An unusually high production of hepatic aflatoxin B1-dihydrodiol, the possible explanation for the high susceptibility of ducks to aflatoxin B1

A study was conducted to determine the enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for the hepatic in vitro production of aflatoxin B1-dihydrodiol (AFB1-dhd) from aflatoxin B1 (AFB1) in four commercial poultry species, ranging in sensitivity to AFB1 from highest (ducks) to lowest (chickens). Significant but small differences were seen for Vmax, while large significant differences were observed for KM. However, the largest inter-species differences were observed for the CLint parameter, with ducks being extraordinarily efficient in converting AFB1 into AFB1-dhd. Since AFB1-dhd is considered the metabolite responsible for the acute toxic effects of AFB1, the high hepatic production of AFB1-dhd from AFB1 in ducks is the possible biochemical explanation for the extraordinary high sensitivity of this poultry species to the adverse effects of AFB1.

parameters of AFB 1 -dhd production in liver microsomes that could explain the different in vivo sensitivity to AFB 1 of resistant (chickens and quail), sensitive (turkeys) and highly sensitive (ducks) poultry species.

Results
Due to the lack of a commercially available AFB 1 -dhd standard, a mass spectrometric analysis of the putative AFB 1 -dhd peak was conducted to determine its monoisotopic mass. The putative peak observed at 6.7 min Fig. 2a corresponded to a compound of 347 Da, which is consistent with the monoisotopic protonated mass of AFB 1 -dhd Fig. 2b.
The enzymatic kinetic parameters for AFB 1 -dhd production by the four poultry species investigated are presented in Fig. 3. Chicken and quail enzymes did not saturate even at the highest AFB 1 concentration evaluated (256 μM) Fig. 3a; however, turkey and duck enzymes seemed to become completely saturated with only 56 μM AFB 1 . The average values for the V max were the highest in Rhode Island Red chickens (11.2 ± 1.48 nmol of dhd-AFB 1 /mg protein/minute) and quail (9.57 ± 3.06 nmol of dhd-AFB 1 /mg protein/minute), while no differences (P > 0.05) were observed among Ross chickens, turkeys and ducks (5.75 ± 1.95, 5.84 ± 2.07 and 5.55 ± 1.33 nmol of dhd-AFB 1 /mg protein/minute, respectively) Fig. 3b. Rhode Island Red chicks had a higher V max value compared with Ross chickens. Regarding differences by sex, only quail and turkeys showed significant differences between males and females. The average values for K M showed large (P < 0.05) differences, with ducks presenting the lowest K M value by far (3.84 ± 1.01 μM of AFB 1 ), followed by turkeys (49.33 ± 7.66 μM of AFB 1 ), quail (77.79 ± 22.14 μM of AFB 1 ) and the chicken breeds Rhode Island Red and Ross (112.5 ± 33.4 and 131.8 ± 26.2 μM of AFB 1 , respectively) Fig. 3c. No differences between males and females were found in any species for this enzyme kinetic parameter. Further, no differences between the chicken breeds were found either. Regarding the CL int parameter, very large differences among the species evaluated were observed, with ducks being extraordinarily efficient in converting AFB 1 into AFB 1 -dhd compared to the other poultry species investigated Fig. 3d. CL int values for ducks, turkey, quail and Rhode Island Red and Ross chickens were 1.64 ± 1.00, 0.12 ± 0.04, 0.14 ± 0.08, 0.11 ± 0.02 and 0.05 ± 0.02 mL/mg protein/minute, respectively. No differences between males and females were observed.

Discussion
Since the discovery of aflatoxins in the early 1960's it was observed that different animal species exhibit very different adverse effects upon exposure to the toxins. For example, ducklings, pigs and dogs die acutely at dietary concentrations that are well tolerated by humans, chickens and rats [22][23][24] . In some animal models, these differences can be explained through a differential hepatic biotransformation of AFB 1 . For instance, in mice and rats, differences in the ability to trap AFB 1 with glutathione (GSH) ultimately determine the degree of AFB 1 -induced liver damage: while rats develop hepatocellular carcinoma upon chronic exposure to AFB 1 , mice are resistant. The reason for this differential response lies in the constitutive expression of high levels of an Alpha-class glutathione transferase (GST) that catalyzes the trapping of AFBO in the mouse that is only expressed at low levels in the rat 25 . Among poultry species exposed chronically to AFB 1 the only one that develops liver cancer is the duck 26 ; however, due to the short life-span of commercial poultry, it is actually the acute effects the ones that are more important. For more than a decade our research group has been searching for a biochemical explanation for the differences in susceptibility to AFB 1 among the main poultry species. We have found that AFB 1 is essentially biotransformed into aflatoxicol and AFB 1 -dhd by chicken, quail, turkey and duck liver microsomes and that at least four CYPs can bioactivate AFB 1 into the epoxide in ducks, whereas CYP2A6 is the main cytochrome responsible for this reaction in chickens, quail and turkeys 8,[18][19][20][21] . However, none of these findings could explain the extraordinarily high sensitivity of the duck compared to other poultry.
In the present study we investigated the in vitro kinetic constants V max and K M , as well as their ratio, also known as intrinsic clearance. Measurement of CL int has been used to predict the hepatic extraction of a compound 27 , and it is considered to be a measure of the total amount of enzyme that is coupled to the substrate and engaged in the conversion of the substrate into the product 28 , in other words it is a means to express enzyme efficiency 29 . Maximal velocity did not differ significantly between duck and turkey (sensitive species) or Ross chickens (highly resistant species); however, large significant differences in K M were seen among the poultry species studied. Duck presented the lowest value: almost 13 times lower that turkey, 20 times lower than quail and 30 times lower than the chicken breeds. The calculation of the CL int values revealed that duck liver microsomes clear AFB 1 as AFB 1 -dhd at rates between 15 and 33 times higher than chickens. These values are due the low duck K M values for AFB 1 -dhd production, which means that duck CYPs require very low concentrations of AFB 1 to reach maximal velocity. More tolerant or resistant species require higher amounts of AFB 1 to reach V max , making their www.nature.com/scientificreports www.nature.com/scientificreports/ CYP enzymes a low performance biotransformation system. Based on these results we propose an order of AFB 1 clearance as AFB 1 -dhd in the poultry species studied as follows: duck ⋙ quail > turkey > chicken (Ross), with values of 1.64, 0.14, 0.12 and 0.05 mL/mg/minute, respectively. In regard to differences between males and females we confirmed previous results obtained in our laboratory, where no significant differences were found by sex.
In summary, the present findings not only provide a biochemical explanation for the large differences in susceptibility to AFB 1 between chickens and ducks, but also provide strong evidence that AFB 1 -dhd is the metabolite  www.nature.com/scientificreports www.nature.com/scientificreports/ responsible for the acute toxicity of AFB 1 . We hypothesize that the large production of AFB 1 -dhd by the duck liver is the cause of the mortality and liver lesions observed with dietary concentrations that do not affect other poultry. Further, the large production of AFB 1 -dhd, which is in turn produced by the AFB 1 -exo-8,9-epoxide, might be related to the fact that ducks are the only poultry species that develop hepatic cancer after AFB 1 exposure.
Microsomal fraction processing. Liver fractions were obtained from 12 healthy birds (6 males and 6 females) from each of the following species and age: seven-week old Ross and Rhode Island Red chickens (Gallus gallus ssp. domesticus), eight-week old turkeys (Meleagris gallopavo), eight-week old quails (Coturnix coturnix japonica) and nine-week old Pekin ducks (Anas platyrhynchos ssp. domesticus). The birds were sacrificed by cervical dislocation, and their livers extracted immediately, washed with cold PBS buffer (50 mM phosphates, pH 7.4, NaCl 150 mM), cut into small pieces and stored at −70 °C until processing. The experiment was conducted following the welfare guidelines of the Poultry Research Facility and was approved by the Bioethics Committee, Faculty of Veterinary Medicine and Zootechnics, National University of Colombia, Bogotá D.C., Colombia (approval document CB-FMVZ-UN-033-18). Frozen liver samples were allowed to thaw, and 2.5 g were minced and homogenized for 1 minute with a tissue homogenizer (Cat X120, Cat Scientific Inc., Paso Robles, CA, USA) with 10 mL of extraction buffer (phosphates 50 mM pH 7.4, EDTA 1 mM, sucrose 250 mM). The homogenates were then centrifuged at 12,000 × g for 30 minutes at 4 °C (IEC CL31R Multispeed Centrifuge, Thermo Scientific, Waltham, MA, USA). After this first centrifugation, the supernatants (approximately 10 mL) were transferred into ultracentrifuge tubes kept at 4 °C and centrifuged for 90 minutes at 100,000 × g (Sorval WX Ultra 100 Centrifuge, Thermo Scientific, Waltham, MA, USA). The resulting pellets (corresponding to the microsomal fraction) were resuspended in 3 mL of storage buffer (phosphates 50 mM pH 7.4, EDTA 1 mM, sucrose 250 mM, 20% glycerol), fractioned in microcentrifuge tubes and stored at −70 °C. An aliquot of each sample was taken to determine its protein content by using the bicinchoninic acid protein quantification method according to Redinbaugh  Chromatographic conditions (HpLC). The production of AFB 1 -dhd in each incubation was quantitated in a Shimadzu Prominence system (Shimadzu Scientific Instruments, Columbia, MD, USA) equipped with a DGU-20A3R degassing unit, two LC-20AD pumps, a SIL-20AC HT autosampler, a CTO-20A column oven, an SPD-20AV UV-Vis detector, an RF-20A XS fluorescence detector, and a CBM-20A bus module, all controlled by "LC Solutions" software. The chromatography was carried out on an Alltech Alltima HP C18, 150 mm × 3.0 mm (Alltech Associates Inc., Deerfield, IL, USA) kept at 40 °C. The mobile phase was a linear gradient of solvent A (water − 0.1% formic acid) and B (acetonitrile:methanol, 1:1-0.1% formic acid), as follows: 0 min: 25% B, 1 min: 25% B, 10 min: 60% B, 10.01 min: 25% B, and 17 min: 25% B. The flow rate was 0.4 mL/min and the fluorescence detector was set at excitation and emission wavelengths of 365 nm and 425 nm, respectively. The in-vial concentration of AFB 1 -dhd was quantitated using an external standard of AFB 2a , since these two compounds share identical spectral properties 9 . Further, the monoisotopic protonated mass of AFB 1 -dhd was determined by HPLC-MS by means of a 3200 QTrap mass spectrometer (Applied Biosystems, Toronto, Canada) using a thermospray ionization probe in positive mode and the following settings: probe voltage: 4,800 V, declustering potential: 140 V, entrance potential: 10 V, curtain gas value: 30, collision energy: 81 V and collision cell exit potential: 5 V. statistical analysis. The enzymatic parameters K M and V max were determined by non-linear regression using the Marquardt method adjusting the data to the Michaelis-Menten enzyme kinetics using the equation: v = V max [S]/K M + [S], where v is the enzyme reaction velocity, [S] represents substrate concentration, V max represents maximal velocity and K M represents the Michaelis-Menten constant. Intrinsic clearance (CL int ) was calculated as the ratio V max /K M . Inter-species differences in enzymatic kinetic parameters were determined by using the Kruskal-Wallis test, while nonparametric multiple comparisons were made by using the Dwass-Steel-Critchlow-Fligner method. All analyses were performed using the Statistical Analysis System software 31 .

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.