ARTICLE pubs.acs.org/est
Tailored Microarray Platform for the Detection of Marine Toxins T. F. H. Bovee,*,† P. J. M. Hendriksen,† L. Portier,† S. Wangz,†,‡ C. T. Elliott,§ H. P. van Egmond,† M. W. F. Nielen,†,|| A. A. C. M. Peijnenburg,† and L. A. P. Hoogenboom† †
)
RIKILT-Institute of Food Safety, Wageningen UR, Business Unit Bioanalysis & Toxicology, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands ‡ Division of Toxicology, Wageningen University and Research Centre, Tuinlaan 5, 6703 HE Wageningen, The Netherlands § Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University, Belfast BT9 5AY, Northern Ireland Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, The Netherlands ABSTRACT: Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave clear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and downregulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRTPCR might a be more suitable approach than a dedicated array.
’ INTRODUCTION Among the fast growing microscopic algae at the base of the marine food chain, there are a few dozen that produce potent toxins and these algae are found worldwide.14 Public health impact from harmful algal blooms occurs when toxic phytoplankton are filtered from the water by fish, crabs, and especially, shellfish that accumulate the algal toxins to levels that are potentially lethal to humans upon consumption.515 Because of the consequences of global and regional climate changes for algae, it is expected that the occurrence, patterns, and chemistries of marine toxins will change. As a result marine toxins present a factor of growing concern.15,16 On the basis of their chemical properties shellfish toxins can be divided into two broad classes: hydrophilic and lipophilic toxins. One the basis of their adverse effects four classes are recognized, amnesic, paralytic, neurotoxic and diarrhetic poisons. The Amnesic and Paralytic Shellfish Poisons, ASPs and PSPs, respectively, are hydrophilic and have a molecular weight (MW) below 500 Da, while the Diarrhetic Shellfish Poisons, DSPs, have strong lipophilic properties and a MW above 600 Da (up to 2000 Da).5,14,17 Their modes of action are different, complex, and not fully understood, but the DSPs okadaic acid (OA) and dinophysistoxins (DTXs) for instance inhibit protein phosphatases, including the serine/threonine phosphatases 1 and 2A (PP1 and PP2A),2,14 while some PSPs affect the nervous transmission by blocking sodium channels.14,1820 PSPs provoke nerve dysfunction and lead to paralysis and death due to r 2011 American Chemical Society
respiratory failure.21 For other sea fish poisons, including ASPs, the modes of action are still unclear.22 Presently the detection of marine toxins is still largely based on in vivo tests in which mice are injected with an extract of the test sample and lethality is the selected end point for a positive test result.5,23 DSPs can also be detected by feeding rats the test sample potentially resulting in diarrhea. Although EU legislation prescribes these in vivo tests for the determination of OA, DTXs, yessotoxins (YTXs), pectenotoxins (PTXs), and AZAs in shellfish, they are considered highly unethical and will be forbidden from 2015 onward.24 Chemical analytical methods are being developed for the known marine toxins 2527 and most recently an LC-MS/MS method has been developed for the detection of the most prominent lipophilic marine toxins.28 However, these methods are expensive and not high-throughput. Even more important is the fact that they are not able to detect presently unknown toxins and cannot correlate concentrations detected to toxicity because of the absence of toxic equivalence factors for individual toxins expressing their relative potency.29 Moreover, the fact that most classes of marine toxins consist of many analogues and the lack of certified standards and reference materials, makes use of Received: April 5, 2011 Accepted: August 19, 2011 Revised: August 19, 2011 Published: August 19, 2011 8965
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Environmental Science & Technology chemical analytical methods for detecting all marine toxins very difficult if not impossible. There are at least 24 PSP type saxitoxins,30 13 DSP type okadaic acid-ester derivatives 2 and 90 DSP type yessotoxin analogues,31 15 different NSP type neurotoxic brevetoxins,32 and around 30 different AZP type azaspiracid analogues.6 The development of suitable alternative test methods to detect marine toxin contaminations, both those that are currently known and those which might emerge, is therefore urgently needed. In vitro bioassays offer the possibility to screen for the presence of whole classes of contaminants and toxins based on their biological effects. As a result, such bioassays are able to detect both known and yet unknown compounds. Well known examples are the use of bacteria to detect antibiotics and the use of genetically modified yeast or mammalian cells for the detection of hormones and dioxin-like compounds.33,34 Toxins of the OA group can be determined by a PP2A enzyme inhibition assay, PTXs and OAs by a cytotoxicity assay based on the BE(2)-M17 neuroblastoma cell line, OAs and YTXs by accumulation of E-cadherin in MCF-7 and Caco-2 cells and SPXs by an inhibition assay based on nicotinic acetylcholine receptor-enriched membranes.3538 However, none of these effect-based in vitro bioassays for marine toxins are suitable enough for routine applications or has been validated successfully. The main objective of the present study was to develop effect-based in vitro bioassays capable of detecting marine toxins by identifying marker genes specifically up- or down regulated in human Caco-2 cells. Human intestinal Caco-2 cells were exposed to pure standards of the lipophilic diarrhetic shellfish poisons AZA1 and DTX1, blank mussel extracts, and to mussel extracts contaminated with AZA1 or DTX1. Seventeen genes were selected that were differentially regulated by examining the gene expression using whole genome Agilent microarrays. Subsequently, specific primers and probes were constructed for the selected 17 marker genes and 2 control genes and used to develop a dedicated low-density microarray which functioning was subsequently tested by exposing Caco-2 cells to pure standards of AZA1, DTX1, and okadaic acid (OA).
’ EXPERIMENTAL SECTION Chemicals. Okadaic acid (OA) and azaspiracid-1 (AZA1) were purchased from the National Research Council, Institute for Marine Biosciences (NRC-CNRC), Halifax, Canada. Dinophysis toxin-1 (DTX1) reference material was kindly donated by Dr P. Hess from the Marine Institute, Oranmore, Ireland. Stock solutions of OA, AZA1 and DTX1 in DMSO were prepared in the concentration range of 150 μM down to 15 nM. DMSO and ethanol were obtained from Merck (Darmstadt, Germany), and β-mercaptoethanol was obtained from Sigma (St. Louis, MO, U.S.A.). Cell Culture and Exposure. The human colorectal adenocarcinoma Caco-2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.) and used at passages 2535 at a seeding density of 60 000 cells/cm2. The BioWhittaker’s Dulbecco modified Eagle’s minimal essential medium with L-glutamine (DMEM, 4.5 g/L glucose) was obtained from Lonza (Verviers, Belgium). Caco-2 cells were grown in DMEM supplemented with nonessential amino acids (NEAA) from MP Biomedicals (Illkirch, France), penicillinstreptomycin from Sigma (St. Louis, MO, U.S.A.), and heat
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inactivated fetal bovine serum (FBS; 10% v/v) from Gibco BRL (Life Technologies Ltd., Paisly, Scotland). For exposure, cells were seeded in 6-wells plates. When cells reached a confluence of approximately 50%, the cells were exposed to one of the three marine toxins o/n. Concentrations tested were 1, 3, 10, 30, and 100 nM for DTX1, 0.03, 0.1, 0.3, 1, 3, 10, and 20 nM for AZA1 and 0.3, 1, 3, 10, 30, and 100 nM for OA for the tube array experiments and 6.25 and 25 nM of DTX1 and AZA1 for the qRT-PCR analysis. Wells contained 2 mL of medium, and the final DMSO concentration was 0.2%. Exposures were performed in triplicate. RNA Extraction, Complementary Strand Synthesis, Amplification, and Biotin-Labeling. The RNA from the exposed Caco-2 cells was extracted using the QIAshredder and RNeasy Mini kits (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocols. In short, the medium was removed from the cells and the cells were lysed with 600 μL f RLT buffer with 1% β-mercaptoethanol. After the extraction of the RNA using the QIAshredder and RNeasy columns, the amount and quality of the RNA was evaluated by UV spectrometry (260 and 280 nm wavelength on the Nanodrop spectrophotometer of Nanodrop technologies). Primerset 1 is a mix of reverse primers for all selected genes and the two control genes. The sequence of these rtprimers is given in Table 4. Multiplex cDNA synthesis was performed with 5 μg of purified RNA and 1.5 μL of 0.1 μM primer set 1 in an end volume of 25 μL using the Omniscript RT kit (Qiagen) according to the manufacturer’s instructions. Tubes were placed into a thermocycler for 30 min at 50 °C and subsequently put on ice. Primerset 2 is a mix of biotin-labeled forward primers (lbprimers) for all selected genes and the two control genes and primerset 3 is a mix of primers complementary to all primers of primerset 1. The sequence of the lbprimers (primer set 2) is given in Table 4. Labeling and a linear amplification was then carried out by adding 1.7 μL 3.0 μM Primer set 2 (with a 50 -biotin label) and 1.7 μL 3.0 μM Primer set 3 (a mix of competitor sequences complementary to the primers in primer set 1 in order to avoid an exponential amplification), followed by a multiplex linear amplification reaction of 40 cycles in a thermocycler (melt 15 min at 95 °C, 40 cycles of 45 s at 94 °C, annealing for 45 s at 56 °C and elongation for 45 s at 76 °C). The purity of the cDNA product was determined by UV spectrometry on the Nanodrop spectrophotometer and the A260/ A280 ratio had to be within the range of 1.82. Low-Density Microarray Hybridizations. Table 3 shows the sequence of the manufactured hybridization probes (hbprobes) that were immobilized on the microchip (microchips of 9 mm2, printed with 156 oligonucleotide probes, manufactured by Alere Technologies GmbH, Jena, Germany), where each gene was represented by two oligonucleotide probes and each oligonucleotide probe was printed in duplicate. Biotin-labeled amplification products were heat-denatured (95 °C for 5 min) and hybridized to the DNA microchips in the array tube format The microchips were equilibrated at 30 °C by rinsing them twice for 5 min with 500 μL hybridization buffer (0.9 M NaCl, 60 mM sodium phosphate, 6 mM EDTA, 0.05% triton X-100, pH 7.4). Hybridizations (60 °C for 1 h) were performed with 16 μg of biotinylated cDNA in 100 μL hybridization buffer. Next, the arrays were washed with 500 μL of 2xSSC containing 0.1% (v/v) Triton X-100 (5 min, 30 °C), with 2xSSC (5 min, 20 °C), and finally with 0.2xSSC (5 min, 30 °C) (1xSSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). In addition the microchip contained spot controls for the biotin-marker. 8966
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Table 1. Qiagen QuantiTect Primer Assay assay name
gene symbol amplified exons amplicon length
Hs_TMEM179B_1_SG TMEM179B
150 bp
Hs_GAPDH_2_SG
GAPDH
1/2/3
Hs_RGS16_1_SG
RGS16
1/2
Hs_DDIT4_1_SG
DDIT4
Hs_NPPB_1SG
NPPB
119 bp 100 bp
Table 2. 2-log-Fold Up- and Down-regulation of 17 Selected Genes and Two Control Genes in Caco-2 Cells Exposed to Blank Mussel Extracts Spiked with Either 6.25 nM AZA1 or DTX1 (Up-regulation Marked with + and Down-regulation Marked with ) as Analyzed on Whole Genome Agilent Microarrays with 60-mer Probes
97 bp 2/3
upon exposure upon exposure
135 bp
Probe Detection and Data Analysis. After blocking with 2% (w/v) milk powder (Bio-Rad, Hercules, CA, U.S.A.) in 500 μL hybridization buffer containing 0.05% (w/v) Triton X-100 (15 min, 30 °C), the arrays were incubated for 15 min at 30 °C with 100 μL of a 1:2500 dilution of horseradish peroxidase-streptavidin conjugates (1 mg/mL; Thermoscientific, Rockford, Canada) in hybridization buffer containing 0.05% (w/v) Triton X-100. Thereafter, the arrays were washed with 500 μL of 2xSSC containing 0.1% (w/v) Triton X-100 (5 min, 30 °C), with 2xSSC (5 min, 20 °C) and 0.2xSSC (5 min, 20 °C). The reaction was initiated by adding 100 μL of TrueBlue Peroxidase substrate (KPL, Gaithersburg, MD, U.S.A.) and the blue precipitates were recorded by light absorption in a dedicated array tube reader ATR01 (Alere Technologies). Data analysis was conducted with the IconoClust and Partisan software version 3.5r (Alere Technologies), and all transcript levels were compared or normalized to the housekeeping control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qRT-PCR Analysis. qRT-PCR controls were performed for DDIT4, RGS16, and NPPB. The RNA from the exposed Caco-2 cells was extracted using the QIAshredder and RNeasy Mini kits as described above. Each sample was processed in triplicate. Synthesis of cDNA was performed on 1 μg RNA isolated from cells exposed to DMSO, DTX1, and AZA1 (both at 6.25 and 25 nM) and from a RNA pool (mix) of these 5 treatments using the BioRad iScript cDNA Synthese Kit with iScript and Reverse Transcript (BioRad 170-8891) in the BioRad iCycler. This cDNA synthesis was performed with the following program: 5 min at 25 °C, 30 min at 42 °C, 5 min at 85 °C, and put on ice. After cDNA synthesis the samples were diluted 100 times and the pool was diluted 10, 31.6, 100, 316, 1000, and 3160 times and used to make a calibration line. Next, qRT-PCR was performed with specific and certified QuantiTect primers (Qiagen, Venlo, The Netherlands) and was carried out in a 15 μL reaction mixture containing 8,5 μL SYBR green (BioRad 170-8880), 2.5 μL of the QuantiTect forward/reverse primer mix, 2 μL RNase free water and 2 μL of diluted cDNA. Reactions were carried out in a BioRad HSP9645 PCR plate. Water and a pool without the addition of the reverse transcript were used as negative controls. The plate was covered with a microseal and centrifuged for 1 min. Thermal cycling was performed in a CFX96 Real-Time System (BioRad) starting with a denaturation step at 95 °C for 3 min, followed by 45 cycles at 65 °C for 35 s for annealing, 95 °C for 10 s for denaturation and extension at 65 °C for 1 min. PCR products were checked by melting curve analysis applying an increment of 0.5 °C per 5 s from 65 to 95 °C. Data were analyzed using BioRad software and GAPDH and TMEM179B as controls for normalization. Expression ratios of the genes under investigation were calculated versus the DMSO control.
gene
spot
intensity rank
intensitya 1 to 100b
to AZA1
to DTX1
AK091132
1.51
0.18
3.4
C21orf129
1.76
1.55
17.8
79
C3orf57 CDKN1C
1.75 0.42
0.12 +2.70
4.7 8.4
56 67
CEACAM1
+2.14
+1.51
22.0
82
DDIT4
+2.40
+1.44
18.7
80
GAPDH control
+0.13
0.14
318.9
99
50
LOC387763
+0.60
+3.52
1.0
22
MAFB
+0.09
+3.50
1.7
34
NPPB
0.07
1.82
52.3
90
OSR2 RGS16
0.85 +0.30
+0.81 +2.79
2.8 1.7
46 34
TFEC
1.55
0.95
2.5
43
TMCC1
+1.16
+0.14
1.9
37
TMEM179B control
0.04
+0.04
3.5
50
TNS4
+2.66
+1.80
1.3
28
TRIB3
+1.11
+0.06
4.7
56
TUBB3
+0.72
+2.25
23.4
83
VASN
0.06
+3.10
4.6
55
a
Spot intensity compared to the median intensity (set on 1) of all spots on the microarray. b The relative intensity of the spot when the spot with highest intensity on the array is set at 100 and the spot with lowest intensity at 1.
’ RESULTS Gene Selection. Caco-2 cells were exposed o/n to the lipophilic diarrhetic shellfish poisons AZA1 and DTX1, both at 6.25 and 25 nM, to blank mussel extracts and to mussel extracts naturally contaminated with AZA1 or DTX1. RNA was extracted, reverse transcribed, labeled and analyzed on whole genome Agilent microarrays containing 44 000 probes of 60 bp representing approximately 25 000 human genes. Seventeen genes were selected based on the response to AZA1 or DTX1 exposure by examining the gene expression using these Agilent microarrays (this microarray study will be described elsewhere). Table 2 shows the 17 selected genes and two control genes (GAPDH and TMEM179B) and their 2 log fold up- or down regulation by either AZA1 or DTX1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transmembrane protein 179B (TMEM179B) were selected as reference genes since these genes were not affected by the treatments according to the Agilent transcriptomics study. The GAPDH is a well-known housekeeping gene that is often used as a control for microarray analysis.31 Table 2 also provides an indication of the expression level of the genes. No matrix effects were observed on the gene expression profile when Caco-2 cells were exposed to blank mussel extracts (data not shown). Indicating that proper marker genes for AZA1 and DTX1 exposure were chosen.As shown in Table 2, genes were selected that were up-regulated by both AZA1 and DTX1, for example, CEACAM1, DDIT4, TNS4, and 8967
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Table 3. Sequence, Length (bp), %GC, and Melt Temperature (Tm) of the Designed Non-Overlapping Hybridization Probes (hbprobes) for the 17 Selected Genes and the Two Control Genes gene
sequence hbprobe
length
% GC
Tm
AK091132-01
CACTAACAGTTCCTTCTGCATCTGTCC
27
48
60.8
AK091132-02
ACACAGTTCCTTTATTTCAGATGTGTTTGTCT
32
34
60.9
C21orf129-01
TCCTCTAGGTGGTATTTCAAAGGGTACG
28
46
60.9
C21orf129-02 C3orf57-01
TCATAAACTGGAGCACATGATCGGGT TCTCCGGTCTAAAGCACAATGTAGCA
26 26
46 46
61.4 61.3
C3orf57-02
ACAGAATATGATTTGTGAGGTAAAGTCACTGT
32
34
60.2
CDKN1C-01
AGTGTACCTTCTCGTGCAGAATACATTTAGA
31
39
61.2
CDKN1C-02
TTTGCACTGAGTTTCAGCAGAGATTAAACA
30
37
61.1
CEACAM1-01
CTGAAAGAGGTACCTGAGTATAGAGAACTCC
31
45
60.6
CEACAM1-02
AGCACAGCATGGATGAGGGAAAG
23
52
60.3
DDIT4-01
AGTAAGATACACAAACCACCTCCACGAC
28
46
61.7
DDIT4-02 GAPDH-01
ACAAACAACAAACACACTTGGTCCCTTC TCTAGACGGCAGGTCAGGTCCA
28 22
43 59
61.9 61.7
GAPDH-02
TCACCACCTTCTTGATGTCATCATATTTGG
30
40
60.7
LOC387763-01
TCAATCCTCCAGACGCAGTAGCAG
24
54
61.5
LOC387763-02
CGTTAAATAAATGCCTTCAGCCATCGC
27
44
61
MAFB-01
CGCCAGCAATTTCAAATGGGAACTT
25
44
60.5
MAFB-02
GCACCATGCGGTTCATACAATCTTGT
26
46
61.7
NPPB-01
CAGCCAGGACTTCCTCTTAATGCC
24
54
60.5
NPPB-02 OSR2-01
CCTTGTGGAATCAGAAGCAGGTGTC ACAAGAAACTAGCATATACTCTTTGTGAAGGG
25 32
52 38
61 60.5
OSR2-02
CAAAGACCCACAACATTATGTACAGAGCT
29
41
60.7
RGS16-01
AGGTTCCCAGCACATTCAGAGGT
23
52
61
RGS16-02
ACAGTGAGGAGTTCCTGACAGGC
23
57
61.2
TFEC-01
TGCTGTTAGATAAGATTCTTGAATTTCGTTTGC
33
33
60.6
TFEC-02
AGCCTTGTATGCCATAAGCAACTGC
25
48
61.4
TMCC1-01
GGTGTGCTCCAGATTGTTGGTTCA
24
50
60.8
TMCC1-02 TMEM179B-01
TGCAGTCCTTCAAGCCACAATTTAGA TGATGGAGTTGCAGAGAGACCTGG
26 24
42 54
60.3 61.2
TMEM179B-02
CTGGGCTTCAGAACAGCTAATTGTAGT
27
44
60.3
TNS4-01
CTGGTCTCAGCCTCTCCTGCAG
22
64
61.6
TNS4-02
CCTGTGACCTTGAGAACCTCATCTCA
26
50
61.1
TRIB3-01
CAGGAGTCCTCCAGGTTCTCCAG
23
61
60.8
TRIB3-02
CAGGGAATCATCTGGCCCAGTCA
23
57
61.4
TUBB3-01
CCTGGAGCTGCAATAAGACAGAGACA
26
50
61.6
TUBB3-02 VASN-01
AGTATCCCCGAAAATATAAACACAAACCAGT TCTTCCCAGAATAAATGGGAAAGGATCTCT
31 30
35 40
60.3 60.7
VASN-02
AAACCAAAGTCCTTGTCTCTGAGTTTGAA
29
38
60.5
TUBB3, up-regulated by AZA1 only, for example, TRIB3, genes that were up-regulated by DTX1 only, for example, CDKN1, MAFB, RGS16, and VASN, down-regulated by both AZA1 and DTX1, for example, C21orf129 and TFEC, down-regulated by AZA1 only, for example, AK091132 and C3orf57, and genes that were down-regulated by DTX1 only, for example, NPPB. DNA Microchip Design. Two nonoverlapping hybridization probes of 2132 bp (hbprobes) were designed for each of the 17 selected genes and the two control genes and these probes were immobilized on the glass chip in the array tube format. Each oligonucleotide hbprobe was printed in duplicates on the microchip. The sequence, %GC and melting temperature (Tm) of the hbprobes are given in Table 3. The advantage of this particular microchip format is that it fits into the bottom of an ordinary reaction tube and, hence, requires no specialized laboratory equipment.
For each target mRNA three types of oligonucleotides were made: (1) a rtprimer, (2) a lbprimer, and (3) a competitor primer complementary to the rtprimer. The rtprimer is used for reverse transcribing the selected mRNA. Subsequently, the lbprimer and competitor primer are used to obtain biotin-labeled sequences. The lbprimer is a forward primer that is 50 -labeled with biotin. This lbprimer is used for the cDNA amplification. The competitor primers are complementary to the rtprimers and are added during the cDNA amplification in order to avoid a PCR-like exponential amplification. The 30 -OH ends of these competitor primers are substituted by an amino group to avoid unwanted extension by DNA polymerase activity. Table 4 shows the sequence, %GC and melt temperature (Tm) of the designed oligonucleotides for the rtprimers and lbprimers. Both the primers and probes were designed as previously described for 8968
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Table 4. Sequence, Length (bp), % GC, and Melt Temperature (Tm) of the Designed Reverse Transcription Primers (rtprimer)a and the 50 -Biotin-Labeled Forward Primers (lbprimer) gene
sequence rtprimer
length % GC 0a
Tm
sequence lbprimer
length % GC
Tm
AK091132
50 -AGTGCAGTTATGAATTGTTAACATGT-3
C21orf129 C3orf57
TTCCGAGTGACGTGCAACT TCTGGTAGGTCTGTTAGGAGTCT
26
31
55.4 50 -CTAAAGCTGACCTAAATGGATCAGT-30
25
40
55.9
19 23
53 48
56.9 CCCTAGGAAAGTAAAACAAGGAGT 56.6 CCTGGAGTTTGAAATACATGTCTCT
24 25
42 40
55.4 56.1
CDKN1C
TCCACGCCTGGACATAAATAGA
22
45
56.2 TGGGACCGTTCATGTAGCAG
20
55
57.1
CEACAM1
TTCAGACATGATCTTAGCCAGGA
23
43
56.2 CCTAAGAGGCTTTCTCCAGGA
21
52
55.8
DDIT4
TCAGTAGTGATGCTCCGATCAG
22
50
56.7 GGTGAAGGAAGAGGCACGT
19
58
56.9
GAPDH
TCAGTGTAGCCCAGGATGC
19
58
56.4 GGAAGCTCACTGGCATGG
18
61
55.4
LOC387763 AGGAGCCTCACAGACATATCC
21
52
55.9 CACACCCATTCACACTCACG
20
55
56.5
MAFB
CTGATGCAGGACAAATATCCACA
23
43
56.1 AGTAAAAGGATTTAAGTTGCACTGAC
26
35
55.6
NPPB OSR2
TGTTGACTTTATTTCACCGTGGA CTAAGTGCATCCAACAAGATCCC
23 23
39 48
55.8 GGATCAGCTCCTCCAGTGG 56.7 ACTGTAGAAAGCCACACACTACT
19 23
63 43
56.5 56.6
RGS16
TCATATCTAGGAAACCAGCTCCA
23
43
55.7 GGTTTCAGCCTGACTGTCTC
20
55
55.5
TFEC
TGTTTTCTTGATCTAACCAATGTCAG
26
35
55.4 AGAAGAGACTGATTTTGCTGAAAGA
25
36
55.9
TMCC1
CCTCATCAGGTTTTCGCATCT
21
48
55.7 TGTAGCATGATTTTCCTTGGGATG
24
42
56.6
TMEM179B TGGAGTAAAACTGCAGAGCAGT
22
45
57.2 GTGTCTGCCTGTATCCTTCGA
21
52
56.8
TNS4
TCATGATAGGAGCTGTAGCAGA
22
45
55.4 GTTGCATTATCTTTGGCCAAGG
22
45
55.8
TRIB3
GAGTATCTCAGGTCCCACGT
20
55
55.5 TCAAGCTGTGTCGCTTTGTC
20
50
56.4
TUBB3 VASN
TTTTAAGGGTATCTGACAGCAATAGA GCCTTCATATCATCGTTTGTCTTACA
26 26
35 38
55.4 CTGCAGTATTTATGGCCTCGT 57 AACTGGAAAGGAAGATGCTTTAGG
21 24
48 42
55.5 56.2
a
Sequences for the competitor primers with the aminolink, in order to prevent an exponential amplification, are complementary to the rtprimer. For example, for the AK091132 marker gene this is as follows: 50 -ACATGTTAACAATTCATAACTGCACT-C7-Aminolink-30 .
the developed low-density tube microarray for the detection of mycotoxins.39 DNA Microchip Results. Caco-2 cells were exposed to different concentrations of AZA1, DTX1, and okadaic acid (OA). After a 24 h exposure, the cells were visually inspected under a microscope and subsequently RNA was extracted from the cells. All cells were viable, except those exposed to 30 and 100 nM DTX1, as these concentrations of DTX1 appeared cytotoxic for the Caco-2 cells. Selected marker genes and control genes in purified RNA (5 μg) were converted to biotin-labeled cDNA (cDNA) using the three sets of oligonucleotide primers targeting the 17 selected mRNA transcripts and 2 control transcripts. The labeled PCR products, 136 to 318 bp in length, were then used for hybridization on the developed low density microarray on the tube format. Staining was performed with a streptavidin coupled with a peroxidase and the addition of tetramethylbenzidine. Figure 1 shows the results on the Array Tube obtained with Caco-2 cells exposed to a DMSO control and standards of 10 nM AZA1, 30 nM DTX1, and 100 nM OA. The square spots represent the biotin controls. The two control genes gave clear spots and five of the 17 selected marker genes gave spot intensities above the spot intensity as obtained with the DMSO control. These 5 marker genes were CEACAM1, DDIT4, TRIB3, TUBB3, and OSR2 (Figure 1 and all indicated in bold in Table 2 for the complementary result on the Agilent array). As shown in Figure 2, the GAPDH control was highly expressed and not affected by any treatment, even not by DTX1 concentrations of 30 and 100 nM that were cytotoxic for the Caco-2 cells (Figure 2a). Similar results were obtained with the second control gene TMEM179B (data not shown). Figure 2 also shows the expression profiles of CEACAM1, DDIT4, TRIB3, TUBB3, and OSR2 in Caco-2 cells exposed to different concentrations of the marine toxins. The CEACAM1 marker gene was up-regulated by AZA1 and DTX1 but less clear
Figure 1. Array tube images obtained on the newly developed array tube for the detection of marine toxins with Caco-2 cells exposed to a DMSO control and standards of DTX1, AZA1, and OA. Spots for GAPDH, CEACAM1, DDIT4, TUBB3, OSR2, and VASN are indicated, while the square spots represent the biotin marker controls.
by OA (figure 2b). The up-regulation of this gene by both AZA1 and DTX1 is in agreement with the outcome of the wholegenome microarray (Table 2). The DDIT4 marker gene was upregulated by AZA1, DTX1, and OA from the lowest exposure 8969
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Figure 2. Expression profiles obtained on the dedicated low-density DNA tube microarray for the GAPDH, CEACAM1, DDIT4, TRIB3, TUBB3, OSR2, and VASN marker genes in Caco-2 cells exposed to different concentrations of marine toxins. Each sample concentration was performed in triplicate and the bars show the mean intensity of the spot as measured by the tube reader (blue precipitation product) and its standard deviation. 8970
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Figure 3. Normalized fold expression of the DDIT4, RGS16, and NPPB marker genes as determined by qRT-PCR of Caco-2 cells exposed to 6.25 and 25 nM of DTX1 or AZA1. Each sample concentration was performed in triplicate and the bars show the fold expression and its standard deviation.
dose onward (Figure 2c). The TRIB3 marker gene was mainly up-regulated by AZA1 and to a much lesser extend by DTX1 (Figure 2d), while TUBB3 was clearly up-regulated by DTX1 and OA but to a much lesser extent by AZA1 (figure 2e). The OSR2 marker gene was not affected by AZA1 but was clearly upregulated by DTX (figure 2f). The array tube results for DDIT4, TRIB3, TUBB3, and OSR2 are also in good agreement with the outcome of the whole-genome microarray (these 5 marker genes are all shown in bold in Table 2). Moreover, Figure 2 shows that the slightly cytotoxic concentration of 30 nM DTX1 inhibited the induction of TRIB3 but not the other genes, while the clear cytotoxic concentration of 100 nM DTX1 also had an inhibiting effect on the expression of CEACAM1, TUBB3, and OSR2. To determine whether the spot intensities on the array tube could be improved for the twelve marker genes that did not reveal spot intensities above background, 2 rounds of linear cDNA amplification were performed instead of one. This gave a slight improvement but only for VASN and TNS4 (data not shown). VASN is a gene up-regulated by DTX1 and OA only (Figure 2g) and TNS4 is a gene that is up-regulated by both AZA1 and DTX1 (Table 2). qRT-PCR Results. Three genes were selected for qRT-PCR analysis, that is, DDIT4, RGS16, and NPPB (underlined in Table 2). DDIT4 was selected as it gave clear spots on the dedicated array tube and RGS16 and NPPB were selected as no spot intensities above the background were obtained for these markers. Figure 3 shows the DDIT4, RGS16, and NPPB qRTPCR results for cells exposed to AZA1 or DTX1. As observed in the microarrays, DDIT4 was clearly up-regulated by AZA1 and DTX1 and both already at the low dose of 6.25 nM, although this was more pronounced for AZA1 than for DTX1. The RGS16 gene was only up-regulated by DTX1 and not by AZA1, whereas NPPB was down-regulated by DTX1 and not by AZA1. These results are completely in line with the whole genome microarray data (underlined in Table 2).
’ DISCUSSION Bioassays can be used as early warning systems for new emerging risks and have already shown their ability to support selection, purification, and identification of unknown compounds in so-called bioassay-directed identification approaches.33,34 Moreover, the combination of receptor- and omics-based assays can also be
helpful to characterize modes of action of newly detected contaminants, for example, as in the case of 2-isopropylthioxanthone. 40 These kind of studies show that chemicalanalytical, bioassay, and -omics-based analyses are complementary and offer the possibility to detect and identify as yet unknown toxic compounds and contaminants. Marine toxins are a problem area, where such an approach is urgently needed. In this study, a tailored microarray platform was designed for the detection of marine toxins based on the differential expression of a set of genes. For this assay an array tube test format was chosen that was previously used for detection of T2/HT2 mycotoxins and estrogens. Exposing Caco-2 cells to different marine toxins resulted in a clear fingerprint on the newly developed dedicated low-density microarray, despite the fact that only 5 out of the 17 selected marker genes gave clear signals. The two control genes were highly expressed, gave clear signals and were not affected by any toxin treatment. The fingerprint of the 5 markers agreed with the whole genome microarray data that were used for the selection of these marker genes: CEACAM1, DDIT4, and TUBB3 were up-regulated by AZA1, DTX1, and OA, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Twelve marker genes gave no detectable signals on the microarray. For the AK091132 gene, it transpired that the forward and backward primers were not properly designed (no PCR product possible). For the other eleven selected markers, the primers and hbprobes or the linear amplification procedure to obtain the labeled PCR product might need further optimization, for example, by using longer hbprobes. The expression level of the genes is very likely another factor that determines the success of detection with the low-density microarray. None of the spots with an intensity below 2.8 times the median intensity of all spots on the whole genome microarray were detected by the dedicated tube array, that is, RGS16, MAFB, LOC387763, TFEC, TMCC1, and TNS4. Moreover, only the up-regulated genes gave clear signals. All genes that were down-regulated, that is, C21orf129 and TFEC by both AZA1 and DTX1, C3orf57 and CDKN1C by AZA1 and NPPB by DTX1, could not be detected by the dedicated array. This left VASN, because it displayed an intensity of 55 on the whole genome microarray and was clearly up-regulated by DTX1 (Table2) but gave no signal on the dedicated array. Figure 2g shows the expression profile of this marker gene and reveals that it was only slightly responsive to 8971
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Environmental Science & Technology DTX1 and less so to OA. Performing 2 rounds of linear cDNA amplification instead of one, only gave a slight improvement for VASN and TNS4. An alternative detection method was therefore setup, qRTPCR analysis. This analysis confirmed that DDIT4 was clearly up-regulated by both AZA1 and DTX1, that RGS16 was upregulated by DTX1 only and that NPPB was down-regulated by DTX1 only. These results were completely in line with the whole genome microarray data, but the up-regulation of RGS16 and the down-regulation of NPPB are not detected by the newly developed tailored microarray. Again, this suggests that either the sensitivity of the tube array format is limited or that the primers and hbprobes in combination with the linear amplification procedure to obtain the labeled PCR product need further optimization. Regarding OA, this toxin is known to elicit effects similar to that of its structure analogue DTX1, and in combination, they act additively.5,14 This is in line with the overall data generated, as these show that OA elicits a pattern on the dedicated array that was more similar to that of DTX1 than that of AZA1. For this reason AZA-like toxicity is often classified as a separate class, although they do show diarrhetic effects in animals and humans. Presently, there are no mode of action based in vitro bioassays for marine toxins that offer the advantage over analytical methods for being capable to detect all, both known and unknown, toxins with a similar mode of action simultaneously. The new dedicated low-density tube microarray for marine toxins requires further improvement. The multiplex PCR targeting 17 marker genes and 2 controls is probably very complicated and might need further optimization while hybridization might be improved by using longer hbprobes. However, the results also showed that the outcomes with these 5 marker genes were comparable with the whole genome microarray data and resulted in clear fingerprints of AZA1, DTX1, and OA. This tube array has the advantage that it can be performed at each lab relatively cheaply: the tube array reader is the only equipment that has to be bought. The tube array, however, also has some disadvantages: the procedure is still labor intensive, and it takes about three days from exposing the cells to shellfish extracts until the evaluation of the tube array results. For testing sea food, results should be provided preferably within 24 h, comparable to the presently used in vivo animal tests, which take 16 to 24 h.5,23 In addition the variation on the results seems quite large (Figure 2). However, the current microarray data enable future developments of other fast in vitro bioassays. One option is the development of a multiplex qRT-PCR. This seems very promising, as the singleplex qRT-PCR analysis targeting the DDIT4, RGS16, and NPPB genes already resulted in a specific pattern for both DTX1 and AZA1 (Figure 3). Other possibilities are the development of molecular beacons targeting one of the marker mRNAs in intact living Caco-2 cells or the development of reporter assays. The promoter region of the DDIT4 marker gene for instance, might be used for the development of such a reporter assay, as DDIT4 was already upregulated by DTX1, AZA1, and OA at the lowest concentrations tested. The current data will thus be helpful to develop methods that can be used for high-throughput quality control of seafood and replacement of the current unethical in vivo tests. The benefits are clear: detection might no longer be dependent on a priori knowledge of the type and chemistry of marine toxins involved, and the output will have a relevance on the bioeffect level.
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’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: +31(0)317480391. Fax: +31(0)317417717.
’ ACKNOWLEDGMENT This work was supported by the Dutch Ministry of Economic Affairs, Agriculture and Innovation (project number WOT-02001-035) and the European Commission, within the sixth Framework “BioCop” project (contract FOOD-CT-2004-06988; www. biocop.org). The authors would like to thank K. Lancova from the University of Z€urich for visiting RIKILT and her help with the software and Arjen Gerssen from RIKILT for the marine toxin standards. ’ REFERENCES (1) Daranas, A. H.; Norte, M.; Fernandez, J. J. Toxic marine microalgae. Toxicon 2001, 39, 1101–1132. (2) Dominguez, H. J.; Paz, B.; Daranas, A. H.; Norte, M.; Franco, J. M.; Fernandez, J. J. Dinoflagellate polyether within the yessotoxin, pectenotoxin, and okadaic acid toxin groups: Characterization, analysis, and human health implications. Toxicon 2010, 56, 191–217. (3) Furey, A.; O’Doherty, S.; O’Callaghan, K.; Lehane, M.; James, K. J. Azaspiracid poisoning (AZP) toxins in shellfish: Toxicological and health considerations. Toxicon 2006, 56, 173–190. (4) Toyofuku, H. Joint FAO/WHO/IOC activities to provide scientific advice on marine biotoxins (research report). Mar. Pollut. Bull. 2006, 52, 1735–1745. (5) EFSA Panel on Contaminants in the Food Chain (CONTAM). Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish—Okadaic acid and analogues. EFSA J. 2008, 589, 162. (6) EFSA Panel on Contaminants in the Food Chain (CONTAM). Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish— Azaspiracids. EFSA J. 2008, 723, 152. (7) EFSA Panel on Contaminants in the Food Chain (CONTAM). Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission on marine biotoxins in shellfish—Yessotoxin group. EFSA J. 2008, 907, 162. (8) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on Marine Biotoxins in Shellfish—Summary on regulated marine biotoxins. EFSA J. 2009, 1306, 123. (9) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on marine biotoxins in shellfish—Domoic acid. . 2009, 1181, 161. (10) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish—Palytoxin group. EFSA J. 2009, 1393, 138. (11) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish—Emerging toxins: Ciguatoxin group. EFSA J. 2010, 1627, 138. (12) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish—Cyclic imines (spirolides, gymnodimines, pinnatoxins, and pteriatoxins). EFSA J. 2010, 1628, 139. (13) EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish Emerging toxins: Brevetoxin group. EFSA J. 2010, 1677, 129. (14) Food and Agriculture Organization. Marine Biotoxins. FAO Food Nutr. Pap. 2004, 80. 8972
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