http://groups.yahoo.com/group/aspartameNM/message/939
aspartame (aspartic acid, phenylalanine) binding to DNA: Karikas July
1998: Murray 1.5.3 rmforall:
FDA Docket 02P-0317 Recall Aspartame as a Neurotoxic Drug

Please post this to the FDA Dockets website.

Rich Murray, MA    Room For All    rmforall@...
1943 Otowi Road, Santa Fe, New Mexico 87505 USA   505-986-9103

Karikas (1998):
"In conclusion, these in vitro findings are of interest because
a widely used compound such as ASP along with its
metabolites gave a measurable molecular interaction with DNA."

This study tests aspartame directly reacting with DNA, and finds very
troubling results.  Karikas also tested the two aspartame components,
aspartic acid and phenylalanine, finding substantial binding with
DNA.  A month earlier, the Trocho study found that formaldehyde from the
methanol component of aspartame also binds with DNA when fed to rats.

So, all three components of aspartame, quickly decomposed in the GI
tract, methanol (11%), aspartic acid (39%), and phenylalanine (50%),
lead to binding  with DNA, the probable results including cell
malfunction and death, mutations, spontaneous abortions, birth defects,
cancers, and chronic complex symptoms for long-term heavy users, over 2
L daily of diet soda.

It is high time for definitive, independently funded studies on the
mutagenic properties of aspartame and its problematic metabolites in
humans.

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 1.1.3 rmforall

http://groups.yahoo.com/group/aspartameNM/message/910
formaldehyde & formic acid from methanol in aspartame:
Murray: 12.9.2 rmforall

It is certain that high levels of aspartame use, above 2 liters daily
for months and years, must lead to chronic formaldehyde-formic acid
toxicity, since 11% of aspartame (1,120 mg in 2L diet soda, 5.6 12-oz
cans) is 123 mg methanol (wood alcohol), immediately released into the
body after drinking (unlike the large levels of methanol locked up in
molecules inside many fruits), then quickly transformed into
formaldehyde, which in turn becomes formic acid, both of which in
time become carbon dioxide and water-- 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.
If 10% of the methanol is retained as formaldehyde, that would give 12
mg daily formaldehyde accumulation, about 60 times more than the 0.2 mg
from 10% retention of the 2 mg EPA daily limit for formaldehyde in
drinking water.

Bear in mind that the EPA limit for formaldehyde in
drinking water is 1 ppm,
or 2 mg daily for a typical daily consumption of 2 L of water.

http://groups.yahoo.com/group/aspartameNM/message/835
RTM: ATSDR: EPA limit 1 ppm formaldehyde in drinking water July 1999
5.30.2 rmforall

This long-term low-level chronic toxic exposure leads to typical
patterns of increasingly severe complex symptoms, starting with
headache, fatigue, joint pain, irritability, memory loss, and leading to
vision and eye problems and even seizures. In many cases there is
addiction.  Probably there are immune system disorders, with a
hypersensitivity to these toxins and other chemicals.

Confirming evidence and a general theory are given by Pall (2002):
http://groups.yahoo.com/group/aspartameNM/message/909
testable theory of MCS type diseases, vicious cycle of nitric oxide &
peroxynitrite: MSG: formaldehyde-methanol-aspartame:
Martin L. Pall: Murray: 12.9.2 rmforall
************************************************************************

"Measurement of Molecular Interaction of Aspartame and Its
Metabolites with DNA"
Clinical Biochemistry, 31 (5); 405-7, July 1998
Manuscript received July 15 1997;
revised and accepted March 16, 1998.
copyright 1998 The Canadian Society of Clinical Chemists
0009-9120/98

George A. Karikas, Pharm D, MD, Lab. of Organic Chemistry,
U. of Athens, Panepistimiopolis, Athens 15771 Greece

George Kokotos, PhD, Assc. Prof., Lab of Organic Chemistry
http://www.chem.uoa.gr/personel/Laboratories/OrganicChem/CVS/kokotos.htm

gkokotos@...    gkokotos@...
++301-7274462   fax ++301-7249101

Kleopatra H. Schulpis, MD, PhD
Pharmacokinetics and Parental Nutrition Unit
Institute of Child Health
"Aghia Sophia" Children's Hospital
Thivon & Levadias Street  GR-11527 Athens,  Greece
(+30 1) 7467 000   fax: (+30 1) 7798 088
http://www.teleremedy.gr/Pages/Members/ag_sophia/aghia.htm

George J. Reclos, PhD    R&D Diagnostics, Ltd
41, Eleftheriou Venizelou street, GR 15561, Holargos, Greece.
+30-1-6537307     fax: +30-1-6537357 & 6548284
reklos@...    reklos@...    reklos@...
http://www.rddiagnostics.com/
http://www.RdDiagnostics.com/cv/page4.html
Dr. George J. Reclos   Curriculum Vitae

Schulpis KH, Karikas GA, Georgala S, Michas T, Tsakiris S.
Elevated plasma homocysteine levels in patients
on isotretinoin therapy for cystic acne.
Int J Dermatol. 2001 Jan;40(1):33-6.
PMID: 11277950

[ Notes by Rich Murray are in square brackets. ]

Following amide and ester hydrolysis ASP is metabolized to
aspartic acid (Asp), methanol and phenylalanine (Phe) with
serum levels of all three metabolites increasing after
ingestion of modest amounts (1,2).

Increases of serum Phe levels have been of concern because of the
pivotal role played by Phe in the transport of precursors of
monoamide neurotransmitters into the brain.  Additionally, Phe is a
diagnostic tool for phenylketonuria, an inborn error of metabolism (3).

Some findings have speculated that ASP molecule might
possess mutagenic potential effect...a promising candidate
to explain the increase in incident and degree of
malignancy of brain tumors (2,4).

In an attempt to reassess by in vitro experiments the
possible carcinogenic potential of ASP we measured its
direct molecular interaction with DNA by using a rapid
reversed phase high-performance liquid chromatography
(HPLC) method (5-7), which has showed a good
correlation with brine shrimp toxicity and tumor inhibition
tests (5).  Asp and Phe were also tested by the same method.
Additionally, a number of synthetic Phe analogues were
used in order to investigate the mechanism of ASP binding
to DNA....

Doxorubicin (Doxo) from Farmacia (1.0 mg/mL) was used
as a typical intercalating agent with major binding (100%) capability...

The column was equilibrated with a H20:MeOH (80:20) solution.
Test samples and DNA solutions were then introduced in a
ratio (1:1, v/v) into the sample loop (20 microL) without
incubation.  The flow rate was maintained at 1 mL/min and the
free DNA eluted from the column in approximately 1 min.
After the appearance of DNA peak, the column was later
washed with MeOH for 20 min to elute the sample mixture.
All samples were tested in triplicate.

ASP and Doxo were tested at three final concentrations
(0.12, 0.25, 0.5 mg/mL) versus DNA at a final
concentration of 0.05 mg/mL.

According to our method a fixed amount of ligand
is added to the elution solvent of the HPLC system
and a known quantify of DNA is then injected.  This
results in a residual DNA peak (% DNA peak size
exclusion) where the exclusion of the peak is
proportional to the amount of bound ligand....

Table 1
Molecular Effect of Doxo and ASP on DNA

Compound (mg/mL)       % DNA Peak Exclusion (% DNA Bonding)

DNA 0.05 + Doxo 0.12          45.8+- 5.4
DNA 0.05 + Doxo 0.25         100
DNA 0.05 + Doxo 0.50         100

DNA 0.05 + ASP  0.12          11.3+-  3.4
DNA 0.05 + ASP  0.25          39.8+-  6.1
DNA 0.25 + ASP  0.50          65.5+- 10.1

[ So, doubling the amount of ligand roughly
doubles the % DNA bound, until 100% saturation
of the DNA. ]

A moderate DNA molecular interaction, expressed as
almost 40% (39.8+- 6.1) DNA peak exclusion
was observed when ASP was tested with DNA
(0.05 mg/mL) at a final concentration of of 0.25 mg/mL.

Analogous effect was exhibited by Phe (31.6+- 8.5%) at a final
concentration of 0.25 mg/mL, whereas a 65.5+- 10.1% DNA peak exclusion
was observed when ASP reached the final concentration of 0.50 mg/mL.

Doxo performed a complete molecular DNA effect
(intercalation plus ionic interaction) (100% DNA
peak size exclusion) at concentration of 0.25 and
0.50 mag/mL.

Table 2
Molecular Effect of Phe and Related Compounds on DNA

Compound (mg/mL)           % DNA Peak Exclusion (% DNA binding)

DNA 0.05 + Phe 0.25        31.6+- 8.5
DNA 0.05 + Asp 0.25        39.3+- 4.2
DNA 0.05 + Ala 0.25        12.3+- 4.1
DNA 0.05 + Z-Phe 0.25       0
DNA 0.05 + I   0.25       100

Phe        L-phenylalanine
Asp        L-aspartic acid
Ala        L-alanine
Z-Phe      benzyloxycarbonyl-L phenylalanine
I          3-phenyl-1,2-propanediamine

A moderate DNA exclusion (39.3+- 4.2%) was found
when L-Asp (conc. 0.25 mg/mL) was tested with
DNA (0.05 mg/mL), whereas Ala performed only a
mild effect on DNA (12.3+- 4.1%) at the same
concentration.

Benzyloxycarbonyl-L-Phe gave no measureable interactions.
On the contrary, the synthetic analogue 3-phenyl-1,2-propanediamine
exhibited complete DNA peak exclusion (100%).

In general, there are three major classes of clinically
important DNA interactive substances:
the alkylators, which react covalently with DNA base,
the DNA strand breakers, which generate reactive radicals
that produce cleavage of the polynucleotide strands, and
the compounds that interact reversibly with DNA (10,11).

Intercalators and cationic polyamines exhibiting
electrostatic interactions are included in the third class...

The ionized amino group was close to the
deoxyribose phosphate chain suggesting that a
strong electrostatic interaction could take place between
the drug and the negatively charged DNA phosphate away
from the interaction site (10). Such phenomena were
successfully detected in previous experiments by using
cationic polyamines (6) and DNA photochemical adducts
(7) as well as in the present study expressed in the
% DNA peak exclusion.

Therefore, ASP along with Phe and Asp exhibited relative binding effect
on DNA due to the presence of their amino groups.

The amino acid Ala, which does not contain the aromatic ring of the Phe
side chain gave a small measureable % DNA peak exclusion due to the
presence of the amino group, whereas no DNA peak exclusion was observed
with benzyloxycarbonyl-L-Phe, a protected amino group Phe analogue.

However, the Phe derivative 3-phenyl-1,2-propanediamine, where the
carbonyl group of Phe has been substituted by an additional amino group
showed a considerable increase of % DNA peak exclusion compared to that
caused by Phe (100% and 31.6% respectively).

This result is obviously related to the presence of the two amino groups
which is in full agreement with previous data (6).

Consequently, the potency of the phenomenon attributed to
ionic interactions is increased when the number of amino
groups increases (Table 2).

Apart from the ionic effect, a possible partial intercalation of the Phe
aromatic ring into the base pairs of DNA is reinforced by recent
findings showing that the Phe residues of TATA box binding protein
interacts with the T-A base pair by Van der Walls contacts (12).

Although Shephard et al (13) found no detectable mutagenicity
of ASP and Phe by using other methods, structural modification
of DNA through covalent and noncovalent interactions have
significant functional consequences such as replication errors,
which could be among the events that start the cellular
processes ultimately yielding malignant tumors (14).

The hypothesis that structural transitions and condensations
in specific DNA sequences caused by polyamines may also
be related to nucleosome formations and the condensation
of DNA into chromatin is gaining experimental support.

In conclusion, these in vitro findings are of interest because
a widely used compound such as ASP along with its
metabolites gave a measurable molecular interaction with DNA.

These DNA effects are evaluated for the first time with a
considerable reproducibility and can serve as a useful
prescreen assay.

1. Partridge WM.
The safety of aspartame.
JAMA 1986; 256: 2678 (letter)

2. Janssen PJ, Heijden CA.
Aspartame: review of recent experimental and observational data.
Toxicology 1986; 50: 1-26.

3. Michals K, Azen C, Acosta P.
Blood Phenylalanine levels and intelligence
of 10 year old children with Phenylketonuria.
J Am Diet Assoc 1988; 88: 1226-9.

4. Olney JW, Farber NB, Spitznagel E, Robins LN.
Increasing brain tumor rates: Is there a link to aspartame?
J Neuropathol Exp Neurol 1996 55/11: 115-23.

5. Gupta MP, Monge A, Karikas GA, et al.
Screening of panamanian medicinal plants
for brine shrimp toxicity, grown gall, tumor inhibition,
cytotoxicity and DNA intercalation.
Int J Pharmacognosy 1996; 34: 19-27.

6. Karikas GA, Constantinou V, Kokotos G.
An HPLC method for the measurement of polyamines
and lipidic amines binding to DNA.
J Liquid Chromatography 1997; 20(11): 1789-96.

7. Karikas GA, Schulpis KH, Kokotos G,
Michas T, Georgala S.
Stoichiometric measurement of DNA damage
caused by 8-Methyl-Psoralen and UVA.
Clin Biochemistry 1997; 30(5): 439-42.

8. Aposhian HV, Kornberg A
Enzymatic synthesis of Deoxyribonucleic acid.
J. Biol Chem 1962; 237: 519-25.

9. Kokotos G, Constantinou V,
Modifid amino acids and peptides.
Part 2. A convenient conversion of amino and
peptide alcohols into amines.
J Chem Res (S) 1992; 391, and
J Chem Res (M) 1992; 3117-32.

10. Silverman RB.
The organic chemistry of drug design and drug action.
DNA 1992; 236-43.

11. Blackburn GM, Gait MJ, eds.
Reversible interactions of nucleic acids with small molecules.
In: Nucleic acids in chemistry and biology. Pp. 297-336.
Oxford: IRL Press, 1990.

12. Burley SK.
The TATA box binding protein.
Curr Opin Struct Biol 1996; 6: 69-75.

13. Shephard SE, Wakabayashi K, Nagao M.
Mutagenic activity of peptides and the artifical
sweetener aspartame after nitrosation.
Food Chem Toxicol 1993; 31/5: 325-9.

14. Wallace S, Van Heuten B, Kow YW.
DNA damage effecting DNA structure and
protein recognitions.
Ann NY Acad Sci 1994; 726: 18.
************************************************************************

http://groups.yahoo.com/group/aspartameNM/message/932
aspartame: methanol, formaldehyde, formic acid toxicity:
brief review: Murray 12.30.2 rmforall:

http://groups.yahoo.com/group/aspartameNM/messages
for 939 posts in a public searchable archive

http://groups.yahoo.com/group/aspartameNM/message/915
formaldehyde toxicity:  Thrasher & Kilburn: Shaham: EPA: Gold: Murray:
Wilson: CIIN: 12.12.2 rmforall

http://www.drthrasher.org/formaldehyde_1990.html  [ full text ]
Arch Environ Health 1990 Jul-Aug; 45(4): 217-23
Immune activation and autoantibodies in humans
with long-term inhalation exposure to formaldehyde.
Thrasher JD, Broughton A, Madison R.
Thrasher & Associates, Northridge, California.

Arch Environ Health 2001 Jul-Aug; 56(4): 300-11
Embryo toxicity and teratogenicity of formaldehyde. [ 100 references]
Thrasher JD, Kilburn KH.
Sam-1 Trust, Alto, New Mexico, USA.
http://www.drthrasher.org/formaldehyde_embryo_toxicity.html
[ 127K full text ] http://www.drthrasher.org
Jack D. Thrasher, PhD   toxicology@...   Sam-1 Trust,
PO Box 874  Alto, New Mexico 88312    505-336-8312  fax 425-675-7379

http://www-hsc.usc.edu/~kilburn/
Kaye H. Kilburn, M.D.      kilburn@...
University of Southern California
Keck School of Medicine
Environmental Sciences Laboratory
2025 Zonal Avenue, CSC 201, Los Angeles, California 90033
text Dec, 1997  "Chemical Brain Injury"

http://groups.yahoo.com/group/aspartameNM/message/925
aspartame puts formaldehyde adducts into tissues, Part 1/2
full text, Trocho & Alemany 6.26.98: Murray 12.22.2 rmforall

http://groups.yahoo.com/group/aspartameNM/message/926
aspartame puts formaldehyde adducts into tissues, Part 2/2
full text, Trocho & Alemany 6.26.98: Murray 12.22.2 rmforall

http://ww.presidiotex.com/barcelona/index.html
Trocho C, Pardo R, Rafecas I, Virgili J, Remesar X,
Fernandez-Lopez JA, Alemany M  ["Trok-ho"]
Formaldehyde derived from dietary aspartame binds to tissue
components in vivo.  Life Sci 1998 Jun 26; 63(5): 337-49.
Departament de Bioquimica i Biologia Molecular, Facultat de Biologia,
Universitat de Barcelona, Spain.
http://www.presidiotex.com/barcelona/index.html
Maria Alemany, PhD (male)  alemany@...

http://groups.yahoo.com/group/aspartameNM/message/864
Murray: Butchko, Tephly, McMartin: Alemany: aspartame formaldehyde
adducts in rats 9.8.2 rmforall
Prof. Alemany vigorously affirms the validity of the Trocho study
against criticism:
Butchko, HH et al [24 authors], Aspartame: review of safety.
Regul. Toxicol. Pharmacol. 2002 April 1; 35 (2 Pt 2): S1-93, review
available for $35, [an industry paid organ].  Butchko:
"When all the research on aspartame, including evaluations in both the
premarketing and postmarketing periods, is examined as a whole, it is
clear that aspartame is safe, and there are no unresolved questions
regarding its safety under conditions of intended use."
[They repeatedly pass on the ageless industry deceit that the methanol
in fruits and vegetables is as as biochemically available as that in
aspartame-- see the 1984 rebuttal by Monte, below.]

http://groups.yahoo.com/group/aspartameNM/message/911
RTP ties to industry criticized by CSPI: Murray: 12.9.2 rmforall

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 1.1.3 rmforall

http://groups.yahoo.com/group/aspartameNM/message/934
24 recent formaldehyde toxicity [Comet assay] reports:
Murray 12.31.2 rmforall

http://groups.yahoo.com/group/aspartameNM/message/622
Rich Murray: Gold: Koehler: Walton: Van Den Eeden: Leon:
aspartame toxicity 6.4.1 rmforall

http://groups.yahoo.com/group/aspartameNM/message/623
Rich Murray: Simmons: Gold: Schiffman: Spiers:
aspartame toxicity 6.4.1 rmforall

http://groups.yahoo.com/group/aspartameNM/message/917
HERP ranking of carcinogens (formaldehyde is very high):
www.berkeley.edu: Murray 12.14.2 rmforall

http://groups.yahoo.com/group/aspartameNM/message/870
Aspartame: Methanol and the Public Interest 1984:
Monte: Murray 9.23.2 rmforall

Dr. Woodrow C. Monte  Aspartame: methanol and the public health.
Journal of Applied Nutrition 1984;  36 (1):  42-54.
(62 references)   Professsor of Food Science
Director of the Food Science and Nutrition Laboratory
Arizona State University,  Tempe, Arizona 85287
6411 South River Drive #61 Tempe, Arizona 85283-3337
602-965-6938    woody.monte@...
The methanol from 2 L of diet soda, 5.6 12-oz cans, 20 mg/can, is
112 mg, 10% of the aspartame.  The EPA limit for water is 7.8 mg daily
for methanol (wood alcohol), a deadly cumulative poison. Many users
drink 1-2 L daily. The reported symptoms are entirely consistent
with chronic methanol toxicity. (Fresh orange juice has 34 mg/L, but,
like all juices, has 16 times more ethanol, which strongly protects
against methanol.)

"Fruit and vegetables contain pectin with variable methyl ester
content. However, the human has no digestive enzymes for pectin (6, 25)
particularly the pectin esterase required
for its hydrolysis to methanol (26).

Fermentation in the gut may cause disappearance of pectin (6) but the
production of free methanol is not guaranteed by fermentation (3).  In
fact, bacteria in the colon probably reduce methanol directly to formic
acid or carbon dioxide (6)  (aspartame is completely absorbed before
reaching the colon). Heating of pectins has been shown to cause
virtually no demethoxylation; even temperatures of 120 deg C produced
only traces of methanol (3).  Methanol evolved during cooking of high
pectin foods (7) has been accounted for in the volatile fraction during
boiling and is quickly lost to the atmosphere (49).
Entrapment of these volatiles probably accounts for the elevation in
methanol levels of certain fruit and vegetable products
during canning (31, 33)."

Recent research [see links at end of post] supports his focus on the
methanol to formaldehyde toxic process:

"The United States Environmental Protection Agency in their Multimedia
Environmental Goals for Environmental Assessment recommends a minimum
acute toxicity concentration of methanol in drinking water at 3.9 parts
per million, with a recommended limit of consumption below 7.8 mg/day
(8). This report clearly indicates that methanol:

"is considered a cumulative poison
due to the low rate of excretion once it is absorbed.
In the body, methanol is oxidized to formaldehyde and
formic acid; both of these metabolites are toxic." (8)....

Recently the toxic role of formaldehyde (in methanol toxicity) has been
questioned (34).  No skeptic can overlook the fact that, metabolically,
formaldehyde must be formed as an intermediate to formic acid
production (54).

Formaldehyde has a high reactivity which may be why it
has not been found in humans or other primates during methanol
poisioning (59)....

If formaldehyde is produced from methanol and does have a reasonable
half life within certain cells in the poisoned organism the chronic
toxicological ramifications could be grave.

Formaldehyde is a known
carcinogen (57) producing squamous-cell carcinomas by inhalation
exposure in experimental animals (22).  The available epidemiological
studies do not provide adequate data for assessing the carcinogenicity
of formaldehyde in man (22, 24, 57).

However, reaction of formaldehyde
with deoxyribonucleic acid (DNA) has resulted in irreversible
denaturation that could interfere with DNA replication and result in
mutation (37)...."

http://groups.yahoo.com/group/aspartameNM/message/936
flawed test for aspartame DNA damage: Jeffrey & Williams 2000: Murray:
1.3.3 rmforall

http://groups.yahoo.com/group/aspartameNM/message/886
aspartame reduces aggression in rats?: Goerss, Wagner, Hill: Aug 2001:
Murray 11.10.2 rmforall
************************************************************************