Posted: 29 June 2005
http://groups.yahoo.com/group/aspartameNM/message/1131
Genotoxicity of aspartame in human lymphocytes 2004.07.29 Full plain text, Rencuzogullari E et al, Cukurova University, Adana, Turkey 2004 Aug
[ Comments and corrections by Rich Murray are in square brackets. Spacing has been added, without changing text, to increase readability and clarity, and add emphasis. ]
Obviously, it hardly is conclusive to simply place aspartame in contact with isolated living cells, without doing detailed explorations to determine the degree of disassociation into phenylalanine, aspartic acid, and methanol, with resulting formation of formaldehyde and formic acid, as well as studying long-term accumulations in animals and humans -- except as a very valuable initial pilot study.
Nevertheless, their evidence and conclusions are devastating.
http://www.HolisticMed.com/aspartame mgold@holisticmed.com
Aspartame Toxicity Information Center Mark D. Gold also Co-Moderator 12 East Side Drive #2-18 Concord, NH 03301 603-225-2110
http://www.holisticmed.com/aspartame/abuse/methanol.html
"Scientific Abuse in Aspartame Research"
http://groups.yahoo.com/group/aspartameNM/message/957
Safety of aspartame Part 1/2 12.4.2: EC HCPD-G SCF: EU Scientific Committee on Food, a whitewash
http://groups.yahoo.com/group/aspartameNM/message/1045
http://www.holisticmed.com/aspartame/scf2002-response.htm
Mark Gold exhaustively critiques European Commission Scientific Committee on Food re aspartame ( 2002.12.04 ): 59 pages, 230 references
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"Schwartz ( 1999 ) also reported that methanol is converted to formaldehyde which then accumulates in the cells. Formaldehyde has been considered an inducer of cancer and acts to alter DNA ( Ewertz, 1993; Ewertz and Gill, 1990 ).
Olney et al. ( 1996 ) reviewed and explained that ASP had mutagenic potential...
In this study, we found that, ASP did appear to have genotoxic potential consistent with potential carcinogenicity.
According to these results, phenyalanine and methanol, which are metabolic products of ASP, have a genotoxic risk for humans.
In contrast, ASP was not found as a mutagen in in vivo studies.
However, in the present study, ASP induced CA and micronuclei in human lyphocytes dose-dependently.
ASP did not change the osmolality of the medium at the maximum concentrations ( 346 milliosmol) when compared with untreated medium (342 milliosmol ).
It was reported that a deviation from physiological osmolality ( approximately 300 milliosmol ) can lead to genotoxic effects ( Nowak, 1984, 1997; Seeberg et al., 1989 ).
According to these results, we can conclude that ASP induced CA and percentage of micronuclei by itself because it did not alter the pH and osmolality of the medium.
As shown, there are several contradictory studies about genotoxicity and carcinogenicity of ASP.
However, it must be taken into account that ASP induced the CA and micronuclei formation in a dose-dependent manner.
It is not possible to conclude that ASP is safe according to these results.
Therefore, it is necessary to be careful when using it in food and beverages as a sweetener."
Genotoxicity of aspartame 2004.07.29 plain text, Rencuzogullari E et al, Cukurova University, Adana, Turkey 2004 Aug
Drug Chem Toxicol. 2004 Aug; 27(3): 257-68.
Genotoxicity of aspartame. reyyup@mail.cu.edu.tr
Rencuzogullari E, Tuylu BA, Topaktas M, Ila HB, Kayraldiz A, Arslan M, Diler SB. Biology Department, Faculty of Arts and Sciences, Natural and Applied Sciences Institute, Cukurova University, Adana, Turkey.
http://www.cu.edu.tr/Content/Asp/English/index.asp
http://rektorluk.cukurova.edu.tr/en/rehber.asp
In the present study, the genotoxic effects of the low-calorie sweetener aspartame (ASP), which is a dipeptide derivative, was investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test, micronucleus test in human lymphocytes and also Ames/Salmonella/ microsome test.
ASP induced CAs at all concentrations (500, 1000 and 2000 microg/ml) and treatment periods (24 and 48 h) dose-dependently, while it did not induce SCEs.
On the other hand, ASP decreased the replication index ( RI ) only at the highest concentration for 48 h treatment period. However, ASP decreased the mitotic index ( MI ) at all concentrations and treatment periods dose-dependently.
In addition, ASP induced micronuclei at the highest concentrations only. This induction was also dose-dependent for 48 hours treatment period. ASP was not mutagenic for Salmonella typhimurium TA98 and TA100 strains in the absence and presence of S9 mix. PMID: 15478947
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Genotoxicity of Aspartame
Published in Drug and Chemical Toxicology , Volume 27 , Issue 3
Print ISSN: 0148-0545 Online ISSN: 1525-6014
Online Article World Price: $24.00
Eyyüp Rencüzoullar *Corresponding reyyup@mail.cu.edu.tr
[http://lokman.cu.edu.tr/ent
Çukurova University Medical School Adana, Turkey
This page was last updated on 07/06/00.
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Çukurova University Department of Biophysics
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Berrin Ayaz Tüylü 3
Mehmet Topakta 1 Mehmet Topaktas mtopaktas@mail.cu.edu.tr
Hasan Basri la [1]
Ahmet Kayraldz [2]
Mehmet Arslan [2]
Songül Budak Diler [2]
Keywords: Sweeteners, Aspartame, Chromosome aberrations, Sister chromatid exchange, Micronuclei, Ames test
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Introduction
Many non-nutritive sweeteners have been used in foods and beverages to allow people to enjoy the sweet taste without consuming sugar-associated calories, and therefore inducing weight gain. One of these sweeteners is aspartame. Aspartame ( alpha-l-aspartyl-l-phenylalanine 1-methylester ) is a dipeptide artificial sweetener that is widely used as a non-nutritive sweetener in foods and drinks. ASP was discovered in 1965. In 1981, ASP became the first low calorie sweetener approved by the Food and Drug Administration (FDA) in more than 26 years.
ASP is used as a sweetener in food products including dry beverage mixes, chewable multi-vitamins, breakfast cereals, chewing gum, puddings and fillings, carbonated beverages, refrigerated and non-refrigerated ready to drink beverages, yogurt-type products and pharmaceuticals.
The ministry of Agricultural (of Turkey) suggested that ASP may be used at a maximum dose of 5500 mg/kg (The ministry of Agricultural of Turkey, 1997).
Butchko et al. (2002) reviewed a study on safety of ASP and reported that ASP is safe.
It was reported that ASP did not induce DNA damage in rat hepatocytes (Jeffrey and Williams, 2000) and was not clastogenic in mice when given orally (Durnev et al., 1995). In addition, Molinary (1984) reported that ASP was not mutagenic in Ames Test and had no genotoxic effect in dominant-lethal and host mediated assay.
However, Shephard et al. ( 1991 ) reported that ASP has a weak mutagenic effect in Salmonella typhimurium TA100 and TA98 strains after nitrosation. As shown, some of the sweeteners have mutagenic effects and some of them such as saccharin caused cancer. Olney et al. ( 1996 ) reported that ASP may be carcinogenic in Sprague-Dawley rats. However, Ishii ( 1981 ) reported that ASP does not cause brain cancer in rats.
As shown, there are several studies suggesting that ASP has genotoxic effects but there are also several studies suggesting that ASP has no genotoxicity.
At present, there is no published data on the induction of CA, SCE and micronuclei by ASP in human lymphocytes or mutagenic effects in S. typhimurium without nitrosation.
These studies have been completed and are presented here.
Materials and Methods
The test substance Aspartame phenylalanine methyl ester was obtained from Sigma ( Cat. no: A5139, CAS no: 22839-47-0, FW: 294, 3 ). Purity of ASP is 99.9%. The osmolality of ASP was measured using Roebling automatic osmometer. The pH of the medium was measured using WTW 315i ( Germany ) pH meter. The chemical structure and formula of ASP is shown in Fig. 1.
Figure 1. N-l-alpha-aspartyl-l-phenylalanine 1-methylester.
CA and SCE Assay
Whole blood ( 0.2 ml ) from four healthy donors ( two male, two female, non-smokers, age: 18-19 ) was added to 2.5 ml chromosome medium B ( Seromed, Biochrom cat. no.: F5023 ) supplemented with 10 µg/ml bromodeoxyuridine ( Sigma cat. no.: B5002 ).
The cultures were incubated at 37°C for 72 h.
The cells were treated with 500, 1000 and 2000 µg/ml concentrations of ASP for 24 h (ASP was added 48 h after initiating the culture) and 48 h (ASP was added 24 h after initiating the culture).
These concentrations were selected according to cytotoxicity of ASP.
A negative control ( untreated cultures ) and a positive control ( mitomycin-C, 0.3 µg/ml, Kyowa, Hakko, Japan ) were used
. The test substance, ASP, and positive control mitomycin-C ( MMC ) were dissolved in bidistilled water. Colchicine ( 0.06 µg/ml, Sigma cat. no.: C9754 ) was present for the last 2 h of culture. To collect the cells, the cultures were centrifuged ( 1200 rpm, 15 min ), treated with hypotonic solution ( 0.4% KCl ) for 20 min at 37°C, and then fixed in cold methanol:glacial acetic acid ( 3:1 ) for 20 min at room temperature. The treatment with fixative was repeated three times.
Then the cells were spread on glass slides and air dried.
The slides were stained with Giemsa according to fluorescence plus Giemsa technique ( Speit and Haupter, 1985 ). 100 well spread metaphases per donor (a total of 400 metaphases per concentration) were examined at 1000 × magnification for occurrence of different types of CA.
The number of abnormal cells per concentration and treatment period were determined and the mean frequency of abnormal cells was calculated.
The number of CA per cell ( CA/cell ) was calculated as well.
For the occurrence of SCEs, a total 100 cells ( 25 cells from each donor ) under second metaphases were examined. The metaphases were examined at 1000 × magnification. The results were used to determine the mean number of SCE ( SCE/cell ).
In addition, a total 400 cells ( 100 cells from each donor ) were scored for the determination of the replication index ( RI ).
The MI was also determined by scoring 3000 cells from each donor. The MI explained the effects of the chemicals on G2 stage of cell cycle, and the RI reflects the effects of chemicals on S and G2 stages of the cycles.
The RI was calculated according to the formula as follows: RI = ( M1 × 1 ) + ( M2 × 2 ) + ( M3 × 3 )/total scored cells.
M1, M2 and M3 are the fraction of cells undergoing the first, second and third mitosis during 72 hours cells culture times.
Micronucleus Assay
The micronucleus test was performed as described by Rothfus et al. (2000).
The peripheral blood from 4 healthy donors was added to 2.5 ml chromosome medium B and incubated at 37°C for 68 h.
The cells were treated with 500, 1000 and 2000 µg/ml concentrations of ASP for 24 and 48 hours as mentioned above.
A negative and a positive control ( 0.3 µg/ml Mitomycin-C ) was also used. Cytochalasin B was added to cultures at a final concentrations of 6 µg/ml 44 hours after initiating of the cultures.
Cultures were harvested 24 hours later.
Then, the cells were treated with hypotonic solution (0.4% KCl), fixed once with fixative ( methanol:glacial acetic acid, 5:1 ) mixed with an equal amount of 0.9% NaCl for 20 min, and then fixed twice with methanol/glacial acetic acid ( 5:1 ) for 20 min. The slides were air-dried and stained with 5% Giemsa.
The micronucleus frequency was determined by analysing 2000 binucleated cells for each donor from blinded slides.
In addition, the ratio of binucleated to mononucleated cells was also scored by analysing 2000 cells for each donor.
Salmonella/Microsome
Test Bacterial Strains
Histidine deficient ( his-) tester strains TA98 and TA100 of Salmonella typhimurium were kindly provided by L. K. Nakamura ( Microbiologist Emeritus, Microbial Properties Research, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois, 61604, USA ).
The TA98 strain is used for the detection of frameshift mutagens and TA100 strain for the detection of base pair substitution mutagens. Prior to use in the assay, each strain was checked for the presence of strain-specific marker as described by Maron and Ames ( 1983 ).
Mutagenicity Assay
The standard plate-incorporation assay was examined with Salmonella typhimurium TA98 and TA100 strains in the presence and absence of S9 mix according to Maron and Ames ( 1983 ). 0.5 ml of S9 mix containing 50 µl of S9 factor per plate was used for the assay.
For the test, ASP was dissolved in distilled water and 50, 100, 250, 500, 1000 and 2000 µg per plate were used.
On the other hand, 4-NPD ( 4-nitrophenylene diamine ) was used as a positive mutagen (200 µg/plate) for TA98 and TA100.
In the presence of S9 mix, 2-AF ( 2-aminoflourene ) was used as positive mutagen ( 20 µg/plate ) for both TA98 and TA100 strains.
Each sample was evaluated with three replicate plates and all experiments were performed twice.
Preparation of S9
The male albino rats ( Rattus norvegicus var. albinos) weighing 200 gr were pre-treated with 80 mg/kg concentration of 3-methylcholanthrene ( Oekanal, Cat. no: 45794 ) ( dissolved in sunflower seed oil ) for 5 days and the S9 fraction and S9 mix were prepared following the procedure of Maron and Ames ( 1983 ). The S9 tablets were purchased from Roche (Cat. no: 1.745.425).
The freshly prepared S9 fraction is distributed in 1-ml portions in small plastic tubes frozen immediately and stored at - 35°C. The S9 mix was prepared fresh for each mutagenicity assay.
Each tablet was dissolved in 18 ml bidistilled water supplemented with 2 ml of S9 fraction.
Statistical Significance
Chromosome and chromatid gaps were not evaluated as CA ( Mace et al., 1978 ).
The significance between the percentage of abnormal cell, CA/cell, mean SCE, percentage of the micronucleated cells, RI and MI in treated cultures and their controls were determined using the t-test.
Dose response relationships were determined from the correlation and regression coefficients and the corresponding regression lines for the percentage of abnormal cell, CA/cell, mean SCE, percentage of micronuclei, RI and MI. The significance between control revertants and revertants of treated groups were also determined using t-test.
Dose-response relationships were also determined using regression and correlation ( r ) test systems.
Result
ASP induced a significant increase of CAs at all concentrations and treatment periods compared to control ( Table 1 ).
However this increase was not dose-dependent.
The potency of ASP on induction of CAs was lower than that caused by the positive control MMC. However, ASP did not induce the SCE ( Table 2 ).
NOTE: For the Tables 1 and 2, see the web site indicated above.
ASP did not decrease the RI except the highest concentration for 48 h treatment period.
However, ASP significantly decreased the MI at all concentrations for 24 and 48 h treatment periods when compared with control ( Table 2 ).
This decrease was dose-dependent ( r = - 0.96 and r = - 0.97, respectively ).
ASP induced micronucleus formation at 2000 µg/ml concentrations for 24 and 48 h treatment periods ( Table 3 ).
This effect was also dose-dependent for 48 hours treatment period ( r = 0.97 ).
ASP also dose-dependently decreased the ratio of binucleated to mononucleated cells for 24- and 48-hour treatment periods ( r = - 0.81 and r = - 0.98, respectively ) ( Table 3 ).
This decrease was clearly related with the decreasing the MI in Table 2.
Table 3. The Frequency of Micronuclei in Cultured Human Lymphocytes Treated with Aspartame # .
NOTE: For the Tables 3, see the web site indicated above.
ASP did not change the pH and the osmolality of the medium. The osmolality was measured as 342 milliosmol in control and 346 milliosmomol in the maximum dose of ASP (2000 µg/ml).
ASP did not increase the number of revertants of TA98 in the absence or presence of S9 mix.
ASP slightly increased the number of revertants of TA100 in the absence of S9 mix but this is not considered to represent a positive response.
There is no dose-dependent effect ( Table 4 ).
The effect of ASP on TA100 strain was greater than TA98 strain.
Table 4. The Mutagenicity of Aspartame on S.typhimurium TA98 and TA100 Strains in the Presence or Absence of S9 Mix.
NOTE: For the Tables 4, see the web site indicated above.
Discussion
In this study, ASP significantly induced CA and micronucleus formation and showed a cytotoxic effect by decreasing the MI.
However, ASP did not induce the SCE and it had no mutagenic effect on the tester strains TA98 and TA100 of S. typhimurium in the presence and absence of S9 mix.
ASP is hydrolysed in the gut to yield aspartic acid, phenylalanine, methanol and cyclised diketopiperazine ( Maher, 1987; Ranney and Oppermann, 1979 ).
There are a lot of studies on genotoxicity of metabolities of ASP ( Austin et al., 1992; Brook and Chandley, 1985; Generoso et al., 1995; Sargentini and Smith, 1986 ).
Shephard et al. ( 1991 ) reported that ASP has a weak mutagenic effect in Ames test after nitrosation.
Shephard et al. ( 1993 ) reported that, in the human stomach, the chain nitrosation of the amino acids (Phe) and aspartame might be more important than the reactions at the primary amino group.
However, Butchko et al. ( 2002 ) reported that ASP is safe and it was also reported that ASP was not mutagenic and clastogenic in animals ( Durnev et al., 1995; Jeffrey and Williams, 2000; Molinary, 1984 ).
In the present study, it was found that ASP had a genotoxic effect using CA and micronuclei test in human lymphocytes but had no mutagenic effect using Ames/Salmonella/microsome test and did not induce SCE, in human lymphocytes.
It has been reported that ASP does not cause brain and bladder tumours ( Hagiwara et al., 1984; Ishii, 1981; Ito et al., 1984 ) however, there are some reports that ASP ( Olney et al., 1996 ) and especially its metabolites, phenylalanine and methanol ( Schwartz, 1999 ) increased the breast, brain and prostate cancer incidence.
The data obtained from a study carried out by FDA supported the report of Olney et al., 1996 in the assay on 320 Sprague-Dawley rats fed with ASP which caused brain cancer [ According to Study E33-34 in master file 134 on ASP from Weihrauch et al. (2001) ].
Schwartz ( 1999 ) also reported that methanol is converted to formaldehyde which then accumulates in the cells. Formaldehyde has been considered an inducer of cancer and acts to alter DNA ( Ewertz, 1993; Ewertz and Gill, 1990 ).
Olney et al. ( 1996 ) reviewed and explained that ASP had mutagenic potential.
Ahmed and Thomas ( 1992 ) reported that cyclamate (sweetener) was not carcinogenic by itself; however, it might have cancer-promoting or carcinogenic activities.
In this study, we found that, ASP did appear to have genotoxic potential consistent with potential carcinogenicity.
According to these results, phenyalanine and methanol, which are metabolic products of ASP, have a genotoxic risk for humans.
In contrast, ASP was not found as a mutagen in in vivo studies.
However, in the present study, ASP induced CA and micronuclei in human lyphocytes dose-dependently.
ASP did not change the osmolality of the medium at the maximum concentrations ( 346 milliosmol ) when compared with untreated medium ( 342 milliosmol ).
It was reported that a deviation from physiological osmolality ( approximately 300 milliosmol ) can lead to genotoxic effects ( Nowak, 1984, 1997; Seeberg et al., 1989 ).
According to these results, we can conclude that ASP induced CA and percentage of micronuclei by itself because it did not alter the pH and osmolality of the medium.
As shown, there are several contradictory studies about genotoxicity and carcinogenicity of ASP.
However, it must be taken into account that ASP induced the CA and micronuclei formation in a dose-dependent manner.
It is not possible to conclude that ASP is safe according to these results.
Therefore, it is necessary to be careful when using it in food and beverages as a sweetener.
This study was funded by Çukurova University Research Fund FEF 2002 BAP 21. We also thank Mr. L.K. Nakamura for his kind collaboration.
Ahmed F. E., Thomas D. B., Assessment of the carcinogenicity of the nonnutritive sweetener cyclamate, Crit. Rev. Toxicol., 22 (2) , (1992) 81-118.
Austin M. J., Han Y. H., Povirk L. F., DNA sequence analysis of mutations induced by melphalan in the CHO aprt locus, Cancer Genet. Cytogenet., 64 (1), (1992) 69-74.
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Butchko H. H., Stargel W. W., Comer C. P., Mayhew D. A., Benninger C., Blackburn G. L., de Sonneville L. M., Geha R. S., Hertelendy Z., Koestner A., Leon A. S., Liepa G. U., McMartin K. E., Mendenhall C. L., Munro I. C., Novotny E. J., Renwick A. G., Schiffman S. S., Schomer D. L., Shaywitz B. A., Spiers P. A., Tephly T. R., Thomas J. A., Trefz F. K., Aspartame: review of safety, Regul. Toxicol. Pharmacol., 35 (2) , (2002), 1-92.
Durnev A. D., Oreshchenko A. V. , Kulakova A. V. , Beresten N. F. , Seredenin S. B., Clastogenic activity of dietary sugar substitutes, Vopr. Med. Khim., 41 (4) , (1995) 31-33.
Ewertz M., Breast cancer in Denmark. Incidence, risk factors, and characteristics of survival, Acta Oncol., 32 (1993) 595-615.
Ewertz M., Gill C., Dietary factors and breast-cancer risk in Denmark, Int. J. Cancer, 46 (1990) 779-784.
Generoso W. M., Witt K. L., Cain K. T., Hughes L., Cacheiro N. L., Lockhart A. M. , Shelby M. D., Dominant lethal and heritable translocation test with chlorambucil and melphalan in male mice, Mutat. Res., 345 (3-4), (1995) 167-180.
Hagiwara A., Fukushima S., Kitaori M., Shibata M., Ito M., Effects of three sweeteners on the rat urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybuthyl)-nitrosamine, Gann, 75 (9) , (1984) 763-768.
Ishii H., Incidence of brain tumors in rats fed aspartame, Toxicol. Lett., 7 (6) , (1981) 433-438.
Ito N., Fukushima S., Shirai T., Hagiwara A., Imaida K., Drugs, food additives and natural products as promoters in rat urinary bladder carcinogenesis, IARC Sci. Publ., 56 (1984) 399-407.
Jeffrey A. M., Williams G. M., Lack of DNA-damaging activity of five non-nutritive sweeteners in the rat hepatocyte/DNA repair assay, Food Chem. Toxicol., 38 (4) , (2000) 335-338.
Mace M. L., Daskal Y., Wray W., Scanning-electron microscopy of chromosome aberrations, Mutat. Res., 52 (1978) 199-206.
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Maron D. M., Ames B. N., Revised method for the Salmonella mutagenicity test, Mutat. Res., 113 (1983) 173-215.
Molinary S. V. Preiclinical Studies of Aspartame in Non-primate Animals, Aspartame, Physiology and Biochemistry, Stegink L. D., Tiler L. S. Marcel Dekker, New York, 1984, pp. 289-306.
Nowak C., Induction of chromosomal aberrations by hypotonic culture conditions is dependent of the S-phase in V79 hamster cells, Environ. Mol. Mutagen., 13 (1) , (1984) 44-49.
Nowak C., Studies on the ability of hypotonic solutions to induce chromosomal aberrations in V79 cells, Teratog. Carcinog. Mutagen., 7 (6) , (1997) 515-525.
Olney J. W., Farber N. B., Spitznagel E., Robins L. N., Increasing brain tumors rates: is there a link to aspartame?, J. Neuropathol. Exp. Neurol., 55 (11), (1996) 1115-1123.
Ranney R. E., Oppermann J. A., A review of the metabolism of the aspartyl moiety of aspartame in experimental animals and man, J. Environ. Pathol. Toxicol., 2 (4) , (1979) 979-985.
Rothfus A., Schütz P., Bochum S., Volm T., Eberhardt E., Kreirenberg R., Vogel W., Speit G., Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families, Cancer Res., 60 (2000) 390-394.
Sargentini N. J., Smith K. C., Mutagenesis by normal metabolites in Escherichia coli: phenylalanine mutagenesis is dependent on error-prone DNA repair, Mutat. Res., 161 (2), (1986) 113-118.
Schwartz G. R., Aspartame and breast and other cancers, West J. Med., 171 (1999) 300-301.
Seeberg A. H., Mosesso P., Forster R., High-dose-level effects in mutagenicity assays utilizing mammalian cells in culture, Mutagenesis, 3 (3), (1989) 213-218.
Shephard E. E., Meier I., Lutz W. K., Alkylating potency of nitrosated amino acids and peptides, IARC Sci. Publ., 105 (1991) 383-387.
Shephard S. E. , Wakabayashi K. , Nagao M., Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation, Food Chem. Toxicol., 31 (5) , (1993) 323-329. Speit G., Haupter S., On the mechanisms of differential giemsa staining of bromodeoxyuridine-substituted chromosomes. II Differences between the demonstration of sister chromatid differentiation and replication patterns, Hum. Genet., 70 (1985) 126-129.
The ministry of Agricultural of Turkey Food Codex, Globus World Publications, Turkey, 1997, pp. 46-47.
Weihrauch M. R., Diehl V., Bohlen H., Künstliche Süstoffe -- Haben sie ein kanzerogenes potential?, Med. Klin., 96 (2001) 670-675.
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PubMed Items 1 - 5 of 5 One page.
The moderated newsgroup, bionet.toxicology , has accepted 24 of my long reviews since March 24:
Dr. Charles "Chuck" A. Miller III rellim@tulane.edu
Associate Professor of Environmental Health Sciences
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Tulane Univ. School of Public Health and Tropical Medicine
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http://groups.yahoo.com/group/aspartameNM/message/1128
Hangover symptoms from methanol from dark wines and liquors or 11% methanol part of aspartame, review of research: Jones AW 1988
Fully 11% of aspartame is methanol -- 1,120 mg aspartame in 2 L diet soda, almost six 12-oz cans, gives 123 mg methanol (wood alcohol). However, about 30% of the methanol remains in the body as cumulative durable toxic metabolites of formaldehyde and formic acid, 37 mg daily, a gram every month, accumulating in and affecting every tissue -- over 60 times the USA EPA limit for formaldehyde in drinking water.
Aspartame is made of phenylalanine (50% by weight) and aspartic acid (39%), both ordinary amino acids, bound loosely together by methanol (wood alcohol, 11%). Similar amounts of methanol in many fruits and vegetables, locked up in complex pectin molecules, and always paired with ethanol, its natural antidote, are not usually released by human digestion and so are harmless. But the readily released methanol from aspartame is within hours largely turned by the liver into formaldehyde and then formic acid, both potent, cumulative toxins.
Red wine contains twice as much methanol as does diet soda as an impurity, about one part in ten thousand. It is the natural conversion by the body of this methanol into formaldehyde and formic acid that is the main cause of the well known "morning after" hangover symptoms: headache, nausea, weakness, impaired memory, irritability, anxiety, "brain fog", body pains -- the same symptoms as aspartame victims.
Jones AW.
Elimination half-life of methanol during hangover.
Pharmacol Toxicol. 1987 Mar; 60(3): 217-20. PMID: 3588516
" But higher blood-methanol concentrations are definitely associated with higher blood-ethanol in this sample of Swedish drinking drivers.
Frequent exposure to methanol and its toxic products of metabolism, formaldehyde and formic acid, might constitute an additional health risk associated with heavy drinking in predisposed individuals. " Jones AW 1988
Forensic Sci Int. 1988 Jun; 37(4): 277-85.
Relationship between the concentration of ethanol and methanol in blood samples from Swedish drinking drivers.
Jones AW, Lowinger H.
Department of Alcohol Toxicology, University Hospital, Linkoping, Sweden.
Jones AW has 341 items in PubMed.
http://groups.yahoo.com/group/aspartameNM/message/1106
Hangover research relevant to toxicity of 11% methanol in aspartame (formaldehyde, formic acid): Calder I (full text): Jones AW: Also some methanol from fruit pectin in colon
http://bmj.bmjjournals.com/search.dtl
Search to get free full text
British Medical Journal 1997 (4 January); 314(7073): 2.
Ian Calder, F.R.C.A. [ Tel/Fax: 0171 720 9279 Consultant Anaesthetist at the National Hospital for Neurology and Neurosurgery, London WCIN 3BG, UK ]
Editorials Hangovers: Not the ethanol - perhaps the methanol
"Pawan compared the hangover produced by different types of drink (but only one brand of each) in his study of 20 volunteers. The severity of hangover symptoms declined in the order of brandy, red wine, rum, whisky, white wine, gin, vodka, and pure ethanol.(6) Vodka and pure ethanol caused only mild headaches in two volunteers."
6. Pawan GL.
Alcoholic drinks and hangover effects.
Proc Nutr Soc 1973 May; 32: 15A. PMID: 4760771
J.A. Oppermann's Searle Co. lab proved that 30% of the methanol in aspartame fed once to monkeys remained -- surely as formaldehyde and formic acid in all tissues (1973, 1976, 1979).
This was confirmed by an expert team at the University of Barcelona (Trocho C, Alemany M, et al, 1998): " ...the binding of methanol-derived carbon [ from low levels of aspartame fed to rats for 10 days ] to tissue proteins was widespread, affecting all systems, fully reaching even sensitive targets such as the brain and retina...These are indeed extremely high levels for adducts of formaldehyde, a substance responsible for chronic deleterious effects (33), that has also been considered carcinogenic. " Prof. Marià Alemany alemany@bio.ub.es Life Sci. 1998 June 26; 63(5): 337-49. PMID: 9714421
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President Bush & formaldehyde (aspartame) toxicity: Ramazzini Foundation carcinogenicity results Dec 2002: Soffritti: Murray 2003.08.03 Ann N Y Acad Sci. 2002 Dec; 982: 87-105. Results of long-term experimental studies on the carcinogenicity of formaldehyde and acetaldehyde in rats. M. Soffritti et al. Cancer Research Center, European Ramazzini Foundation for Oncology and Environmental Sciences, Bologna, Italy. crcfr@tin.it p. 88 " The sweetening agent aspartame hydrolyzes in the gastrointestinal tract to become free methyl alcohol, which is metabolized in the liver to formaldehyde, formic acid, and CO2. (11) " Ref. (11) is: Medinsky MA & Dorman DC. 1994; Assessing risks of low-level methanol exposure. CIIT Act. 14: 1-7.
http://groups.yahoo.com/group/aspartameNM/message/1131
Genotoxicity of aspartame in human lymphocytes 2004.07.29 full plain text, Rencuzogullari E et al, Cukurova University, Adana, Turkey 2004 Aug
Drug Chem Toxicol. 2004 Aug; 27(3): 257-68. Genotoxicity of aspartame. Rencuzogullari E, Tuylu BA, Topaktas M, Ila HB, Kayraldiz A, Arslan M, Diler SB. Biology Dept, Faculty of Arts and Sciences, Natural and Applied Sciences Institute, Cukurova University, Adana, Turkey. reyyup@mail.cu.edu.tr PMID: 15478947 This expert team found DNA damage in human lymphocytes. Rencuzogullari E has 4 other similar studies in PubMed.
http://groups.yahoo.com/group/aspartameNM/message/1088
Full plain text & critique: chronic aspartame in rats affects memory, brain cholinergic receptors, and brain chemistry, Christian B, McConnaughey M et al, 2004 May: 2004.06.05 Pharmacol Biochem Behav. 2004 May; 78(1): 121-7. PMID: 15159141 Mona M. McConnaughey, Ph.D. Research Assistant Professor 252-744-2756 mcconnaugheym@mail.ecu.edu Twelve rats fed aspartame at otherwise nontoxic levels for 4 months forgot how to turn right to get a treat, and had specific brain changes.
http://groups.yahoo.com/group/aspartameNM/message/1067
Eyelid contact dermatitis by formaldehyde from aspartame, Hill AM & Belsito DV, Nov 2003: Murray 2004.03.30 Contact Dermatitis. 2003 Nov; 49(5): 258-9. PMID: 14996049 A mysterious dermatitis was caused by a dose the same as two packets Equal daily.
http://groups.yahoo.com/group/aspartameNM/message/846
Aspartame in Merck Maxalt-MLT worsens migraine, AstraZeneca Zomig, Eli Lilly Zyprexa, J&J Merck Pepcid AC (Famotidine 10mg) Chewable Tab, Pfizer Cool Mint Listerine Pocketpaks
Migraine MLT-Down: an unusual presentation of migraine in patients with aspartame-triggered headaches.
Newman LC, Lipton RB Headache 2001 Oct; 41(9): 899-901.
[ Merck 10-mg Maxalt-MLT, for migraine, has 3.75 mg aspartame, while 12 oz diet soda has 200 mg. ]
Headache Institute, St. Lukes-Roosevelt Hospital Center, New York, NY Department of Neurology newmanache@aol.com Albert Einstein College of Medicine, Bronx, NY Innovative Medical Research RLipton@aecom.yu.edu
http://groups.yahoo.com/group/aspartameNM/message/855
Blumenthall & Vance: aspartame chewing gum headaches Nov 1997
Harvey J. Blumenthal, MD, Dwight A Vance, RPh
Chewing Gum Headaches. Headache 1997 Nov-Dec; 37(10): 665-6.
Department of Neurology, University of Oklahoma College of Medicine, Tulsa, USA.
neurotulsa@aol.com
Aspartame, a popular dietetic sweetener, may provoke headache in some susceptible individuals. Herein, we describe three cases of young women with migraine who reported their headaches could be provoked by chewing gum sweetened with aspartame. [ 6-8 mg aspartame per stick chewing gum ]
http://groups.yahoo.com/group/aspartameNM/message/1077
Eight depressed people react strongly to aspartame, Prof. Ralph G. Walton, MD, 1993 double-blind study, full text: Murray 2004.04.26 rmforall They reported with aspartame, compared to placebo, much higher levels of anxiety, poor memory, nausea, depression, anger, and malaise. A 60 kg subject would have had 1800 mg aspartame, the same as nine 12-oz diet sodas, daily for 7 days. 330-740-3621 rwalton193@aol.com PMID: 8373935
http://groups.yahoo.com/group/aspartameNM/message/1133
Mark Gold, most recent of 14 Rapid Responses to Aspartame and its effects on health, BMJ
http://groups.yahoo.com/group/aspartameNM/message/1124
8 more Rapid Responses to Aspartame and its effects on health, BMJ
http://groups.yahoo.com/group/aspartameNM/message/1120
5 critical Rapid Responses to Aspartame and its effects on health, Michael E J Lean and Catherine R Hankey, BMJ 2004; 329: 755-756
http://groups.yahoo.com/group/aspartameNM/message/1117
Aspartame and its effects on health, Michael E.J. Lean, Catherine R. Hankey, Glasgow UK, British Medical Journal: 11% methanol component of aspartame, and same level of methanol in dark wines and liquors, turns to formaldehyde and formic acid, the main cause of chronic hangover symptoms
http://bmj.bmjjournals.com/cgi/eletters/329/7469/755#76712
http://groups.yahoo.com/group/aspartameNM/message/927
Donald Rumsfeld, 1977 head of Searle Corp., got aspartame FDA approval
http://groups.yahoo.com/group/aspartameNM/message/1065
Politicians and celebrities hooked on diet sodas (aspartame): Murray 2004.03.24 Senator John Edwards still has a dozen Diet Cokes daily -- a gallon, which gives as much methanol as a half-gallon of red wine.
http://groups.yahoo.com/group/aspartameNM/message/1071
Aspartame (NutraSweet, Equal, Canderel, E951), after eight years of controversy, was suddenly and capriciously approved in July 1981 by a new FDA commissioner, Arthur Hull Hayes, Jr (just appointed by President Reagan), disregarding the negative vote of his own Scientific Board of Inquiry.
http://www.dorway.com/enclosur.html
http://groups.yahoo.com/group/aspartameNM/message/53
Aspartame history Part 1/4 1964-1976: Gold: Murray 1999.11.06
http://groups.yahoo.com/group/aspartameNM/message/957
Safety of aspartame Part 1/2 12.4.2: EC HCPD-G SCF: Murray 2003.01.12 rmforall EU Scientific Committee on Food, a whitewash
http://www.holisticmed.com/aspartame/scf2002-response.htm
A detailed critique of European Commission Scientific Committee on Food re aspartame (2002.12.04)
http://www.HolisticMed.com/aspartame mgold@holisticmed.com
Aspartame Toxicity Information Center
Mark D. Gold also Co-Moderator
12 East Side Drive #2-18
Concord, NH 03301
603-225-2110
http://www.holisticmed.com/aspartame/abuse/methanol.html
"Scientific Abuse in Aspartame Research"
Many scientific studies and case histories report: * headaches * many body and joint pains (or burning, tingling, tremors, twitching, spasms, cramps, stiffness, numbness, difficulty swallowing) * fever, fatigue, swollen glands * "mind fog", "feel unreal", poor memory, confusion, anxiety, irritability, depression, mania, insomnia, dizziness, slurred speech, sexual problems, poor vision, hearing (deafness, tinnitus), or taste * red face, itching, rashes, allergic dermatitis, hair loss, burning eyes or throat, dry eyes or mouth, mouth sores, burning tongue * obesity, bloating, edema, anorexia, poor appetite or excessive hunger or thirst * breathing problems, shortness of breath * nausea, diarrhea or constipation * coldness * sweating * racing heart, low or high blood pressure, erratic blood sugar levels * hypothryroidism or hyperthyroidism * seizures * birth defects * brain cancers * addiction * aggrivates diabetes, autism, allergies, lupus, ADHD, fibromyalgia, chronic fatigue syndrome, multiple chemical sensitivity, multiple sclerosis, pseudotumor cerebri, Grave's disease, Parkinson's disease, and interstitial cystitis (bladder pain).
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Finally, an intrepid and much published team in Japan has found DNA damage in 8 tissues from single non-lethal doses of aspartame (near-significant high levels of DNA damage in 5 tissues) and many other additives in groups of just 4 mice:
Mutat Res 2002 Aug 26; 519(1-2): 103-19
The comet assay with 8 mouse organs: results with 39 currently used food additives.
Sasaki YF, Kawaguchi S, Kamaya A, Ohshita M, Kabasawa K, Iwama K, Taniguchi K, Tsuda S.
Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Aomori 039-1192, Japan.
yfsasaki-c@hachinohe-ct.ac.jp ; s.tsuda@iwate-u.ac.jp
We determined the genotoxicity of 39 chemicals currently in use as food additives. They fell into six categories-dyes, color fixatives and preservatives, preservatives, antioxidants, fungicides, and sweeteners.
We tested groups of four male ddY mice once orally with each additive at up to 0.5xLD(50) or the limit dose (2000 mg/kg) and performed the comet assay on the glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24 h after treatment.
Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine, Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal organs at a low dose (10 or 100 mg/kg).
Among them, Amaranth, Allura Red, New Coccine, and Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs).
Two antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides (biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate, saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs.
Based on these results, we believe that more extensive assessment of food additives in current use is warranted. PMID: 12160896
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24 recent formaldehyde toxicity [Comet assay] reports
http://groups.yahoo.com/group/aspartameNM/message/935
Comet assay finds DNA damage from sucralose, cyclamate, saccharin in mice: Sasaki YF & Tsuda S Aug 2002: Murray 2003.01.01 rmforall [ Also borderline evidence, in this pilot study of 39 food additives, using test groups of 4 mice, for DNA damage from for stomach, colon, liver, bladder, and lung 3 hr after oral dose of 2000 mg/kg aspartame-- a very high dose. Methanol is the only component of aspartame that can lead to DNA damage. ]
http://groups.yahoo.com/group/aspartameNM/message/961
Genotoxins, Comet assay in mice: Ace-K, stevia fine; aspartame poor; sucralose, cyclamate, saccharin bad: Y.F. Sasaki Aug 2002: Murray 2003.01.27 rmforall [A detailed look at the data] ]
J Toxicol Sci. 2002 Dec; 27 Suppl 1: 1-8.
[Genotoxicity studies of stevia extract and steviol by the comet assay]
[Article in Japanese]
Sekihashi K, Saitoh H, Sasaki Y. yfsasaki-c@hachinohe-ct.ac.jp
Safety Research Institute for Chemical Compounds Co., Ltd., 363-24 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan.
The genotoxicity of steviol, a metabolite of stevia extract, was evaluated for its genotoxic potential using the comet assay.
In an in vitro study, steviol at 62.5, 125, 250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells in the presence and absence of S9 mix. In vivo studies of steviol were conducted by two independent organizations. Mice were sacrificed 3 and 24 hr after one oral administration of steviol at 250, 500, 1000, and 2000 mg/kg.
DNA damage in multiple mouse organs was measured by the comet assay as modified by us. After oral treatment, stomach, colon, liver, kidney and testis DNA were not damaged.
The in vivo genotoxicity of stevia extract was also evaluated for its genotoxic potential using the comet assay. Mice were sacrificed 3 and 24 hr after oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg.
Stomach, colon and liver DNA were not damaged.
As all studies showed negative responses, stevia extract and steviol are concluded to not have DNA-damaging activity in cultured cells and mouse organs. PMID: 12533916
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http://groups.yahoo.com/group/aspartameNM/message/939
Aspartame (aspartic acid, phenylalanine) binding to DNA:
Karikas July 1998
Karikas GA, Schulpis KH, Reclos GJ, Kokotos G Measurement of molecular interaction of aspartame and its metabolites with DNA. Clin Biochem 1998 Jul; 31(5): 405-7.
Dept. of Chemistry, University of Athens, Greece
http://www.chem.uoa.gr gkokotos@atlas.uoa.gr
"K.H. Schulpis" inchildh@otenet.gr "G.J. Reclos" reklos@otenet.gr
http://groups.yahoo.com/group/aspartameNM/message/938
Aspartame harms mice brain cells: Hetle & Eltervaag: 2001 thesis abstract: Sonnewald 1995 study, full text
http://groups.yahoo.com/group/aspartameNM/message/760
Kovatsi L, Tsouggas M
The effect of oral aspartame administration on the balance of magnesium in the rat.
Magnes Res 2001 Sep;14(3): 189-94.
Laboratory of Forensic Medicine & Toxicology, Faculty of Medicine
Aristotle University of Thessaloniki, Greece kovatsi@med.auth.gr
http://groups.yahoo.com/group/aspartameNM/message/915
Formaldehyde toxicity: Thrasher & Kilburn: Shaham: EPA: Gold: Wilson: CIIN: Murray 2002.12.12
Thrasher (2001): "The major difference is that the Japanese demonstrated the incorporation of FA and its metabolites into the placenta and fetus.
The quantity of radioactivity remaining in maternal and fetal tissues at 48 hours was 26.9% of the administered dose." [ Ref. 14-16 ]
Arch Environ Health 2001 Jul-Aug; 56(4): 300-11.
Embryo toxicity and teratogenicity of formaldehyde. [100 references]
Thrasher JD, Kilburn KH. toxicology@drthrasher.org
Sam-1 Trust, Alto, New Mexico, USA.
http://www.drthrasher.org/formaldehyde_embryo_toxicity.html full text
http://www.drthrasher.org/formaldehyde_1990.html
Full text Jack Dwayne Thrasher, Alan Broughton, Roberta Madison. Immune activation and autoantibodies in humans with long-term inhalation exposure to formaldehyde. Archives of Environmental Health. 1990; 45: 217-223. "Immune activation, autoantibodies, and anti-HCHO-HSA antibodies are associated with long-term formaldehyde inhalation." PMID: 2400243
Confirming evidence and a general theory are given by Pall (2002):
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Testable theory of MCS type diseases, vicious cycle of nitric oxide & peroxynitrite: MSG: formaldehyde-methanol-aspartame: Martin L. Pall: Murray: 2002.12.09
Environ Health Perspect. 2003 Sep; 111(12): 1461-4.
Elevated nitric oxide/peroxynitrite theory of multiple chemical sensitivity: central role of N-methyl-D-aspartate receptors in the sensitivity mechanism. Pall ML. School of Molecular Biosciences, 301 Abelson Hall, Washington State University, Pullman, WA 99164, USA. martin_pall@wsu.edu
The elevated nitric oxide/peroxynitrite and the neural sensitization theories of multiple chemical sensitivity (MCS) are extended here to propose a central mechanism for the exquisite sensitivity to organic solvents apparently induced by previous chemical exposure in MCS.
This mechanism is centered on the activation of N-methyl-D-aspartate (NMDA) receptors by organic solvents producing elevated nitric oxide and peroxynitrite, leading in turn to increased stimulating of and hypersensitivity of NMDA receptors. In this way, organic solvent exposure may produce progressive sensitivity to organic solvents.
Pesticides such as organophosphates and carbamates may act via muscarinic stimulation to produce a similar biochemical and sensitivity response. Accessory mechanisms of sensitivity may involve both increased blood-brain barrier permeability, induced by peroxynitrite, and cytochrome P450 inhibition by nitric oxide.
The NMDA hyperactivity/hypersensitivity and excessive nitric oxide/peroxynitrite view of MCS provides answers to many of the most puzzling aspects of MCS while building on previous studies and views of this condition. PMID: 12948884
Prof. Pall describes processes by which an initial trigger exposure, such as carbon monoxide or formaldehyde, can generate hypersensitivity to many substances. He himself had recovered from a sudden, debilitating attack of multiple chemical sensitivity in June/July 1997.
http://groups.yahoo.com/group/aspartameNM/message/1055
Hormesis: possible benefits of low-level aspartame (methanol, formaldehyde) use: Calabrese: Soffritti: Murray 3.11.4
http://groups.yahoo.com/group/aspartameNM/message/1056
Disorders of NMDA glutamate receptors in brain range from high activity (MCS, CF, PTSD, FM, from carbon monoxide or formaldehyde (methanol, aspartame)-- Pall) to low activity (schizophrenia-- Coyle, Goff, Javitts): Murray 3.13.4
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http://www.readthelabel.org.ukAdditives Survivors' Network (UK)
Geoff Brewer geoffbrewer@eurobell.co.uk
http://www.chem.ox.ac.uk/mom/aspartame/aspartame.html
http://www.chm.bris.ac.uk/webprojects2000/srogers/sarah.html
Sarah Rogers sr8442@bristol.ac.uk
http://www.react.ie/Health/Nutrition/Aspartame.htm Ireland
http://members.tripod.com/~mission_possible/scotland_branch.html
http://www.aspartame.ca/indexa.html
John T. Linnell admin@aspartame.ca
http://www.cybernaute.com/earthconcert2000/AspartaMalcache.htm
http://www.fedupwithfoodadditives.info Australia FAILSAFE diet
http://www.bradymax.com/nzaa New Zealand
http://www.reseauproteus.net/therapies/nutritio/aspartame.htm France
http://ww2.grn.es/avalls/aspa1.htm Spain
http://www.geocities.com/HotSprings/Falls/8669 Brazil
http://www.phd.com.br/aspartame.htm http://hem.passagen.se/mission.possible.sweden
http://home.online.no/~dusan/foods/aspartame.html Norway
http://www.ostara.org/aspartam/#menue Germany
http://www.aspartaam.nl/info/product.html Holland, in Dutch
http://www.laleva.org archimede@laleva.cc Italy 9 languages
http://www.laleva.cc/alimenti/alimenti.html Aspartame vs stevia 4.17.03
http://users.westnet.gr/~cgian/aspartame.htm Greece
http://www.cseindia.org/html/cola-indepth/index.htm India
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http://www.vegsource.com
Extensive vegan information
http://www.drmcdougall.com
Practical, delicious healthy diet guidance
http://www.vegsource.com/articles/kradjian_milk.htm
Robert Kradjian MD Discusses Milk
http://groups.yahoo.com/group/aspartameNM/message/971
Joel Fuhrman critique of Atkins diet in "Eat To Live": Murray 2003.03.01
http://www.hyp.ac.uk/cash/index.htm
Consensus Action on Salt and Health
Substitute stevia (at health food stores).
Avoid all products with aspartame and MSG.
Gradually reduce alcohol, sugar, caffeine (coffee, cocoa, and teas), meat, fish, eggs, milk, butter, and cheese, hydrogenated oils, food additives and colors, fluoride, city water, salt and sodium.
Enjoy organic rice, potatoes, vegetables, fruits, beans, nuts, almond butter, garlic, tumeric, with modest use of soy products and sprouted grain breads, flax seed and olive oils, vitamins and minerals, 4-8 1,000 mg fish oil capsules, and fill your jugs with deionized water.