Gerald E. Zaroogian

Vitellogenin-induced pathology in male summer flounder (Paralichthys dentatus)

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17beta-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1-10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells

Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.

Comparison of cadmium, nickel and benzo(a)pyrene uptake into cultured brown cells of the hard shell clam, Mercenaria mercenaria

Abstract Cadmium and nickel uptake into M. mercenaria brown cells increased curvilinearly with solute concentration, whereas benzo(a)pyrene [B(a)P] uptake is linear at concentrations studied. Uptake appeared to be a process of facilitated diffusion which is more efficient at lower Cd2+ and Ni2+ concentrations. Buthionine-(S,R)-sulfoximine, which blocks glutathione synthesis significantly inhibited Cd2+, Ni2+ and B(a)P uptake. It appears that sites other than Ca2+ channels and a sulfhydryl sensitive system are involved in Cd2+, Ni2+ and B(a)P uptake into Mercenaria brown cells.

Effect of selected inhibitors on cadmium, nickel, and benzo(a)pyrene uptake into brown cells of Mercenaria mercenaria

Abstract Uptake and inhibition studies were used to evaluate mechanisms of uptake of Ni 2+ , Cd 2+ , and B(a)P in the brown cells of M. mercenaria. Brown cells contain one or more vesicles that have been shown to be lysosomes. Cd 2+ , Ni 2+ , and B(a)P accumulation by brown cells was concentration-dependent and independent of time and temperature at 5°C. Metabolic inhibitors such as carbonyl cyanide- m -chlorophenyl hydrazone and NaF did not inhibit their uptake. N -ethylmaleimide facilitated Ni 2+ and Cd 2+ uptake, but inhibited B(a)P uptake. Buthionine-(S,R)-sulfoximine inhibited Ni 2+ , Cd 2+ , and B(a)P uptake in a dose-dependent manner, and diethylmaleate had no effect on Cd 2+ and B(a)P uptake, but increased Ni 2+ uptake. Chloroquine and copper, which accumulate in lysosomes, inhibited Ni 2+ , Cd 2+ , and B(a)P uptake. Verapamil inhibited Ni 2+ and B(a)P uptake, whereas it increased Cd uptake. Our results suggest that the brown cells of M. mercenaria are capable of accumulation of soluble foreign material and that membrane sulfhydryl groups, glutathione, and Ca 2+ channels are active in these processes.

Studies on the depuration of cadmium and copper by the American oyster Crassostrea virginica.

This study was an attempt to establish under laboratory conditions whether treated oysters would depurate accumulated cadmium and copper when returned to cleaner waters containing natural concentrations of these metals. In addition, an attempt was made to determine if cadmium accumulation would promote copper loss in the oyster.

Estimation of toxicity to marine species with structure-activity models developed to estimate toxicity to freshwater fish☆

Abstract Structure-activity models which were developed to estimate toxicity of chemicals to freshwater fish were tested for use with an estuarine fish ( Cyprinodon variegatus ) and mysids ( Mysidopsis bahia ). Significant linear and polynomial relationships that correlated well existed between reported 96-h LC 50 values for each marine species and log P (log octanol/water partition coefficient). Good linear relationships were obtained when the 96-h LC 50 values for C. variegatus and M. bahia were regressed on water solubility (μmol/l). These models were compared to models developed for freshwater fish using log P and log S . Models using log P to estimate acute toxicity for two freshwater fish produced 96-h LC 50 values similar to those measured for C. variegatus and M. bahia , whereas, those models developed with water solubility produced 96-h LC 50 values similar to those for C. variegatus , but not for M. bahia . The data indicated that models developed with log P for freshwater fish can be used to estimate toxicity to C. variegatus for a minimum of 58% of the chemicals, whereas models using water solubility estimated toxicity to C. variegatus for a minimum of 77% of the chemicals within an order of magnitude for screening purposes. The calculated 96-h LC 50 values were compared to the measured values for each marine species and those measured for Pimephales promelas (fathead minnow) and Poecilia reticulata (guppy). Tests indicated generally that calculated 96-h LC 50 values were overestimates of the measured 96-h LC 50 values when models for freshwater fish were used to estimate toxicity to each marine species. More data are required for marine species to determine if highly significant relationships between marine and freshwater fish exist with comparisons using larger sample sizes.

Modulation of aromatase activity as a mode of action for endocrine disrupting chemicals in a marine fish

The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E2), ethynylestradiol (EE2), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E2, EE2, and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E2 and EE2, male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E2 and EE2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact aromatase activity also affect reproductive output in spawning cunner.

Individual and combined cytotoxic effects of cadmium, copper, and nickel on brown cells of Mercenaria mercenaria

An assay based on the lysosomal incorporation of neutral red dye by brown cells of Mercenaria mercenaria (Bivalvia) was used to measure the cytotoxicity of Cd2+, Cu2+, and Ni2+ singly and in combination. Cytotoxicity was a linear function of Cd2+ concentration 0.1-1.5 mM and Cu2+ concentration between 10 and 100 microM. Nickel was not cytotoxic at concentrations as high as 10 mM. The presence of Cu2+ lessened the cytotoxic effect of Cd2+. Ni2+ did not affect cytotoxicity in combination with either Cu2+ or Cd2+.Ni2+ inflated estimates of cell survival by the neutral red assay in this study. Cells exposed to Ni2+ yielded measured quantities of neutral red dye in excess of those measured in cells from control treatments.

The nature and function of the brown cell in Crassostrea virginica

Abstract This study was undertaken to determine the role of the brown cell in Crassostrea virginica in degradative and detoxification processes. Histopathological and biochemical methods were used to study brown cells in vivo and in vitro before and after treatment with organic and inorganic compounds. Histopathological examination indicated that brown cells in the connective tissue of healthy animals were sparse, and found primarily around sinusoids and in the intertubular connective tissue of the digestive diverticula. Brown cells in the auricle were derived from connective tissue of the auricular muscle bundles and occurred on the surface and within the muscle bundles. In addition, the surface of the pericardial wall was lined with brown cells. At sites of inflammation, an increase in the number and size of brown cells occurred as well as an increase in the number and color density of brown vesicles in the cytoplasm. Brown cell isolates were separated into fractions on a Percoll discontinuous gradient. Fraction 2 contained primarily what appeared to be young brown cells (nonpigmented to lightly pigmented and granular in appearance). The majority of the cells in fraction 3 were brown cells (small to large pigmented vesicles) and fraction 4 was entirely brown vesicles (devoid of a cell membrane). Toluidine blue (soluble dye) accumulated in brown cells in vivo after injection into the visceral mass and in vitro , after addition to brown cell isolates. In com-parison, carmine red (colloidal dye) did not accumulate under the same conditions. It appeared that only soluble substances accumulate in brown cells, and that brown cells are incapable of phago- cytosing yeast cells. Brown cell vesicles fluoresced within 2 hours after addition of FITC-bovine serum albumen and acridine orange to cell isolates and within 24 h after whole animal injection. Cadmium and nickel accumulated in brown cell isolates curvilinearly with solute concentration and uptake was by passive diffusion. Brown cells and brown vesicles possess glutathione reductase, acid phosphatase and lysozyme which indicate the vesicles are lysosomes. Brown cells accumulate soluble foreign material and have the potential to function in detoxification and degradative processes.

The role of the red gland in Mercenaria mercenaria in detoxification

Abstract Brown cells of the red gland in Mercenaria mercenaria are involved in the excretory process. We studied their involvement in the removal of foreign substances from the tissues of Mercenaria . Toluidine blue (soluble dye) accumulated in brown cells after dye injection into the foot muscle and after addition to brown cell isolates. In comparison, carmine red (particulate dye) did not accumulate under the same conditions. FITC-labeled bovine albumen accumulated in brown cells when introduced in vivo and to cell isolates. Kinetic studies indicated uptake of FITC-albumen occurred within 2 h when added to cell isolates and within 24 h after injection into the foot muscle. Brown cell isolates were separated into three fractions on a discontinuous Percoll gradient. Fraction 1 contained primarily what appeared to be precursor brown cells. The majority of the cells in fraction 2 were brown cells. Fraction 3 was entirely brown globular material. Assays indicated the presence of acid phosphatase, glutathione reductase and lysozyme in lysates from each fraction. Thus it appears that brown cells of the red gland contain enzymes involved in detoxification and degradative processes.