Effects of Trace Acrylamide Intake in Wistar Rats

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1 Journal of Oleo Science Copyright 2007 by Japan Oil Chemists Society Effects of Trace Acrylamide Intake in Wistar Rats Nagao Totani 1, Miho Yawata 2, Yuko Ojiri 1 and Yoshio Fujioka 1 1 Faculty of Nutrition, Kobe-Gakuin University (518 Arise, Ikawadani-cho, Nishi-ku, Kobe , JAPAN) 2 Department of Home Economics, Shukugawa Gakuin College (6-58 Koshikiiwa-cho, Nishinomiya, Hyogo , JAPAN) Abstract: Frying oil in use of cooking may contain acrylamide formed from frying foodstuffs. We have reported that administration of a diet containing 7% practically used frying oil for 12 weeks damaged liver and kidneys severely in Wistar rats. Then, male Wistar rats were fed ad libitum for 12 weeks a powdered diet (AIN93G; no fat) containing 7 wt% of fresh oil (control group) or frying oil heated with Asn + glucose for 20 h at 180 under a nitrogen flow in order to form acrylamide under the least thermal deterioration (experimental group). The rats were subjected to anthropometric measurements, hematological analyses, and observations of the liver and kidneys. All of the rats grew well, and no gross symptoms attributable to the experimental oil were observed. But the experimental rats had significantly low insulin and triacylglycerol levels. The liver and kidneys from the experimental rats had damages, but the degree of the histological changes looked lighter than that of the rats fed practically used frying oil described above. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also not much increased. Thus, it was suggested that continuous intake of trace acrylamide induced characteristically low serum insulin level and that the effects of the used frying oil on the liver and kidneys were hardly attributable to acrylamide possibly contained therein. Key words: acrylamide, frying oil, insulin, thermally oxidized oil, AST, ALT 1 INTRODUCTION Acrylamide, the potential for carcinogenicity, toxicities of heredity and generation to animals, is known to exist in a considerable amount in a variety of foods ingested daily 1-4) ; ppm in potato chips and ppm in French fries. We reported that content of acrylamide in the used frying oil recovered from food manufacturing companies in Japan (recovered oil) was below detection limit of GC/MS-SIM determination (<0.02 ppm) 5). But trace amounts of acrylamide can be harmful and remarkable existence of acrylamide in frying oil in use cannot be denied because acrylamide is easily polymerized by peroxides after frying practice. On the other hand, in the animal experiment feeding male adult Wistar rats diets containing 7wt% recovered oil and fresh oils, respectively 6, 7), all of the rats grew well, and no gross symptoms attributable to recovered oil were observed. In the serum of the experimental group, no difference was detected in the levels of glucose, triacylglycerol, and phospholipids, but the levels of AST and ALT were significantly higher than those of the controls. And many dark-red patches, and severe necrosis and bleeding were found in the livers of 75% of the experimental rats. The renal cells were also heavily damaged. It is a question if these symptoms are attributable to continuous intake of trace acrylamide. In the present study, frying oil containing trace acrylamide synthesized with Asn and glucose was administered to rats for 12 weeks and the effects on the living body was compared with those by recovered oil described above. 2 EXPERIMENTAL Oil The reference oil (fresh oil: AV 0.1, COV 1.9, POV 4.0 meq/kg, PC 4.2%, Gardner color 1) was made of fresh soybean and rapeseed oils. To 1 L of this oil Asn (Nacalai Tesque, Inc., Kyoto) + glucose (Wako Pure Chemical Industries Ltd., Osaka), 500 ppm each were added, and the mixture was heated at 180 for 20 h in a 2-L four-necked flask Correspondence to: Nagao Totani, Dept. of Nutritional Physiology, Faculty of Nutrition, Kobe-Gakuin University, 518 Arise, Ikawadanicho, Nishi-ku, Kobe , JAPAN totani@nutr.kobegakuin.ac.jp Accepted June 18, 2007 (received for review February 22, 2007) Journal of Oleo Science ISSN print / ISSN online 501

2 N. Totani, M. Yawata, Y. Ojiri and Y. Fujioka under a stream of nitrogen at 3 L/m in order to reduce thermal deterioration. The amount of each nutrient added was based on the minimum amount possible to come in contact with oil constantly during frying practice 8-12). Experimental oil, thus obtained, was allowed to stand at a room temperature for a day to precipitate solid materials, and supernatant was employed for an animal experiment Diets A commercial pelleted diet (Labo MR Stock, Nihon Nosan Kohgyo, Japan) and a powdered AIN93G diet without fat (Japan Clea, Tokyo) were purchased. Using a blender (Kenmix Major, Aicoh, Saitama, Japan), the latter was mixed uniformly with 7 wt% experimental oil or fresh oil. The two kinds of diets, thus prepared, were divided into 12 portions (1120 g/portion) for the 12-week experiment, packed in plastic bags, and sealed under nitrogen flow. The diets were then sterilized by radiant rays within their bags and kept in a refrigerator until use. Fresh diet was provided daily. Bags opened for administration were kept in a refrigerator. Unused diet in the opened bag was discarded after the seventh administration Chemical Analyses Methods for chemical analyses of oil were the same as in our previous papers 6-7). The fatty acid compositions of the fresh oil analyzed as previously 6) were as follows: myristic acid 0.1%; palmitic acid 8.4%; palmitoleic acid 0.1%; stearic acid 3.3%; oleic acid 38.2%; linoleic acid 39.2%; and a- linolenic acid 8.3%; eicosenoic acid 0.6%; others 1.8%. Determination of acrylamide content in the experimental and fresh oils was carried out by Japan Food Research Laboratories as in our previous paper 5), and the contents were found to be 0.28 ppm and below detection limit, respectively. 2 2 Male Wistar rats aged 9 weeks were obtained from Japan SLC, Inc., Shizuoka, Japan, and were housed separately in wire cages at 24 2 and humidity 50 10%, with light from 7:00 to 19:00 at Japan SLC, Inc., Animal Experiment Center, Shizuoka, Japan. Animal care and handling were in accordance with the Ethical Agreement Concerning Care and Use of Laboratory Animals for Research and Education, Kobe-Gakuin University. 2 3 Sixteen animals were maintained on radio-sterilized Labo MR Stock for 1 week of adaptation; animals were then divided into two groups (8 rats/group) by Statlight System, Yukms, Tokyo, Japan so as to make average body weights similar and minimize standard deviation in body weight. One group was fed a diet containing 7 wt% experimental oils, and another group was fed a diet containing 7 wt% fresh oil. All animals were allowed feed and water ad libitum throughout the experiment. Body weight and the amount of feed ingested were determined weekly. After 12 weeks, a fasting period of 18 h was imposed prior to the administration of anesthesia with pentobarbital. Serum was obtained from blood drawn from the abdominal aorta using conventional methods. Livers, kidneys, and adipose tissue were excised, rinsed with cold saline, weighed, and examined microscopically. 2 4 Concentrations of insulin, and serum triacylglycerol and cholesterol (Lipo Search, a high-sensitivity lipoprotein profiling system) were determined by Skylight Biotech Inc., Japan. Determinations of glucose, free fatty acids, and phospholipids were made with Glucose C II-test Wako, NEFA C-test Wako, and Phospholipid C-test Wako (Wako Pure Chemical Industries, Osaka, Japan), respectively. AST and ALT were assayed with Autosera S AST and Autosera S ALT (Daiichi-Kagaku-Yakuhin Co. Ltd., Tokyo, Japan), respectively. 2 5 Fixed liver and kidneys were embedded with paraffin, and microscopic specimens were sliced, then subjected to hematoxylin and eosin stain according to conventional methods. Histological evaluation was made at a magnification of 100 and 400 according to circumstances. 2 6 All the values obtained from animals are revealed as mean SD. Data from 8 animals each for experimental and control groups were analyzed using Student s t-test for unpaired observations and results were considered significant at p< RESULTS 3 1 As shown in, the experimental oil contained 0.28ppm acrylamide. Although Asn, forming colored materials by amino-carbonyl reaction, was heated in the oil, the color score was low because the heating was carried out under nitrogen flow 10, 11). Thermal oxidation was not inhibited completely by flowing nitrogen into the flask but content of polar compounds (PC), and acid value of experimental oil were lower than those of recovered and fresh oils. Carbonyl value (COV) and peroxide value (POV) were higher in the experimental oil than in the recovered oil. The reason why recovered oil had lower COV than the experimental oil was explained in our paper 12) ; briefly, carbonyl compounds vaporize from oil with steam generated from water in frying foodstuffs. The same seems to be applied to the low POV in the recovered oil in addition to unstability 502

3 Effects of Acrylamide of peroxides. As fresh oil is added intermittently in the frying practice, TG content of recovered oil was relatively high. 3 2 Rats did not exhibit diarrhea, seborrhea, dermatitis, or excessive hair loss after the administration of the diet containing the experimental oil described above. The amount of food ingested and body weight increase did not differ among experimental and control groups and the former gradually decreased with slowdown of body weight increase ( ). The weights of organs excised are shown in. No difference in the weights of liver, kidneys, and retroperitoneal fat was detected between groups. 3 3 As shows, there were no difference in levels of glucose, fatty acids, phospholipids, and cholesterol, but triacylglycerol and insulin levels were significantly lower in the experimental group than in the control group. AST and ALT were higher in the experimental group, although not to a significant extent. Fig. 1 Growth Curves of Wistar Rats Fed a Diet Containing 7 wt% Experimental Oil or Fresh Oil for 12 Weeks. Table 1 Chemical Properties of Oils. Experimental oil Fresh oil Recovered oil PC (%) Gardner color COV POV (meq/kg) AV TG (%) Acrylamide(ppm) 0.28 n.d. n.d. B grade recovered vegetable oil, Miyoshi Oil & Fat Co., Ltd., oil used at food-manufacturing companies, data from the reference 7. n.d. : not detected Table 2 Weights of Organs from Wistar Rats Fed a Diet Containing 7% Oil for 12 Weeks. Organs(% body wt.) Experimental oil Fresh oil Recovered oil Liver Kidney left Kidney right Retroperitoneal fat Values are means SD for eight animals. B grade recovered vegetable oil, Miyoshi Oil & Fat Co., Ltd., oil used at food-manufacturing companies, data from the reference

4 N. Totani, M. Yawata, Y. Ojiri and Y. Fujioka The distribution of triacylglycerol and cholesterol in each lipoprotein is shown in. Triacylglycerol content in VLDL and LDL, and cholesterol content in VLDL for the experimental group were significantly different from those of the fresh oil group. This is different from the result of the experiment using the recovered oil 7). 3 4 Histological evaluation of the livers showed light but frequent necrosis and bleeding in the experimental group. No differences in color and size were observed in the kidneys of experimental vs. control rats. But, hydropic degeneration and damages, such as adhesion and narrow space between the glomerulus and Bowman s capsule were observed in the kidneys besides bleeding. 3 5 shows serum levels of AST and ALT, and results of histological observation of liver and kidneys from individual rats. Levels of AST and ALT of experimental rats were normal except those of one rat (Rat No. 1). However, light but frequent histological degeneration and damages were found in the organs from the experimental group as Table 3 Hematological Values in Wistar Rats Fed for 12 Weeks a Diet Containing 7 wt% Oil. Experimental oil Fresh oil Recovered oil Glucose (mg/dl) Triacylglycerol (mg/dl) Free fatty acids (meq/l) Phospholipids (mg/dl) Cholesterol (mg/dl) AST ALT Insulin (ng/ml) Values are means SD for eight animals. p<0.05, significantly different from the value of the fresh oil group (unpaired t-test). B grade recovered vegetable oil, Miyoshi Oil & Fat Co., Ltd., oil used at foodmanufacturing companies, data from the reference 7. Table 4 Distribution of Triacylglycerol and Cholesterol in Lipoproteins of Wistar Rats Fed for 12 Weeks a Diet Containing 7 wt% Experimental Oil or Fresh Oil. Experimental oil Fresh oil Total triacylglycerol (mg/dl) (100%) (100%) Chylomicron (mg/dl) (0.3%) (0.2%) VLDL (mg/dl) (89.8%) (89.9%) LDL (mg/dl) (7.6%) (7.8%) HDL (mg/dl) (2.3%) (2.1%) Total Cholesterol (mg/dl) (100%) (100%) Chylomicron (mg/dl) (0.0%) (0.0%) VLDL (mg/dl) (8.3%) (9.8%) LDL (mg/dl) (12.0%) (10.0%) HDL (mg/dl) (79.7%) (80.1%) Values are means SD for eight animals. p<0.05, significantly different from the value of the fresh oil group (unpaired t-test). 504

5 Effects of Acrylamide Table 5 Correlation between AST, ALT, and Histological Changes in Liver and Kidneys from Wistar Rats Fed for 12 Weeks a Diet Containing 7 wt% Oil. Rat No. 1 2 Experimental oil Fresh oil Recovered oil AST ALT Rat No. 1 2 AST ALT Rat No. 1 2 AST EO1 N + B FO RO1 N + B EO2 N B FO RO EO3 B FO RO3 N + B EO4 N FO4 N + B RO4 B EO5 N FO RO5 N + B EO6 N B FO RO6 N + B B EO7 N + B B FO RO7 B EO8 B FO RO8 B B : Necrosis (N)/bleeding (B) in liver, 2: Bleeding (B) in kidneys. B grade recovered vegetable oil, Miyoshi Oil & Fat Co., Ltd., oil used at food-manufacturing companies, data from the reference 7. ALT described in DISCUSSION Effects of acrylamide in an amount possible to be formed in oil during frying practice on serum compositions and organs of rats were investigated in order to clarify the cause of the damages to the liver and kidneys when deteriorated oil was ingested. COV and PC of the experimental oil were lower than those of the oil heated under air (COV 31.9, PC 20.5%) 5), but thermal oxidation was not fully inhibited probably because oxygen dissolved in the oil before heating was not removed. Still the chemical properties of the experimental oil were comparable to, or better than those of the recovered oil except acrylamide content. Asn is very reactive with carbonyl compounds in oxidized frying oil 8, 11) and forms browning substances (amino-carbonyl reaction), but color deterioration of the experimental oil, in addition to triacylglycerol decrease, was not drastic due to the heating under a nitrogen flow 8, 10-13). In contrast to the practical frying taking water through frying foodstuffs into oil, AV of the experimental oil did not increase at all. Acrylamide content, 0.28 ppm, was at the lower end of the content in potato chips described above. According to the Swedish and American surveys on ingestion amount of acrylamide per capita, it sums up about mg/day 14). When a man takes 1000 g foods per day, average intake of acrylamide is calculated to be ppm. In this study, a rat ate about 16 g/day of the diet containing 7 wt% experimental oil so that the rat ingested about the same level of acrylamide as humans do (16 g 7% 0.28 ppm/16 g = 0.02 ppm). The experimental oil had characteristically unpleasant smell but influenced neither the appetite of rats nor the ingestion amount of the diet. No difference was found in weights of body, liver, kidneys and retroperitoneal fat of the experimental and the control groups, suggesting that experimental oil was the same as fresh oil in nutrition efficiency, although the chemical properties of the experimental oil were inferior to those of fresh oil. In our another experiment feeding Wistar rats a diet containing oils heated with gluten (OG) 15) or wheat starch (OS) 15), weights of body, liver, kidneys and retroperitoneal fat of the experimental and the control groups also showed no difference. The properties of the oils administered were as follows; PC, COV and POV of the heated oils were 21.6%, 34.1 and 63.4 meq/kg for OG, and 24.7%, 34.2 and 80.0 meq/kg for OS. Thus, COV and POV of the experimental oil in the present study, higher than those of recovered oil, are not likely to influence the following translation of the result. The significant decrease of serum insulin level in the experimental group was very characteristic, while the level was not changed when fresh oil, recovered oil, OG, and OS were administered to rats; see for recovered oil, for OG 15) and ) for OS. When all the results of the experimental group were compared with those from rats fed a diet containing recovered oil, OG, and OS, it was strongly supported that ingestion of acrylamide induced a low insulin level and that the damages to liver and kidneys were related to thermal deterioration products of oil, such as PC 16) and others. A low level of insulin decreases intake of serum glucose into cells, resulting in a high serum glucose level. But in the present study no difference was found in the serum glucose level 505

6 N. Totani, M. Yawata, Y. Ojiri and Y. Fujioka between the experimental and control groups. This is probably because the fasting period prior to the administration of anesthesia was long enough for low insulin level to decrease glucose level to the level shown in. The significantly low triacylglycerol content seems to be due to a low level of insulin which inhibits the production of camp followed by inhibitions of both camp-dependant protein kinase activity and triacyglycerol decomposition 17). In conclusion, it was found that intake of acrylamide decreased serum insulin level drastically and that the effects of recovered oil, severe damages to the liver and kidneys, were hardly attributable to acrylamide possibly contained therein. ACKNOWLEDGMENT We are very grateful to Mr. Nobuzoh Iwa, Pathology Department, Municipal Hospital, Kashihara, Osaka, for the interpretation of histological specimens of liver and kidneys. 1. Rosen, J.; Hellenas, K. Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 127, (2002). 2. Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Tornqvist, M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem. 50, (2002). 3. Tareke, E.; Rydberg, P.; Karlsson P.; Eriksson, S.; Tornqvist, M. Acrylamide: A cooking carcinogen? Chem. REs. Toxicol. 13, (2000). 4. Ahn, J.S.; Castle, L.; Lloyd D.B.A.S.; Philo, M.R.; Speck, D.R. Verification of the findings of acrylamide in heated foods. Food Addit. Contam. 19, (2002). 5. Totani, N.; Yawata, M.; Takada, M.; Moriya, M. Acrylamide content of commercial frying oil. J. Oleo Sci. 56, (2007). 6. Totani, N.; Satoh, K.; Tsuji, S.; Yamaguchi, A. Effects of deteriorated frying oil in Wister rats. J. Oleo Sci. 55, (2006). 7. Totani, N.; Ojiri, Y. Mild ingestion of used frying oil damages hepatic and renal cells in Wistar rats. J. Oleo Sci. 56, (2007). 8. Totani, N.; Kuzume, T.; Yamaguchi, A.; Takada, M.; Moriya, M. Amino acids brown oil during frying. J. Oleo Sci. 55, (2006). 9. Totani, N.; Ohno, C.; Yamaguchi, A. Is the frying oil in deep-fried foods safe? J. Oleo Sci. 55, (2006). 10. Totani, N. Thermal deterioration of edible oil used in food manufacturing companies. J. Oleo Sci. 6, (2006). 11. Totani, N. A small reduction in atmospheric oxygen decreases thermal deterioration of oil during frying. J. Oleo Sci. 55, (2006). 12. Totani, N.; Ono, M.; Burenjargal, M.; Ojiri, Y. Carbonyl compounds vaporize from oil with stream during deepfrying. J. Oleo Sci. 56, (2007). 13. Totani, N.; Yamaguchi, A.; Takada, M.; Moriya, M. Color deterioration of oil during frying. J. Oleo Sci. 55, (2006). 14. Mucci, L.A.; Dickman, P.W.; Steineck, G.; Adami, H.O.; Augustsson, K. Dietary acrylamide and cancer risk: additional data on coffee. Brit. J. Cancer. 89, (2003). 15. Totani, N.; Yawata, M.; Ojiri, Y.; Takada, M. Thermal deterioration of oil and frying foodstuffs. J. Oleo Sci. 56 (2007). 16. Soriguer, F.; Rojo-Martinez, G.; Dobarganes, M.C.; Garcia-Almeida, J.M.; Esteva, I.; Beltran, M.; Ruiz deadana, M.S.; Tinahones, F.; Gomez-Zumaquero, J.M.; Garcia-Fuentes, E.; Gonzalez-Romero, S. Hypertension is related to the degradation of dietary frying oils. Am. J. Clin. Nutr. 78, (2003). 17. Oku, T.; Takahashi, M. Biochemistry. Nankodo, Tokyo p.120 (2001). 506

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