Salmonid sexual development is not consistently altered by embryonic exposure to endocrine-active chemicals.

Fish sexual development is sensitive to exogenous hormone manipulation, and salmonids have been used extensively as environmental sentinels and models for biomedical research. We simulated maternal transfer of contaminants by microinjecting rainbow trout (Oncorhynchus mykiss) and chinook salmon (Oncorhynchus tshawytscha) embryos. Fish were reared for 6 months and sexed, and gonads were removed for histology and measurement of in vitro steroid production. Analysis of fat samples showed that dichlorodiphenylethylene (DDE) levels, o, p'M-DDE and p,o, p'-DDE isomers, were elevated 6 months after treatment. A preliminary study showed an increased ratio of males to females after treatment with 80 mg/kg and 160 mg/kg of the xenoestrogen o,o, p'-DDE. One fish treated with 160 mg/kg o,o, p'-DDE had gonads with cells typical of both males and females. A follow-up study, using more fish and excluding the highly toxic 160 mg/kg o,o, p'-DDE dose, showed no effect on sex ratio or gonadal histology. Embryonic exposure of monosex male trout, monosex female trout, and mixed sex salmon to o, o, p'-DDE, p,o, p'-DDE, mixtures of DDE isomers, and octylphenol failed to alter sexual development. We observed no treatment-dependent changes in in vitro gonadal steroid production in any experiments. Trout exposed in ovo and reared to maturity spawned successfully. These results suggest that mortality attributable to the xenoestrogens o,o, p'-DDE, chlordecone, and octylphenol, and the antiandrogen p,o, p'-DDE, is likely to occur before the appearance of subtle changes in sexual development. Because trout appeared to be sensitive to endocrine disruption, we cannot dismiss the threat of heavily contaminated sites or complex mixtures to normal sexual development of salmonids or other aquatic organisms.

Distribution of chlordecone to liver plasma membranes and recovery from hepatobiliary dysfunction in rats.

Chlordecone (CD) impairs biliary excretion of organic anions (including phenolphthalein glucuronide (PG), imipramine polar metabolites, and taurocholate) without evidence of hepatocellular necrosis in rats. In this study we investigated the hypothesis that CD-induced hepatobiliary dysfunction is dependent on CD concentration in liver plasma membranes where it inhibits active transport in vitro. Rats were treated by gavage (0 or 60 mg CD/kg in corn oil) 24 or 72 h prior to bile duct cannulation. Biliary excretion of PG, a marker of hepatic organic anion transport, and [14C]mannitol, a marker of passive transcellular permeability, was determined. Biliary excretion of PG decreased approximately 25% in rats 24 h after CD treatment, however rats recovered control PG excretion rates 72 h after CD treatment. Recovery of PG excretion occurred despite higher liver homogenate [14C]CD concentrations at 72 h than at 24 h after [14C]CD treatment. Biliary clearance of [14C]mannitol decreased both 24 h and 72 h after treatment. Even though the amount of [14C]CD retained in the liver was greater at 72 h than at 24 h after treatment, the concentration of [14C]CD in isolated liver plasma membranes (LPM) was the same (3.5-3.9 nmol/mg protein) at both times. There was a significant reduction in 5'-nucleotidase activity of LPM at 24 h but not at 72 h after CD. This study demonstrated no correlation between recovery from CD-induced hepatobiliary dysfunction and whole liver accumulation. Altered subcellular [14C]CD distribution (reduced LPM-to-homogenate concentration ratio was coincident with recovery.

Dieldrin induces cytosolic [3H]7, 12-dimethylbenz[a]anthracene binding but not multidrug resistance proteins in rainbow trout liver.

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Interaction of 2,2',4,4',5,5'-hexachlorobiphenyl with hepatic cytochrome P-4501A in rainbow trout (Oncorhynchus mykiss).

Di-ortho polychlorinated biphenyls (PCBs) are prominent environmental contaminants and their biological activity in fish may be more significant than previously thought. Four weeks after intraperitoneal (i.p.) injection with 50 or 250 microg 2,2',4,4',5,5'-hexachlorobiphenyl (2HxCB)/g fish, rainbow trout livers were removed and frozen at -80 degrees C or microsomes were prepared. Microsomal ethoxyresorufin O-deethylase (EROD) activity was approximately one and two orders of magnitude greater than controls in fish treated with 50 and 250 microg 2HxCB/g fish, respectively. Cytochrome P4501A (CYP1A) Western immunoblot relative optical density increased with 2HxCB dose. Hepatic CYP1A1 mRNA levels were approximately threefold greater in fish treated with 250 microg 2HxCB/g fish than in controls, while hepatic CYP1A1 mRNA levels in fish treated with 50 microg 2HxCB/g fish were not significantly induced. There was no increase of CYP1A3 mRNA in 2HxCB-treated fish. The study showed 2HxCB induced hepatic EROD activity, CYP1A protein, and CYP1A1 mRNA content in rainbow trout.

Modulation of 7,12-dimethylbenz[a]anthracene disposition and hepatocarcinogenesis by dieldrin and chlordecone in rainbow trout.

The present study examined whether modified xenobiotic transport, resulting from chlordecone (CD) or dieldrin pretreatment, would alter polycyclic aromatic hydrocarbon (PAH) or organochlorine (OC) target organ doses and subsequent tumor organospecificity or incidence rates in rainbow trout. Additionally, the potential for exposure to dieldrin or CD, following PAH exposure, to enhance tumor incidence was assessed. Evaluation of CD pretreatment effects on [14C]CD disposition in trout was conducted following two i.p. (0-15 mg/kg) and two dietary (0-0.4 mg/kg/d) pretreatment regimes. To assess the influence of OC pretreatment on cancer induced by the PAH 7,12-dimethylbenz[a]anthracene (DMBA), juvenile trout were fed control, CD (0.1, 0.4 mg/kg/d), or dieldrin (0.1, 0.3 mg/kg/d) diets for 9 wk, received a waterborne [3H]DMBA exposure (1 mg/L, 20 h), and resumed control, CD, or dieldrin diets for 33 wk. [3H]DMBA disposition and hepatic [3H]DMBA binding were examined immediately and 24 h after exposure. Hepatic and stomach tumor incidences were determined 33 wk after DMBA exposure. CD pretreatment did not influence [14C]CD or [3H]DMBA hepatic concentrations, hepatic [3H]DMBA DNA binding, or hepatic/stomach tumor incidence. It did, however, elevate bile [14C]CD and [3H]DMBA concentrations. Postinitiation exposure to CD weakly enhanced DMBA-induced hepatic tumor incidence at the low but not the high CD dose. Dieldrin pretreatment did not influence stomach [3H]DMBA equivalents or stomach tumor incidence but did cause an elevation in biliary and hepatic concentrations of [3H]DMBA equivalents. [3H]DMBA binding to liver DNA was significantly increased and hepatic tumor incidence was elevated by dieldrin pretreatment. Dieldrin treatment following DMBA initiation did not enhance hepatic or stomach tumor incidence. Ecoepidemiology studies, to date, have reported correlations between the co-occurrence of PAHs and OCs and elevated tumor incidence in feral fish, but cause-and-effect relationships have been difficult to establish. The results of the present study confirm that OCs, such as dieldrin and CD, play a role in modifying PAH-induced carcinogenesis in fish.

Formation of nitro musk adducts of rainbow trout hemoglobin for potential use as biomarkers of exposure.

The high use of nitro musk xylene (MX) and musk ketone (MK) as fragrances, and their persistence and bioaccumulation potential make them ubiquitous environmental contaminants. The 4-amino-MX (AMX) and 2-amino-MK (AMK) metabolites have been detected in trout fish hemoglobin (Hb) samples by gas chromatography-ion trap-mass spectrometry (GC-MS). Twelve Hb samples prepared from rainbow trout that were exposed to MX and MK, over a period of 24 and 72 h, were analyzed. Amino metabolites were liberated by basic hydrolysis and extracted from the fish Hb into n-hexane. The extract was concentrated, analyzed, and spiked with a standard solution (80 pg/microl) of AMX or AMK and reanalyzed. Concentrations of AMX from 10 to 25 ng/g were detected in Hb from fish taken 24 and 72 h after MX exposure. At 24 and 72 h after MK exposure, the concentration of AMK was found to be 25-51 ng/g and 9.5-25 ng/g, respectively. Concentrations of AMK in Hb from two of the three trout were substantially lower after 72 h compared with 24 h exposure. The AMX and AMK metabolites were not detected in four control samples. Average recoveries exceeding 89 and 86% could be achieved for AMX and AMK, respectively, with a coefficient of variation (CV) around 5%.

Roles of uptake, biotransformation, and target site sensitivity in determining the differential toxicity of chlorpyrifos to second to fourth instar Chironomous riparius (Meigen).

Early life stages of aquatic organisms tend to be more sensitive to various chemical contaminants than later life stages. This research attempted to identify the key biological factors that determined sensitivity differences among life stages of the aquatic insect Chironomous riparius. Specifically, second to fourth instar larvae were exposed in vivo to both low and high waterborne concentrations of chlorpyrifos to examine differences in accumulation rates, chlorpyrifos biotransformation, and overall sensitivity among instars. In vitro acetylcholinesterase (AChE) assays were performed with chlorpyrifos and the metabolite, chlorpyrifos-oxon, to investigate potential target site sensitivity differences among instars. Earlier instars accumulated chlorpyrifos more rapidly than later instars. There were no major differences among instars in the biotransformation rates of chlorpyrifos to the more polar metabolites, chlorpyrifos-oxon, and chlorpyridinol (TCP). Homogenate AChE activities from second to fourth instar larvae were refractory to chlorpyrifos, even at high concentrations. In contrast, homogenate AChE activities were responsive in a dose-dependent manner to chlorpyrifos-oxon. In general, it appeared that chlorpyrifos sensitivity differences among second to fourth instar C. riparius were largely determined by differences in uptake rates. In terms of AChE depression, fourth instar homogenates were more sensitive to chlorpyrifos and chlorpyrifos-oxon than earlier instars. However, basal AChE activity in fourth instar larvae was significantly higher than basal AChE activity in second to third instar larvae, which could potentially offset the apparent increased sensitivity to the oxon.

Vitamin and thyroid status in arctic grayling (Thymallus arcticus) exposed to doses of 3,3',4,4'-tetrachlorobiphenyl that induce the phase I enzyme system.

Induction of phase I biotransformation enzymes is recognized as a hallmark response in fish exposed to coplanar PCBs. Depletions of vitamins A and E and disrupted thyroid hormone and glandular structure secondary to this induction have not yet been examined in an arctic fish species. Arctic grayling were exposed to a single oral dose of 0 (control), 10, 100 or 1000 ng 3,3',4,4'-tetrachlorobiphenyl (TCB) g(-1) bodyweight, a contaminant found in most arctic fish. After 30 and 90 days of exposure, TCB concentrations in tissues, hepatic phase I activity (as ethoxyresorufin-O-deethylase (EROD)), plasma and tissue vitamin A and E concentrations, plasma thyroid hormone levels and thyroid glandular structure were examined. Total plasma osmolality, as an indicator of overall fish health was also monitored. TCB recovery in tissues was low and extremely variable, making comparisons between intended dose groups inappropriate. Therefore, correlation analysis between actual recovered TCB concentrations and biochemical responses was employed. Hepatic EROD activity correlated strongly with liver TCB concentrations. Liver concentrations of vitamin A were altered as a function of TCB concentrations and EROD activity, but plasma vitamin A status was not affected. Vitamin E was depleted by TCB accumulation in blood and EROD induction in liver of males only at 90 days postexposure. Thyroid hormones status and glandular structure were not affected by the short duration TCB exposures used in this experiment. TCB concentrations were correlated with an elevation in plasma osmolality. Results from this experiment indicate that the vitamin status and osmoregulation of arctic grayling exposed to TCB can be compromised. Further studies of field populations exposed to this type of contaminant are warranted.

Libido, menopause, and estrogen replacement therapy.

Patients may be embarrassed to communicate their concerns regarding a decrease in sexual desire to their physician, let alone request treatment. Drs Beard and Curtis recommend that patients be asked specific questions to elicit symptoms of sexual dysfunction. They outline a program of hormone replacement therapy for women who have sexual dysfunction secondary to genitourinary atrophy and for those whose loss of libido is secondary to declining levels of estrogen.

Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture.

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility.

Chlordecone is a potent in vitro inhibitor of oligomycin-insensitive Mg2+ -ATPase of rat bile canaliculi-enriched fraction.

The oligomycin-insensitive Mg2+ -ATPase (OIMATPase) of rat bile canaliculi-enriched fraction (BCEF) was inhibited by chlordecone (CD) in vitro (IC-50 = 25 microM). Kinetic analysis indicated noncompetitive inhibition. Inhibition of OIMATPase by filipin but not by atractyloside verified plasma membrane origin of activity. The cholestatic agents alpha-naphthyl isothiocyanate (ANIT) and taurolithocholate (TLC) decreased OIMATPase activity at in vitro concentrations of 33 and 162 microM, while taurocholate (a choleretic bile salt), ethynylestradiol, and manganese did not. Cholestatic drugs with primary intracellular sites of action (colchicine and phalloidin) were ineffective OIMATPase inhibitors in this concentration range. Inhibition of OIMATPase by N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD) indicated some H+ -ATPase activity in BCEF. In vitro sensitivity of OIMATPase of BCEF to CD, ANIT, and TLC suggested the bile canaliculus as a subcellular-level target for their cholestatic actions.

Glucuronidation and biliary excretion of phenolphthalein in temperature-acclimated steelhead trout (Salmo gairdneri).

Conjugative metabolism and biliary excretion of phenolphthalein (PP) were studied in steelhead trout (Salmo gairdneri) acclimated to 10, 14 or 18 degrees C. Significant seasonal effects were found on liver weight to body weight ratio and conjugative metabolism but not biliary excretion of free plus conjugate of PP. Temperature did not significantly interact with these relationships. PP was excreted in bile as parent compound and the glucuronide conjugate at all temperatures. Saturation of the excretory process was apparent at a dose of approximately 10 mumol/kg (i.p.) at 10 and 14, but not 18 degrees C.

A characterization of chlordecone pretreatment-altered pharmacokinetics in mice.

Lipophilic chlorinated hydrocarbons pose a potential health hazard to humans and animals and the toxicity of a number of these compounds has been well documented. Despite the low environmental concentrations of most of these chemicals, much of the research conducted to date has used maximally tolerated doses. Our research, conducted with low, apparently nontoxic, doses of the insecticide chlordecone (CD), showed that the administration of CD (5 mg/kg ip) to mice (C57BL/6N and DBA/2N strains) caused a time-dependent alteration in the pattern of distribution of a subsequently administered dose of [14C]CD. Livers of CD-pretreated animals contained less label than did those from controls and CD pretreatment increased amounts of label in kidney, lung, fat, and muscle. Changes did not appear to be due to an altered rate of metaboLism and analysis of total CD in tissues (unlabeled plus [14C]CD) indicated that these responses were not due to a simple redistribution phenomenon. We have termed this preexposure effect a pretreatment disposition response (PDR) and feel it may reflect an important cellular response to lipophilic compounds. CD-induced PDR is dose related, exhibits a threshold, and is saturable at a given level of induction. In addition, PDR exhibits some specificity, inasmuch as pretreating mice with CD (5 mg/kg) does not alter the distribution of subsequently administered [14C]dieldrin. The characteristics of threshold, saturability, and specificity are consistent with the premise that CD-induced PDR is a protein-mediated phenomenon.

Low dose chlordecone pretreatment altered cholesterol disposition without induction of cytochrome P-450.

Pretreatment of mice with low doses of chlordecone (CD) alters the pattern of distribution of a subsequent tracer dose of [14C]CD. We call this preexposure effect a pretreatment disposition response (PDR) and suggest that it reflects important cellular responses to lipophilic compounds. The present study examined three possible mechanisms for CD-induced PDR (CD-PDR). The first was that CD-PDR occurred with induction of the cytochrome P-450 system. A cumulative dose of 45 mg/kg CD caused a PDR, increased the content of cytochrome P-450, and elevated the activities of ethoxyresorufin- and ethoxycoumarin-O-deethylases (EROD and ECOD). A cumulative dose of 10 mg/kg caused a PDR, but did not affect cytochrome P-450, EROD, or ECOD, indicating that an induction of the cytochrome P-450 system in not necessary for PDR. A second possibility examined was that CD-PDR resulted because of an altered affinity of a subcellular fraction. Following a pretreatment regimen designed to produce PDR, amounts of [14C]CD in each fraction paralleled homogenate values in the liver and the kidney. However, when values were calculated as percentages of total label recovered, it was apparent that [14C]CD levels were higher in the microsomal fraction of the liver. Finally, the possibility that CD-PDR occurred because of an interaction of CD with proteins involved in cholesterol synthesis and transport was addressed. CD pretreatment increased disposition of a dose of [14C]cholesterol to the fat at the expense of [14C]cholesterol in the liver and kidney.