This review is a comprehensive introduction to the effects of poultry exposure to the toxic and carcinogenic mycotoxin aflatoxin B₁ (AFB₁). The relationship between AFB₁ sensitivity and metabolism, major direct and indirect effects of AFB₁, recent studies of gene expression and transcriptome responses to exposure, and mitigation strategies to reduce toxicity are discussed. Exposure to AFB₁ primarily occurs by consumption of contaminated corn, grain or other feed components. Low levels of residual AFB₁ in poultry feeds can cause reduction in growth, feed conversion, egg production, and compromised immune functions, resulting in significant economic costs to producers. Thus, AFB₁ acts as a “force multiplier” synergizing the adverse effects of microbial pathogens and other agents, and factors detrimental to poultry health. Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB₁ due, in large part, to a combination of efficient hepatic bioactivation by cytochromes P450 1A5 and 3A37, and deficient hepatic glutathione-S-transferase (GST)-mediated detoxification. Because of their sensitivity, turkeys are a good model to investigate chemopreventive treatments and feed additives for their ability to reduce AFB₁ toxicity. Transcriptome analysis (RNA-seq) of turkey poults (liver and spleen) has identified AFB₁-induced gene expression changes in pathways of apoptosis, carcinogenesis, lipid regulation, antimicrobial activity, cytotoxicity and antigen presentation. Current research focuses on further identifying the molecular mechanisms underlying AFB₁ toxicity with the goal of reducing aflatoxicosis and improving poultry health.

Because of its presumed adverse health effects, particulate air pollution (PM) has received growing attention, but the cellular mechanisms by which PM exerts toxicity are not well elucidated. PM has been associated with early mortality from illnesses that share endoplasmic reticulum (ER) stress as a mechanism of pathogenesis. In this study, we examined whether PM would induce the unfolded protein response (UPR) which is a cellular response to ER stress. Coarse (PM₁₀) and fine (PM_2.5) PM was collected from a single location in Northern Utah's Cache Valley during atmospheric inversions occurring in January 2002 and January 2003. Extracts of PM samples were added (12.5 and 25 μg/ml) to cultured human bronchial epithelial (BEAS-2B) cells for 24 h. At these concentrations neither PM nor LPS exhibited demonstrable cytotoxicity by the neutral red assay. However, PM elicited significant increases of unfolded protein response (UPR)–related post-translational modifications, such as S6 ribosomal protein, heat-shock protein (Hsp)27, and protein kinase related protein phosphorylation and cleavage of activating transcription factor (ATF)-6. PM exposure also resulted in significant increases in the UPR-associated proteins ATF-4, Hsp70, Hsp90, and binding immunoglobulin protein. PM also interfered with the export of Hsp70 from the cells in a concentration-dependent manner and resulted in release of C-reactive protein. Calpain was upregulated and activated in PM-treated cultures, though these events were not proapoptotic. This study demonstrates that PM is capable of inducing ER stress and the UPR in vitro and may be a mechanism by which PM exerts toxicity.

It has been known since the early days of the discovery of aflatoxin B1 (AFB1) that there were large species differences in susceptibility to AFB1. It was also evident early on that AFB1 itself was not toxic but required bioactivation to a reactive form. Over the past 60 years there have been thousands of studies to delineate the role of ~10 specific biotransformation pathways of AFB1, both phase I (oxidation, reduction) and phase II (hydrolysis, conjugation, secondary oxidations, and reductions of phase I metabolites). This review provides a historical context and substantive analysis of each of these pathways as contributors to species differences in AFB1 hepatoxicity and carcinogenicity. Since the discovery of AFB1 as the toxic contaminant in groundnut meal that led to Turkey X diseases in 1960, there have been over 15,000 publications related to aflatoxins, of which nearly 8000 have addressed the significance of biotransformation (metabolism, in the older literature) of AFB1. While it is impossible to give justice to all of these studies, this review provides a historical perspective on the major discoveries related to species differences in the biotransformation of AFB1 and sets the stage for discussion of other papers in this Special Issue of the important role that AFB1 metabolites have played as biomarkers of exposure and effect in thousands of human studies on the toxic effects of aflatoxins. Dr. John Groopman has played a leading role in every step of the way—from initial laboratory studies on specific AFB1 metabolites to the application of molecular biomarkers in epidemiological studies associating dietary AFB1 exposure with liver cancer, and the design and conduct of chemoprevention clinical trials to reduce cancer risk from unavoidable aflatoxin exposures by alteration of specific AFB1 biotransformation pathways. This article is written in honor of Dr. Groopman’s many contributions in this area.

The mycotoxin, aflatoxin B₁ (AFB₁) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB₁ can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are likely more resistant. In ovo exposure provided a controlled AFB₁ challenge and comparison of domesticated and wild turkeys. Gene expression responses to AFB₁ in the embryonic hepatic transcriptome were examined using RNA-sequencing (RNA-seq). Eggs were injected with AFB₁ (1 μg) or sham control and dissected for liver tissue after 1 day or 5 days of exposure. Libraries from domesticated turkey (n = 24) and wild turkey (n = 15) produced 89.2 Gb of sequence. Approximately 670 M reads were mapped to a turkey gene set. Differential expression analysis identified 1535 significant genes with |log₂ fold change| ≥ 1.0 in at least one pair-wise comparison. AFB₁ effects were dependent on exposure time and turkey type, occurred more rapidly in domesticated turkeys, and led to notable up-regulation in cell cycle regulators, NRF2-mediated response genes and coagulation factors. Further investigation of NRF2-response genes may identify targets to improve poultry resistance.

Domestic turkeys ( Meleagris gallopavo ) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B 1 (AFB 1 ), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo -AFB 1 -8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB 1 -resistant, possess hepatic GST-mediated AFBO conjugating activity . Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes ( GSTA ) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli -expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB 1 -protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB 1 , and may also have the benefit of identifying genetic markers which could be used to enhance AFB 1 resistance in modern domestic strains.