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Sunday, February 27, 2011

ASPARTAME TRIGGERS: LIVER CANCER and LUNG CANCER IN LATEST STUDIES

______________________________________________

Am J Ind Med. 2010 Sep 30. 
Aspartame administered in feed through life span, induces cancers of the liver and lung in mice.
Soffritti M, Belpoggi F, Manservigi M, Tibaldi E, Lauriola M, Falcioni L, Bua L.
Cesare Maltoni Cancer Research Center, Ramazzini Institute,
Bentivoglio, Bologna, Italy.

Abstract

BACKGROUND:
Aspartame (APM) is a well-known intense artificial sweetener used in more
than 6,000 products. Among the major users of aspartame are children and women of childbearing
age. In previous lifespan experiments conducted on Sprague-Dawley rats we have
shown that APM is a carcinogenic agent in multiple sites and that its
effects are increased when exposure starts from prenatal life.

OBJECTIVE:
The aim of this study is to evaluate the potential of APM to induce
carcinogenic effects in mice.

APM is metabolized in the gastrointestinal tract by esterases and
peptidases into three components: the amino acids phenylanine and
aspartic acid, and methanol
[Ranney et al., 1976].

APM can be also absorbed into the mucosal cells prior to hydrolysis
and then metabolized within the cell to its three components which
then enter circulation [Mattews, 1984].

Methanol is not subject to metabolism within the enterocyte and
rapidly enters the portal circulation and is oxidized in the liver to
formaldehyde, an highly reactive chemical which strongly binds to
proteins [Haschemeyer and Haschemeyer, 1973] and nucleic acids
[Metzler, 1977] forming formaldehyde adducts.

In a study, in which APM, 14 C-labeled in the methanol carbon, was
given orally to adult male Wistar rats for 10 days, it was shown that
the carbon adducts of protein and DNA could have been generated only
from formaldehyde derived from APM methanol.
Moreover, it was suggested that the amount of formaldehyde adducts may
be cumulative [Trocho et al., 1998].

Several reviews conclude that APM is digested in all species in the
same way [Ranney et al., 1976].
Since APM is metabolized before entering the blood stream, there is no
distribution of APM outside the gastrointestinal tract.

Epidemiological studies conducted among users of artificial sweeteners
(including APM) did not show an increased carcinogenic risk, except in
one study which postulated an association of increased risk of brain
cancer and use of APM [Olney et al., 1996].

Studies performed by the US National ToxicologyProgram (NTP) in which
groups of 15 males and 15 females of transgenic mice, p53
haploinsufficient strain (p53) and Tg.AC homozygous strain (Tg.AC)
dermal exposure model were treated with diets containing 0, 3, 125,
6,250, 12,500, 25,000, or 50,000 ppm of APM for 40 weeks and then
sacrificed did not show any carcinogenic responses [NTP, 2005].
Overall there was no evidence of a positive response for tumors in
animals treated with APM in feed up to 50,000 ppm.
Although the studies did not show carcinogenic response, it should be
noted that altered genetic mice were evaluated by NTP with the intent
to develop faster, less costly and more predictive in vivo models for
identifying potential chemical carcinogenic agents and that APM was
selected as a presumed non-carcinogen.
Pritchard et al. [2003] evaluated the NTP findings regarding the
potential of transgenic mouse models to identify carcinogenic agents.
The authors concluded that the Tg.AC dermal exposure model and p53
oral exposure model had an overall accuracy of 74% in correctly
predicting chemicals that are listed by the International Agency for
Research on Cancer (IARC) and/or NTP in their respective lists of
chemicals classified carcinogenic or probably carcinogenic in humans.
The study concluded that the transgenic mouse models missed a number
of known or probable human carcinogens, whereas long-term rodent
bioassays missed none of these chemicals.

Indeed, the authors of the studies performed by NTP concluded that the
negative findings were of uncertain value:
''because this is a new model, there is uncertainty whether the
(aspartame) study possessed sufficient sensitivity to detect a
carcinogenic effect'' [NTP, 2005].
In fact the P53 deficient transgenic model does not respond to
non-genotoxic carcinogenic chemicals, and hence choosing that model
confirmed this fact with APM.
The NTP has since virtually discontinued the use of genetically
modified models for identifying carcinogens.

Long-term carcinogenicity bioassays performed on rats and mice in the
early 1970s by industry did not show any carcinogenic effects.
In female p53 haploinsufficient mice, the results of the micronucleus
test were judged to be positive, based on a significant trend test and
a small but statistically significant increased frequency of
micronucleated erythrocytes in the 50,000 ppm group
(P = 0.028) [NTP, 2005].

A detailed review and comments on the genotoxicity, long-term
carcinogenicity studies in rodents and epidemiological studies
available today on APM has been reported previously [Soffritti et al.,
2005, 2006, 2007].
Overall, we believe that the potential long-term toxic effects of APM,
and in particular the carcinogenic effects, had not been adequately
demonstrated by the long-term bioassays on rats and mice, mainly
because of the small number of animals used per sex per group and the
duration of the experiments (in which rodents were sacrificed at 110
weeks of age, corresponding to the two-thirds of the lifespan).

For these reasons we started a project encompassing several
experiments on rats and mice in which APM was administered in feed at
various doses to a large number of rats or mice per group per sex.
Treatment started at different ages and lasted for different periods;
rodents were always kept under observation until natural death to
allow APM to express all its full carcinogenic potential.

In the first experiment we demonstrated that APM, administered from 8
weeks of age for the lifespan to Sprague–Dawley rats, induced a
significantly increased incidence of lymphomas/leukemias and of
neoplastic lesions of the renal pelvis and ureter in females, and a
significantly increased incidence of malignant Schwannomas of the
peripheral nerves in males
[Soffritti et al., 2006].

In a second experiment we showed that APM, administered from fetal
life until natural death, caused lymphomas/leukemias in male and
female rats and, for the first time, cancers of the mammary glands in
females
[Soffritti et al., 2007].
Furthermore, this study demonstrated that when lifespan exposure
starts during fetal life, the incidences of lymphomas/leukemias were
increased in comparison to the treatment starting postnatally.
Neither cranial Schwannomas nor neoplasms of the renal pelvis and
ureter were observed in the second experiment.
This result may be explained by the fact that the number of rats per
sex per group in this study was lower and therefore the sensitivity of
the study for this type of tumors may have been reduced....

.... APM was pulverized in a standard pelleted diet at concentrations of
0, 2,000, 8,000, 16,000, or 32,000
to simulate an assumed daily APM intake of
0, 250, 1,000, 2,000, and 4,000 mg/kg b.w.,
and was administered to groups of 62–122 male and female Swiss mice
from the 12th day of fetal life until death.
The dose levels of APM were chosen on the basis of available data
reported in the literature.
The standard ''Corticella diet'' was provided by Laboratorio Dottori
Piccioni, Milan, Italy;
the same diet used for more than 30 years at the laboratory of the
Cesare Maltoni Cancer Research Center (CMCRC).
Fresh tap water was provided daily.
The major constituents of the diet were: water 12%; raw protein 24%;
raw fat 3.50%; raw fibers 5.50%; ashes 10.50%; non-nitrogenous extracts
56.50%.
The diet was analyzed for nutritional components, microorganisms, and
possible contaminants (pesticides, metals, estrogen activity,
nitrosamines, and aflatoxins) every 6 months, and disposed of if older
than 3 months from the date of manufacture.
The diet was formulated every 40–50 days.
At room temperature APM is stable in food and liquid.
The stability of APM in the feed was analyzed periodically during the
experiment.
Feed and water were supplied ad libitum.....

...DISCUSSION
The present study, in which APM was administered in feed at the dose
levels of 0, 2,000, 8,000, 16,000, or 32,000 ppm to Swiss mice from
prenatal life until death, further confirms that APM induces
carcinogenic effects in rodents.
The study shows:
(a) significant dose-related increase of hepatocellular carcinomas in
males (P < 0.01).
Incidences were also significantly increased at the two top dietary
concentrations of 32,000 ppm (P < 0.01) and 16,000 ppm (P < 0.05);
(b) a significant dose-related increase of the incidence of lung
alveolar/bronchiolar carcinomas
(P < 0.01), and at 32,000 ppm (P <
0.05).
Since the survival of the males was not affected by APM exposure, we
used logistic analysis to evaluate the combined adenoma/carcinoma
results of the liver and of the lung
.
The incidence of HCA and HCC combined resulted significantly increased
(P < 0.05) in the group treated at 16,000 ppm.
No significant dose–response was observed.
The reason for the lack of significance is that the dose–response is
flat over the exposure groups while the controls are lower (i.e., 12.8,
21.4, 21.0, 25.0, and 20.5).
It is noticeable that until 98 weeks of age 8/55 deceased males
(14.6%) treated at 32,000 ppm had HCC and no HCA.
On the contrary, three HCA and no HCC were observed among the 60
controls deceased in the same period.
This may depend on a more rapid progression of preneoplastic lesions to HCC.
However, others suggest that the response to carcinogens differ, and
that both HCA and HCC may develop de novo, without going through the
stage of foci of cellular alterations [Frith et al., 1979].
A significant dose-related trend (P < 0.05) of A/BA and A/BC combined
was observed among males.
Moreover, the incidence of A/BA plus A/BC in males treated at 32,000
ppm was significantly increased (P < 0.05) compared to controls.

Both liver and lung carcinomas in all exposure groups of males were
within the historical control range of these neoplasms in the CMCRC
laboratory.

Concerning the HCC, the concurrent control (5.1%) falls within the
lower range of our historical controls (0–26.3%) and because the
incidences of HCC in the groups treated at 32,000 (18.1%) and 16,000
(15.6%) were over three and two times the concurrent control, we
considered this effect related to the treatment.

Concerning A/BC, the concurrent  control (6.0%) falls also within the
lower range of our
historical controls (0–14.3%) and because the incidence observed at
the highest dose was more than double the concurrent control we
considered these effects to be related
to APM exposure [Haseman et al., 1984; Haseman, 1992, 1995].

No differences were observed in the incidences of liver and lung
tumors among the females of treated and control groups.
It has been reported that both spontaneously occurring and treatment
induced hepatocellular tumors occur with significantly greater
frequency and multiplicity in males
than in females even though occasionally exceptions do occur [Maronpot, 2009].
Male mice are also more susceptible to develop A/BA and A/BC than
females [Hahn et al., 2007; Dixon et al., 2008].

The carcinogenic effects observed in our mouse bioassay do not support
the negative outcome obtained with the CD-1 mouse study performed at
the Searle Laboratory in 1974 [Molinary, 1984].
In that experiment one group of 72 male and female CD-1 mice (control)
and three groups of 32 males and 32 females were treated,
respectively, with APM in feed at the dose levels of 0, 1, 2, 4 g/kg
from prenatal life for 2 years.
These studies are not comparable for two reasons:
(a) the number of the treated animals per sex per group is smaller in
comparison to the number in our experiment and to the number requested
by the current standard for carcinogenic bioassays (at least 50
animals per sex per group) used by NTP
and most others and
(b) the length of observation is much shorter (110 weeks compared to
130 weeks).
Both of these factors result in a loss of sensitivity for detecting a
carcinogenic effect.

As already reported [Soffritti et al., 1999; Haseman et al., 2001;
Huff et al., 2008; Soffritti et al., 2008], in longterm
carcinogenicity bioassays the number of animals per sex/group and life
span observation are critical points for identification and assessment
of diffuse carcinogenic risks, defined as the exposure to a single or
multiple agents or to mixtures that are expected to have limited
carcinogenic potential because of the agent type (weak carcinogen)
and/or dose/concentration (low), but that involve large group of the
population (as is the case with APM).

Concerning the prolonged (over 110 weeks of age) or lifespan duration
of the experiment, we must consider that neoplastic response depends
not only on the chemical–physical characteristics of the agent and its
toxicological properties, the mode of exposure, and the type of
animals, but also, to a greater extent, on the latency of the tumor
which varies and may be very long
.
Truncating an experiment after 2 years (more or less two-thirds of the
natural life of rodents) as requested by several regulatory agencies
(and as practiced by NTP), may mask a possible carcinogenic response.
This has been shown by us in experiments on benzene, Mancozeb (a
widely used fungicide), vinyl acetate, toluene, and xylenes [Soffritti
et al., 2002].
It should be noted that in the experiment on toluene and xylenes
performed by NTP, in which the rats were sacrificed after 104 weeks of
treatment, no carcinogenic effects were found [Huff, 2002, 2003; Huff
et al., 2010], whereas lifetime studies conducted in the CMCRC showed
unequivocal carcinogenicity after 104 weeks [Maltoni et al., 1997;
Soffritti et al., 2004].
These two factors in our opinion makes the Searle study less sensitive
than ours.

Overall, the results of our integrated project of lifespan
carcinogenic bioassays on APM conducted on Sprague–Dawley rats and
Swiss mice are consistent in showing that under our experimental
conditions APM must be considered a trans-species carcinogenic agent
in multiple sites (Table V), inducing a significantly increased
incidence of malignant tumors in:
(a) multiple tissues in male and female rats;
(b) multiple organs in male mice;
(c) an earlier occurrence in treated animals and an higher incidence
and an anticipated onset of cancers when the treatment starts from
fetal life [Soffritti et al., 2007].

Finally, the carcinogenic effects of APM in rats were shown also at
dose levels of 100 and 20 mg/kg b.w. to which humans could be exposed
[Soffritti et al., 2006, 2007].


CONCLUSIONS

The present study demonstrates for the first time that APM administered
in feed to Swiss mice at doses of 32,000, 16,000, 8,000, 2,000, or 0
ppm, starting the dietary exposure on day 12 of gestation and lasting
until death, induces significant dose-related increases of
hepatocellular carcinomas (P<0.01) and of alveolar/bronchiolar
carcinomas (P < 0.05) in males.
In particular, the significant increased incidences of hepatocellular
carcinomas were observed at the dietary levels of 32,000 ppm (P<0.01)
and 16,000 ppm (P<0.05) and of lung alveolar/bronchiolar carcinomas at
32,000 ppm (P<0.05).
HCA and HCC (combined) resulted significantly increased (P<0.05) in the
male group treated at 16,000 ppm.
A/BA and A/BC (combined) resulted significantly increased (P<0.05) in
the male group treated at 32,000 ppm.
A significant dose-related trend (P<0.05) was also observed.

Given that APM is completely metabolized in the gastrointestinal tract
to phenylalanine, aspartic acid, and methanol, it may be concluded
that the observed carcinogenic effects were caused not by APM itself
but rather by its metabolites.

In particular, it cannot be disregarded that the conversion of APM
methanol into formaldehyde in the liver may result in a generation of
formaldehyde adducts [Trocho et al., 1998], which could explain the
plausibility of hepatocarcinogenic effects of APM in male mice.

The fact that females did not develop a significantly increased
incidence of liver tumors may be explained by the gender resistance,
as already reported.

On the basis of these results, together with previous carcinogenicity
bioassays conducted on rats in our laboratories, APM should be
considered a multiple site, transspecies carcinogenic agent.

A re-evaluation of the current regulations on APM remains, in our
opinion, urgent.

ACKNOWLEDGMENTS

This research was supported entirely by the Ramazzini Institute.
The authors declare that they have no competing financial interests.
The authors thank Dr. David Hoel for his great support in the
statistical evaluation of the results.
A special thanks to the U.S. National Toxicology Program for having
organized a meeting of a group of pathologists at NIEHS in order to
provide a second opinion regarding the pathological lesions observed
in the APM Swiss mice study.
Ten pathologists participated in the NTP histopathology review.
The number of slides reviewed was 100 of which 26 were the subject of
discussion.
In the remaining cases, the original Ramazzini Institute diagnoses
were confirmed.
The lesions reviewed were liver adenomas/carcinomas and angiosarcomas;
lung adenomas/carcinomas; lymphomas; skin fibrosarcomas; and a few
miscellaneous lesions.


With regard to the data presented in this manuscript (liver and lung
tumors
) there were no discrepancies between NTP and RI pathology
evaluation.

A special thanks to Luana De Angelis and to all the CRCCM staff who
were involved in the study.

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"CONCLUSIONS
The present study demonstrates for the first time that APM (Aspartame), administered
in feed to Swiss mice at doses of 32,000, 16,000, 8,000, 2,000, or 0
ppm, starting the dietary exposure on day 12 of gestation and lasting
until death, induces significant dose-related increases of:
hepatocellular carcinomas (P<0.01) and of alveolar/bronchiolar
carcinomas (P < 0.05) in males.
In particular, the significant increased incidences of hepatocellular
carcinomas were observed at the dietary levels of 32,000 ppm (P<0.01)
and 16,000 ppm (P<0.05) and of lung alveolar/bronchiolar carcinomas at
32,000 ppm (P<0.05).
HCA and HCC (combined) resulted significantly increased (P<0.05) in the
male group treated at 16,000 ppm.
A/BA and A/BC (combined) resulted significantly increased (P<0.05) in
the male group treated at 32,000 ppm.
A significant dose-related trend (P<0.05) was also observed.

Given that APM is completely metabolized in the gastrointestinal tract
to phenylalanine, aspartic acid, and methanol, it may be concluded
that the observed carcinogenic effects were caused not by APM itself
but rather by its metabolites.

In particular, it cannot be disregarded that the conversion of APM
methanol into formaldehyde in the liver may result in a generation of
formaldehyde adducts [Trocho et al., 1998], which could explain the
plausibility of hepatocarcinogenic effects of APM in male mice.

The fact that females did not develop a significantly increased
incidence of liver tumors may be explained by the gender resistance,
as already reported.

On the basis of these results, together with previous carcinogenicity
bioassays conducted on rats in our laboratories, APM should be
considered a multiple site, transspecies carcinogenic agent.

*A re-evaluation of the current regulations on APM remains, in our
opinion, urgent. *

http://www.ncbi.nlm.nih.gov/pubmed/20886530


ACKNOWLEDGMENTS

This research was supported entirely by the Ramazzini Institute.
The authors declare that they have no competing financial interests.
The authors thank Dr. David Hoel for his great support in the
statistical evaluation of the results.
A special thanks to the U.S. National Toxicology Program for having
organized a meeting of a group of pathologists at NIEHS in order to
provide a second opinion regarding the pathological lesions observed
in the APM Swiss mice study.
Ten pathologists participated in the NTP histopathology review.
The number of slides reviewed was 100 of which 26 were the subject of
discussion.
In the remaining cases, the original Ramazzini Institute diagnoses
were confirmed.
The lesions reviewed were liver adenomas/carcinomas and angiosarcomas;
lung adenomas/carcinomas; lymphomas; skin fibrosarcomas; and a few
miscellaneous lesions.

With regard to the data presented in this manuscript (liver and lung
tumors) there were no discrepancies between NTP and RI pathology
evaluation."

METHODS:
Six groups of 62-122 male and female Swiss mice were treated with APM in
feed at doses of 32,000, 16,000, 8,000, 2,000, or 0 ppm from prenatal life
(12 days of gestation) until death.
At death each animal underwent complete necropsy and all tissues and organs
of all animals in the experiment were microscopically examined.
RESULTS:
APM in our experimental conditions induces in males a significant
dose-related increased incidence of hepatocellular carcinomas (P<0.01),
and a significant increase at the dose levels of 32,000?ppm (P<0.01) and
16,000 ppm (P<0.05).
Moreover, the results show a significant dose-related increased incidence of
alveolar/bronchiolar carcinomas in males (P<0.05),
and a significant increase at 32,000?ppm (P<0.05).
CONCLUSIONS:
The results of the present study confirm that APM is a carcinogenic agent in
multiple sites in rodents,

and that this effect is induced in two species,
rats (males and females) and mice (males).
No carcinogenic effects were observed in female mice.
Am. J. Ind. Med. © 2010 Wiley-Liss, Inc.
PMID: 20886530

American Journal of Industrial Medicine
Copyright © 2010 Wiley-Liss, Inc., A Wiley Company
Edited by: Steven B. Markowitz
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Occupational Health)
Online ISSN: 1097-0274

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Abbreviations:
APM, aspartame;
CMCRC/RI, Cesare Maltoni Cancer Research Center/Ramazzini Institute;
EFSA, European Food Safety Authority;
EU, European Union;
FDA, Food and Drug Administration.

Cesare Maltoni Cancer Research Center, Ramazzini Institute,
Bentivoglio, Bologna, Italy
Contract grant sponsor: Ramazzini Institute.
*Correspondence to: Morando Soffritti, Cesare Maltoni Cancer Research
Center, Ramazzini Institute, Castello di Bentivoglio,Via Saliceto, 3,
40010 Bentivoglio, Bologna, Italy. 
Accepted 30 July 2010
DOI 10.1002/ajim.20896.
Published online 30 September 2010 in Wiley Online Library
(wileyonlinelibrary.com)

(Aspartame Consumer Safety Network and Pilot Hotline since 1987: http://www.aspartamesafety.com/); (marystod.blogspot.com); (marystod.Twitter.com); (marystod@youtube.com)