Exploration of Cytochrome P450-Related Interactions between Aflatoxin B1 and Tiamulin in Broiler Chickens

Poultry may face simultaneous exposure to aflatoxin B1 (AFB1) and tiamulin (TIA), given mycotoxin contamination and antibiotic use. As both mycotoxins and antibiotics can affect cytochrome P450 enzymes (CYP450), our study aimed to explore their interaction. We developed UHPLC-MS/MS methods for the first-time determination of the interaction between TIA and AFB1 in vitro and in vivo in broiler chickens. The inhibition assay showed the half maximal inhibitory concentration (IC50) values of AFB1 and TIA in chicken liver microsomes are more than 7.6 μM, indicating an extremely weak inhibitory effect on hepatic enzymes. Nevertheless, the oral TIA pharmacokinetic results indicated that AFB1 significantly increased the area under the plasma concentration-time curve (AUClast) of TIA by 167% (p < 0.01). Additionally, the oral AFB1 pharmacokinetics revealed that TIA increased the AUClast and mean residence time (MRT) of AFB1 by 194% (p < 0.01) and 136%, respectively. These results suggested that the observed inhibition may be influenced by other factors, such as transport. Therefore, it is meaningful to further explore transport and other enzymes, involved in the interaction between AFB1 and TIA. Furthermore, additional clinical studies are necessary to thoroughly assess the safety of co-exposure with mycotoxins and antibiotics.


Introduction
Mycotoxins are toxic secondary metabolites produced by various types of fungi, including Alternaria, Aspergillus, Fusarium, and Penicillium.More than 400 mycotoxins have been identified so far, such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FUMs), and zearalenone (ZEN), which are hazardous to human and domestic animals [1,2].Among them, aflatoxin B1 (AFB1) is known as one of the most toxic mycotoxins due to genotoxic, mutagenic, teratogenic, and carcinogenic effects [3,4].It is commonly reported that these toxic compounds are frequently detected in poultry feed, constituting a great threat to the health of both animals and humans [2,5,6].The global surveys revealed the worldwide occurrence of myco-toxins regarding tested samples from Europe, Africa, and Asia, and more seriously, samples were contaminated with at least one mycotoxin [2,7].AFB1 levels ranged from 0.5-36.4µg/kg feed, indicating that mycotoxin contamination in China urgently requires more attention and monitoring [8].Ma et al. also demonstrated that AFB1 is commonly found in feed ingredients and commercial feed in China, ranging from 1.3 to 10 µg/kg [9].In Spain, AFB1 was found at a maximum concentration of 6.9 µg/kg in poultry feed [10].More seriously, AFB1 levels were as high as 39.9 µg/kg in Pakistan [11], where 100% of grower broiler feed was contaminated with AFB1 [12].In Egypt, it was observed that AFB1 and aflatoxin B2 (AFB2) were both present in animal feedstuff ranging from 0.851 to 1.363 µg/kg [13].Even, AFB1 concentrations of 1067 µg/kg have been reported in poultry feed in West Africa, highlighting the widespread presence of AFs in feed commodities [14].In sub-Saharan Africa, poultry feeds were co-contaminated with more than 21 mycotoxins and/or metabolites [15].In the United States, most of the corn samples had co-occurrences of at least two detectable mycotoxin (≥2 mycotoxins) [16].Similarly, co-occurrence of mycotoxins has also been observed mainly with AFs and FUMs in Latin American countries [17].AFB1 can induce liver cell damage in poultry through a variety of molecular mechanisms, and the main of damage mechanisms that have been discovered so far include oxidative damage, promoting apoptosis, influencing hepatocyte gene expression, interfering with hepatocyte autophagy, pyroptosis and necroptosis [18].As reported, it is well documented that AFs are mainly metabolized by CYP450 enzymes [15,19].In broilers, it was summarized that hepatic CYP1A1, CYP1A2, CYP2A6 and CYP3A4 enzymes are mainly involved in AFB1 biotransformation [20].The gene expressions of CYP3A4 and CYP3A7 in poultry were influenced [21].Antibiotics such as sulfonamides, tetracyclines, quinolones, macrolides, aminoglycosides, and β-lactam antibiotics, have been widely used as supplements or as additives in animal feed to promote animal growth and prevent diseases [22][23][24].Because of the wide usage, a variety of antibiotics were found in livestock products, potentially posing health risks to the public due to antibiotics residues [25].A study implied that lincomycin inhibits CYP1A, CYP2A, CYP2C, CYP2B, CYP2E and CYP3A in porcine liver microsomes at a concentration of 3 µM [26].Amoxicillin inhibited CYP2C8 with an IC 50 of 830 µM by recombinant Escherichia coli [27].Trimethoprim also shows a weak inhibitory effect on CYP2C8 [28,29].Meanwhile, enrofloxacin was found to inhibit CYP3A activity in chickens at doses of 25 and 125 mg/kg body weight (BW) [30].Antonissen et al. showed that the pharmacokinetics of CYP1A4 substrate drugs, such as enrofloxacin might be altered by simultaneous FUMs exposure in chickens [31].In addition, a significant upregulation of CYP1A4 was observed in chickens fed a diet contaminated with FUMs [31].The traditional medicine curcumin, with its broad spectrum of antibacterial actions, was found to inhibit CYP2A6 enzyme activity at a dose of 450 mg/kg feed in the detoxification of AFB1 [32].The metabolism of enrofloxacin was affected by tilmicosin via inhibition of CYP3A4 [33].Besides, tilmicosin increased the residual concentrations of enrofloxacin in broilers.Among the pleuromutilin antibiotics, tiamulin (TIA) has been commonly used in poultry over 30 years, and its use has not largely led to a general increase of resistance to many pathogens [34] TIA administered at therapeutic levels is relatively quickly absorbed, metabolized in the liver, and rapidly eliminated from the chickens' body.Moreover, it showed a strong interaction with the ionophore antibiotics (coccidiostats) monensin, narasin, and salinomycin at therapeutic levels [34].In vitro, TIA was an effective inhibitor of CYP450 activities in goat and cattle microsomes [35], where a stable metabolic intermediate complex was formed with TIA.The inhibition phenomenon was also observed in pig liver microsomes [36].In another study, it was clarified that TIA acted as an inhibitor of porcine hepatic CYP3A [26].Importantly, TIA also influenced certain CYP450 enzymes in chickens, such as CYP1A, 2A, 2B, 2E, and 3A [37,38].In summary, TIA was found to inhibit various CYP450 enzyme activities in rats, pigs, chickens, and goats, although species-or dose-related differences were reported [35,36,39].
Importantly, Lootens et al. suggested that drugs are more likely to be perpetrators of CYP450-mediated drug-mycotoxin interactions in animals, while mycotoxins are more likely to be considered the victims, based on the usually higher systemic levels of drugs compared to mycotoxins [40].Therefore, the assumption has been made that antibiotics could influence the disposition of AFs in poultry.In general, few research has been conducted on the CYP450-related mycotoxin and antibiotics interaction [31].Therefore, the objective of this study was to determine the interaction of the antibiotic TIA and the mycotoxin AFB1 (Figure 1) in vitro and in vivo in broiler chickens using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and pharmacokinetic methods.
(coccidiostats) monensin, narasin, and salinomycin at therapeutic levels [34].In vitro, T was an effective inhibitor of CYP450 activities in goat and cattle microsomes [35], wh a stable metabolic intermediate complex was formed with TIA.The inhibit phenomenon was also observed in pig liver microsomes [36].In another study, it w clarified that TIA acted as an inhibitor of porcine hepatic CYP3A [26].Importantly, T also influenced certain CYP450 enzymes in chickens, such as CYP1A, 2A, 2B, 2E, and [37,38].In summary, TIA was found to inhibit various CYP450 enzyme activities in r pigs, chickens, and goats, although species-or dose-related differences were repor [35,36,39].
Importantly, Lootens et al. suggested that drugs are more likely to be perpetrator CYP450-mediated drug-mycotoxin interactions in animals, while mycotoxins are m likely to be considered the victims, based on the usually higher systemic levels of dr compared to mycotoxins [40].Therefore, the assumption has been made that antibio could influence the disposition of AFs in poultry.In general, few research has b conducted on the CYP450-related mycotoxin and antibiotics interaction [31].Theref the objective of this study was to determine the interaction of the antibiotic TIA and mycotoxin AFB1 (Figure 1) in vitro and in vivo in broiler chickens using ultra-h performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) pharmacokinetic methods.

Validation of the Analytical Methods
The established methods for the six probe substrates, TIA and AFB1 in CLM, and TIA and AFB1 in chicken plasma were thoroughly validated, including assess sensitivity, linearity range, accuracy, and precision.In this study, the limits of detect (LODs) for 4-acetaminophen (ACE, ACE is the hydroxylated form of P hydroxybupropion (OH-BP), 6-hydroxychlorzoxazone (OH-CLN), hydroxytolbutamide (OH-TBD), 7-hydroxycoumarin (OH-CAN), and hydroxymidazolam (OH-MDZ) were 2 ng/mL in CLM.The obtained limits quantification (LOQs) for ACE, OH-BP, OH-CLN, OH-TBD, OH-CAN, and OH-M were 5 ng/mL in CLM.The LOQs for AFB1 and TIA analysis in chicken plasma were ng/mL and 5 ng/mL, respectively.The LODs for AFB1 and TIA analysis in chicken plas were 0.2 ng/mL and 2 ng/mL, respectively.The calibration curves for all analytes w linear over the concentration ranges with a correlation coefficient (R 2 ) > 0.99.The obtai accuracy and precision of the methods were evaluated using recoveries and coefficien variation (CV) for intra-day and inter-day measurements.The mean recoveries ran from 90.4 to 108.6%, with CVs lower than 13.8%, and are summarized in Tables 1 an which suggested that the methods had suitable accuracy and precision.

Validation of the Analytical Methods
The established methods for the six probe substrates, TIA and AFB1 in CLM, and for TIA and AFB1 in chicken plasma were thoroughly validated, including assessing sensitivity, linearity range, accuracy, and precision.In this study, the limits of detection (LODs) for 4acetaminophen (ACE, ACE is the hydroxylated form of PH), hydroxybupropion (OH-BP), 6hydroxychlorzoxazone (OH-CLN), 4-hydroxytolbutamide (OH-TBD), 7-hydroxycoumarin (OH-CAN), and 1-hydroxymidazolam (OH-MDZ) were 2 ng/mL in CLM.The obtained limits of quantification (LOQs) for ACE, OH-BP, OH-CLN, OH-TBD, OH-CAN, and OH-MDZ were 5 ng/mL in CLM.The LOQs for AFB1 and TIA analysis in chicken plasma were 0.5 ng/mL and 5 ng/mL, respectively.The LODs for AFB1 and TIA analysis in chicken plasma were 0.2 ng/mL and 2 ng/mL, respectively.The calibration curves for all analytes were linear over the concentration ranges with a correlation coefficient (R 2 ) > 0.99.The obtained accuracy and precision of the methods were evaluated using recoveries and coefficient of variation (CV) for intra-day and inter-day measurements.The mean recoveries ranged from 90.4 to 108.6%, with CVs lower than 13.8%, and are summarized in Tables 1 and 2, which suggested that the methods had suitable accuracy and precision.

Enzyme Kinetics Profile of the Probe Substrates
An enzyme kinetic study was conducted to optimize the probe substrate concentrations.The Michaelis-Menten equation was used to calculate the maximal velocity (V max ) and Michaelis-Menten constant (K m ) for PH, BP, CLN, TBD, CAN and MDZ in CLM.The kinetic profiles of six probes are represented in Figure 2.Meanwhile, the optimal incubation time, microsomal protein concentration, and substrate concentration for the probes are listed in Table 3.As shown in Table 3, the K m values of PH, BP, CLN, TBD, CAN, and MDZ were 20.7 µM, 9.4 µM, 21.8 µM, 14.4 µM, 56.1 µM, and 3.3 µM, respectively.

Pharmacokinetics of Aflatoxin B1 and Tiamulin in Chickens
No significant adverse health effects were observed in broilers when AFB1 and TIA were co-administrated.However, it is noteworthy to mention that during the course of the experiment, one bird (n° T5) died in the group that received AFB1 in the feed (on day 8 after start of the trial).The histological examination results indicated that the cause of death was not related to AFB1 exposure.Plasma concentration data (>LOQ) were analyzed using software WinNonlin (v.8.3.4) via noncompartmental modeling to obtain primary pharmacokinetic parameters.An overview of the plasma concentrations of TIA and AFB1 after oral administration is shown in Supplementary Materials (Table S3).The pharmacokinetic parameters are shown in Tables 4 and 5.

Pharmacokinetics of Aflatoxin B1 and Tiamulin in Chickens
No significant adverse health effects were observed in broilers when AFB1 and TIA were co-administrated.However, it is noteworthy to mention that during the course of the experiment, one bird (n° T5) died in the group that received AFB1 in the feed (on day 8 after start of the trial).The histological examination results indicated that the cause of death was not related to AFB1 exposure.Plasma concentration data (>LOQ) were analyzed using software WinNonlin (v.8.3.4) via noncompartmental modeling to obtain primary pharmacokinetic parameters.An overview of the plasma concentrations of TIA and AFB1 after oral administration is shown in Supplementary Materials (Table S3).The pharmacokinetic parameters are shown in Tables 4 and 5.

Pharmacokinetics of Aflatoxin B1 and Tiamulin in Chickens
No significant adverse health effects were observed in broilers when AFB1 and TIA were co-administrated.However, it is noteworthy to mention that during the course of the experiment, one bird (n • T5) died in the group that received AFB1 in the feed (on day 8 after start of the trial).The histological examination results indicated that the cause of death was not related to AFB1 exposure.Plasma concentration data (>LOQ) were analyzed using software WinNonlin (v.8.3.4) via noncompartmental modeling to obtain primary pharmacokinetic parameters.An overview of the plasma concentrations of TIA and AFB1 after oral administration is shown in Supplementary Materials (Table S3).The pharmacokinetic parameters are shown in Tables 4 and 5.The pharmacokinetic profile of TIA is demonstrated in Figure 5A.Liao et al. implicated exposure to airborne AFB1 of humans was a relatively high-risk during livestock feeding and storage bin cleaning [41].Therefore, a safe AFB1 concentration of 20 µg/kg in feed was used as officially recommended as maximum level by EU [42].Following oral administration of TIA at a dose of 25 mg/kg BW, the drug was rapidly absorbed in AFB1-unexposed broiler chickens.The plasma drug concentration reached its peak concentration of 89.9 ± 45.9 ng/mL at 0.44 h post administration.As reported, CYP1A and CYP3A protein levels were decreased in piglets exposed to a very high level of AFB1 contamination (1807 µg/kg AFB1 in feed) [43].For the mean pharmacokinetic parameters of TIA, the elimination half-life time (T 1/2 ) and mean residence time (MRT) were 7.0 ± 2.9 h and 6.8 ± 1.4 h in AFB1-unexposed broilers, respectively.The AUC last and peak concentration (C max ) in the AFB1-uncontaminated group were 294.5 ± 63.3 h•ng/mL and 89.9 ± 45.96 ng/mL, respectively.AFB1 exposure increased the values of AUC last and C max of TIA by 167% and 155%, respectively.Additionally, apparent volume of distribution (V d_F_obs ) and apparent body clearance (CL _F_obs ) in AFB1-exposed group were 57% and 63% lower than in the control group (p < 0.01).
plasma concentration description.The obtained AUClast and MRT were 170 ± 103.6 h•ng/mL and 6.4 ± 3.2 h for TIA-exposed chickens, respectively, and 87.5 ± 39.6 h•ng/mL (p < 0.01) and 4.7 ± 0.7 h for TIA-unexposed broilers, respectively.The Vd_F_obs and CL_F_obs values were 18.4 ± 7.7 L/kg and 9.4 ± 6.5 L/h/kg in TIA-unexposed broilers, and 14.8 ± 4.8 L/kg and 14.3 ± 6.7 L/h/kg in TIA-exposed chickens.Table 5 shows that there was no significant difference between the treatment and control groups in terms of T1/2 or apparent body clearance (CL).However, there was a significant difference between the groups in terms of Cmax and the area under the plasma concentration time curve from 0 to the last time point AUClast.In addition, as shown in Figure 4B, the curves exhibited a similar trend during the initial phase from 0.5 h to 4 h, which suggested that type of interaction is formation-limited.
The CYP450 isoenzymes of CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, and CYP3A were measured by commercially available antibodies in broilers [46].CYP3A activity was studied in vitro using MDZ as a substrate, showing high CYP3A activity in the livers of broilers [47].It was pointed out that the enzyme CYP3A37 has the same activity of CYP3A4 in humans [48].It was summarized that CYP2A6, CYP3A37, CYP1A5, and CYP1A1 were responsible for bioactivation of AFB1 in poultry species [49].The total CYP450 content in chickens was 0.296 ± 0.04 nmol/mg [50] Meanwhile, the biotransformation activity of CYP2D6, CYP1A4, CYP2C19, and CYP3A4 in chickens was lower than in dogs, while the activity of CYP2C9 was higher, with a Km of 627.61 ± 145.61 μM in chickens [50].Hu et al. demonstrated a similar Km value of 678 ± 137 μM in Ross 708 The pharmacokinetic profile of AFB1 is shown in Figure 5B, following oral administration of AFB1 at a dose of 1 mg/kg BW.A noncompartmental model was used for AFB1 plasma concentration description.The obtained AUC last and MRT were 170 ± 103.6 h•ng/mL and 6.4 ± 3.2 h for TIA-exposed chickens, respectively, and 87.5 ± 39.6 h•ng/mL (p < 0.01) and 4.7 ± 0.7 h for TIA-unexposed broilers, respectively.The V d_F_obs and CL _F_obs values were 18.4 ± 7.7 L/kg and 9.4 ± 6.5 L/h/kg in TIA-unexposed broilers, and 14.8 ± 4.8 L/kg and 14.3 ± 6.7 L/h/kg in TIA-exposed chickens.Table 5 shows that there was no significant difference between the treatment and control groups in terms of T 1/2 or apparent body clearance (CL).However, there was a significant difference between the groups in terms of C max and the area under the plasma concentration time curve from 0 to the last time point AUC last .In addition, as shown in Figure 4B, the curves exhibited a similar trend during the initial phase from 0.5 h to 4 h, which suggested that type of interaction is formation-limited.
The CYP450 isoenzymes of CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, and CYP3A were measured by commercially available antibodies in broilers [46].CYP3A activity was studied in vitro using MDZ as a substrate, showing high CYP3A activity in the livers of broilers [47].It was pointed out that the enzyme CYP3A37 has the same activity of CYP3A4 in humans [48].It was summarized that CYP2A6, CYP3A37, CYP1A5, and CYP1A1 were responsible for bioactivation of AFB1 in poultry species [49].The total CYP450 content in chickens was 0.296 ± 0.04 nmol/mg [50] Meanwhile, the biotransformation activity of CYP2D6, CYP1A4, CYP2C19, and CYP3A4 in chickens was lower than in dogs, while the activity of CYP2C9 was higher, with a K m of 627.61 ± 145.61 µM in chickens [50].Hu et al. demonstrated a similar K m value of 678 ± 137 µM in Ross 708 chickens [51].However, the K m of CYP2C9 was 14.42 µM in CLM in our study, while the V max was 159.4 nmol/(min•mg).Additionally, the K m values of CYP1A2 and CYP3A4 were 98.1 ± 18.6 µM and 1.30 ± 0.67 µM in chickens, respectively, using PH and MDZ [51].CYP1A1and CYP2A6 are important for biotransformation in chickens [52].By contrast, a K m of 2.1 ± 0.81 µM using MDZ in chicken was demonstrated [53].The K m for CYP1A2 and CYP3A4 in current study were 20.69 µM and 3.31 µM, respectively.The K m of CYP2A6 for turkey using CAN as substrate was 33.6 ±8.52 µM [44].In contrast, the K m of CYP2A6 was 56.08 µM for chickens in our study using CAN as probe substrate.
It has been demonstrated that gene expressions of CYP3A4 and CYP3A7 in poultry was impacted by AFB1 [21].Also, a significant decrease of CYP450 activity in rabbits was observed at an oral dose of 0.10 mg/kg/day [54].Our results were broadly in line with above studies, which implied that AFB1 inhibited the CYP450 enzymes activity.The IC 50 values of AFB1 for CYP2A6 and CYP3A4 were 24.11 µM and 41.37 µM in CLM, respectively, whereas there was extremely weak inhibition for CYP1A2, CYP2B6, CYP2E1, and CYP2C9 with IC 50 values of 147.0 µM, 662.6 µM, 271.2 µM, and 526.7 µM, respectively.
As reported, TIA inhibited CYP450 enzyme activities in rats, pigs, and goats [35,36,39].On the other hand, another study confirmed that TIA could induce some CYP450-related catalytic activities in poultry [50].However, our results rather excluded the possibility of TIA inhibiting the activities of enzymes in CLM.The IC 50 values of CYP2E1 and CYP2A6 being 47.6 µM and 7.6 µM, respectively, and for CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were 65.83, 101.9, 65.62, 334.2 µM, suggested weak inhibition activity in current study.A more recent study has shown that TIA was an effective inhibitor of CYP450 activities in goat and cattle microsomes [35].This is consistent with what has been found in previous studies that TIA acted as inhibitor of porcine hepatic CYP3A and CYP2C, respectively [26].Moreover, the CYP450 content in broiler chickens was lower in contrast with horses, pigs, cattle, rabbits, and rats [55,56].Similarly, it was found that the rate of glucuronidation of either 1-naphthol or p-nitrophenol was in the order pigs~rabbits > horses >> cattle > broiler chicks [57].
Pharmacokinetic parameters of enrofloxacin were modified after exposure to AFB1 (750 µg/kg) in chickens [58] However, the values of T 1/2a , T max and AUC 0-∞ of enrofloxacin were nonsignificantly increased by 17%, 26% and 17%, respectively.Also, AFB1 decreased the bioavailability and delayed the elimination of doxycycline in chickens [59].After oral administration, the T max of doxycycline in aflatoxin-exposed chickens was 2.37 h compared to 1.97 h for healthy ones.In this study, results revealed that AFB1 significantly increased the AUC last of TIA when broiler chickens were given either a control or AFB1 diet for 2 weeks at a rather low contamination level (20 µg/kg feed).Hence, higher AFB1 contamination levels and/or a longer exposure period may more likely result in negative effects than a two-week exposure at a low AFB1 level of 20 µg/kg in this study [58,60].The IC 50 values of AFB1 and TIA were higher than 7.6 µM as reported in our in vitro results.The highest IC 50 for TIA and AFB1 was higher than 100 µM, surpassing plasma concentrations obtained with the recommended dose for TIA and safe dose for AFB1.Meanwhile, the influence of TIA and AFB1 on the in vivo pharmacokinetics of substrates of CYP450 may not be predicted by the in vitro cocktail incubation method.Therefore, we studied the pharmacokinetic interactions between TIA and AFB1 in vivo in broilers, which indicated an interaction takes place but probably CYP450 did not play a major role in the interaction between AFB1 and TIA and other factors may be responsible for the effects observed.These findings suggested that longer exposure periods and multiple doses of AFB1 should be considered for further investigation.However, practicality and safety constraints currently limit the feasibility of such experiments.Nevertheless, a pharmacokinetic interaction between AFB1 and TIA was observed in broilers.Despite the IC 50 of AFB1 indicating weak inhibition on CYP450 enzymes in CLM, this study provides insight that future research should focus on other factors involved in the mycotoxin-drug interaction.
The C max of AFB1 in chickens reached 29.3 ng/mL at 3.2 h after oral administration at a dose of 1 mg/kg BW, indicating rapid absorption of the mycotoxin in blood.Similarly, according to literature, in rats, the T max of AFB1 was about 0.15 h, with a C max of 90 ± 20 ng/mL following oral administration at dose of 1 mg/kg BW [61].Notably, the T 1/2 was about 22.3 h in chicken following oral administration of a dose of 1 mg/kg BW.Jubert et al. reported a T 1/2α of 2.86 h and T 1/2β of 64.4 h in humans at a dose of 30 ng AFB1 [62].In addition, in the latter study, the C max and T max were 0.941 ± 0.154 pg/mL and 1.02 ± 0.31 h, respectively.The AUC 0-t was 25.6 ± 6.3 h•pg/mL.Moreover, AFB1 shows a rapid absorption and metabolism in rats and cows compared to human [61,63].A C max of 93.42 ± 23.01 ng/mL was observed at approximately at 0.15 h in rats after a single dose of 1 mg/kg BW.The T 1/2e was 7.62 h in rats after oral administration, suggesting rather rapid elimination.The AUC 0-24 was 45.59 ± 17.19 h•ng/mL, and the CL and V d were 22.33 ± 9.79 L/h/kg and 226.46 ± 100.76 L/kg in rats, respectively.Guo et al. calculated the toxicokinetic parameters of AFB1 in plasma of cows after oral administration of 40 µg/kg BW, the results indicated that AFB1 was rapidly absorbed in cows with a C max of 3.8 ± 0.9 ng/mL and a T max of 0.58 ± 0.17 h.In addition, the T 1/2e and MRT were 15.52 ± 0.51 h and 11.73 ± 0.94 h, respectively, indicating that AFB1 was rather gradually eliminated in cow [64].It was found that the AFB1 depletion in chicken liver, kidney, and muscle may take up to 10-19 days at a dose of 5 mg/kg feed [65].In our study AFB1 had a rather short MRT of 4.7 ± 0.7 h in chickens, and the T 1/2e was approximately 22.3 h in broilers, which is long in contrast to rats of 7.62 h, but shorter than in humans with a T 1/2β of 64.4 h [62].The primary kinetic parameters in present study were different from other animals, such as rat, cow, and human [61,62,64,66].Overall, the difference might be due to factors such as dietary AFB1 levels, sex, age, species, duration of administration of AFB1 [65].The limitation of this study is that only the AFB1 parent compound was analyzed, thus in future studies, the metabolites should be taken into account too, such as the highly toxic metabolite AFBO.For TIA, IC 50 values for CYP1A2, CYP2B6, CYP2E1, CYP2C9, CYP2A6, and CYP3A4 were 65.8 µM, 101.9 µM, 47.6 µM, 65.6 µM, 7.6 µM, and 334.2 µM, respectively.The IC 50 of AFB1 on CYP2A6, CYP3A4, CYP1A2, CYP2B6, CYP2E1 and CYP2C9 were 24.1 µM, 41.4 µM, 147.0 µM, 662.6 µM, 271.2 µM, and 526.7 µM, respectively.The C max of AFB1 and TIA in broilers was 141.7 ng/mL and 44.4 ng/mL, respectively, suggesting that they were significantly lower than the IC 50 .This indicated that the concentration may not reach levels sufficient to induce an inhibitory effect.However, predicting in vivo results from in vitro data is challenging due to the complexity of CYP450 [67].An in vivo pharmacokinetic interaction between AFB1 and TIA was observed in this study.Moreover, residues of AFB1 were detected in the liver [60] and eggs [68] at the low-level dietary exposure.Therefore, even minor changes in the pharmacokinetic characteristics of AFB1 in broilers should be carefully considered.
Pleuromutilin derivatives like TIA can inhibit CYP3A4 activity with an IC 50 of 1.6 µM and showed strong inhibition to CYP450 enzymes [69].In contrast, the IC 50 values for CYP2A6 and CYP2E1 were 7.6 µM and 47.6 µM in CLM in this study, respectively.MRT (6.4 ± 3.2 h) and AUC last (170 ± 103.6 h•ng/mL) of AFB1 in TIA-exposed chickens were significantly higher than TIA-unexposed chickens.Besides, the V d_F_obs and CL _F_obs were 18.4 ± 7.7 L/kg and 9.4 ± 6.5 L/h/kg with TIA administration, respectively, while they were 14.8 ± 4.8 L/kg and 14.3 ± 6.7 L/h/kg, respectively, in broilers without TIA administration.Furthermore, Anadon et al. revealed that TIA could influence the drug metabolism rate in chickens because the pharmacokinetics of antipyrine were also altered when co-administered with TIA [70].Of note, TIA did not have a strong inhibition on CYP1A and CYP2A in CLM in the present study.However, the metabolism of AFB1 was significantly inhibited when co-administered with TIA at a dose of 25 mg/kg in broilers as compared to AUC last in the two groups.It should be noted that results obtained with microsomes cannot be used for quantitative estimations of in vivo biotransformation [71].As TIA was reported to have a high inhibitory effect on CYP3A4 in human liver microsomes with an IC 50 of 1.6 µM [69], data obtained in the present study exhibited that the pharmacokinetic parameters, such as AUC but not V d , CL, MRT, of AFB1 were altered after oral co-administration with TIA in chicken.
A pharmacokinetic interaction between AFB1 and TIA was observed, with TIA increasing the AUC of AFB1 in broilers.Considering the prominence of chicken as a global meat product, further investigations may be necessary to assess AFB1 residues in chicken tissues following exposure to TIA.Additionally, mycotoxin sorbent additives represent a type of AFB1 detoxication [72].The combination of curcumin and cellulosic polymers have proved to be an effective approach to address poultry health issues associated with AFB1 consumption [73].Baker yeast has been identified as partially alleviating specific toxic effects induced by AFB1 in growing chicks [74].In an upcoming study, two types of modified general biochar will be utilized to assess their effectiveness in detoxifying AFB1.

Conclusions
The purpose of this study was to investigate the CYP450-related drug-mycotoxin interactions both in vitro and in vivo, for TIA and AFB1.The IC 50 values of AFB1 and TIA on CYP450 in CLM suggested a weak inhibitory effect on hepatic enzymes.Nonetheless, the pharmacokinetic profiles of AFB1 and TIA were somewhat altered with co-exposure, which indicated that CYP450 did not play a major role in the interaction between AFB1 and TIA.It is crucial to delve further into exploring additional factors involved in the interaction.Furthermore, it is essential to manage the use of antibiotics and feed contaminated with mycotoxins in poultry production to minimize potential risks to both poultry health and food safety.

Microsomal Incubation Procedure
A cocktail-approach for elucidating the metabolites profiling and CYP450 inhibition was conducted as in Lin et al. with minor changes [75].The incubation mixture contained 50 µL of 0.2 mg/mL of CLM, 10 µL of 0.1 M phosphate buffer (pH 7.4), 40 µL of 1 mM NADPH and six probe substrates with a total volume of 200 µL.The specific probe substrates used for each CYP450 isoenzyme in the incubation mixture were as follows: PH (CYP1A2), BP (CYP2B6), CLN (CYP2E1), TBD (CYP2C9), CAN (CYP2A6), and MDZ (CYP3A4).Probes substrates were dissolved in methanol as stock solution (1 mg/mL).All of them were diluted to the final concentrations (see Supplemental Materials) with incubation buffer and the percentage of organic solvent in the final incubation volume was below 1% (v/v) [76].Briefly, the mixture was preincubated in a thermoshaker (Biosan, Geraardsbergen, Belgium) for 5 min at 37 • C prior to the initiation of the reaction with NADPH.4Na.After incubation, the mixture was terminated by 400 µL ice-cold acetonitrile.Each sample was centrifuged (5810R, Eppendorf, Germany) at 14,000 rpm for 10 min.A volume of 5 µL of supernatant was analysed using UHPLC-MS/MS.Enzyme kinetic studies were conducted for the six CYP450-related probes reactions.For each probe, the maximum rate of the V max and K m were obtained using GraphPad Prism v.8.0.2 software via Michaelis-Menten Kinetic model (La Jolla, CA, USA).The incubation times of 5, 10, 15, 30, 45 and 60 min were tested individually.A series of protein concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL) of CLM in the incubation mixture were conducted.Triplicate analyses were performed for each incubation system.The optimal values for the incubation time and CLM protein concentration were found to be 15 min for PH, BP, and CAN, 20 min for CLN, 5 min for MDZ, and TBD, respectively, and 0.4 mg/mL protein concentration for PH, BP, MDZ, and CLN, 0.2 mg/mL for TBD, and CAN, respectively.
Inhibition assays of TIA and AFB1 on CYP450 isoforms in CLM were performed with a fixed concentration of 11.2 µM for the model probes substrate CYP1A2, 4.2 µM for CYP2B6, 35.4 µM for CYP2E1, 14.7 µM for CYP2C9, 136.8 µM for CYP2A6, and 2.5 µM for 3A4, respectively.For the inhibition assays, the final concentration of AFB1 and TIA in the incubation mixture ranged from 0-25 µM and 0-40 µM, respectively.
The mass spectrometer contained an electrospray ionization (ESI) source.The ESI source was operated in both positive and negative ionization mode with capillary voltage, 3.0 kV; source temperature, 120 • C; desolvation temperature, 500 • C; desolvation gas flow rate, 100 L/h; and cone gas flow rate, 150 L/h.The MS conditions for all the analytes are shown in the Table 6.).The smaller number of chickens was used due to the limited availability of AFB1.A commercial broiler grower diet (511, CP feed, Tianjin, China) was used, that contained no antibiotics and AFB1 was not found in the feed.Animals were handled in accordance with the guideline for Care and Use of laboratory Animals.Chickens were selected randomly and numbered according to random numbers generated in Excel.They were housed individually under controlled conditions (22 • C, 18:6 h light/dark cycle) with free access to feed and water throughout the experiment.All broiler chickens went through 8 h fasting before each oral gavage dosing.The treatment group was given TIA at a dose of 25 mg/kg BW by gavage for 4 consecutive days.The control group was given the same volume of saline as the TIA solution for 4 days.After TIA administration at day 4, two groups were orally administered AFB1 at a dose of 1 mg/kg BW at 30 min after TIA administration.The chickens received feed again after oral administration of AFB1.At 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h after oral administration (p.a.), blood was obtained (0.5 mL) via direct venepuncture of the leg vein and put into heparinized tubes.This was followed with centrifugation (TGL16M, Xiangli, China) (4000 rpm, 10 min) to obtain plasma, which was stored at −20 • C until analysis for AFB1.

Pharmacokinetic Study of TIA with or without AFB1
A commercial broiler grower diet (Farmer Mash, Aveve, Belgium) was fed to all chickens.Screening of this feed was executed by a multi-mycotoxin LC-MS/MS method, indicating it was free of AFB1.Chickens were fed either the grower diet without AFB1, or a diet experimentally contaminated with 20 µg/kg AFB1 [45].To produce the grower diet experimentally contaminated with AFB1, first, 100 µL of AFB1 working solution (200 µg/mL in edible grade ethanol) was mixed with 1 g of feed for every batch.In total, 40 batches of contaminated feed were prepared.Then, this premix was blended with 1 kg of feed for 30 min to ensure a homogenous distribution of AFB1, with the results confirmed by UHPLC-MS/MS that the samples were contaminated with AFB1 in a range from 9.3 to 25.4 µg/kg (see Supplementary Material Table S1).Moreover, daily feed consumption was recorded.
Eighteen Ross 308 chickens (2-week-old, male) weighing 0.5-1.1 kg were provided by a commercial farm (Ghent, Belgium) and were randomly divided in 2 groups of 9 birds each.Chickens were selected randomly and numbered according to random numbers generated in Excel.The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine and of Bioscience Engineering of Ghent University on 23 November 2022 (EC 2022-068).They were housed individually under controlled conditions (22 • C, 18:6 h light/dark cycle) with free access to feed and water throughout the experiment.All broiler chickens went through 8 h fasting before the bolus administration of TIA, and were fed again 4 h p.a.While the treatment group was fed with an AFB1-contaminated diet for 2 weeks, the control group was given an AFB1-free grower diet.Each animal was orally administered with a single bolus of TIA at a dose of 25 mg/kg BW after two consecutive weeks of AFB1 exposure.Furthermore, chickens were given TIA by drinking water (25 mg/kg BW) for four consecutive days after the oral bolus administration of TIA.New medicated drinking water was prepared daily.To calibrate the dose, the volume of the drinking water was noted.After bolus dosing, blood was collected into heparinized tubes by direct venepuncture of the leg vein at 0.25, 0.5, 1, 2, 3,4,6,8,12,24,36,48,60,72,84,96,108,120,132,144,156,168, and 180 h p.a. and put into heparinized tubes.Then, the blood samples were centrifuged (5810R, Eppendorf, Germany) at 4000 rpm for 10 min to obtain the plasma, which was stored at −20 • C until analysis for TIA.After the last blood sampling, birds were euthanized and liver samples were collected for histopathological analysis.

Plasma Sample Preparation and UHPLC-MS/MS Analysis
A volume of 100 µL of chicken plasma was added to 150 µL of ACN in a 1.5 mL centrifuge tube.The samples were vortexed for 5 min and then centrifuged (5810R, Eppendorf,

Figure 5 .
Figure 5. Tiamulin plasma concentration-time profile after oral administration in chickens (n = 8 in the treatment group, n = 9 in the control group) with (treatment) or without (control) aflatoxin B1 contaminated diet (20 μg/kg feed for 2 weeks) (A); aflatoxin B1 plasma concentration-time profile after oral administration in chickens (n = 4 in each group) with (treatment) or without (control) tiamulin oral gavage administration (25 mg/kg BW for 4 days) (B).The mean ± SD is shown.

Figure 5 .
Figure 5. Tiamulin plasma concentration-time profile after oral administration in chickens (n = 8 in the treatment group, n = 9 in the control group) with (treatment) or without (control) aflatoxin B1 contaminated diet (20 µg/kg feed for 2 weeks) (A); aflatoxin B1 plasma concentration-time profile after oral administration in chickens (n = 4 in each group) with (treatment) or without (control) tiamulin oral gavage administration (25 mg/kg BW for 4 days) (B).The mean ± SD is shown.

Table 1 .
The intra-day and inter-day accuracy and precision of six probe substrates chicken liver microsomes.

Table 2 .
The intra-day and inter-day accuracy and precision of tiamulin and aflatoxin B1 in chicken plasma.

Table 3 .
Incubation conditions for six probe substrates for the in vitro CYP450 inhibition studyAnalytesProtein Concentration (mg/

Table 3 .
Incubation conditions for six probe substrates for the in vitro CYP450 inhibition study.
1 Tiamulin pharmacokinetic parameters in chickens pretreated with aflatoxin B1 contaminated diet (20 µg/kg feed for 2 weeks) (n = 8); 2 Tiamulin pharmacokinetic parameters in chickens that were not pretreated with aflatoxin B1 contaminated diet (control feed for 2 weeks) (n = 9).The AUC last of tiamulin was calculated until last sampling point of 180 h.** Significantly different from control, p < 0.01.*** Significantly different from control, p < 0.001.The mean ± SD is shown.T 1/2λz : elimination half-life; T max : time to reach peak plasma concentration; C max : plasma peak concentration; AUC last : area under the concentration-time curve from 0 to the last time point; AUC INF_obs : area under the concentration-time curve from 0 to infinity; V d_F_obs : apparent volume of distribution; CL _F_obs : apparent body clearance; MRT last : mean residence time.

Table 5 .
Pharmacokinetic parameters of aflatoxin B1 (AFB1) in chickens pretreated with or without tiamulin (25 mg/kg of BW for 4 days).Aflatoxin B1 pharmacokinetic parameters in chickens that were not pretreated with tiamulin (25 mg/kg BW saline for 4 days) (n = 4).The AUC last of aflatoxin B1 was calculated until last sampling point of 24 h.** Significantly different from control, p < 0.01.The mean ± SD is shown.T 1/2λz : elimination half-life; T max : time to reach peak plasma concentration; C max : plasma peak concentration; AUC last : area under the concentration-time curve from 0 to the last time point; AUC INF_obs : area under the concentration-time curve from 0 to infinity; V d_F_obs : apparent volume of distribution; CL _F_obs : apparent body clearance; MRT last : mean residence time.

Table 6 .
Mass spectrometry parameters for six probes substrates, tiamulin, and aflatoxin B1.College of Veterinary Medicine (Beijing, China), and authorization was approved by the Animal Care and Use Committee of China Agricultural University on 12 April 2022 (39905-23-G-001