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Validation of an in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression
Toxicology in Vitro 24 (2010) 995–1001
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Validation of an in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression H. Maeshima *, K. Ohno, S. Nakano, T. Yamada Food Safety Research Institute, Nissin Foods Holdings Co., Ltd., 2247 Noji-cho, Kusatsu, Shiga 525-0055, Japan
a r t i c l e
i n f o
Article history: Received 28 August 2009 Accepted 11 December 2009 Available online 16 December 2009 Keywords: In vitro screening Molecular marker Transformation assay Tumor promoter Genotoxicity Carcinogen
a b s t r a c t Chemical carcinogenesis is a multifactorial process comprising two main stages: initiation and promotion. Tumor promoters cause the development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The identification of tumor promoters is important for preventing cancer. We previously identified 22 specific gene markers using a global gene expression analysis of chemically induced tumor promotion and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells. Our in vitro tumor promoter screening test, based on these marker genes, enables earlier assessment, and is easier to conduct than classical methods. The general applicability of these markers, however, was unknown. In this study, to evaluate the performance of a set of markers, we independently validated a separate sample set, which had various structures and properties. Independent validation of the signature of 63 test chemicals showed an accuracy, sensitivity, and specificity of the assay of 96.8%, 97.0% and 96.7%, respectively. These results indicate that the tumor promoting activity assay, based on the expression of 22 marker genes, will become a valuable tool for rapid screening of potential tumor promoters. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Chemical carcinogenesis is a multistage, multifactorial process, comprising two main stages: initiation and promotion (Barrett, 1993; Hennings and Boutwell, 1970; Slaga, 1983; Weisburger and Wiliams, 1983). Initiators (genotoxic agents) can be detected by various short-term genotoxicity screening tests (Ames et al., 1973; Clive and Spector, 1975; Oda et al., 1985). However, many carcinogenic chemicals are negative in the genotoxicity assays. The majority of these non-genotoxic carcinogens are assumed to be tumor promoters. Tumor promoters differ from genotoxic carcinogens, and have various biological activities and chemical structures. In vivo rodent models remain the only reliable way for experimental investigation of the tumor promotion activity of chemicals to humans. However, the rodent carcinogenicity assay is expensive, time-consuming, uses large amounts of the compound, and involves ethical issues. While many in vitro tumor promotion assays have been developed as screening methods (Combes et al., 1999; Kakunaga, 1973; Reznikoff et al., 1973), their low
Abbreviations: TPA, 12-O-Tetradecanoylphorbol-13-acetate; MeIQ, 2-amino3,4-dimethylimidazo[4,5-f]quinoline; PhIP, 2-amino-1-methyl-6-phenylimidazo(4,5-B)pyridine. * Corresponding author. Tel.: +81 77 561 9115; fax: +81 77 561 9140. E-mail address: [email protected] (H. Maeshima). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.12.013
throughputs have rendered them impractical as rapid screening tools. In a previously published pilot study, we explored the performance of a microarray in predicting the tumor promoting potential of chemicals in BALB/c 3T3 cells (Maeshima et al., 2009). We investigated whether gene expression analysis could be used to distinguish between a tumor promoter and a non-tumor promoter at 48 h after chemical addition. The results showed that 22 gene markers were differentially expressed between the tumor promoter and the non-tumor promoter. To establish a gene expression score, we counted the number of marker genes that were up-regulated by more than 1.5-fold by the chemical treatment. The gene expression score was compared with the results of a cell transformation assay. There was a correlation between the gene expression score and the results of the cell transformation assay using BALB/c 3T3 cells. These results indicated that gene expression analysis could be used to predict the prognosis of a tumor promoter at an early time point. However, this study was not a sufficient validation of the proposed test modification; an independent evaluation was required to give an unbiased estimate of its general applicability. In the present study, our goal was to evaluate whether an independent study could confirm the applicability of this QRT-PCR assay for the tumor promoters. Independent validation using a separate sample set, that has various structure and properties, is one way to determine the true performance of a set of markers. We describe the results of an independent evaluation of models for
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Table 1 List of chemicals and their respective results in the in vitro two-stage transformation assay using BALB/c 3T3 cells, the gene expression assay, and published data. Chemicals
Dose (lg/mL)
BALB/c 3T3 cell transformation assayc
Twenty-two genes expression assayd
In vivo tumor promoting activities
W N T W W W S F W W W L W W S W O A W W S W W N S W J W N M
79-06-1 9011-18-1 335-67-1 57-97-6 7705-08-0 7758-94-3 302-79-4 16423-68-0 58-08-2 123-91-1 120-80-9 1143-38-0 637-07-0 81-25-4 50892-23-4 103-90-2 223419-20-3 96-09-3 51-03-6 132-27-4 7632-00-0 11077-03-5 110-44-1 65-85-0 50-28-2 521-18-6 7647-14-5
+++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + +
9005-65-6 59-02-9
100.0 30.0 45.0 0.05 100.0 100.0 3.0 100.0 100.0 300.0 0.6 0.1 60.0 100.0 30.0 20.0 30.0 30.0 0.3 10.0 2000.0 0.0001 300.0 300.0 1.0 10.0 3000.0 0.005 100.0 10.0
S W S S W N W N J W S W W S W S J W
446-72-0 79-09-4 7235-40-7 79902-63-9 95-53-4 94-26-8 4247-02-3 62-53-3 71-43-2 99-56-9 120-12-7 77094-11-2 110-86-1 56-53-1 51-79-6 66-27-3 67-63-0 50-32-8
0.3 100.0 10.0 0.03 2.0 3.0 1.0 0.9 30.0 3.0 3.0 10.0 300.0 0.3 100.0 10.0 300.0 0.1
++ + +
Acrylamide Dextran sulfate sodium salt Perfluorooctanoic acid Dimethylbenz[a]anthracene Ferric chloride Ferrous chloride Trans-retinoic acid Erythrosine Caffeine 1,4-Dioxane Catechol Anthralin Clofibrate Cholic acid Wy-14643 Acetaminophen Profluthrin Styrene oxide Piperonyl butoxide o-Phenylphenol sodium Sodium nitrite Palytoxin Sorbic acid Benzoic acid 17b-Estradiol 5a-Dihydrotestosterone Sodium chloride Tumor necrosis factor a Tween80 Genistein Propionic acid b-Carotene Simvastatin o-Toluidine Butyl-p-hydroxybenzoate Isobutyl-p-hydroxybenzoate Aniline Benzene 4-Nitro-o-phenylenediamine Anthracene MeIQ Pyridine Diethylstilbestrol Ethyl carbamate Methylmethansulfonate 2-Propanol Benzo[a]pyrene
+ + +
Mutagenicity (Ames test)
Carcinogenicity in rodents
IARC 2A
+
+ + + +
+ ± + + + + + + +
+ + + +
+
3 2B 2B 3 3
+ +
3
+ +
+
+ + + ±
+ + +
+ +
+
±
2A 3
+ + +
+ + w+ +
+ +
+ +
+ +
+
+ + + + +
+
+
1
3 1 3 3 2B 3 1 2A 3 1
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
Source
a-Tocopherol
b
CAS number
Name
D-
Published datae
In vitro tumor promoting activities a
1 +
30.0 1.0 100.0 10.0 3.0 3.0 100.0 3.0 100.0 300.0 3.0 3.0 3.0 1.5% 0.03 56-23-5 136-77-6 60-35-5 137-66-6 5307-14-2 94-13-3 100-42-5 120-47-8 108-90-7 96-24-2 105650-23-5 13311-84-7 192-97-2 67-68-5 33419-42-0
Source
N W W W T W W W W T W S A W C
Name
Carbon tetrachloride 4-Hexylresorcinol Acetamide Ascorbyl palmitate 2-Nitro-p-phenylenediamine Propyl-p-hydroxybenzoate Styrene Ethyl-p-hydroxybenzoate Chlorobenzene 3-Chloro-1,2-propanediol PhIP Flutamide Benzo[e]pyrene Dimethyl sulfoxide Etoposide
These chemicals were purchased from the following sources: A, Aldrich Chemical Co., Inc. (Milwaukee, WI, USA); C, Calbiochem, Merk (Darmstadt, Germany); F, San-Ei Gen F.F.I., Inc. (Osaka, Japan); I, ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA); J, Sigma–Aldrich Japan (Tokyo, Japan); L, Alfa Aesar (Ward Hill, MA, USA); M, MP Biomedicals (Illkirch, France); N, Nacalai Tesque Inc. (Kyoto, Japan); O, Chemical Synthesis; S, Sigma–Aldrich Co. (St. Louis, MI, USA); T, Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); V, Avocado Research Chemicals Ltd. (Lancashire, UK); W, Wako Pure Chemical Industries Ltd. (Osaka, Japan). b The dose that produced the maximum number of foci (positive chemicals) or the highest dose tested (negative chemicals) were presented. c Results of BALB/c 3T3 cell transformation assay were scored on a scale of 0–3 (+++, severe; ++, moderate; +, slight; , negative). d The results for the assessment of 22 genes expression assay are as follows: – = negative, 1.5-fold up-regulated gene number under 2; + = positive, 1.5-fold up-regulated gene number more than 2. e These data were referred to in the published database: Chemical Carcinogenesis Research Information System (CCRIS), Carcinogenic Potency Database (CPDB), and the National Toxicology Program (NTP). Published results of several tumor related tests are shown as +, , ±.
a
3 +
2B ± + + + + +
2B + +
3 + +
2B
2B
+ ± + +
+
IARC Carcinogenicity in rodents Mutagenicity (Ames test) In vivo tumor promoting activities Dose (lg/mL)
BALB/c 3T3 cell transformation assayc
Twenty-two genes expression assayd
Published datae
CAS number
b
In vitro tumor promoting activities
a
Chemicals
Table 1 (continued)
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
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predicting tumor promoting potential of a sample set of 63 chemicals, built on the quantitative real-time PCR method used in the pilot study. 2. Materials and methods 2.1. Chemicals The 63 chemicals tested in this study are listed in Table 1. All chemicals used were of the highest purity grade available. Tumor promoters and non-tumor promoters in the test set were identified by previously published data and our results of cell transformation assays using BALB/c 3T3 cells. Published data were obtained from the Carcinogenicity Potency Database (http://potency.berkeley.edu), the National Library of Medicine Chemical Carcinogenesis Research Information System (http://toxnet.nlm.nih.gov/cgi-bin/ sis/htmlgen?CCRIS), the National Toxicology Program Database (http://ntp.niehs.nih.gov). 2.2. Cell culture BALB/c 3T3 cells (clone A31-1-1, JCRB0601) were provided by the Health Science Research Resources Bank (Osaka, Japan). Cells were maintained in Eagle’s minimum essential medium (MEM, GIBCO, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Cell viability assay A cell viability assay, using the standard crystal-violet absorption method, was applied to dose range finding for the cell transformation assay. BALB/c 3T3 cells were harvested in MEM/10% FBS at a density of 2 105 cells/60 mm dish, and cultured for two days. Test chemicals were then added and cells treated for another four days. Cells were fixed with 10% formalin and stained with 1% crystal-violet (CV) solution. After extraction of the stained CV with 0.02 N HCl– 50% ethanol, OD570 values were measured. The highest concentration that could be used in a transformation assay was that which did not inhibit cell growth less than 50% of the control or promoted cell viability more than 110% of the control. 2.4. Short-term, two-stage cell transformation assay The chemical-induced transformation assay was carried out as previously reported (Maeshima et al., 2009; Tsuchiya and Umeda, 1995). Exponentially growing BALB/c 3T3 cells were harvested in MEM/10% FBS at a density of 104 cells/60 mm from 10 plates per condition (day 0). After 24 h incubation, 3-methylcholanthrene (MCA; 0.2 lg/mL) was added as the initiation treatment (day 1). On day 4, the medium was changed to DMEM/F-12 (DFI2F) supplemented with 2% FBS, 0.2% ITS-X and 0.9 lg/mL transferrin medium (DFI2F). Thereafter, the DFI2F medium was changed twice per week. The test chemical, as the promotion treatment, was added to the medium on day 7, 11, and 14. On day 25, cells were fixed with methanol and stained with Giemsa solution. Data were analyzed statistically using the Wilcoxon’s rank sum test to determine the statistical significance between group differences, with P < 0.05 considered significant. Assessments of tumor promotion activity were as follows: = negative, no significant differences and under 200% of the solvent control activity; + = slight, no significant differences and more than 200% of the solvent control activity; ++ = moderate, significant differences and under 50% of the positive control (TPA) activity; +++ = severe, significant differences and more than 50% of the positive control (TPA) activity.
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H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
Table 2 List of 22 markers for tumor promoting activity and sequence. Gene symbol
Gene title
Accession number
Forward primer
Reverse primer
Ccnb1 Rif1 Mcm3
NM_172301 NM_175238 NM_008563
GCAGCACCTGGCTAAGAATGT CAGGACTGTCTCCACGGATGA CCCAGGACTCCCAGAAAGTG
TTCTTGACAGTCATGTGCTTTGTG GGGTATCTAGGGTCACAGGTTCA GAGGGCCGCCTTAAAAGC
NM_007691
CCGACTTTCTAAGGGTGATGGA
CGCTGAGCTTCCCTTTAATCTTC
NM_010591 NM_010235 NM_008234 NM_009505 NM_019641 NM_011118 NM_016741 NM_011077
ATTGCTTCTGTAGTGCTCCTTAACAC CCGAAGAAAGGAGCTGACAGA TCTAGAATTACTGTTGGATCGAAGTGA TGCACCCACGACAGAAGGA CCCACAAAATGGAGGCTAACA GCCACAGACATAAAGAAAAAGATCAAC GCCAAGCTATAGGGTCCTGAAG GCCAAGAGAAATGGGAAAGCT
TGCAGTCTAGCCTGGCACTTAC CGATTTCTCATCCTCCAATTTGT TCCCTGTCTTCCCTTTAATTGG TCGCTGGTAGACATCCATGAAC TCCACGTGCTTGTCCTTCTCT TCTTCTTTTCTTCATCTCCATTCTGA GACTGGGTGGCTGGTCTGA AGCACAAAACCTGTCCTTCCA
NM_008768 NM_011016 NM_183392 NM_011400
ACTCCACCCATCTAGGATTCCA ACCTTACCCCCAACTTGATAAATG AGATGCAGACCTGTTACGAGAAATC TCCAACTGGACCTCAAACTTCA
GCAAAGGTTTCTACTCCTCCTTCA ACAGTGGTCATCTATGGTGTGATACTC TCAAGTGGCTAAGGCCTTCCT CCGCACAGTTGCTCCACATA
Il1rl1
Cyclin B1 Rap1 interacting factor 1 homolog (yeast) Minichromosome maintenance deficient 3 (Saccharomyces cerevisiae) Checkpoint kinase 1 homolog (Schizosaccharomyces pombe) Jun oncogene Fos-like antigen 1 Helicase, lymphoid specific Vascular endothelial growth factor A Stathmin 1 Prolactin family 2, subfamily c, member 3 Scavenger receptor class B, member 1 Phosphate regulating gene with homologies to endopeptidases on the X chromosome (hypophosphatemia, vitamin D resistant rickets) Orosomucoid 1 Orosomucoid 2 Nucleoporin 54 Solute carrier family 2 (facilitated glucose transporter), member 1 Interleukin 1 receptor-like 1
CTGCAGGAAAAGAGAATCCAAAC
GGAAGGCATTGTGGAATCAAG
Rad51ap1 Tfrc Ab1 Car13 Pik3r5
RAD51 associated protein 1 Transferrin receptor Abl-interactor 1 Carbonic anhydrase 13 Phosphoinositide-3-kinase, regulatory subunit 5, p101
NM_001025602; NM_010743 NM_009013 NM_011638 NM_019501 NM_024495 NM_177320
TGAAAGCAAGAGGCCCAAGT TTGAGGCAGACCTTGCACTCT AAAATTCTCTGACCTTTAATCCTATGGT TTGAGAGTGTCACGTGGATTGTT GCAGAGTGTGGTCAGGTGTGA
AATGCATTGCTGCTAGAGTTCCT AAAGCCAGGTGTGTATGGATCA TGCCCACATGTAAAGCCATTAC CACAAGAGGCTTCGGAATCTG GGTGGCAAGCTGCTCTTCTC
Chek1 Jun Fosl1 Hells Vegfa Stmn1 Prl2c3 Scarb1 Phex
Orm1 Orm2 Nup54 Slc2a1
2.5. In vitro screening test for predicting tumor promoting potential of chemicals based on gene expression 2.5.1. Chemical treatment The in vitro screening test for predicting tumor promoting potential of chemicals based on gene expression was carried out as previously reported (Maeshima et al., 2009). The test chemical, as the promotion treatment, was added to the medium on day 7 of a short-term two-stage cell transformation assay, using MCA and BALB/c 3T3 cells as described in Section 2.4. The highest test concentration of each chemical was determined by its effect on cell viability and its solubility. 2.5.2. RNA extraction Total RNA was extracted from cells using the Qiagen RNeasy protocol (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, 48 h after test chemicals or solvent were added in the promotion phase of the assay as described in Section 2.4. Before in vitro transcription, residual genomic DNA was removed from the total RNA by DNase I treatment (RNase-free DNase set; QIAGEN). 2.5.3. Reverse transcription Total RNA samples (200 ng) were reverse transcribed to yield first-strand cDNA using the Applied Biosystems Reverse Transcription Reagents protocol (Applied Biosystems). The reverse transcription reactions were then diluted 1:10 in distilled H2O. 2.5.4. Quantitative real-time PCR The 22 markers for tumor promoting activity, and the sequences of the primers, are listed in Table 2. For individual reactions, 5 lL of each sample were combined with 20 lL of SYBR Green PCR Master Mix (Applied Biosystems), containing the appropriate primer pair at 6.5 lM. SYBR Green PCR reactions were performed in 96 well optical plates and run in an Applied Biosystems 7500 Real-Time PCR System for 40 cycles at 95 °C for 15 s, 60 °C for 60 s. To quantify the results obtained by real-time
PCR, we used a relative standard curve method. The amount of expression of both the target gene and an endogenous gene (b-actin) in a sample were measured by comparison with the standard curve. The target amount was divided by the endogenous reference amount to obtain a normalized target value. Each of the normalized target values was divided by the solvent control (DMSO) normalized target value to generate the relative expression levels. The number of marker genes that were up-regulated more than 1.5fold by a chemical treatment was used as the gene expression score. The results for the assessment of 22 genes expression assay are as follows: = negative, 1.5-fold up-regulated gene number under 2; + = positive, 1.5-fold up-regulated gene number more than 2.
3. Results 3.1. Chemical classification in BALB/c 3T3 cell transformation assay The objective this study was to investigate whether our assay using 22 marker genes could classify tumor promoters and non-tumor promoters. The test set for validation was selected based on the results of the cell transformation assay and the carcinogen classification available in public databases (CCRIS, NTP and CPDB). The criterion for classify tumor promoters and non-tumor promoters was the results of tumor promoting activities in BALB/c 3T3 cell transformation assay. As a result of the cell transformation assay, 33 chemicals induced transformed foci and were judged positive (tumor promoter). The other 30 chemicals were judged negative (non-tumor promoter). Of the 33 positive chemicals, 8 chemicals showed severe activity (significant differences and more than 50% of the positive control activity), 23 chemicals showed moderate activity (significant differences and under 50% of the positive control activity), and 2 chemicals showed slight activity (no significant differences and more than 200% of the solvent control activity) (Table 1).
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H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
(A) Severe tumor promoters 24
14
***
Up-regulated gene numbers 12
Foci/dish
20
***
18
***
***
16
***
14
*** ***
*** ***
12
10
***
*** ***
10
***
8
***
*
6
6
**
**
**
8
Foci/dish
Up-regulated gene numbers
22
4
4
2
2
Solvent control
TPA
Acrylamide
Perfluorooctanoic acid
Dimethyl benz[a]anthracene
Ferric chloride
Ferrous chloride
Trans-retinoic acid
30
100
10
3
1
0.3
30
100
10
30
100
10
0.05
0.03
0.01
45
30
3
Dextran sulfate sodium salt
10
30
10
3
30
100
10
0.1
3
0
0 Conc. (μg/mL)
Erythrosine
(B) Moderate tumor promoters 1 14
22
Up-regulated gene numbers
20
Foci/dish
12
18
10
16 14
8
12 6
10
*
8 6
*
*
4
*
**
***
*
**
*
***
*
**
Foci/dish
Up-regulated gene numbers
24
4
** 2
2
Solvent control
Caffeine
1,4-Dioxane
Catechol
Anthralin
Clofibrate
Cholic acid
Wy-14643
30
3
Acetaminophen
10
20
10
3
30
10
3
100
30
10
60
30
0.1
0.05
0.03
0.01
0.6
0.3
0.15
0.08
300
100
30
10
100
30
10
0
0 Conc. (μg/mL)
Profluthrin
14 Up-regulated gene numbers 12
Foci/dish
8
***
Solvent control
Styrene oxide
Piperonyl butoxide
Sodium nitrite
Palytoxin
**
Sorbic acid
Benzoic acid
17β-Estradiol
4
10
3
2
1
0.3
*
0.1
300
*
1
*
100
30
100
0.0001
*
300
***
0.00003
300
10
3
1
o-Phenyl phenol Na
**
2000
*
*
1000
**
0.3
**
0.1
30
10
**
6
Foci/dish
10
3
Up-regulated gene numbers
(C) Moderate tumor promoters 2 24 22 20 18 16 14 12 10 8 6 4 2 0
0 Conc. (μg/mL)
5αDihydrotestosterone
Fig. 1. The results of the in vitro two-stage transformation assay and gene expression assay. (A) Severe tumor promoters, (B) Moderate tumor promoters 1, (C) Moderate tumor promoters 2. Each bar represents the number of genes that were more than 1.5-fold up-regulated compared to the control, as assayed by our gene expression assay. Line graph represents the number of foci/dish in the cell transformation assay. The transformed foci value presented are means ± SD (n = 10). ***P < 0.001, **P < 0.01, *P < 0.05 (vs. control, Wilcoxon rank sum).
3.2. Independent QRT-PCR assay of 63 chemicals To validate the reliability and robustness of this screening assay, the 22 gene markers were tested on the 63 independent test chemicals (test set). The performance of this assay was measured by examining how well the classifier predicted the non-tumor promoter (cell transformation assay negative) or tumor promoter samples (cell transformation assay positive) in the test set. Table 1 and Figs. 1 and 2 show the relative gene expression levels of the 22 markers 48 h after treatment with each of the 63 chemicals and the results of the cell transformation assays. Most of the mod-
erate to severe tumor promoters, as classified by the cell transformation assay, had a high gene expression score, which increased in a dose dependent manner. Some of these chemicals increased the gene expression score at lower doses than the dose determined by the cell transformation assay. Genistein, which was judged to be a moderate tumor promoter by the cell transformation assay, did not score more than two in the gene expression score. Among the 30 non-tumor promoters, 29 did not induce a gene expression score of more than two, and were judged negative. However, simvastatin was judged positive in the screening assay based on gene expression.
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H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
(A) Moderate to slight tumor promoters and simvastatin 14 Up-regulated gene numbers
22 20
12
Foci/dish
18
10
16 14
8
12 6
10 8 6
*
**
*
*
4 2
** **
**
4
*
2
Sodium chloride
Tumor necrosis factor α
Tween80
D-α-tocopherol
Genistein
Propionic acid
β-Carotene
0.03
0.01
0.003
3
10
1
30
100
10
0.3
0.1
0.03
3
10
1
30
100
10
0.005
0.0025
0.001
3000
1000
300
0
Solvent control
Foci/dish
Up-regulated gene numbers
24
0 Conc. (μg/mL)
Simvastatin
(B) Non-tumor promoters 14
22
Up-regulated gene numbers
20
Foci/dish
12
18
10
16 14
8
12 6
10 8
Foci/dish
Up-regulated gene numbers
24
4
6 4
2
2
Solvent control
MeIQ
Pyridine
Benzo[a]pyrene
4-Hexylresorcinol
Acetamide
Ascorbyl palmitate
3-Chloro-1,2propanediol
3
1
0.3
300
100
30
10
3
1
100
30
10
1
0.3
0.1
0.1
0.03
0.01
0.003
300
100
30
10
10
3
1
0
0 Conc. (μg/mL)
PhIP
Fig. 2. The results of the in vitro two-stage transformation assay and gene expression assay. (A) Moderate to slight tumor promoters and simvastatin, (B) Non-tumor promoters. Each bar represents the number of genes that were more than 1.5-fold up-regulated compared to the control, as assayed by our gene expression assay. Line graph represents the number of foci/dish in the cell transformation assay. The transformed foci value presented are means ± SD (n = 10). ***P < 0.001, **P < 0.01, *P < 0.05 (vs. control, Wilcoxon rank sum).
3.3. Accuracy, specificity and sensitivity of the assay Accuracy is defined as the ratio of the number of correctly predicted samples to the total number of samples. Sensitivity is the ratio of the number of correctly predicted (tumor promoter) samples to the total number of that type of sample (tumor promoter). Specificity is the ratio of the number of correctly predicted (non-tumor promoter) samples to the total number of non-tumor promoter. This assay consistently achieves high accuracy, sensitivity, and specificity on the test set. The accuracy, sensitivity, and specificity of our assay system are 96.8%, 97.0% and 96.7%, respectively. Of the moderate or severe tumor promoters, 96.8% (30/31) were detected. 4. Discussion In a previous study we selected 22 gene markers as potential biomarkers for the early detection of tumor promoters, and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells (Maeshima et al., 2009). However, the study did not have a completely independent test set, therefore, these markers required further validation. The most straightforward approach for properly evaluating a classifier is to base the evaluation on a separate test set (Dupuy and Simon, 2007). Over-fitting can be detected when using proper cross-validation techniques or independent test set analysis. Only an independent test set, which is not used for determining pre-processing parameters, selection of differentially expressed gene model building or model selection, can be used to estimate the true performance of models. Therefore, we carried
out a new study to estimate the performance of the 22 marker quantitative PCR assay in an unbiased way. Sixty-three chemicals were tested by QRT-PCR and a cell transformation assay. Thirty-three chemicals were positive in the cell transformation assay, and the others were negative. Our gene expression assay judged 32 to be positive among 33 chemicals that were positive in the cell transformation assay, and judged 29 to be negative among 30 that were negative in the cell transformation assay (Table 1). For severe or moderate tumor promoters, our assay showed dose dependent increases in the gene expression scores (Figs. 1 and 2). There was a correlation between the results of our assay and the cell transformation assay results, in BALB/c 3T3 cells. This assay consistently achieved high accuracy, sensitivity, and specificity (96.8%, 97.0% and 96.7%). Our in vitro tumor promotion assay failed to identify genistein as positive (Fig. 2A). Genistein is an isoflavone whose chemical structure resembles estrogen and it interacts with animal and human estrogen receptors. In a previous report, genistein induced apoptosis (Katdare et al., 2002), inhibited cell proliferation (Xiang et al., 2002), or had tumor promoting activity (Murata et al., 2004) in vitro. In in vivo studies of genistein, both a cancer prevention effect (Fritz et al., 1998; Lamartiniere et al., 1995) and a cancer enhancement effect (Rao et al., 1997; Seike et al., 2003) have been reported. However, despite many studies, the effectiveness and the safety of genistein have not been established. Although BALB/c 3T3 cells have been reported not to express estrogen receptors at measurable levels (Gaben and Mester, 1991), genistein (also 17-b-estradiol) was judged to be positive by the cell transformation assay. In a previous report, estrogens and estrogen-like chem-
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
icals were shown to have the ability to directly transform cells by multiple mechanisms unrelated to their estrogenicity (Newbold et al., 1990; Tsutsui and Barrett, 1997). Thus, the enhancement of transformed foci by these chemicals might not be mediated through estrogen receptors in this assay. Simvastatin was judged positive on our gene expression assay, but was judged negative by cell transformation assay (Fig. 2A). Simvastatin is a hypolipidemic drug and is considered a non-genotoxic rodent carcinogen (Snyder and Green, 2001). Therefore, it is not surprising that simvastatin was judged positive on our assay. Tumor promoters have been postulated to act via a number of mechanisms, such as inhibition of apoptosis, inhibition of gapjunction intercellular communication, and induction of cell proliferation (Combes, 2000; Tomatis, 1993; Trosko and Ruch, 1998; Wright et al., 1994). This study showed that our assay could detect various types of tumor promoters (metal compounds, peroxisome proliferators, hormonal agent, and marine toxins, Table 1). It may be important to know the mode of action of tumor promoter’s effect. However, as mentioned above, it is not enough to focus on one mode of action for the detection of a tumor promoter, because tumor promoters have various modes of action. Our 22 gene markers are involved in various pathways (cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation) (Table 2). On this point, it seems that our gene expression assay is reasonable. The new in vitro assay must be evaluated with many materials. We have already evaluated 101 chemicals, including 38 chemicals used in a previous study (Maeshima et al., 2009). Our gene expression assay judged 54 to be positive among 55 tumor promoters, and judged 43 to be negative among 46 non-tumor promoters. In addition, our gene expression assay does not require the continued administration of the test chemicals, unlike other in vitro assays, thereby reducing the consumption of the test chemicals. However, more validation is necessary. In the future, a device for the handling of cells, PCR reagents, and test chemicals should be developed to simplify the assay. Tumor promoters often produce tumors only at high doses, and some tumor promoters may even be anticancer agents at low doses (Kinoshita et al., 2006). In addition, it is recognized that chemicals inducing carcinogenesis in rodents may not induce carcinogenesis in humans due to species differences. (Cunningham, 2002; Fung et al., 1995; Ward, 2007). Therefore, our in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression should be considered as a starting point for further investigations of carcinogenesis. In summary, we used an independent test set to evaluate 22 previously discovered biomarkers for the tumor promoting potential of chemicals. The accuracy, sensitivity, and specificity of our assay system are 96.8%, 97.0%, and 96.7%, respectively. To our knowledge, this study represents the first multigene predictive model for in vitro tumor promotion in BALB/c 3T3 cells. We are confident that this 22 marker gene-based tumor promoting activity test system will become a valuable tool for rapid screening of potential tumor promoters. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tiv.2009.12.013. References Ames, B.N., Lee, F.D., Durston, W.E., 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Sciences of the United States of America 70, 782–786. Barrett, J.C., 1993. Mechanisms of multistep carcinogenesis and carcinogen risk assessment. Environmental Health Perspectives 100, 9–20.
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Clive, D., Spector, J.F.S., 1975. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research 31, 17–29. Combes, R.D., 2000. The use of structure–activity relationships and markers of cell toxicity to detect non-genotoxic carcinogens. Toxicology In Vitro 14, 387–399. Combes, R., Balls, M., Curren, R., Fischbach, M., Fusenig, N., Kirkland, D., Lasne, C., Landolph, J., LeBoeuf, R., Marquardt, H., McCormick, J., Muller, L., Rivedal, E., Sabbioni, E., Tanaka, N., Vasseur, P., Yamasaki, H., 1999. Cell transformation assays as predictors of human carcinogenicity. In: The Report and Recommendations of ECVAM Workshop 39, vol. 27. ATLA, pp. 745–767. Cunningham, M.L., 2002. A mouse is not a rat is not a human: Species differences exist. Toxicological Sciences 70, 157–158. Dupuy, A., Simon, R.M., 2007. Critical review of published microarray studies for cancer outcome and guidelines on statistical analysis and reporting. Journal of the National Cancer Institute 99, 147–157. Fritz, W.A., Coward, L., Wang, J., Lamartiniere, C.A., 1998. Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis 19, 2151–2158. Fung, V.A., Barrett, J.C., Huff, J., 1995. The carcinogenesis bioassay in perspective: application in identifying human cancer hazards. Environmental Health Perspectives 103, 680–683. Gaben, A.M., Mester, J., 1991. BALB/C mouse 3T3 fibroblasts expressing human estrogen receptor: effect of estradiol on cell growth. Biochemical and Biophysical Research Communications 176, 1473–1481. Hennings, H., Boutwell, R.K., 1970. Studies on the mechanism of skin tumor promotion. Cancer Research 30, 312–320. Kakunaga, T., 1973. A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB/3T3. International Journal of Cancer 12, 463–473. Katdare, M., Osborne, M., Telang, N.T., 2002. Soy isoflavone genistein modulates cell cycle progression and induces apoptosis in HER-2/neu oncogene expressing human breast epithelial cells. International Journal of Oncology 21, 809–815. Kinoshita, A., Wanibuchi, H., Wei, M., Fukushima, S., 2006. Hormesis in carcinogenicity of non-genotoxic carcinogens. Journal of Toxicologic Pathology 19, 111–122. Lamartiniere, C.A., Moore, J.B., Brown, N.M., Thompson, R., Hardin, M.J., Barnes, S., 1995. Genistein suppresses mammary cancer in rats. Carcinogenesis 16, 2833– 2840. Maeshima, H., Ohno, K., Tanaka-Azuma, Y., Nakano, S., Yamada, T., 2009. Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells. Toxicology In Vitro 23, 148–157. Murata, M., Midorikawa, K., Koh, M., Umezawa, K., Kawanishi, S., 2004. Genistein and daizein induce cell proliferation and their metabolites cause oxidative DNA damage in relation to isoflavone-induced cancer of estrogen-sensitive organs. Biochemistry 43, 2569–2577. Newbold, R.R., Bullock, B.C., McLachlan, J.A., 1990. Uterine adenocarcinoma in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis. Cancer Research 50, 7677–7681. Oda, Y., Nakamura, S., Shimada, T., Oki, I., Kato, T., Shinagawa, H., 1985. Evaluation of the new system (umu-test) for the detection of environmental mutagens and cariconogens. Mutation Research 147, 219–229. Rao, C.V., Wang, C.X., Simi, B., Lubet, R., Kelloff, G., Steele, V., Reddy, B.S., 1997. Enhancement of experimental colon cancer by genistein. Cancer Research 57, 3717–3722. Reznikoff, C.A., Brankow, D.W., Heidelberger, C., 1973. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Research 33, 3231–3238. Seike, N., Wanibuchi, H., Morimura, K., Wei, M., Nishikawa, T., Hirata, K., Yoshikawa, J., Fukushima, S., 2003. Enhancement of lung carcinogenesis by nonylphenol and genistein in a F344 rat multiorgan carcinogenesis model. Cancer Letters 192, 25–36. Slaga, T.J., 1983. Overview of tumor promotion in animals. Environmental Health Perspectives 50, 3–14. Snyder, R.D., Green, J.W., 2001. A review of the genotoxicity of marketed pharmaceuticals. Mutation Research 488, 151–169. Tomatis, L., 1993. Cell proliferation and carcinogenesis: a brief history and current view based on an IARC workshop report. International agency for research on cancer. Environmental Health Perspectives 101, 149–151. Trosko, J.E., Ruch, R.J., 1998. Cell–cell communication in carcinogenesis. Frontiers in Bioscience 3, 208–236. Tsuchiya, T., Umeda, M., 1995. Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis 16, 1887–1894. Tsutsui, T., Barrett, J.C., 1997. Neoplastic transformation of cultured mammalian cells by estrogens and estrogen-like chemicals. Environmental Health Perspectives 105, 619–624. Ward, J.M., 2007. The two-year rodent carcinogenesis bioassay – will it survive? Journal of Toxicologic Pathology 20, 13–19. Weisburger, J.H., Wiliams, G.M., 1983. The distinct health risk analyses required for genotoxic carcinogens and promoting agents. Environmental Health Perspectives 50, 233–245. Wright, S.C., Zhong, J., Larrick, J.W., 1994. Inhibition of apoptosis as a mechanism of tumor promotion. The FASEB Journal 8, 654–660. Xiang, H., Schevzov, G., Gunning, P., Williams, H.M., Silink, M., 2002. A comparative study of growth-inhibitory effects of isoflavones and their metabolites on human breast and prostate cancer cell lines. Nutrition and Cancer 42, 224–232.
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Validation of an in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression H. Maeshima *, K. Ohno, S. Nakano, T. Yamada Food Safety Research Institute, Nissin Foods Holdings Co., Ltd., 2247 Noji-cho, Kusatsu, Shiga 525-0055, Japan
a r t i c l e
i n f o
Article history: Received 28 August 2009 Accepted 11 December 2009 Available online 16 December 2009 Keywords: In vitro screening Molecular marker Transformation assay Tumor promoter Genotoxicity Carcinogen
a b s t r a c t Chemical carcinogenesis is a multifactorial process comprising two main stages: initiation and promotion. Tumor promoters cause the development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The identification of tumor promoters is important for preventing cancer. We previously identified 22 specific gene markers using a global gene expression analysis of chemically induced tumor promotion and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells. Our in vitro tumor promoter screening test, based on these marker genes, enables earlier assessment, and is easier to conduct than classical methods. The general applicability of these markers, however, was unknown. In this study, to evaluate the performance of a set of markers, we independently validated a separate sample set, which had various structures and properties. Independent validation of the signature of 63 test chemicals showed an accuracy, sensitivity, and specificity of the assay of 96.8%, 97.0% and 96.7%, respectively. These results indicate that the tumor promoting activity assay, based on the expression of 22 marker genes, will become a valuable tool for rapid screening of potential tumor promoters. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Chemical carcinogenesis is a multistage, multifactorial process, comprising two main stages: initiation and promotion (Barrett, 1993; Hennings and Boutwell, 1970; Slaga, 1983; Weisburger and Wiliams, 1983). Initiators (genotoxic agents) can be detected by various short-term genotoxicity screening tests (Ames et al., 1973; Clive and Spector, 1975; Oda et al., 1985). However, many carcinogenic chemicals are negative in the genotoxicity assays. The majority of these non-genotoxic carcinogens are assumed to be tumor promoters. Tumor promoters differ from genotoxic carcinogens, and have various biological activities and chemical structures. In vivo rodent models remain the only reliable way for experimental investigation of the tumor promotion activity of chemicals to humans. However, the rodent carcinogenicity assay is expensive, time-consuming, uses large amounts of the compound, and involves ethical issues. While many in vitro tumor promotion assays have been developed as screening methods (Combes et al., 1999; Kakunaga, 1973; Reznikoff et al., 1973), their low
Abbreviations: TPA, 12-O-Tetradecanoylphorbol-13-acetate; MeIQ, 2-amino3,4-dimethylimidazo[4,5-f]quinoline; PhIP, 2-amino-1-methyl-6-phenylimidazo(4,5-B)pyridine. * Corresponding author. Tel.: +81 77 561 9115; fax: +81 77 561 9140. E-mail address: [email protected] (H. Maeshima). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.12.013
throughputs have rendered them impractical as rapid screening tools. In a previously published pilot study, we explored the performance of a microarray in predicting the tumor promoting potential of chemicals in BALB/c 3T3 cells (Maeshima et al., 2009). We investigated whether gene expression analysis could be used to distinguish between a tumor promoter and a non-tumor promoter at 48 h after chemical addition. The results showed that 22 gene markers were differentially expressed between the tumor promoter and the non-tumor promoter. To establish a gene expression score, we counted the number of marker genes that were up-regulated by more than 1.5-fold by the chemical treatment. The gene expression score was compared with the results of a cell transformation assay. There was a correlation between the gene expression score and the results of the cell transformation assay using BALB/c 3T3 cells. These results indicated that gene expression analysis could be used to predict the prognosis of a tumor promoter at an early time point. However, this study was not a sufficient validation of the proposed test modification; an independent evaluation was required to give an unbiased estimate of its general applicability. In the present study, our goal was to evaluate whether an independent study could confirm the applicability of this QRT-PCR assay for the tumor promoters. Independent validation using a separate sample set, that has various structure and properties, is one way to determine the true performance of a set of markers. We describe the results of an independent evaluation of models for
996
Table 1 List of chemicals and their respective results in the in vitro two-stage transformation assay using BALB/c 3T3 cells, the gene expression assay, and published data. Chemicals
Dose (lg/mL)
BALB/c 3T3 cell transformation assayc
Twenty-two genes expression assayd
In vivo tumor promoting activities
W N T W W W S F W W W L W W S W O A W W S W W N S W J W N M
79-06-1 9011-18-1 335-67-1 57-97-6 7705-08-0 7758-94-3 302-79-4 16423-68-0 58-08-2 123-91-1 120-80-9 1143-38-0 637-07-0 81-25-4 50892-23-4 103-90-2 223419-20-3 96-09-3 51-03-6 132-27-4 7632-00-0 11077-03-5 110-44-1 65-85-0 50-28-2 521-18-6 7647-14-5
+++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + +
9005-65-6 59-02-9
100.0 30.0 45.0 0.05 100.0 100.0 3.0 100.0 100.0 300.0 0.6 0.1 60.0 100.0 30.0 20.0 30.0 30.0 0.3 10.0 2000.0 0.0001 300.0 300.0 1.0 10.0 3000.0 0.005 100.0 10.0
S W S S W N W N J W S W W S W S J W
446-72-0 79-09-4 7235-40-7 79902-63-9 95-53-4 94-26-8 4247-02-3 62-53-3 71-43-2 99-56-9 120-12-7 77094-11-2 110-86-1 56-53-1 51-79-6 66-27-3 67-63-0 50-32-8
0.3 100.0 10.0 0.03 2.0 3.0 1.0 0.9 30.0 3.0 3.0 10.0 300.0 0.3 100.0 10.0 300.0 0.1
++ + +
Acrylamide Dextran sulfate sodium salt Perfluorooctanoic acid Dimethylbenz[a]anthracene Ferric chloride Ferrous chloride Trans-retinoic acid Erythrosine Caffeine 1,4-Dioxane Catechol Anthralin Clofibrate Cholic acid Wy-14643 Acetaminophen Profluthrin Styrene oxide Piperonyl butoxide o-Phenylphenol sodium Sodium nitrite Palytoxin Sorbic acid Benzoic acid 17b-Estradiol 5a-Dihydrotestosterone Sodium chloride Tumor necrosis factor a Tween80 Genistein Propionic acid b-Carotene Simvastatin o-Toluidine Butyl-p-hydroxybenzoate Isobutyl-p-hydroxybenzoate Aniline Benzene 4-Nitro-o-phenylenediamine Anthracene MeIQ Pyridine Diethylstilbestrol Ethyl carbamate Methylmethansulfonate 2-Propanol Benzo[a]pyrene
+ + +
Mutagenicity (Ames test)
Carcinogenicity in rodents
IARC 2A
+
+ + + +
+ ± + + + + + + +
+ + + +
+
3 2B 2B 3 3
+ +
3
+ +
+
+ + + ±
+ + +
+ +
+
±
2A 3
+ + +
+ + w+ +
+ +
+ +
+ +
+
+ + + + +
+
+
1
3 1 3 3 2B 3 1 2A 3 1
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
Source
a-Tocopherol
b
CAS number
Name
D-
Published datae
In vitro tumor promoting activities a
1 +
30.0 1.0 100.0 10.0 3.0 3.0 100.0 3.0 100.0 300.0 3.0 3.0 3.0 1.5% 0.03 56-23-5 136-77-6 60-35-5 137-66-6 5307-14-2 94-13-3 100-42-5 120-47-8 108-90-7 96-24-2 105650-23-5 13311-84-7 192-97-2 67-68-5 33419-42-0
Source
N W W W T W W W W T W S A W C
Name
Carbon tetrachloride 4-Hexylresorcinol Acetamide Ascorbyl palmitate 2-Nitro-p-phenylenediamine Propyl-p-hydroxybenzoate Styrene Ethyl-p-hydroxybenzoate Chlorobenzene 3-Chloro-1,2-propanediol PhIP Flutamide Benzo[e]pyrene Dimethyl sulfoxide Etoposide
These chemicals were purchased from the following sources: A, Aldrich Chemical Co., Inc. (Milwaukee, WI, USA); C, Calbiochem, Merk (Darmstadt, Germany); F, San-Ei Gen F.F.I., Inc. (Osaka, Japan); I, ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA); J, Sigma–Aldrich Japan (Tokyo, Japan); L, Alfa Aesar (Ward Hill, MA, USA); M, MP Biomedicals (Illkirch, France); N, Nacalai Tesque Inc. (Kyoto, Japan); O, Chemical Synthesis; S, Sigma–Aldrich Co. (St. Louis, MI, USA); T, Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); V, Avocado Research Chemicals Ltd. (Lancashire, UK); W, Wako Pure Chemical Industries Ltd. (Osaka, Japan). b The dose that produced the maximum number of foci (positive chemicals) or the highest dose tested (negative chemicals) were presented. c Results of BALB/c 3T3 cell transformation assay were scored on a scale of 0–3 (+++, severe; ++, moderate; +, slight; , negative). d The results for the assessment of 22 genes expression assay are as follows: – = negative, 1.5-fold up-regulated gene number under 2; + = positive, 1.5-fold up-regulated gene number more than 2. e These data were referred to in the published database: Chemical Carcinogenesis Research Information System (CCRIS), Carcinogenic Potency Database (CPDB), and the National Toxicology Program (NTP). Published results of several tumor related tests are shown as +, , ±.
a
3 +
2B ± + + + + +
2B + +
3 + +
2B
2B
+ ± + +
+
IARC Carcinogenicity in rodents Mutagenicity (Ames test) In vivo tumor promoting activities Dose (lg/mL)
BALB/c 3T3 cell transformation assayc
Twenty-two genes expression assayd
Published datae
CAS number
b
In vitro tumor promoting activities
a
Chemicals
Table 1 (continued)
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
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predicting tumor promoting potential of a sample set of 63 chemicals, built on the quantitative real-time PCR method used in the pilot study. 2. Materials and methods 2.1. Chemicals The 63 chemicals tested in this study are listed in Table 1. All chemicals used were of the highest purity grade available. Tumor promoters and non-tumor promoters in the test set were identified by previously published data and our results of cell transformation assays using BALB/c 3T3 cells. Published data were obtained from the Carcinogenicity Potency Database (http://potency.berkeley.edu), the National Library of Medicine Chemical Carcinogenesis Research Information System (http://toxnet.nlm.nih.gov/cgi-bin/ sis/htmlgen?CCRIS), the National Toxicology Program Database (http://ntp.niehs.nih.gov). 2.2. Cell culture BALB/c 3T3 cells (clone A31-1-1, JCRB0601) were provided by the Health Science Research Resources Bank (Osaka, Japan). Cells were maintained in Eagle’s minimum essential medium (MEM, GIBCO, Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Cell viability assay A cell viability assay, using the standard crystal-violet absorption method, was applied to dose range finding for the cell transformation assay. BALB/c 3T3 cells were harvested in MEM/10% FBS at a density of 2 105 cells/60 mm dish, and cultured for two days. Test chemicals were then added and cells treated for another four days. Cells were fixed with 10% formalin and stained with 1% crystal-violet (CV) solution. After extraction of the stained CV with 0.02 N HCl– 50% ethanol, OD570 values were measured. The highest concentration that could be used in a transformation assay was that which did not inhibit cell growth less than 50% of the control or promoted cell viability more than 110% of the control. 2.4. Short-term, two-stage cell transformation assay The chemical-induced transformation assay was carried out as previously reported (Maeshima et al., 2009; Tsuchiya and Umeda, 1995). Exponentially growing BALB/c 3T3 cells were harvested in MEM/10% FBS at a density of 104 cells/60 mm from 10 plates per condition (day 0). After 24 h incubation, 3-methylcholanthrene (MCA; 0.2 lg/mL) was added as the initiation treatment (day 1). On day 4, the medium was changed to DMEM/F-12 (DFI2F) supplemented with 2% FBS, 0.2% ITS-X and 0.9 lg/mL transferrin medium (DFI2F). Thereafter, the DFI2F medium was changed twice per week. The test chemical, as the promotion treatment, was added to the medium on day 7, 11, and 14. On day 25, cells were fixed with methanol and stained with Giemsa solution. Data were analyzed statistically using the Wilcoxon’s rank sum test to determine the statistical significance between group differences, with P < 0.05 considered significant. Assessments of tumor promotion activity were as follows: = negative, no significant differences and under 200% of the solvent control activity; + = slight, no significant differences and more than 200% of the solvent control activity; ++ = moderate, significant differences and under 50% of the positive control (TPA) activity; +++ = severe, significant differences and more than 50% of the positive control (TPA) activity.
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H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
Table 2 List of 22 markers for tumor promoting activity and sequence. Gene symbol
Gene title
Accession number
Forward primer
Reverse primer
Ccnb1 Rif1 Mcm3
NM_172301 NM_175238 NM_008563
GCAGCACCTGGCTAAGAATGT CAGGACTGTCTCCACGGATGA CCCAGGACTCCCAGAAAGTG
TTCTTGACAGTCATGTGCTTTGTG GGGTATCTAGGGTCACAGGTTCA GAGGGCCGCCTTAAAAGC
NM_007691
CCGACTTTCTAAGGGTGATGGA
CGCTGAGCTTCCCTTTAATCTTC
NM_010591 NM_010235 NM_008234 NM_009505 NM_019641 NM_011118 NM_016741 NM_011077
ATTGCTTCTGTAGTGCTCCTTAACAC CCGAAGAAAGGAGCTGACAGA TCTAGAATTACTGTTGGATCGAAGTGA TGCACCCACGACAGAAGGA CCCACAAAATGGAGGCTAACA GCCACAGACATAAAGAAAAAGATCAAC GCCAAGCTATAGGGTCCTGAAG GCCAAGAGAAATGGGAAAGCT
TGCAGTCTAGCCTGGCACTTAC CGATTTCTCATCCTCCAATTTGT TCCCTGTCTTCCCTTTAATTGG TCGCTGGTAGACATCCATGAAC TCCACGTGCTTGTCCTTCTCT TCTTCTTTTCTTCATCTCCATTCTGA GACTGGGTGGCTGGTCTGA AGCACAAAACCTGTCCTTCCA
NM_008768 NM_011016 NM_183392 NM_011400
ACTCCACCCATCTAGGATTCCA ACCTTACCCCCAACTTGATAAATG AGATGCAGACCTGTTACGAGAAATC TCCAACTGGACCTCAAACTTCA
GCAAAGGTTTCTACTCCTCCTTCA ACAGTGGTCATCTATGGTGTGATACTC TCAAGTGGCTAAGGCCTTCCT CCGCACAGTTGCTCCACATA
Il1rl1
Cyclin B1 Rap1 interacting factor 1 homolog (yeast) Minichromosome maintenance deficient 3 (Saccharomyces cerevisiae) Checkpoint kinase 1 homolog (Schizosaccharomyces pombe) Jun oncogene Fos-like antigen 1 Helicase, lymphoid specific Vascular endothelial growth factor A Stathmin 1 Prolactin family 2, subfamily c, member 3 Scavenger receptor class B, member 1 Phosphate regulating gene with homologies to endopeptidases on the X chromosome (hypophosphatemia, vitamin D resistant rickets) Orosomucoid 1 Orosomucoid 2 Nucleoporin 54 Solute carrier family 2 (facilitated glucose transporter), member 1 Interleukin 1 receptor-like 1
CTGCAGGAAAAGAGAATCCAAAC
GGAAGGCATTGTGGAATCAAG
Rad51ap1 Tfrc Ab1 Car13 Pik3r5
RAD51 associated protein 1 Transferrin receptor Abl-interactor 1 Carbonic anhydrase 13 Phosphoinositide-3-kinase, regulatory subunit 5, p101
NM_001025602; NM_010743 NM_009013 NM_011638 NM_019501 NM_024495 NM_177320
TGAAAGCAAGAGGCCCAAGT TTGAGGCAGACCTTGCACTCT AAAATTCTCTGACCTTTAATCCTATGGT TTGAGAGTGTCACGTGGATTGTT GCAGAGTGTGGTCAGGTGTGA
AATGCATTGCTGCTAGAGTTCCT AAAGCCAGGTGTGTATGGATCA TGCCCACATGTAAAGCCATTAC CACAAGAGGCTTCGGAATCTG GGTGGCAAGCTGCTCTTCTC
Chek1 Jun Fosl1 Hells Vegfa Stmn1 Prl2c3 Scarb1 Phex
Orm1 Orm2 Nup54 Slc2a1
2.5. In vitro screening test for predicting tumor promoting potential of chemicals based on gene expression 2.5.1. Chemical treatment The in vitro screening test for predicting tumor promoting potential of chemicals based on gene expression was carried out as previously reported (Maeshima et al., 2009). The test chemical, as the promotion treatment, was added to the medium on day 7 of a short-term two-stage cell transformation assay, using MCA and BALB/c 3T3 cells as described in Section 2.4. The highest test concentration of each chemical was determined by its effect on cell viability and its solubility. 2.5.2. RNA extraction Total RNA was extracted from cells using the Qiagen RNeasy protocol (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, 48 h after test chemicals or solvent were added in the promotion phase of the assay as described in Section 2.4. Before in vitro transcription, residual genomic DNA was removed from the total RNA by DNase I treatment (RNase-free DNase set; QIAGEN). 2.5.3. Reverse transcription Total RNA samples (200 ng) were reverse transcribed to yield first-strand cDNA using the Applied Biosystems Reverse Transcription Reagents protocol (Applied Biosystems). The reverse transcription reactions were then diluted 1:10 in distilled H2O. 2.5.4. Quantitative real-time PCR The 22 markers for tumor promoting activity, and the sequences of the primers, are listed in Table 2. For individual reactions, 5 lL of each sample were combined with 20 lL of SYBR Green PCR Master Mix (Applied Biosystems), containing the appropriate primer pair at 6.5 lM. SYBR Green PCR reactions were performed in 96 well optical plates and run in an Applied Biosystems 7500 Real-Time PCR System for 40 cycles at 95 °C for 15 s, 60 °C for 60 s. To quantify the results obtained by real-time
PCR, we used a relative standard curve method. The amount of expression of both the target gene and an endogenous gene (b-actin) in a sample were measured by comparison with the standard curve. The target amount was divided by the endogenous reference amount to obtain a normalized target value. Each of the normalized target values was divided by the solvent control (DMSO) normalized target value to generate the relative expression levels. The number of marker genes that were up-regulated more than 1.5fold by a chemical treatment was used as the gene expression score. The results for the assessment of 22 genes expression assay are as follows: = negative, 1.5-fold up-regulated gene number under 2; + = positive, 1.5-fold up-regulated gene number more than 2.
3. Results 3.1. Chemical classification in BALB/c 3T3 cell transformation assay The objective this study was to investigate whether our assay using 22 marker genes could classify tumor promoters and non-tumor promoters. The test set for validation was selected based on the results of the cell transformation assay and the carcinogen classification available in public databases (CCRIS, NTP and CPDB). The criterion for classify tumor promoters and non-tumor promoters was the results of tumor promoting activities in BALB/c 3T3 cell transformation assay. As a result of the cell transformation assay, 33 chemicals induced transformed foci and were judged positive (tumor promoter). The other 30 chemicals were judged negative (non-tumor promoter). Of the 33 positive chemicals, 8 chemicals showed severe activity (significant differences and more than 50% of the positive control activity), 23 chemicals showed moderate activity (significant differences and under 50% of the positive control activity), and 2 chemicals showed slight activity (no significant differences and more than 200% of the solvent control activity) (Table 1).
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(A) Severe tumor promoters 24
14
***
Up-regulated gene numbers 12
Foci/dish
20
***
18
***
***
16
***
14
*** ***
*** ***
12
10
***
*** ***
10
***
8
***
*
6
6
**
**
**
8
Foci/dish
Up-regulated gene numbers
22
4
4
2
2
Solvent control
TPA
Acrylamide
Perfluorooctanoic acid
Dimethyl benz[a]anthracene
Ferric chloride
Ferrous chloride
Trans-retinoic acid
30
100
10
3
1
0.3
30
100
10
30
100
10
0.05
0.03
0.01
45
30
3
Dextran sulfate sodium salt
10
30
10
3
30
100
10
0.1
3
0
0 Conc. (μg/mL)
Erythrosine
(B) Moderate tumor promoters 1 14
22
Up-regulated gene numbers
20
Foci/dish
12
18
10
16 14
8
12 6
10
*
8 6
*
*
4
*
**
***
*
**
*
***
*
**
Foci/dish
Up-regulated gene numbers
24
4
** 2
2
Solvent control
Caffeine
1,4-Dioxane
Catechol
Anthralin
Clofibrate
Cholic acid
Wy-14643
30
3
Acetaminophen
10
20
10
3
30
10
3
100
30
10
60
30
0.1
0.05
0.03
0.01
0.6
0.3
0.15
0.08
300
100
30
10
100
30
10
0
0 Conc. (μg/mL)
Profluthrin
14 Up-regulated gene numbers 12
Foci/dish
8
***
Solvent control
Styrene oxide
Piperonyl butoxide
Sodium nitrite
Palytoxin
**
Sorbic acid
Benzoic acid
17β-Estradiol
4
10
3
2
1
0.3
*
0.1
300
*
1
*
100
30
100
0.0001
*
300
***
0.00003
300
10
3
1
o-Phenyl phenol Na
**
2000
*
*
1000
**
0.3
**
0.1
30
10
**
6
Foci/dish
10
3
Up-regulated gene numbers
(C) Moderate tumor promoters 2 24 22 20 18 16 14 12 10 8 6 4 2 0
0 Conc. (μg/mL)
5αDihydrotestosterone
Fig. 1. The results of the in vitro two-stage transformation assay and gene expression assay. (A) Severe tumor promoters, (B) Moderate tumor promoters 1, (C) Moderate tumor promoters 2. Each bar represents the number of genes that were more than 1.5-fold up-regulated compared to the control, as assayed by our gene expression assay. Line graph represents the number of foci/dish in the cell transformation assay. The transformed foci value presented are means ± SD (n = 10). ***P < 0.001, **P < 0.01, *P < 0.05 (vs. control, Wilcoxon rank sum).
3.2. Independent QRT-PCR assay of 63 chemicals To validate the reliability and robustness of this screening assay, the 22 gene markers were tested on the 63 independent test chemicals (test set). The performance of this assay was measured by examining how well the classifier predicted the non-tumor promoter (cell transformation assay negative) or tumor promoter samples (cell transformation assay positive) in the test set. Table 1 and Figs. 1 and 2 show the relative gene expression levels of the 22 markers 48 h after treatment with each of the 63 chemicals and the results of the cell transformation assays. Most of the mod-
erate to severe tumor promoters, as classified by the cell transformation assay, had a high gene expression score, which increased in a dose dependent manner. Some of these chemicals increased the gene expression score at lower doses than the dose determined by the cell transformation assay. Genistein, which was judged to be a moderate tumor promoter by the cell transformation assay, did not score more than two in the gene expression score. Among the 30 non-tumor promoters, 29 did not induce a gene expression score of more than two, and were judged negative. However, simvastatin was judged positive in the screening assay based on gene expression.
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(A) Moderate to slight tumor promoters and simvastatin 14 Up-regulated gene numbers
22 20
12
Foci/dish
18
10
16 14
8
12 6
10 8 6
*
**
*
*
4 2
** **
**
4
*
2
Sodium chloride
Tumor necrosis factor α
Tween80
D-α-tocopherol
Genistein
Propionic acid
β-Carotene
0.03
0.01
0.003
3
10
1
30
100
10
0.3
0.1
0.03
3
10
1
30
100
10
0.005
0.0025
0.001
3000
1000
300
0
Solvent control
Foci/dish
Up-regulated gene numbers
24
0 Conc. (μg/mL)
Simvastatin
(B) Non-tumor promoters 14
22
Up-regulated gene numbers
20
Foci/dish
12
18
10
16 14
8
12 6
10 8
Foci/dish
Up-regulated gene numbers
24
4
6 4
2
2
Solvent control
MeIQ
Pyridine
Benzo[a]pyrene
4-Hexylresorcinol
Acetamide
Ascorbyl palmitate
3-Chloro-1,2propanediol
3
1
0.3
300
100
30
10
3
1
100
30
10
1
0.3
0.1
0.1
0.03
0.01
0.003
300
100
30
10
10
3
1
0
0 Conc. (μg/mL)
PhIP
Fig. 2. The results of the in vitro two-stage transformation assay and gene expression assay. (A) Moderate to slight tumor promoters and simvastatin, (B) Non-tumor promoters. Each bar represents the number of genes that were more than 1.5-fold up-regulated compared to the control, as assayed by our gene expression assay. Line graph represents the number of foci/dish in the cell transformation assay. The transformed foci value presented are means ± SD (n = 10). ***P < 0.001, **P < 0.01, *P < 0.05 (vs. control, Wilcoxon rank sum).
3.3. Accuracy, specificity and sensitivity of the assay Accuracy is defined as the ratio of the number of correctly predicted samples to the total number of samples. Sensitivity is the ratio of the number of correctly predicted (tumor promoter) samples to the total number of that type of sample (tumor promoter). Specificity is the ratio of the number of correctly predicted (non-tumor promoter) samples to the total number of non-tumor promoter. This assay consistently achieves high accuracy, sensitivity, and specificity on the test set. The accuracy, sensitivity, and specificity of our assay system are 96.8%, 97.0% and 96.7%, respectively. Of the moderate or severe tumor promoters, 96.8% (30/31) were detected. 4. Discussion In a previous study we selected 22 gene markers as potential biomarkers for the early detection of tumor promoters, and established an in vitro real-time PCR screening assay for the assessment of the tumor promoting potential of chemicals in BALB/c 3T3 cells (Maeshima et al., 2009). However, the study did not have a completely independent test set, therefore, these markers required further validation. The most straightforward approach for properly evaluating a classifier is to base the evaluation on a separate test set (Dupuy and Simon, 2007). Over-fitting can be detected when using proper cross-validation techniques or independent test set analysis. Only an independent test set, which is not used for determining pre-processing parameters, selection of differentially expressed gene model building or model selection, can be used to estimate the true performance of models. Therefore, we carried
out a new study to estimate the performance of the 22 marker quantitative PCR assay in an unbiased way. Sixty-three chemicals were tested by QRT-PCR and a cell transformation assay. Thirty-three chemicals were positive in the cell transformation assay, and the others were negative. Our gene expression assay judged 32 to be positive among 33 chemicals that were positive in the cell transformation assay, and judged 29 to be negative among 30 that were negative in the cell transformation assay (Table 1). For severe or moderate tumor promoters, our assay showed dose dependent increases in the gene expression scores (Figs. 1 and 2). There was a correlation between the results of our assay and the cell transformation assay results, in BALB/c 3T3 cells. This assay consistently achieved high accuracy, sensitivity, and specificity (96.8%, 97.0% and 96.7%). Our in vitro tumor promotion assay failed to identify genistein as positive (Fig. 2A). Genistein is an isoflavone whose chemical structure resembles estrogen and it interacts with animal and human estrogen receptors. In a previous report, genistein induced apoptosis (Katdare et al., 2002), inhibited cell proliferation (Xiang et al., 2002), or had tumor promoting activity (Murata et al., 2004) in vitro. In in vivo studies of genistein, both a cancer prevention effect (Fritz et al., 1998; Lamartiniere et al., 1995) and a cancer enhancement effect (Rao et al., 1997; Seike et al., 2003) have been reported. However, despite many studies, the effectiveness and the safety of genistein have not been established. Although BALB/c 3T3 cells have been reported not to express estrogen receptors at measurable levels (Gaben and Mester, 1991), genistein (also 17-b-estradiol) was judged to be positive by the cell transformation assay. In a previous report, estrogens and estrogen-like chem-
H. Maeshima et al. / Toxicology in Vitro 24 (2010) 995–1001
icals were shown to have the ability to directly transform cells by multiple mechanisms unrelated to their estrogenicity (Newbold et al., 1990; Tsutsui and Barrett, 1997). Thus, the enhancement of transformed foci by these chemicals might not be mediated through estrogen receptors in this assay. Simvastatin was judged positive on our gene expression assay, but was judged negative by cell transformation assay (Fig. 2A). Simvastatin is a hypolipidemic drug and is considered a non-genotoxic rodent carcinogen (Snyder and Green, 2001). Therefore, it is not surprising that simvastatin was judged positive on our assay. Tumor promoters have been postulated to act via a number of mechanisms, such as inhibition of apoptosis, inhibition of gapjunction intercellular communication, and induction of cell proliferation (Combes, 2000; Tomatis, 1993; Trosko and Ruch, 1998; Wright et al., 1994). This study showed that our assay could detect various types of tumor promoters (metal compounds, peroxisome proliferators, hormonal agent, and marine toxins, Table 1). It may be important to know the mode of action of tumor promoter’s effect. However, as mentioned above, it is not enough to focus on one mode of action for the detection of a tumor promoter, because tumor promoters have various modes of action. Our 22 gene markers are involved in various pathways (cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation) (Table 2). On this point, it seems that our gene expression assay is reasonable. The new in vitro assay must be evaluated with many materials. We have already evaluated 101 chemicals, including 38 chemicals used in a previous study (Maeshima et al., 2009). Our gene expression assay judged 54 to be positive among 55 tumor promoters, and judged 43 to be negative among 46 non-tumor promoters. In addition, our gene expression assay does not require the continued administration of the test chemicals, unlike other in vitro assays, thereby reducing the consumption of the test chemicals. However, more validation is necessary. In the future, a device for the handling of cells, PCR reagents, and test chemicals should be developed to simplify the assay. Tumor promoters often produce tumors only at high doses, and some tumor promoters may even be anticancer agents at low doses (Kinoshita et al., 2006). In addition, it is recognized that chemicals inducing carcinogenesis in rodents may not induce carcinogenesis in humans due to species differences. (Cunningham, 2002; Fung et al., 1995; Ward, 2007). Therefore, our in vitro screening test for predicting the tumor promoting potential of chemicals based on gene expression should be considered as a starting point for further investigations of carcinogenesis. In summary, we used an independent test set to evaluate 22 previously discovered biomarkers for the tumor promoting potential of chemicals. The accuracy, sensitivity, and specificity of our assay system are 96.8%, 97.0%, and 96.7%, respectively. To our knowledge, this study represents the first multigene predictive model for in vitro tumor promotion in BALB/c 3T3 cells. We are confident that this 22 marker gene-based tumor promoting activity test system will become a valuable tool for rapid screening of potential tumor promoters. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tiv.2009.12.013. References Ames, B.N., Lee, F.D., Durston, W.E., 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Sciences of the United States of America 70, 782–786. Barrett, J.C., 1993. Mechanisms of multistep carcinogenesis and carcinogen risk assessment. Environmental Health Perspectives 100, 9–20.
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