Embryonic effects of an environmentally relevant PCB mixture in the domestic chicken.

Studies were conducted to develop methods to assess the effects of a complex mixture of polychlorinated biphenyls (PCBs) in the domestic chicken (Gallus domesticus). Treatments were administered by egg injection to compare embryonic effects of an environmentally relevant PCB congener mixture in the domestic chicken over a range of doses. Chicken eggs were injected with the PCB mixture with a profile similar to that found in avian eggs collected on the upper Hudson River, New York, USA, at doses that spanned 0 to 98 µg/g egg. Eggs were hatched in the laboratory to ascertain hatching success. In the domestic chicken, the median lethal dose was 0.3 µg/g. These data demonstrate adverse effects of an environmentally relevant PCB mixture and provide the basis for further work using in vitro and other models to characterize the potential risk to avian populations. Environ Toxicol Chem 2018;37:2513-2522. © 2018 SETAC.

Differential expression of hepatic genes with embryonic exposure to an environmentally relevant PCB mixture in Japanese quail (Coturnix japonica).

The upper Hudson River was contaminated with polychlorinated biphenyls (PCB) Aroclor mixtures from the 1940s until the late 1970s. Several well-established biomarkers, such as induction of hepatic cytochrome P450 monooxygenases, were used to measure exposure to PCBs and similar contaminants in birds. In the present study, Japanese quail eggs were injected with a PCB mixture based upon a congener profile found in spotted sandpiper eggs at the upper Hudson River and subsequently, RNA was extracted from hatchling liver tissue for hybridization to a customized chicken cDNA microarray. Nominal concentrations of the mixture used for microarray hybridization were 0, 6, 12, or 49 µg/g egg. Hepatic gene expression profiles were analyzed using cluster and pathway analyses. Results showed potentially useful biomarkers of both exposure and effect attributed to PCB mixture. Biorag and Ingenuity Pathway Analysis® analyses revealed differentially expressed genes including those involved in glycolysis, xenobiotic metabolism, replication, protein degradation, and tumor regulation. These genes included cytochrome P450 1A5 (CYP1A5), cytochrome b5 (CYB5), NADH-cytochrome b5 reductase, glutathione S-transferase (GSTA), fructose bisphosphate aldolase (ALDOB), glycogen phosphorylase, carbonic anhydrase, and DNA topoisomerase II. CYP1A5, CYB5, GSTA, and ALDOB were chosen for quantitative real-time polymerase chain reaction confirmation, as these genes exhibited a clear dose response on the array. Data demonstrated that an initial transcriptional profile associated with an environmentally relevant PCB mixture in Japanese quail occurred.

An overview of dioxin-like compounds, PCB, and pesticide exposures associated with sexual differentiation of neuroendocrine systems, fluctuating asymmetry, and behavioral effects in birds.

Dioxin, polychlorinated biphenyls (PCBs), and pesticides impact neural systems in birds due to interference with sexual differentiation. Early endocrine disrupting chemical (EDC) effects may delay maturation and have long-term effects on lifetime reproduction, especially in precocial birds that complete sexual differentiation prior to hatch. Semi-altricial and altricial species appear more resilient to EDC effects and show a gradient in sensitivity, especially in the neuroplastic song system. Embryonic steroid exposure occurs via maternally deposited steroids followed by embryo produced hormones; EDCs potentially affect these developing systems. As such, EDCs can impact lifelong fitness by acting on neural systems that regulate reproduction, metabolism, and behavior.

Is the gonadotropin releasing hormone system vulnerable to endocrine disruption in birds?

Endocrine disrupting chemicals (EDCs) from a variety of sources occur widely in the environment, but relationships between exposure to EDCs and long term effects on bird populations can be difficult to prove. Embryonic exposure to EDCs may be particularly detrimental, with potential long-term effects on reproduction and ultimately individual fitness. Because many EDCs may have subtle sublethal effects, it is necessary to establish sensitive end points as biomarkers of EDC exposure in birds. Because the effects of EDCs may be both short- and long-term, it is important to determine if embryonic exposure impacts sexual differentiation and development of the reproductive axis in hatchlings and if there are effects on reproductive function in adults. Our studies have focused on the effects of estrogen- and androgen-active EDCs on the hypothalamic gonadotropin releasing hormone-I (GnRH-I) system in an avian model of precocial species, the Japanese quail. Estrogen- or androgen-active EDCs were administered between 0 and embryonic day 4, and hypothalamic GnRH-I was measured in hatchlings and adults. Treatment with vinclozolin and PCB126 depressed the concentration of embryonic GnRH-I peptide while methoxyclor had an inconsistent stimulatory effect. Treatment with atrazine or trenbolone had no significant effects on hypothalamic GnRH-I in adults. Overall these observations support the view that the developing avian GnRH-I neural system may be vulnerable to EDCs with potential to alter lifelong reproductive function.

Effects of estradiol on the development of the bursa of Fabricius in Japanese quail.

Effects of androgens on the development of the bursa of Fabricius are better understood than those of estradiol, despite the known sensitivity of the bursa to estradiol early in embryogenesis. The goal of this study was to determine the effects of one-time yolk injections of estradiol at day 4 of incubation on the development of the bursa and spleen as indices of treatment effects on the immune system. Follicle size and numbers in hatchling bursas were significantly reduced at 50 and 500 microg/egg, respectively. Additionally, distorted plicae and thicker epithelial layers surrounding the plicae were observed in day-old chicks at the same treatment levels. Adult bursas from birds embryonically exposed to estrogen were significantly larger than controls, suggesting an inhibition of natural bursal regression. Although estradiol altered the development of the bursa, the spleen appeared to be unaffected. The observed effects of estradiol on the development of the bursa indicate that this lymphoid organ may be a target for developmental disruption by estrogenic endocrine disrupting chemicals, though long-term consequences of embryonic exposure on immune function remain unknown.

Activity patterns of biotransformation enzymes in juvenile chickens after in ovo dosage of PCB126.

Little is known about the correlations between biotransformation enzymes in juvenile birds after exposure to environmental toxicants like PCBs. In this study eggs of domestic chicken (Gallus domesticus) were dosed with PCB126 in concentrations of 0.175-0.325 ng/g egg weight. Liver subcellular fractions were analyzed for activities of Phase 1 and Phase 2 biotransformation enzymes 2 and 5 weeks post-hatch. Ethoxyresorufin O-deethylase (EROD) activity was increased in both the 2-week and 5-week samples. Glutathione-S-transferase (GST) activity was increased in the 2-week samples only, but the 5-week samples showed an overall much higher GST activity, probably as a result of a still developing enzyme expression in maturing chickens. The same pattern was seen in the phenol-type UDP-glucuronosyltransferase (UGT) activity of the control animals. The week two samples showed a positive dose-response relationship for the UGT activity, but after 5 weeks this was reversed, possibly caused by inhibition of hydroxylated PCB metabolites. Phenol-type sulfotransferase (SULT) activities were not significantly correlated with time or dose. There was a strong positive regression between the Ah-receptor mediated EROD and UGT activities. The EROD activities were also positively correlated to the GST activities. Most interesting was a negative correlation between the UGT and SULT activities: an inhibited UGT activity appeared to be compensated by an increased SULT activity.

Immunotoxicity of trenbolone acetate in Japanese quail.

Trenbolone acetate is a synthetic androgen that is currently used as a growth promoter in many meat-exporting countries. Despite industry laboratories classifying trenbolone as nonteratogenic, data showed that embryonic exposure to this androgenic chemical altered development of the immune system in Japanese quail. Trenbolone is lipophilic, persistent, and released into the environment in manure used as soil fertilizer. This is the first study to date to assess this chemical's immunotoxic effects in an avian species. A one-time injection of trenbolone into yolks was administered to mimic maternal deposition, and subsequent effects on the development and function of the immune system were determined in chicks and adults. Development of the bursa of Fabricius, an organ responsible for development of the humoral arm of the immune system, was disrupted, as indicated by lower masse, and smaller and fewer follicles at day 1 of hatch. Morphological differences in the bursas persisted in adults, although no differences in either two measures of immune function were observed. Total numbers of circulating leukocytes were reduced and heterophil-lymphocyte ratios were elevated in chicks but not adults. This study shows that trenbolone acetate is teratogenic and immunotoxic in Japanese quail, and provides evidence that the quail immune system may be fairly resilient to embryonic endocrine-disrupting chemical-induced alterations following no further exposure posthatch.

Immunosenescence and age-related susceptibility to influenza virus in Japanese quail.

We evaluated juvenile, pubescent, reproductive adult, and aged Japanese quail (Coturnix coturnix japonica) to determine if there were age-related differences in immune function with the hypothesis that aged birds would have weaker immune responses. Immune responses were measured using phytohemagglutinin (PHA) skin test, antibody response to foreign red blood cells and exposure to an H9N2 influenza virus. Adult birds consistently had stronger immune responses than young and aged birds. Aged quail had skin responses 38% lower than adults. Pubescent birds' mean anti-red blood cell response was four-fold lower than adult birds. Adults had greater increase in total anti-viral antibody between primary and secondary infections than all other groups. Our data demonstrate an age-related difference in immune function in Japanese quail that has similarities to age-related immunity in humans; younger and older animals had weaker immune responses compared to young adults.

Reproductive toxicity of trenbolone acetate in embryonically exposed Japanese quail.

This study was conducted to assess the effects of a one time embryonic exposure to trenbolone acetate on reproductive development and function in Japanese quail (Coturnix japonica). Embryos were exposed to either 0.05, 0.5, 5, or 50microg trenbolone or a sesame oil vehicle control at embryonic day 4. Onset of puberty, gonadal histopathology, sperm motility, cloacal gland size, and male copulatory behavior were assessed in adults. Trenbolone delayed onset of puberty in males, inhibited cloacal gland development, and reduced male reproductive behaviors. Industry laboratories have shown trenbolone acetate to be non-teratogenic in mammalian studies. Our study, however, shows that this one time in ovo exposure delayed onset of puberty in and suppressed adult copulatory behavior in quail males. These results suggest that this one time embryonic exposure to trenbolone may have disrupted development of either the central nervous system or the hypothalamic-pituitary-gonadal axis. This is the first study to demonstrate a demasculinizing effect on copulatory behavior in Japanese quail from embryonic exposure to a non-aromatizable androgenic chemical. More studies are needed to determine the mechanisms behind the observed effects.

Avian immunosenescence.

Immunosenescence, the aging of the immune system, is well studied in humans. Mammalian immune systems become less capable of fighting pathogens and individuals become more susceptible to infection and cancer in their elder years. Little is currently published on avian immunosenescence even though avian immune function has been well characterized and birds have been critical models in the study of immunology. The value of birds in the study of aging has been well established. Evidence demonstrates a decline in cell-mediated and (or) humoral immune function with age in four species: the barn swallow, collared flycatcher, ruff and Japanese quail. These studies suggest that birds may experience age-related changes in immune function similar to humans. Therefore, avian aging models should be evaluated for changes in immune function and comparisons made between short- and long-lived birds. Specifically, data on age-related changes in innate immunity are lacking for birds and should be characterized. These data will strengthen the use of avian models for the study of aging.