Abstract
Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B1 (AFB1), 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-AFB1-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB1-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 AFB1-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 AFB1, and may also have the benefit of identifying genetic markers which could be used to enhance AFB1 resistance in modern domestic strains.
Highlights
Aflatoxin B1 (AFB1), produced by Aspergillus fungi, is a ubiquitous dietary hepatotoxin and hepatocarcinogen, and a major public health concern worldwide, especially in locales where contaminated commodities, such as corn and peanuts, are consumed as a dietary staple [1,2]
Each GSTA was identical in length to the corresponding homologs from domestic turkey (DT) (GSTA1.1, 663 bp; GSTA1.2, 666 bp; GSTA1.3, 666 bp; GSTA2, 669 bp; GSTA3, 672 bp; GST4, 690 bp) (Figures S1, S2, S3, S4, S5, S6)
The GSTA1.3 variant in EW, RGW and RP possessed 3 single nucleotide polymorphisms (SNPs) compared to DT
Summary
Aflatoxin B1 (AFB1), produced by Aspergillus fungi, is a ubiquitous dietary hepatotoxin and hepatocarcinogen, and a major public health concern worldwide, especially in locales where contaminated commodities, such as corn and peanuts, are consumed as a dietary staple [1,2]. There are considerable species-specific differences with respect to susceptibility to the toxic effects of AFB1, and domestic turkeys (Meleagris gallopavo) are one of the most susceptible species known. Because of their extreme sensitivity, turkeys have been posited as effective surrogates to study AFB1 susceptible human populations [6]. Turkeys bioactivate AFB1 primarily by high-efficiency P450 s 1A5 and 3A37 which we have recently cloned, sequenced and functionally characterized [6,7,8]. At pharmacological AFB1 concentrations, P450 1A5 is responsible for more than 98% of the bioactivation in turkey liver [6]
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