Chinese Chemical Letters  2014, Vol.25 Issue (08):1107-1111   PDF    
Identification of in vitro and in vivo metabolites of 12β-hydroxylveratroylzygadenine associated with neurotoxicity by using HPLC-MS/MS
Yue Conga,b, Jing-Gong Guoc, Zhi Tangb, Qing-Chun Zhanga, Zong-Wei Caib     
a Institute of Pharmacy, Pharmaceutical College, Henan University, Jinming Road, Kaifeng 475004, China;
b State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China;
c The Key Laboratory of Plant Stress Biology, Henan University, Jinming Road, Kaifeng 475004, China
Abstract: Metabolism study was carried out on 12β-hydroxylveratroylzygadenine (VOG) that is a cevine-type alkaloid existing in Veratrum nigrum L. and a neurotoxic component. In order to better understand the potential mechanism of neurotoxicity of VOG, this study measured VOG-induced DNA damage in the cerebellum and cerebral cortex of mice after 7 days repetitive oral dose by using single-cell gel electrophoresis (Comet assay). High performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and applied to separate and identify in vitro and in vivo metabolites of VOG for investing the possible relationship of metabolism and neurotoxicity. In vitro experiment was carried out using rat liver microsomes, while the in vivo study was conducted on rats. The obtained results indicated that VOG might cause DNA damage in cerebellum and cerebral cortex of mice in a dosedependent manner. Hydrolysis of ester bond and O-demethylation were proposed to be themain in vivo metabolic pathways of VOG, while the major in vitro metabolic pathways were proposed as methyl oxidation to aldehyde, dehydrogenation, hydrolysis of ester bond, hydrolysis of ester bond together with acetylation, and methoxylation. O-Demethylation reaction was likely to be associated with reactive oxygen species production, leading to the DNA damage.
Key words: 12β-Hydroxylveratroylzygadenine     Metabolites     O-Demethylation     LC-MS    
1. Introduction

Veratrum nigrum L. has been used traditionally in Chinese medicine for treating hypertension,blood-stroke,excessive phlegm and epilepsy for centuries [1]. However,it is well-known that V. nigrum L. as a neurotoxic plant [2] can induce DNA damage in mice brain [3],result in persistent depolarization of excitable biomemberances by activating voltage-dependent Na+ channels [4] and have teratogenic effects in several laboratory animals [5]. The alkaloidal extracts of Veratrum species are well known for their antihypertensive and antimanic effects [6, 7]. Veratrum alkaloids were widely prescribed until late nineteen century when emetic side effects greatly curtailed its use [8]. In addition,it was also proved that Veratrum alkaloids possessed genotoxicity [3, 9, 10], reproductive toxicity [11] and neurotoxicity [3, 12].

A high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) method was developed for the simultaneous determination of ten steroidal alkaloids in V. nigrum L. [13]. The results indicated that 12b-hydroxylveratroylzygadenine (VOG) could be one of the markers with high content in the original herb. The chemical belongs to isosteroidal alkaloid’s sub-classes of cevine group (Fig. 1) [14]. VOG has showed cytotoxicity [15] and neurotoxicity that is an agonist of voltagegated sodium channel [16]. Recent studies have suggested that Veratrum alkaloids can cause DNA damage in the cerebellum and cerebral cortex of mice and some toxic chemicals can be transformed by phase I enzymes into DNA-reactive metabolites [3, 12, 17, 18, 19]. For example,one of the Veratrum alkaloids,veratridine, and some of its metabolites were presumed to mediate the neurotoxicity due to the formation of catechol structures in the in vitro metabolism of veratridine [20]. Inspired by this,we suspected that VOG and/or its metabolites might have the neurotoxic effect. To better understand the potential mechanism of neurotoxic effect, we performed the alkaline comet assay to detect DNA damage and to delineate the genotoxic effect of VOG on brain cells in mice. Because of inadequate biological samples available from the mice, in vivo and in vitro metabolism studies were performed by using rat as animal model due to its similarity to mice. The metabolites of VOG were separated and identified using liquid chromatography- ion trap tandem mass spectrometry (LC-MS/MS) that provided reliable analytical approach with high sensitivity for identification of metabolites in the biological samples. The metabolic transformation was proposed for the investigation of possible metabolites responsible for neurotoxicity of VOG,which might be helpful to understand the mechanism of the neurotoxic action of VOG.

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Fig. 1.The structure and LC-ESI-MS spectrum of VOG.
2. Experimental

VOG (>98%) was separated and purified according to procedures reported previously [15]. NADPH was purchased from Oriental Yeast Co.,Ltd. (Tokyo,Japan). Male Swiss mice (22 ± 2 g) were obtained from the Experimental Animal Center of Zhengzhou University. Male Sprague-Dawley rats (~8-week old and average weight of 220 g) were purchased from Laboratory Animal Services Center,the Chinese University of Hong Kong. All animal experiments were conducted according to guidelines established by the NIH Guide for the Care and Use of Laboratory Animals.

Mice were randomly divided into four groups of 4 mice in each group,which included control,positive,VOG at 0.25 mmol/kg and VOG at 2.50 mmol/kg. The animals in the VOG groups were treated by gavage with VOG at 0.25 and 2.50 mmol/kg every day for consecutive 7 days. The control group was treated with distilled water for 7 days. At the last day,the positive group received an i.p. injection of DMS at 20 mg/kg [21]. The comet assay was performed according to the previous reports [3, 12]. Tail moment was used as parameter to evaluate the DNA damage. Data were expressed as mean ± S.E.M. calculated from four mice of every group. Statistical comparisons were made by means of one-way analysis of variance (ANOVA),followed by the Fisher’s least significant difference (LSD) test (SPSS13.0 software,SPSS,USA).

For the in vitro metabolism experiment,rat liver microsomes were prepared according to protocol described previously [22]. The rat liver microsomal CYP and protein concentrations were determined by the method reported by Omura [23] and Bradford [24]. The incubation was performed in the presence of NADPH according to the previous report [25]. For the in vivo metabolism experiment,rats were fasted 12 h prior to administration of the dose and were fed 12 h after the dose,which were separated into two groups (n = 3). The rats received sample solution by gavage administration with a single dosage of 3 mg/kg body weight. Blood samples were collected in heparinized tubes via caudal vein predose and at 2.5 h postdose. Rat plasma (300 mL) was diluted with three volume of acetonitrile. The mixture was then centrifuged at 11,000 rpm,and the supernatant was pooled and blown to dry by using N2 at 35 8C,then reconstituted in 20 mL acetonitrile and transferred to a clean tube.

Urine and feces predose samples,i.e. blanks,were collected at the end of interval of 0-24 h after a 12 h fasting period. Urine and feces postdose samples were collected into clean container for 24 h interval postdose. The pooled urine samples (5 mL) were diluted with three volume of acetonitrile. The mixture was centrifuged at 8000 rpm,and the supernatant was pooled and blown to dry by N2 at 35 8C. The residue was dissolved in acetonitrile and then centrifuged at 11,000 rpm. The supernatant was pooled and transferred to a clean tube. Feces samples (10 g) were added to acetonitrile (3 mL/g feces) and mixed to generate the fecal homogenates and let stand for 4 h at room temperature. Feces samples were extracted two times. After filtration,the filtrates were evaporated to dryness at 35 8C by using N2. The residue was dissolved in acetonitrile and then centrifuged at 11,000 rpm. The supernatant was pooled and transferred to a clean tube. The biological samples were analyzed on a Bruker Esquire-4000 ion-trap mass spectrometer (Bruker-Fransen,Germany) equipped with electrospray ionization source couple to an Agilent 1100 HPLC system (Agilent Technologies,USA). Chromatographic separations were accomplished by using a Waters Symmetry ODS column (150 mm × 2.1 mm,3.5 mm) (Waters Corp.,USA). 3. Results and discussion After the oral administration of VOG at the doses of 0.25 and 2.50 mmol/kg every day for 7 consecutive days,comet assay was performed in the cells obtained from the cerebellum and cerebral cortex region in mice. The extent of DNA damage was calculated from relative changes in tail moment length. The obtained results showed that VOG significantly increased the values of tail moment length when compared to control group (one-way ANOVA; P < 0.001) (Fig. 2),suggesting that VOG induced DNA damage in mouse cerebellum and cerebral cortex following a 7 day repetitive dose. In addition,dose dependent increase in DNA damage indicated that this damage is chemically related. These two brain regions could be targeted for neurotoxicity of VOG and/or its metabolites.

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Fig. 2.Effects of VOG on brain cells DNA strand breaks in the cerebellum and cerebral cortex of mice. Two hundred cells were examined in duplicate for each condition and the tail moments are expressed as mean ± S.E.M.,***P < 0.001.

VOG showed a protonated ion at m/z 674 at the retention of 16.5 min in the LC-MS ion chromatogram(Fig. S1A in Supporting information). The characteristic fragment ion at m/z 474 was observed in MS/MS spectrum of VOG (Fig. 1). According to the MS/MS spectra,steric and electronic effects may activate the hydroxyl group at site 4,which is expelled aswater. After the loss of water at site 4,an unsaturated band is formed at sites 4 and 5, which further activates the ester group at site 3. When the 3-ester group is lost from C-3,an unsaturated band is formed at sites 2 and 3,and a stable intermediate at m/z 474 could be formed [26].

In vivo and in vitro samples were analyzed for possible metabolites by using LC-ion trap MS system (Figs. S1 and S2 in Supporting information). By careful mining of the chromatograms and data collected,nine metabolites were found in rat plasma, urine,feces and liver microsomes incubation samples. The LC-MS ion chromatograms of all in vivo and in vitro detected metabolites (M1-M9) were shown in Fig. S1. The metabolites were confirmed in LC-MS/MS system with structure elucidation.

M1 was observed at 2.9 min,with protonated molecule at m/z 510,which was 164 Da lower than that of VOG. The major fragment ion at m/z 474 was the same as that of VOG in the MS/MS spectrum of M1 (Fig. S3),indicating that the core structure of six in VOG was intact and the veratroyl group at site 3 of VOG was hydrolyzed to be 12b-hydroxyl-zygadenine. M2 at 15.8 min showed a protonated molecule at m/z 660,which was 14 Da lower than that of VOG. The characteristic fragment ion at m/z 474 was the same as that of VOG (Fig. S3),suggesting that Odemethylation may occur on methoxyl group at position 40 or 50. M3 at 13.9 min showed a protonated molecule at m/z 508,which was 2 Da lower than that of M1. The main fragment ion at m/z 472 was 2 Da lower than that of M1 atm/z 474 (Fig. S3),suggesting that dehydrogenation had occurred on hydroxyl group at site 3,15 or 16 of M1 to be ketone [27]. M4 at 14.5 min showed a protonated molecule atm/z 552,which was 122 Da lower than that of VOG and 42 Da higher than that of M1. The consecutive losses of three H2O moieties from m/z 552 [M+H]+ and the characteristic fragment ion at m/z 474 were both similar to those of VOG in the MS/MS spectrum (Fig. S3),implying that hydrolysis of ester bond at site 3 was followed by acetylation to be 3b-acetylzygadenine due to steric and electronic effects.M5 at 14.9 min showed a protonated molecule at m/z 646,which was 28 Da less than that of VOG. The characteristic fragment ion atm/z 474 was the same as that of VOG in the MS/MS spectrum (Fig. S3),revealing that O-didemethylation likely occurred at sites 40 and 50 on two methoxyl groups. Therefore,it was presumed to be 40,50-o-didemethyl-12bhydroxylveratroylzygadenine. M6 at 16.7 min was eluted with longer retention time than that of VOG. M6 showed a protonated molecule at m/z 672 and a main fragment ion at m/z 472,which were 2 Da lower than that of VOG and its main fragment ion at m/z 474 (Fig. S3),revealing that dehydrogenation may occurred on the core structure of VOG at site 15 or 16 on hydroxyl group to be ketone. Therefore,the metabolite was presumed to be 12bhydroxylveratroylzygadenine- 15-one or 12b-hydroxylveratroylzygadenine- 16-one. M7 was observed at 15.3 min and showed a protonated molecule at m/z 690 and a characteristic fragment ion at m/z 490,which were 16 Da higher than that of VOG and its fragment ion at m/z 474 (Fig. S3),indicating that hydroxylation occurred on the core structure of VOG. The exact position of hydroxylation was not characterized. M8 at 16.8 min was eluted with longer retention time than that of VOG. M8 showed a protonated molecule at m/z 704,which was 30 Da higher than that of VOG,and the characteristic fragment ion at m/z 474 was the same as that of VOG (Fig. S3),revealing that the core structure of VOG was intact and the methoxylation did not occur on the core structure of VOG. In addition,the methoxylation might occur at site 70 due to steric hindrance and electronic effect. M9 at 15.4 min showed a protonated molecule at m/z 688 and a characteristic fragment ion at m/z 488,which was 14 Da higher than that of VOG and its fragment ion at m/z 474 (Fig. S3),revealing that the modification occurred on the core structure of VOG and methyl group at site 27 of VOG may be oxidized into aldehyde [27] due to steric hindrance.

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Fig. 3.Proposed in vivo and in vitro metabolic pathways of VOG.

To the best of our knowledge,this is the first investigation of in vivo metabolites of cevine-type Veratrum alkaloid by using LC-MS/ MS. Based on the metabolite profile described above,major in vivo metabolic pathways of VOG were proposed as O-demethylation and hydrolysis of ester bond,while the major in vitro metabolic pathways of VOG were proposed as dehydrogenation,hydrolysis of ester bond,hydrolysis of ester bond together with acetylation, methoxylation and methyl oxidation to aldehyde (Fig. 3). The putative identification of the VOG metabolites showed that metabolic pathways were different between in vitro and in vivo. The microsomes incubation could not represent the in vivo physiological conditions,as lipophilic molecules may be attached to fractions of serum protein,e.g. albumin. Thus,the in vitro metabolism of substrates could differ significantly from the in vivo metabolism [28]. For example,M9 was only observed in the incubation with the rat liver microsomes and M2,M3 and M5 were only observed in vivo.

The metabolites M2 and M5 were both O-demethylation of VOG which could generate a catechol structure. The catechol structure easily form highly electrophilic ortho-quinone as toxic reactive metabolites catalyzed by mitochondrial dysfunction,inflammation, oxidative stress and dysfunction of the ubiquitin-proteasome system [29],which is responsible for neurotoxicity by neuron oxidative stress [30, 31]. ortho-Quinones are highly redox active molecules,as their semiquinone radicals can induce redox cycle, leading to formation of reactive oxygen species (ROS) to oxidized DNA and proteins [32]. Eletrophile quinones could also form covalent adducts with crucial cellular protein orDNA. Thus,itwould be likely that thesequinonemetabolitesmight be responsible for the VOG-induced DNA damages detected in mouse brain cells from cerebellumand cerebral cortex. In addition,hydrolysis of ester bond reaction may reduce the neurotoxicity of VOG due to the loss of a veratroyl group at site 3 [16],for example,to form M1 and M3. 4. Conclusion

In this study,eight metabolites were detected in rat feces samples,two were detected in urine,and three were detected in plasma samples from the in vivo experiment,while six metabolites were detected in the in vitro samples with rat liver microsomes. Low- and high-dose VOG could induce DNA damage in mouse brain cells of cerebellum and cerebral cortex. The O-demethylation of benzene ring in VOG might contribute to VOG-induced DNA damage by forming reactive oxygen species. The investigation on metabolic profiles of VOG might reveal in vivo biotransformation characteristic of cevine-type alkaloids and provide perspectives for their potential pharmaceutical applications and toxicity study. Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 21102035). Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.05.016.

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