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We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82μg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC 30 ) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An abandoned cinnabar mining and roasting site is in the major sub-basin of the watershed for Cottage Grove Reservoir, Oregon. Average surface sediment total mercury concentration in the river draining this sub-basin (0.61 ± 0.52 μg/g) was about ten-fold higher than three smaller tributaries to the reservoir. Total mercury in reservoir surface sediments averaged 1.66 ± 0.70 μg/g. Stratigraphy for two sediment cores indicated generally decreased reservoir mercury loading from 1963 to 2002 but two pronounced peaks in mercury deposition. Years of extreme precipitation immediately prior to these peaks at least partially explained them. Epaxial muscle total mercury concentrations of largemouth bass increased with body weight up to 2.5 μg/g. A gradient of mercury concentrations in soils from a 3.3 km diameter grid indicated condensation of mercury vapors from the mine site polluted the sub-basin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2nM [(3)H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2α. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50days of feeding 3μg of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3days and remained elevated for up to 28days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14days of feeding both PAHs that was not significant after 50days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28days. There was no clear association between AHR2α affinity and BaP and BeP-induced oxidative stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 SETAC.)

Fish in agricultural and remote areas may be exposed to endosulfan and its degradation products as a result of direct runoff, atmospheric transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degradation product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the endosulfan I or endosulfan sulfate from 6 to 120h post-fertilization (hpf). Endosulfan I exposure concentrations ranged from 0.01 to 10microg/L and endosulfan sulfate from 1 to 100microg/L. Water solutions were renewed every 24h and fish were scored for overt developmental and behavioral abnormalities. Chemical analysis was performed on water, whole embryo, and larvae samples to determine waterborne exposure concentrations and tissue concentrations throughout the 5-day period. The most sensitive toxicity endpoint for both endosulfan I and endosulfan sulfate was an abnormal response of the embryo/larvae to touch, suggesting that endosulfan I and sulfate are developmentally neurotoxic. The waterborne exposure EC(50)s for inhibition of touch response for endosulfan I and endosulfan sulfate were 2.2microg/L and 23microg/L, respectively. The endosulfans were highly concentrated by the organisms, and the inhibition of touch response tissue EC(50), determined from the measured tissue concentrations, was 367ng/g for endosulfan I and 4552ng/g for endosulfan sulfate.

Pretreatment of male C57BL/6 mice with low doses of the persistent organochlorine pesticide, chlordecone (CD), stimulated biliary excretion of exogenous cholesterol (CH) up to 3-fold. Increased biliary excretion occurred without changes in hepatic ATP-binding cassette transporter G8 (ABCG8) of the bile canaliculus or scavenger receptor class B type I (SR-BI) of the sinusoidal surface. A variety of tissues express scavenger receptor class B type II (SR-BII) and this protein was identified as a splice variant from the SR-BI gene. Although the function of SR-BII has not been elucidated it may play a role in CH homeostasis and trafficking distinctly different than SR-BI. Western blotting demonstrated that a single dose of CD promoted subcellular distribution of SR-BII to murine hepatic microsomes about 2.2-fold when compared to controls without effect on liver crude membrane SR-BII content. This was consistent with increased vesicular CH trafficking. Relative quantification of hepatic cytosolic proteins in a fraction that sequestered [(14)C]CH by mass spectrometry (MS) indicated no role for cytosolic CH binding proteins in CD altered CH homeostasis. Western blotting verified no effect of CD on liver fatty acid-binding protein (L-FABP) in cytosol. MS detected a statistically significant increase in myosin-9, which was also consistent with increased vesicular trafficking.

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [(14)C]CD or [(14)C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor alpha (ERalpha) in a concentration-dependent manner (0-50 muM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice.

The use of isobaric tagging for relative and absolute quantification (iTRAQ) has increased dramatically over the past few years. Many factors can affect the accuracy of quantification. Some of these include the number of biological/technical replicates, sample complexity, instrumentation, method of peptide/protein identification and the statistical techniques used for data analysis. It has been observed that the low collision energies normally used in electrospray ionization quadrupole time-of-flight (ESI QTOF) can result in low iTRAQ reporter ion abundances. We used two-way analysis of variance (ANOVA) to compare the iTRAQ ratios that were generated on an ESI QTOF and a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF). It appears that iTRAQ analyses performed on an ESI QTOF without any special modifications to instrumental parameters produce essentially the same protein ratios as those obtained on a MALDI TOF/TOF.

Organochlorine (OC) insecticides continue to occur in tissues of humans and wildlife throughout the world although they were banned in the United States a few decades ago. Low doses of the OC insecticide chlordecone (CD) alter hepatic disposition of lipophilic xenobiotics and perturb lipid homeostasis in rainbow trout, mice and rats. CD pretreatment altered tissue and hepatic subcellular distribution of exogenous [(14)C]cholesterol (CH) equivalents 4 and 16 h after a bolus intraperitoneal (ip) injection of 5 ml corn oil/kg that contained 10 mg CH/kg. CD pretreatment altered tissue distribution of exogenously administered [(14)C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [(14)C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [(14)C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [(14)C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [(14)C]CH equivalents in the non-HDL fraction 4 h after a bolus lipid dose. CD pretreatment increased plasma non-HDL total CH by 80% 4 h after a bolus lipid dose. Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G8 (ABCG8) proteins were quantified by western blotting in hepatic membranes from control and CD treated mice. Liver membrane contents of SR-BI and ABCG8 proteins were unchanged by CD pretreatment. The data demonstrated that a single dose of CD altered CH homeostasis and lipoprotein metabolism.

Prometon is one of the most consistently detected herbicides in the U.S. environment. However, no previous assessment of the potential for prometon or related methoxytriazine herbicides to act as endocrine-disrupting chemicals has been conducted. This study used an array of in vitro bioassays to assess whether prometon, atraton, terbumeton, or secbumeton might act as potent (ant)agonists of the aryl hydrocarbon, estrogen, androgen, or glucocorticoid receptors or as aromatase inhibitors or inducers in vitro. Potential effects of prometon were also evaluated using a 21-d fathead minnow reproduction assay. Concentrations of methoxytriazines, as great as 1 mg/L (4.4 microM), did not induce significant dioxin-like responses in H4IIE-luc cells, estrogenic responses in MVLN cells, or androgen or glucocorticoid receptor-mediated responses in MDA-kb2 cells, nor did the methoxytriazines significantly affect aromatase activity in vitro. In the fathead minnow assay, exposure to 20, 200, or 1,000 microg prometon/L significantly reduced the weight of the male fat pad (an androgen-responsive tissue) relative to body weight. Exposure to 20 microg prometon/L significantly increased female plasma testosterone concentrations, but the effect was not observed at greater concentrations. Overall, prometon did not significantly reduce fecundity over the 21-d exposure, nor were other endpoints, including plasma vitellogenin and estradiol concentrations, brain and ovary aromatase activity, and male tubercle index, significantly affected. Evidence from our work suggests that prometon may cause subtle endocrine and/or reproductive effects in fathead minnows, but no clear mechanism of action was observed. The relevance of these effects to hazard assessment for the pesticide is uncertain.

The Willamette River, one of 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of the Willamette River detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (Newberg pool [NP], rivermile [RM] 26 - 55) and middle (Wheatland Ferry [WF], RM 72 - 74) river, relative to those in the upper river (Corvallis [CV], RM 125-138). The objective of this study was to determine the likely cause of these skeletal deformities. In 2002 and 2003, deformity loads in Willamette River fishes were 2-3 times greater at the NP and WF locations than at the CV location. There were some differences in water quality parameters between the NP and CV sites, but they did not readily explain the difference in deformity loads. Concentrations of bioavailable metals were below detection limits (0.6 - 1 microg/ L). Concentrations of bioavailable polychlorinated biphenyls (PCBs) and chlorinated pesticides were generally below 0.25 ng/L. Concentrations of bioavailable polycyclic aromatic hydrocarbons were generally less than 5 ng/L. Concentrations of most persistent organic pollutants were below detection limits in ovary/oocyte tissue samples and sediments, and those that were detected were not significantly different among sites. Bioassay of Willamette River water extracts provided no evidence that unidentified compounds or the complex mixture of compounds present in the extracts could induce skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large percentage of deformities detected in two Willamette River fishes, and similar deformities were reproduced in laboratoryfathead minnows exposed to cercariae extracted from Willamette River snails. Thus, the weight of evidence suggests that parasitic infection, not chemical contaminants, was the primary cause of skeletal deformities observed in Willamette River fish.

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%.

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.

Aquatic insects have evolved diverse respiratory strategies that range from breathing atmospheric air to breathing dissolved oxygen. These strategies result in vast morphological differences among taxa in terms of exchange epithelial surface areas that are in direct contact with the surrounding water that, in turn, affect physiological processes. This paper examines the effects of acute temperature shifts on water permeability and chlorpyrifos uptake in aquatic insects with different respiratory strategies. While considerable differences existed in water permeability among the species tested, acute temperature shifts raised water influx rates similarly in air-breathing and gill-bearing taxa. This contrasts significantly with temperature-shift effects on chlorpyrifos uptake. Temperature shifts of 4.5 degrees C increased 14C-chlorpyrifos accumulation rates in the gill-bearing mayfly Cinygma sp. and in the air-breathing hemipteran Sigara washingtonensis. However, the temperature-induced increase in 14C-chlorpyrifos uptake after 8 h of exposure was 2.75-fold higher in Cinygma than in Sigara. Uptake of 14C-chlorpyrifos was uniformly higher in Cinygma than in Sigara in all experiments. These findings suggest that organisms with relatively large exchange epithelial surface areas are potentially more vulnerable to both osmoregulatory distress as well as contaminant accumulation. Temperature increases appear more likely to impact organisms that have relatively large exchange epithelial surface areas, both as an individual stressor and in combination with additional stressors such as contaminants.

Activities of hepatic microsomal and cytosolic epoxide hydrolases, accumulation of dieldrin in liver, and in vivo metabolism and disposition of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BP), were examined in rainbow trout pretreated with dieldrin, a chlorinated cyclodiene insecticide. Rainbow trout were fed 0.3 mg dieldrin/kg/day for 9 weeks and the same dose of dieldrin for 9 weeks, followed by 3 weeks on control diet (12 weeks). Fish then received an intraperitoneal (ip) challenge dose of 14C-BP (10 micromol/kg). Dieldrin pretreatment significantly elevated the concentration of 14C-BP in bile (142% and 200% at 9 and 12 weeks, respectively), but not liver or fat. Extraction of bile subsamples confirmed dieldrin pretreatment significantly stimulated total biliary excretion of 14C-BP polar metabolites (244% and 221% at week 9 and 12, respectively). The complex metabolism of BP characterized the in vivo state of the CYP system, UDP-glucuronyltransferases, and sulfotransferases. Bile was extracted and then hydrolyzed by beta-glucuronidase and arylsulfatase to regenerate BP metabolites conjugated by phase II enzymes. Evaluation of biliary polar metabolite profiles of 14C-BP revealed no significant differences between control and dieldrin-fed fish. There was no selective enhancement of any particular metabolite, or formation of a novel metabolite with dieldrin pretreatment. This research confirmed that enhanced biliary excretion, following chronic dieldrin exposure, was not explained by induction of xenobiotic metabolizing enzymes. The results are consistent with induction of hepatic intracellular trafficking proteins in dieldrin-fed fish.

Temperature-modulated hepatic disposition, covalent binding of radiolabeled genotoxin to hepatic DNA, and cancer incidence in rainbow trout (Oncorhyncus mykiss) were assessed after a single exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Fish (2 g) were acclimated at 10, 14, or 18 degrees C for 1 mo and then exposed to 1 ppm DMBA in their water for 20 h. Exposures were at respective acclimation temperatures, or 10 and 18 degrees C acclimated fish were shifted to 14 degrees C for DMBA exposures. After 4 but not 20 h of exposure, hepatic [(3)H]DMBA equivalents increased with temperature for fish exposed at their respective acclimation temperatures (10 or 18 degrees C). Covalent binding of [(3)H]DMBA to hepatic DNA was similar after 3 d in fish exposed at their respective acclimation temperatures. However, in fish exposed at 14 degrees C, after 3 d the concentration of [(3)H]DMBA covalently bound to hepatic DNA was higher in 10 degrees C than 18 degrees C acclimated fish. After 21 d, covalent binding of [(3)H]DMBA to hepatic DNA was less persistent in 18 degrees C than 10 degrees C acclimated, exposed, and reared fish. There were no differences between temperature-shifted groups at that time. Temperature effects on tumor incidence were assessed 9 mo after DMBA waterborne exposures in fish that were reared at (1) their respective acclimation and exposure temperatures, (2) 14 degrees C after exposure at their respective acclimation temperature, and (3) 14 degrees C after 14 degrees C exposures. Incidence of stomach, liver, and swimbladder cancer increased dramatically with rearing temperature. Differences in tumor incidence were less marked in fish reared at the same temperature (14 degrees C). A strong negative correlation between liver tumor incidence and persistence of [(3)H]DMBA equivalents covalently bound to hepatic DNA suggested increased error-prone DNA repair at warmer temperature played an important role in increased tumor incidence.

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.

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.

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.

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.

Organochlorine (OC) concentrations in surface sediment, snails (Lymnea sp.), and two freshwater fish species (grayling, Thymallus arcticus; and lake trout, Salvelinus namaycush) from four lakes in the US Arctic were determined. In surface sediment, chlorinated benzenes (including hexachlorobenzene, HCB), and p,p'-DDT were the primary analytes detected (max = 0.7 ng/g dry wt), while individual polychlorinated biphenyl (PCB) congeners were always below 0.1 ng/g. A wider range of compounds and higher concentrations were found in lake trout, the top predatory fish species in the same lakes. The concentration ranges for hexachlorocyclohexanes (HCHs), chlordane-related compounds (CHLORs), DDTs, and PCBs in lake trout and grayling were similar to those reported for other arctic freshwater fish (1-100 ng/g wet wt), but one to two orders of magnitude lower than Great Lakes salmonids. Nitrogen isotope analysis confirmed that differences in OC concentrations between grayling and lake trout are explained partly by differences in food web position.

Mercury pollution was compared in two Oregon reservoirs of similar size and age, located within the same ecoregion. Cottage Grove Reservoir was distinguished by a history of mercury mining and processing within its watershed, while Dorena Reservoir was not. Mercury concentrations in sediments of the reservoirs, tributary streams, and three species of fish were measured. Sediment mercury concentrations in the main tributary of Cottage Grove Reservoir, which drains the subbasin where past mercury mining occurred, was tenfold higher than mercury in sediments from other reservoir tributaries. There were no significant differences between sediment mercury concentrations in the tributaries of Dorena Reservoir. The average mercury concentration in the basin sediment of Cottage Grove Reservoir (0.67 +/- 0.05 microg/g dry wt) was higher than for Dorena Reservoir (0.12 +/- 0.01 microg/g dry wt). At Cottage Grove Reservoir, maximum mercury concentrations were near or exceeded 1 microg/g wet wt for largemouth bass (Micropterus salmonides) and bluegill (Lepomis macrochirus) epaxial muscle. Muscle mercury concentrations in largemouth bass and crappie (Pomoxis nigromaculatus) from Cottage Grove Reservoir were significantly higher than from the same species from Dorena Reservoir. Numbers of bluegill of the same age available from both reservoirs were too small for statistical comparisons. Mercury concentrations in largemouth bass muscle fluctuated annually in both reservoirs. Fish ages were consistently positively correlated with muscle mercury concentrations in only the point-source-impacted reservoir. These results indicated that a point source, Black Butte Mine, contributed amounts of mercury greatly in excess of mobilization from natural deposits, atmospheric deposition, and small-scale uses of the metal as an amalgamating agent in gold mining.

Dehydroepiandrosterone (DHEA) is an adrenal steroid with chemoprotective effects against a wide variety of conditions including cancer, obesity, diabetes, and cardiovascular disease. However, DHEA is also a carcinogen in laboratory animals, possibly through its function as a precursor of sex steroids or peroxisome proliferation. The structural analog 16 alpha-fluoro-5-androsten-17-one (8354) has been reported to have enhanced chemopreventive activity without the steroid precursor and peroxisome proliferating effects of DHEA. This study compares DHEA and 8354 in rainbow trout, a species that is resistant to peroxisome proliferation but is highly susceptible to the carcinogenic and tumor enhancing effects of DHEA. Trout were exposed as fry to aflatoxin B1 (AFB1) or given a sham exposure, then were fed diets containing 444 ppm DHEA or 8354 for 6 months. Postinitiation treatment with DHEA significantly increased liver tumor incidence, multiplicity, and size compared to initiated controls. The analog 8354 slightly increased tumor incidence (p = 0.06) but had no effect on multiplicity or size. Six percent of trout treated with DHEA alone developed tumors, whereas no tumors occurred in noninitiated trout fed control or 8354-containing diets. Serum levels of androstenedione were elevated by DHEA (48-fold) or 8354 (6-fold) treatment. Serum beta-estradiol titers were increased in DHEA- but not 8354-treated trout. Vitellogenin was induced significantly by either DHEA (434-fold) or 8354 (21-fold). Peroxisomal beta-oxidation was not increased by either compound and catalase activity was decreased in DHEA-treated animals. Both steroids were potent inhibitors in vitro of trout liver glucose-6-phosphate dehydrogenase with IC50s of 24 and 0.5 microM for DHEA and 8354, respectively. This research suggests that in trout the tumor enhancing effects of DHEA may be due to its function as a sex steroid precursor and are unrelated to peroxisome proliferation. These carcinogenic properties are reduced in the analog 8354 which has been advocated as an alternative to DHEA for chemoprevention.

Pretreatment of mice with chlordecone (CD) reduced hepatic accumulation of a subsequent dose of [14C]CD without significantly changing [14C]CD biotransformation. To determine if CD-induced changes in hepatic [14C]CD accumulation were coincident with altered cell composition, we examined the effects of CD on hepatic protein and lipid content, on fatty acid profiles of liver and kidney, and on the ultrastructure of hepatocytes. SDS-polyacrylamide gel electrophoresis detected an apparent CD dose-related increase in a microsomal protein with a molecular weight of about 23 kDa. Total liver or kidney lipid contents were not altered by CD but relative amounts of several hepatic fatty acids were changed. CD caused marked hepatic mitochondrial swelling, increased amounts of endoplasmic reticulum, apparently increased numbers of peroxisome-like structures, and decreased numbers of lipid droplets in cytoplasm of hepatocytes. Numbers of lipid droplets were not decreased in perisinusoidal fat storage cells. In addition, the numbers of cytoplasmic lipoprotein vesicles were apparently increased in some hepatocytes. Overall these changes indicated an increased hepatocyte secretory activity and suggested that CD changed hepatocellular lipid transport, storage, and metabolism pathways.

As a consequence of the 1991 Gulf War, a substantial amount of crude oil (CO) and partially combusted crude oil (PCO) were emitted into the environment. Therefore, the study objective was to evaluate the toxicity of the water soluble fraction (WSF) of CO and PCO on a fish, Menidia beryllina, and an invertebrate, Palaemonetes pugio, in 16-d flow-through tests. Specific growth rate (SGR) was studied as a function of total petroleum hydrocarbon (TPHC) concentration in water. Reductions in SGR were observed in fish exposed to PCO and CO WSFs, with TPHC water concentration being 10-fold higher in CO exposures (67-145 microg/L) than in PCO exposures (4-12 microg/L). Significant negative correlations were observed between TPHC concentration and fish SGR in both CO (r2=0.730) and PCO (r2=0.867) exposures, with the slope being significantly lower for PCO exposures (-0.169) than CO exposures (-0.009). Differences between CO and PCO toxicity were not as clear in shrimp exposures due to slow growth rates and variability in TPHC concentrations. Qualitative PAH analysis indicated that naphthalene was present in the CO WSF whereas chrysene and benzo(a)pyrene were present in the PCO WSF. Heavy metal analysis of concentrated stock solutions indicated that the PCO WSF had substantially higher concentrations of some metals (Sr=2,521 microg/L, B=556 microg/L, and Ba=130 microg/L) than the CO WSF in which concentrations were less than 55 microg/L. Fish and shrimp tissue analysis did not reveal any uptake of parent PAH compounds from the water, which may be attributed to the formation of PAH metabolites.

We previously demonstrated that pretreatment of rainbow trout with the organochlorine insecticide dieldrin altered in vivo disposition of a subsequent [14C]dieldrin dose. This was not explained by changes in total lipid content or the activity of common xenobiotic metabolizing enzymes. We hypothesized that dieldrin induced hepatic proteins responsible for organochlorine (OC) sequestration, transport, or excretion and that these changes reflected an adaptive response of trout to OC exposure. Here, uptake of 1.18 microM [14C]-dieldrin by precision cut liver slices was increased by dieldrin pretreatment of rainbow trout. Uptake of 0.118 and 1.18 microM [3H]-7,12-dimethylbenz[a]anthracene (DMBA) and efflux of 0.118 microM [3H]DMBA were significantly increased in slices from dieldrin-pretreated trout. Liver slice uptake of 10 but not 1.18 microM [3H]-estradiol and [3H]cholic acid was significantly increased by dieldrin pretreatment. There were no such significant differences for [3H]cholesterol, [3H]cholesterol-oleate, or [3H]oleic acid uptake. Dieldrin pretreatment did not alter hepatic microsomal metabolism of [3H]DMBA or [14C]benzo[a]pyrene or content of six cytochrome P450 isozymes, as quantitated by Western Blot analysis. These results provide further evidence that altered disposition of [14C]dieldrin and [3H]DMBA in dieldrin-pretreated trout was not explained by microsomal enzyme induction but reflected altered processes integral to hepatocellular transmembrane kinetics. These changes may have important implications for OC bioaccumulation by rainbow trout and demonstrate an interaction between dieldrin and DMBA in the absence of cytochrome P450 system induction.

Rainbow trout (initial weight of 4 or 5 g) were acclimated at a cool, 11.0 degrees C (C), a warm, 18.0 degrees C (W), or an intermediate temperature 14.5 degrees C (I) for 1 month. There was a slight difference in hepatic microsomal content of one of six cytochrome P450 isozymes between acclimation groups. Monounsaturated fatty acids in hepatic phosphotidylethanolamine but not phosphotidylcholine increased at lower acclimation temperatures. Saturated fatty acid content decreased with temperature for both phospholipid classes. Fish were exposed to 0.08-0.12 ppm waterborne aflatoxin B1 (AFB1) for 30 min at respective acclimation temperatures or after acute temperature shifts (24 hr) and reared for 9 months at C, I, or W. With exposure concentrations which delivered equivalent target organ doses, trout acclimated, exposed, and reared at C, I, or W had liver tumor incidences of 4, 35, and 61%, respectively. The average number of tumors per liver increased from 1.25-1.34 at C to 2.46-2.66 at W. There were no temperature-dependent differences in tumor diameter. When C- and W-acclimated fish were AFB1 exposed and reared at I, tumor incidence was 12.5% for W-I-shifted fish and 26.5% for C-I-shifted fish. This was consistent with previous work which demonstrated acute downward temperature shift reduced [3H]AFB1 adduction to hepatic DNA. Tumor incidence and multiplicity data suggested manipulation of temperature permitted selective modulation of cancer initiation and promotion in rainbow trout.

Alterations in membrane lipid composition during temperature acclimation of poikilotherms is hypothesized to compensate for direct effects of temperature on membrane fluidity. Temperature also influences disposition and actions of some xenobiotics. This suggests the potential for complex interactions between temperature and metabolism of chemical carcinogens. Whole livers and hepatic microsomes from rainbow trout acclimated at 18 degrees C have more saturated fatty acids and less mono- and polyunsaturated fatty acids than those from fish acclimated at 10 degrees C. Such changes are consistent with a role for membrane lipid fluidity in temperature compensation. When 10 and 18 degrees C acclimated fish are ip injected with 0.4 mg/kg [3H]aflatoxin B1 (AFB1) at their respective acclimation temperatures, hepatic disposition of AFB1, DNA adduction, and biliary metabolites are similar. An acute shift of 18 degrees C acclimated trout to 14 degrees C reduces [3H]AFB-DNA adduct formation, while [3H]AFB1 adduction after acute shift of 10 degrees C acclimated fish to 14 degrees C is no different than in non-shifted fish. Hepatic microsomes isolated from 10 or 18 degrees C acclimated trout, incubated with 10 microM [3H]AFB1 and calf thymus DNA between 6 and 22 degrees C exhibit no differences in the "break points" of Arrhenius plots (16 degrees C in both groups). There is, however, more in vitro DNA adduction of [3H]AFB1 by microsomes from 18 degrees C acclimated fish, a difference abolished by 0.5 mM alpha-naphthoflavone (ANF). These results suggest that temperature acclimation of trout differentially modifies activities of cytochrome P-450 isozymes. When assayed at respective acclimation temperatures, hepatic cytosol from 18 degrees C fish produces more aflatoxicol, a detoxication product of AFB1, than cytosol from 10 degree C fish. Therefore, this soluble enzyme does not exhibit ideal temperature compensation. Such temperature-induced differences in microsomal cytochrome P-450 isozymes and cytosolic dehydrogenase partially explain temperature-modulated AFB1 genotoxicity.

This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle-specific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil-mediated, periportal inflammation appears to play a primary role in progression of alpha-naphthylisothiocyanate-induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane-induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down-regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant-induced cell injury. In oxidative injury, a pH-mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.

Female mink (Mustela vison) are highly sensitive to organochlorine (OC)-induced reproductive impairment. However, mechanisms of this reproductive toxicity are unknown. We have investigated the possible role of steroid receptors in embryotoxicity and reduced neonate weights. Anestrous, juvenile female mink and pregnant adult mink were exposed to 3,3',4,4',5,5'-hexachlorobiphenyl (3HCB), a coplanar polychlorinated biphenyl (PCB), or 2,2',4,4',5,5'-hexachlorobiphenyl (2HCB), a noncoplanar PCB congener. Both congeners impaired 17 beta-estradiol-stimulated (24 hr after ip administration of 100 micrograms E2 beta ip) up-regulation of uterine nuclear estrogen receptors (ERn) in anestrous mink. Embryotoxicity and reduced embryo growth were first observed 14 days after exposure to 0.4 mg 3HCB/kg > 0.8 mg 3HCB/kg > 20 mg 2HCB/kg. In pregnant mink, all 3HCB treatments significantly increased progesterone receptor dissociation constants (PR Kd). ER concentration and PR total receptor number (Rt) were increased by 20 mg 2HCB/kg > 0.8 mg 3HCB/kg, but were unaffected by 0.4 mg 3HCB/kg. Serum E2 beta was below assay detection limits. Progesterone (P) concentrations were increased by 2HCB, decreased by 0.8 mg 3HCB/kg, and unchanged by 0.4 mg 3HCB/kg. Hepatic cytochrome P450 (P450) was induced 1.8-fold in anestrous and 2.2-fold in pregnant mink by 3HCB. Ethoxyresorufin-O-deethylase (EROD) was induced 13- and 4-fold in anestrous and pregnant mink, respectively. 2HCB exposure resulted in decreased P450 concentration in anestrous juveniles, but had no effect on P450 during gestation or EROD activity at any time. We propose that embryotoxicity and retarded embryo growth result from impairment of PR function and that differences in the efficacy of HCB treatments are a result of their dose-dependent, partial estrogenic actions which increase PR Rt via up-regulation of ER.

Previous work demonstrated that pretreatment of mice with low doses of the organochlorine insecticide chlordecone (CD) altered the tissue disposition of a subsequent [14C]CD or [14C]cholesterol challenge dose. The profile of these changes was consistent with the induction of a protein integral to hepatic CD/cholesterol turnover. The present study was undertaken to confirm similar in vivo effects in the rat and to analyze potential CD-induced changes in hepatic transport kinetics in the perfused rat liver. For in vivo experiments, male, Sprague-Dawley rats were treated with CD (5, 15, or 40 mg/kg) and challenged 3 or 7 days later with a 5 mg/kg [14C]CD tracer dose. Rats challenged 3 days after treatment and evaluated 16 hr later showed a dose-dependent decrease in hepatic [14C]CD relative to controls. This decrease could not be attributed to alterations in liver mass or total liver lipid. For kinetics studies, rats received 15 mg/kg CD and livers were perfused 3 days later. Following a brief (5-7 min) single-pass perfusion, the perfusate was replaced with recirculating buffer containing albumin-bound [3H]oleic acid or high-density lipoprotein-bound [14C]CD or [14C]cholesterol. Livers from pretreated animals had significantly decreased rates of [14C]CD and [14C]cholesterol uptake. Efflux of [14C]CD and biliary excretion of [14C]cholesterol were increased. No changes were observed in uptake or biliary excretion of [3H]oleic acid. SDS-PAGE of hepatic cytosol revealed an enhanced band intensity corresponding to a M(r) of 25,600 in livers from pretreated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Previous work demonstrated that exposure of laboratory animals including fish to certain organochlorine (OC) insecticides altered the tissue distribution of a subsequent tracer dose of the same [14C]OC. In the present study, 10- to 20-g rainbow trout were exposed to 15 ppm dieldrin in the diet. Fish were subsequently challenged at 2-week intervals with an intraperitoneal injection of 0.1 mg/kg [14C]dieldrin and viscera (liver, bile, mesenteric fat, kidney, and intestine) analyzed for radioactivity, 24 hr later. After 10 and 12 weeks of dieldrin pretreatment, [14C]dieldrin was significantly elevated relative to controls in liver (200%), bile (500%), and fat (500 and 1200% for 10 and 12 weeks, respectively) of pretreated fish. Other tissues were unchanged. Chloroform/methanol extractions revealed a time-dependent increase in label disposition to carcass lipid in controls but not in pretreated fish. Altered disposition could not be explained by changes in total body lipid or induction of total cytochrome P-450 or ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase, glutathione S-transferase, or UDP glucuronosyltransferase activities. In vivo assessment of [14C]dieldrin metabolism revealed no increase in hepatic and only a slight (22%) increase in biliary polar:nonpolar concentration ratio after 9 weeks 20 ppm dieldrin pretreatment. Results suggest that constitutive changes in liver integral to dieldrin sequestration, transport, or excretion may be an adaptive response of trout to chronic OC exposure.

Previous work showed that the incidence of chemically induced tumors in fish increased with environmental temperature. The present study assessed genotoxicity as a mechanism to explain such results. Rainbow trout (2 g) were acclimated to 10 or 18 degrees C for 1 month and then immersed in 0.1 ppm [3H]aflatoxin B1 (AFB1) solutions for 30 min at their respective acclimation temperatures, or at 14 degrees C. The total radioactivity in liver immediately after immersions was 50% higher for 18- than 10 degrees C-acclimated and exposed fish. Conversely, adduction of [3H]AFB1 to hepatic DNA of 10 degrees C-acclimated fish was higher than that of 18 degrees C-acclimated fish after exposure. A similar DNA adduction result was observed after [3H]AFB1 immersion concentrations were varied to achieve similar hepatic [3H]AFB1 equivalents. After acute shifts in temperature (10 to 14 degrees C or 18 to 14 degrees C), no differences were found in hepatic [3H]AFB1 equivalents. However, adduction of [3H]AFB1 to hepatic DNA was higher for 10- than for 18 degrees C-acclimated fish 1 day after exposures at 14 degrees C. This was probably explained by effects of temperature acclimation on the status of the membrane-bound cytochrome P-450 system, affecting its competition with a detoxifying cytosolic enzyme. After [3H]AFB1 immersions at respective acclimation temperatures, or acute temperature shifts to 14 degrees C, DNA adducts were less persistent in fish maintained for 21 days at 18 degrees C than at 10 degrees C. Our data demonstrated that temperature-modulated AFB1 genotoxicity occurred via three mechanisms: hepatic disposition and formation and persistence of DNA adducts.

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.

Previous work demonstrated that mixed function oxidase activities of hepatic microsomes from cold- and warm-acclimated rainbow trout were similar when assayed at temperatures to which fish were acclimated. This "ideal temperature compensation" was partially explained by constitutive differences in microsomes. In the work reported here, rainbow trout were acclimated at 10 or 18 degrees C for 4 weeks and then ip injected with 10 mumol [3H] or [14C]benzo[a]pyrene (BP)/kg in one of two temperature regimens. First, fish were acclimated and exposed at the same temperature and killed after 4, 24, or 48 hr. Concentrations of [3H]BP equivalents in liver, bile, and fat but not in plasma, muscle, intestine, gill, or kidney increased with time. There were no differences in hexane or ethyl acetate extractable [3H] or [14C]BP tissue concentrations in 10 and 18 degrees C-acclimated fish exposed at their acclimation temperatures. At 24 hr after injection, biliary excretion of [3H]BP equivalents was about twofold higher at 18 degrees C than at 10 degrees C. Therefore, warmer temperature stimulated biliary excretion without a marked effect on in vivo BP metabolism. In the second regimen, 10 and 18 degrees C-acclimated fish were shifted to 14 degrees C, injected with [3H] or [14C]BP 1 hr later, and killed after an additional 24 hr. There were no differences in tissue concentrations of total [3H]BP equivalents between acclimation groups at 14 degrees C. However, the biliary concentration of [14C]BP not extracted by ethyl acetate was significantly higher in bile from 10 degrees C-acclimated fish than from 18 degrees C-acclimated fish when both groups were exposed at 14 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlordecone (CD) treatment of rat liver plasma membranes (LPM) provided in vitro evidence for mechanisms of in vivo liver dysfunction caused by CD. LPM preparations enriched 14- to 19-fold in the bile canalicular markers gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were isolated from male Sprague-Dawley rats. CD inhibited the bile canalicular-specific active transport of Na(+)-stimulated L-[3H]glutamate in LPM vesicles. CD (0.08 and 0.5 mumol/mg protein) reduced both the initial velocity and the maximum level of Na(+)-stimulated L-[3H]glutamate uptake without significantly reducing Na(+)-independent uptake. In vitro treatment of LPM with CD (0.2-1.0 mumols/mg protein) also reduced the mobility of a 16-doxyl stearate spin label probe in a concentration-dependent manner. No change in mobility was apparent at CD concentrations below 0.2 mumol/mg protein. These results demonstrated that CD impaired a bile canalicular-specific transport system and induced liver plasma membrane perturbation. Na(+)-stimulated L-[3H]glutamate uptake was more sensitive to CD than was detectable immobilization of the spin label probe.

Lipid partitioning of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inadequately explains its tissue distribution since higher concentrations occur in liver than fat except at high doses. This study provides in vivo evidence that an inducible, saturable system plays a predominant role in disposition of [14C]TCDD in female mice at doses between 5 and 20 micrograms/kg. Female C57BL/6J mice were gavaged with 0, 5, or 15 micrograms TCDD/kg, received a subsequent gavage of 5 or 20 micrograms [14C]TCDD after 6 days, and were killed 1 day later. In mice pretreated with 5 and 15 micrograms TCDD/kg and subsequently dosed with 20 micrograms [14C]TCDD/kg, liver weight and [14C]TCDD concentration increased. Total liver [14C]TCDD burden increased about 50% in both pretreatment groups. Concentrations of [14C]TCDD in kidney, fat, heart, lung, gastrointestinal tract, but not plasma or splenic lymphocytes, decreased in a reciprocal manner. Alterations in absorption, concentrations of polar metabolites of [14C]TCDD in liver, and hepatic lipid content failed to explain these results. About 97% of hepatic 14C was hexane extractable. HPLC of this extract indicated [14C]TCDD was the only significant nonpolar form of radiolabel in liver. In mice pretreated with 5 micrograms TCDD/kg and subsequently dosed with 5 micrograms [14C]TCDD/kg, a more marked pretreatment disposition response was observed. These results are consistent with a predominant role for an inducible, high affinity, low capacity system in whole animal pharmacokinetics of TCDD.

1. The possibility that temperature acclimation (to 10 or 18 degrees C for 28 days) would alter the cytochromes P-450 of rainbow trout was addressed. 2. The specific content of LM4b (P-450 IA1), the trout isozyme responsible for activation of polynuclear aromatic hydrocarbons, was lower in 18 degrees C fish than it was in 10 degrees C fish. 3. Kinetic analysis of aryl hydrocarbon hydroxylase indicated that, while thermal acclimation caused no change in Vmax, it lowered the apparent Km of this enzyme for benzo[a]pyrene when assayed at acutely shifted temperatures. 4. Thermal acclimation of fish may have significance when feral populations are subjected to acute temperature shifts.

Sexually immature Shasta strain rainbow trout (Salmo gairdneri) of either sex were acclimated at 10, 14 or 18 degrees C for at least 4 weeks and plasma pharmacokinetics and biliary excretion of i.p. injected [14C]taurocholate (TC) examined in spinally transected or free-swimming fish, respectively. Plasma elimination half-lives but not absorption rate constants for [14C]TC (10 mumol/kg) were about two-fold reduced in 18 as compared to 10 or 14 degrees C acclimated fish. Distribution of [14C]TC to tissues other than plasma, liver, bile and small intestine was not different in 10, 14 or 18 degrees C acclimated free-swimming fish at 1 or 4 hr post-injection. Biliary excretion of [14C]TC (7.5-10 mumol/kg) at 1 hr post-injection was significantly higher in 14 and 18 as compared to 10 degrees C acclimated fish.

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.

Environmental toxicant exposure commonly vary in terms of duration and concentration. However, laboratory toxicity tests usually entail continuous exposures to constant concentrations. We compared survival, growth, and toxicant accumulation in early life-stage steelhead trout intermittently or continuously exposed to fenvalerate (FV) for 70 d after fertilization. Acute lethality was assessed in ancillary 96-h LC50 determinations with juvenile fish. Intermittent exposures were daily 4.5-h introductions of toxicant, and continuous exposures were to constant concentrations. All tests were conducted in a flow-through dilution apparatus, and mean concentrations for the entire exposure period were calculated for comparisons between regimens. The respective 96-h LC50 values for intermittently and continuously exposed were 88 and 172 ng/l. In the subchronic study, marked lethality (32%) and reduced terminal weight (50%) were found following exposures to cyclic FV concentrations that yielded an average of 80 ng/l (peak of 461). Continuous exposure to 80 ng FV/l did not affect these parameters. At mean FV concentrations above 20 ng/l, bioaccumulation was greater following intermittent than continuous exposure. Interaction of partitioning and elimination processes may partially explain differences in FV accumulation and subsequent toxicity.

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.