Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Environmentally enriched male mink gain more copulations than stereotypic, barren-reared competitors.

Wild carnivores in zoos, conservation breeding centres, and farms commonly live in relatively small, unstimulating enclosures. Under these captive conditions, in a range of species including giant pandas, black-footed ferrets, and European mink, male reproductive abilities are often poor. Such problems have long been hypothesized to be caused by these animals' housing conditions. We show for the first time that rearing under welfare-improving (i.e., highly valued and stress-reducing) environmental enrichments enhances male carnivores' copulatory performance: in mate choice competitions, enriched male American mink (Neovison vison) mated more often than non-enriched males. We screened for several potential mediators of this effect. First was physiological stress and its impact on reproductive physiology; second, stress-mediated changes in morphology and variables related to immunocompetence that could influence male attractiveness; and third, behavioural changes likely to affect social competence, particularly autistic-like excessive routine and repetition ('perseveration') as is reflected in the stereotypies common in captive animals. Consistent with physiological stress, excreted steroid metabolites revealed that non-enriched males had higher cortisol levels and lower androgen levels than enriched conspecifics. Their os penises (bacula) also tended to be less developed. Consistent with reduced attractiveness, non-enriched males were lighter, with comparatively small spleens and a trend to greater fluctuating asymmetry. Consistent with impaired social competence, non-enriched males performed more stereotypic behaviour (e.g., pacing) in their home cages. Of all these effects, the only significant predictor of copulation number was stereotypy (a trend suggesting that low bodyweights may also be influential): highly stereotypic males gained the fewest copulations. The neurophysiological changes underlying stereotypy thus handicap males sexually. We hypothesise that such males are abnormally perseverative when interacting with females. Investigating similar problems in other taxa would be worthwhile, since many vertebrates, wild and domestic, live in conditions that cause stereotypic behaviour and/or impair neurological development.

Development and use of real-time reverse transcription-polymerase chain reaction assay to quantify cytochrome P4501A1 expression in American mink.

The distribution of natural populations of American mink is restricted to locales that are in proximity to aquatic ecosystems. Because of the lipophilicity and persistence of polychlorinated biphenyls (PCBs) and reliance of mink on aquatic-based diets, mink at contaminated locales often bioacccumulate high levels of PCBs. In addition, in controlled laboratory studies, mink are highly sensitive at reproductive and developmental end points to the toxic effects of environmental PCB mixtures. It is believed that most, if not all, toxic effects of PCBs occur through activation of the aryl hydrocarbon receptor (AHR) pathway. Transcription of cytochrome P4501A1 (CYP1A1) by PCBs is also mediated through activation of AHR. Thus, levels of CYP1A1 mRNA provide a quantitative assay of exposure to and early biologic effect of PCBs on mink and may be predictive of toxicity at higher levels of biologic organization. We developed polymerase chain reaction (PCR) primers to amplify CYP1A1 as well as identified a housekeeping gene from mink cDNA. We used real-time reverse transcription-PCR to quantify and compare levels of hepatic CYP1A mRNA among groups of ranched mink kits and juveniles, which were fed diets or exposed in utero to fish that were low in PCBs (Atlantic herring) or to diets that were contaminated with three different levels of PCBs (carp) from Saginaw Bay, Lake Michigan. We found significant differences in CYP1A1 mRNA expression between mink fed the control diet and those fed a PCB-contaminated carp diet at all three treatment levels and exposure times. CYP1A1 mRNA was significantly induced 5.3- to 6.6-fold and 3.7- to 4.7-fold at 6 and 27 weeks, respectively. In previous studies, dietary exposures to PCB-contaminated carp were shown to cause mild to moderate lesions in the mandible and maxilla of these animals. This study demonstrates that hepatic CYP1A1 mRNA may be a sensitive biomarker of exposure of mink to environmentally relevant levels of PCBs and may be predictive of their effects in natural populations.

Chronic effects of polychlorinated dibenzofurans on mink in laboratory and field environments.

Mink are often used as a sentinel species in ecological risk assessments of chemicals such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) that cause toxicity mediated through the aromatic hydrocarbon receptor. Considerable toxicological information is available on the effects of PCBs and PCDDs on mink, but limited toxicological information is available for PCDFs. Thus, exposure concentrations at which adverse effects occur could not be determined reliably for complex mixtures in which PCDFs dominate the total calculated concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (TEQ). Two studies were conducted to evaluate the potential toxicity of PCDFs to mink. The first was a chronic exposure, conducted under controlled laboratory conditions, in which mink were exposed to 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF) concentrations as great as 2.4 x 10(3) ng 2,3,7,8-TCDF/kg wet-weight (ww) diet or 2.4 x 10(2) ng TEQ(2006-WHO-mammal)/kg ww diet. In that study, transient decreases in body masses of kits relative to the controls was the only statistically significant effect observed. The second study was a 3-y field study during which indicators of individual health, including hematological and morphological parameters, were determined for mink exposed chronically to a mixture of PCDDs and PCDFs under field conditions. In the field study, there were no statistically significant differences in any of the measured parameters between mink exposed to a median estimated dietary dose of 31 ng TEQ(2006-WHO-mammal)/kg ww and mink from an upstream reference area where they had a median dietary exposure of 0.68 ng TEQ(2006-WHO-mammal)/kg ww. In both studies, concentrations of TEQ(2006-WHO-mammal) to which the mink were exposed exceeded those at which adverse effects, based on studies with PCDD and PCB congeners, would have been expected. Yet in both instances where PCDF congeners were the sole or predominant source of the TEQ(2006-WHO-mammal), predicted adverse effects were not observed. Taken together, the results of these studies suggest that the values of the mammalian-specific toxicity equivalency factors suggested by the World Health Organization overestimate the toxic potency of PCDFs to mink. Therefore, hazard cannot be accurately predicted by making comparisons to toxicity reference values derived from exposure studies conducted with PCBs or PCDDs in situations where mink are exposed to TEQ mixtures dominated by PCDFs.

Dietary accumulation, disposition, and metabolism of technical pentabrominated diphenyl ether (de-71) in pregnant mink (Mustela vison) and their offspring.

Concentrations of polybrominated diphenyl ethers (PBDEs) in humans and wildlife suggest significant bioaccumulation potential in mammals, but no quantitative biomagnification data from controlled experiments are currently available. As part of a larger study examining the effects of PBDEs on mink (Mustela vison) reproduction and development, we examined congener-specific tissue distribution, maternal transfer, biotransformation, and biomagnification of the technical penta-BDE mixture, DE-71, in farmed mink. Adult female mink were fed one of four concentrations of DE-71 (0-2.5 microg/g) in the diet from breeding through gestation and until weaning at 6 weeks postparturition. Parent PBDEs were measured in tissues and excreta of adult mink, kits, and juveniles, whereas hydroxylated PBDEs (OH-PBDEs) were measured in juveniles only. Similar lipid-normalized concentrations of PBDEs were detected in most tissues of adult mink with the exception of brain, in which concentrations were significantly lower. Kits, however, had a higher proportion of PBDEs in brain compared with adults, presumably because of incomplete development of the blood-brain barrier. Maternal transfer favored lower-brominated PBDE congeners, and the bulk of the body burden in kits at weaning resulted from lactational rather than transplacental transfer. Lipid-normalized, whole-body biomagnification factors ranged from 0.5 to 5.2 for the major congeners and were highest for BDEs 47 and 153. Metabolism clearly limited the biomagnification of some PBDEs, and OH-PBDEs were detectable in plasma, liver, and feces. On a mass basis, OH-PBDEs accounted for 28 to 32% of the excreted fraction, indicating metabolism was an important elimination pathway. Further studies are required to understand the mechanisms of PBDE biotransformation.

Nondestructive scat sampling in assessment of mink (Mustela vison) exposed to polychlorinated dibenzofurans (PCDFs).

The mink (Mustela vison) is often utilized as a sentinel species for ecological assessments at sites where contaminants of concern include dioxins and dioxin-like compounds. Utilizing mink scat as a nondestructive tool to determine internal exposure to dioxin-like compounds may allow for rapid, accurate estimates of exposure without the need to capture mink or their prey. To determine the relationships between concentrations of polychlorinated dibenzofurans (PCDFs) in tissues (liver and adipose) and those in scat, mink were fed PCDFs in scat during a controlled laboratory study for 180 days. Mink were fed a control diet, diets with three doses of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) or 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF), and a diet with an environmentally relevant mixture of the two congeners. Concentrations of PCDFs in liver and adipose were measured after 0, 90, and 180 days of exposure. Concentrations of the two PCDF congeners in mink scat were determined after 2, 23, 45, 90, and 180 days of exposure. Concentrations of both PCDF congeners in scat were significantly correlated with those in liver and adipose tissue (r(2) = 0.94-0.97, p < 0.01). This indicates that measurements of concentrations of both PCDFs in scat can be used to predict concentrations of PCDFs in liver and adipose. Assimilation and elimination characteristics of 2,3,4,7,8-PeCDF or 2,3,7,8,-TCDF and a mixture of the two congeners by mink could be predicted from concentrations of these congeners in scat. Overall, concentrations of PCDFs in mink scat can be used as a rapid and inexpensive nondestructive method to predict concentrations of PCDFs in mink when certain assumptions are met.

Changes in thyroid and vitamin A status in mink fed polyhalogenated-aromatic-hydrocarbon-contaminated carp from the Saginaw River, Michigan, USA.

A study was conducted to determine whether changes in thyroid and vitamin A dynamics were induced in ranch mink exposed to environmentally relevant concentrations of polyhalogenated aromatic hydrocarbons. Adult female mink were fed diets that contained 0% (control), 10%, 20%, or 30% wild carp (Cyprinus carpio) collected from the Saginaw River, Michigan, USA. Total polychlorinated biphenyls concentrations were 0.03, 0.83, 1.05, and 1.69 mg/kg feed, respectively; the 2,3,7,8-tetrachlorodibenzo-p-diozin toxic equivalents were 3.4, 27.9, 47.6, and 73.2 ng/kg, respectively. Diets were fed 3 weeks prior to breeding and throughout gestation and lactation. When the kits were weaned at 6 weeks of age, they were continued on their respective diets until 27 weeks of age. Plasma thyroid hormone concentrations, thyroid gland activity and structure, and vitamin A dynamics were assessed in young mink at 6 and 27 weeks of age. Plasma total T4 and free T4 in 6-week-old female and male kits fed the 10% carp diet were significantly higher than those of controls, while kits fed the 20% and 30% carp diet had nonsignificant decreases relative to the control mink. Plasma total T3 concentrations in 27-week-old juvenile males fed the 30% carp diet were significantly lower than those in individuals fed the 10% carp diet. No overt thyroid toxicity was apparent as thyroid weight, activity, and structure in kits and juveniles of both sexes were similar among diet groups. Plasma retinol and total ester concentrations in both kits and juveniles were reduced in mink fed the 30% carp diet relative to controls. The ratio of retinol to retinyl palmitate in livers of juveniles fed the 30% carp diet was two times higher than that in control mink. Significant reductions in kidney retinol and fatty acyl retinyl esters were observed in kits and juveniles fed the 30% carp diet relative to control values.