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.

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.

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.

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.

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.

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.

Organochlorine pesticides and polychlorinated biphenyls (PCBs) in sediments and biota from four US Arctic lakes.

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 distribution in sediments and bioaccumulation by fish in two oregon reservoirs: point-source and nonpoint-source impacted systems.

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.

Comparison of the enhancing effects of dehydroepiandrosterone with the structural analog 16 alpha-fluoro-5-androsten-17-one on aflatoxin B1 hepatocarcinogenesis in rainbow trout.

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.

Ultrastructural, protein, and lipid changes in liver associated with chlordecone treatment of mice.

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.

Subacute toxicity of the water-soluble fractions of Kuwait crude oil and partially combusted crude oil on Menidia beryllina and Palaemonetes pugio.

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.

Dieldrin pretreatment alters [14C]dieldrin and [3H]7,12-dimethylbenz[a]anthracene uptake in rainbow trout liver slices.

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.

Temperature-modulated incidence of aflatoxin B1-initiated liver cancer in rainbow trout.

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.

In vitro and in vivo temperature modulation of hepatic metabolism and DNA adduction of aflatoxin B1 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.

Novel mechanisms in chemically induced hepatotoxicity.

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.

2,2',4,4',5,5'- and 3,3',4,4',5,5'-hexachlorobiphenyl alteration of uterine progesterone and estrogen receptors coincides with embryotoxicity in mink (Mustela vision).

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.

Chlordecone pretreatment alters [14C]chlordecone and [14C]cholesterol transport kinetics in the perfused rat liver.

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)