COMPARATIVE EVALUATION OF DIABETOGENIC AND MUTAGENIC POTENTIAL OF ARTIFICIAL SWEETENERS - ASPARTAME, ACESULFAME-K AND SUCRALOSE

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1 Review Article COMPARATIVE EVALUATION OF DIABETOGENIC AND MUTAGENIC POTENTIAL OF ARTIFICIAL SWEETENERS - ASPARTAME, ACESULFAME-K AND SUCRALOSE Shstry C.S., Ytheesh C.K., Aswthnryn B.J. 1 Deprtment of Phrmcology, N.G.S.M. Institute of Phrmceuticl Sciences, Pneer, Derlktte, Mnglore, Krntk Indi Correspondence: Shstry C.S. Deprtment of Phrmcology, NGSM Institute of Phrmceuticl Sciences, Derlktte, Mnglore , Krntk, Indi Mobile : E-mil : drcshstry@gmil.com Abstrct : Objectives: Artificil sweeteners provide the sweetness of sugr without clories. Since from discovery, sfety of rtificil sweeteners hs been controversil s they directly or indirectly link to induce crcinogenic nd genotoxic risks. Hence the present study ws undertken to compre the dibetogenic nd mutgenic potentil of most widely using rtificil sweeteners; sprtme, cesulfme-k, nd sucrlose. Methods: Dibetic potentil is ssessed by scending repeted dose study in which cceptble dily intke (ADI) dose of rtificil sweeteners fter converting humn dose to niml dose using stndrd reference tble nd dministered up to 13 weeks with 3 different phses in n scending mnner on experimentl rts. Mutgenic potentil ws ccessed by Ames test with nd without metbolic ctivtion using Slmonell typhimurium strins TA 97 nd TA1535. Results: At ADI doses between 0-3 weeks, no significnt chnges but fter 13 weeks significnt increse ws observed in the levels of fsting blood glucose, glycted hemoglobin, totl cholesterol, triglyceride, LDL nd VLDL in ll rtificil sweetener groups. Sucrlose showed comprtively less increse which ws supported by histology reports. In Ames mutgenic ssy sprtme, cesulfme-k nd sucrlose gve negtive results. Conclusion: Asprtme, cesulfme-k nd sucrlose were found to exhibit dibetogenic effect t higher dose levels but they were sfer to use t ADI doses nd non mutgenic compounds. Comprtively sucrlose is sfer thn sprtme nd cesulfme-k. Hence these rtificil sweeteners should be used with cution nd over usge is not pprecited. Keywords : Artificil sweeteners, sprtme, cesulfme-k, sucrlose, dibetogenic, mutgenic. NUJHS Vol. 2, No.3, September 2012, Nitte University Journl of Helth Science Introduction : Artificil sweeteners include substnces from severl different chemicl clsses tht interct with tste receptors nd typiclly exceed the sweetness of sucrose by fctor of 30 to 13,000 times but hve no or low clories. They provide only sweetness but not the dily clorie needs [1]. Due to their intense sweetness they re needed in smll Access this rticle online Quick Response Code quntity nd hence re economicl. Currently FDA p p r o v e d r t i f i c i l s w e e t e n e r s f o r c o n s u m p t i o n r e cesulfme-k, sprtme, neotme, scchrin, nd sucrlose, out of which most extensively used sweeteners re sprtme, sucrlose nd cesulfme-k [2]. As the rtificil sweeteners provide the sweetness of sugr without clories, public helth ttention hs turned to reversing the obesity epidemics in the individuls of ll ges by choosing to use the products contining rtificil sweeteners. Hence the use of low-clorie, sugr-free products tripled in the lst two decdes of the 20th century. In the United Sttes lone, more thn 150 million people use these products regulrly. However sfety of these rtificil sweeteners is unresolved nd controversil. Dibetes mellitus hs now ssumed epidemic proportions in mny countries of the world. With the present popultion of 19.4 million dibetics, nd pproximtely 60 COMPARATIVE EVALUATION - Shstry C.S. 80

2 Nitte University Journl of Helth Science million by the yer 2025, Indi would rnk first in its shre of the globl burden of dibetes. When portion of the popultion suffers from disese in which sucrose is the inititing culprit, the tretment choices re to either eliminte the source of glucose or dd/regulte the mount of insulin vilble to the bloodstrem [3]. So, in lieu of ridding the diet of sweet, science went looking for sweet replcement by the rtificil sweeteners. But, however recent epidemiologic studies showed the ssocition between diet sod consumption (which contins rtificil sweeteners) nd the risk of development of obesity, metbolic syndrome nd Type 2 dibetes [4,5]. The erlier studies linked rtificil sweeteners to crcinogenic nd genotoxic risk. Asprtme exhibited crcinogenicity on prolonged use, sucrlose in mouse lymphom ssy showed positive muttion frequency t higher doses nd cesulfme-k cused slight chromosoml berrtion indicting tht these rtificil sweeteners not entirely sfe even though they re FDA pproved [6-9]. Asprtme, cesulfme-k nd sucrlose re not entirely sfe s they were rtificilly synthesized nd their metbolites my yield to toxic chemicls. Most importntly, the risk-benefit rtio of rtificil sweeteners is uncler. Recent study lso shows helth risk even below the cceptble dily intke (ADI) doses fter the long term consumption. So, further studies re essentil to ssess the sfety of these three rtificil sweeteners. Hence present study ws undertken to ccess the dibetogenic nd mutgenic potentils of the rtificil sweetenerssprtme, cesulfme-k nd sucrlose. Mterils nd Methods : Chemicls Asprtme, cesulfme-k nd sucrlose were procured by Highmedi Bomby, Indi. HbA1C (glycted hemoglobin) kit, totl cholesterol kit, triglyceride kit, etc, were procured from Agppe dignostics Ltd, Kerl. And ll other chemicls were of nlyticl grde nd used s received. Animls All the experiments were crried out with Sprgue-Dwley rts weighing g. Animls were kept in the niml house of NGSM Institute of Phrmceuticl Sciences, Mnglore under controlled conditions of temperture (23±2ºC), humidity (50±5%) nd 12 h light-drk cycle. Animls were fed pellet diet (Venkteshwr enterprises, Bnglore) nd wter d libitum. All the nimls were cclimtized for seven dys before the study. The experimentl protocol ws pproved by institutionl niml ethicl committee (pprovl number: Reg.No.KSHEMA/AEC/39/2010) Selection of Drug Doses The humn ADI of sprtme, sucrlose nd cesulfme-k ws 50 mg/kg, 15 mg/kg nd 15 mg/kg respectively. The ADI doses were converted to niml doses s per the conversion chrt nd used for the study [10]. Assessment of dibetogenic potentil [11,12] Experimentl design: Dibetic potentil ccessed by scending repeted dose study up to 13 week in 3 phses on rts. Study involved 4 groups with 6 nimls ech. Drugs were dministered orlly through orl gvge. ª Control group ª Asprtme treted group ª Acesulfme-K treted group ª Sucrlose treted group Phse?(0-3 weeks- ADI dose): Control group: dministered with distilled wter for 0-3 weeks. Asprtme treted group: dministered with 315 mg/kg rt Acesulfme-K treted group: dministered with 94.5 mg/kg rt Sucrlose treted group: dministered with 94.5 mg/kg rt Phse ІІ (3-7 weeks- 2 x ADI dose): Control group: dministered with distilled wter Asprtme treted group: dministered with 630 mg/kg rt Acesulfme-K treted group: dministered with 75.6 COMPARATIVE EVALUATION - Shstry C.S. 81

3 Nitte University Journl of Helth Science mg/kg rt Sucrlose treted group: dministered with 75.6 mg/kg rt Phse ІІІ (7-13 weeks- 4 x ADI dose): Control group: dministered with distilled wter Asprtme treted group: dministered with 1260 mg/kg rt Acesulfme-K treted group: dministered with mg/kg rt Sucrlose treted group: dministered with mg/kg rt At the end of 3rd, 7th, 13th week rts were fsted for 18 hour nd the fsting blood glucose (FBG) levels were mesured. Blood ws collected by retro orbitl sinus method [13] nd centrifuged t 2500 rpm. Serum ws seprted nd lipid profiles like totl cholesterol, triglyceride, LDL nd VLDL ws mesured long with the HbA1C levels which re mesured only t the end of the 13th week. Assessment of mutgenic potentil by Ames test [14,15] For comprtive evlution of mutgenic potentil, smples of sprtme, cesulfme-k, nd sucrlose were sent to Shree Dhnwntry Phrmceuticl Anlysis nd Reserch Centre (SDPARC) t Kim, Surt, Gujrt. The potentil of mutgenic effects of sprtme, cesulfme-k nd sucrlose were evluted on two Slmonell typhimurium strins TA97 (Detects frme shift muttions) nd TA1535. (Detects bse pir substitution muttions). with nd without metbolic ctivtion. Slmonell/ microsome reversion ssy ws conducted using the plte incorportion procedure described by Mron nd Ames nd s per 471-OECD guidelines for testing of chemicls. Sttisticl Anlysis : The dt were expressed s men ± stndrd error of the men (S.E.M.) of 6 nimls per group. Prmetric one wy nlysis of vrince (ANOVA) followed by Dunnett's test. Sttisticl nlysis ws performed using Grph pd prism 5.0. The miniml level of significnce ws identified t P<0.05. Results : Assessment of dibetogenic potentil Fsting blood glucose level In Phse I, t ADI doses of sprtme, cesulfme-k nd sucrlose, FBG level were found to be similr/slightly lower thn control group vlues in ll the drug treted groups. In Phse II (t 2 x ADI doses) nd phse III (t 4 x ADI doses) of sprtme, cesulfme-k nd sucrlose, the FBG level ws significntly rised (p<0.001) in ll the drug treted groups compred control group indicting induction of dibetes. In comprison with sprtme nd cesulfme-k, sucrlose shows less increse in FBG levels. (Tble 1) HbA1c Level After the 13 week, HbA1C levels were rised significntly (p<0.001) in ll three drug treted groups (sprtme, cesulfme-k nd sucrlose) compred to control group. However the sucrlose shows less increse compred to other two rtificil sweetener groups. (Tble 2) Lipid profile In Phse I, lipid profiles like Totl cholesterol, Triglyceride level, LDL nd VLDL levels were norml s control group. But t phse II nd phse III there ws significnt (p<0.001) increse in lipid profile of ll rtificil sweetener treted groups compred to control group. (Tble 3) Assessment of mutgenic potentil by Ames test Ames test with nd without metbolic ctivtion results reveled Asprtme, Acesulfme-K nd Sucrlose were non mutgenic. Histology Histology of norml rt pncres showed no rchitecturl chnges. Asprtme treted rt pncres showed diffused necrotic chnges. Acesulfme-K treted rt pncres showed focl lymphocytic ggregtion/focl chronic infiltrte in the form of lymphocyte indicting utoimmune response ginst β cells. Histology of sucrlose treted rt pncres showed no significnt rchitecturl chnges which is comprble with norml rt pncres. (Figure 1) COMPARATIVE EVALUATION - Shstry C.S. 82

4 Nitte University Journl of Helth Science Tble 1: Effect of sprtme, cesulfme-k nd sucrlose on FBG level Group Fsting Glucose Level (mg/dl) 0-3 weeks 3-7 weeks 7-13 weeks Control group Asprtme treted Acesulfme-K treted Sucrlose treted The Vlues re expressed s Men ± SEM, n=6 rts in one group. significnt compred with control group (p<0.001). Tble 2: Effect of Asprtme, Acesulfme-K nd Sucrlose on HbA1c level Group HbA1c level (%) Control group Asprtme treted Acesulfme-K treted Sucrlose treted The Vlues re expressed s Men ± SEM, n=6 rts in one group. significnt compred with control group (p<0.001). Tble 3: Effect of sprtme, cesulfme-k nd sucrlose on Lipid profiles Group Totl cholesterol (mg/dl) Triglyceride level (mg/dl) 0-3 weeks 3-7 weeks 7-13 weeks 0-3 weeks 3-7 weeks 7-13 weeks Control group Asprtme treted Acesulfme-K treted Sucrlose treted Group LDL cholesterol (mg/dl) VLDL-C (mg/dl) 0-3 weeks 3-7 weeks 7-13 weeks 0-3 weeks 3-7 weeks 7-13 weeks Control group Asprtme treted Acesulfme-K treted Sucrlose treted The Vlues re expressed s Men ± SEM, n=6 rts in one group. significnt compred with control group (p<0.001). Discussion : In the present dibetogenic study, scending repeted dose 13 weeks with three phses gve the cler indiction of effect of these rtificil sweeteners t vrious dose levels. The doses strted t ADI doses since recent long term studies indicted sprtme not sfe even t dily cceptble doses [7]. Mutgenic potentil ws ccessed by Ames test with nd without metbolic ctivtion using Slmonell typhimurium strins TA 97 nd TA1535. Present study reveled tht the rtificil sweeteners cuse significnt increse in FBG levels, HbA1C levels nd lipid profile. The sucrlose group showed lesser increse compred to other two rtificil sweeteners. The pncretic histology report supports the sme. The Ames test ws negtive for ll three rtificil sweeteners. Conclusion : From the present study it ws confirmed tht sprtme, cesulfme-k nd sucrlose exhibit dibetogenic effect t higher dose levels but they were sfer to use t ADI doses. They were non mutgenic compounds, confirmed from the Ames mutgenic test with nd without metbolic ctivtion. So study indicted these rtificil sweeteners cn be used with cution to limited extent. Over usge bove the ADI doses nd long term usge of these rtificil sweeteners is not dvisble. Overll comprtive evlution showed tht sucrlose found to be sfer thn sprtme nd cesulfme-k. The study lso pprecites the crrying similr long term study for these sweeteners for further sfety ssessment on helth risks. Acknowledgements : The uthors re grteful to Nitte Eduction Trust, Nitte University nd Deprtment of Phrmcology, NGSM Institute of Phrmceuticl Sciences, Derlktte, Pneer, Mnglore for providing the necessry fcility nd their full co-opertion to crry out the reserch work. COMPARATIVE EVALUATION - Shstry C.S. 83

5 Nitte University Journl of Helth Science Conflict of interest The uthors declre tht they hve no conflicts of interest to disclose. Funding : This study received no specific grnt from ny funding gency in the public, commercil or not for profit sectors. Figure 1 : Histology report Diffused necrotic chnges Control group Asprtme treted group Focl lymphocytic ggregtion Acesulfme-K treted group Sucrlose treted group References 1. Erin EVH. Artificil Sweeteners: their origin nd mechnism. Cited on Avilble from: URL: Artificil%20Sweeteners.pdf 2. Duffy VB, Grnt SM. Position of the mericn dietetic ssocition: use of nutritive nd nonnutritive sweeteners. J Am Diet Assoc 2004;104(2): Tripthi BK, Srivstv AK. Dibetes mellitus: complictions nd therpeutics. Med Sci Monit 2006;12(7): Nettleton JA, Lutsey PL, Wng Y, Lim JA, Michos ED, Jcobs DR. Diet sod intke nd risk of incident metbolic syndrome nd type 2 dibetes in the multi-ethnic study of therosclerosis (MESA). Dibetes Cre 2009;32(4): Fowler SP, Willims K, Resendez RG, Hunt KJ, Hzud HP, Stern MP. Fueling the obesity epidemic? rtificilly sweetened beverge use nd long-term weight gin. Obesity 2008;16(8): Soffritti M, Belpoggi F, Esposti DD, Lmbertini L, Tibldi E, Rigno A. First experimentl demonstrtion of the multipotentil crcinogenic effects of sprtme dministered in the feed to sprgue-dwley rts. Environ Helth Perspect 2006;114: Soffritti M, Belpoggi F, Esposti DD, Tibldi E, Luriol M Life-spn exposure to low doses of sprtme beginning during prentl life increses cncer effects in rts. Environ Helth Perspect 2007;115: Bndyopdhyy A, Ghoshl S, Mukherjee A. Genotoxicity Testing of low- clorie sweeteners: sprtme, cesulfme-k, nd scchrin. Drug Chem Toxicol 2008;31(4): Mukherjee A, Chkrbrti J. In vivo cytogenetic studies on mice exposed to cesulfme-k non-nutritive sweetener. Food Chem Toxicol 1997;35(12): Ghosh MN. Fundmentls of experimentl phrmcology. 4th ed. Kolkt; Hilton nd compny: pp: Nfis PCF, Chkrdh VL, Vndn SP, Suresh RN. An experimentl evlution of the ntidibetic nd ntilipidemic properties of stndrdized Memodic chrnti fruit extrct. BMC Complemen Alter Med 2007;7(29): McLen BI, Shephrd NW, Merrit RJ, Hildick-Smith G. Repeted dose study of sucrlose tolernce in humn subjects. Food Chem Toxicol 2000;38(2): Kkdiy J, Shh M, Shh NJ. Effect of nobivolol on serum dibetic mrker nd lipid profile in norml nd streptozotocin-nicotinmide induced dibetic rts. Biol Chem Sci 2010;1: Mortelmns K, Zeiger E. The mes slmonell/microsome mutgenicity ssy. Mutt Res 2009;455(1-2): Mron DM, Ames BN. Revised methods for the slmonell mutgenicity test. Mutt Res 1983;113: COMPARATIVE EVALUATION - Shstry C.S. 84

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