskolin (FOR), and/or iso-methylbutylxanthine (IBMX) for 15 min, then co-incubated with the cytokine tumor necrosis factor (TNF) for 4 h. HDMEC surface expression of E-selectin and VCAM-1 was evaluated by flow cytometry and ELISA. Adherence of 51Cr-labeled colon carcinoma cells to HDMEC monolayers was then determined. In parallel assays, HDMECs were incubated with anti-E-selectin and anti-VCAM-1 monoclonal antibody (1:100) prior to the addition of tumor cells. Prostacyclins, its analogs, and mimics significantly reduced E-selectin expression by HDMEC, while the reduction of VCAM-1 expression was much less pronounced. Prostacyclins also significantly decreased colon carcinoma adherence to stimulated HDMECs. The inhibition of E-selectin expression, but not VCAM-1 expression, corresponded to the reduction of tumor cell adherence. Prostacyclin's effects on tumor adhesion were nullified by pre-incubation with E-selectin antibody. The inhibition of colon carcinoma adherence to cytokine-stimulated endothelial cells treated with prostacyclin, its analogs, and mimics appears to result from blocking endothelial E-selectin, but not VCAM-1, expression. These data support the hypothesis that prostacyclins may exert their anti-metastatic effect, in part, by inhibiting CAM-mediated adherence of colon carcinoma to endothelial cells in metastatic target organs.

Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy. Experimental studies of optimal dosages and therapeutic durations.

Ethanehydroxydiphosphonate therapy was studied for prevention of calcification of bioprosthetic heart valve cusps (from glutaraldehyde-preserved porcine aortic valves) implanted subcutaneously in 3-week-old male rats. Animals received daily subcutaneous injections of the drug (1, 5, 10, 15, or 25 mg/kg/24 hr) for 21 days with maximal inhibition of bioprosthetic heart valve calcification at a dosage of 15 mg/kg/24 hr (calcium level of diphosphonate-treated bioprostheses 3.5 +/- 0.5 micrograms/ml; calcium level of control bioprostheses, 161.2 +/- 5.0 micrograms/mg), but with irreversibly diminished bone and somatic growth. A dosage optimum was observed at 10 mg/kg/24 hr with significant inhibition of bioprosthetic heart valve calcification (at 21 days, the calcium level was 16.4 +/- 3.6 micrograms/mg) and an absence of adverse effects on epiphyseal development and overall growth. Bioprosthetic heart valves retrieved from animal receiving ethanehydroxydiphosphonate (15 mg/kg/24 hr) for only the first week after implantation had significantly more calcification after 21 days than did bioprostheses from animals treated for 2 or 3 weeks. Bioprostheses explanted after 110 days from animals receiving the drug (15 mg/kg/24 hr) for the first 3 weeks had calcification equivalent to that of untreated control rats. Diphosphonate (15 mg/kg/24 hr) was most efficacious when initiated within 48 hours of bioprosthesis implantation, but was totally ineffective if administered after 1 week. It is concluded that ethanehydroxydiphosphonate optimally prevents bioprosthesis calcification without significant adverse effects on epiphyseal development and overall somatic growth at a dosage of 10 mg/kg/24 hr in rat subdermal implants, but it must be administered by continuous daily injections beginning within 48 hours of the implantation; this approach should be pursued in further long-term circulatory experimental studies because of its possible clinical relevance.

Calcification of subcutaneously implanted type I collagen sponges. Effects of formaldehyde and glutaraldehyde pretreatments.

Although collagen-containing implants are widely used in various surgical applications, there has been relatively little attention paid to the possibility that this type of biomaterial may undergo pathologic calcification which could compromise its function. The present study reports for the first time the calcification of a series of implants of purified collagen sponges prepared with graded degrees of aldehyde-induced cross-linkages (assessed by shrinkage-temperature, wetting time, and collagenase digestibility). Type I collagen sponges were pretreated with either glutaraldehyde (0.1% to 2.0% aqueous solution, for 5-180 minutes) or formaldehyde (as vapors for 15 minutes to 15 hours), and implanted subcutaneously for 21 days in weanling rats. Although specimens not pretreated with either aldehyde reagent and the formaldehyde sponges pretreated for 15 minutes were resorbed without evidence of calcification, all other aldehyde-pretreated implants mineralized. The degree of calcification did not correlate with extent of cross-linking. Formaldehyde-pretreated implants calcified more extensively (Ca2+ = 87.8 +/- 2.8 micrograms/mg, mean +/- standard error of the mean; n = 58) than did glutaraldehyde-pretreated implants (Ca2+ = 40.9 +/- 1.4 micrograms/mg; n = 52). It is concluded that both glutaraldehyde- and formaldehyde-pretreated Type I collagen sponges calcify after subdermal implantation in young rats. Although aldehyde pretreatment of Type I collagen sponge implants is a prerequisite for their eventual mineralization, the threshold level of aldehyde-induced cross-linking required to potentiate their maximal pathologic calcification is low.

Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate.

Bioprostheses fabricated from porcine aortic valves are widely used to replace diseased heart valves. Calcification is the principal cause of the clinical failure of these devices. In the present study, inhibition of the calcification of bioprosthetic heart valve cusps implanted subcutaneously in rats was achieved through the adjacent implantation of controlled-release matrices containing the anticalcification agent ethanehydroxydiphosphonate dispersed in a copolymer of ethylene-vinyl acetate. Prevention of calcification was virtually complete, without the adverse effects of retarded bone and somatic growth that accompany systemic administration of ethanehydroxydiphosphonate.

Inhibition by diphosphonate compounds of calcification of porcine bioprosthetic heart valve cusps implanted subcutaneously in rats.

Calcification limits the long-term success of heart valve bioprostheses fabricated from glutaraldehyde cross-linked porcine aortic valves. The pathophysiology of calcification of bioprostheses has been studied experimentally with subcutaneous implants of the valve cusps in rats; in this preparation, the accumulation of calcific deposits is biochemically and morphologically identical to that occurring in clinical specimens. The objective of the present study was to determine whether mineralization of bioprosthetic valve cusps (BC) subcutaneously implanted in 3-week-old male rats could be inhibited through the use of diphosphonate compounds. Ethanehydroxydiphosphonate (EHDP), administered by daily subcutaneous injection (25 mg/kg/24 hr) for 21 days inhibited calcification (BC Ca++ = 154.9 +/- 4.1), but caused somatic growth retardation and disruption of epiphyseal development. However, local administration of EHDP by osmotic pump (5 mg/kg/24 hr) implanted in direct contact with the cuspal tissue for 14 days prevented BC calcification (BC CA++ = 4.3 +/- 0.7) without adverse effects. Furthermore, EHDP given by osmotic pump had a prolonged effect on reducing calcification, as demonstrated by implants harvested 21 days (BC CA++ = 12.2 +/- 6.4) after the drug supply was exhausted. Finally, BC preincubated in aminopropanehydroxydiphosphonate for 24 hr before 21 day implantation underwent less calcification (CA++ = 24.2 +/- 7.4) than control valves (BC CA++ 126.6 +/- 7.5) with no adverse effects. We conclude that diphosphonates inhibit BC calcification, and that adverse effects of systemic therapy can be avoided by local administration.

Toxaphene residues in the bovine after oral exposure.

Toxaphene was administered orally to 19 mixed-breed heifers at levels of 50, 100, or 150 mg/kg. Thirteen of 19 died with deaths in all 3 dosage levels. Liver, kidney, and brain samples were collected and analyzed for toxaphene residues by GLC. Liver residues varied logarithmically with dosage from 1.2 to 38 ppm, kidney residues were from 0.8 to 12 ppm, and brain residues were from 0.3 to 7.0 ppm. No detectable amounts of toxaphene (< 0.1 ppm) were observed in tissues collected from 2 control calves which received no toxaphene.