Alan L. Blankenship

Toxicity Reference Values for the Toxic Effects of Polychlorinated Biphenyls to Aquatic Mammals

Threshold tissue residue concentrations of polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) were derived from the published results of semi-field (i.e., field collected food items were used as a medium of exposure to PCBs in laboratory reared animals) or field toxicity studies conducted with seals, European otters and mink. Based on biomagnification factors (BMFs) and concentrations of PCBs or TEQs measured in fish fed in the diet of experimental aquatic mammals, dietary threshold concentrations were estimated. Hepatic vitamin A, thyroid hormone concentration, suppression of natural killer (NK) cell activity and proliferative response of lymphocytes to mitogens were the toxicity endpoints measured in aquatic mammals. Threshold concentrations for PCBs or TEQs in livers of aquatic mammals to elicit the physiological effects ranged from 6.6 to 11 µg PCBs/g (geometric mean: 8.7 µ/g) and 160 to 1400 pg TEQs/g (geometric mean: 520 pg/g), lipid weight, respectively. The BMFs for PCBs and TEQs varied depending on the marine mammal species, and therefore the dietary threshold concentrations could be referred only by a range of values (rather than a mean value), which were 10 to 150 ng PCBs/g and 1.4 to 1.9 pg TEQs/g, wet weight, for PCBs and TEQs, respectively.

Interactions between aryl hydrocarbon receptor (AhR) and hypoxia signaling pathways

Most if not all of the toxic responses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated through the AhR, which requires ARNT to regulate gene expression. ARNT is also required by HIF-1alpha to enhance the expression of various genes in response to hypoxia. Since both the AhR and hypoxia transcriptional pathways require ARNT, some of the effects of TCDD and similar types of ligands could be explained by interaction between the AhR and hypoxia pathways involving ARNT. The studies on which we report here were conducted to test the hypothesis that there is cross talk between AhR- and HIF-1-mediated transcription pathways. TCDD significantly reduced the hypoxia-mediated reporter gene activity in B-1 cells. Reciprocally, the hypoxia response inducers desferrioxamine or CoCl(2) inhibited AhR-mediated CYP1A1 enzyme activity in B-1 and Hepa 1 cells, and the AhR-mediated luciferase reporter gene activity in H1L1.1c2 cells. The inhibition of AhR-mediated transcription by hypoxia inducers, however, was not observed in H4IIE-luc cells. The interaction between the AhR- and HIF-1-mediated transcription can be attributed to changes in DNA binding activities. TCDD-induced protein binding to dioxin responsive element (DRE) was diminished by desferrioxamine, and TCDD reduced the binding activity to HIF-1 binding site in desferrioxamine-treated Hepa 1 cells. This mutual repression may provide an underlying mechanism for many TCDD-induced toxic responses. The results reported here indicate that there is cross talk between ARNT-requiring pathways. Since ARNT is possibly required by a number of pathways, this type of interaction may explain some of the pleiotropic effects caused by TCDD.

Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in birds has been well-established in laboratory and field studies. Observed effects of TCDD and related chemicals in birds include developmental deformities, reproductive failure, liver damage, wasting syndrome and death. The mechanism of action of TCDD at the cellular level is primarily mediated through the aryl hydrocarbon receptor (AhR). However, the mechanism of toxic action at the organism level is poorly understood. In this study, the role of radical oxygen species and mixed function oxidize (MFO; cytochrome P4501A) in the mechanism of TCDD-induced abnormalities and lethality were examined by co-injecting radical scavengers and an MFO inhibitor (piperonyl butoxide). Egg injection studies were conducted to determine if in ovo TCDD exposure can cause oxidative stress in white leghorn chicken eggs. Test agents were injected into the yolk prior to incubation. Treatments included TCDD (150 ng/kg), triolein (vehicle control), and various co-treatments including MnTBAP (a mimetic of superoxide dismutase), piperonyl butoxide, piroxicam, vitamin A acetate, and vitamin E succinate. Phenytoin, which is known to cause teratogenesis through oxidative stress was used as a positive control. Eggs were incubated until hatch and then the following parameters were assessed: mortality, hatching success, abnormalities, weights for whole body, liver, heart and brain, and biochemical endpoints for oxidative stress. As a measure of exposure, concentrations of TCDD and ethoxyresorufin-O-deethylase (EROD) activities were measured in tissues of hatchlings. While greater mortality and abnormalities were observed in the TCDD treatment groups, the number of the replicates were not great enough to detect statistically significant differences in abnormality rates for the co-treatments. Some of the observed developmental abnormalities included edema, liver necrosis and bill, eye and limb deformities with TCDD treatments, bill and brain deformities with phenytoin treatments, eye abnormalities with Vitamin E treatments, and abnormal feather pigmentation with piperonyl butoxide treatments.

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.

Tree swallow (Tachycineta bicolor) exposure to polychlorinated biphenyls at the Kalamazoo River superfund site, Michigan, USA

In 1990, a portion of the Kalamazoo River in Michigan, USA, was designated a Superfund site because of the presence of polychlorinated biphenyls (PCBs) in the sediment and floodplain soils. During a four-year period from 2000 to 2003, several avian species were monitored for reproductive effects and concentrations of PCBs in tissues attributed to food chain transfer from contaminated sediments. The tree swallow (Tachycineta bicolor) was chosen as a model receptor for contamination of passerine species. A top-down methodology was used to evaluate the bioaccumulation of PCBs, including non-ortho and mono-ortho congeners, in tree swallow eggs, nestlings, and adults at the Kalamazoo River area of concern (KRAOC) and at an upstream reference site. Generally, a sixfold difference in tissue concentrations of total PCBs was observed between the two sites with concentrations in eggs and nestlings at the KRAOC ranging from 0.95 to 15 microg PCB/g wet weight. Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQsWHO Avian) for PCBs, based on bird-specific World Health Organization toxic equivalence factors, were 10- to 30-fold greater in the KRAOC than at the reference location. Egg and nestling TEQsWHO-Avian ranged from 0.21 to 2.4 ng TEQ/g wet weight at the KRAOC. Hazard quotients calculated from literature-derived toxicity reference values were below 1.0 at both the target and the reference site based on the no-observed-adverse-effect level and the lowest-observed-adverse-effect level.

Productivity of Tree Swallows (Tachycineta bicolor) Exposed to PCBs at the Kalamazoo River Superfund Site

A 123-km stretch of the Kalamazoo River in Michigan, was designated a Superfund site in 1990 due to historical releases of effluent containing polychlorinated biphenyl (PCB)-contaminated paper waste. Risk to bird species in the river ecosystem was evaluated using the tree swallow (Tachycineta bicolor) as a monitor for possible effects due to PCB exposure at two nesting locations, one in the Superfund site and one in an upstream reference location that is less contaminated with PCBs. In 2 of the 3 years of the study, clutch size at the contaminated location was 3.7 ± 1.4 and 4.8 ± 0.73 eggs per nest (mean ± SD), which was significantly less than the clutch size at the reference location (5.0 ± 1.1 and 5.3 ± 1.1 eggs per nest). However, there were no statistically significant differences in fledging success, predicted brood size, predicted number of fledglings, or growth of nestlings between the Kalamazoo River Superfund site and an upstream reference location with lesser concentrations of PCBs in the sediments and riparian soils. Productivity and hatching success comparisons between these same sites were also not significantly different; however, the power of these conclusions was less (p < .10). The reduction in clutch size at the co-contaminated location could not be attributed to PCBs due to a number of confounding factors, including Co-cocontaminants, habitat structure, and food availability. Other reproductive parameters were not significantly impaired, and the size of the newly established colony at the Kalamazoo River Superfund site continued to grow over the period of the study. These site-specific observations, combined with multiple lines of evidence approach that considered results reported for the effects of both total PCBs and 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents (TEQ) on tree swallows at other locations, suggest that there were no significant population-level effects of PCBs on tree swallows at the Kalamazoo River Superfund site.

Accumulation of polychlorinated biphenyls from floodplain soils by passerine birds

Eggs, nestlings, and adults of the eastern bluebird (Sialia sialis) and house wren (Troglodytes aedon) were collected at a polychlorinated biphenyl (PCB)-contaminated site and a reference location on the Kalamazoo River (MI, USA). Eggs and nestlings of eastern bluebirds at the more contaminated location contained concentrations of 8.3 and 1.3 mg/kg, respectively, of total PCBs and 77 and 6.3 ng/kg, respectively, of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs). Eggs, nestlings, and adults of house wrens from the contaminated location contained 6.3, 0.77, and 3.2 mg/kg, respectively, of PCBs and 400, 63, and 110 ng/kg, respectively, of TEQs. Concentrations of total PCBs and TEQs in tissues at the more contaminated location were significantly greater than concentrations in tissues at the reference site for all tissue types of both species. Exposures of the two species studied were different, which suggests that terrestrial-based insectivorous passerine species, foraging in the same area, may have differential exposure to PCBs depending on specific foraging techniques and the insect orders that are targeted. Despite the greater accumulation of PCBs at the more contaminated location, the risk of exposure to PCBs did not exceed the threshold for adverse effects at either location.

Exposure and Multiple Lines of Evidence Assessment of Risk for PCBs Found in the Diets of Passerine Birds at the Kalamazoo River Superfund Site, Michigan

ABSTRACT Dietary exposures of passerine birds at the Kalamazoo River, Michigan, were examined due to the presence of polychlorinated biphenyls (PCBs) in the terrestrial and aquatic food webs. Average potential daily doses in diets were 6- to 29-fold and 16- to 35-fold greater at a contaminated location than at a reference location for PCB exposures quantified as total PCBs and 2,3,7,8–tetrachlorodibenzo-p-dioxin equivalents (TEQs), respectively. Birds with diets comprised of primarily aquatic insects had greater dietary exposure than birds with diets of primarily terrestrial insects. Risk associated with dietary exposure varied with the selection of the threshold for effects including hazard quotients, which exceeded 1 in instances where the most conservative toxicity reference values were utilized. Risk based on concentrations of PCBs in the tissues indicated little risk to avian species, and co-located studies evaluating reproductive health did not suggest that observed incidences of diminished reproductive success were related to PCB exposure. Measures of risk based on comparison to toxicity reference values (TRVs) were consistent with direct measures of ecologically relevant endpoints of reproductive fitness, but uncertainty exists in the selection of threshold values for effects in these species especially based on TEQs. This is largely due to the absence of species-specific, dose-response relationships. Therefore, the best estimate of risk is through the application of multiple lines of evidence.

Risk assessment methodologies for exposure of great Horned Owls (Bubo virginianus) to PCBs on the Kalamazoo River, Michigan

ABSTRACT Dietary exposures of great horned owls (GHO; Bubo virginianus) to polychlorinated biphenyls (PCBs) in the terrestrial food web at the Kalamazoo River, Michigan, USA, were examined. Average potential daily doses (APDD) in GHO diets were 7- to 10-fold and 3-fold greater at the more contaminated location versus a reference location for site-specific exposures quantified as total PCBs and 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQWHO-Avian), respectively. Wetland/aquatic prey contributed significantly to PCB exposure and APDD. Estimates of risk based on comparison of modeled dietary intake (e.g., APDD) to toxicity reference values (TRVs), using a hazard quotient (HQ) methodology, varied between diet composition methods (mass basis vs numeric basis). Mass-basis compositions yielded greater HQs at all sites. Potential risks associated with dietary exposures (“bottom-up” risk assessment methodology) were less than (HQ < 1) benchmarks for effects,. This result is consistent with risk estimates based on concentrations in tissues (“top-down” risk assessment methodology), and indicated PCBs posed no significant risk to terrestrial raptor species. Colocated and concurrent studies that evaluated GHO reproductive performance (nestling productivity) and relative abundance were consistent with results of the risk assessment. Measures of risk based on HQs were consistent with direct measures of ecologically relevant endpoints (reproductive fitness). Uncertainty in risk estimates is contributed during the selection of TRVs for effects in GHO based on TEQWHO-Avian because of the absence of species-specific, dose-response thresholds. This evaluation indicated that a multiple-lines-of-evidence approach provided the best estimate of risk.

Site-Specific Assessments of Environmental Risk and Natural Resource Damage Based on Great Horned Owls

ABSTRACT Selection of receptors is a key element of ecological risk and natural resource damage assessments. The great horned owl (Bubo virginianus; GHO) has advantages as a tertiary terrestrial receptor and integrated measure of exposure to chemical residues in a multiple-lines-of-evidence approach that includes elucidation of contaminant exposure by measured (tissue-based) and predicted (dietary) methodologies, and population-level measures of potential adverse effects (i.e., productivity, abundance). Methods described herein exploited attributes of GHO behavior, including its propensity to nest in artificial nesting platforms. This approach allowed better control of experimental conditions, minimized uncertainty in assessment endpoints, and maximized data utility for testing hypotheses. During 5 years, 54 GHO nests (14 active territories) along 38 km of river floodplain were monitored at the Kalamazoo River Superfund Site (Kalamazoo/Allegan Counties, Michigan). Concentrations of polychlorinated biphenyls (PCBs) and otho-, para-substituted isomers of dichlorodiphenyltrichloroethane (DDT), including DDD/DDE (Σ DDTs), were measured in 24 eggs and 16 samples of nestling blood plasma. Dietary PCB exposure was predicted by determining site-specific dietary composition and sampling and quantifying PCB concentrations in 171 prey items collected within active GHO territories. The convergence of exposure and effects assessments improved confidence in resulting predictions of minimal risk to resident GHO populations (Hazard Quotients ≤1.5). Repeated GHO use of nesting platforms minimized temporal and spatial variability.