Environmental survey in the Tuul and Orkhon River basins of north-central Mongolia, 2010: metals and other elements in streambed sediment and floodplain soil.

Streambed sediment and subsurface floodplain soil were sampled for elemental analyses from 15 locations in river basins of north-central Mongolia during August 2010. Our primary objective was to conduct a reconnaissance-level assessment of potential inputs of toxicologically important metals and metalloids to Lake Baikal, Russia, that might originate from mining and urban activities within tributaries of the Selenga River in Mongolia. Samples were collected in triplicate from all sites, then dried, and sieved to <2 mm for analysis by portable X-ray florescence spectroscopy and by inductively coupled plasma mass spectrometry after digestion with concentrated nitric and hydrochloric acids. Arsenic, copper, and mercury were greatly elevated in sediment and floodplain soil collected from tributary streams located near two major mining operations. Lead and zinc were moderately elevated in streambed sediment and in floodplain soil obtained from a small tributary in the capital city of Ulaanbaatar, but those concentrations were considerably less than probable effects benchmarks. Historical and possibly present mining activities have led to considerable metal contamination in certain tributaries of the Orkhon River in north-central Mongolia; however, metals originating from those sources did not appear to be accumulating in sediments at our downstream-most sampling sites located near the border between Mongolia and Russia.

Behavioral response and kinetics of terrestrial atrazine exposure in American toads (Bufo americanus).

Amphibians in terrestrial environments obtain water through a highly vascularized pelvic patch of skin. Chemicals can also be exchanged across this patch. Atrazine (ATZ), a widespread herbicide, continues to be a concern among amphibian ecologists based on potential exposure and toxicity. Few studies have examined its impact on the terrestrial juvenile or adult stages of toads. In the current study, we asked the following questions: (1) Will juvenile American toads (Bufo americanus) avoid soils contaminated with ATZ? (2) Can they absorb ATZ across the pelvic patch? (3) If so, how is it distributed among the organs and eventually eliminated? We conducted a behavioral choice test between control soil and soil dosed with ecologically relevant concentrations of ATZ. In addition, we examined the uptake, distribution, and elimination of water dosed with (14)C-labeled ATZ. Our data demonstrate that toads do not avoid ATZ-laden soils. ATZ crossed the pelvic patch rapidly and reached an apparent equilibrium within 5 h. The majority of the radiolabeled ATZ ended up in the intestines, whereas the greatest concentrations were observed in the gall bladder. Thus, exposure of adult life stages of amphibians through direct uptake of ATZ from soils and runoff water should be considered in risk evaluations.

Accumulation of environmental contaminants in wood duck (Aix sponsa) eggs, with emphasis on polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans.

We measured polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), organochlorine pesticides, and mercury in wood duck (Aix sponsa) eggs collected near a North Carolina (USA) bleached kraft paper mill. Samples were taken a decade after the mill stopped using molecular chlorine. Using avian toxic equivalency factors, 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalent (TEQ) concentrations were 1-30 pg/g fresh wet weight in eggs (n = 48) collected near the mill in 2002-2005 and were significantly higher than those from a reference site (<1 pg/g) 25 km away. Geometric mean wood duck egg TEQs (6 pg/g) were one-fifth those measured at this site prior to the cessation of molecular chlorine bleaching. Concentrations of mercury in wood duck eggs from nests of the Roanoke River sites ranged from 0.01 to 0.14 microg/g (geometric mean, 0.04 microg/g) and were significantly higher than those from the reference site, where concentrations did not exceed 0.04 microg/g (geometric mean, 0.02 mug/g). All concentrations were lower than those associated with adverse effects in birds. The congener profiles, lack of contamination in reference site eggs, and decline in contaminant concentrations after process changes at the mill provide strong evidence that mill discharges influenced contamination of local wood duck eggs. Collectively, the results indicate that the wood duck is an effective sentinel of the spatial and temporal extent of PCDD, PCDF, and mercury contamination.

Embryo toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the wood duck (Aix sponsa).

We examined the sensitivity of the wood duck (Aix sponsa) embryo to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by injecting the toxicant into their eggs. Six groups of wood duck eggs (n = 35 to 211 per trial) were injected with 0 to 4600 pg TCDD/g egg between 2003 and 2005. Injections were made into yolk prior to incubation, and eggs were subsequently incubated and assessed weekly for mortality. Significant TCDD-induced mortality was not observed through day 25 (90% of incubation). Liver, heart, eye, and brain histology were generally unremarkable. Hepatic ethoxyresorufin-O-deethylase activity, a biomarker of dioxin-like compound exposure, was induced by 12-fold in the 4600 pg/g treatment relative to controls. The median lethal dose for chicken (Gallus domesticus) eggs we dosed identically to wood duck eggs was about 100 pg/g, similar to other assessments of chickens. Among dioxin-like compound embryo lethality data for 15 avian genera, the wood duck 4600 pg/g no-observed-effect level ranks near the middle. Because no higher doses were tested, wood ducks may be like other waterfowl (order Anseriformes), which are comparatively tolerant to embryo mortality from polychlorinated dibenzo-p-dioxins and dibenzofurans when exposed by egg injection.

Effect of In Ovo exposure to an organochlorine mixture extracted from double crested cormorant eggs (Phalacrocorax auritus) and PCB 126 on immune function of juvenile chickens.

Organochlorine (OC) contaminants including polychlorinated biphenyls (PCBs) and p, p'-dichlorodiphenyldichloroethylene (DDE) have been associated with immune modulation in wild fish-eating birds from the Great Lakes. The objective of this study was to evaluate the immune function of juvenile chickens after in ovo exposure to PCB 126 or an environmentally relevant OC mixture extracted from eggs of double crested cormorants (Phalacrocorax auritus) from Green Bay, Lake Michigan, USA. Fertile white leghorn chicken (Gallus domesticus) eggs were injected before incubation with 0.55-1.79 ng TCDD equivalents (TEQ)/egg PCB 126 and 1.2-4.9 ng TEQs/egg of cormorant egg extract into the air cell in two separate experiments. After hatching, the immune function was tested using in vivo phytohemagglutinin (PHA) skin response in 11-day-old chicks, antibody titers to immunization with sheep red blood cells (SRBC) in 28-day-old chicks, and, at necropsy, thymus and bursal mass and cellularity. PCB 126 decreased antibody titers at all doses and decreased the thymus and bursa index but not cellularity at 1.79 ng TEQ/egg. The cormorant egg extract caused no significant alterations in immune function even though it has been demonstrated as immunotoxic in chicken embryos. However, twofold to threefold increases in total anti-SRBC titers in 28-day-old chicks exposed to 1.2 or 2.4 ng TEQ/egg of cormorant extract were similar to elevations in anti-SRBC titer observed in Caspian tern (Sterna caspia) chicks from a highly OC-contaminated site in Saginaw Bay, Lake Huron. Posthatch exposure to OC through fish consumption in addition to in ovo OC exposure might be associated with the immune modulation reported in wild birds. Chicks in this study might have begun to compensate for embryonic immunotoxicity by the ages at which we studied them.

Biomarkers of contaminant exposure in Northern Pike (Esox lucius) from the Yukon River Basin, Alaska.

As part of a larger investigation, northern pike (n = 158; Esox lucius) were collected from ten sites in the Yukon River Basin (YRB), Alaska, to document biomarkers and their correlations with organochlorine pesticide (total p,p'-DDT, total chlordane, dieldrin, and toxaphene), total polychlorinated biphenyls (PCBs), and elemental contaminant (arsenic, cadmium, copper, lead, total mercury, selenium, and zinc) concentrations. A suite of biomarkers including somatic indices, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, vitellogenin concentrations, steroid hormone (17B- ustradiol and 16-kebtestosteront) concentrations, splenic macrophage aggregates (MAs), oocyte atresia, and other microscopic anomalies in various tissues were documented in YRB pike. Mean condition factor (0.50 to 0.68), hepatosomatic index (1.00% to 3.56%), and splenosomatic index (0.09% to 0.18%) were not anomalous at any site nor correlated with any contaminant concentration. Mean EROD activity (0.71 to 17.51 pmol/min/mg protein) was similar to basal activity levels previously measured in pike and was positively correlated with selenium concentrations (r = 0.88, P < 0.01). Vitellogenin concentrations in female (0.09 to 5.32 mg/mL) and male (<0.0005 to 0.097 mg/mL) pike were not correlated with any contaminant, but vitellogenin concentrations >0.01 mg/mL in male pike from multiple sites indicated exposure to estrogenic compounds. Mean steroid hormone concentrations and percent oocyte atresia were not anomalous in pike from any YRB site. Few site differences were significant for mean MA density (1.86 to 6.42 MA/mm(2)), size (812 to 1481 microm(2)), and tissue occupied (MA-%; 0.24% to 0.75%). A linear regression between MA-% and total PCBs was significant, although PCB concentrations were generally low in YRB pike (< or =63 ng/g), and MA-% values in female pike (0.24% to 0.54%) were lower than in male pike (0.32% to 0.75%) at similar PCB concentrations. Greater numbers of MAs were found as zinc concentrations increased in YRB female pike, but it is unlikely that this is a causative relationship. Histological abnormalities observed in gill, liver, spleen, and kidney tissues were not likely a result of contaminant exposure but provide information on the general health of YRB pike. The most common histologic anomalies were parasitic infestations in various organs and developing nephrons and nephrocalcinosis in posterior kidney tissues. Overall, few biomarker responses in YRB pike were correlated with chemical contaminant concentrations, and YRB pike generally appeared to be healthy with no site having multiple anomalous biomarker responses.

The H4IIE cell bioassay as an indicator of dioxin-like chemicals in wildlife and the environment.

The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

Cytochrome P450 1A expression in midwater fishes: potential effects of chemical contaminants in remote oceanic zones.

Cytochrome P450 1A (CYP1A) induction is a robust marker for exposure to polynuclear aromatic hydrocarbons and planar halogenated aromatic hydrocarbons that are aryl hydrocarbon receptor agonists. We examined CYP1A expression in mesopelagic fishes from the western North Atlantic. Individuals in 22 species were obtained from slope water and the Sargasso Sea in 1977, 1978, and 1993. Aryl hydrocarbon hydroxylase (AHH), a CYP1A activity, was detected in liver from all species in 1977/78. In some, including Gonostoma elongatum, AHH was inhibited by the CYP1A inhibitor alpha-naphthoflavone. CYP1A-dependent ethoxyresorufin O-deethylase (EROD) was detected in liver microsomes of all species in 1993; rates were highest in G. elongatum and Argyropelecus aculeatus. Immunoblot analysis with the CYP1A-specific monoclonal antibody 1-12-3 detected a single microsomal protein band in most 1993 samples; the highest content was in G. elongatum. Immunohistochemical analysis showed CYP1A staining in gill, heart, kidney, and/or liver of several species. Extracts of the 1993 G. elongatum and A. aculeatus, when applied to fish hepatoma cells (PLHC-1) in culture, elicited a significant induction of EROD in those cells. The capacity of the extracts to induce CYP1A correlated with the content of PCBs measured in the same fish (2-4.6 ng/g total body weight). Mesopelagic fish in the western North Atlantic, which experience no direct exposure to surface waters or sediments, are exposed chronically to inducers of CYP1A at levels that appear to be biochemically active in those fish.

Accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin by rainbow trout (Onchorhynchus mykiss) at environmentally relevant dietary concentrations.

Rainbow trout were fed a diet containing 1.8, 18, or 90 pg/g 3H-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for up to 320 d. Concentrations of TCDD were determined in muscle, liver, and ovaries at 100, 150, 200, and 250 d. Concentrations of TCDD reached an apparent steady-state concentration in liver after 100 d of exposure, whereas concentrations in other tissues continued to increase until 150 d of exposure. The greatest portion of the total mass of TCDD was present in the muscle tissue with lesser proportions in other organs. As the ovaries developed before spawning, an increase occurred in the total mass of TCDD present in this tissue. The assimilation rate of TCDD during the initial 100 d of the exposure was determined to be between 10 and 30%. This is somewhat less than estimates derived based on both uptake and elimination constants determined during shorter exposures. Biomagnification factors (BMFs) were estimated for all tissues and exposure concentrations, and at all exposure periods. Lipid-normalized BMFs for muscle ranged from 0.38 to 1.51, which is consistent with the value of 1.0 predicted from fugacity theory. Uptake and depuration rate constants were determined and used to predict individual organ TCDD concentrations. Comparison with observed values indicated that the model could be used to predict tissue concentrations from the known concentrations of TCDD in food. This model will allow more refined risk assessments by predicting TCDD concentrations in sensitive tissues such as developing eggs.

Regulation of subcellular localization of the aryl hydrocarbon receptor (AhR).

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and other xenobiotics. In the absence of exogenous ligand, AhR is cytosolic. We investigated how AhR is retained in the cytosol and how dioxin induces AhR to move to the nucleus. Disruption of nuclear export of AhR by the nuclear export inhibitor leptomycin B (LMB) or by mutation of the AhR nuclear export signal resulted in nuclear accumulation of AhR in the absence of exogenous ligand. Mutation of the AhR nuclear localization signal resulted in defects in nuclear import of AhR in both the presence and the absence of exogenous ligand. Dioxin treatment caused a more rapid accumulation of AhR in the nucleus than LMB treatment. In the presence of both dioxin and LMB, nuclear accumulation of AhR was more rapid than in the presence of dioxin alone. Our results show that AhR shuttles between the nucleus and the cytosol in the absence of exogenous ligand. Binding of ligand induces an increase in the rate of nuclear import of AhR but does not eliminate nuclear export of AhR.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos.

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC(50) and LD(50) of TCDD for Fundulus embryos were approximately 19.7+/-9.5 pg TCDD/microl (water bath) and 0.25+/-0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction.

Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure.

This review compiles and evaluates existing scientific information on the use, limitations, and procedural considerations for EROD activity (a catalytic measurement of cytochrome P4501A induction) as a biomarker in fish. A multitude of chemicals induce EROD activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit EROD induction/activity, this interference is generally not a drawback to the use of EROD induction as a biomarker. The various methods of EROD analysis currently in use yield comparable results, particularly when data are expressed as relative rates of EROD activity. EROD induction in fish is well characterized, the most important modifying factors being fish species, reproductive status and age, all of which can be controlled through proper study design. Good candidate species for biomonitoring should have a wide range between basal and induced EROD activity (e.g., common carp, channel catfish, and mummichog). EROD activity has proven value as a biomarker in a number of field investigations of bleached kraft mill and industrial effluents, contaminated sediments, and chemical spills. Research on mechanisms of CYP1A-induced toxicity suggests that EROD activity may not only indicate chemical exposure, but also may also precede effects at various levels of biological organization. A current research need is the development of chemical exposure-response relationships for EROD activity in fish. In addition, routine reporting in the literature of EROD activity in standard positive and negative control material will enhance confidence in comparing results from different studies using this biomarker.

Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC)--measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts.

The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree of chlorination.

Effects of methyl testosterone exposure on sexual differentiation in medaka, Oryzias latipes.

Studies were conducted to characterize effects of a known androgen on sexual differentiation and development of medaka, Oryzias latipes (d-rR strain), at two life stages. Embryos were injected with graded doses of methyl testosterone (MT) prior to epiboly. The occurrence of sex-reversal, and the gonadosomatic index (GSI) were evaluated in adults. Primary germ cells were counted and gonad volumes calculated for larvae to determine if sex-reversal could be detected at an early life stage. Sex-reversal of genetic females to phenotypic males was observed at both life stages. The GSI for phenotypic females was greater than for phenotypic males, while the GSI in XX males was similar to XY males. MT appeared to reduce the GSI of XX females exposed to MT but not sex-reversed. Our results indicate that embryonic exposure to androgens influences sexual development in medaka. Utilizing the d-rR strain of medaka allows detection of an effect as early as 2 weeks after chemical exposure making this a useful tool to screen chemicals for effects on sexual differentiation.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Aryl hydrocarbon receptor function in early vertebrates: inducibility of cytochrome P450 1A in agnathan and elasmobranch fish.

The mammalian aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that controls the expression of cytochrome P450 1A (CYP1A) genes in response to halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand and normal physiologic function of this protein are as yet unknown. One approach to understanding AHR function and significance is to determine the evolutionary history of this receptor and of processes such as CYP1A induction that are controlled by the AHR in mammals. In these studies, AHR function was evaluated in representative cartilaginous fish (little skate, Raja erinacea) and jawless fish (sea lamprey, Petromyzon marinus and Atlantic hagfish, Myxine glutinosa), using CYP1A induction as a model AHR-dependent response. Treatment of skate with beta-naphthoflavone (BNF) caused an 8-fold increase in hepatic ethoxyresorufin O-deethylase (EROD) activity as well as a 37-fold increase in the content of immunodetectable CYP1A protein. Evidence of CYP1A inducibility was also obtained for another cartilaginous fish, the smooth dogfish Mustelus canis. In contrast, hepatic EROD activity was not detected in untreated lamprey nor in lamprey treated with 3,3'4,4'-tetrachlorobiphenyl (TCB), a potent AHR agonist in teleosts. A possible CYP1A homolog was detected in lamprey hepatic microsomes by one of three antibodies to teleost CYP1A, but expression of this protein was not altered by TCB treatment. CYP1A protein and catalytic activity were measurable in hagfish, but neither was induced after treatment with TCB. These results suggest that the AHR-CYP1A signal transduction pathway is highly conserved in gnathostomes, but that there may be fundamental differences in AHR signaling or AHR-CYP1A coupling in agnathan fish. Agnathan fish such as hagfish and lamprey may be interesting model species for examining possible ancestral AHR functions not related to CYP1A regulation.

Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptotic cell death in the embryonic vasculature with embryotoxicity.

Vertebrate embryos are particularly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for understanding the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apoptosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis indicated that TCDD-induced programmed cell death in the embryonic medial yolk vein was mechanistically linked to embryo mortality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic development. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A expression and TCDD-induced programmed cell death. TCDD-induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post-hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds.

Carrier effects of dosing the H4IIE cells with 3,3',4,4'-tetrachlorobiphenyl (PCB77) in dimethyl sulfoxide or isooctane.

A rat hepatoma cell line, H4IIE, serves as a bioassay tool to assess the potential toxicity of dioxin-like chemicals, including polychlorinated biphenyls (PCB) in environmental samples. PCB exposure to these cells induces cytochrome (CYP) P4501A1 activity in a dose-dependent fashion, thus allowing assessment of mixtures. The objective of this study was to determine the effect of different carriers, dimethyl sulfoxide (DMSO) and isooctane on the concentrations of PCBs in the H4IIE cells and induction of CYP1A1 activity as measured by ethoxyresorufin O-deethylase (EROD) activity. H4IIE cells were dosed with three micrograms of UL-14C-PCB77/plate dissolved in DMSO or isooctane, and were harvested at sequential time periods for 4 days. PCB77 concentration and EROD activity were measured in the cells. EROD activity was greater when using DMSO as compared to isooctane, while there was no difference in the distribution of PCB77-derived radioactivities within the cell culture system based upon the carrier solvent used to deliver PCB77.

Retinoids in eggs and embryos of birds fed fish from the Great Lakes.

Retinoids were analyzed in 11-day chick embryos and eggs from white Leghorn hens (Gallus domesticus) fed environmentally-derived polychlorinated biphenyls (PCB) in carp (Cyprinus carpio) from Saginaw Bay. The yolks and the embryos contained all-trans-retinol, 3,4-didehydroretinol and retinyl esters. There was no significant difference in the total retinoid content in the yolks of 11-day incubated eggs among hens fed for 7 weeks diets containing 0.5-6.6 mg PCB/kg diet. However, the proportional amount of retinols in the high (6.6 mg) PCB group was significantly less than that in low (0.5 mg) PCB controls, while the amount of retinyl palmitate in the high PCB group was significantly greater than that in the controls. Retinoids in the embryos were not affected by any of the PCB levels fed to hens for 7 weeks prior to laying the eggs. The 50% reduction in the molar ratio of retinols to retinyl palmitate in the yolks of eggs as the result of the high PCB level fed to hens for 7 weeks can serve as an indicator for chronic exposure to PCB contamination at the level of 6.6 mg or higher PCB/kg diet.

In vivo/in vitro comparison of the pharmacokinetics and pharmacodynamics of 3,3',4,4'-tetrachlorobiphenyl (PCB77).

The rat hepatoma cell line, H4IIE, serves as a useful tool to assess potential biological effects such as induction of cytochrome P4501A1 expression. The objectives of this study were twofold: to investigate the kinetic time course and dosimetry of PCB77 in rat hepatoma cells dosed with PCB77 and in liver of rats given i.p. doses of PCB77, and to compare in vitro and in vivo P4501A1 enzyme induction responses. For the 4-day time-course study, H4IIE cells were exposed with two doses of [14C]PCB77 (0.9 and 3 microg/plate) and harvested at 15 and 30 min, 1, 2, 4, 8, and 12 hr, and 1, 2, 3, and 4 days. PCB77-derived radioactivity was detected in the cells as early as 15 min postdosing. For the dose-response study, the cells were dosed with various concentrations of PCB77 (0.00316-5.37 microg/plate) and harvested on Day 3 since ethoxyresorufin O-deethylase (EROD) activity in vitro reached its maximum on the third day postdosing. Time-course and dose-response studies revealed that only 1-3% of the total delivered dose was found in the cells, with the remainder in the media and adhering to the culture plates. For the dose-response study in vivo, male Fischer rats were dosed with a single i.p. injection of various concentrations of PCB77 (0.1-50 mg/kg body wt) and euthanized on Day 3. PCB77-derived radioactivity and EROD induction in vivo were measured. When EROD activity and PCB77-derived radioactivity in the rat hepatoma cells and in the rat liver were compared on an equivalent weight basis, there was a significant correlation (r2 = 0.985) between them. Prior to this study, no information on quantitative dosimetry and EROD activities of PCB77 has been reported to validate the in vitro assay with in vivo data.