http://groups.yahoo.com/group/aspartameNM/message/1173
five recent abstracts on formaldehyde damage to cells -- vitamin E and
selenium protect, Yoshiro Saito and Yasukazu Yoshida et al, O'Brien PJ et
al, Costa M et al: Murray 2005.06.07

From: "twoggletheturtle" <twoggletheturtle@...>
To: <WNHO-Aspartame-Information@yahoogroups.com>
Subject: [WNHO-Aspartame-Information] Re: From Mission Possible Aviation,
June is Save A Pilot Month
Date: Sunday, June 05, 2005 11:26 PM

--- In WNHO-Aspartame-Information@yahoogroups.com,
"aspartamehelp@j..." <aspartamehelp@j...> wrote:
>: 1. The toxic effects of toxic methyl esters like Aspartame are far
>: greater than just merely the toxicity from that same amount of
>: ingested methanol.

Dr. Bowen,

As a specific example of increased toxicity, I will post an abstract
below.
As everyone here knows, when they ingest aspartame they get
exposed to enough formaldehyde alone to eventually cause chronic poisoning.

But the abstract below shows that even when the formaldehyde levels
alone are not enough to cause cell death, formaldehyde combined with a
chemical to increase free radicals can dramatically reduce cell
viability (and increase cell death).
The researchers used AIPH to increase free radicals.
As you may know, excitotoxins can also increase free radical production
significantly.
As Dr. Blaylock says:
"Excitotoxins destroy neurons partly by stimulating the generation of
large numbers of free radicals."
See: http://www.dorway.com/blayart1.txt
***************************************************************

[Rich Murray: Here is the abstract and four more that show that many
groups are intensively studying cell damage and death from formaldehyde, and
the role of chemicals that increase or decrease this damage from
formaldehyde. ]

"It is well known that formaldehyde (HCHO) and reactive oxygen species
(ROS), such as free radicals, are cytotoxic as well as potentially
carcinogenic.
Although the individual effects of these reactants on
cells have been investigated, the cytotoxicity exerted by the
coexistence of HCHO and reactive radicals is poorly understood."

Toxicology. 2005 Jun 1; 210(2-3): 235-45.
Cytotoxic effect of formaldehyde with free radicals via increment of
cellular reactive oxygen species.
Saito Y, Nishio K, Yoshida Y, Niki E.

It is well known that formaldehyde (HCHO) and reactive oxygen species
(ROS), such as free radicals, are cytotoxic as well as potentially
carcinogenic.
Although the individual effects of these reactants on
cells have been investigated, the cytotoxicity exerted by the
coexistence of HCHO and reactive radicals is poorly understood.
The present study using Jurkat cells demonstrated that the coexistence of
HCHO with water-soluble radical initiator,
2,2'-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH)
dramatically decreased cell viability,
and that under such conditions
scant cell death was observable induced by either of the reactants alone.
Based on the results of phosphatidylserine exposure and caspase
activation, this observed cell death, in fact,
was apparently necrotic rather than apoptotic.
To understand the mechanisms of the cell toxicity of HCHO and AIPH, we
assessed two kinds of oxidative stress markers
such as cellular glutathione (GSH) content and cellular ROS,
and the DNA-protein cross-links,
which formed as the result of HCHO treatment.
A marked decrease in total cellular GSH was observed not only in the case of
the coexistence conditions but also with AIPH alone.
Dichlorodihydrofluorescein (DCF) assay revealed that cellular ROS
were synergistically increased before cell death.
The formation of DNA-protein cross-links was observed in the presence
of HCHO and AIPH,
and the extent was similar to HCHO alone.
Co-incubation with semicarbazide, which inactivates HCHO,
prevented this cell death induced by a combination of HCHO and AIPH.
Semicarbazide also exhibited an inhibitory effect on the synergistic
increment of
cellular ROS and the formation of DNA-protein cross-links.
These results suggest that the free radicals from AIPH induced GSH
reduction, while HCHO resulted in the formation of DNA-protein cross-links,
eventuating in a synergistic, incremental increase
of cellular ROS and cell death brought about by this combination.
****************************************************************


Free Radic Res. 2004 Apr; 38(4): 375-84.
Application of water-soluble radical initiator,
2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of
oxidative stress.
Yoshida Y, Itoh N, Saito Y, Hayakawa M, Niki E.
Human Stress Signal Research Center, National Institute of Advanced
Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka
563-8577, Japan. yoshida-ya@...

It is essential to generate free radicals at a controled and constant rate
for specific duration and at specific site to study the dynamics of
oxidation and also antioxidation.
Both hydrophilic and lipophilic azo compounds have been used for such
purpose.
In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane]
dihydrochloride (AIPH) was examined and compared with those of
2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and
2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP).
The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x
10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH
gives 3.8 times more free radicals than AAPH under the same conditions.
It was found that the dynamics of oxidation and antioxidation induced by
AIPH can be studied satisfactorily in the oxidation in micelles, LDL and
erythrocyte suspensions, plasma, and cultured cells.
The extent of cell death induced by AIPH and AAPH was directly proportional
to the total free radicals formed.
Interestingly, it was found that rats would not drink water containing AAPH,
but they drank water containing AIPH.
The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol
(FCOH), lysophosphatidylcholine in the plasma of rats given water containing
50 mM AIPH for 1 month increased compared with those of control rats which
drank water without AIPH.
It may be concluded that AIPH is useful for kinetic and mechanistic studies
on oxidative stress to membranes, lipoproteins, cells, and even animal
models.
PMID: 15190934
****************************************************************

"Many reports have shown significant correlations between selenium
deficiency and the incidence of cancer (51, 52).
It has been also reported that selenium supplementation prevents the
generation of cancer (2, 53).
As shown in this study, selenium deficiency caused a significant increase in
ROS and peroxidation inside cells.
It is therefore considered that selenium-deficient conditions cause
oxidative DNA damage that eventually leads to cancer formation.
In conclusion, the present study clearly shows that selenium deficiency
decreased the activities of cGPx, PH-GPx, and TR, increased lipid
peroxidation in the membranes, and eventually induced cell death.
The cell death was inhibited by other types of antioxidants with different
functions, such as tocopherols and deferoxamine, which inhibit lipid
peroxidation in the membranes and sequester redox-active iron, respectively.
These results strongly indicate that the lipid hydroperoxides play a
causative role in the oxidative damage to cells induced by selenium
deficiency."

http://www.jbc.org/cgi/content/full/278/41/39428 free full text

[ Selections ]

Originally published In Press as doi:10.1074/jbc.M305542200 on July 29, 2003
J. Biol. Chem., Vol. 278, Issue 41, 39428-39434, October 10, 2003

Cell Death Caused by Selenium Deficiency and Protective Effect of
Antioxidants*
Yoshiro Saito * , yoshiro-saito@...
Yasukazu Yoshida * , yoshida-ya@...
Takashi Akazawa ¶,
Kazuhiko Takahashi ¶
and Etsuo Niki *

* From the Human Stress Signal Research Center (HSSRC), National Institute
of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka,
Ikeda, Osaka 563-8577, Japan
and the ¶ Department of Immunology, Graduate School of Pharmaceutical
Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo, 060-0812,
Japan

Received for publication, May 27, 2003 , and in revised form, July 28, 2003.

ABSTRACT

Selenium is an essential trace element and it is well known that selenium is
necessary for cell culture.
However, the mechanism underlying the role of selenium in cellular
proliferation and survival is still unknown.
The present study using Jurkat cells showed that selenium deficiency in a
serum-free medium decreased the selenium-dependent enzyme activity
(glutathione peroxidases and thioredoxin reductase) within cells and cell
viability.
To understand the mechanism of this effect of selenium, we examined the
effect of other antioxidants, which act by different mechanisms.
Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely
blocked selenium deficiency-induced cell death, although -tocopherol
(biologically the most active form of vitamin E) could not preserve
selenium-dependent enzyme activity. Other antioxidants, such as different
isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate,
also exerted an inhibitory effect.
However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl
cysteine, and glutathione, displayed no such effect.
Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive
oxygen species (ROS) increased before cell death, and sodium selenite
and -tocopherol inhibited ROS increase in a dose-dependent manner.
The generation of lipid hydroperoxides was observed by fluorescence probe
diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in
selenium-deficient cells.
These results suggest that the ROS, especially lipid hydroperoxides, are
involved in the cell death caused by selenium deficiency and that selenium
and vitamin E cooperate in the defense against oxidative stress upon cells
by detoxifying and inhibiting the formation of lipid hydroperoxides.

Selenium is an essential trace element for humans and many other forms of
life, and a deficiency of this element induces some pathological conditions,
such as cancer, coronary heart disease, and liver necrosis (1-5).
Selenium deficiency is also accompanied by a loss of immunocompetence (6),
and both cell-mediated immunity and B-cell function can be impaired (7).
Supplementation with selenium has marked immunostimulant effects, including
an enhancement of activated T-cell proliferation (8).
Selenium is an essential component of several enzymes such as glutathione
peroxidase (GPx)1 (9), thioredoxin reductase (TR) (10), and selenoprotein P
(SeP) (11), which contain selenium as selenocysteine.
It is also well known that selenium is essential for cell culture when a
serum-free medium is used (12).
Serum-free media, especially for immune cells and neurons, contain insulin,
transferrin, and sodium selenite.
Without selenium, cells can neither proliferate nor survive.
However, the underlying mechanism for the role of selenium in cell
proliferation is still unknown.

Vitamin E, a generic term for tocopherols and tocotrienols, is one of the
most potent lipid-soluble antioxidants (13).
Vitamin E occurs in nature in at least eight different isoforms: -, -, -,
and -tocopherols and -, -, -, and -tocotrienols (14).
Tocotrienols differ from the corresponding tocopherols only in their
aliphatic tail. Vitamin E deficiencies have been implicated in some
pathologic conditions, such as cancer, coronary heart disease, and liver
necrosis (15, 16) and are also accompanied by a loss of immunocompetence
(17).
It is well known that selenium and vitamin E show compensative effects and
that a deficiency of both elements causes massive injury in some cases
(18-20).
In the present study, we characterize the nature of cell death caused by
selenium deficiency and the cell death inhibitory effect of antioxidants
including vitamin E.
We also demonstrate the involvement of reactive oxygen species (ROS),
especially lipid hydroperoxides, on the cell death.


DISCUSSION

The essential role of selenium in nutrition has been well established.
It is also well known that selenium is necessary for cell culture when using
a serum-free medium.
Insulin (as a growth factor), transferrin (as an iron source), and selenite
are added to the serum-free media for immune and neuronal cells.
Although the effects of selenium on cell viability and the cellcycle
progression has been reported (12, 32), the underlying mechanism of the
protective effect of this element has not yet been elucidated.
In the present study using Jurkat cells, a model of proliferating T lymphoma
cells, the decrease in cell viability was observed when applying a
serum-free medium without selenium.
This cell death was completely blocked by selenium-containing materials,
except for ebselen, in a dose-dependent manner.
It has been reported that these selenium-containing materials are
incorporated and can be the cellular source of selenium used for synthesis
of selenoprotein (22). SeP, which is a selenium-rich extracellular
glycoprotein (11, 33, 34) that functions as selenium transport protein (22,
35, 36), was the most effective of the materials tested (Table I).
Although we observed a loss of cell viability after 24 h, Jurkat cells
duplicated for 20 h under the serum-free culture conditions.
This observation suggests that cell death occurred after a single division.
We speculate that the proliferating cells became selenium-deficient, and the
divided cells contained almost half of the selenoenzyme activities, such as
cGPx, PH-GPx, and TR.
In the presence of sodium selenite, these enzyme activies were retained or
up-regulated in cells.
The decrease in TR activity was lower than that of cGPx and PH-GPx
activities in the Jurkat cells cultured with selenium-deficient medium for
72 h (Fig. 5). Selenoproteins have been proposed to follow a hierarchy for
selenium supply in that the amounts of certain selenoproteins decrease more
rapidly under selenium-deficient conditions (37).
A previous study suggested that this was due in part to differences in the
SECIS elements.
Gasdaska et al. (38) demonstrated that the element of TR was highly active;
therefore, TR levels would be better preserved when the selenium supply was
limited as in a selenium-deficient medium.
It has been reported that these selenoproteins play an important role in the
defense against oxidative stress (1, 39).
In the case of lipid hydroperoxides, it is known that PH-GPx, but not cGPx,
is able to reduce lipid hydroperoxides, including phospholipid hydroperoxide
and cholesterol hydroperoxide, directly (40, 41).
It has also been proved that overexpression of PH-GPx suppresses cell death
due to oxidative damage induced by radical initiator and lipid hydroperoxide
(42, 43). Moreover, Lewin et al. (44) reported that TR also plays a role in
preventing oxidative damage induced by tert-butyl hydroperoxide and oxidized
LDL, but the mechanism of the protection afforded by this selenoprotein
against oxidative stress induced by lipid hydroperoxides is still unclear.
At present, these selenoproteins are assumed to play an important role in
the defense against oxidative stress related to lipid hydroperoxides.
Under selenium-deficient conditions, the decrease in these selenoproteins is
speculated to cause peroxidation in the lipid layer inside cells.
It is noteworthy that radical-scavenging antioxidants, such as -tocopherol,
completely blocked the cell death caused by selenium deficiency,
although -tocopherol did not affect the enzyme activity of selenoproteins.
-Tocopherol-supplemented Jurkat cells did not show any loss of cell
viability despite the undetectable levels of cGPx and PH-GPx activity.
Other isoforms of vitamin E, such as -, -, and -tocopherols and -, -, -,
and -tocotrienols, were also effective.
Tocotrienols were more effective than the corresponding tocopherols (Table
I), which may be ascribed primarily, if not solely, to the differences in
the rate of cellular uptake.
A higher uptake of -tocotrienol than -tocopherol into culture cells has been
reported (45, 46).
Such a difference was also observed for liposomal membranes (47).
Vitamin E derivatives, such as PMC and its water-soluble analogue Trolox,
also showed an inhibitory effect on cell death, but their higher ED50 values
(Table I) suggest that the antioxidant incorporated into cell membranes is
more effective than that localized outside the membranes.
Interestingly, a synthetic radical-scavenging antioxidant, BO-653, also
showed an inhibitory effect, suggesting the importance of the inhibition of
lipid peroxidation in the cell membranes.
The fact that selenium deficiency, which results in a decrease in the
capacity to reduce lipid hydroperoxides, induced cell death and that this
cell death could be inhibited by radical-scavenging antioxidants that
suppress the formation of lipid hydroperoxides strongly indicates the
causative role of lipid hydroperoxides in cell death.
The ED50 value of -tocopherol was as low as 36 nM (Table I).
One may argue that this concentration is quite low compared with the
physiological concentration; for example, 30 µM in human plasma.
It should be pointed out, however, that the concentration of -tocopherol in
the membrane is of more importance than that in the bulk phase.
In the present study, the lipid concentrations were measured as follows; FC,
4.4; PC, 12; PE, 5.2 nmol/106 cells.
Thus, the molar ratio of -tocopherol to total lipids in the cell culture
system (106 cells/ml) was 36 x 10-9/22 x 10-6 M = 1/610 mol/mol, which is
similar to that in human plasma;
that is, 30 x 10-6/11 x 10-3 M = 1/370 mol/ mol.
It may be noted that the micromolar ranges of -tocopherol applied to many
cell culture systems are not always physiological, but that the
concentration in the membranes should be considered.
Deferoxamine mesylate, a well-known iron chelator, was also found to be a
potent inhibitor of cell death induced by selenium deficiency.
It has been suggested that iron plays an important role in oxidative damage
to cells (48, 49), by reacting with hydrogen peroxide or lipid
hydroperoxides to form reactive oxygen radicals.
It was found that the removal of selenium from the culture medium induced
ROS production as measured by DCF fluorescence and also lipid hydroperoxides
as measured by DPPP fluorescence and HPLC chemiluminescence.
We are aware of the inherent drawbacks of DCFH, but it can be a useful probe
for estimating semi-quantitatively the generation of ROS under specific
conditions (50). Both selenium and -tocopherol suppressed ROS by apparently
different mechanisms, the former by enhancing the reduction of
hydroperoxides, while the latter by inhibiting their formation.
FC-OOH was detected as a major lipid hydroperoxide, which was unexpected
since polyunsaturated lipids in PC and PE are more susceptible to oxidation
than free cholesterol.
One possible reason could be that the reduction of FC-OOH by PH-GPx is at
least six times slower than that of phospholipid hydroperoxides (41).
Many reports have shown significant correlations between selenium deficiency
and the incidence of cancer (51, 52).
It has been also reported that selenium supplementation prevents the
generation of cancer (2, 53).
As shown in this study, selenium deficiency caused a significant increase in
ROS and peroxidation inside cells.
It is therefore considered that selenium-deficient conditions cause
oxidative DNA damage that eventually leads to cancer formation.
In conclusion, the present study clearly shows that selenium deficiency
decreased the activities of cGPx, PH-GPx, and TR, increased lipid
peroxidation in the membranes, and eventually induced cell death.
The cell death was inhibited by other types of antioxidants with different
functions, such as tocopherols and deferoxamine, which inhibit lipid
peroxidation in the membranes and sequester redox-active iron, respectively.
These results strongly indicate that the lipid hydroperoxides play a
causative role in the oxidative damage to cells induced by selenium
deficiency.

FOOTNOTES

* The costs of publication of this article were defrayed in part by the
payment of page charges.
This article must therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Human Stress Signal Research
Center (HSSRC), National Institute of Advanced Industrial Science and
Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. Tel.:
81-72-751-8293; Fax: 81-72-751-9964; E-mail: yoshiro-saito@...

1 The abbreviations used are:
GPx, glutathione peroxidase;
ROS, reactive oxygen species;
PBS, phosphate-buffered saline;
HPLC, high pressure liquid chromatography;
DCFH-DA, dichlorofluorescin diacetate;
TR, thioredoxin reductase; pNA, p-nitroanilide;
FITC, fluorescein isothiocyanate;
SeP, selenoprotein P.

ACKNOWLEDGMENTS

We express cordial thanks to Dr. Shuichi Shimakawa, Nanako Itoh, and Mieko
Hayakawa in HSSRC, AIST for the total lipid analysis,
and also to Dr. Kohji Ichimori in HSSRC, AIST for technical support.


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This Article

All ASBMB Journals Molecular and Cellular Proteomics
Journal of Lipid Research Biochemistry and Molecular Biology Education
Copyright © 2003 by the American Society for Biochemistry and Molecular
Biology.
****************************************************************

"The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed
to its ability to form adducts with DNA and proteins."

"This work demonstrates that DNA-protein cross-links can be formed in vitro
following exposure to a variety of industrial compounds and that most
cross-links are formed at cytotoxic concentrations."

"At higher HCHO concentrations, lipid peroxidation ensued followed by cell
death."

"Antioxidants and iron chelators protected against HCHO cytotoxicity."

Chem Biol Interact. 2001 Jan 30; 130-132(1-3): 285-96.
The formaldehyde metabolic detoxification enzyme systems and molecular
cytotoxic mechanism in isolated rat hepatocytes.
Teng S, Beard K, Pourahmad J, Moridani M, Easson E, Poon R, O'Brien PJ.
Faculty of Pharmacy, University of Toronto, 19 Russell St., Ont., M5S 2S2,
Toronto, Canada.
[ O'Brien Prof Peter J THE LESLIE DAN FACULTY OF PHARMACY
416-978-2716 fax 416-978-8511 peter.obrien@... ]

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed
to its ability to form adducts with DNA and proteins.
A marked decrease in mitochondrial membrane potential and inhibition of
mitochondrial respiration that was accompanied by reactive oxygen species
formation occurred when isolated rat hepatocytes were incubated with low
concentrations of HCHO in a dose-dependent manner.
Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner.
At higher HCHO concentrations, lipid peroxidation ensued followed by cell
death. Cytotoxicity studies were conducted in which isolated hepatocytes
exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes.
There was a marked increase in HCHO cytotoxicity when either alcohol
dehydrogenase or aldehyde dehydrogenase was inhibited.
Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion
of GSH markedly increased hepatocyte susceptibility to HCHO.
In each case, cytotoxicity was dose-dependent and corresponded with a
decrease in hepatocyte HCHO metabolism and increased lipid peroxidation.
Antioxidants and iron chelators protected against HCHO cytotoxicity.
Cytotoxicity was also prevented, when cyclosporine or carnitine was added to
prevent the opening of the mitochondrial permeability transition pore which
further suggests that HCHO targets the mitochondria.
Thus, HCHO-metabolising gene polymorphisms would be expected to have
toxicological consequences on an individual's susceptibility to HCHO
toxicity and carcinogenesis. PMID: 11306052
****************************************************************

"Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain
arsenic compounds have been shown to produce DNA-protein cross-links in
human in vitro cell systems at high doses, such as those in the cytotoxic
range."

J Toxicol Environ Health. 1997 Apr 11; 50(5): 433-49.
DNA-protein cross-links produced by various chemicals in cultured human
lymphoma cells.
Costa M, Zhitkovich A, Harris M, Paustenbach D, Gargas M.
Institute of Environmental Medicine, New York University Medical Center, New
York 10016, USA. costam@...

Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain
arsenic compounds have been shown to produce DNA-protein cross-links in
human in vitro cell systems at high doses, such as those in the cytotoxic
range.
Thus far there have only been a limited number of other chemicals evaluated
for their ability to produce cross-links.
The purpose of the work described here was to evaluate whether select
industrial chemicals can form DNA-protein cross-links in human cells in
vitro.
We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde,
2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a
deodorant tablet [Mega Blue; hazardous component listed as
tris(hydroxymethyl)nitromethane].
Short- and long-term cytotoxicity was evaluated and used to select
appropriate doses for in vitro testing. DNA-protein cross-linking was
evaluated at no fewer than three doses and two cell lysate washing
temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human
Burkitt's lymphoma cells.
The two washing temperatures were used to assess the heat stability of the
DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde
produced statistically significant increases in DNA-protein cross-links at
washing temperatures of 45 degrees C, but not 65 degrees C, and at or above
concentrations of 5, 17.5, and 75 mM, respectively.
Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced
statistically significant increases in DNA-protein cross-links washed at 45
and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and
0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce
significantly increased DNA-protein cross-links at either temperature nor at
any dose tested.
Excluding paraformaldehyde and 2-furaldehyde treatments, significant
increases in DNA-protein cross-links were observed only at doses that
resulted in complete cell death within 4 d following dosing.
This work demonstrates that DNA-protein cross-links can be formed in vitro
following exposure to a variety of industrial compounds and that most
cross-links are formed at cytotoxic concentrations. PMID: 9140463
****************************************************************

Rich Murray, MA Room For All rmforall@... 505-501-2298
1943 Otowi Road Santa Fe, New Mexico 87505 USA
http://groups.yahoo.com/group/aspartameNM/messages
group with 185 members, 1,173 posts in a public, searchable archive

http://groups.yahoo.com/group/aspartameNM/message/1165
short review: research on aspartame (methanol, formaldehyde, formic acid)
toxicity: Murray 2005.06.04 rmforall

http://groups.yahoo.com/group/aspartameNM/message/1071
research on aspartame (methanol, formaldehyde, formic acid) toxicity: Murray
2004.04.29 rmforall

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). If 30% of
the methanol is turned into formaldehyde, the amount of formaldehyde is 18
times the USA EPA limit for daily formaldehyde in drinking water, 2 mg in 2
L water.
****************************************************************

http://groups.yahoo.com/group/aspartameNM/message/1143
methanol (formaldehyde, formic acid) disposition: Bouchard M et al, full
plain text, 2001: substantial sources are degradation of fruit pectins,
liquors, aspartame, smoke: Murray 2005.04.02 rmforall

"That substantial amounts of methanol metabolites or by-products are
retained for a long time is verified by Horton et al. (1992) who estimated
that 18 h following an iv injection of 100 mg/kg of 14C-methanol in male
Fischer-344 rats, only 57% of the dose was eliminated from the body.

From the data of Dorman et al. (1994) and Medinsky et al. (1997), it can
further be calculated that 48 h following the start of a 2-h inhalation
exposure to 900 ppm of 14C-methanol vapors in female cynomolgus monkeys,
only 23% of the absorbed 14C-methanol was eliminated from the body.

These findings are corroborated by the data of Heck et al. (1983) showing
that 40% of a 14C-formaldehyde inhalation dose remained in the body 70 h
postexposure."

"Exposure to methanol also results from the consumption of certain
foodstuffs (fruits, fruit juices, certain vegetables, aspartame sweetener,
roasted coffee, honey) and alcoholic beverages (Health Effects Institute,
1987; Jacobsen et al., 1988)."



"However, the severe toxic effects are usually associated with the
production and accumulation of formic acid, which causes metabolic acidosis
and visual impairment that can lead to blindness and death at blood
concentrations of methanol above 31 mmol/l (Røe, 1982; Tephly and McMartin,
1984; U.S. DHHS, 1993).

Although the acute toxic effects of methanol in humans are well documented,
little is known about the chronic effects of low exposure doses, which are
of interest in view of the potential use of methanol as an engine fuel and
current use as a solvent and chemical intermediate.

Gestational exposure studies in pregnant rodents (mice and rats) have also
shown that high methanol inhalation exposures (5000 or 10,000 ppm and more,
7 h/day during days 6 or 7 to 15 of gestation) can induce birth defects
(Bolon et al., 1993; IPCS, 1997; Nelson et al., 1985)."

"The corresponding average elimination half-life of absorbed methanol
through metabolism to formaldehyde was estimated to be 1.3, 0.7-3.2, and 1.7
h."



"Inversely, in monkeys and in humans, a larger fraction of body burden of
formaldehyde is rapidly transferred to a long-term component.
The latter represents the formaldehyde that (directly or after oxidation to
formate) binds to various endogenous molecules..."

"Animal studies have reported that systemic methanol is eliminated mainly by
metabolism (70 to 97% of absorbed dose) and only a small fraction is
eliminated as unchanged methanol in urine and in the expired air (< 3-4%)
(Dorman et al., 1994; Horton et al., 1992).

Systemic methanol is extensively metabolized by liver alcohol dehydrogenase
and catalase-peroxidase enzymes to formaldehyde, which is in turn rapidly
oxidized to formic acid by formaldehyde dehydrogenase enzymes (Goodman and
Tephly, 1968; Heck et al., 1983; Røe, 1982; Tephly and McMartin, 1984).

Under physiological conditions, formic acid dissociates to formate and
hydrogen ions.

Current evidence indicates that, in rodents, methanol is converted mainly by
the catalase-peroxidase system whereas monkeys and humans metabolize
methanol mainly through the alcohol dehydrogenase system (Goodman and
Tephly, 1968; Tephly and McMartin, 1984).

Formaldehyde, as it is highly reactive, forms relatively stable adducts with
cellular constituents (Heck et al., 1983; Røe, 1982)."

"The whole body loads of methanol, formaldehyde, formate, and unobserved
by-products of formaldehyde metabolism were followed.

Since methanol distributes quite evenly in the total body water, detailed
compartmental representation of body tissue loads was not deemed necessary."

"According to model predictions, congruent with the data in the literature
(Dorman et al., 1994; Horton et al., 1992), a certain fraction of
formaldehyde is readily oxidized to formate, a major fraction of which is
rapidly converted to CO2 and exhaled, whereas a small fraction is excreted
as formic acid in urine.

However, fits to the available data in rats and monkeys of Horton et al.
(1992) and Dorman et al. (1994) show that, once formed, a substantial
fraction of formaldehyde is converted to unobserved forms.

This pathway contributes to a long-term unobserved compartment.

The latter, most plausibly, represents either the formaldehyde that
(directly or after oxidation to formate) binds to various endogenous
molecules (Heck et al., 1983; Røe, 1982)
or is incorporated in the tetrahydrofolic-acid-dependent one-carbon pathway
to become the building block of a number of synthetic pathways (Røe, 1982;
Tephly and McMartin, 1984).

That substantial amounts of methanol metabolites or by-products are retained
for a long time is verified by Horton et al. (1992) who estimated that 18 h
following an iv injection of 100 mg/kg of 14C-methanol in male Fischer-344
rats, only 57% of the dose was eliminated from the body.

From the data of Dorman et al. (1994) and Medinsky et al. (1997), it can
further be calculated that 48 h following the start of a 2-h inhalation
exposure to 900 ppm of 14C-methanol vapors in female cynomolgus monkeys,
only 23% of the absorbed 14C-methanol was eliminated from the body.

These findings are corroborated by the data of Heck et al. (1983) showing
that 40% of a 14C-formaldehyde inhalation dose remained in the body 70 h
postexposure.

In the present study, the model proposed rests on acute exposure data, where
the time profiles of methanol and its metabolites were determined only over
short time periods (a maximum of 6 h of exposure and a maximum of 48 h
postexposure).

This does not allow observation of the slow release from the long-term
components.

It is to be noted that most of the published studies on the detailed
disposition kinetics of methanol regard controlled short-term (iv injection
or continuous inhalation exposure over a few hours) methanol exposures in
rats, primates, and humans (Batterman et al., 1998; Damian and Raabe, 1996;
Dorman et al., 1994; Ferry et al., 1980; Fisher et al., 2000; Franzblau et
al., 1995; Horton et al., 1992; Jacobsen et al., 1988; Osterloh et al.,
1996; Pollack et al., 1993; Sedivec et al., 1981; Ward et al., 1995; Ward
and Pollack, 1996).

Experimental studies on the detailed time profiles following controlled
repeated exposures to methanol are lacking."

"Thus, in monkeys and plausibly humans, a much larger fraction of body
formaldehyde is rapidly converted to unobserved forms rather than passed on
to formate and eventually CO2."

"However, the volume of distribution of formate was larger than that of
methanol, which strongly suggests that formate distributes in body
constituents other than water, such as proteins.

The closeness of our simulations to the available experimental data on the
time course of formate blood concentrations is consistent with the volume of
distribution concept (i.e., rapid exchanges between the nonblood pool of
formate and blood formate)."

"Also, background concentrations of formate are subject to wide
interindividual variations (Baumann and Angerer, 1979; D'Alessandro et al.,
1994; Franzblau et al., 1995; Heinrich and Angerer, 1982; Lee et al., 1992;
Osterloh et al., 1996; Sedivec et al., 1981)."

http://www.toxsci.oupjournals.org/cgi/content/full/64/2/169

Toxicological Sciences 64, 169-184 (2001)
Copyright © 2001 by the Society of Toxicology

BIOTRANSFORMATION AND TOXICOKINETIC

A Biologically Based Dynamic Model for Predicting the Disposition of
Methanol and Its Metabolites in Animals and Humans

Michèle Bouchard *, #,1, bouchmic@...

Robert C. Brunet, # brunet@...

Pierre-Olivier Droz, #

and Gaétan Carrier* gaetan.carrier@...

* Department of Environmental and Occupational Health, Faculty of Medicine,
Université de Montréal, P.O. Box 6128, Main Station, Montréal, Québec,
Canada, H3C 3J7;

# Institut Universitaire romand de Santé au Travail, rue du Bugnon 19,
CH-1005, Lausanne, Switzerland, and

# Département de Mathématiques et de Statistique and Centre de Recherches
Mathématiques, Faculté des arts et des sciences, Université de Montréal,
P.O. Box 6128, Main Station, Montréal, Québec, Canada, H3C 3J7

NOTES

1 To whom correspondence should be addressed at Département de santé
environnementale et santé au travail, Université de Montréal, P.O. Box 6128,
Main Station, Montréal, Québec, H3C 3J7, Canada. Fax: (514) 343-2200.
E-mail: bouchmic@...
****************************************************************