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Title page

Changes in Expression of Drug-Metabolizing Enzymes by Single-walled Carbon nanotubes in

Human Respiratory Tract Cells

Kotaro Hitoshi, Miki Katoh, Tomoko Suzuki, Yoshinori Ando, and Masayuki Nadai

Pharmaceutics, Faculty of Pharmacy, Meijo University; 150 Yagotoyama, Tenpaku-ku, Nagoya

468–8503, Japan (K. H., M. K., M. N.)

Department of Materials Science and Engineering, Faculty of Science and Technology, Meijo

University; 1-501 Shiogamaguchi, Tenpaku-ku, Nagoya 468-8502, Japan (T. S., Y. A.)

DMD Fast Forward. Published on December 20, 2011 as doi:10.1124/dmd.111.043455

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 20, 2011 as DOI: 10.1124/dmd.111.043455

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Running title page

a) Running title: Down-regulation of CYP1A1 and CYP1B1 by SWCNTs

b) Correspondence author: Miki Katoh, Ph. D., Pharmaceutics, Faculty of Pharmacy, Meijo

University; 150 Yagotoyama, Tenpaku-ku, Nagoya 468–8503, Japan. E-mail: [email protected],

Tel: +81-52-832-1151, Fax: +81-52-834-8090

c)

Number of text pages: 28

Number of tables: 3

Number of figures: 9

Number of references: 40

Number of words in Abstract: 221

Number of words in Introduction: 501

Number of words in Discussion: 1434

d) Nonstandard abbreviations

SWCNT, single walled carbon nanotube; NHBE, normal human bronchial epithelial; DMSO,

dimethyl sulfoxide; TCDD, tetrachlorodibenzo-p-dioxin; ChIP, chromatin immunoprecipitation


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Abstract

Single-walled carbon nanotubes (SWCNTs) have attracted attention for biomedical and

biotechnological applications, such as drug delivery. However, there are concerns about the safety of

SWCNTs for use in humans. To investigate the potential use of SWCNTs for targeted drug delivery to

the lung, we examined their effect on drug-metabolizing enzymes in primary normal human bronchial

epithelial (NHBE) cells from 2 donors and the lung carcinoma A549 cell line. Exposure of NHBE and

A549 cells to SWCNTs dysregulated some of the important drug-metabolizing enzymes expressed in

the human respiratory organs. Exposure of NHBE cells to SWCNTs for 24 h had a pronounced effect

on expression of CYP1A1 and CYP1B1 mRNAs, which were reduced to less than 1% of control cells.

These effects were also observed in A549 cells. Exposure of A549, HepG2 hepatic carcinoma cells,

and MCF-7 breast carcinoma cells to tetrachlorodibenzo-p-dioxin induced the expression and

enzymatic activity of CYP1A1 and CYP1B1, which were also suppressed by SWCNTs, suggesting

that SWCNTs downregulated both basal and induced CYP1A1 and CYP1B1 activities. Chromatin

immunoprecipitation assays revealed that the downregulatory effect of SWCNTs may be due to

inhibition of activated aryl hydrocarbon receptor binding to the enhancer regions of the CYP1A1 and

CYP1B1 genes. Downregulation of CYP1A1 and CYP1B1 genes by SWCNTs may affect the defense

mechanisms by reducing procarcinogen bio-activation in the human lung.


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Introduction

Nanometer-length materials, or nanomaterials, exhibit extraordinary physical and chemical

properties, which has motivated interest in their biomedical and biotechnological applications, such as

for drug delivery, gene delivery, and disease diagnosis (Lowe, 2000).

Carbon nanotubes, discovered by Iijima in 1991, are cylindrical molecules consisting of

hexagonally arranged carbon atoms (graphene sheet). They are hollow cylinders formed by rolling

single (for single-walled carbon nanotubes; SWCNTs) or multiple layers of graphene sheets. The

diameters of SWCNTs are between 0.4 and 2 nm and their lengths can reach several micrometers. The

distinct structural properties of SWCNTs allow molecules such as antibodies or drugs to be loaded

along the length of the nanotube sidewall (Liu et al., 2007).

Despite the potential advantages of SWCNTs as drug delivery carriers, there are many reports of

their negative side effects such as cytotoxicity towards cultured cells, inhalation toxicity in animals,

and effects on expression of genes involved in stress-responses and apoptosis (Alazzam et al., 2010;

Cui et al., 2005). These deleterious effects vary with the manufacturing method and the degree of

purification to remove residual catalytic metals, which may also be toxicants. We have previously

investigated the cytotoxic effect of the SWCNTs used in the present study on pulmonary carcinoma

cell lines. The results of that study showed that SWCNT exposure did not induce apoptosis or

oxidative stress at 1.0 mg/ml, the maximum concentration dispersible in cell culture medium (Hitoshi

et al., 2011).

In assessing the capacity of SWCNTs to serve as drug delivery carriers, we focused on

drug-metabolizing enzymes expressed in the human respiratory organs. Drug-metabolizing enzymes

play a central role in the metabolism, detoxification, and elimination of xenobiotics, including drugs

(Xu et al., 2005). The bronchi and lungs contains phase I enzymes such as cytochrome P450 (P450),

cyclooxygenases, and flavin-dependent monooxygenases, as well as phase II conjugating enzymes

such as glutathione S-transferases (GSTs) and UDP-glucuronyltransferases. Phase I enzymes in the

respiratory organs are mainly involved in activation of carcinogens, while phase II enzymes are

involved in their detoxification; thus, all of these enzymes play a role in early defense against


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pulmonary toxicity (Hukkanen et al., 2002). Targeted delivery of drugs for the treatment of lung

metastases and non-small cell lung cancer has been described (Liu et al., 2011). In addition, there is

an interesting report that intravenously injected non-functionalized SWCNTs lodged in lung tissue as

large aggregates, although polyethylene-functionalized SWCNTs did not accumulate in the lung

(Bhirde et al., 2010). Although functionalized CNTs are the mainly used for application as drug

delivery to improve dispersion and solubilization (Klumpp et al., 2006), the present study was

conducted with non-functionalized SWCNTs to clarify direct effect of SWCNTs.

The purpose of the present study was to clarify the effect of SWCNTs on drug-metabolizing

enzymes in primary normal human bronchial epithelial (NHBE) cells and A549 cells derived from

lung carcinoma. We found that CYP1A1 and CYP1B1 were the genes most affected by SWCNT

exposure, and we further examined the mechanism underlying their downregulation in basal and

induced status of these genes.


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Materials and methods

Nanomaterials

The SWCNTs used in the present study were provided by Meijo Nano Carbon Co. Ltd (Nagoya,

Japan). SWCNTs were synthesized by the arc electrical discharge method, using nickel and yttrium as

catalysts (Ando et al., 2000). The physicochemical properties of SWCNTs are given in Table 1. The

diameters were measured by Raman spectrometry (HoloSpec, Kaiser Optical Systems, Ann Arbor,

MI), and the lengths were determined using a transmission electron microscope (TEM) (H-7000,

Hitachi, Tokyo, Japan). SWCNT purity was determined by thermogravimetric-differential thermal

analysis (DTG-60, Shimadzu, Kyoto, Japan). The residual metal catalyst content was determined with

a scanning electron microscope (ABT-150F, Topcon, Tokyo, Japan) equipped with an energy

dispersive X-ray analysis system (EMAX-5770W, HORIBA, Kyoto, Japan). SWCNTs were dispersed

in cell culture medium, supplemented with 10% Fetal bovine serum (FBS), at a concentration of 0.1

mg/ml using an ultrasonic homogenizer (VC-505, Sonics & Materials, Newtown, CT). A probe tip

with a diameter of 13 mm was placed into 100 ml of the SWCNT dispersion, which was then

sonicated at 40% amplitude (200 W) for 10 min.

Chemicals

FBS and Minimum Essential Medium were purchased from Invitrogen (Carlsbad, CA). Dulbecco’s

modified Eagle medium was purchased from Nissui Pharmaceutical (Tokyo, Japan). The RNeasy Plus

Mini Kit, RT2 First Strand Kit, and RT2 Profiler PCR array (Human Drug Metabolism) were obtained

from Qiagen (Hilden, Germany). The FastPure RNA Kit and SYBR Premix Ex Taq II were obtained

from Takara Bio (Shiga, Japan), and the ReverTra Ace qPCR RT Kit was from Toyobo (Osaka, Japan).

Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from AccuStandard (New Haven, CT). Anti-aryl

hydrocarbon receptor (AhR) antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz,

CA).

Cell culture

Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Basel, Switzerland).

The cells were isolated from 2 healthy male donors, both 43 years of age (referred to as donors 1 and


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2), and were cultured in bronchial epithelial growth medium (Lonza), as recommended by the supplier.

Human lung carcinoma A549 cells (DS Pharma Biomedical, Osaka, Japan) were cultured in

Dulbecco’s modified Eagle medium supplemented with 10% FBS. The human hepatic carcinoma cell

line HepG2 and the human breast carcinoma cell line MCF-7 (American Type Culture Collection,

Rockville, MD), were cultured in Minimum Essential Medium supplemented with 10% FBS. All cells

were cultured at 37°C in a humidified 5% CO2 atmosphere.

SWCNT internalization into NHBE cells

Where present, the SWCNT concentration for all the experiments in the present study was 0.1

mg/ml, which we previously determined to have no effect on cell viability. NHBE cells (donor 2)

were harvested to 12-well culture plate. After 24-hr exposure to 0.1 mg/ml SWCNT, the cells were

pre-fixed with 2% glutaraldehyde in 30 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid

buffer, post-fixed using 2% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in

epoxy resin. Ultrathin (60−80 nm) sections were cut with a LEICA-UCT ultramicrotome, post-stained

with 2% uranyl acetate and lead citrate, and viewed using a JOEL JEM-2000 EX electron microscope.

NHBE cell viability after exposure to SWCNTs

The viability of NHBE cells (donor 2) was measured after 24 h exposure to SWCNTs by evaluating

their metabolic capacity using the CellTiter-Blue viability assay (Promega, Madison, WI), as

described previously (Hitoshi et al., 2011).

NHBE RNA extraction

NHBE cells were exposed to SWCNTs at 0.1 mg/ml for 24 h. Cell culture medium without

SWCNT was used as the untreated control. Three independent experiments were performed for each

concentration. Total RNA was extracted from the cells by using the RNeasy Plus Mini Kit

immediately after the 24-h exposure. Cells from 3 wells were pooled before RNA extraction.

PCR array analysis for NHBE cells

One microgram of total RNA isolated from NHBE cells (donor 1) was used for synthesis of cDNA

using the RT2 First Strand Kit. Gene analysis was carried out using The Human Drug Metabolism

RT2 Profiler PCR array with an ABI 7500 instrument (Applied Biosystems, Foster City, CA),


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according to the manufacturer’s instructions. Eighty-four genes involved in drug metabolism were

analyzed. The fold change in expression of a target gene between the untreated control group and the

SWCNT-exposed group, normalized to the level of glyceraldehyde-3-phosphate dehydrogenase

(GAPDH), was determined by the comparative Ct method using the following equation: Fold change

= 2-∆(∆Ct), where ∆Ct = Ct (target gene) – Ct (GAPDH), and ∆(∆Ct) = ∆Ct (SWCNT exposed) – ∆Ct

(untreated control).

Real-time PCR analysis for NHBE and A549 cells

One microgram of total RNA was used for the reverse transcription reactions with the ReverTra

Ace qPCR RT Kit. Eleven genes of interest identified in the PCR array were selected for analysis by

real-time PCR, which was carried out with the Thermal Cycler Dice (Takara Bio) using SYBR Premix

Ex Taq II. Specific primers used in the present study are described in table 2. PCR amplification

conditions were an initial 30-s denaturation step at 95°C followed by 40 cycles of denaturation at

95°C for 5 s, and annealing and extension steps as described in Table 2. The relative expression level

of each gene was calculated using the standard curve method. All data were derived from 3

independent measurements and were normalized to the expression level of GAPDH.

Dose-responsive effect of SWCNTs on CYP1A1 and CYP1B1 mRNA expression in NHBE and

A549 cells

Total RNAs were extracted from cells incubated for 24 h with medium alone or 0.00001, 0.0001,

0.001, 0.01, and 0.1 mg/ml SWCNTs for NHBE cells (donor 2), and 0.001, 0.005, 0.01, 0.05, and 0.1

mg/ml SWCNTs for A549 cells. The reverse transcription and real-time PCR was performed

according to the methods described above. IC50 value was calculated using KaleidaGraph (HULINKS,

Tokyo, Japan)

Effect of SWCNTs on basal CYP1A1, CYP1B1, and AhR mRNAs in A549, HepG2, and MCF-7

cell lines

Total RNAs were extracted from cells incubated for 24 h with medium alone or with SWCNTs. The

reverse transcription and real-time PCR was performed according to the methods described above.

Since Beedanagari et al. (2009) has reported that CYP1B1 expression was undetectable in HepG2

cells, in the present study only CYP1A1 mRNA was investigated in HepG2 cells.


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Effect of SWCNTs on TCDD-induced CYP1A1 and CYP1B1 in A549, HepG2, and MCF-7 cell

lines

RNA was extracted from cells after incubation for 24 h with 0.1% dimethyl sulfoxide (DMSO;

TCDD vehicle), 10 nM TCDD, or SWCNT + 10 nM TCDD. The reverse transcription and real-time

PCR was performed according to the methods described above.

Enzymatic activity

For analysis of P450 enzymatic activity, HepG2 and MCF-7 cells were allowed to attach to culture

wells for 24 h, and were then exposed to 0.1% DMSO (control), 10 nM TCDD, or 10 nM TCDD +

SWCNT for a further 24 h. After 24-h exposure, CYP1A1 and CYP1B1 activity was measured

according to the protocol of the P450-Glo CYP1A1 Assay (Promega). The substrate provided in this

assay is metabolized by both CYP1A1 and CYP1B1 to generate luciferin, and the assay therefore

reflects the combined activities of the 2 isoforms.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed to assess specific binding of AhR to

the CYP1A1 and CYP1B1 enhancers in HepG2 and MCF-7 cells. Assays were performed according

to the protocol of ChIP-IT Express (Active Motif, Carlsbad, CA). MCF-7 and HepG2 cells were

treated with 0.1% DMSO (control), 10 nM TCDD, or 10 nM TCDD + SWCNT for 24 h.

Immunoprecipitations were performed with anti-AhR antibodies overnight at 4˚C. The PCR primers

for the CYP1A1 and CYP1B1 enhancers were described previously (Matthews et al., 2005; Okino et

al., 2006).

MicroRNA analysis

MicroRNA (miRNA) was extracted with a Nucleospin miRNA isolation kit (Takara Bio) from

MCF-7 cells after treatment with 0.1% DMSO (control), 10 nM TCDD, or 10 nM TCDD + SWCNT

for 24 h. The expression of RNU38B and hsa-miR-27b was quantified using ABI 7700 (Applied

Biosystems) according to the TaqMan MicroRNA Assay protocol. All data were determined in 3

independent measurements and were normalized to the expression of RNU38B.

Statistical analysis

Statistical analyses were performed by either Student’s unpaired t-test or Bonferroni correction


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following ANOVA (dose-response study) using StatView software (HULINKS).


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Results

Characterization of SWCNTs

The diameter and the length of SWCNTs were 1.36−1.42 nm and 1−5 µm, respectively, provided as

a referential value from the manufacturer. Purity of the SWCNT was over 90% with metal catalyst

residue of nickel (0.56 atom %) and yttrium (0.16 atom %) (Table 1). Rest of the component is

amorphous carbon that does not form tubular structure. The dispersion status of 1.0 mg/ml SWCNT

has been characterized by TEM and light microscope in our previous report (Hitoshi et al., 2011).

SWCNTs could be dispersed in culture medium, supplemented with 10 % FBS, since protein

improves its dispersibility and stability.

SWCNT internalization into NHBE cells

Electron microscopy revealed dense black aggregated material in NHBE cells (donor 2) that had

been exposed to SWCNTs, but not in untreated cells (Fig. 1). The aggregate seemed to be localized in

pinocytotic vesicles (representative section indicated by the circle). We presume this material is

bundled SWCNTs. In addition, no black aggregates were detected in the nucleus of the cell and the

cell morphology was unaffected by exposure to SWCNTs. Exposure to 0.1 mg/ml SWCNTs for 24 h

had no effect on NHBE cell viability.

Changes in expression of drug metabolizing genes in NHBE and A549 cells after SWCNT

exposure

PCR array analysis was performed to assess the expression of drug-metabolizing enzymes in

NHBE cells (donor 1) after a 24-h exposure to SWCNTs. The results are shown in Fig. 2. Exposure to

SWCNTs upregulated 4 genes by greater than 2-fold and 15 were downregulated to less than 50% of

the levels in untreated control cells (Table 3). The effects of SWCNTs on the remaining genes are

shown in supplemental Table 1. Among the 84 genes, 13 genes were below the limit of detection.

Eight genes were selected from the PCR array results for further validation using real-time PCR.

Three of the selected genes were upregulated by SWCNT exposure: aldehyde dehydrogenase 1 family,

member A1 (ALDH1A1) (NM_000689), GSTA4 (NM_001512), and hydroxysteroid (17-beta)


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dehydrogenase 3 (HSD17B3) (NM_000197), and 5 genes were downregulated: CYP1A1

(NM_000499), CYP19A1 (NM_000103), carboxylesterase 2 (CES2) (NM_198061), glutathione

reductase (GSR) ( NM_000637), and GSTM3 (NM_000849). The results of the real-time PCR

analysis in NHBE cells (donor 1) were in good agreement with the results of the PCR array, with the

exception that GSTM3 was slightly upregulated in the real-time PCR analysis but was downregulated

in the PCR array (Table 3 and Fig. 3). In addition to these 8 genes, the expression levels of CYP1B1

(NM_000104), CYP2S1 (NM_030622), and AhR (NM_001621) were analyzed by real-time PCR.

CYP1B1 and CYP2S1 were significantly downregulated after exposure of NHBE cells to SWCNTs

and AhR was slightly upregulated. Real-time PCR analysis of these 11 genes was also performed with

NHBE (donor 2) and A549 cells. For both cell types, most of the selected genes were downregulated

after SWCNT exposure and none were significantly upregulated. SWCNT-induced downregulation of

CYP1A1, CYP1B1, CYP2S1, and CYP19A1 expression was consistent in all 3 cell types. Moreover,

the degree of CYP1A1 and CYP1B1 downregulation (to less than 1% of control cells) was consistent

in both NHBEs (donor 1 and donor 2). The degree of CYP1A1 and CYP1B1 downregulation in A549

cells was moderate compared with NHBE cells, but these isoforms were still the most affected by

SWCNTs among the selected genes.

Dose-responsive effect of SWCNTs on CYP1A1 and CYP1B1 mRNA expression in NHBE and

A549 cells

Significant dose-responsive effect of SWCNTs on CYP1A1 and CYP1B1 mRNA expression was

observed in NHBE and A549 cells at concentrations lower than 0.1 mg/ml (Fig. 4). IC50 value of

SWCNTs on expression level of CYP1A1 was 0.0024 and 0.033 mg/ml for NHBE and A549 cells,

respectively and that on CYP1B1 was 0.0025 and 0.16 mg/ml, respectively. No-observed-adverse-

effect-level on CYP1A1 and CYP1B1 mRNA expression was 0.00001 mg/ml (10 ng/ml) for NHBE

cells, based on ANOVA and Bonferroni’s test.

Effect of SWCNTs on basal and TCDD-induced CYP1A1 and CYP1B1 in A549, HepG2, and

MCF-7 cells

SWCNT exposure reduced basal CYP1A1 mRNA levels in A549, HepG2, and MCF-7 cells to 23%,

19%, and 6% of the levels in untreated control cells, respectively. In A549 and MCF-7 cells, basal


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CYP1B1 mRNA levels were reduced to 30% and 35%, respectively. In contrast, SWCNTs had little

effect on AhR expression in the 3 cell lines (Fig. 5).

Expression of CYP1A1 and CYP1B1 mRNA was quantified in TCDD-treated A549, HepG2, and

MCF-7 cells (Fig. 6). Addition of SWCNTs reduced the CYP1A1 mRNA levels to 3%, 8%, and 1%

of the TCDD-induced levels in A549, HepG2, and MCF-7 cells, respectively. TCDD-induced

CYP1B1 mRNA levels were also reduced by the addition of SWCNTs to 24% and 5% in A549 and

MCF-7 cells, respectively. Overall, the suppressive effect of SWCNTs on the TCDD-induced

expression of CYP1A1 and CYP1B1 was greater than on basal expression.

Enzymatic activity

The effects of SWCNTs and TCDD on the combined enzymatic activity of CYP1A1 and CYP1B1

were determined in living cells using P450-Glo analysis (Fig. 7). TCDD treatment induced the

combined enzymatic activity in HepG2 and MCF-7 cells and this was reduced to 24% and 1% of

induced levels, respectively, by the addition of SWCNTs. The enzyme activity in A549 cells was

below the limit of quantification, even after induction with TCDD.

Chromatin immunoprecipitation assay

The ChIP assay was used to examine the effects of SWCNTs and TCDD on recruitment of AhR to

the enhancer region of CYP1A1 in HepG2 cells, or to those of CYP1A1 and CYP1B1 in MCF-7 cells

(Fig. 8). Recruitment of AhR to the enhancer region of CYP1A1 was reduced to 37% by exposure of

SWCNTs. Likewise, the TCDD-induced recruitment of AhR to the enhancer regions of CYP1A1 and

CYP1B1 in MCF-7 cells were reduced to 30% and 15%, respectively. However, recruitment of AhR

to the enhancer region of CYP1A1 and CYP1B1 could not be detected in A549 cells, even after

treatment with TCDD.

MicroRNA analysis

SWCNT exposure had no significant effect on hsa-miR-27b expression in MCF-7 treated with

TCDD (Fig. 9).


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Discussion

The concentration of SWCNTs used in the present study was based on our previous report in which

0.1 mg/ml SWCNT had no significant effect on A549 cell viability, as assessed by metabolic capacity

and intracellular ATP content (Hitoshi et al., 2011). Here, we confirmed that SWCNT exposure also

had no effect on the viability of NHBE cells. Lower concentrations of SWCNTs have been reported to

stimulate a stress response in various cell types. For example, Sarkar et al. (2007) reported increased

expression of stress-responsive genes measured by PCR array after exposure of BJ foreskin cells to

0.06 mg/ml SWCNT for 24 h. Alazzam et al. (2010) investigated the effect on NHBE cells of a 48-h

incubation with 0.1 mg/ml SWCNT, which are the same concentration and cell type as used in the

present study. Using DNA microarray analysis, they observed dysregulation of genes involved in the

cell cycle, apoptosis, cell survival, and cell adhesion. However, NHBE cell morphology and viability

were not significantly affected by SWCNTs in the present study. In TEM analysis, visible larger

bundles of SWCNT dispersion was located only in the pinocytotic vesicles of the cells, suggesting

that these are likely to be engulfed into the cell, not penetrated the cell membrane. There are also

reports that SWCNT did not reach inside the cell (Davoren et al., 2007). However, since Porter et al.

(2007) have reported that different non-functionalized SWCNTs could be observed in lysosomes,

cytoplasm, and nucleus, using TEM and confocal microscopy. The localization of SWCNT in cells

still remains controversial and further study is needed for clarification.

Concerning P450s that are reported to be expressed in human bronchial tissue (Leclerc et al., 2010),

mRNA expression of 4 P450 isoforms, CYP1A1, CYP1B1, CYP2S1, and CYP19A1 were

significantly downregulated by SWCNTs in A549 cells, and in NHBE cells from both donors.

CYP1A1 is by far the most actively studied human pulmonary P450s due to its importance in the

metabolism of polycyclic aromatic hydrocarbons (PAH), and CYP1B1 also plays a role in activation

of PAHs (Shimada et al., 1996). In human lung tissue, expression of CYP1A1 mRNA and protein was

shown to correlate positively with the aromatic/hydrophobic DNA adduct levels (Cheng et al., 2000).

Therefore, the SWCNT-induced decreases in CYP1A1 and CYP1B1 mRNA expression observed here

may affect detoxification of xenobiotics such as PAH. CYP2S1 is also expressed in epithelial tissues

frequently exposed to xenobiotics, such as the respiratory and gastrointestinal tracts, and its


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expression pattern resembles that reported for CYP1B1. CYP2S1 exhibits features typical of the

CYP1 family, such as TCDD inducibility. Although the biological function of CYP2S1 is not yet fully

understood, it has been suggested to play a role in the metabolism of carcinogens (Saarikoski et al.,

2005). Therefore, we surmise that the SWCNT-induced downregulation of CYP2S1 expression

observed here may have similar significance to CYP1A1 and CYP1B1 downregulation. CYP19A1

expression was also downregulated by SWCNTs in the cell lines studied here. CYP19A1 is an

aromatase responsible for the final step in the biosynthesis of the estrogens, estradiol and estrone

(Chen et al., 2009). Dysregulation of CYP19A1 gene expression not only disrupts the balance

between estrogens and androgens, but also alters many other biochemical processes in both males and

females (Li, 2007). The observed downregulation of CYP19A1 by SWCNTs might therefore have

implications for these pathways, although further study is necessary. In contrast to CYP1A1, CYP1B1,

CYP2S1, and CYP19A1, there was no effect of SWCNTs on the expression of CYP2E1, CYP2J2, and

CYP3A5 mRNA in NHBE cells (donor 1) (supplemental Table 1), and CYP2B6 and CYP2F1 mRNA

were not detected in the PCR array analysis.

Effect of SWCNT on drug-metabolizing enzymes in NHBE cells was greater than in A549 cells,

indicating that normal cells may be more susceptible to SWCNT exposure. IC50 value of SWCNTs on

expression level of CYP1A1 was 13 times lower in NHBE cells (0.0024 and 0.033 mg/ml) that on

CYP1B1 was 64 times lower in NHBE cells (0.0025 and 0.16 mg/ml), respectively. In addition,

no-observed-adverse-effect-level on CYP1A1 and CYP1B1 mRNA expression was determined as

0.00001 mg/ml (10 ng/ml) for NHBE cells. Pacurari et al. (2008) have reported that the effect of

SWCNTs was greater in normal human mesothelial cells than in malignant mesothelial cells,

concerning genes involved in molecular signaling. However, the downregulatory effect on the P450

isoforms specifically was comparable between NHBE and A549 cells, which justifies the use of A549

cells for investigation of the mechanism of SWCNT action.

We further analyzed the mechanism of CYP1A1 and CYP1B1 downregulation because these genes

were most affected by SWCNT exposure. Since AhR is reported to be involved in the constitutive

expression of CYP1A1 (Zhang and Walker, 2007), the effect of SWCNTs on expression of AhR

mRNA was investigated. However, the effect of SWCNTs on basal AhR expression was negligible in


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NHBE and A549 cells, indicating that the downregulatory effect of SWCNTs on CYP1A1 mRNA was

not due to the suppression of AhR expression.

TCDD is the most potent ligand of the AhR receptor, and in the present study it was used to

characterize the effect of SWCNTs on the TCDD-induced expression of CYP1A1 and CYP1B1 using

A549, HepG2, and MCF-7 cells. HepG2 and MCF-7 cells were studied because they have been

extensively used in studies of CYP1A1 and CYP1B1 induction, and have well-characterized AhR

pathways. As previously reported, the AhR-mediated induction of CYP1A1 and CYP1B1 was greater

in HepG2 and MCF-7 cells than in A549 cells (Iwanari et al., 2002). Addition of SWCNTs

downregulated the TCDD-induced CYP1A1 and CYP1B1 mRNA expression in all 3 carcinoma cell

lines. The combined enzymatic activity of CYP1A1 and CYP1B1 was also reduced by SWCNTs in

HepG2 and MCF-7 cells, as measured using P450-Glo analysis (Fig. 7). Since HepG2 cells do not

express CYP1B1 mRNA (Beedanagari et al., 2009), the observed enzymatic activity in HepG2 cells is

presumably due to CYP1A1. The effect of SWCNTs on CYP1A1 and CYP1B1 mRNA and enzymatic

activity was more potent in TCDD-induced cells, indicating that an AhR-modulated pathway is

involved in the effect of SWCNTs on these genes.

Resveratrol, a phenolic phytoalexin, downregulates both constitutive and PAH-induced CYP1A1

expression, and thus has a chemopreventive effect by inhibiting procarcinogen activation (Mollerup et

al., 2001). Resveratrol has been reported to mediate this effect by preventing binding of activated

nuclear AhR to the xenobiotic responsive element of CYP1A1 (Beedanagari et al., 2009). Using ChIP

assays, SWCNTs repressed AhR binding to the CYP1A1 enhancer region in HepG2 cells, and to both

CYP1A1 and CYP1B1 enhancer regions in MCF-7 cells as in the case with resveratrol (Fig. 8). These

results indicate that inhibition of binding of activated AhR to the enhancer regions of these genes is at

least one of the mechanisms in downregulation of CYP1A1 and CYP1B1 mRNA and enzymatic

activity. It is notable that SWCNTs were not detected in the nucleus of NHBE cells (Fig. 1),

suggesting they were unlikely to directly compete with AhR binding to DNA.

A significant inverse relationship has been reported between the expression levels of miR-27b and

CYP1B1 protein (Tsuchiya et al., 2006), prompting us to investigate the effect of SWCNTs on this

microRNA. However, miR-27b expression was not changed by exposure to SWCNTs, indicating that


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the observed downregulation of CYP1B1 activity was not due to post-transcriptional regulation by

miR-27b (Fig. 9).

The phase II enzymes GSTA4 and GSTM3 were significantly upregulated in donor 1 NHBE cells

but not in NHBE cells derived from donor 2. The induction of phase II enzymes results in the

detoxification of carcinogens, leading to the protective effect of chemopreventive agents (Guidice and

Montella, 2006). Given the different effects of SWCNTs on phase II enzyme induction in our 2 NHBE

cell populations, it may be useful to investigate this further in cells derived from a larger group of

donors.

In conclusion, we clarified that exposure of primary cultured NHBE and A549 cells to SWCNTs

dysregulated some important drug-metabolizing enzymes expressed in human respiratory organs.

Exposure of SWCNTs downregulated the mRNA expression and enzymatic activity of CYP1A1 and

CYP1B1 in HepG2 and MCF-7 cells, as a consequence of preventing the binding of activated AhR to

the enhancer region of these genes. Downregulation of CYP1A1 and CYP1B1 genes by SWCNTs

may affect the defense mechanisms by reducing procarcinogen bio-activation in the human lung, and

additionally may alter the metabolism of drugs delivered to the respiratory organ. The present study

has provided some basic information on the effect of SWCNTs on drug-metabolizing enzymes in

respiratory organs. Clearly, the results support examination of the effects of SWCNTs on

drug-metabolizing enzymes at the target organ before their clinical application as drug carriers.


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Authorship contributions

Participation in research design: Hitoshi, Katoh, and Nadai

Conducted experiments: Hitoshi and Katoh

Contributed new reagents or analytic tools: Suzuki and Ando

Performed data analysis: Hitoshi and Katoh

Wrote or contributed to the writing of the manuscript: Hitoshi, Katoh, and Nadai


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Footnotes

This work was supported by the Grant-in-Aid for Young Scientists (B)[Grant 21790167] and the

Grant-in-Aid for Scientific Research (C) [Grant 21590183] from Japan Society for the Promotion of

Science.

Reprint request to Miki Katoh, Ph. D., Pharmaceutics, Faculty of Pharmacy, Meijo University; 150

Yagotoyama, Tenpaku-ku, Nagoya 468–8503, Japan. E-mail: [email protected]


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Figure legends

Fig. 1. TEM micrograph of NHBE cells after exposure to 0.1 mg/ml SWCNTs for 24 h. (A)

untreated, (B) SWCNT exposed cells. Representative pinocytotic vesicles are indicated by the circle.

Fig. 2. PCR array analysis of 84 drug-metabolizing enzymes after 24-h exposure of NHBE cells to

SWCNTs. Closed circles represent genes that were upregulated (●) or downregulated (●) by more

than 2-fold compared to untreated control cells.

Fig. 3. Real-time PCR analysis of drug-metabolizing enzymes in NHBE (donor 1) (A), NHBE

(donor 2) (B), and A549 cells (C). The open and filled columns represent untreated control and

SWCNT exposure, respectively. Each column represents mean ± S.D. (n = 3). * p < 0.05 compared to

untreated control cells.

Fig. 4. Dose-responsive effects of SWCNTs on CYP1A1 and CYP1B1 mRNA expression in NHBE

and A549 cells. Expression levels of CYP1A1 (closed symbols) and CYP1B1 (open symbols) are

represented by squares (NHBE cells) and circles (A549 cells), respectively. *, p < 0.05 compared to

untreated control.

Fig. 5. Basal CYP1A1, CYP1B1, and AhR mRNA expression in A549 (A), HepG2 (B), and MCF-7

(C) cells after exposure to SWCNTs for 24 h. Each column represents mean ± S.D. (n = 3). *, p < 0.05

compared to untreated control.

Fig. 6. TCDD-induced CYP1A1 and CYP1B1 mRNA expression in A549 (A), HepG2 (B), and

MCF-7 (C) cells after exposure to SWCNTs for 24 h. Cells were exposed to 10 nM TCDD for

induction of CYP1A1 and CYP1B1. Each column represents mean ± S.D. (n = 3). *, p < 0.05

compared to TCDD-induced cells.


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Fig. 7. CYP1A1 and CYP1B1 enzymatic activity in TCDD-induced HepG2 (A) and MCF-7 (B)

cells after exposure to SWCNTs for 24 h. Cells were exposed to 10 nM TCDD for induction of

CYP1A1 and CYP1B1. Each column represents mean ± S.D. (n = 3). *, p < 0.05 compared to

TCDD-induced cells.

Fig. 8. Effect of SWCNTs on TCDD-induced recruitment of AhR to the enhancer region of

CYP1A1 in HepG2 cells (A) or to the enhancer regions of CYP1A1 and CYP1B1 in MCF-7 cells (B).

Cells were exposed to 10 nM TCDD for induction of CYP1A1 and CYP1B1. The results were

expressed relative to those of the total input controls. Each column represents mean ± S.D. (n = 3). *,

p < 0.05 compared to TCDD-induced cells.

Fig. 9. MiR-27b expression in TCDD-induced MCF-7 cells after exposure to SWCNTs for 24 h.

Cells were exposed to 10 nM TCDD for induction of CYP1B1. Each column represents mean ± S.D.

(n = 3).


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TABLE 1 Physicochemical properties of SWCNTs used in the present study.

Diametera Lengthb SWCNT purityb Metal catalyst residuec (nm) (µm) (%) (atom %)

SO-SWCNT 1.36–1.42 1–5 >90 Nickel: 0.56 ± 0.09 Yttrium: 0.16 ± 0.05 a Range (n = 5). b Referential value provided from the manufacturer. c Mean ± SD (n = 10).


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TABLE 2

Sequences of primers and PCR conditions used in the present study

Gene Primer sequence Annealing temp. (°C)

Annealing time (sec)

Amplicon size (bp)

References

AhR Sense Antisense

5'-ATA CTG AAG CAG AGC TGT GC-3' 5'-AAA GCA GGC GTG CAT TAG AC -3'

60 60 183 Ikuta et al. (2010)

ALDH1A1 Sense Antisense

5'-AGC CTT CAC AGG ATC AAC AGA-3' 5'-GTC GGC ATC AGC TAA CAC AA-3'

60 60 124 Li et al. (2007)

CES2 Sense Antisense

5'-AGT GGT GTG AGG GAT GGA AC-3' 5'-TGG CTA AGA AAC TCT GAC TCC A-3'

60 60 80 Inoue et al. (2006)

CYP1A1 Sense Antisense

5'-TTC GTC CCC TTC ACC ATC-3' 5'-CTG AAT TCC ACC CGT TGC-3'

60 90 302 Wilkening et al. (2003)

CYP1B1 Sense Antisense

5'-GAG AAC GTA CCG GCC ACT ATC-3' 5'-CGG GTT AGG CCA CTT CAG T-3'

60 60 357

CYP2S1 Sense Antisense

5'-ACC CCA ACA TCT TCA AGC AC-3' 5'-TTC ATC TGG TCT GCG TGG T-3'

62 60 312 Thum et al. (2006)

CYP19A1 Sense Antisense

5'-AGG AGG TGA CCA ATG AAT CG-3' 5'-CAC GAT AGC ACT TTC GTC CA-3'

62 60 116 Pal et al. (2008)

GSR Sense Antisense

5'-CAG TGG GAC TCA CGG AAG AT-3' 5'-TTC ACT GCA ACA GCA AAA CC-3'

60 60 219 Schmidt et al. (2009)

GSTA4 Sense Antisense

5'-CCG GAT GGA GTC CGT GAG-3' 5'-CTT CGG GTC TGT ACC AAC TT-3'

60 60 168

GSTM3 Sense Antisense

5'-CCT GGA TGG GAA GAA CAA GA-3' 5'-TTG TGT GCG GAA ATC CAT TA-3'

60 60 142 Zieker et al. (2005)

HSD17B3 Sense Antisense

5'-ACC TTC TCC CAA GCC ATT TC-3' 5'-AAC GCC TTG GAA GCT GAG TA-3'

62 60 202 Kasai et al. (2004)

GAPDH Sense Antisense

5'-CCA GGG CTG CTT TTA ACT C-3' 5'-GCT CCC CCC TGC AAA TGA-3'

60 60 289 Tsuchiya et al. (2004)

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TABLE 3

Changes in the expression of drug-metabolizing enzymes in NHBE cells by PCR array

Gene Fold change Up-regulated ALDH1A1 2.99 GSTA4 2.12 HSD17B3 2.05 Alcohol dehydrogenase 1C (class I), gamma polypeptide 2.01 Down-regulated CYP1A1 0.002 Alcohol dehydrogenase 4 (class II), pi polypeptide 0.11 Amiloride binding protein 1 (amine oxidase (copper-containing)) 0.16 CYP19A1 0.21 Glucokinase (hexokinase 4) regulator 0.27 Pyruvate kinase, liver and RBC 0.31 GSR 0.32 CYP11B2 0.33 GSTM3 0.35 ATP-binding cassette, sub-family C (CFTR/MRP), member 1 0.43 CES2 0.44 Metallothionein 3 0.46 Hexokinase 2 0.47 Hydroxysteroid (17-beta) dehydrogenase 2 0.48 5,10-methylenetetrahydrofolate reductase (NADPH) 0.49


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