The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC(50) values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied, and differences at amino acid sites 256, 257, 297, 324, 337, and 380 were identified. Site-directed mutagenesis, the LRG assay, and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of the 86 avian species to DLCs and (2) in vitro luciferase activity of AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r (2) = 0.93, p < 0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.

Carp (Cyprinus carpio) collected from Saginaw Bay, Michigan, containing 8.4 mg total polychlorinated biphenyls (PCBs)/kg and 194 ng of 2,3,7,8-tetrachloro-dibenzo-p-dioxin equivalents (TEQs)/kg, were substituted for marine fish at levels of 0, 10, 20, or 40% in the diets of adult ranch mink (Mustela vison). The diets, containing 0.015, 0.72, 1.53, and 2.56 mg PCBs/kg diet, or 1.03, 19.41, 40.02, and 80.76 ng TEQs/kg diet, respectively, were fed to mink prior to and throughout the reproductive period to evaluate the effects of a naturally-contaminated prey species on their survival and reproductive performance. The total quantities of PCBs ingested by the mink fed 0, 10, 20, or 40% carp over the 85-day treatment period were 0.34, 13.2, 25.3, and 32.3 mg PCBs/mink. respectively. The corresponding quantities of TEQs ingested by the mink over the same treatment period were 23, 356, 661, and 1,019 ng TEQs/mink, respectively. Consumption of feed by mink was inversely proportional to the PCB and TEQ content of the diet. The diet containing Saginaw Bay carp caused impaired reproduction and/or reduced survival of the kits. Compared to controls, body weights of kits at birth were significantly reduced in the 20 and 40% carp groups, and kit body weights and survival in the 10 and 20% carp groups were significantly reduced at three and six weeks of age. The females fed 40% carp whelped the fewest number of kits, all of which were stillborn or died within 24 hours. Lowest observable adverse effect levels (LOAEL) of 0.134 mg PCBs/kg body weight/day or 3.6 ng TEQs/kg body weight/day for adult female mink were determined. The potential effects of exposure of wild mink to contaminated Great Lakes fish were assessed by calculating "maximum allowable daily intakes" and "hazard indices" based on total concentrations of PCB residues in several species of Great Lakes fish and mink toxicity data derived from the study.

Mink are known to be very sensitive to the toxic effects of planar polychlorinated biphenyls (pPCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs), collectively known as planar halogenated hydrocarbons (PHHs). Previously, we reported the reproductive effects in mink fed a diet containing 10, 20, or 40% fish taken from Saginaw Bay, Lake Huron. The present study reports the chemical characterization of the diets and the adult mink livers, along with a comparison of an additive model of toxicity with the results of the H4IIE bioassay on these samples. The assessment of dietary or tissue-based exposure of the mink to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds revealed that TCDD equivalents of the PHH mixtures largely followed an additive model of toxicity as compared with the H4IIE bioassay. Consistent dietary and liver tissue-based threshold concentrations for reproductive toxicity in mink were determined regardless of whether PHHs were quantified as TEQs (additive toxicity) or TCDD-EQs (H4IIE bioassay). Significant reproductive effects were observed in the lowest treatment group (10% fish or 19.4 pg of H4IIE bioassay-derived TCDD-EQs/g). Consumptionnormalized mink liver biomagnification factors (BMFs) were 6.4-74.2 for PCDDs, <1-75.8 for PCDFs, <1-15.9 for PCBs, and in general, increased with degree of chlorination within each class. Based on TEQs or TCDD-EQ, this study confirms that mink are among the most, if not the most, sensitive mammalian species to the reproductive toxicity of TCDD and related compounds.

No abstract

There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324_Ser380 vs common tern--250-fold less sensitive than chicken; Val325_Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324_Ala380 and Val324_Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324_Ala380) and Japanese quail (Coturnix japonica; Val324_Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.

Relative potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were determined in vitro in primary hepatocyte cultures of chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), and Japanese quail (Coturnix japonica) embryos. Concentration-dependent effects on ethoxyresorufin O-deethylase (EROD) activity and expression of cytochrome P4501A4 and cytochrome P4501A5 (CYP1A4 and CYP1A5) messenger RNA (mRNA) were determined in hepatocytes exposed to serial dilutions of TCDD, PeCDF, or TCDF for 24 h. In chicken hepatocytes, the three compounds were equipotent inducers of EROD activity and CYP1A4/CYP1A5 mRNA expression. However, in ring-necked pheasant and Japanese quail hepatocytes, PeCDF was more potent than TCDD (3- to 5-fold in ring-necked pheasant and 13- to 30-fold in Japanese quail). Among species, the rank order of sensitivity (most to least) to EROD and CYP1A4/CYP1A5 mRNA induction for TCDD and TCDF was chicken > ring-necked pheasant > Japanese quail. In contrast, the three species were approximately equisensitive to EROD and CYP1A4/CYP1A5 mRNA induction by PeCDF. It has generally been assumed that TCDD is the most potent "dioxin-like compound" (DLC) and that the chicken is the most sensitive avian species to CYP1A induction by all DLCs. This study indicates that PeCDF is more potent than TCDD in ring-necked pheasant and Japanese quail hepatocytes and that ring-necked pheasant, Japanese quail, and chicken hepatocytes are equally sensitive to CYP1A induction by PeCDF.

Idiosyncratic reactions occur in a small fraction (typically Ͻ5%) of the population taking therapeutic drugs. Chlorpromazine (CPZ) is a phenothiazine, antipsychotic drug that has caused several idiosyncratic responses during its therapeutic use. Clinical evidence suggests that conditions associated with inflammation are risk factors for the appearance of these responses. Accordingly, we tested the hypothesis that an inflammatory stimulus, bacterial lipopolysaccharide (LPS), renders animals susceptible to CPZ-induced idiosyncratic reactions seen in humans. Male Sprague-Dawley rats (200 -250 g) were fasted for 24 h. A small dose of LPS (7.4 ϫ 10 6 EU/kg from Escherichia coli) or its vehicle (saline) was administered by tail vein 2 h before an intraperitoneal injection of CPZ (70 mg/kg) or its vehicle (saline). Cholestasis and hepatocellular necrosis were evaluated as increased concentrations of serum bile acids and bilirubin and increased activities of alkaline phosphatase, ␥-glutamyltransferase, alanine aminotransferase, and aspartate aminotransferase. With the exception of bile acids, these serum markers were elevated in animals treated with LPS/CPZ. Histopathological lesions in liver sections were consistent with these findings. Elevated serum creatine kinase activity, which is associated with human idiosyncratic responses to phenothiazines, was also found in animals treated with LPS/CPZ, but not with either LPS or CPZ alone. These results raise the possibility that concurrent, modest inflammation may underlie susceptibility of individuals to certain idiosyncratic reactions and may form the basis for an animal model with which to understand and predict drug idiosyncrasy.Drug idiosyncrasy is an untoward biological response to a therapeutic agent occurring in a small percentage of individuals. Idiosyncratic responses appear to occur independently of dose and have an inconsistent temporal relationship to the course of drug administration (Hollister, 1957). Although drug metabolism polymorphisms and drug allergy are widely presumed to underlie idiosyncratic responses, convincing evidence to support these as causes is lacking for the majority of drugs. Reproducing such responses in animals has been met with little success. Inasmuch as drug idiosyncrasy results in human suffering and considerable cost to pharmaceutical companies, animal models that are able to predict such responses in people before a drug is marketed could have great benefit.Among the many drugs that have caused idiosyncratic reactions in people are aliphatic phenothiazines. For example, chlorpromazine (CPZ) (Thorazine, 10-[3-dimethylaminopropyl]-2-chlorphenothiazine) is a tricyclic antidepressant that has been used as a sedative and antiemetic and for the management of psychotic disorders. Two types of adverse reactions result from phenothiazine usage. First, extrapyramidal side effects such as pseudoparkinsonism, dystonic reactions, and akathisia are common, dose-related side effects that likely result from the blockade of dopamine recepto...

Egg injection studies were performed to confirm a proposed model of relative sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this model, species are classified as belonging to one of three categories of sensitivity based on amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon receptor. Embryo lethality and relative potencies of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were compared with TCDD for Japanese quail (Coturnix japonica; least sensitive), Common pheasant (Phasianus colchicus; moderately sensitive), and White Leghorn chicken (Gallus gallus domesticus; most sensitive). Doses ranging from 0.044 to 37 pmol/g egg (0.015-12 ng/g egg) were injected into the air cell of eggs prior to incubation. LD(50) (95% confidence intervals) values, based on rate of hatching for TCDD, PeCDF, and TCDF, were 30 (25-36), 4.9 (2.3-9.2), and 15 (11-24) pmol/g egg for the quail, 3.5 (2.3-6.3), 0.61 (0.28-1.2), and 1.2 (0.62-2.2) pmol/g egg for pheasant, and 0.66 (0.47-0.90), 0.75 (0.64-0.87), and 0.33 (0.23-0.45) pmol/g egg for chicken, respectively. LD(50)-based relative potencies of PeCDF and TCDF were 6.1 and 2.0 for quail, 5.7 and 2.9 for pheasant, and 0.88 and 2.0 for chicken, respectively. TCDD was not the most potent compound among the species tested, with PeCDF and TCDF being more potent than TCDD in the quail and pheasant. TCDF was the most potent in chicken. Species sensitivity was as expected for TCDD and TCDF, whereas for PeCDF, the chicken and pheasant were similar in sensitivity and both were more sensitive than the quail. Results from companion in vitro studies are generally similar to those reported here with a few exceptions.