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genotoxins, Comet assay in mice: Ace-K, stevia fine; aspartame poor; sucralose, cyclamate, saccharin bad: Y.F. Sasaki Aug 2002: Murray 1.27.3 rmforall

Rich Murray rmforall at att.net
Wed Jan 29 00:25:51 EST 2003

genotoxins, Comet assay in mice: Ace-K, stevia fine; aspartame poor;
sucralose, cyclamate, saccharin bad: Y.F. Sasaki Aug 2002:
Murray 1.27.3 rmforall 

Jan 27 2003

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

Summary of Review by Rich Murray:  This study tests 39 common food
additives for DNA damage, comparing a control group of 4 mice against
test groups of 4 mice each, killed 3 hr and 24 hr after oral
ingestion of up to 2000 mg/kg.
However, there are only 21 unique control groups, with widely varying
values.  By using the averages for all 21 control groups to make
comparison with the groups exposed to the food additives, it is easy
to see that many additives cause about 140% to about 180% to over 300%
of the averages of all control groups for the 8 organs measured.  By
using more mice, statistical significance may be easily proved for most
of these easily noticable high values, which are not significant for
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.   Yu F. Sasaki
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 at hachinohe-ct.ac.jp  s.tsuda at 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 100mg/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    

Also tested were acesulfame K, aspartame, stevia, and glycyrrhizin--
which all came out nonsignificant, while, as the abstract mentions, 
sodium cyclamate had 4, saccharin 3, sucralose 3, and sodium saccharin 5
significant results.

Each test condition had just 4 mice, and, according to the text, each
additive had its own control group of 4 mice.  However, there are only
21 unique sets of control groups, with 8 sets used once, 10 sets used
twice, 2 sets used 3 times, and 1 set used 4 times, a total of 38 food
additives listed [Sodium erythorbic acid was left out of Table 2, while
mentioned in the report 3 times, "...erythorbic acid and its sodium salt
did not increase DNA damage in any of the organs studied."].

Aspartame was assigned the control group that had the highest levels of
Migration of damaged nuclear DNA for Liver and  Bladder, and the second
highest for Brain.  The same control group was used for the xanthene
dye, erythrosinc, which had Migration as high as 42.4+-2.17 um
[micro-meter], measured on 50 nuclei from stomach cells, 3 hours
after ingestion.  So, the high control groups values had no effect on
the statistical analysis for erythrosinc.

The available range of the 21 control groups ranged for the Liver from 
1.1 to 3.6 um.  For aspartame, the Liver Migration, the average length
of the "comet" tail of damaged, broken DNA pulled out of 50 Liver cell
nuclei by an electric field for 15 minutes, was, average of 4 mice:

control value used  3.59+-0.50 um [1.1 to 3.6 range in 21 controls]
2000 mg/kg  3 hr    3.26+-0.16 um   
2000 mg/kg 24 hr    0.57+-0.22 um

The 3 hr aspartame test value was about the same as the control value.
This may be discordant with the Trocho (1998) findings that rats given
200 mg/kg oral doses of aspartame for 11 days, about the same total
dose, had accumulation of formaldehyde adducts, bound to DNA, RNA, and
proteins, in liver, kidneys, brain, retinas, and other tissues, at about
the same total dose, spread over 11 days. 

Appying the lowest available control group liver level 1.06+-0.12 um
would make the aspartame level of 3.26+-0.16 um significant [ratio 3.1].

How significant is a ratio of about 2?
I found two examples in the data, where P<.05 existed for BHT, Bladder,
1000 mg/kg, 3 hr:
10.9+-1.32 vs control 4.77+-0.40 [range 3.6 to 7.1 for 21 controls],
[ratio 2.3]
and sodium cyclamate, Stomach, 1000 mg/kg, 3 hr:
12.2+-1.38 vs control 6.37+-0.57 [range 4.3 to 8.6 for 21 controls]
[ratio 1.9].
However, not significant was:
sodium saccharin, Liver, 2000 mg/kg, 3 hr:
5.95+-2.42 vs control 1.94+-0.36  [range 1.1 to 3.6 for 21 controls]
[ratio 3.1], since the +- error was 33% of the test value. So, if the
data for 4 mice is scattered, then the mean value of the test group has
to be over 3 times that of the control group to be significant.

For Liver, 5 of the 21 control groups, with values 1.67, 1.63, 1.29,
1.06, 1.65 would make some 3 hr aspartame values approach or reach

Ratios about 2 for different tissues with aspartame that would be close
to significant would exist for many of the 21 control groups:
Stomach 1  Colon 5   Liver 5   Bladder 11   Lung 5 .

The aspartame values at 3 hr are compared with 
the mean values for the 21 control groups:

Somach      Colon       Liver       Kidney       Bladder      Lung
DNA Migration at 3 hr from 2000 mg/kg dose
8.49+-0.48  9.18+-0.56  3.26+-0.16  1.91+-0.26   10.7+-2.77   4.13+-1.26
mean of 21 control groups
6.31        5.81        2.15        2.25          5.40        2.61
range of values for 21 control groups
4.3--8.6    4.0--8.1    1.1--3.6    1.2--2.9      3.6--7.1    1.6--4.7
ratio = DNA Migration/control mean
1.4         1.6         1.5         0.9           2.0         1.6

Brain       Bone [marrow]
0.37+-0.70  1.01+-0.59  DNA Migration at 3 hr from 2000 mg/kg dose

1.48        1.12        mean of 21 control groups

0.8--2.6    0.6--1.9    range of values for 21 control groups

0.3         0.9         ratio = DNA Migration/control mean

Wouldn't the average of all the 21 control groups be the best control
values to use?  What would then be the appropriate statistical test? 
How many mice would it take to reach significance for the 5 tissues with
ratios over 1.4: Stomach, Colon, Liver, Bladder, Lung? 

Aspartame at 24 hours had levels too low to reach significance with any
of the 21 control groups.

However, people who are heavy users of aspartame for years are bound
to accumulate toxic metabolites of the three components of aspartame:
methanol 11%, phenylalanine 50%, aspartic acid 29%, all genotoxic
[Trocho (1998), Karakis (1998)].

Comparing the mean control values to the values for the other 7
Best is acesulfame K, with no significant or high values.

Good is glycyrrhizin (derived from licorice), two 1.4 ratios for Stomach
and Brain. 

Next is stevia, with one high value [above ratio 1.4],
9.48+-1.99 for Bladder, 2000 mg 3 hr,  ratio 1.8 .

Aspartame has high values for 2000 mg 3 hr for Stomach, Colon, Liver,
Bladder, Lung.

Sucralose has 3 significant values and 13 high values, for Stomach,
Colon, Kidney, Bladder, Lung, Brain.

Sodium cyclamate has 4 significant values and 10 high values for
Stomach, Colon, Liver, Kidney, Bladder, Lung, Brain, Bone.

Saccharin has 3 highly significant values for Colon, and 13 high values
for Stomach, Colon, Kidney, Lung, Brain, Bone.

Sodium saccharin has 5 highly significant values for Stomach and Colon,
and 14 high values for Stomach, Liver, Kidney, Bladder, Lung, Brain,

We should keep in mind that toxicity in humans involves many vulnerable
groups, years of daily use, often evolution of hypersensitivity, and
complex interactions with a multitude of foods, additives, other toxins,
and foods.

Some of the dye data was earlier published in Tsuda (2001):
Toxicol Sci 2001 May; 61(1): 92-9
DNA damage induced by red food dyes orally administered to pregnant
and male mice.
Tsuda S, Murakami M, Matsusaka N, Kano K, Taniguchi K, Sasaki YF.
Laboratory of Veterinary Public Health, Department of Veterinary
Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8,
Morioka, Iwate 020-8550, Japan.  s.tsuda at iwate-u.ac.jp

We determined the genotoxicity of synthetic red tar dyes currently used
as food color additives in many countries, including JAPAN: For the
preliminary assessment, we treated groups of 4 pregnant mice
(gestational day 11) once orally at the limit dose (2000 mg/kg) of
amaranth (food red No. 2), allura red (food red No. 40), or acid red
(food red No. 106), and we sampled brain, lung, liver, kidney, glandular
stomach, colon, urinary bladder, and embryo 3, 6, and 24 h after

We used the comet (alkaline single cell gel electrophoresis) assay to
measure DNA damage. The assay was positive in the colon 3 h after the
administration of amaranth and allura red and weakly positive in the
lung 6 h after the administration of amaranth.

Acid red did not induce DNA damage in any sample at any sampling time. 

None of the dyes damaged DNA in other organs or the embryo.

We then tested male mice with amaranth, allura red, and a related
color additive, new coccine (food red No. 18). The 3 dyes induced DNA
damage in the colon starting at 10 mg/kg.

Twenty ml/kg of soaking liquid from commercial red ginger pickles, which
contained 6.5 mg/10 ml of new coccine, induced DNA damage in colon,
glandular stomach, and bladder.

The potencies were compared to those of other rodent carcinogens. The
rodent hepatocarcinogen p-dimethylaminoazobenzene induced colon DNA
damage at 1 mg/kg, whereas it damaged liver DNA only at 500 mg/kg. 

Although 1 mg/kg of N-nitrosodimethylamine induced DNA damage in liver
and bladder, it did not induce colon DNA damage. N-nitrosodiethylamine
at 14 mg/kg did not induce DNA damage in any organs examined. Because
the 3 azo additives we examined induced colon DNA damage at a very low
dose, more extensive assessment of azo additives is warranted.
PMID: 11294979

comet assay finds DNA damage from sucralose, cyclamate, saccharin in
mice: Sasaki YF & Tsuda S  Aug 2002: Murray 1.1.3 rmforall

The Single Cell Gel Assay is able to detect single-strand and
double-strand DNA breaks in individual eukaryotic cells; requires small
numbers of cells (<20,000 per sample); can detect DNA damage from low
levels of toxic or physical insults; and is rapid, simple and efficient.
In this assay, cells are treated with the agent of interest, embedded in
agarose on a histological slide, the cell membranes are lysed, and the
slides are placed in an electric field. If the DNA has single or
double-strand breaks, it will flow out of the cells and move toward the
anode, causing the cell and its DNA to resemble a comet. The more DNA
released from the cell, the greater the DNA damage. A computerized
imaging system is used to score and measure the comets.  The Comet assay
is not FDA approved as a human medical test, so it is not covered by
insurance.  It is used in many human research studies.

http://cometassay.com/  Comet Assay Interest Group



http://www.ems-us.org/   Environmental Mutagen Society

DNA repair Interest Group  about a thousand members

Integrated Laboratory Systems    Comet assays for $155-300

http://www.mdbiotechinc.com/  MD Biotech, Inc.
Comet assays on four 10 ml blood samples for $800

Here I summarize the mean +- variation values for the 21 control groups,
for each tissue, giving the smallest variation and the largest.

Stomach     Colon       Liver       Kidney      Bladder     Lung 
4.90+-0.26  4.49+-0.19  1.91+-0.19  1.81+-0.13  5.89+-0.24  2.44+-0.17
      5%          4%         10%          7%          4%          7%
5.55+-1.26  7.91+-1.95  1.29+-0.69  1.73+-0.96  5.68+-1.30  2.56+-1.04
     23%         25%         53%         56%         23%         41%

Brain       Bone [marrow]  
2.58+-0.40  1.16+-0.15  
     16%         13%
1.09+-1.09  0.75+-0.75
    100%        100%

We have +- mean variation, for the 21 control groups of 4 mice, from 4
to 100%.  What causes this variation, for a specific strain of mice,
with the same diet, environment, and age?  Are there a number of
genotoxins in the laboratory diet, with the mice exhibiting many genetic

aspartame: methanol, formaldehyde, formic acid toxicity:
brief review: Murray 1.17.3 rmforall

for 961 posts in a public searchable archive

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.

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):
testable theory of MCS type diseases, vicious cycle of nitric oxide &
peroxynitrite: MSG: formaldehyde-methanol-aspartame:
Martin L. Pall: Murray: 12.9.2 rmforall

Functional Therapeutics in Neurodegenerative Disease Part 1/2:
Perlmutter 7.15.99: Murray 1.10.3 rmforall

formaldehyde toxicity:  Thrasher & Kilburn: Shaham: EPA: Gold: Murray:
Wilson: CIIN: 12.12.2 rmforall

24 recent formaldehyde toxicity [Comet assay] reports:
Murray 12.31.2 rmforall

comet assay finds DNA damage from sucralose, cyclamate, saccharin in
mice: Sasaki YF & Tsuda S  Aug 2002: Murray 1.1.3 rmforall

aspartame (aspartic acid, phenylalanine) binding to DNA:
Karikas July 1998: Murray 1.5.3 rmforall
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 at atlas.uoa.gr

http://www.dorway.com/tldaddic.html  5-page review
Roberts HJ Aspartame (NutraSweet) addiction.
Townsend Letter  2000 Jan;  HJRobertsMD at aol.com
http://www.sunsentpress.com/    sunsentpress at aol.com
Sunshine Sentinel Press  P.O.Box 17799  West Palm Beach, FL 33416
800-814-9800 561-588-7628 561-547-8008 fax

1038-page medical text   "Aspartame Disease: An Ignored Epidemic"
published May 30  2001    $ 85.00 postpaid    data from 1200 cases
available at  http://www.amazon.com
over 600 references from standard medical research

aspartame puts formaldehyde adducts into tissues, Part 1/2
full text, Trocho & Alemany 6.26.98: Murray 12.22.2 rmforall

aspartame puts formaldehyde adducts into tissues, Part 2/2
full text, Trocho & Alemany 6.26.98: Murray 12.22.2 rmforall

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.
Maria Alemany, PhD (male)  alemany at porthos.bio.ub.es

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.]

RTP ties to industry criticized by CSPI: Murray: 12.9.2 rmforall


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