analytica chimica actaprobe 7-ethoxyresoruﬁn (7-er) under the same procedures. moreover, based on...

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Analytica Chimica Acta xxx (2018) 1e6

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Analytica Chimica Acta

journal homepage: www.elsevier .com/locate/aca

Highly sensitive fluorescent bioassay of 2,3,7,8-tetrachloro-dibenzo-p-dioxin based on abnormal expression of cytochrome P450 1A2 inhuman cells

Li Shangguan a, b, Yuanqing Wei a, Kan Wang a, Yuanjian Zhang a, *, Songqin Liu a, **

a Key Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials andDevice, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, PR Chinab School of Chemistry and Environmental Engineering, Jiangsu University of Technology, Changzhou, 213001, Jiangsu Province, PR China

h i g h l i g h t s

* Corresponding author.** Correspondin author.

E-mail addresses: [email protected] ((S. Liu).

https://doi.org/10.1016/j.aca.2018.08.0060003-2670/© 2018 Elsevier B.V. All rights reserved.

Please cite this article in press as: L. Shanggabnormal expression of cytochrome P450 1

g r a p h i c a l a b s t r a c t

� HBMN demonstrated ca. 60 timeshigher affinity to CYP1A2 than the-state-of-art 7-ER probe.

� The in vitro bioassay was constructedby HBMN probe in human cells.

� The sensing strategy had 10 timesimproved sensitivity TCDD detectioncompared to previous reports using7-ER probe.

� Based on HBMN, the higher abnormalexpression of CYP1A2 by TCDD in-duction in human hepatic cells wasfound than that in lung cells.

a r t i c l e i n f o

Article history:Received 21 May 2018Received in revised form10 July 2018Accepted 2 August 2018Available online xxx

Keywords:BiosensorsTCDDCytochrome P450FluorescenceHuman cells

a b s t r a c t

Current in vitro bioassays of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a major threat carcinogen) arerelied on murine cells and fluorescent probe 7-ethoxyresorufin (7-ER), in which TCDD mostly causesabnormal expression of cytochrome P450 1A1 (CYP1A1). However, for human cells, TCDD mainly leads toa distinct abnormal expression of cytochrome P450 1A2 (CYP1A2). The poor response of 7-ER to CYP1A2limits the traditional bioassay for human cells. Herein, we report a fluorescent probe N-(3-hydroxybutyl)-4-methoxy-1,8-naphthalimide (HBMN) for in vitro bioassay of TCDD with human cells.HBMN had ca. 60 times higher affinity to CYP1A2 than 7-ER. As such, the sensing sensitivity increased by10 times, and different expression of CYP1A2 by TCDD induction in different human cells was found.Besides, HBMN was also feasible in rapid screening of TCDD concentration by naked eye. It would open anew way to highly sensitive detect TCDD and understand the pathogenesis of TCDD in different humanorgans.

© 2018 Elsevier B.V. All rights reserved.

Y. Zhang), [email protected]

uan, et al., Highly sensitive fl

A2 in human cells, Analytica

1. Introduction

As an environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is considered as a major threat carcinogen indioxin-like compounds [1e3]. Hence, the sensitive detection ofTCDD is vital to evaluate the toxicity of TCDD for human being

uorescent bioassay of 2,3,7,8-tetrachloro-dibenzo-p-dioxin based onChimica Acta (2018), https://doi.org/10.1016/j.aca.2018.08.006

mailto:[email protected]

mailto:[email protected]

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L. Shangguan et al. / Analytica Chimica Acta xxx (2018) 1e62

[4e6]. High resolution gas-chromatography combined with highresolution mass-spectrometry (HRGC-HRMS) is considered as the“golden standard” method for the quantification of TCDD [7], butrequires a sophisticated platform and highly trained personnel [8].Recently, some biological methods, namely in vivo and in vitrobioassays, have been developed for sensing TCDD. In general, theformer is used as a defensible screening method of the potentialhealth effects in environmental mixtures [9e11], while the latter isused for quantitative realistic detection of TCDD [12e16]. Theprinciple of in vitro bioassays lies in that TCDD directly induces theexpression of cytochrome P450 1 A (CYP1A) or indirectly inducesCYP1A-correlated luciferase expression in cultural cells, and TCDDwas then quantitatively detected by the increased expression of theenzymes. The advantages of these methods with aforementionedcounterparts are simple, rapid, of low cost, sensitivity and on-siteanalysis [4,8]. Recent toxicity studies reveal that TCDD inducesdifferent expressions of CYP1A in animal and human cells, e.g.,mostly inducing expression of CYP1A1 in murine hepatocytes andwhile CYP1A2 in human hepatocytes [17e20]. Nevertheless, ofnote, nearly all previous vitro bioassays are built on the expressionof CYP1A1 in the murine cell lines [14e16]. In this regard, the vitrobioassays, merely relying on the expression of CYP1A1 in murinecell lines, would significantly restrict the development of thesemethods in comprehensively understanding the pathogenesis ofTCDD, especially for human.

Herein, we report the fluorescent probe HBMN has a significanthigher affinity to CYP1A2 than CYP1A1. As a result of this advan-tage, the proposed in vitro bioassay exhibited a much highersensitivity up to 10 times for TCDD detection in human hepatocytescell lines (HepG2 cells) with respect to conventional fluorescentprobe 7-ethoxyresorufin (7-ER) under the same procedures.Moreover, based on the fluorescent probe HBMN, the differentdistribution and the expression of CYP1A2 were also firstlyobserved by the confocal microscope in different human cells bythe induction of TCDD.

2. Experiment section

2.1. Chemicals and materials

Cytochrome P450 1A1 (CYP1A1), cytochrome P450 1A2(CYP1A2), cytochrome P450 2C9 (CYP2C9), cytochrome P450 2D6(CYP2D6), cytochrome P450 2E1 (CYP2E1), cytochrome P450 3A4(CYP3A4) recombinant human isozyme microsomes with CYPreductase (CPR) and cytochrome b5 reductase were obtained fromBD Biosciences (USA). The concentration of all isozymes was 1.0 mM2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD, 50 mgmL�1 in toluene)was obtained from J&K Scientific Ltd. (Beijing, China). Glucose-6-phosphate dehydrogenase (G6PD, 550e1100 units mg�1 protein),glucose-6-phosphate (G6P), b-nicotinamide adenine dinucleotidephosphate hydrate (NADPþ) were purchased from Sigma-Aldrich(Shanghai, China). Human hepatoma cell lines (HepG2 cells), Hu-man lung cancer cell lines (A549 cells), dulbecco's modified eagle'smedium (DMEM), DMEM without phenol red, fetal bovine serum(FBS), paraformaldehyde and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) kit were obtained from Key-GEN BioTECH Co. Ltd. (Nanjing, China). N-(3-hydroxybutyl)-4-methoxy-1,8-naphthalimide (HBMN), HBMN-h, and esterifiedHBMN were homemade according to previous report (See the de-tails in SI) [21]. High concentration stock solution of them wasfirstly prepared in ethanol, and then diluted in PBS. The percentageof ethanol was less than 0.1% in the final solution, which wassupposed to have negligible influences for enzymes and cells in thisstudy. Other chemicals were of analytical grade and used withoutfurther purification unless otherwise specified. Phosphate buffer

Please cite this article in press as: L. Shangguan, et al., Highly sensitive fl

abnormal expression of cytochrome P450 1A2 in human cells, Analytica

(PBS, 0.1M pH 7.4) was prepared by mixing 0.1M NaH2PO4 andNa2HPO4. Ultrapure water (Thermo Smart2 Pure 3, resis-tance� 18MU cm) was used throughout experiments.

2.2. Apparatus

UVevis absorption spectra were obtained from Cary 100 (Agi-lent, Singapore). The photoluminescence spectra were collected bya fluorescent spectrometer (Fluoromax-4, Horiba Jobin Yvon,Japan). 1H NMR and 13C NMR spectra were recorded on a Brukerinstrument (Germany) with tetramethylsilane as an internal stan-dard. Mass spectra (MS) were obtained from a time-of-flight massspectrometer (TOF-MS, Agilent Technologies, USA). High perfor-mance liquid chromatography (HPLC) measurements were con-ducted by Shimadzu-2010 system (Japan). All the samples wereextracted by dichloromethane, dried by N2 blowing and re-dissolved in methanol. 10 mL of samples were injected into Inert-Sustain C18 column (250mm� 4.6mm, 5 mm). The isocratic mobilephase was the mixture of acetonitrile and water with volume ratioof 7 to 1. Flow rate of mobile phase was 1.0mLmin�1. The fluo-rescence images of cells were taken using a FV1000 MPF confocallaser microscopy (Olympus, Japan) with an objective lens.

2.3. Metabolism kinetics of HBMN by cytochrome P450

To monitor the metabolic performance, the NADPH regeneratedsystem (0.5 U G6PD, 3.3mMG6P, 1.3mM NADPþ) [22] was mixedwith 2.0 mM HBMN and the common cytochrome P450 includingCYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, respec-tively. Here, NADPH provided the eletron and proton to cytochromeP450 that catalyzed HBMNmetabolism. The amount of NADPHwaskept the same in all catalysis reactions. The possible reaction pro-cess was that G6PD catalyzed the oxidation of G6P, accompanyingby the reduction of NADPþ to NADPH [22]. After incubation at 37 �Cfor 60min, the mixture solution was centrifuged at 13000 rpm for10min. The fluorescence spectra of supernatant were measured atthe excitation wavelengths of 370 and 465 nm. The ratio fluores-cence intensity at ca. 550 nm for metabolite and ca. 460 nm forsubstrate (I550/I460) was used to represent the metabolic ability. Thefluorescence emission peak of NADPH was also observed at ca.460 nm, which was overlapped with HBMN at 460 nm. However,the fluorescence intensity of NADPH regenerated system couldreach the equilibrium in several minutes [23]. Therefore, the fluo-rescence intensity of HBMN was determined by the total fluores-cence intensity of the mixture of HBMN and NADPH deducting thebackground signal of NADPH in equilibrium condition. The meta-bolic behavior of HBMN with CYP1A1 or CYP1A2 at different con-centrations (10, 20, 50, 100, 150, 200, 300, 400 and 500 nM) wereconducted by the same process as aforementioned.

2.4. Cytotoxicity assays

The cytotoxicity of HBMN and esterified HBMNwas investigatedby MTT kit [24]. In this case, 200 mL 2� 104 cells mL�1 HepG2 cellssuspension in DMEDmediumwith 10% FBS was seeded in a 96 wellplate. After cultured at 37 �C in a 5% CO2 incubator for 24 h, theHepG2 cells were treated by removing the culture medium andwashing with PBS. Then, HBMN or esterified HBMN with concen-trations of 0, 0.5, 1, 2, 4, 6, 8, 10, 20 and 30 mM were added to thewells of 96 well plate, respectively, and incubated for another 48 h.After washing with PBS, 100 mL of 0.05% MTT were added to eachwell and incubated for 4 h. Then, the culture mediumwas removedand 50 mL of DMSO was added to each well to solubilize the for-mazan. The plate was shaken gently for 10min at room tempera-ture and the absorbance was measured at 490 nm.


Administrator

高亮

Administrator

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Fig. 2. (a) Fluorescence excitation and emission spectra of HBMN (2 mM) in aqueoussolution. (b) The fluorescence spectra of HBMN (2 mM) before (dash) and after (solid)metabolized by 500 nM CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4,respectively. (c) Fluorescence spectra of HBMN metabolized by different concentra-tions of CYP1A2 ranging from 0 to 500 nM, and (d) the linear correlation of fluores-cence intensity ratio (I550/I460) and different concentrations of CYP1A1 and CYP1A2.

L. Shangguan et al. / Analytica Chimica Acta xxx (2018) 1e6 3

2.5. Detection of TCDD with HepG2 cells

200 mL of 1� 105 cells mL�1 HepG2 cell suspension wasdispensed into a 96 well plate and cultured for 24 h. Then, 200 mLTCDDwith different concentrations of 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80,100, 120, 140 pgmL�1 were added into 96 well plate, respectively,and incubated at 37 �C for 24 h. After washing with PBS for threetimes, 200 mL of 10 mM esterified HBMN in DMEM without phenolred was added into each well and cultured for 12 h. Then, 50 mL ofmethanol was added into each well and jiggling for 10min at roomtemperature, the fluorescence and UVevis spectra of each wellwere measured with micro-cuvettes.

2.6. Fluorescence imaging

For fluorescent cell imaging, 0.5mL of 1� 105 cells mL�1 HepG2cells or A549 cells were seeded in each confocal dish and culturedin DMEM containing 10% fetal bovine serum for 24 h. After washingwith 0.1M pH 7.4 PBS for twice, 10 mM of esterified HBMN wasadded into the dish and incubated at 37 �C for 12 h. To remove theincubation solution of HepG2 cells or A549 cells and washed with0.1M pH 7.4 PBS for three times. Then 4% paraformaldehyde wasadded to fixed cells for 15min for fluorescent confocal imaging[25].

3. Results and discussion

3.1. Synthesis and characterization of HBMN

Fig. 1a showed the general synthetic procedurals and reactionsof HBMN which was firstly reported by Yang and co-workers [21].In a typical in vitro fluorescent bioassay process, TCDD firstly acti-vated the intracellular transcription factor aryl hydrocarbon re-ceptor (AhR), which later transferred into cell nucleus andcombined with AhR nuclear translocator (ARNT) forming a heter-odimer [26e28]. It would trigger the abnormal expression of cy-tochrome P450 1 A (CYP1A1 and CYP1A2) [29,30], after which, thefluorescent properties of HBMN (the substrate) were significantlyaltered (Fig. 1b). The fluorescent excitation and emission spectra oforiginal HBMN were showed in Fig. 2a. The optimized excitation

Fig. 1. (a) Typical preparation procedures and reactions of N-(3-hydroxy butyl)-4-methoxy-1, 8-naphthalimide (HBMN). (b) General in vitro fluorescent bioassay pro-cess for detecting TCDD by HBMN.



wavelength was at 370 nm (black line) and the emission peakappeared at ca. 460 nm (red line), which demonstrated a largestoke-shift up to 90 nm, much preferred for biosensing to minimizethe fluorescence background [31]. The utility of HBMN as thefluorescent probe for the metabolic reaction of common cyto-chrome P450 including CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP2E1and CYP3A4 [32], was investigated using NADPH regenerated sys-tem. The ability of HBMN as the fluorescent probe for the metabolicreaction of CYP1A was investigated by fluorescence method. Afterthe metabolic de-methylation reaction, a new emission peak at ca.550 nm appeared at 465 nm excitation was observed (Fig. 2b). Twoemission peaks were stable in a broad biologically relevant pHranging from 5 to 10 (Fig. S2), other preferred features for bio-sensing in the physiological conditions [21]. The ratio of fluores-cence intensity of HBMN before and after metabolized at 550 nmand 460 nm (I550/I460) was used to evaluate the metabolic ability[21,33e35]. It was observed that the I550/I460 value of HBMNmetabolized by CYP1A2 was nearly 25 times higher than that ofCYP1A1, and more strikingly, over 220 times higher than that othertypes of cytochrome P450. The HPLC measurements also showedthat HBMN was more easily metabolized by CYP1A2 than that byCYP1A1 (Fig. S1). These facts jointly revealed that the fluorescentprobe HBMN had excellent specificity to CYP1A2 among cyto-chrome P450 family.

3.2. Affinity of HBMN to CYP1A

The metabolized affinity of HBMN by CYP1A1 and CYP1A2 wasfurther investigated, and it was found the catalytic reactions couldreach equilibrium within 60min (Fig. S3). Upon this optimal reac-tion time, the corrections of 2 mM HBMN in the different concen-tration of CYP1A1 and CYP1A2 were studied. With increasing of theenzymes concentration, the fluorescence intensity at 460 nmdecreased and that at 550 nm increased (Fig. 2c and Fig. S4). Asshown in Fig. 1d, the ratio of I550/I460 was proportional to the twoenzymes concentration in the range from 10 nM to 500 nM. Of note,


Fig. 3. (a) The calibration curve of I550/I460 with the concentration of TCDD rangingfrom 0.1 to 140 pgmL�1 in HepG2 cells. (b) UVevis spectra and photos of HBMN-containing HepG2 cells metabolism solution. Inset: calibration curve of the absor-bance ratio at 450 nm and 370 nm (A450/A370) versus the concentration of TCDD. (c)Confocal fluorescence images of HepG2 cells and A549 cells using HBMN as the fluo-rescent probe before and after exposed to TCDD. Scale bar: 20 mm.


the calibration curve for CYP1A2 had two segments (the concen-tration range of 10e200 nM with the slope of 0.08 and the con-centration range of 200e500 nMwith the slope of 0.23), the slopesof both of which, were higher than that for CYP1A1 (the slope ofcalibration curve was 0.04), especially in the high concentrationregion (up to ca. 6-fold). It clearly demonstrated that HBMN hadstronger affinity with CYP1A2 than that with CYP1A1, which issupposed to greatly boost the biosensing sensitivity of CYP1A2-dominated cells. Moreover, for obtaining deeper insight into themetabolism rate of HBMN by CYP1A1 and CYP1A2, the kinetic pa-rameters of enzymes catalytic reaction, including the apparentMichaelis constant (Km

app), catalytic rate constant (kcat) and the ratioof kcat/Km

app were extracted by using Michaelis�Menten model(Fig. S5). The Km

app, kcat and kcat/Kmapp were summarized in Table S1.

Among them, kcat/Kmapp represents the apparent second-order rate

constant for the association of substrate and free enzyme to formproduct and free enzyme [36]. It was found that kcat/Km

app value ofCYP1A2 (3.41� 106M�1min�1) was near 6 times higher than thatof CYP1A1 (5.70� 105M�1min�1), indicating that CYP1A2 showedhigher reactivity to HBMN de-methylation reaction. Therefore,HBMN probe was a promising candidate for in vitro bioassay ofTCDD with high sensitivity to CYP1A2 expression.

3.3. Detection performance of in vitro bioassay for TCDD

To explore the potential of HBMN for in vitro bioassay of TCDD inbiologic system, the standard TCDD treated HepG2 cells wereselected in the first set of experiments. For enhancing the cellpermeability, HBMN was esterified, which would be recovered byesterase in the cytomembrane [21,37]. Of note, HBMNwith/withoutesterification had negligible toxicity toward HepG2 cells at a con-centration of 30 mM for 48 h (Fig. S6). In the presence of TCDD, theadditional emission peak of cell metabolism solution presentingTCDD increased remarkably at ca. 550 nm, while the emission peakof original HBMN decreased at ca. 460 nm (Fig. S7). It indicated thatTCDD induced the abnormal higher expression of CYP1A2, whichenhanced the metabolism of HBMN in HepG2 cells. The inducedexpression of cytochrome P450 by different concentration of TCDDreached a plateau in 24 h (Fig. S8). Under this optimized time, thecorrelation between I550/I460 and concentrations of TCDD rangingfrom 0.1 to 140 pgmL�1 was investigated (Figs. S9 and 3a). The limitof detection (LOD, S/N¼ 3, S is the detection signal and N is theblank signal) for TCDD was 0.05 pgmL�1 that was already 10 timeslower than the-state-of-art one by using the 7-ER as the fluorescentprobe in the same HepG2 cells [15,38]. Control experimentsshowed it was because that compared to 7-ER, HBMN had higheraffinity (ca. 60-fold) toward CYP1A2 than CYP1A1 (see Fig. S10 andmore discussion). Therefore, the fluorescent HBMN probe wasmuch superior to the traditional 7-ER for sensing TCDD in HepG2cells.

In addition, the UVevis spectra of HBMN-containing cell meta-bolism solution showed that the absorbance gradually decreased at370 nm but increased at 450 nm with the increase of TCDD con-centration (Fig. 2b). The ratio of the absorption peak at 450 and370 nm (A450/A370) was found to be proportional to the TCDDconcentration. In these sense, HBMN could also be used for therapid preliminary screening of TCDD concentration in realisticsamples by naked eye.

3.4. Expression of CYP1A in different human cells exposed to TCDD

The induced expression of cytochrome P450 by TCDD in HepG2cells were further investigated by fluorescence imaging (Fig. 3c).After incubation of HepG2 cells with esterified HBMN for 12 h, theconfocal microscope images displayed obvious blue light and



relatively weak green light, corresponding to the fluorescence ofsubstrate HBMN at ca. 460 nm and that of metabolic product ofHBMN (i.e., HBMN-h) at 550 nm. The activity of abnormal expres-sion of CYP1A in HepG2 cells could be measured by monitoring thegreen light intensity. After incubation with TCDD for 24 h, HepG2cells showed the similar blue light, but the green light was brighterthan that before TCDD pretreating. It was because that TCDDinduced the expression of CYP1A, especially for CYP1A2, whichwould enhance the metabolism of HBMN into HBMN-h. As anothertype of biological system, A549 cells were incubated with esterifiedHBMN at the same condition. The confocal microscope imagesdisplayed the near blue light intensity with HepG2 cells, but thegreen light intensity was relatively weak. It indicted that moreCYP1A2was distributed in HepG2 cells than that in A549 cells. AfterTCDD inducing, the blue light and green light intensity seemssimilar, suggesting TCDD induced weakly the expression of CYP1Ain A549 cells. The above results demonstrated that the distributionand the induction expression of CYP1A varied significantly indifferent cells. Therefore, the in vitro bioassays based on TCDDinducing the expression of CYP1A in different tissues and cells,would not only improve the detection precision but also coulddeepen the understanding of the pathogenesis of TCDD in differentorgans. Besides, it should be noted that 16 fluorescent probes bychanging N-substitutes of 4-methoxy-1,8-naphthalimide had been


L. Shangguan et al. / Analytica Chimica Acta xxx (2018) 1e6 5

developed in previous work [21], but only N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide (NCMN) was comprehensively inves-tigated, which displayed good reactivity and high selectivity towardcytochrome P450 1 A enzyme (CYP1A). In this study, we demon-strated their new applications in highly sensitive detection of2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD).

4. Conclusion

In summary, we report a promising bioassay for TCDD usingHBMN as the fluorescent probe based on the differentiatedabnormal expression of CYP1A in different human cells whenexposing to TCDD. HBMN had a large stroke-shift, good stability,low toxicity, high specificity and selectivity to CYP1A2. As a results,HBMN demonstrated ca. 60 times higher affinity to CYP1A2 thanthe-state-of-art 7-ER probe, leading to a 10 times improved sensi-tivity for sensing TCDD in HepG2 cells compared to previous re-ports. Besides, the color change of HBMN before and aftermetabolism could also be used for rapid screening of TCDD con-centration by naked eye. This workwould provide new thoughts forTCDD detection and this new method presented great potential inthe study of different human cells toxicity.

Acknowledgments

We gratefully appreciate the support from National NaturalScience Foundation of China. (21375014, 21635004, 21627806, and21775018).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.aca.2018.08.006.

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analytica chimica actaprobe 7-ethoxyresoruﬁn (7-er) under the same procedures. moreover, based on...

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