A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide

A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide

Available online at www.sciencedirect.com Toxicology Letters 175 (2007) 82–88 A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food Koichi Hasegawa a,b , Satsuki Miwa a , Tomoko Tajima c , Kaname Tsutsumiuchi a,c , Hajime Taniguchi d , Johji Miwa a,c,∗ a b Institute for Biological Function, Chubu University, 1200 Matsumoto, Kasugai 487-8501, Japan Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan c Graduate School of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto, Kasugai 487-8501, Japan d Department of Environmental Bioresource, Ishikawa Prefectural University, 308-1 Suematsu, Nonoichi, Ishikawa 921-8836, Japan Received 20 August 2007; received in revised form 25 September 2007; accepted 25 September 2007 Available online October 2007 Abstract By DNA microarray and protein 2-DE screens for Caenorhabditis elegans genes up-regulated by acrylamide, we selected the gst-4 gene and constructed a gst::gfp fusion gene, which was used to transform C elegans into a biosensor for acrylamide This biosensor detects acrylamide as a GFP-expression signal in a dose- and time-dependent manner When the biosensor was exposed to acrylamide together with commercially available powdered green tea, GFP levels decreased to the control level, suggestive of acrylamide detoxification or prevention of GST induction The present methodology should be applicable for screening of not only harmful substances but also substances that reduce or counteract their harmfulness or action, with appropriately constructed visible biosensors © 2007 Elsevier Ireland Ltd All rights reserved Keywords: Biosensor; Acrylamide; GST; Food; Caenorhabditis elegans Introduction In April 2002, the Swedish National Food Agency and Stockholm University reported that acrylamide was formed in high concentrations of 30–2300 ␮g/kg during the frying or baking of carbohydrate-rich foods ∗ Corresponding author at: Institute for Biological Function, Chubu University, 1200 Matsumoto, Kasugai 487-8501, Japan Tel.: +81 568 51 6218; fax: +81 568 51 6218 E-mail address: miwa@isc.chubu.ac.jp (J Miwa) (Tareke et al., 2002); 64–5000 ␮g/kg were reported in potato-based snacks (the Japanese National Food Research Institute (http://aa.iacfc.affrc.go.jp/en/), 2002; Tsutsumiuchi et al., 2004) The compound is produced by the Maillard reaction during heat treatment of foods (Mottram et al., 2002; Stadler et al., 2002) The nontoxic acrylamide polymer is commonly used in chemical paper and fibers, soil stabilizers, plastic materials, gel electrophoresis, and so on In contrast, the acrylamide monomer, a known industrial hazard, has long been studied and shown to exhibit neurotoxicity in vertebrates, 0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd All rights reserved doi:10.1016/j.toxlet.2007.09.013 K Hasegawa et al / Toxicology Letters 175 (2007) 82–88 mutagenicity in somatic and germ cells, and carcinogenicity in experimental animals Thus, the finding of monomeric acrylamide in our daily diet focused the world’s attention on this shocking public health problem (Friedman, 2003) In this report, we selected the gst-4 gene, which encodes a human homologue of glutathione-requiring prostaglandin d synthase and was most strongly upregulated after exposure to acrylamide (to be published elsewhere), and constructed a gst-4::gfp translational fusion gene to transform Caenorhabditis elegans into a biosensor for acrylamide (MJCU017) As a whole animal system, it can directly demonstrate the active and perhaps harmful dose of a substance, such as acrylamide Here we report the use of this biosensor and CL2166 (transcriptional reporter transgenic C elegans) to establish a rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food 83 of S medium containing × 109 cells/mL of E coli OP50, 500 mg/L of acrylamide with or without several common foods and grown at 25 ◦ C The common foods used were powdered green tea (Ohi Ocha Koiaji, Itoen, Tokyo, Japan), instant coffee (Nescafe Gold Blend, Nestle Japan, Kobe, Japan), tomato juice (Delmonte Tomato Juice, Kikkoman, Chiba, Japan), and powdered sports drink (Pocari Sweat, Otsuka Seiyaku, Tokyo, Japan) After h, h, and h of incubation, the transgenic animals in mL of S medium were collected, washed with M9 buffer, and re-suspended in M9 buffer at 50 animals per 100 ␮L, which was put into each well (100 ␮L per well × 8–12) of a B & W IsoPlate-96 Plates were monitored for the GFP-expression signal as was done for GFP signal kinetics 2.4 Statistical analysis Paired Student’s t-test (Microsoft Excel) was used to determine the significance of differences in the mean GFP signal values Results Materials and methods 2.1 Nematode strains and culturing C elegans, unc-119 (ed3) (Maduro and Pilgrim, 1996) and CL2166 (dvIs19[pAF15(gst-4(727 bp)::gfp::NLS)]) (Link and Johnson, 2002) were obtained from the Caenorhabditis elegans Genetics Center (University of Minnesota, Minnesota, Minneapolis, MN, USA) MJCU017 (Is [gst-4(1491 bp)::gfp unc-119+ ]) was also used (to be published elsewhere) The nematodes were cultured and handled essentially as described by Brenner (1974) 2.2 GFP signal kinetics Synchronized L1-stage animals were transferred onto NGM plates seeded with E coli OP50 and grown at 20 ◦ C for 48 h to reach late L4 stage Fifty L4-synchronized (CL2166) or 100 (MJCU017) animals were placed into each well of a B & W IsoPlate-96 (PerkinElmer, Massachusetts, MA, USA) containing E coli OP50 (109 cells/mL) and acrylamide (1 g/L, 500 mg/L, 400 mg/L, 300 mg/L, 200 mg/L, 100 mg/L, 50 mg/L) or control without acrylamide in S medium (Stiernagle, 1999), at a total volume of 100 ␮L per well Plates were sealed with optical adhesive covers (ABI Prism, Foster City, CA, USA) and incubated at 20 ◦ C or 25 ◦ C The GFP fluorescence was measured once every hour with a Wallac 1420 ARVOsx multilabel counter (PerkinElmer) The GFP signal value for each well was the mean value of three measurements; whereas the value for each acrylamide concentration and the control was the mean value from to 12 wells 2.3 Co-feeding experiment About 5000 transgenic animals (CL2166), synchronized at late L4 stage as described above, were transferred into 10 mL 3.1 gst-4 expression pattern in vivo We constructed a gst-4::gfp translational fusion gene and made the transgenic line MJCU017 having a chromosomally integrated gst-4::gfp fusion gene This line, hereinafter called an acrylamide biosensor, emitted no detectable GFP signal in the absence of acrylamide, but emitted a very strong GFP signal from the whole body with 500 mg/L of acrylamide (Fig 1) In contrast, CL2166 line, which contains a chromosomally integrated transcriptional reporter (gst-4 promoter drives gfp transcription) (Link and Johnson, 2002), constitutively emitted GFP signals in the body-wall muscle without acrylamide, although its GFP signal pattern was the same as that for MJCU017 upon exposure to acrylamide (Fig 1) 3.2 gst-4 expression increases by acrylamide in a dose- and time-dependent manner Fig shows the kinetics of GST expression in response to acrylamide exposure at concentrations from 50 mg/L to g/L Columns indicate the mean values ± S.E.M of GFP signals at various acrylamide concentrations compared with that of the control (100%) from four independent experiments (or plates) GFP signals increased in a dose- and time-dependent manner in both transgenic nematodes, and the signals increased faster and their peaks were higher at 25 ◦ C than at 20 ◦ C Because CL2166 recorded much higher sensitivity and signal peaks than did MJCU017, we chose the more sen- 84 K Hasegawa et al / Toxicology Letters 175 (2007) 82–88 Fig GST-expression patterns in MJCU017 (A and B) and CL2166 (C and D) (A) MJCU017 without acrylamide (B) MJCU017 with 500 mg/L of acrylamide (C) CL2166 without acrylamide (D) CL2166 with 500 mg/L of acrylamide Scale bars, 200 ␮m sitive and faster combination of CL2166 at 25 ◦ C for screening of common foods that reduce the action of acrylamide 3.3 Powdered tea prevents gst-4 induction To screen for common foods that reduce the action of acrylamide, the acrylamide biosensors were grown on S media containing 500 mg/L of acrylamide, with or without common foods Fig shows the results of such “co-feeding” experiments with commercially available powdered green tea, instant coffee, tomato juice, and powdered sports drink Columns indicate the mean values ± S.E.M of GFP signals from six (green tea) or three (coffee, tomato juice, and sports drink) independent experiments, as compared with that for the control (100%) In Fig 3A–C, Tea was g/L (powdered green tea), Coffee was 14 g/L (instant coffee), and PS was 74 g/L (powdered sports drink), all at the makers’ recom- mended concentrations for drinking; and Tea 2, Coffee 2, and PS or Tea 3, Coffee 3, and PS were the 1/10 or 1/100 diluted concentrations, respectively In Fig 3D, 1% tomato contained 100 ␮L of tomato juice in 10 mL of S medium, and 0.1% tomato and 0.01% tomato were 1/10 and 1/100 diluted, respectively When the acrylamide biosensors were cultured in S medium containing several common foods only, the GFP-expression signals differed little from that of the control without acrylamide (Fig 3A–D); and with acrylamide only, the GFP signals increased time-dependently (Fig 3A–D) However, when the acrylamide biosensors were cultured in S medium containing acrylamide together with g/L of powdered green tea (Tea + AA) and 74 g/L of powdered sports drink (PS + AA), the GFP-expression signals decreased to the control level (Fig 3A and C) After the signal measurements, the acrylamide biosensors were collected from each of their corresponding wells and checked for their viability and their GFP expression K Hasegawa et al / Toxicology Letters 175 (2007) 82–88 85 Fig Kinetics of GST expression by MJCU017 (A and C) and CL2166 (B and D) acrylamide biosensors exposed to acrylamide at concentrations from 50 mg/L to g/L Columns indicate the mean values ± S.E.M of five independent experiments (plates) GFP signals were measured at 20 ◦ C (A and B) or 25 ◦ C (C and D) All nematodes were alive after measurement The significant differences in GFP signal relative to the control are indicated for the first appearance at each acrylamide concentration as determined by the paired Student’s t-test; *P < 0.05 under a fluorescence dissection microscope The biosensors in the 74 g/L of powdered sports drink (PS and PS + AA) were all shriveled and dead (Fig 3C), and the death of the animals should explain the lack of GFP signal All acrylamide biosensors, however, were alive in the g/L of powdered tea (Tea and Tea + AA) The co-feeding result thus suggests that the powdered tea used must have contained some substance(s) that prevented acrylamide from inducing GST and renders this tea a candidate for an acrylamide detoxifier or modifier Discussion Glutathione S-transferases (GSTs) comprise a large family of enzymes whose members generally exist in every organism from bacteria to humans (Vuilleumier and Pagni, 2002) GSTs are considered one of the major players in the phase II detoxification of both endogenous products of oxidative stress and electrophilic xenobi- otics GST binds with glutathione (GSH), a tripeptide thiol, and catalyzes its conjugation with target substrates to enable their excretion from cells About 50 gst genes have been identified in the C elegans genome (WormBase, http://www.wormbase.org/), and gst-4 expression was reported to be dramatically induced by oxidative stressors such as paraquat, juglon, plumbagin, hyperbaric oxygen treatment, or the endocrine active substances diethylstilbestrol and progesterone (Link and Johnson, 2002; Tawe et al., 1998; Cutodia et al., 2001; Leiers et al., 2003; Reichert and Menzel, 2005) Our DNA microarray and protein 2-DE data obtained for acrylamide also showed that gst-4 was the most highly up-regulated gene (to be published elsewhere) For these reasons, we selected the gst-4 gene as the most suitable molecule for the acrylamide biosensor The differences in GFP signal sensitivity between MJCU017 and CL2166 might be caused by their transgenic structures: CL2166 had a transcriptional reporter without the 86 K Hasegawa et al / Toxicology Letters 175 (2007) 82–88 Fig Co-feeding experiments Columns indicate the mean values ± S.E.M of six (A) or three (B–D) independent experiments (plates) (A) Cofeeding with powdered green tea: AA, 500 mg/L of acrylamide; Tea 1, g/L; Tea 2, 0.8 g/L; Tea 3, 0.08 g/L When biosensors were cultured in acrylamide with g/L of powdered green tea (Tea + AA), GFP-expression signals remained at the control level (B) Co-feeding with instant coffee: AA, 500 mg/L of acrylamide; Coffee 1, 14 g/L; Coffee 2, 1.4 g/L; Coffee 3, 0.14 g/L (C) Co-feeding with powdered sports drink: AA, 500 mg/L of acrylamide; PS 1, 74 g/L; PS 2, 7.4 g/L; PS 3, 0.74 g/L GFP-expression signals did not increase in 74 g/L of powdered drink (PS + AA), but all biosensors died apparently from osmotic stress (D) Co-feeding with tomato juice: AA, 500 mg/L of acrylamide; Tomato 1%, 100 ␮L/10 mL; Tomato 0.1%, 10 ␮L/10 mL The significant difference in GFP signal of co-fed biosensors relative to the AA plate at each time point was determined by the paired Student’s t-test; *P < 0.05 gst-4 coding region, whereas MJCU017 comprised a translational reporter with it The acrylamide biosensor introduced here provides an easily usable and available, rapid and inexpensive method to detect acrylamide, a harmful substance in food, not to mention that this C elegans-based biosensor reproduces rapidly, whereby one biosensor reproduces to become million in a week Although man-made machines offer superior sensitivity to detect lower concentrations of chemicals (Tsutsumiuchi et al., 2004), they can only tell us the amounts of known chemical compounds In contrast to machines, our biosensor not only tells us what these known chemicals to living forms, but it can also the same for totally unknown chemicals Since our method utilizes natural biological responses of whole live animals against a xenobiotic toxin, it also tells us the threshold concentration of a toxin that is capable of triggering the animal’s natural response against it, as reported here There are also other reports that transgenic C elegans were developed as biosensors against xenobiotics For example, a heat shock protein promoter-driven lacZ was used as a reporter to detect a fungicide (captan) (Jones et al., 1996), heavy metal pollution in water (Mutwakil et al., 1997), dithiocarbamate fungicides (Guven et al., 1999), and pharmaceutical compounds (EGFR kinase inhibitors) (Dengg and van Meel, 2004), and a constitutive promoter let-858-driven lacZ was used for detecting K Hasegawa et al / Toxicology Letters 175 (2007) 82–88 heavy metal and xenobiotics (3, 5-DCP) (Lagido et al., 2001) By using the gfp reporter, gene expression patterns can be rapidly and directly identified in living animals without the need for fixing or staining A small heat shock promoter-driven gfp was used to detect heat and oxidative stress, beta amyloid peptide (Link et al., 1999), and microwave stress by a nonthermal mechanism (de Pomerai et al., 2000), and a gst-4 promoter-driven gfp was used for detection of oxidative stresses (Link and Johnson, 2002; Leiers et al., 2003) In addition to the acrylamide-detection method presented here, we have also shown that the same method can be used to find common foods that may reduce or inactivate the action of acrylamide, which was used as a model compound of oxidative-stress-producing xenobiotics By this method, we found one candidate from four commercially available common foods examined (Fig 3) When biosensors were cultured in S medium containing acrylamide at 500 mg/L together with g/L of powdered green tea, the GFP-expression signals remained at the levels of the control while almost all biosensors in g/L of powdered green tea were healthy This result apparently suggests that the green tea used prevented GST induction despite the presence of the very high concentration of acrylamide, which usually makes biosensors (nematodes) very sick Although the animals showed significantly increased GFP signals in 100 mg/L of acrylamide after 6–9 h of exposure (Fig 2), for these experiments, we chose the acrylamide concentration of 500 mg/L, which is 100 times the level of acrylamide in some potato-based snacks and elicits its significant response in only 3–5 h, as a suitably severe condition under which to detect acrylamide-counteracting foods After such screening, we can take the next steps of identifying acting components in the tea and finding the exact mechanism of how they work as well The major components in the powdered green tea we used are catechins, which comprise (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epigallocatechin-3-gallate (EGCG) They have very high-antioxidant activity and reportedly have a protective effect against a variety of cancers, such as lung, prostate, and breast cancers as well as common diseases (Yang et al., 2002) Other components such as vitamin C, caffeine, or the additives of dextrin and cyclodextrin might also have taken part in inhibiting GST induction We have presented the acrylamide-detecting biosensor that not only detects acrylamide but also and importantly finds candidates for common foods that may reduce or inactivate the action of acrylamide, as 87 a model compound of oxidative-stress-producing xenobiotics One of the essential features of the present methodology is that it requires no a priori knowledge about either the substances to test for their effects on the animal or the genes to be used for constructing biosensors; we simply choose those genes that are up-regulated by any given substances tested, whether they are known or unknown Appropriately constructed visible biosensors can thus be tailor-made for specific substances we want to detect, or whose harmful effects we want to reduce, as demonstrated in this report The methodology should be applicable to detect a wide range of harmful substances in our diet or environment and to screen foods or other edible substances that could reduce their harmful effects and counteract their actions Acknowledgement This work was supported by a grant from the HighTech Research Center Establishment Project from the Japanese Ministry of Education, Culture, Sports, Science and Technology References Brenner, S., 1974 The genetics of the nematode Caenorhabditis elegans Genetics 77, 71–94 Cutodia, N., Won, S.J., Novillo, A., Wieland, M., Li, C., Callard, I.P., 2001 Caenorhabditis elegans as an environmental monitor using DNA microarray analysis Ann N.Y Acad Sci 948, 32–42 de Pomerai, D., Daniells, C., David, H., Allan, J., Duce, 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action of acrylamide, a harmful substance... and PS + AA) were all shriveled and dead (Fig 3C), and the death of the animals should explain the lack of GFP signal All acrylamide biosensors, however, were alive in the g/L of powdered tea