Dealing with aflatoxin B1 dihydrodiol acute effects: Impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects.

Aflatoxin B1 aldehyde reductase (AFAR) enzyme activity has been associated to a higher resistance to the aflatoxin B1 (AFB1) toxicity in ethoxyquin-fed rats. However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species (chicken, quail, turkey and duck). Specifically, the kinetic parameters Vmax, Km and intrinsic clearance (CLint) were determined for AFB1 dialdehyde reductase (AFB1-monoalcohol production) and AFB1 monoalcohol reductase (AFB1-dialcohol production). In all cases, AFB1 monoalcohol reductase activity saturated at the highest aflatoxin B1 dialdehyde concentration tested (66.4 μM), whereas AFB1 dialdehyde reductase did not. Both activities were highly and significantly correlated and therefore are most likely catalyzed by the same AFAR enzyme. However, it appears that production of the AFB1 monoalcohol is favored over the AFB1 dialcohol. The production of alcohols from aflatoxin dialdehyde showed the highest enzymatic efficiency (highest CLint value) in chickens, a species resistant to AFB1; however, it was also high in the turkey, a species with intermediate sensitivity; further, CLint values were lowest in another tolerant species (quail) and in the most sensitive poultry species (the duck). These results suggest that AFAR activity is related to resistance to the acute toxic effects of AFB1 only in chickens and ducks. Genetic selection of ducks for high AFAR activity could be a means to control aflatoxin sensitivity in this poultry species.

Introduction

Aflatoxin B1 (AFB1) is a secondary metabolite produced by some strains of Aspergillus fungi, including Aspergillus flavus, A. parasiticus, A. nomius and A. pseudonomius. Hepatic biotransformation of AFB1 by cytochrome P450 (CYP) enzymes produces aflatoxin B1-8,9-epoxide (AFBO) with two possible stereoisomers: aflatoxin B1-8,9-exo-epoxide and aflatoxin B1-8,9-endo-epoxide; only aflatoxin B1-8,9-exo-epoxide can react with DNA, producing adducts at position 7 of guanine leading to carcinogenesis [1, 2]. Once formed, AFBO is highly electrophilic and quickly reacts with water (t1/2 = <1 second) forming AFB1-8,9-dihydrodiol (AFB1-dhd) [3-5]. AFB1-dhd has a pH dependent equilibrium with another species known as AFB1 dialdehyde, which can produce Schiff bases with lysine [6], affecting protein synthesis and causing cytotoxic effects [7]. AFB1 dialdehyde can be reduced by aflatoxin B1-aldehyde reductase (AFAR, EC 1.1.1.2; Fig 1), a cytosolic enzyme of the aldo-keto reductase superfamily, that was first described in liver extracts from ethoxyquin-fed rats [8]. A DNA sequence associated with AFAR enzyme activity [9] was later identified as the inducible isoform of aflatoxin B1-aldehyde reductase 1 (AFAR1, currently known as AKR7A1), which strongly interacts with chemopreventive agents such as ethoxyquin [10]. After the discovery of the AKR7A1 enzyme, a second encoding region was found corresponding to the enzyme AKR7A3 (formerly known as AFAR29) [11, 12]. Concerning inducible gene expression, it has been observed that both AFAR and glutathione sulfotransferases (GSTs) are inducible by compounds like ethoxyquin, making it difficult to discriminate the relevance of these two enzymatic activities on AFB1 toxicity [13, 14].

Fig 1

Bioactivation of aflatoxin B1 into aflatoxin B1 8,9-epoxide through cytochrome P450 enzymes (CYPs).

Spontaneous hydrolysis of the epoxide or the enzymatic activity of epoxide hydrolase (EPHX), produce aflatoxin B1 8,9-dihydrodiol which in a pH dependent manner equilibrates with aflatoxin B1 dialdehyde. Enzymatic reduction of aflatoxin B1 dialdehyde into aflatoxin B1 C6-monoalcohol and aflatoxin B1 C8-monoalcohol is carried out by aflatoxin B1 aldehyde reductase (AFAR), which in turn can also reduce these monoalcohols into aflatoxin B1 dialcohol.

Information on AFAR enzyme activity in poultry is scarce; however, sequences corresponding to AFAR enzymes have been reported for poultry species. In the National Center for Biotechnology Information [15] there is a chicken DNA sequence which corresponds to the AKR7A2 gene. Furthermore, in the Kegg Pathways database [16-18] the AKR7A2 enzyme is associated with aflatoxin B1 dialdehyde reduction to AFB1-C6-monoalcohol phenolate and AFB1-C8-monoalcohol phenolate; these two monoalcohols can be further reduced to AFB1-dialcohol phenolate. Other poultry DNA sequences with functional annotations found in the NCBI [15] include a sequence in turkeys that corresponds to aflatoxin B1 aldehyde reductase member 2-like, and sequences in ducks and quail that correspond to AKR7A2 aldo-keto reductase family 7, member A2.

Because it has been suggested that AFAR activity is related to a higher resistance to AFB1, especially through ameliorating the acute effects caused by aflatoxin dialdehyde [8], the present study was conducted to investigate the enzyme kinetic parameters of aflatoxin B1-monoalcohol and aflatoxin B1-dialcohol production, and to relate them with the known sensitivity to AFB1 in chickens, turkeys, ducks and quail.

Materials and methods

Reagents

Glucose 6-phosphate sodium salt, glucose 6-phosphate dehydrogenase, nicotinamide dinucleotide phosphate (NADP+), ethylenediaminetetraacetic acid (EDTA), bicinchoninic acid solution (sodium carbonate, sodium tartrate, sodium bicarbonate and sodium hydroxide 0.1 N pH 11.25), copper sulphate pentahydrate, formic acid, sucrose, bovine serum albumin, sodium borohydride, m-chloroperbenzoic acid, ethanol (spectrophotometric grade), isopropyl alcohol, 2-(cyclohexylamino) ethane sulfonic acid (CHES), aflatoxin B2 and N,N-dimethylformamide were from Sigma-Aldrich (St. Louis, MO, USA). Aflatoxin B1 was from Fermentek Ltd. (Jerusalem, Israel). Sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous and sodium chloride were from Merck (Darmstadt, Germany). Methanol, acetonitrile and water were all HPLC grade.

AFB1-dhd synthesis and purification

AFB1-dhd was produced based on the method of Fringuelli [19] with some modifications. To a 2 mL of a water:acetonitrile mix (1:1, v/v), 5 mg of AFB1 and 5.38 mg of m-chloroperbenzoic acid ≤ 70% were added and mixed. The mix was stirred at room temperature for 30 minutes, after which the AFB1-dhd formed was purified by using a μBondapack C18 125 Å, 10 μm, 7.8 x 300 mm preparative column (Waters Corporation, Milford, MA, USA) kept at 50°C. The chromatograph was an Agilent Technologies InfinityLab LC system (Agilent, Santa Clara, CA, USA) equipped with a G1314B 1260 VWD VL variable wavelength UV/Vis detector, a G1316A 1260 TCC thermostated column compartment, a G1329B 1260 ALS standard autosampler, and a G1311C 1260 Quaternary Pump VL, all modules controlled by “LC Openlab CDS ChemStation Edition” software. The mobile phase was a linear gradient of solvents A (water 0.1% formic acid) and B (isopropil alcohol 20% in acetonitrile, 0.1% formic acid) as follows: 0 min: 18% B, 10 min: 18% B, 13 min: 100% B, 15 min: 100% B, 15.01 min: 18% B, 17 min: 18% B. The flow rate was 2.5 mL/min and the UV detector was set at 365 nm. Aliquots of 50 μL of the synthesis solution were injected until all the volume was run into the HPLC system. The AFB1-dhd-containing fractions were collected, taken to dryness in a rotary evaporator (Hei-Vap Advantage, Heidolph Instruments GmbH & CO, Schwabach, Germany) and resuspended in ultrapure water. The purified AFB1-dhd was quantitated using an external standard of AFB2, since these two compounds share identical spectral properties [20].

AFB1 monoalcohol and dialcohol synthesis and purification

The synthesis of AFB1 monoalcohol and AFB1 dialcohol was made according to the method of Guengerich [21]. To a 1 mL of a 63 μM solution of AFB1-dhd in water acetonitrile 1:1 (v/v) (adjusted to pH 10 with buffer CHES 250 mM), 60 μL of an 8.9 mM solution of NaBH4 in N,N-dimethylformamide was added and let stir for 30 minutes. After this, 10 μL of formic acid was added to neutralize the mixture. Purification of the synthesis products was made by preparative HPLC on a Phenomenex Prodigy LC Column C18 ODS-3V 100 Å, 250 x 4.6 mm 5 μm (Phenomenex, Torrance CA. USA) kept at 40°C. The mobile phase was a linear gradient of solvents A (water 0.1% formic acid) and B (acetonitrile 0.1% formic acid) as follows: 0 min: 15% B, 5 min: 15% B, 15 min: 40% B, 15.01 min: 100% B, 17 min: 100% B, 17.01min: 15% B, 27 min: 15% B. The flow rate was set at 0.6 mL/min and the UV detector was set at 365 nm. Aliquots of 10 μL were injected until the whole synthesis volume was run in the HPLC system. The fractions containing the compounds were collected, taken to dryness in a rotary evaporator (Hei-Vap Advantage, Heidolph Instruments GmbH & CO, Schwabach, Germany) and suspended in ethanol for UV quantitation. The concentrations of the AFB1 monoalcohol and AFB1 dialcohol were estimated by using the AFB1 extinction coefficient in ethanol (ϵ = 21800 M-1 cm-1; [22]). To confirm their identities, the monoisotopic protonated masses of both compounds were determined by HPLC-MS on a Sciex 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. Since the molecular masses of both AFB1 C6-monoalcohol and AFB1 C8-monoalcohol are the same, and only one chromatographic peak was found, the enzyme kinetics analyses were done for both analytes under the term AFB1 monoalcohol.

Microsomal and cytosolic 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 Nicholas turkeys (Meleagris gallopavo), eight-week old Japanese quails (Coturnix coturnix japonica) and nine-week old meat-type Pekin ducks (Anas platyrhynchos ssp. domesticus). No additives or medication were added to the diets provided to the birds. The diets were formulated with the same ingredients (corn, extruded full-fat soybeans, soybean meal, vegetable oil, calcium phosphate, calcium carbonate, sodium chloride, lysine, methionine, tryptophan, choline, vitamin and mineral premix) formulated to reach or exceed the nutrient requirements of each poultry species studied. Poultry were obtained from local commercial poultry suppliers and at the moment of sacrifice no noticeable clinical signs were observed. 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, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional de 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) after adding 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). The resulting 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). An aliquot of each of the ultracentrifuged supernatants (corresponding to the cytosolic fraction) was taken to determine its protein content by the bicinchoninic acid protein quantification method according to Redinbaugh and Turley [23]. The remaining supernatant was fractioned into microcentrifuge tubes and stored at −70°C until used for the enzyme kinetic studies. No further enzyme purification was carried out and the incubations were carried out with the cytosolic fractions obtained as previously described.

Aflatoxin B1 monoalcohol and AFB1 dialcohol enzyme kinetics

To determine the enzyme kinetics of AFB1 monoalcohol and AFB1 dialcohol production (reduction of AFB1 dialdehyde by AFAR: AFB1 dialdehyde + NADH + H+ → AFB1 monoalcohol + NAD+; reduction of AFB1 monoalcohol by AFAR: AFB1 monoalcohol + NADH + H+ → AFB1 dialcohol + NAD+), the method proposed by Judah et al. [8] was used with some modifications. AFB1 dialdehyde was obtained by adjusting to pH 10 (with buffer CHES 25 mM) the chemically synthetized AFB1-dhd [4]. For AFB1 dialdehyde and AFB1 monoalcohol reductase enzyme kinetics, a discontinuous direct assay was carried out in 1.5 mL microcentrifuge tubes kept at 39°C (the normal body temperature for the age of the birds used) containing 5 mM glucose 6-phosphate, 0.5 IU of glucose 6-phosphate dehydrogenase, 0.5 mM NADP+ and 30 μg of cytosolic protein for chicken breeds or turkey, 50 μg for duck and 70 μg for quail. All volumes were completed with incubation buffer (phosphates 50 mM pH 7.4). After 3 minutes of preincubation, 4 μL of AFB1-dhd in buffer CHES 25 mM pH 10 (AFB1 dialdehyde form) at concentrations ranging from 3.38 to 66.4 μM was added and the reaction stopped after 90 seconds with 250 μL of ice-cold acetonitrile. The stopped incubations were centrifuged at 15,000 × g for 10 minutes and 5 μL were analyzed by HPLC. The amount of AFB1 monoalcohol and AFB1 dialcohol found 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-20ACHT autosampler with cooling system, a CTO-20A column oven, an RF-20AXS 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 solvents A (water − 0.1% formic acid) and B (acetonitrile − 0.1% formic acid), as follows: 0 min: 5% B, 1 min: 5% B, 15 min: 15% B, 15.01 min: 5% B, 20 min: 5% B. The flow rate was 0.6 mL/min and the fluorescence detector was set at excitation and emission wavelengths of 360 nm and 440 nm, respectively. The in-vial concentrations of AFB1 monoalcohol and AFB1 dialcohol were quantitated by using the standards of AFB1 monoalcohol and AFB1 dialcohol chemically synthetized as previously described.

Statistical analysis

The enzymatic parameters K and V were determined by non-linear regression using the Marquardt method adjusting the data to the Michaelis-Menten enzyme kinetics using the equation: v = V[S]/K + [S], where v is the enzyme reaction velocity, [S] represents substrate concentration, V represents maximal velocity and K represents the Michaelis-Menten constant. Intrinsic clearance (CL—mL/mg protein/minute) was calculated as the ratio V/K. The calculated CL only applies for the selected enzymatic activity and not for the hepatic clearance, since AFAR enzyme was not purified from liver extracts. In all cases the kinetic parameters are “apparent” because hepatic extracts and not purified enzymes were used. 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, with a significance level of 5% (p <0.05). Correlations were estimated by using the Spearman’s rank-order correlation coefficient. All analyses were performed using the Statistical Analysis System software [24].

Results

The molecular mass of the chemically-synthetized AFB1 monoalcohol was confirmed by mass spectrometry, since the peak eluting at t = 13.92 minutes (Fig 2A) had the expected protonated monoisotopic mass of the compound (349.2 Da; Fig 2B). Similarly, the molecular mass of the AFB1 dialcohol was also confirmed, given that the mass of the peak eluting at t = 12.29 minutes (Fig 3A) corresponded to the expected protonated monoisotopic mass (351.0 Da; Fig 3B).

Fig 2

Identification of AFB1 monoalcohol by HPLC-MS.

(A) Chromatogram of the purified AFB1 monoalcohol product obtained from the reduction of AFB1 dialdehyde with NaBH4. The peak at t = 13.92 shows the putative AFB1 monoalcohol product. (B) Protonated monoisotopic mass found in the 13.92 min peak, corresponding to a value of 349.2 Da.

Fig 3

Identification of AFB1 dialcohol by HPLC-MS.

(A) Chromatogram of the purified AFB1 dialcohol product obtained from the reduction of AFB1 dialdehyde with NaBH4. The peak at t = 12.29 shows the putative AFB1 dialcohol product. (B) Protonated monoisotopic mass found in the 12.29 min peak, corresponding to a value of 351.0 Da.

The enzyme kinetics of AFB1-monoalcohol production (AFB1-dialdehyde reductase activity) is shown in Fig 4. The AFAR enzyme activity does not seem to saturate in any of the species studied, even at the highest AFB1-dialdehyde concentration tested (66.4 μM; Fig 4A). Further, large differences in biotransformation rates were observed, with Rhode Island Red chickens and turkey showing the highest rates, quail and duck the lowest, and the Ross chickens showing an intermediate rate. In regard to the V kinetic parameter, the Rhode Island Red chickens showed a significantly higher value (10.1 ± 5.52 nmol AFB1 monoalcohol/mg protein/minute), which was about 5 times higher than the values obtained with other poultry (p <0.0001; Fig 4B). No differences in V were found among the Ross chickens, turkey, quail and duck; however, differences for this parameter were found between sexes for the Ross chickens (1.21 ± 0.38 and 3.06 ± 0.77 nmol AFB1 monoalcohol/mg protein/minute for females and males, respectively, p = 0.0039) and the turkey (1.75 ± 0.93 and 3.52 ± 1.55 nmol AFB1 monoalcohol/mg protein/minute for females and males, respectively; p = 0.025). The K parameter value (Fig 4C) was significantly higher (p <0.0001) in Rhode Island Red chickens (393.5 ± 227.8 μM of AFB1 dialdehyde) compared with the duck (139.4 ± 176.5 μM), the Ross chicken breed (80.2 ± 46.53 μM), and the turkey (72.7 ± 45.9 μM); however, it did not differ significantly from the quail (231.0 ± 206.1 μM). Differences in K between sexes were found only for the Ross chickens, with values of 42.08 ± 23.8 and 118.4 ± 26.4 μM for females and males, respectively (p = 0.0039). The calculated intrinsic clearance (CL) for AFB1-monoalcohol production was the highest in the turkey, and the Ross and Rhode Island Red chickens (0.038 ± 0.008, 0.031 ± 0.011, and 0.026 ± 0.004 mL/mg protein/minute, respectively). Significantly lower CL values (p <0.0001) were observed for quail (0.011 ± 0.003 mL/mg protein/minute) and duck (0.019 ± 0.010 mL/mg protein/minute; Fig 4D). Only the duck showed differences between sexes (0.02 ± 0.01 and 0.01 ± 0.004 mL/mg protein/minute for female and male, respectively; p = 0.0374).

Fig 4

Enzyme kinetic parameters of cytosolic in vitro AFB1 monoalcohol production from AFB1 dialdehyde.

(A) Saturation curve at AFB1 dialdehyde concentrations of 3.4 to 66.4 μM (B) Maximal velocity (V). (C) Michaelis-Menten constant (K). (D) Intrinsic Clearance (CL; V/K). Species mean values sharing the same letter do not differ significantly. Statistical differences (P <0.05) were calculated using the Kruskal-Wallis test and nonparametric multiple comparisons were done by the Dwass-Steel-Critchlow-Fligner method. Values are means ± SEM of 12 birds.

AFB1-dialcohol enzyme production kinetics is presented in Fig 5. In contrast to AFB1-monoalcohol production activity, AFB1-dialcohol production reached a plateau (enzyme saturation due to substrate concentration) below the highest AFB1 dialdehyde concentration tested (66.4 μM); however, duck and quail reached the plateau at a lower substrate concentration compared with the two strains of chickens and the turkey (Fig 5A). A large difference in V was found between the turkey, and the Ross and Rhode Island Red chickens (0.38 ± 0.17, 0.33 ± 0.18 and 0.30 ± 0.17 nmol AFB1 dialcohol/mg protein/minute, respectively) and the duck (0.18 ± 0.09 nmol AFB1 dialcohol/mg protein/minute) and quail (0.11 ± 0.06 nmol AFB1 dialcohol/mg protein/minute) (p <0.0001; Fig 5B). Differences by sex for this parameter were found in Rhode Island Red chickens (0.41 ± 0.17 and 0.20 ± 0.05 nmol AFB1 dialcohol/mg protein/minute for females and males respectively, p = 0.0374), Ross chickens (0.21 ± 0.06 and 0.46 ± 0.18 nmol AFB1 dialcohol/mg protein/minute for females and males respectively, p = 0.0104), and turkey (0.23 ± 0.03 and 0.53 ± 0.08 nmol AFB1 dialcohol/mg protein/minute for females and males respectively, p = 0.0039). Even though there were numerical differences in K values among the different poultry species, the differences failed to reach statistical significance (p = 0.4216). Values of K were 21.57 ± 14.32 μM for Rhode Island Red chickens, 19.43 ± 11.62 μM for Ross chickens, 15.63 ± 5.47 μM for ducks, 13.97 ± 6.22 μM for quail, and 12.60 ± 6.12 μM for turkeys (Fig 5C). Only Ross chickens (10.62 ± 4.97 and 28.22 ± 9.33 μM for females and males, respectively, p = 0.0104) and turkey (7.20 ± 2.10 and 18.02 ± 2.77 μM for females and males, respectively, p = 0.0039) showed significant differences between sexes. Finally, the CL value for AFB1 dialcohol production (Fig 5D) was highest for the turkey (0.032 ± 0.008 mL/mg protein/minute) followed by Ross and Rhode Island Red chickens (0.020 ± 0.009 and 0.015 ± 0.005 mL/mg protein/minute, respectively), duck (0.012 ± 0.005 mL/mg protein/minute) and quail (0.008 ± 0.002 mL/mg protein/minute) with a p <0.0001. There were no significant differences between sexes.

Fig 5

Enzyme kinetic parameters of cytosolic in vitro AFB1 dialcohol production from AFB1 dialdehyde.

(A) Saturation curve at AFB1 dialdehyde concentrations of 3.4 to 66.4 μM (B) Maximal velocity (V). (C) Michaelis-Menten constant (K). (D) Intrinsic Clearance (CL; V/K). Species mean values sharing the same letter do not differ significantly. Statistical differences (P <0.05) were calculated using the Kruskal-Wallis test and nonparametric multiple comparisons were done by the Dwass-Steel-Critchlow-Fligner method. No differences were found for K enzyme activity parameter between poultry species. Values are means ± SEM of 12 birds.

When the two enzymatic activities were compared, it was found that AFB1-monoalcohol and AFB1-dialdehyde reductase activities were significantly correlated (p <0.05) for all poultry species. Spearman´s rank correlation coefficients for these two activities were 0.87 for Ross chickens, 0.88 for Rhode Island Red chickens, 0.84 for quail, 0.91 for turkey and 0.78 for duck. When the data obtained from all species were combined, the Spearman´s rank correlation coefficient value was 0.86 (Fig 6).

Fig 6

Correlation of AFB1 monoalcohol reductase enzyme activity vs AFB1 dialdehyde reductase enzyme activity velocities.

Cytosolic fractions from 12 birds were used and concentrations of AFB1 dialdehyde from 3.6 to 66.4 μM. Spearman’s rank-order correlation coefficient was calculated for A: Ross chicken breed (0.87), B: Rhode Island Red Chicken breed (0.88), C: quail (0.84), D: turkey (0.91), E: duck (0.78) and F: all species (0.86).

Finally, the ratios of CL for AFB1-monoalcohol production / CL for AFB1-dialcohol production and CL for AFB1-dialcohol production / CL for AFB1-monoalcohol production did not show significant differences among poultry species (p = 0.0846 and 0.0881, respectively; Table 1). However, when the ratios of CLint for AFB1-dhd production / CL for AFB1 monoalcohol production and CL for AFB1-dhd production / CL for AFB1 dialcohol production were calculated (based on AFB1-dhd production data from the same set of samples [5]) significant differences were observed among the different species evaluated (p <0.0001; Table 1).

Table 1

Comparison of CL AFB1-dhd enzyme production / CL AFB1 dialcohol enzyme production (dhd/dial), CL AFB1-dhd enzyme production / CL AFB1 monoalcohol enzyme production (dhd/mono), CL AFB1 monoalcohol enzyme production / CL AFB1 dialcohol enzyme production (mono/dial) and the inverse (dial/mono) ratios.

Among the different poultry species means were compared by using the Dwass-Steel-Critchlow-Fligner method. Values are means ± SD of 12 birds. Mean values with the same superscript do not differ significantly.

Species	dhd/dial	dhd/mono	mono/dial	dial/mono
Ross chickens	2.77 ± 1.84a	1.65 ± 0.80a	1.68 ± 0.68a	0.67 ± 0.22a
Rhode Island Red chickens	7.44 ± 2.37b	4.01 ± 0.79b	1.90 ± 0.65a	0.59 ± 0.21a
Quail	19.92 ± 11.89c	12.99 ± 7.79c	1.59 ± 0.63a	0.70 ± 0.21a
Turkey	3.89 ± 1.91a	3.30 ± 1.55b	1.22 ± 0.30a	0.87 ± 0.23a
Duck	167.80 ± 105.57d	99.20 ± 62.08d	1.92 ± 1.15a	0.70 ± 0.39a

Discussion

The in vivo sensitivity to AFB1 in poultry species follows the order ducklings >>turkey poults >goslings >pheasant chicks >quail chicks >chicks [25]. Recent research conducted in our laboratory has shown that in poultry species the hepatic in vitro AFB1-dhd production is related to the in vivo sensitivity, and we have hypothesized that AFB1-dhd is the metabolite responsible for the acute toxic effects of AFB1 [5]. AFB1-dhd can exist in a pH-dependent equilibrium with AFB1-dialdehyde (pKa value of 8.29) [4] and at a physiological pH of 7.2, AFB1-dialdehyde can adduct lysine residues in proteins, leading to cytotoxicity. AFB1-dialdehyde can be reduced to AFB1-monoalcohol and AFB1-diacohol by the AFAR enzyme. In vitro assays have shown that AFAR activity inhibits the formation of adducts with proteins [8], and therefore the alcohols can be considered detoxification products (there is no evidence that they can form adducts). Further, it has been observed that toxic dialdehydes like malondialdehyde (MDA) can be oxidized by mitochondrial aldehyde reductase, reducing its capacity to form adducts with nucleophile compounds like thiobarbituric acid [26]. In the present study, the V for AFB1 dialdehyde reductase enzyme activity (AFB1-monoalcohol production) was highest for Rhode Island chickens (a resistant species), while the K was the lowest for both resistant (Ross chickens) and susceptible species (turkey and duck). However, it is important to note that the K parameter only reflects the enzyme-substrate complex dissociation constant [27]; in fact, the K enzyme parameter is a collection of rate constants and not the binding constant for the interaction between enzyme and substrate, as it has been misunderstood [28]. Therefore, this parameter only indicates that Ross chickens, turkey and duck AFAR enzymes reach the V at lower concentrations than Rhode Island Red chickens and quail. In the case of enzyme efficiency, measured as intrinsic clearance (CL), it was observed that highly resistant species (both chicken breeds) have the most efficient AFB1-dialdehyde reductase enzyme activities. However, the turkey, which has an intermediate sensitivity between chickens and ducks, also had a high AFB1-dialdehyde reductase activity; further, the quail, which is almost as resistant to AFB1 as the chicken, had an AFB1-dialdehyde reductase activity comparable to that of the duck. These results suggest that AFB1-dhd detoxification by AFAR is related to poultry species resistance only in chickens and ducks. It is important to highlight that AFAR enzyme activity cannot be considered as the only reaction capable of explaining the differences in sensitivity among different poultry species. For example, glutathione sulfotransferase (GST) enzyme activity is capable of affecting the production of AFB1 dihydrodiol (and therefore the production of AFB1 dialdehyde) through AFBO nucleophilic trapping. Therefore, the toxicity of AFB1 should be considered as a multifactorial mechanism in which different metabolic pathways in AFB1 biotransformation are interconnected, including AFAR activity. Regarding the AFB1-monoalcohol reductase V value, it was found that it does seem to be associated with species sensitivity since the chicken breeds had a higher value and the ducks a low value; however, the turkey is again an exception with a V value similar to those found for the chicken breeds. Due to the fact that the K value for this reaction did not differ significantly among poultry species, the AFB1-monoalcohol reductase CL values were dependent on the differences found in V. Therefore, the CL for AFAR AFB1-monoalcohol reductase showed differences between tolerant species (chicken breeds and quail) and the duck, but not for the turkey. Consideration must be given to the fact that AFAR expression and activity change with age [29] and this is probably the explanation for the higher AFAR activity found in the turkeys since they were older than 41-days (56 days-old), the age at which they are at the peak of AFAR activity.

The highly significant correlation found between AFB1-dialdehyde and AFB1-monoalcohol reductase activities strongly suggests that the same enzyme catalyzes both activities. This fact explains why the AFB1-dialdehyde reductase activity did not saturate, even at the highest AFB1-dialdehyde concentration used (66.4 μM of AFB1-dialdehyde), whereas AFB1-monoalcohol activity saturated completely. As the reduction of AFB1-dialdehyde into AFB1-monoalcohol moves forward, and the concentration of AFB1-monoalcohol increases, the reduction of AFB1-monoalcohol into the dialcohol saturates at lower AFB1-dialdehyde concentrations, since both activities are being carried out by the same AFAR enzyme. This scenario is substantiated by the ratios of CL for AFB1-monoalcohol production / CL for AFB1-dialcohol production, which ranged from 1.22 to 1.92 (Table 1); these ratios indicate that AFB1-monoalcohol production is favored over AFB1-dialcohol production. Concerning the specific enzyme responsible for these reactions, it is most likely that the AFAR enzyme aldo-keto reductase AKR7A2 member is the one responsible for these two activities in the poultry species studied, according to the information provided by the NCBI [15] and the Kegg pathways [16-18] databases.

Recent findings have shown that AFB1-dhd is probably the metabolite responsible for the acute toxic effects of AFB1 since its hepatic in vitro production in related to the known in vivo sensitivity in poultry [5]. When the ratios of CL for AFB1-dhd production / CL for AFB1-monoalcohol production were compared it was found that AFB1-dhd production is highly favored over AFB1-monoalcohol production in the most sensitive species (the duck) compared with the other poultry species. The calculated ratios followed the order duck >>>quail >Rhode Island Red chickens = Turkey >Ross chickens. The ratios of CL for AFB1-dhd production / CL for AFB1 dialcohol production also followed the same pattern, suggesting that the cytotoxic effects of AFB1 exposure in ducks are due to the lack of a detoxification pathway for the large amounts of AFB1-dhd produced by their cytochrome P450 enzymes. A previous study failed to find an association between AFAR enzyme activity and animal resistance to AFB1 exposure [30]. This apparent discrepancy could be the result of using the inappropriate model to determine V and K (the Lineweaver-Burk linearization method) since it is widely accepted that a nonlinear regression is a more accurate and precise method to estimate these parameters [31, 32].

Conclusion

The present study provides, for the first-time, experimental evidence for the role of AFAR activity in the resistance to the acute toxic effects of AFB1-dhd in different poultry species. AFB1 dialdehyde and AFB1 monoalcohol reductase enzyme activities (probably catalyzed by the AKR7A2 aldo-keto reductase family member) are higher in resistant species like the chickens, but also in less resistant like the turkey. Interestingly it was found that the ratio of CL for AFB1-dhd production / CL for AFB1-dialcohol production is more than a hundred times higher in the duck than in the chicken; this finding suggests that the duck is unable to cope with the highly unstable metabolite AFB1-dhd, which results in acute toxic liver damage upon AFB1 exposure. Finally, the correlation analysis between AFB1-dialdehyde and AFB1-monoalcohol reductase activities shows that some individuals posses high activity for both enzyme reactions; this fact suggests the possibility of selecting individuals with high rates of AFAR activity for the genetic selection of resistance, especially in sensitive species like the duck (S3 Table). The present trial is limited to the use of only one duck breed (Pekin breed), the number of individuals and the flock source of birds. Therefore, in order to identify possible tolerant individuals, a larger variety of birds should be assessed to validate more clearly the population effect across a wider diversity of bird sources. Additionally, possible interbreed differences should also be considered, since significant histopathological differences have been reported in different duck breeds after AFB1 exposure [33].

Feed ingredients and nutritional content of the diets fed to the experimental birds.

(DOCX)

Click here for additional data file.

Final body weight and total feed intake at the time of sacrifice of the experimental birds.

(DOCX)

Click here for additional data file.

Values of CL AFB1-dhd enzyme production / CL AFB1 dialcohol enzyme production (dhd/dial), CL AFB1-dhd enzyme production / CL AFB1 monoalcohol enzyme production (dhd/mono), CLint AFB1 monoalcohol enzyme production / CL AFB1 dialcohol enzyme production (mono/dial) and the inverse (dial/mono) ratios per individuals.

Values in bold represent individuals which ratio value is below SD.

(DOCX)

Click here for additional data file.

19 Mar 2020

PONE-D-19-32966

Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerance to AFB1 toxic effects

PLOS ONE

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Reviewer #1: This manuscript provides an evidence that differences in the resistance of poultry species to aflatoxin B1 toxicity are associated with differences in AFAR activity.

Please consider the following suggestions:

Klein et al (Comp Biochem Physiol C Toxicol Pharmacol. 2002 Jun; 132 (2): 193-201.) show that AFAR acivity increases with age.

In this study, chickens were 7 weeks old, turkeys and quails were 8 weeks old, and Peking Duck was 9 weeks old. Please explain whether these age differences affect AFAR activity.

Different poultry species may have different feeds.

Please explain the potential effects of different feed types, compositions, and feeding patterns on bioenzymatic activity.

Ethoxyquin is an ingredient found in commercial feeds. It should be explained whether the diet used in this study contained ethoxyquin.

L307-309: The present study... in chickens and ducks.

...in different poultry species?

Reviewer #2: GENERAL COMMENTS

The MS provides a description of a study with potentially very important results. However, before the suggestions of selecting ducks with high AFAR activity, a larger variety of birds may need to be assessed to validate more clearly the population effect across a wider diversity of bird sources.

Is there any evidence already that some ducks may be dramatically less susceptible to AF exposure than others?

The limitation of the study should be included in the discussion.

It is not clear why other routes of detoxification were not discussed and this should be addressed. For example, enzyme activity in other organs may add to that of the activity in the liver (eg GST activity in the kidney).

SPECIFIC COMMENTS

TITLE

Change tolerance to tolerant: “… in poultry species TOLERANT to AFB1 toxic effects”

ABSTRACT

I suggest that the abstract would invite greater interest from potential readers if it stated more clearly WHAT IS KNOWN and WHAT IS ADDED BY THIS STUDY. It is an important and interesting subject, but one that is familiar to a very limited audience, an audience that could be increased by acknowledging that they are not abreast of the AFB1 literature.

As captured later, please consider and respond to the following points:

Intrinsic clearance: from a pharmacological perspective, clearance relates to a particular organ. In the biochemical context that is described in the MS it applies only to the selected enzyme. This should be made very clear, as hepatic clearance may be far higher than a single enzyme clearance if more than one enzyme is involved.

Highest AF dialdehyde concentration tested (66mcM): how does this concentration relate to the expected exposure concentration in intoxicated birds?

The same AFAR enzyme catalyses mono- and di-alcohol AFB1: is this the same enzyme or the same or related or linked enzyme?

AFAR activity related to toxic effects in chickens and ducks – not quail and turkey: does not this imply that the relationship is complex and potentially multifactorial in all species?

INTRODUCTION

No comments (all good)

MATERIALS AND METHODS

Lines 100-104

Details of the breed/source of the turkeys, quail, and Pekin ducks should be provided, especially to allow comparison with any further studies of in vitro or in vivo activity of AFB1.

In addition, the composition of the diet of the birds prior to sacrifice should be described, especially the presence of any medications in the diet. For example, it would be common practice to include anticoccidial agents in the diet of chickens and special anticoccidial agents can cause changes in hepatic enzyme activity. Similarly, the main nutritional ingredients in the diet should be identified.

Line 128

Is there a normal avian body temperature? Measurement of adult/mature bird body temperature has found a temperature of 41 for chickens, 41 for turkeys and 42 for ducks. Your comments on choice of a single temperature for all species and justification for the selection of 39 would be welcome. Younger birds may have a temperature closer to 39.

Lines 150-160

The statistical significance level should be presented and the actual value in each test presented later in the MS

Line 155

Intrinsic clearance – the units should be provided. In addition, as each species has a different mass of liver tissue, the total liver clearance for the enzyme of interest is a useful parameter.

RESULTS

Fig 2 and Fig 3 Captions

Why is the difference in MW of monoalcohol and dialcohol 2 units and not 1 unit?

DISCUSSION

Line 248

… its capacity to FORM adduct …

Line 264

The fact that there is an apparent relationship between AFAR activity and AFB1 susceptibility only in ducks and chickens, and not in quail and turkeys, suggests that toxicity may be multifactorial. Some discussion of this would add increased credibility to the study.

Line 273-274

Does the correlation described indicate that the same enzyme is involved or suggest that the enzyme activities are related or linked? Why is the only conclusion that they are the same enzyme?

Line 276

How representative of the concentration present in the liver of intoxicated birds is the highest concentration of AFBI-dialdehyde of 66mcM?

Line 298

Is the cytotoxic effect of AFB1 exposure in ducks DUE TO or ASSOCIATED WITH the lack of a detoxification pathway? What is the status of GST detoxification pathways in ducks?

CONCLUSION

Line 317

The mention of individuals with high activity may relate to the SD values in Table 1. It would be useful to provide the individual bird values from which the Table was compiled – in this way the variation by individual birds and the number of birds with significant variation can be seen – this could be included in a supplement.

Line 318-Line 319

Genetic selection would only be useful if there was a relationship between intoxication and enzyme activity. This would need to be established for the duck and may require challenge of ducks at the extremes (ie high and low) of enzyme activity with AF to see if in fact there is an in vitro in vivo (IVIV) relationship. Furthermore, only a very small sample of ducks from an unknown source has been studied and described herein.

**********

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Reviewer #1: Yes: Takeshi Kawasaki

Reviewer #2: Yes: Stephen Page

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15 Apr 2020

Reviewer #1:

This manuscript provides an evidence that differences in the resistance of poultry species to aflatoxin B1 toxicity are associated with differences in AFAR activity.

Please consider the following suggestions:

Klein et al (Comp Biochem Physiol C Toxicol Pharmacol. 2002 Jun; 132 (2): 193-201.) show that AFAR activity increases with age.

In this study, chickens were 7 weeks old, turkeys and quails were 8 weeks old, and Peking Duck was 9 weeks old. Please explain whether these age differences affect AFAR activity.

Reply: We are in complete agreement with the reviewer about the changes on the expression levels of AFAR enzyme associated with the bird’s age, as it was reported by Klein et al. (2002). Unfortunately, the study of Klein et al. (2002) does not present western immunoblot densitometry values to compare between ages; however, their data suggest that AFAR expression reaches a maximum level in turkeys at 41 days of age. Further, other two key enzymes involved in xenobiotic biotransformation (cytochrome P450s and glutathione sulfotransferases) also seem to reach a maximum enzyme activity at 41 days of age in turkeys.

Since the turkeys used in our study were 8 weeks old (56 day-old), they were expected to have reached their maximum AFAR activity. To our knowledge there are no data regarding the relationship between age and AFAR activity in the other poultry species studied. A sentence has been inserted indicating the fact that AFAR expression and activity change with age and that consideration must be given to this fact when analyzing the results of the present trial (lines 297 - 300).

Different poultry species may have different feeds.

Please explain the potential effects of different feed types, compositions, and feeding patterns on bioenzymatic activity.

Reply: We agree with the reviewer in that different types of feed may affect the patterns of bioenzymatic activity. However, all diets were formulated with the same macro-ingredients (corn, extruded full-fat soybeans, soybean meal, vegetable oil, calcium phosphate, calcium carbonate, etc.), adjusted for the specific requirements of each poultry species. Therefore, we do not expect an effect of the diet on the results obtained.

Ethoxyquin is an ingredient found in commercial feeds. It should be explained whether the diet used in this study contained ethoxyquin.

Reply: No ethoxyquin was added to the diets provided to the experimental birds.

L307-309: The present study... in chickens and ducks. ...in different poultry species?

Reply: The words “in chickens and ducks” have been replaced by “in different poultry species” (lines 335 - 336).

Reviewer #2: GENERAL COMMENTS

The MS provides a description of a study with potentially very important results. However, before the suggestions of selecting ducks with high AFAR activity, a larger variety of birds may need to be assessed to validate more clearly the population effect across a wider diversity of bird sources. Is there any evidence already that some ducks may be dramatically less susceptible to AF exposure than others?

The limitation of the study should be included in the discussion.

Reply: We agree with the reviewer in that the population of ducks used in our study is very limited. However, we chose the white Pekin duck in our study, which it is the type of duck most commonly used in commercial duck meat and egg production. We have added a sentence addressing this issue in the revised manuscript (lines 347 - 353).

It is not clear why other routes of detoxification were not discussed and this should be addressed. For example, enzyme activity in other organs may add to that of the activity in the liver (eg GST activity in the kidney).

Reply: We completely agree with the reviewer in that there are other routes of detoxication that are also important in AF metabolism. However, due to the fact that aflatoxin exposure is through the gastrointestinal tract and because all of the absorbed compounds from the GIT must pass first through the liver (because of the portal circulation), it is the liver the organ that is most important in AF biotransformation and detoxication. GST detoxication of the electrophilic metabolite of AFB1 (the 8,9-exo-epoxide, AFBO) is a key detoxication pathway that is also being investigated by our research group. What we want to point out in the present study is that tolerant poultry species like the chicken have a higher AFAR enzyme activity than sensitive poultry species like the duck. We do not intend to ignore the possible role of other metabolic pathways in AFB1 sensitivity. We only want to postulate that AFAR activity is probably contributing to the higher resistance of chickens to the adverse effects of AFB1.

SPECIFIC COMMENTS

TITLE

Change tolerance to tolerant: “… in poultry species TOLERANT to AFB1 toxic effects”

Reply: “Tolerance” has been replaced by “tolerant” in the title.

ABSTRACT

I suggest that the abstract would invite greater interest from potential readers if it stated more clearly WHAT IS KNOWN and WHAT IS ADDED BY THIS STUDY. It is an important and interesting subject, but one that is familiar to a very limited audience, an audience that could be increased by acknowledging that they are not abreast of the AFB1 literature.

Reply: This is a very important suggestion and we would like to thank the reviewer for it. In order to create a greater interest in the article the following sentence has been added at the beginning of the abstract: “Aflatoxin B1 aldehyde reductase (AFAR) enzyme activity has been associated to a higher resistance to the aflatoxin B1 (AFB1) toxicity in ethoxyquin-fed rats. However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species (chicken, quail, turkey and duck)”

As captured later, please consider and respond to the following points:

Intrinsic clearance: from a pharmacological perspective, clearance relates to a particular organ. In the biochemical context that is described in the MS it applies only to the selected enzyme. This should be made very clear, as hepatic clearance may be far higher than a single enzyme clearance if more than one enzyme is involved.

Reply: We are in complete agreement with the reviewer’s comments regarding CLint.

We have inserted a sentence explaining that the calculated intrinsic clearance only applies for the selected enzymatic activity (not for the liver) and that due to the fact that we did not purify AFAR enzyme from hepatic cytosolic extracts, the calculated CLint values are only “apparent” [Apparent Intrinsic Clearance (CLintapp)] (lines 128 – 129, 167 – 170).

Highest AF dialdehyde concentration tested (66mcM): how does this concentration relate to the expected exposure concentration in intoxicated birds?

Reply: The AFB1 concentration expected to occur in the hepatocyte upon AFB1 exposure is in the nanomolar or even the femtomolar order (Ch’in, J. & Devlin, T. The distribution and intracellular translocation of aflatoxin B1 in isolated hepatocytes. Biochem. Biophys. Res. Commun. 122, 1–8). In chickens dosed with a single intra-crop bolus of 2 mg of AFB1 per kg of body weight (a very large amount compared to what it is expected from a contaminated diet) AFB1 plasma concentrations have been only 96 nM (Lauwers, M., Croubels, S., De Baere, S., Sevastiyanova, M. 2019. Supplementary materials: Assessment of dried blood spots for multi-mycotoxin biomarker analysis in pigs and broiler chickens. Toxins 11: S1 – S8). Therefore, it is likely that under natural conditions AFB1 dialdehyde concentrations are not going to reach the 66 µM concentration, because the cytosolic AFB1 concentration is expected to be in the nanomolar range. We used high AFB1 dialdehyde concentrations in our in vitro model because of the need to saturate the enzymes; also we needed to be able to detect the analytes (the limit of quantitation of the analytical technique is 0.4 ng/g for AFB1).

The same AFAR enzyme catalyzes mono- and di-alcohol AFB1: is this the same enzyme or the same or related or linked enzyme?

Reply: The search conducted in databases like the NCBI or Kegg pathways indicates that the poultry AKR7A2 enzyme is responsible for the reduction of AFB1 dialdehyde and AFB1 monoalcohol. Because we found a strong relationship between these two enzyme activities (high Spearman’s rank-order correlation coefficient), we postulated that both activities are carried out by the same enzyme.

AFAR activity related to toxic effects in chickens and ducks – not quail and turkey: does not this imply that the relationship is complex and potentially multifactorial in all species?

Reply: We completely agree with the reviewer. AFAR activity is expected to depend on other reactions like glutathione sulfotransferase (GST) enzyme activity. In separate studies conducted by our research group we have found significant differences in GST activity among the same poultry species studied. Differences in the enzymatic nucleophilic trapping of AFBO will affect the production of AFB1 dihydrodiol and therefore the concentration of AFB1 dialdehyde. The overall metabolism of AFB1 most likely depends on several enzymatic reactions. A sentence has been added to clarify the multifactorial nature of the AFB1 detoxication pathways (lines 282 - 289).

INTRODUCTION

No comments (all good)

MATERIALS AND METHODS

Lines 100-104

Details of the breed/source of the turkeys, quail, and Pekin ducks should be provided, especially to allow comparison with any further studies of in vitro or in vivo activity of AFB1.

Reply: Details of the poultry used in the study have been provide as follows: Nicholas turkeys, Japanese quail (Coturnix coturnix japonica) (there are no Bobwhite quail -Colinus virginianus- in Colombia) and meat-type Pekin ducks (there are both meat-type and egg-type Pekin ducks (lines 102 - 104).

In addition, the composition of the diet of the birds prior to sacrifice should be described, especially the presence of any medications in the diet. For example, it would be common practice to include anticoccidial agents in the diet of chickens and special anticoccidial agents can cause changes in hepatic enzyme activity. Similarly, the main nutritional ingredients in the diet should be identified.

Reply: No additives or medication were added to the diets provided to the birds. The diets were formulated with the same ingredients (corn, extruded full-fat soybeans, soybean meal, vegetable oil, calcium phosphate, calcium carbonate, sodium chloride, lysine, methionine, tryptophan, choline, vitamin and mineral premix) formulated to reach or exceed the nutrient requirements of each poultry species studied. The ingredients and lack of use of non-nutritive additives have been indicated in the revised manuscript (lines 104 - 109).

Line 128

Is there a normal avian body temperature? Measurement of adult/mature bird body temperature has found a temperature of 41 for chickens, 41 for turkeys and 42 for ducks. Your comments on choice of a single temperature for all species and justification for the selection of 39 would be welcome. Younger birds may have a temperature closer to 39.

Reply: We agree with reviewer comment. Because the birds used in this trial were 7–9 weeks old birds, the expected temperature was around 39 degrees Celsius. The revised manuscript has been added with the words “the normal body temperature for the age of the birds used” (line 138 - 139).

Lines 150-160

The statistical significance level should be presented and the actual value in each test presented later in the MS

Reply: The following sentence has been added to the “Statistical analysis” section: “with a significance level of 5% (p<0.05).” (line 173). The actual p-value obtained in each test has been presented in the “Results” section where appropriate.

Line 155

Intrinsic clearance – the units should be provided. In addition, as each species has a different mass of liver tissue, the total liver clearance for the enzyme of interest is a useful parameter.

Reply: The CLint units have been indicated: “mL/mg protein/minute” (lines 166-167). We agree with the reviewer comment about the concept of total liver clearance; however, since we did not purify the hepatic AFAR enzyme, we are not able to calculate the total liver clearance. We used the CLint value to express the enzyme efficiency in the same way that it has been reported in previous studies [Guengerich, F. P. Analysis and characterization of enzymes and nucleic acids relevant to toxicology. In Hayes’s Principles and Methods of Toxicology Sixth Edition (eds Hayes, A. W. & Kruger, C. L.) 1939, ISBN-13: 978-1842145364 (CRC Press, 2014)]. Further, as it was mentioned previously, the CLint value obtained corresponds to the apparent CLint.

RESULTS

Fig 2 and Fig 3 Captions

Why is the difference in MW of monoalcohol and dialcohol 2 units and not 1 unit?

Reply: AFB1 monoalcohol has an aldehyde function (R – CHO) that is reduced to alcohol, thus producing AFB1 dialcohol. In this carbonyl function, there is a double bond between carbon and oxygen (R – CH = O). When the carbonyl function is reduced, this C = O double bond becomes R – CH – O. One hydrogen is received by oxygen to produce the alcohol function (R – CH – OH) and the carbon atom receives the other hydrogen to complete its 4 bonds (R – CH2 – OH). That is why the molecular weight difference between the monoalcohol and the dialcohol is 2 units.

DISCUSSION

Line 248

… its capacity to FORM adduct …

Reply: The word “form” has been inserted as it was suggested (line 265)

Line 264

The fact that there is an apparent relationship between AFAR activity and AFB1 susceptibility only in ducks and chickens, and not in quail and turkeys, suggests that toxicity may be multifactorial. Some discussion of this would add increased credibility to the study.

Reply: We completely agree with the reviewer. The following paragraph has been added to the Discussion section: “It is important to highlight that AFAR enzyme activity cannot be considered as the only reaction capable of explaining the differences in sensitivity among different poultry species. For example, glutathione sulfotransferase (GST) enzyme activity is capable of affecting the production of AFB1 dihydrodiol (and therefore the production of AFB1 dialdehyde) through AFBO nucleophilic trapping. Therefore, the toxicity of AFB1 should be considered as a multifactorial mechanism in which different metabolic pathways in AFB1 biotransformation are interconnected, including AFAR activity” (lines 282 - 289).

Line 273-274

Does the correlation described indicate that the same enzyme is involved or suggest that the enzyme activities are related or linked? Why is the only conclusion that they are the same enzyme?

Reply: A linear correlation between two enzyme activities has been used in different experimental models to assume that a single enzyme is responsible for both enzyme activities (Eaton, D.L., Ramsdell, H.S., Neal, G. E. 1994. Biotransformation of aflatoxins. In: The toxicology of aflatoxins. Human health, veterinary, and agricultural significance. Eds. Eaton, D.L. and Groopman, J.D. Academic Press. 53 – 55; Shou, M., Dai, R., Cui, D., Korzekwa, K.R., Baillie, T.A., Rushmore, T.H. 2001. A kinetic model for the metabolic interaction of two substrates at the active site of cytochrome P450 3A4. J. Biol. Chem. 276: 2256 – 2262). Because of the high correlation between AFB1 dialdehyde reduction and AFB1 monoaldehyde reduction, we have hypothesized that the AKR7A2 member is likely responsible for both activities.

Line 276

How representative of the concentration present in the liver of intoxicated birds is the highest concentration of AFBI-dialdehyde of 66mcM?

Reply: As it was explained previously, the 66 µM concentration of AFB1 dialdehyde was used due to the conditions required by the experimental model (although the expected values are in the nanomolar range, not the micromolar range). However, because reduction products are normalized to the amount of cytosolic protein used, the kinetic parameters obtained are expected to reflect what actually happens in the hepatocyte.

Line 298

Is the cytotoxic effect of AFB1 exposure in ducks DUE TO or ASSOCIATED WITH the lack of a detoxification pathway? What is the status of GST detoxification pathways in ducks?

Reply: Currently there is no information on the enzyme kinetic parameters of GST activity against AFB1 or its metabolite AFBO in ducks. Kinetic parameters for this enzyme activity have been determined by our research group and these results are currently under consideration for publication. These unpublished studies have shown that the duck GST activity against AFBO is so low that the cytotoxic effects of AFB1 are most likely due to the lack of an appropriate detoxication pathway.

CONCLUSION

Line 317

The mention of individuals with high activity may relate to the SD values in Table 1. It would be useful to provide the individual bird values from which the Table was compiled – in this way the variation by individual birds and the number of birds with significant variation can be seen – this could be included in a supplement.

Reply: We agree with the reviewer regarding the identification of possible “high AFAR activity” individuals. Individuals with a dhd/dial or dhd/mono ratio value below average minus SD reflect individuals with the highest AFB1 dialdehyde reduction or AFB1 monoalcohol reduction efficiency (inactivation reactions) relative to the AFB1-dhd efficiency (activation reaction). The following chart has been added as supplementary material:

Species Sex Individual dhd/dial dhd/mono dial/mono mono/dial

ross female 1 2.73 2.72 1.00 1.00

ross female 2 1.09 0.74 0.67 1.48

ross female 3 1.74 1.57 0.90 1.11

ross female 4 1.40 0.64 0.46 2.20

ross female 5 2.88 1.29 0.45 2.23

ross female 6 3.99 2.31 0.58 1.73

ross male 1 1.27 0.94 0.74 1.36

ross male 2 1.21 1.16 0.96 1.04

ross male 3 7.27 2.13 0.29 3.41

ross male 4 4.73 2.70 0.57 1.75

ross male 5 1.69 1.04 0.62 1.62

ross male 6 3.25 2.66 0.82 1.22

island female 1 11.14 5.02 0.45 2.22

island female 2 11.13 3.49 0.31 3.19

island female 3 7.97 3.06 0.38 2.61

island female 4 5.74 4.04 0.70 1.42

island female 5 6.11 4.53 0.74 1.35

island female 6 6.16 3.73 0.61 1.65

island male 1 5.25 3.29 0.63 1.60

island male 2 3.53 3.84 1.09 0.92

island male 3 6.83 4.00 0.59 1.71

island male 4 10.08 4.23 0.42 2.38

island male 5 7.45 3.15 0.42 2.37

island male 6 7.93 5.76 0.73 1.38

quail female 1 18.22 18.44 1.01 0.99

quail female 2 6.73 6.22 0.92 1.08

quail female 3 9.67 9.28 0.96 1.04

quail female 4 8.18 6.94 0.85 1.18

quail female 5 29.19 17.06 0.58 1.71

quail female 6 6.34 3.76 0.59 1.68

quail male 1 40.84 29.43 0.72 1.39

quail male 2 17.77 12.99 0.73 1.37

quail male 3 14.70 7.25 0.49 2.03

quail male 4 34.83 10.65 0.31 3.27

quail male 5 19.59 9.87 0.50 1.98

quail male 6 33.03 24.02 0.73 1.38

turkey female 1 3.58 3.92 1.09 0.92

turkey female 2 9.48 7.64 0.81 1.24

turkey female 3 2.59 3.08 1.19 0.84

turkey female 4 3.63 2.85 0.78 1.27

turkey female 5 3.15 3.90 1.24 0.81

turkey female 6 3.05 3.38 1.11 0.90

turkey male 1 3.95 2.33 0.59 1.69

turkey male 2 3.63 2.36 0.65 1.54

turkey male 3 2.69 1.67 0.62 1.61

turkey male 4 2.83 2.31 0.82 1.22

turkey male 5 5.32 3.82 0.72 1.39

turkey male 6 2.81 2.35 0.84 1.20

duck female 1 208.75 81.43 0.39 2.56

duck female 2 228.83 59.49 0.26 3.85

duck female 3 361.73 84.35 0.23 4.29

duck female 4 177.11 85.12 0.48 2.08

duck female 5 44.56 23.13 0.52 1.93

duck female 6 101.50 82.99 0.82 1.22

duck male 1 181.93 182.60 1.00 1.00

duck male 2 60.95 87.24 1.43 0.70

duck male 3 102.01 139.39 1.37 0.73

duck male 4 58.63 38.46 0.66 1.52

duck male 5 142.59 81.06 0.57 1.76

duck male 6 345.03 245.13 0.71 1.41

S1 table. Values of CLint AFB1-dhd enzyme production / CLint AFB1 dialcohol enzyme production (dhd/dial), CLint AFB1-dhd enzyme production / CLint AFB1 monoalcohol enzyme production (dhd/mono), CLint AFB1 monoalcohol enzyme production / CLint AFB1 dialcohol enzyme production (mono/dial) and the inverse (dial/mono) ratios. Values in bold represent individuals which ratio value is below SD.

Line 318-Line 319

Genetic selection would only be useful if there was a relationship between intoxication and enzyme activity. This would need to be established for the duck and may require challenge of ducks at the extremes (ie high and low) of enzyme activity with AF to see if in fact there is an in vitro in vivo (IVIV) relationship. Furthermore, only a very small sample of ducks from an unknown source has been studied and described herein.

Reply: We agree with the reviewer comment. We are fully aware of the limitations of the present study. Nonetheless we believe that we can postulate that research about genetic selection could potentially be used as a possible means of enhancing species tolerance. S1 Table gives additional support regarding the possibility of selecting individuals with a particularly high AFAR activity that could be selected for potentially higher tolerance to AFB1. As it has been mentioned in the first comment of reviewer #2, a sentence has been added to clarify the limitations of the present study in regard to the population studied (lines 347 - 353).

Submitted filename: Response to reviewers.docx

Click here for additional data file.

19 May 2020

PONE-D-19-32966R1

Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects

PLOS ONE

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Reviewer #1: Thank you for responding to my comments and taking appropriate opinions.

However, in order to objectively understand the views expressed in this paper, there seems to be insufficient information on the experimental conditions as background.

To the author's opinion that dietary effects on the results obtained are not expected, it is necessary to be objective by presenting as much evidence as possible of each poultry rearing status in this study.

The authors should give supplemental data on the actual feed ingredients (mixing ratio and ME), feed intake, body weight, origin of rearing and notable clinical symptoms.

Reviewer #2: Thankyou for your detailed responses to the various questions raised.

The only matter you may wish to consider (and it is minor) is the use of DETOXICATION or DETOXIFICATION. The latter term is by far the most common term (a pubmed or a google search has around 10X as many entries for DETOXIFICATION compared with DETOXICATION).

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Reviewer #1: Yes: Takeshi Kawasaki

Reviewer #2: Yes: Stephen W. Page

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2 Jun 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for responding to my comments and taking appropriate opinions. However, in order to objectively understand the views expressed in this paper, there seems to be insufficient information on the experimental conditions as background. To the author's opinion that dietary effects on the results obtained are not expected, it is necessary to be objective by presenting as much evidence as possible of each poultry rearing status in this study. The authors should give supplemental data on the actual feed ingredients (mixing ratio and ME), feed intake, body weight, origin of rearing and notable clinical symptoms.

Reply: We want to thank again reviewer #1 for his constructive comments. In order to clarify rearing origin and poultry clinical signs (the word symptoms is usually restricted to human patients), the following phrase has been added to manuscript: “Poultry were obtained from local commercial poultry suppliers and at the moment of sacrifice no noticeable clinical signs were observed “ (lines 109 - 111).

In regard to the feed ingredient’s composition and calculated analysis (mixing ratio and ME), feed intake and body weight, tables S1 and S2 have been added as supplementary information. Former S1 table has been renumbered as S3 Table.

Feed ingredients (%) Chicken diet Turkey diet Duck diet Quail diet

Corn 55.0 49.1 51.5 47.5

Corn gluten meal --- 4.5 5.0 3.5

Wheat bran --- --- 18.0 ---

Full-fat soybean (extruded) 11.7 6.0 2.0 6.8

Soybean meal (48%) 30.0 34.9 20.5 37.5

Vegetable oil 0.1 2.8 --- 1.0

Calcium carbonate 0.92 1.00 1.25 1.40

Calcium phosphate (20% P) 1.48 1.00 1.30 1.75

Iodized salt 0.3 0.2 0.2 0.35

Vitamin:mineral premix 1.0 1.0 1.0 1.0

Methionine 0.32 0.12 0.05 0.15

Lysine 0.10 0.25 0.12 ---

Threonine 0.03 --- --- ---

Calculated analysis (%)

Crude protein 25.1 25.5 20.1 26.1

ME (kcal/kg) 3103 3120 2800 2909

Ether extract 5.13 3.95 3.61 4.07

Crude fiber 2.77 3.24 4.08 3.33

Linoleic acid 1.25 1.49 1.42 1.29

�-Linolenic acid 0.22 0.15 0.08 0.16

Calcium 0.95 0.88 0.99 1.24

Total phosphorus 0.62 0.54 0.66 0.69

Available phosphorus 0.30 0.25 0.33 0.34

Digestible lysine 0.74 1.41 0.96 1.50

Digestible methionine 1.36 0.48 0.38 0.49

Total sulphur amino acids 0.48 0.83 0.72 0.85

S1 Table. Feed ingredients and nutritional content of the diets fed to the experimental birds.

Species Sex Body weight (g) Feed Intake (kg/bird) Species Sex Body weight (g) Feed Intake (kg/bird)

Rhode Island Red chicks Female 750 1,07 Quail Female 89,1 0,84

720 1,19 80,8 0,86

625 0,99 79,2 0,86

590 1,16 80,5 0,81

630 1,18 83,0 0,85

630 1,26 83,6 0,81

Male 865 1,29 Male 92,9 0,81

850 1,41 86,8 0,79

830 1,37 96,1 0,82

750 1,43 78,5 0,78

810 1,27 73,6 0,81

790 1,26 73,4 0,85

Ross chicks Female 1600 4,25 Turkey Female 2613 4,15

2850 4,15 2674 4,25

3175 4,23 3167 4,28

2175 4,48 2600 4,26

2450 4,15 2817 4,22

3200 3,81 2627 4,26

Male 3180 4,61 Male 3269 3,37

2900 4,55 2912 3,28

2750 4,78 2892 3,65

2850 4,50 3036 3,56

2550 4,78 2740 3,02

2760 4,53 3197 3,44

Duck Female 1850 8,82

2640 8,69

2170 8,89

2300 8,92

2000 8,64

2100 8,70

Male 2700 7,77

2500 7,65

3100 7,79

2800 7,68

2600 7,95

2350 7,71

S2 Table. Final body weight and total feed intake at the time of sacrifice of the experimental birds.

Reviewer #2: Thank you for your detailed responses to the various questions raised.

The only matter you may wish to consider (and it is minor) is the use of DETOXICATION or DETOXIFICATION. The latter term is by far the most common term (a pubmed or a google search has around 10X as many entries for DETOXIFICATION compared with DETOXICATION).

Reply: We want to also thank again reviewer #2 for his suggestion. The word “detoxication” has been replaced by “detoxification” were appropriate.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

9 Jun 2020

Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects

PONE-D-19-32966R2

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PLOS ONE

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Reviewers' comments:

11 Jun 2020

PONE-D-19-32966R2

Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects

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