| Quinoxalines are animal special drugs of chemical synthesis which are widely used as antibacterial growth promoters. Carbadox and olaquindox are the well-known members, which have been banned or limited to be used in food animal for their potential side effects. Mequindox and quinocetone have been successively sanctioned to be used in food animals in China, and the safer medicines such as cyadox which belongs to quinoxalines family are being developed. Studies have demonstrated that the toxicities of carbadox and olaquindox are closely associated with their metabolism. Some metabolites of quinoxalines have been detected in various tissues, but parent drugs have not been. Currently, the major identified metabolites of olaquindox, carbadox and cyadox are their reduced metabolites and residue markers; most of their metabolites have not yet been identified. The metabolism of mequindox and quinocetone has not been investigated till now. Moreover, it has been demonstrated that the metabolism of cyadox has species difference. But, it is unknown whether the metabolism of other quinoxalines has also the species difference As drug for food producing animals, food safety has become a hot problem in the filed of food processing science. Drug residue is one of the main factors to endanger food safety, while drug metabolism is the scientific groundwork to avoid drug residue. Therefore, the quinoxalines were used as objects in this study. The comparative metabolism of olaquindox in rat, swine and chicken liver microsomes and hepatoctyes was investigated using high-performance liquid chromatography combined with IT/TOF mass spectrometry (LC/MS-ITTOF). Based on these results, the metabolic profiles of other quinoxalines in liver microsomes of the three species were compared. The metabolism of olaquindox in rats, swine and chicken was also examined in detail. In addition, the species differences of metabolic enzymes involved in the N→O group reduction, hydroxyl oxidation and N-dehydroxyethylation were studied. The aims of these studies were to clarify the metabolic characteristics and relationship between structure and metabolism of quinoxalines. Moreover, the biotransformation and reasons of species difference of olaquindox were revealed comprehensively in the study. 1. The establishment of in vitro liver systems of animal subjectLiver microsomes from rats, swine and chicken were prepared using differential centrifugation. The enzymatic activities of microsomes were evaluated by the carbon monoxide (CO) difference spectrum and the metabolism of coumarin as probe drug. Hepatocytes from rats, swine and chicken were separated by a modification of a collagenase perfusion method. The metabolic activities of hepatocytes were measured by the metabolism of coumarin after cell counting and cell viability assay. The results showed a maximum absorption peak of microsomes at 450nm. Coumarin could be metabolized to 7-hydroxycoumarin in liver microsomes and hepatocytes. Therefore, the in vitro liver systems had the activities of drug metabolic enzymes.The enzymatic kinetic of CYP2A activity in swine liver microsomes was further examined using coumarin as a probe drug. The inhibitory effects on swine CYP2A activity were also evaluated using five commonly used human CYP inhibitors. The results demonstrated that the Km and Vmax for coumarin 7-hydroxylase (CYP2A) in swine liver microsomes were estimated to be 1μmol/L and 0.26nmol/mg/min, respectively. The following human CYP inhibitors caused litter or no inhibition of CYP2A as defined by a Ki>200umol/L: quinidine (QUI), troleandomycin (TAO), and sulfaphenazole (SUL).α-Naphthoflavone (ANF) inhibited the 7-hydroxylation of coumarin with a Ki value of 32μmol/L, which did not increase ability to inhibit CYP2A when ANF was preincubated with swine liver microsomes for 3 min. In the absence of a preincubation period, 8-methoxypsoralen (MOP) inhibited the 7-hydroxylation of coumarin with a Ki value of 1.1μmol/L, which decreased to 0.1μmol/L when MOP was preincubated with swine liver microsomes for 3 min. These results indicated that MOP may be used as a specific uncompetitive inhibitor of mechanism-based inactive of swine CYP2A in liver microsomes.2. Comparative metabolism of olaquindox in vitro liver systemsThe purpose of this study was to compare the metabolic pathways of olaquindox and its metabolite desoxyolaquindox (DES) using reliable systems of liver microsomes and hepatocytes. The metabolites were characterized rapidly and accurately using LC/MS-ITTOF with MSn capability, high sensitivity, high resolution and mass accuracy. Thirteen metabolites of olaquindox were detected in rat liver microsomes. However, only seven metabolites were formed in swine and chicken liver microsomes. Among the identified metabolites, beside three reduced metabolites (01, 02, 09) and two carboxylic acid derivatives (08, 010) were consistent with the early report, five hydroxylation metabolites (O3～O7), two N-dehydroxyethylation metabolites (O11,O12) and a aldehyde metabolite (O13) were found for the first time in liver microsomes. O2 was the major metabolite in swine and chicken, but O1, O2 and O9 were the major metabolites in rats. Six metabolites of DES were found in rat liver microsomes and identified as O2, O10, O13, O12 and two hydroxylation metabolites (O14, O15). Except metabolite O15, other five metabolites were also measured in swine and chicken liver microsomes. O10 was the major metabolite of DES in the three species. Five metabolites were detected in rat and swine hepatocytes, and identified as O1, O2, O5, O8 and O9. Except metabolite O8, other four metabolites were observed in chicken hepatocytes. The present results revealed that N→O group reduction and oxidation of hydroxyl group were the main metabolic pathways of olaquindox in all three species. The ability of N→O group reduction and hydroxylation in rat was higher than other two species, but the oxidation ability in chicken was highest among the three species. The N→O group reduction and N-oxidation of olaquindox could interconvert in liver microsomes. The results also suggested that the O13, O15 and three reduced metabolites were related to the toxicity of olaquindox.3. Comparative metabolism of mequindox, quinocetone, carbadox and cyadox in liver microsomesTo investigate the metabolic characteristics of quinoxalines and reveal the relation between structure and metabolism of quinoxalines, the metabolism of other quinoxalines in liver microsomes from the three species was compared in this study. The results showed that fourteen metabolites of mequindox were firstly detected in chicken liver microsomes, including three reduction metabolites (M1, M2, M6), five carbonyl reduction metabolites (M10～M14) and six hydroxylation metabolites (M3, M4, M5, M7～M9). Ten metabolites (M1～M4, M6, M7, M10～M12, M14) were found in rat and swine liver microsomes. Moreover, a additional metabolite M13 was detected in swine liver microsomes. M2, M10 and M12 were the major metabolites in swine. M2, M6 and M12 were the major metabolites in chicken, but M2 and M6 were the major metabolites in rats.A total of twenty-seven metabolites (Q1～Q27) of quinocetone were tentatively identified in the rat liver microsomes. Twenty-three and twenty-five metabolites were found in chicken and swine liver mircrosomes, respectively. Compared with metabolites found in rat liver microsmes, except Q8, Q11, Q14, Q15, Q19, Q25, Q26 and Q27, other metabolites were detected in chicken liver microsomes. Four additional metabolites (Q28～Q31) were observed in chicken liver microsomes. Compared with metabolites found in rat liver microsmes, except metabolites Q8, Q9, Q10, Q11, and Q14, other metabolites were also deteced in swine liver microsomes. Three additional metabolites (Q28, Q32 and Q33) were measured in swine liver microsomes. Except metabolite Q6, other metabolites were firstly found and identified in liver microsomes. Q2 and Q4 were the major metabolites in rats. Q2, Q4, Q17 and Q20 were the major metabolites in chicken, but Q2 and Q6 were the major metabolites in swine.Six metabolites of carbadox were detected in rat liver microsomes, and identified as three reduced metabolites (Cb1～Cb3), three hydroxylation metabolites (Cb4～Cb6). Only four metabolites (Cb1～Cb4) were measured in chicken and swine liver microsomes. Cb1 was the major metabolites in rats. Cb1, Cb3 and Cb4 were the major metabolites in chicken, but Cb1 and CM were the major metabolites in swine. Among the identified metabolites, beside Cb3 was consistent with the early report, other five metabolites of carbadox were found for the first time in liver microsomes.Except metabolite Cy7 only formed in swine liver microsomes, other six metabolites (Cy1～Cy6) were detected in rat, swine and chicken liver microsmoes. Metabolites were identified as three reduced metabolites (Cy1, Cy2, Cy4), a hydroxylation metabolite (Cy3) and three hydrolysis metabolites on the amide bond (Cy5～Cy7). Cy1, Cy4, Cy5 and Cy6 were the major metabolites in swine. Cy1 and Cy4 were the major metabolites in rats, but only Cy1 was the major metabolite in chicken. Except Cy2 and Cy4, other five minor metabolites were firstly found in liver microsomes.The metabolic studies of quinoxalines in different species liver microsomes show that the N→O group reduction and hydroxlation are the same two metabolic pathways of quinoxalines. Moreover, the toxicities of quinoxlaines are related to the N→O group reduction. Drugs have obvious metabolism differences owing to the differences on the side chain of quinoxalines. Almost no qualitative species difference in the metabolic pathways of quinoxalines is measured. There are metabolic rate and minor metabolites species difference in the metabolism of quinoxalines among the three species due to enzymes difference. The abilities of N→O group reduction and hydroxylation in rat are higher than other two species, while the oxidation of hydroxyl in chicken and the carbonyl reduction and hydrolysis of amide bond in swine are highest among the three species, respectively.4. Comparative metabolism of olaquindox in rats, swine and chickenRats, swine and chicken were administered olaquindox oral gavages with a single dosage of 10, 5 and 30 mg/kg b.w, respectively. The method using LC/MS-ITTOF had been developed for the analysis olaquindox and its metabolites in urine, plasma, feces, muscle, liver, kidney and contents of intestinum cecum from female and male rats, swine and chicken. A total of eighteen metabolites in rat urine were detected after administration of olaquindox. O1 and O2 were the major metabolites in female rats, but O2, O9 and O13 were the major metabolites in male rat. Six (O2, O9, O10, O13, O16 and O19), three (O10, O13, O19), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female rat feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were also found in male swine feces, only O9 was detected in male rat tissues.A total of sixteen metabolites in swine urine were detected after administration of olaquindox. O2 and O9 were the major metabolites in swine. Six (O2, O9, O10, O13, O16 and O19), one (O13), two (O9 and O19), two (O9 and O19) and three (O9, O12 and O19) metabolites were found in female swine feces, content of intestinum caecum, muscle, kidney and liver, respectively. However, six metabolites were found in male swine feces, only O9 was detected in male swine tissues.A total of fifteen metabolites in female and male chicken were deteced at 2h after administration of olaquindox. O2, O8 and O9 were the major metabolites in female chicken, but O8 and O13 were the major metabolites in male chicken. Beside all metabolites detected in plasma, O16, O25 and O26 were also found in chicken feces. One (O9), six (O2, O8, O9, O10, O11, O12) and three (O9, O12 and O19) metabolites were found in female chicken muscle, kidney and liver, respectively. One (O9), seven (O2, O8, O9, O10, O11, O12, O19) and four (O9, O10, O12 and O19) metabolites were found in male chicken muscle, kidney and liver, respectively.In conclusion, except three reduced metabolites (O1, O2 and O9), four carboxylic acid derivatives (O8, O10, O20 and O21) and MQCA (O19) were consistent with the early report, other fifteen metabolites of olaquindox were found for the first time in vivo. These results demonstrate that, except O6 and O7, all other metabolites of olaquindox formed in liver microsomes and hepatocytes are also detected in vivo. The results show that the major metabolic pathways are similar to the three species, but the minor metabolites have obvious species difference, which are closely with enzymes difference. The excretion rate of olaquindox in three species is: rat> swine > chicken. The rates of excretion and metabolism of olaquindox in male rat and swine are faster than those in female's, but gender difference is opposite in chicken.5. Species differences of metabolic enzymes involved in the reduction, oxidation and N-dehydroxyethylation of olaquindoxTo understand the metabolic mechanism and species difference of olaquindox, the reductive mechanisms of N1 and N4 reduction of olaquindox in liver microsomes and cytosol for rats, swine and chicken were investigated. Then, the CYP and non-CYP enzymes involved in the oxidation and N-dehydroxyethylation of DES in the three species were also examined.Olaquindox was reduced to O2 by liver microsomes and cytosol from rats, swine and chicken under hypoxic conditions. The N1 -reducing activity was inhibited in the presence of NADH by air and CO. However, the activity was stimulated by the addition of riboflavin under hypoxic conditions. When the liver microsomes and cytosol were boiled, these activities were not abolished, but were enhanced in the presence of NADH and riboflavin. The NADH-linked activities of the rat and swine live cytosol were enhanced significantly by the addition of menadione, but chicken liver cytosol did not. Swine liver cytosol exhibited a significant reductase activity toward N1-reduction of olaquindox to form O2 in the presence of menadione under hypoxic conditions, but rat and chicken did not. These results suggest that the N1 -reduction of olaquindox may be enzymatic and non-enzymatic involvement in liver microsomes and cytosol of the three species. The enzymatic reduction of olaquindox in liver microsomes has not species difference, but the enzymatic reduction of olaquindox in liver cytosol has obvious species difference. Rat and swine liver cytosol exhibited N4-reducing activity toward olaquindox to form N4-reduced olaquindox (O1) in the presence of benzaldehyde (BZA) under hypoxic conditions. The N4-reducing activity was inhibited in rat and swine liver cytosol by inhibitors of aldehyde oxidase. The N1 -reducing activity was inhibited in rat liver cytosol by xanthine oxidase inhibitor, but swine liver cytsol did not. These results suggest that N4-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase and xanthine oxidase. The N4-reducing activity may be catalyzed by aldehyde oxidase in swine cytosol. However, N1-reduction of olaquindox in rat cytosol may be catalyzed by aldehyde oxidase. Chicken liver cytosol had not the N4-reducing activity toward olaquindox to form O1 in the presence of BZA, suggesting chicken may be devoid of the activities of aldehyde oxidase or xanthine oxidase.In this study, various concentrations of DES were incubated with rat, swine and chicken liver microsomes, respectively. The results showed that the formation of O10 in rat liver microsomes exhibited a single CYP isoform involved, but chicken and swine liver microsomes would be involved in this metabolic pathway by at least two CYP isoforms. The formation of O12 in all species microsomes exhibited at least two CYP isoforms involvement in the N-dehydroxyethylation of DES. The rank orders of intrinsic clearance rates for O10 and O12 with liver microsomes obtained from the three species were chicken>swine>rat and rat>swine>chicken, respectively. Species difference of CYP enzymes involved in the oxidation of hydroxyl group and N-dehydroxyethylation of DES were investigated using selective chemical inhibitors. The results indicated that MOP, MP and ANF inhibited the formation of O10 in all species liver microsomes. However, the degree to MOP inhibited O10 was not markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. DDTC and DIS inhibited the formation O10 in swine and chicken liver microsomes. In addition, TAO and QUI could inhibit the formation of O10 in chicken liver micrsomes. MOP, MP, ANF, DDTC and DIS inhibited the N-dehydroxyethylation of DES to O12 in all species liver microsomes. However, the degree to DDTC and DIS inhibited O12 were markedly increased when it was preincubated with liver microsomes for 10 min prior to the addition of substrate. TAO and QUI inhibited the formation O12 in chicken liver microsomes, but rat and swine liver microsomes did not. The results suggest that CYP1A may involve in the formation of O10 in rat liver microsomes, CYP1A and CYP2E may involve in the formation of O10 in swine liver microsomes. The formation of O12 may be catalyzed by CYP1A and CYP2E in rat liver microsomes. CYP2A and CYP2E may involve in the formation of O12 in swine liver microsomes. The results suggested chicken various CYP isoforms may catalyze the two reactions.DES was incubated with MP, DIS, DDTC chlorpromazine (CPZ), promethazine (PZ), 7-hydroxycoumarin (HCO) in rat, swine and chicken liver cytosol, respectively. In rat liver cytosol, except HCO, other inhibitors all inhibited the formation of O10. MP, CPZ and MEN inhibited the formation of O10 in swine liver cytosol. However, all inhibitors did not inhibit the formation of O10 in chicken liver cytosol. The results indicate that alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and aldehyde oxidase (AO) may all involve in the oxidation of alcohol of DES in rat liver cytosol. The formation of O10 may be carried out by ADH and AO in swine liver cytosol. However, it is possible that the inhibitors have not selectively in chicken liver cytosol or other enzymes may involve in the reaction.In conclusion, the metabolic pathways and characteristics of quinoxalines in different species were illustrated for the first time circled the comparative metabolism. The application of LC/MS-ITTOF in the qualitative and relative quantitative metabolites was developed firstly. Many new metabolites of quinoxalins were identified, and the relationship between structure and metabolism of quinoxalines were revealed in this study. At the same time, the metabolic fate of olaquindox in vivo from various species was investigated in detail. The species differences of metabolic enzymes involved in the metabolism of olaquindox were examined in depth for the first time in this study. The results demonstrate that the use of LC/MS-ITTOF approach in structural characterization of drug metabolites appears rapid, efficient and reliable. These results will provide comprehensive data to clarify the metabolism of olaquindox, and give scientific guidance for the identification of metabolic locations, the explanation of pharmacokinetics and cause of species difference of olaquindox. The study will contribute valuable information to the following mechanism study of toxicity of olaquindox, the development of analytical methods of residues and identification of residue marker of olaquindox. Moreover, the study will also give useful information for the studies of N→O group reduced mechanism, metabolic enzymes and in vivo metabolism of other quinoxalines.
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