aspartame rat brain toxicity re cytochrome P450 enzymes, expecially
CYP2E1, Vences-Mejia A, Espinosa-Aguirre JJ et al, 2006 Aug, Hum Exp
Toxicol: relevant abstracts re formaldehyde from methanol in alcohol
drinks: Murray 2006.09.29
http://groups.yahoo.com/group/aspartameNM/message/1373

[Rich Murray notes:
As a medical layman, noting that all readers are laymen for any topic
outside the bounds of their
specific expertise, I found related abstracts that illucidate the role
in cytochrome P450 enzymes,
especially the one most affected by aspartame, CYP2E1, in brain
toxicity processes involving ethanol and methanol, suggesting
avenues of research for alcohol addiction and hangover, and the
possibilies of aspartame liver and brain toxicity from its 11% methanol
component.]

"A major finding in this study was that the daily
consumption of ASP at the two doses considered
leads to an increment in the concentration and activity
of CYP2B1/2, CYP2E1 and CYP3A2
in rat cerebral and cerebellar microsomes....

The highest increment (up to 25-fold over controls)
in a CYP-associated activity induced by ASP in brain
was that of 4-NPH corresponding to CYP2E1.

The results mentioned above must be reproduced
using a broad range of ASP concentrations in order
to define the existence of a dose-related effect.

As far as we know, this is the first report regarding
modulation of brain CYPs by the widely used
sweetener ASP.

Specific induction of brain CYPs could constitute
a local regulatory mechanism of enzyme activity,
thus influencing drug response;
for tissues exhibiting low regenerative capacity,
such as the brain,
such modulation would probably be of major toxicological
significance....

It has already been said that once ASP enters the
organism, it is rapidly metabolized by intestinal
esterases and dipeptidases to
aspartic acid,
phenylalanine
and methanol,
substances normally found in the diet and body. 37

One hour after ASP intake at a dose of 200 mg/kg body weight by rats,
corresponding to the acceptable FDA daily intake for the
sweetener after species correction,
increased plasma and brain phenylalanine levels by 62% and 192%
respectively. 6

With regard to methanol,
it accounts for about 10% of the ASP weight administered. 38

We can hypothesize that the exposure to methanol at
the two regimens used in this study
about 7.5 and 12.5 mg/kg from the doses of 75 and 125 mg/kg)
could induce xenobiotic-metabolizing enzymes in a
similar way to that of the chronic administration of
ethanol. 39....

If methanol is the metabolite
responsible for the induction of brain CYP2E1 seen in this work,
the question of why the hepatic CYP2E1 was not altered remains.

Experiments with the three metabolites resulting from ASP
metabolism are currently being undertaken in our
laboratory in order to address this question.

In conclusion, data obtained demonstrated that a
daily consumption of ASP at doses of 75 and 125 mg/kg body weight
over 30 days provokes
a substantial increment in CYP enzymes
involved in endogenous and exogenous molecules metabolism
in the CNS of the rat.

Biological consequences of this
phenomenon should be investigated in view of
the high number of humans exposed to this artificial
sweetener and because of the recent data
indicating the potential carcinogenic effects of this
compound. 41"

Hum Exp Toxicol. 2006 Aug; 25(8): 453-9.
The effect of aspartame on rat brain xenobiotic-metabolizing enzymes.
Vences-Mejia A 1,
Labra-Ruiz N 1,
Hernandez-Martinez N 1,
Dorado-Gonzalez V 1,
Gomez-Garduno J 1,
Perez-Lopez I 1,
Nosti-Palacios R 1,
Camacho Carranza R 2,
Espinosa-Aguirre JJ 2.
Laboratorio de Toxicologia Genetica,
1: Instituto Nacional de Pediatria, Insurgentes Sur, 3700-C,
04530 Mexico, DF Mexico.
2: Instituto de Investigaciones Biomédicas, UNAM, Apartado postal
70228,
Ciudad Universitaria 04510 México, D.F., México
http://www.biomedicas.unam.mx/index.asp
*Correspondence: JJ Espinosa-Aguirre, Instituto de Investigaciones
Biome´dicas, UNAM, Apartado postal 70228, Ciudad
Universitaria 04510 Me´xico, D.F., Me´xico
Human & Experimental Toxicology (2006) 25(8): 453 - 459.
www.sagepublications.com
c 2006 SAGE Publications 10.1191/0960327106het646oa

[ Dra. Araceli Vences M
Jefa de Laboratorio de Toxicologia Genetica
6° P de Hospital Laboratorios
10 84 09 00 Ext.1410 -1448   aritaven@yahoo.com.mx

ISRAEL PÉREZ LÓPEZ,

JAVIER J. ESPINOSA AGUIRRE, jjea@servidor.unam.mx
http://www.biomedicas.unam.mx/investigacionFrame.asp?ID=MG ]

Abstract

This study demonstrates that chronic aspartame (ASP) consumption leads
to an increase of phase I metabolizing enzymes (cytochrome P450 (CYP))
in rat brain.

Wistar rats were treated by gavage with ASP
at daily doses of 75 and 125 mg/kg body weight for 30 days.

Cerebrum and cerebellum were used to obtain microsomal fractions to
analyse activity and protein levels of seven cytochrome P450 enzymes.

Increases in activity were consistently found with the 75 mg/kg dose
both in cerebrum and cerebellum for all seven enzymes,
although not at the same levels:

CYP2E1-associated 4-nitrophenol hydroxylase (4-NPH) activity was
increased 1.5-fold in cerebrum and 25-fold in cerebellum;

likewise, CYP2B1-associated penthoxyresorufin O-dealkylase (PROD)
activity increased 2.9- and 1.7-fold respectively,

CYP2B2-associated benzyloxyresorufin O-dealkylase (BROD)
4.5- and 1.1-fold,

CYP3A-associated erythromycin N-demethylase (END) 1.4- and 3.3-fold,

CYP1A1-associated ethoxyresorufin O-deethylase (EROD) 5.5- and
2.8-fold,

and CYP1A2-associated methoxyresorufin O-demethylase (MROD)
3.7- and 1.3-fold.

Furthermore, the pattern of induction of CYP immunoreactive proteins by
ASP paralleled that of 4-NHP-, PROD-, BROD-, END-, EROD- and
MROD-related activities only in the cerebellum.

Conversely, no differences in CYP concentration and activity
were detected in hepatic microsomes of treated animals
with respect to the controls,
suggesting a brain-specific response to ASP treatment.
PMID: 16937917
Aug 14 2006 08:07:58
Key words: aspartame; brain; cytochrome P450; enzyme induction

Introduction

Sweeteners are paid special attention among food additives,
as their use enables a sharp reduction in sugar consumption
and a significant decrease in caloric intake
while maintaining the desirable palatability
of foods and soft drinks.

Sweeteners are also of primary importance
as part of nutritional guidance for diabetes,
a disease with increasing incidence in developed countries. 1-3

Aspartame (L-asparthyl-L-phenylalanine methyl ester, ASP)
is one of the most widely used artificial sweeteners;
it is a high-intensity sweetener added to
a large variety of foods, most commonly found in
low-calorie beverages, desserts and tabletop sweeteners
added to tea or coffee.

It does not enter into the bloodstream intact,
but is hydrolyzed in the intestine
to form aspartate,
phenylalanine
and methanol,
which are then absorbed into the circulation,
elevating their levels in plasma and in brain phenylalanine
and tyrosine levels as well. 4-6

Aspartate is a highly excitatory neurotransmitter 7
and phenylalanine is a precursor of catecholamines in the brain; 8
increased levels of these molecules could change the
basic activity level of the brain to an unhealthy,
constantly stimulated state.

Short-term studies on ASP consumption and
memory loss have been conducted in humans and rodents
and no relationship was found. 9-11

On the other hand, chronic studies have implicated ASP
consumption in learning and memory.

Consumption of 9% ASP in the diet for 13 weeks affected learning
behaviour in male rats, 12
while ASP exposure of guinea pigs to 500 mg/kg during gestation
disrupted odour-associative learning in pups. 13

Recently, Christian et al. reported that chronic ASP consumption
lengthened the time it took rats to find the reward in a T-maze
and increased the number of muscarinic receptors
in specific brain areas. 14

Despite numerous toxicological studies of ASP and its components,
its effects on metabolic and detoxification enzyme systems
have received little attention.

Metabolic enzymes are of special interest
as changes in their function could lead to an
increased susceptibility of the organisms to the
harmful effects of a variety of contaminants found
in the environment and in food products. 15,16

The presence of cytochrome P450 (CYP) in the
central nervous system (CNS) opens the question of
whether metabolism in endothelial cells may regulate
the penetration of the xenobiotics into the
brain compartment. 17,18

The role of CYP in brain includes such diverse functions as
aromatization of androgens to oestrogens,
formation of catechols,
and it may also participate in the metabolism of
neurotransmitters and of xenobiotics. 17,19

Moreover, lipophilic xenobiotics can diffuse
through the endothelial cells of the brain capillaries
and enter the neuronal cells.

Thus, in situ activation in the neuronal cell
could have far-reaching consequences
by causing irreversible disruption of the neuronal function.

The brain is the target not only for a number of toxic compounds
but also for several psychoactive drugs.

The metabolism of drugs in the brain can lead
to local pharmacological modulation at the site of action
and can result in variable drug response. 17

The purpose of this work is to study the effect of
orally administered ASP on the activity of CYP in
the CNS of the rat.

The characterization of brainspecific CYP
and its regulation and localization within the CNS
is gaining importance for the understanding
of the potential role of these enzymes in the
pathogenesis of neurodegenerative disorders and in
the psychopharmacological modulation of drugs
acting on the CNS. 17

Methods [ technical details omitted ]

Animal protocol

Twenty-four male Wistar rats
(Research Unit at the National Institute of Pediatrics, Mexico),
21 days old (at weaning),
were housed in polypropylene cages
with unlimited access to laboratory chow and water,
and kept in a 12-h light/dark cycle.
Body weight was registered daily over the 30 days of the experiment.
Three groups were formed (n=8)
for oral treatment by gavage:
a group was given ASP in a dose of 75 mg/kg/day;
the second group was given a dose of 125 mg/kg/day;
and controls were given distilled water, 200 mL/day.
After 30 days of treatment, all animals
were euthanized and decapitated.
The liver,
cerebrum and cerebellum were immediately and
aseptically removed from each animal.

Statistics
Statistical analysis was made comparing each ASP
dose group with the control group using Student's
t-test and ANOVA test (a/0.05).

Results

Mean body weight changes during the 4-week treatment
are presented in Figure 1.

After the third week, the ASP-treated animals (75 and 125 mg/kg)
showed diminished body weight gain compared to controls,
even though this difference had no statistical significance.

Immunoblots for hepatic microsomes revealed
the presence of CYPs 1A1/2, 2B1/B2, 2E1 and 3A2
in control and ASP treated animals
without appreciable differences in concentration (Figure 2).

This lack of effect of ASP on hepatic microsomal enzymes
was further demonstrated when selected
activities were determined:

4-nitrophenol hydroxylase (4-NPH; associated to CYP2E1),

penthoxyresorufin O-dealkylase (PROD; associated to CYP2B1),

benzyloxyresorufin O-dealkylase (BROD; associated to CYP2B2),
erythromycin N-demethylase (END; associated to CYP3A),

ethoxyresorufin O-deethylase (EROD; associated to CYP1A1)

and methoxyresorufin O-demethylase (MROD; associated to CYP1A2)

remained unaffected by ASP treatment (Table 1).

With the exemption of CYP2E1 (Figures 3 and 4),
we were unable to demonstrate the presence of CYP
immunoreactive proteins in microsomes of cerebrum
or cerebellum of control rats.

Conversely, an elevated protein concentration over controls
of all the CYP families considered in this study,
including CYP2E1, was detected in cerebellum microsomes of
animals treated with either dose of ASP (Figure 4).

CYP2E1 was also induced in cerebral microsomes of
rats treated with ASP at both concentrations but
CYP2B, and 3A families were clearly induced only
at 125 mg/kg treatment dose (Figure 3).

Immunoreactive proteins belonging to the CYP1A family were
not detected in the control or in the treated groups of
animals (Figures 3 and 4).

Enzyme activities found in cerebral and cerebellar
tissues are shown in Tables 2 and 3.
Compared to controls, cerebral CYP1A1 EROD-associated
activity was enhanced 6-fold by ASP treatment
at the dose of 75 mg/kg.

The higher dose tested failed to modulate EROD activity.

Cerebral CYP2B PROD- and BROD-associated activities were increased
3- and 4.5-fold respectively with the 75 mg/kg ASP treatment
and 3- and 4.5-fold after 125 mg/kg ASP treatment.

With respect to
cerebral CYP3A END- and CYP2E1 4-NPH- associated activities,
a significant enhancement of 1.3 and 1.5 times
respectively over controls was detected in the 75 mg/kg dosed group.

The same activities were
also increased 1.7- and 1.6-fold respectively after
exposure to 125 mg/kg ASP.

Cerebellar microsomes obtained from animals
under ASP treatment at both concentrations tested
showed increased levels of all the enzyme-associated
activities considered here (Table 3);
the highest increment of 26- and 16-fold over controls
corresponded to CYP2E1 from rats treated with
75 and 125 mg/kg ASP respectively,
followed by CYP3A2 (3.3- and 3.2-fold),
CYP1A1 (2.8- and 3.8-fold),
CYP2B1 (1.7- and 1.1-fold),
1A2 (1.3- and 1.5-fold)
and 2B2 (1.1-fold for the two doses tested).

Discussion

Following oral administration to humans and experimental
animals, ASP is rapidly and completely
metabolized by intestinal esterases and dipeptidases
to aspartic acid, phenylalanine and methanol, substances
normally found in the diet and body. 28,29

These three naturally occurring metabolites are
absorbed and subjected to biotransformation that
normally occurs when they are consumed in food.

Tutelyan et al. examined the effect of ASP ingestion
on the inhibition or induction of CYP in rat hepatic
tissues but failed to explore its effects on extrahepatic
organs such as the brain. 19

In this respect,
enzyme systems for the metabolism and detoxification
of foreign compounds are of special interest
as changes in their function could lead to changes in
the susceptibility of an organ to the harmful effects
of the increasing variety of contaminants found in
the environment and in food products.

Recently, the extent of P450-mediated metabolism
in extrahepatic organs and the pharmacological and toxicological
consequences of in situ metabolism in target organs
have been recognized in laboratory animals. 30

The CNS is an important potential target for certain
environmental pro-toxins, but relatively little is
known regarding the specific expression of biotransformation
enzyme systems.

The aim of this study was to explore the effect of orally ingested ASP
upon the expression and activity of CYP families
involved in the CNS of rats.

The chosen ASP doses of 75 and 125 mg/kg body
weight used in this study are within the limits of
human consumption after species factor correction.

Because rats metabolize ASP faster than humans, 31
dose comparisons between them have usually been
corrected by a factor of 5.

After correction, doses used here
are below the FDA (50 mg/kg body weight)
and Health and Welfare Canada (40 mg/kg body weight)
acceptable daily intake for ASP. 32,33

Although the dose and length of treatment were
different, our results agreed with those reported by
Molinary and Tutelyan et al., 19,34
in demonstrating that, compared with controls,
neither differences in mean body weight nor hepatic CYP modulation
after rat exposure to ASP were observed
(Figures 1 and 2; Table 1).

These results suggest that ASP metabolites:
aspartic acid,
phenylalanine
and methanol, are not
CYP-inducing agents in the liver.

On the other hand,
we were unable to detect
constitutive expression of CYP1A1/2, CYP2B1/2 and CYP3A2
in cerebrum or cerebellum of control rats
by western blot analysis (Figures 3 and 4).

This could be due to the fact that in these animals
CYP levels in the whole brain account for 1-10 %
those of the hepatic protein 35,
and the methodology used in this study is not sensitive enough
to detect these levels.

Another point to consider is that
brain is not a homogeneous organ and it is known
that the levels of CYPs in specific neurons can be
higher than the levels in hepatocytes. 36

A major finding in this study was that the daily
consumption of ASP at the two doses considered
leads to an increment in the concentration and activity
of CYP2B1/2, CYP2E1 and CYP3A2
in rat cerebral and cerebellar microsomes.

Activity of CYP1A1/2 was also induced in both cerebrum and
cerebellum but an enhancement in protein concentration
was seen only in cerebellum (Figures 3 and 4; Tables 2 and 3).

The highest increment (up to 25-fold over controls)
in a CYP-associated activity induced by ASP in brain
was that of 4-NPH corresponding to CYP2E1.

The results mentioned above must be reproduced
using a broad range of ASP concentrations in order
to define the existence of a dose-related effect.

As far as we know, this is the first report regarding
modulation of brain CYPs by the widely used
sweetener ASP.

Specific induction of brain CYPs could constitute
a local regulatory mechanism of enzyme activity,
thus influencing drug response;
for tissues exhibiting low regenerative capacity,
such as the brain,
such modulation would probably be of major toxicological significance.

For instance, clinically relevant psychoactive drugs
undergo CYP1A2 metabolism.
Substrates for this enzyme include, among others,
amitriptyline,
caffeine,
imipramine,
fluvoxamine,
clozapine
and olanzapine. 35

It has already been said that once ASP enters the
organism, it is rapidly metabolized by intestinal
esterases and dipeptidases to
aspartic acid,
phenylalanine
and methanol,
substances normally found in the diet and body. 37

One hour after ASP intake at a dose of 200 mg/kg body weight by rats,
corresponding to the acceptable FDA daily intake for the
sweetener after species correction,
increased plasma and brain phenylalanine levels by 62% and 192%
respectively. 6

With regard to methanol,
it accounts for about 10% of the ASP weight administered. 38

We can hypothesize that the exposure to methanol at
the two regimens used in this study
about 7.5 and 12.5 mg/kg from the doses of 75 and 125 mg/kg)
could induce xenobiotic-metabolizing enzymes in a
similar way to that of the chronic administration of
ethanol. 39

In fact, hepatic microsomes prepared from rats
exposed to methanol showed increased
p-nitrophenol hydroxylase activity. 40

If methanol is the metabolite
responsible for the induction of brain CYP2E1 seen in this work,
the question of why the hepatic CYP2E1 was not altered remains.

Experiments with the three metabolites resulting from ASP
metabolism are currently being undertaken in our
laboratory in order to address this question.

In conclusion, data obtained demonstrated that a
daily consumption of ASP at doses of 75 and 125 mg/kg body weight
over 30 days provokes
a substantial increment in CYP enzymes
involved in endogenous and exogenous molecules metabolism
in the CNS of the rat.

Biological consequences of this
phenomenon should be investigated in view of
the high number of humans exposed to this artificial
sweetener and because of the recent data
indicating the potential carcinogenic effects of this
compound. 41

Acknowledgements

We thank Sandra Luz Hernandez for her excellent
technical assistance.

References

1 Meyer H.
Aspartame as part of the solution.
Food Sci Technol 2005; 19: 4345.

2 Gougeon R, Spidel M, Lee K, Field CJ.
Canadian diabetes association
national nutrition committee technical review:
Non-nutritive intense sweeteners in diabetes management.
Can J Diabetes 2004; 128: 38599.

3 Halimi S.
Diabetes in 2005.
Nephrologie et Therapeutique 2006; 2: S27.

4 Maher TJ, Wurtman RJ.
Possible neurologic effects of aspartame,
a widely used food additive.
Environ Health Perspectives 1987; 75: 5357.

5 Pinto JM, Maher TJ.
Administration of aspartame
potentiates pentylenetetrazole- and fluorothyl-induced
seizures in mice.
Neuropharmacology 1988; 27: 51-55.

6 Yokogoshi H, Roberts CH, Caballero B, Wurtman RJ.
Effects of aspartame and glucose administration on
brain and plasma levels of large neutral amino acids
and brain 5-hydroxyindioles.
Am J Clin Nutr 1984; 40: 17.

7 Krebs MO.
Excitatory amino acids: a new class of neurotransmitters:
pharmacology and functional properties.
Encephale 1992; 18: 27179.

8 Milner JD, Wurtman RJ.
Tyrosine availability:
a presynaptic factor controlling catecholamine release.
Adv Exp Med Biol 1987; 221: 211 21.

9 Tilson HA, Hong JS, Sobotka TJ.
High doses of aspartame have no effects on sensorimotor function
or learning and memory in rats.
Neurotoxicol Teratol 1991; 13: 2735.

10 Shaywitz BA, Sullivan CM, Anderson GM, Gillespie
SM, Sullivan B, Shaywitz SE.
Aspartame, behaviour, and cognitive function
in children with attention deficit disorder.
Pediatrics 1994; 93: 7075.

11 Spiers PA, Sabounjian L, Reiner A, Myers DK,
Wurtman J, Schomer DL.
Aspartame: neuropsychologic and neurophysiologic evaluation
of acute and chronic effects.
Am J Clin Nutr 1998; 68: 53137.

12 Potts WJ, Bloss JL, Nutting EF.
Biological properties of aspartame:
I. Evaluation of central nervous system effects.
J Environ Pathol Toxicol 1980; 3: 34153.

13 Dow-Edwards DL, Scribani LA, Riley EP.
Impaired performance on odor-aversion testing
following prenatal aspartame exposure in the guinea pig.
Neurotoxicol Teratol 1989; 11: 41316.

14 Christian B, McConnaughey K, Bethea E, Brantley S,
Coffey A, Hammond L, et al.
Chronic aspartame affects
T-maze performance, brain cholinergic receptors
and
Na, K-ATPase in rats. P
Pharmacol Biochem Behav 2004; 78: 12127.

15 Guengerich FP, Shimada T.
Oxidation of toxic and carcinogenic chemicals
by human cytochrome P-450 enzymes.
Chem Res Toxicol 1991; 4: 391407.

16 Guengerich FP.
Influence of nutrients and other dietary materials
on cytochrome P450 enzymes.
J Clin Nutr 1995; 61: 651S58S.

17 Ravindranath V.
Metabolism of xenobiotics in the central nervous system.
Implications and changes.
Biochem Pharmacol 1998; 56: 54751.

18 Pelkonen O, Raunnio H.
Metabolic activation of toxins:
tissue-specific expression and metabolism in target organs.
Environ Health Perspect 1997; 105: 76774.

19 Tutelyan VA, Kravchenko LV, Kuzmina EE.
The effect of aspartame on the rat activity of rat liver
xenobiotic metabolizing enzymes.
Drug Metab Dispos 1990; 18: 22325.

20 Maron DM and Ames BN.
Revised methods for the Salmonella mutagenicity test.
Mutat Res 1983; 113: 173215.

21 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem 1951; 193: 26575.

22 Burke MD, Thompson S, Elcombe CR, Halpert J,
Haaparanta T, Mayer RT.
Ethoxy-, penthoxy-, and
benxyloxyphenoxazones and homologues:
a series of substrates to distinguish
between different induced cytochromes P450.
Biochem Pharmacol 1985; 34; 333745.

23 Burke MD, Thompson S, Weaver RJ,
Wolf CR, Mayer RT.
Cytochrome P450 specificities
of alkoxyresorufin O-dealkylation in human and rat liver.
Biochem Pharmacol 1994; 48: 92336.

24 Koop DR.
Hydroxylation of p-nitrophenol by rabbit
ethanol-inducible cytochrome P450 isozyme 3a.
Mol Pharmacol 1986; 29: 399404.

25 Alexidis AN, Commandeur JN, Rekka EA.
Novel piperidine derivatives:
inhibitory properties towards cytochrome P450 isoforms and
cytoprotective and cytotoxic characteristics,
Environ Toxicol Pharmacol 1996; 1: 8188.

26 Laemmli UK.
Maturation of the head of bacteriophage T4 I DNA packaging events.
Nature 1970; 227: 68085.

27 Towbin H, Staehelin T, Gordon J.
Electrophoretic transfer of proteins from polyacrylamide gels
to nitrocellulose sheets: Procedure and some applications.
Proc Natl Acad Sci U S A 1979; 76: 435054.

28 Ranney RE, Oppermann JA.
A review of the metabolism of the aspartyl moiety
of aspartame in experimental animals and man.
J Environ Pathol Toxicol 1979; 2: 97985.

29 Burgert SL, Andersen DW, Stegink LD,
Takeuchi H, Schedl HP.
Metabolism of aspartame and its L-phenylalanine
methyl ester decomposition product by porcine gut.
Metabolism 1991; 40: 61218.

30 Gram TE, Okine LR, Gram RA.
The metabolism of xenobiotics
by certain extrahepatic organs and its relation to toxicity.
Annu Rev Pharmacol Toxicol 1986; 26: 25991.

31 Fernstrom JD.
Oral aspartame and plasma phenylalanine:
pharmacokinetic difference between rodents and man,
and relevance to CNS effects of phenylalanine.
J Neural Transm 1989; 75: 15964.

32 FDA (Food and Drug Administration).
Food additives permitted for direct addition to food
for human consumption; aspartame.
Fed Regist 1996; 61: 3365456.

33 Health Protection Branch.
Information letter: aspartame.
Health and Welfare Canada, 1987.

34 Molinary V.
Preclinical studies of aspartame in non primate animals.
In Stegink LD, Filer LJ Jr eds.
Aspartame, physiology and biochemistry.
Marcel Dekker, Inc., 1984: 289 306.

35 Gervasini G, Carrillo JA, Benitez J.
Potential role of cerebral cytochrome P450
in clinical pharmacokinetics.
Modulation by endogenous compounds.
Clin Pharmacokinet 2004; 43: 693706.

36 Miksys S, Hoffmann E, Tyndale RF.
Regional and cellular induction
of nicotine-metab olizing CYP2B1
in rat brain by chronic nicotine treatment.
Biochem Pharmacol 2000; 59: 150111.

37 Oppermann JA.
Aspartame metabolism in animals.
In Stegink LD, Filer LJ Jr eds.
Aspartame, physiology and biochemistry.
Marcel Dekker, Inc., 1984: 14159.

38 Roak-Foltz R, Leveille GA.
Projected aspartame intake:
daily ingestion of aspartic acid, phenylalanine, and methanol.
In Stegink LD, Filer LJ Jr eds.
Aspartame, Physiology and Biochemistry.
Marcel Dekker, Inc., 1984: 201205.

39 Doita HK, Simanowsky UA.
In Hathcock JN ed.
Nutritional Toxicology.
Academic Press, 1987.

40 Allis JW, Brown BL, Simmons JE, Hatch AM, House DE.
Methanol potentiation of carbon tetrachloride hepatotoxicity:
the central role of cytochrome P450.
Toxicology 1996; 112: 13140.

41 Soffritti M, Belpoggi F, Esposti DD, Lambertini L.
Aspartame induces lymphomas and leukaemias in
rats. Eur J Oncol 2005; 10: 10716.
*******************************************************

Drug Metab Rev. 2004 May; 36(2): 313-33.
The unique regulation of brain cytochrome P450 2 (CYP2) family enzymes
by drugs and genetics.
Miksys S,
Tyndale RF.  r.tyndale@utoronto.ca
Centre for Addiction and Mental Health, Department of Pharmacology,
University of Toronto, Toronto, Ontario, Canada.
http://www.camh.net/  416-595-6015 or public_affairs@camh.net
http://www.camh.net/Research/Research_publications/Research_AR_2005/quitsmoking_
rar2005.html
Rachel F. Tyndale, Ph.D., Assistant Professor,
Department of Pharmacology, University of Toronto
Medical Sciences Building   Kings College Circle
Toronto, Ontario M5S 1A8 CANADA (416) 978-6374; Fax: (416) 978-6395

Cytochrome P450 (CYP) enzymes in the brain may have a role in the
activation or inactivation of centrally acting drugs,
in the metabolism of endogenous compounds,
and in the generation of damaging toxic metabolites
and/or oxygen stress.

CYPs are distributed unevenly among brain regions,
and are found in neurons, glial cells and at the blood-brain interface.
They have been observed in mitochondrial membranes,
in neuronal processes and in the plasma membrane,
as well as in endoplastic reticulum.

Brain CYPs are inducible by many common hepatic inducers,
however many compounds affect liver and brain CYP expression
differently,
and some CYPs which are constitutively expressed in liver
are inducible in brain.

CYP induction is isozyme-, brain region-, cell type- and
inducer-specific.

While it is unlikely that brain CYPs
contribute to overall clearance of xenobiotics,
their punctate, region- and cell-specific expression suggests that CNS
CYPs may create micro-environments in the brain with differing drug and
metabolite levels (not detected or predicted by plasma drug
monitoring).

Coupled with the sensitivity of CNS CYPs to induction, this may in part
account for inter-individual variation in response to centrally acting
drugs and neurotoxins, and may have implications for individual
variation in receptor adaptation and cross-tolerance to different
drugs.

In addition, genetic variation in brain CYPs, depending on the type of
polymorphism (structural versus regulatory), will alter enzyme
activity. These aspects of brain CYP expression regulation and genetic
influences are illustrated in this review using mRNA, protein, and
enzyme activity data for CYP 2D1/6, CYP 2E1 and CYP 2B1/6 in rat and
human brain.

The role of CYP-mediated metabolism in the brain,
a highly heterogeneous and complex organ,
is a new and relatively unexplored field of scientific enquiry.
It holds promise for furthering our undestanding of inter-individual
variability in response to centrally acting drugs
as well as risk for neurological diseases and pathogies.
PMID: 15237857

Br J Pharmacol. 2003 Apr; 138(7): 1376-86.
Brain CYP2E1 is induced by nicotine and ethanol in rat and is higher in
smokers and alcoholics.
Howard LA,
Miksys S,
Hoffmann E,
Mash D,
Tyndale RF. r.tyndale@utoronto.ca
Department of Pharmacology, University of Toronto, Toronto, Ontario,
Canada, M5S 1A8.

1. Ethanol and nicotine are commonly coabused drugs.

Cytochrome P450 2E1 (CYP2E1) metabolizes ethanol and bioactivates
tobacco-derived procarcinogens.

Ethanol and nicotine can induce hepatic CYP2E1
and we hypothesized that both centrally active drugs could also induce
CYP2E1 within the brain.

2. Male rats were treated with saline, ethanol (3.0 g kg(-1) by gavage)
or nicotine (1.0 mg kg(-1) s.c.) for 7 days.

Ethanol treatment significantly increased CYP2E1 in olfactory bulbs
(1.7-fold), frontal cortex (2.0-fold), hippocampus (1.9-fold) and
cerebellum (1.8-fold),
while nicotine induced CYP2E1 in olfactory bulbs (2.3-fold), frontal
cortex (3.0-fold), olfactory tubercle (3.1-fold), cerebellum (2.5-fold)
and brainstem (2.0-fold).

Immunocytochemical analysis revealed that the induction was cell-type
specific.

3. Consistent with the increased CYP2E1 found in rat brain following
drug treatments,
brains from alcoholics and alcoholic smokers showed greater staining of
granular cells of the dentate gyrus and the pyramidal cells of CA2 and
CA3 hippocampal regions as well as of cerebellar Purkinje cells
compared to nonalcoholic nonsmokers.

Moreover, greater CYP2E1 immunoreactivity was observed in the frontal
cortices in the alcoholic smokers in comparison to nonalcoholic
nonsmokers and alcoholic nonsmokers.

4. To investigate if nicotine could contribute to the increased CYP2E1
observed in alcoholic smokers, we treated human neuroblastoma IMR-32
cells in culture and found significantly higher CYP2E1 immunostaining
in nicotine-treated cells (0.1-10 nM).

5. CYP2E1 induction in the brain, by ethanol or nicotine, may influence
the central effects of ethanol and the development of nervous tissue
pathologies observed in alcoholics and smokers.
PMID: 12711639

J Pharmacol Exp Ther. 2003 Sep; 306(3): 941-7. Epub 2003 May 15.
Rat hepatic CYP2E1 is induced by very low nicotine doses:
an investigation of induction, time course,
dose response, and mechanism.
Micu AL,
Miksys S,
Sellers EM,  Edward M. Sellers
Koop DR,
Tyndale RF.  r.tyndale@utoronto.ca
Department of Pharmacology, University of Toronto, Canada.

CYP2E1 is an ethanol- and drug-metabolizing enzyme that can also
activate procarcinogens and hepatotoxicants
and generate reactive oxygen species;
it has been implicated in the pathogenesis of liver diseases and
cancer.

Cigarette smoke increases CYP2E1 activity in rodents and in humans
and we have shown that nicotine (0.1-1.0 mg/kg s.c. x 7 days)
increases CYP2E1 protein and activity in the rat liver.

In the current study, we have shown that the induction peaks at 4 h
postnicotine (1 mg/kg s.c. x 7 days) treatment and recovers within 24
h.

No induction was observed after a single injection, and 18 days of
treatment did not increase the levels beyond that found at 7 days.

We found that CYP2E1 is induced by very low doses of chronic (x 7 days)
nicotine with an ED50 value of 0.01 mg/kg s.c.; 0.01 mg/kg in a rat
model results in peak cotinine levels (nicotine metabolite)
similar to those found in people exposed to environmental tobacco smoke
(passive smokers; 2-7 ng/ml).

Previously, we have shown no change in CYP2E1 mRNA,
and our current mechanistic study indicates
that nicotine does not regulate CYP2E1 expression
by protein stabilization.

We postulated that a nicotine metabolite could be causing the induction
but found that cotinine (1 mg/kg x 7 days) did not increase CYP2E1.

Our findings indicate that nicotine increases CYP2E1 at very low doses
and may enhance CYP2E1-related toxicity in smokers, passive smokers,
and people treated with nicotine (e.g., smokers, patients with
Alzheimer's disease, ulcerative colitis or Parkinson's disease).
PMID: 12750430

Environ. Toxicol. Pharmacol. 2000 Dec; 9(1-2): 31-37.
Induction of cytochrome P450 enzymes
by albendazole treatment in the rat.
Asteinza J,
Camacho-Carranza R,
Reyes-Reyes RE,
Dorado-Gonzalez V V,
Espinosa-Aguirre JJ.
Instituto de Investigaciones Biomedicas,
Universidad Nacional Autonoma de Mexico,
Apartado Postal 70228, Ciudad Universitaria,
DF 04510, Mexico, Mexico

The anthelmintic drug albendazole (ABZ),
methyl(5-(propylthio)-1H-benzimidazol-2-yl)carbamate,
is a benzimidazole
highly efficient in the treatment of neurocysticercosis.
The effects of ABZ treatment (i.p. and p.o. administration)
on the expression of several cytochrome P450 (CYP) enzymes were
evaluated in rat liver in order to characterize the spectrum
of altered CYP enzymes involved in the metabolism of environmental
mutagens and carcinogens, after drug intake.
Intraperitoneal administration of ABZ
(50 mg/kg body weight/day/three days in corn oil) to rats,
caused an induction of hepatic activities of
CYP1A1-associated ethoxyresorufin O-deethylase (EROD) 65 fold,
CYP1A2-associated methoxyresorufin O-demethylase (MROD) 6 fold,
CYP2B1-associated penthoxyresorufin O-dealkylase (PROD) 4 fold,
CYP2B2-associated benzyloxyresorufin O-dealkylase (BROD) 14 fold,
as well as a partial reduction of CYP2E1-associated
4-nitrophenol hydroxylase (4-NPH) activity.
CYP3A-associated erythromycin N-demethylase (END) activity was not
modified under the same treatment conditions.
Western blot analysis was conducted to explore if the increased
catalytic activity was a result of an increased protein content;
only CYP1A1/2 showed a visible increase in protein concentration after
ABZ inoculation,
therefore, the increased PROD and BROD activities could be attributed
to
the induction of CYP1A1/2.
Results with the two main metabolites of ABZ (15 mg/kg body
weight/day/three days, i.p.) indicated that ABZ sulfoxide (ABZSO)
but not ABZ sulfone (ABZSO(2))
displayed the same pattern of CYP induction than ABZ.
Oral administration of ABZ at the human therapeutic dose of 20 mg/kg
body weight/day/three days, produced an increase in CYP1A1/2 protein
content 24 h after the first intake.
The protein level remained high during the treatment,
and up to 72 h after the last administration;
basal protein levels were almost recovered 48 h later.
PMID: 11137466

Exp Toxicol Pathol. 2006 Jul; 57(5-6): 427-35. Epub 2006 Apr 17.
Effect of L-carnitine supplementation on xenobiotic-metabolizing
hepatic enzymes exposed to methanol.

Olszowy Z,
Plewka A,
Czech E,
Nowicka J,
Plewka D,
Nowaczyk G,
Kaminski M.
Department of Forensic Medicine, Medical University of Silesia,
ul. Medykow 18, 40-752 Katowice, Poland.

The study aimed to evaluate the effect of L-carnitine on hepatic
cytochrome P450-dependent monooxygenases exposed to methanol.

Male Spraque-Dawley rats were given methanol (1/4 LD50 and 1/2 LD50)
together with L-carnitine (1g/kg body weight).

The parameters of microsome electron transport chains I and II
and the levels of CYP2E1, CYP2B1/2 and CYP1A2
were measured 8, 12, 24, 48, 72 and 96 h after exposure.

L-carnitine did not affect cytochrome P450
but it significantly increased at 72 and 96 h
NADPH-cytochrome P450 reductase.

It stimulated cytochrome b5 at 48 and 96 h
and NADH-cytochrome b5 reductase activity at 12, 72 and 96 h.

Methanol, especially the lower dose,
inhibited cytochrome P450 after 48 h,
but the higher methanol dose inhibited
NADH-cytochrome b5 reductase activity in this time.

L-carnitine, combined with the lower dose of methanol,
stimulated NADPH-cytochrome P450 reductase after 48 h
and cytochrome b5 and NADH-cytochrome b5 reductase
over the whole period of observation.

L-carnitine stimulated CYP2B1/2 but not CYP2E1 and CYP1A2.

Methanol stimulated CYP2E1 at 24 h,
but CYP1A2 at 96 h in the studied doses.

CYP2B1/2 was induced by the lower dose of methanol at 24 h
but by the higher one at 96 h.

When given together,
L-carnitine and methanol (1/2 LD50)
significantly stimulated CYP2E1 up to 170% at 24 h and 145% at 96 h.
PMID: 166164651:

"Another ethanol-metabolising enzyme, cytochrome P450 2E1,
has a higher Km (0.5-0.8 g/L) and is also inducible,
so that the clearance of ethanol is increased in heavy drinkers."

Clin Pharmacokinet. 2003; 42(1): 1-31.
Role of variability in explaining ethanol pharmacokinetics: research
and forensic applications.
Norberg A,
Jones AW,
Hahn RG,
Gabrielsson JL.
Department of Anaesthesia and Intensive Care,
Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden.

Variability in the rate and extent of absorption, distribution and
elimination of ethanol has important ramifications
in clinical and legal medicine.

The speed of absorption of ethanol from the gut depends on time of day,
drinking pattern, dosage form, concentration of ethanol in the
beverage, and particularly the fed or fasting state of the individual.

During the absorption phase, a concentration gradient exists between
the stomach, portal vein and the peripheral venous circulation.

First-pass metabolism and bioavailability are difficult to assess
because of dose-, time- and flow-dependent kinetics.

Ethanol is transported by the bloodstream to all parts of the body.

The rate of equilibration is governed by the ratio
of blood flow to tissue mass.

Arterial and venous concentrations differ
as a function of time after drinking.

Ethanol has low solubility in lipids
and does not bind to plasma proteins,
so volume of distribution is closely related to the amount of water in
the body,
contributing to sex- and age-related differences in disposition.

The bulk of ethanol ingested (95-98%) is metabolised
and the remainder is excreted in breath, urine and sweat.

The rate-limiting step in oxidation is conversion of ethanol into
acetaldehyde by cytosolic alcohol dehydrogenase (ADH),
which has a low Michaelis-Menten constant (Km) of 0.05-0.1 g/L.

Moreover, this enzyme displays polymorphism,
which accounts for racial and ethnic variations in pharmacokinetics.

When a moderate dose is ingested,
zero-order elimination operates for a large part of the
blood-concentration time course,
since ADH quickly becomes saturated.

Another ethanol-metabolising enzyme, cytochrome P450 2E1,
has a higher Km (0.5-0.8 g/L) and is also inducible,
so that the clearance of ethanol is increased in heavy drinkers.

Study design influences variability in blood ethanol pharmacokinetics.

Oral or intravenous administration, or fed or fasted state,
might require different pharmacokinetic models.

Recent work supports the need for multicompartment models to describe
the disposition of ethanol instead of the traditional one-compartment
model with zero-order elimination.

Moreover, appropriate statistical analysis is needed to isolate
between- and within-subject components of variation.

Samples at low blood ethanol concentrations improve the estimation of
parameters and reduce variability.

Variability in ethanol pharmacokinetics stems from a combination of
both genetic and environmental factors,
and also from the nonlinear nature of ethanol disposition,
experimental design, subject selection strategy and dose dependency.

More work is needed to document variability in ethanol pharmacokinetics
in real-world situations.
PMID: 12489977

Free Radic Res. 1997 Oct; 27(4): 369-75.
Decreased antioxidant defense mechanisms in rat liver after methanol
intoxication.
Skrzydlewska E,
Farbiszewski R.
Department of Instrumental Analysis, Medical Academy, Poland.

The primary metabolic fate of methanol
is oxidation to formaldehyde and then to formate
by enzymes of the liver.

Cytochrome P-450 and a role for the hydroxyl radical have been
implicated in this process.

The aim of the paper was to study the liver antioxidant defense system
in methanol intoxication, in doses of 1.5, 3.0 and 6.0 g/kg b.w.,
after methanol administration to rats.

In liver homogenates,
the activities of Cu,Zn-superoxide dismutase and catalase were
significantly increased after 6 h following methanol ingestion
in doses of 3.0 and 6.0 g/kg b.w. and persisted up to 2-5 days,
accompanied by significant decrease of glutathione reductase
and glutathione peroxidase activities.

The content of GSH was significantly decreased during 6 hours to 5
days.

The liver ascorbate level was significantly diminished, too,
while MDA levels were considerably increased
after 1.5, 3.0 and 6.0 g/kg b.w. methanol intoxication.

Changes due to methanol ingestion may indicate impaired antioxidant
defense mechanisms in the liver tissue.
PMID: 9416465

"Lipid peroxidation and superoxide production correlate
with the amount of cytochrome P450 2E1."

J Biomed Sci. 2001 Jan-Feb; 8(1): 59-70.
Oxidative stress, metabolism of ethanol and alcohol-related diseases.
Zima T,
Fialova L,
Mestek O,
Janebova M,
Crkovska J,
Malbohan I,
Stipek S,
Mikulikova L,
Popov P.
Institute of Clinical Chemistry, First Faculty of Medicine, Charles
University, Karlovo nam. 32, CZ-121 11 Prague 2, Czech Republic.
zimatom@mbox.cesnet.cz

Alcohol-induced oxidative stress is linked to the metabolism of
ethanol.

Three metabolic pathways of ethanol have been described in the human
body so far.

They involve the following enzymes:
alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS)
and catalase.

Each of these pathways could produce free radicals which affect the
antioxidant system.

Ethanol per se, hyperlactacidemia and elevated NADH
increase xanthine oxidase activity,
which results in the production of superoxide.

Lipid peroxidation and superoxide production correlate
with the amount of cytochrome P450 2E1.

MEOS aggravates the oxidative stress directly as well as indirectly by
impairing the defense systems.

Hydroxyethyl radicals are probably involved in the alkylation of
hepatic proteins.

Nitric oxide (NO) is one of the key factors contributing to the vessel
wall homeostasis,
an important mediator of the vascular tone and neuronal transduction,
and has cytotoxic effects.

Stable metabolites -- nitrites and nitrates -- were increased in
alcoholics (34.3 ± 2.6 vs. 22.7 ± 1.2 micromol/l, p < 0.001).

High NO concentration could be discussed for its excitotoxicity and may
be linked to cytotoxicity in neurons, glia and myelin.

Formation of NO has been linked to an increased preference for and
tolerance to alcohol in recent studies.

Increased NO biosynthesis also via inducible NO synthase (NOS, chronic
stimulation) may contribute to platelet and endothelial dysfunctions.

Comparison of chronically ethanol-fed rats and controls
demonstrates that exposure to ethanol causes a decrease in NADPH
diaphorase activity (neuronal NOS) in neurons
and fibers of the cerebellar cortex
and superior colliculus (stratum griseum superficiale and intermedium)
in rats.

These changes in the highly organized structure
contribute to the motor disturbances,
which are associated with alcohol abuse.

Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect
membrane lesions, impairment of immunological reactivity,
liver disease progression,
and they correlate significantly with the disease severity.

The low-density lipoprotein (LDL) oxidation is supposed to be one of
the most important pathogenic mechanisms of atherogenesis,
and antibodies against oxidized LDL (oxLDL) are some kind of
epiphenomenon of this process.

We studied IgG oxLDL and four APA
(anticardiolipin, antiphosphatidylserine,
antiphosphatidylethanolamine and antiphosphatidylcholine antibodies).

The IgG oxLDL (406.4 ± 52.5 vs. 499.9 ± 52.5 mU/ml) was not affected
in alcoholic patients,
but oxLDL was higher
(71.6 ± 4.1 vs. 44.2 ± 2.7 micromol/l, p < 0.001).

The prevalence of studied APA in alcoholics with mildly affected liver
function was higher than in controls, but not significantly.

On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide
range of IgG oxLDL titers in a healthy population.

These parameters do not appear to be very promising for the evaluation
of the risk of atherosclerosis.

Free radicals increase the oxidative modification of LDL.

This is one of the most important mechanisms, which increases
cardiovascular risk in chronic alcoholic patients.

Important enzymatic antioxidant systems -- superoxide dismutase and
glutathione peroxidase -- are decreased in alcoholics.

We did not find any changes of serum retinol and tocopherol
concentrations in alcoholics,
and blood and plasma selenium and copper levels were unchanged as well.

Only the zinc concentration was decreased in plasma.

It could be related to the impairment of the immune system in
alcoholics.

Measurement of these parameters in blood compartments does not seem to
indicate a possible organ, e.g. liver deficiency.
Copyright 2001 National Science Council, ROC and S. Karger AG, Basel
PMID: 11173977

"The dramatically increased instability
in the presence of methanol of these three compounds,
each with 1,2-diamino or 1,2-amino hydroxy functional groups,
was due to the formation of [M + 12] products resulting from
condensation reaction of the substrates with formaldehyde."

Drug Metab Dispos. 2001 Feb; 29(2): 185-93.
Methanol solvent may cause increased apparent metabolic instability in
in vitro assays.
Yin H,
Tran P,
Greenberg GE,
Fischer V.
Drug Metabolism and Pharmacokinetics,
Novartis Biomedical Research Institute,
59 Route 10, East Hanover, NJ 07936, USA.
heqin.yin@pharma.novartis.com

Methanol was widely used as a substrate-delivering solvent in in vitro
metabolic stability screenings.

Its interaction with enzyme activities,
particularly those of cytochrome P450s,
has been investigated extensively in the past.

Little was known about the interaction of methanol,
whether direct or indirect, with substrates.

The present study provided data for the first time to show that use of
methanol may result in the formation of artifacts,
which could mislead the metabolic stability information.
The disappearance of LAQ094, metaraminol, and (-)-isoproterenol
following 1-h incubation with human liver microsomes
was 73, 85, and 66%, respectively,
in the presence of 1% methanol,
but was only 3, 15, and 24%,
respectively, in the absence of organic solvent.

The dramatically increased instability
in the presence of methanol of these three compounds,
each with 1,2-diamino or 1,2-amino hydroxy functional groups,
was due to the formation of [M + 12] products resulting from
condensation reaction of the substrates with formaldehyde.

Formaldehyde was formed from methanol
by human liver microsomal enzymes
with an apparent K(m) of 35 mM
and a V(max) of 7.9 nmol/min/mg of protein.

The concentration of formaldehyde reached as high as 600 microM
following a 60-min incubation.

The [M + 12] products were characterized as five-membered heterocycles
by liquid chromatography and tandem mass spectrometry analysis.

Inclusion of 10 mM glutathione prevented the formation of such
artifacts and is therefore suggested for future in vitro screenings.

Our study also documented the novel finding of enzyme-dependent
conversion of NADPH to nicotinamide in microsomal incubations.
PMID: 11159810

Biochem J. 1999 Jun 1; 340 ( Pt 2): 453-8.
Relationship between cytochrome P450 catalytic cycling and stability:
fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in
hepatoma cells is abolished by inactivation of its electron donor
NADPH-cytochrome P450 reductase.
Zhukov A,
Ingelman-Sundberg M.
Division of Molecular Toxicology, Institute of Environmental Medicine,
Karolinska Institutet, S-171 77 Stockholm, Sweden. andzhu@ki.se

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the
metabolism of gluconeogenetic precursors and some cytotoxins is
distinguished from other cytochrome P450 enzymes by its rapid turnover
(in vivo half-life of 4-7 h), with ligands to the haem iron, both
substrates and inhibitors, stabilizing the protein.

CYP2E1 is also known to have a high oxidase activity in the absence of
substrate, resulting in the production of reactive oxygen radicals.

We suggested that the rapid intracellular turnover of the enzyme may be
partly due to covalent modifications by such radicals or to other
changes during catalytic cycling,
in which case the inhibition of electron supply from NADPH-cytochrome
P450 reductase would be expected to stabilize the protein.

Fao hepatoma cells,
where CYP2E1 showed a half-life of 4 h upon serum withdrawal,
were treated for 1 h with 0.3 microM diphenylene iodonium (DPI),
a suicide inhibitor of flavoenzymes,
which resulted in approximately 90% inhibition of the microsomal
NADPH-cytochrome P450 reductase
and CYP2E1-dependent chlorzoxazone hydroxylase activities.

Subsequent cycloheximide chase revealed that the CYP2E1 half-life
increased to 26 h.

Neither the degradation rates of total protein, CYP2B1 and
NADPH-cytochrome P450 reductase
nor the cellular ATP level were affected by DPI
under the conditions employed.

These results demonstrate for the first time that the short half-life
of CYP2E1 in vivo may be largely due to the rapid destabilization
of the enzyme during catalytic cycling
rather than to the intrinsic instability of the protein molecule.
PMID: 10333489

Drug Metab Dispos. 1998 Jan; 26(1): 1-4.
Effect of common organic solvents on in vitro cytochrome P450-mediated
metabolic activities in human liver microsomes.
Chauret N,
Gauthier A,
Nicoll-Griffith DA.
Merck Frosst Center for Therapeutic Research, Pointe-Claire Dorval,
Quebec H9R 4P8, Canada.

In this study,
we report the effect of methanol, dimethyl sulfoxide (DMSO), and
acetonitrile on the cytochrome P450 (P450)-mediated metabolism
of several substrates in human liver microsomes:
phenacetin O-deethylation for P4501A2,
coumarin 7-hydroxylation for P4502A6,
tolbutamide hydroxylation for P4502C8/2C9,
S-mephenytoin 4'-hydroxylation for P4502C19,
dextromethorphan O-demethylation for P4502D6,
chlorzoxazone 6-hydroxylation for P4502E1,
and testosterone 6beta-hydroxylation for P4503A4.

DMSO was found to inhibit several P450-mediated reactions (2C8/2C9,
2C19, 2E1, and 3A4) even at low concentrations (0.2%).

There was no measurable effect on the catalytic activity of the various
P450s when methanol was present at levels </=1%,
except for P4502C8/9 and 2E1.

Acetonitrile did not noticeably change the catalytic activity of the
P4502C8/2C9, 2C19, 2D6, and 2E1 enzymes at concentrations </=1%.

It was found that the content level of the organic solvents should be
kept lower than 1% because, for all three solvents,
a concentration of 5% strongly affected the metabolism of the various
probes.

These findings should be taken into consideration when designing in
vitro metabolism studies of new chemical entities.
PMID: 9443844

"In addition to ADH, ethanol can be oxidized by liver microsomes:
studies over the last 20 years have culminated in the molecular
elucidation of the ethanol-inducible cytochrome P450 (P450 2E1)
which contributes not only to ethanol metabolism and tolerance,
but also to the selective hepatic perivenular toxicity
of various xenobiotics.

Their activation by P4502E1 now provides an understanding for the
increased susceptibility of the heavy drinker to the toxicity of
industrial solvents, anesthetic agents, commonly prescribed drugs,
over-the-counter analgesics, chemical carcinogens, and even nutritional
factors such as vitamin A."

Semin Liver Dis. 1993 May; 13(2): 136-53.
Biochemical factors in alcoholic liver disease.
Lieber CS.
Section of Liver Disease, Bronx Veterans Affairs Medical Center,
Bronx, NY 10468.

Three decades of research in ethanol metabolism have established that
alcohol is hepatotoxic not only because of secondary malnutrition,
but also through metabolic disturbances
associated with the oxidation of ethanol.

Some of these alterations are due to redox changes produced by the NADH
generated via the liver ADH pathway,
which in turn affects the metabolism
of lipids, carbohydrates, proteins, and purines.

Exaggeration of the redox change by the relative hypoxia,
which prevails physiologically in the perivenular zone,
contributes to the exacerbation of the ethanol-induced lesions
in zone III.

Gastric ADH also explains first-pass metabolism by ethanol;
its activity is low in alcoholics and in females
and is decreased by some H2 blockers.

In addition to ADH, ethanol can be oxidized by liver microsomes:
studies over the last 20 years have culminated in the molecular
elucidation of the ethanol-inducible cytochrome P450 (P4502E1)
which contributes not only to ethanol metabolism and tolerance,
but also to the selective hepatic perivenular toxicity
of various xenobiotics.

Their activation by P450 2E1 now provides an understanding for the
increased susceptibility of the heavy drinker to the toxicity of
industrial solvents, anesthetic agents, commonly prescribed drugs,
over-the-counter analgesics, chemical carcinogens, and even nutritional
factors such as vitamin A.

Ethanol causes not only vitamin A depletion, but it also enhances its
hepatotoxicity.

Furthermore, induction of the microsomal pathway contributes to
increased acetaldehyde generation, with formation of protein adducts,
resulting in antibody production, enzyme inactivation, decreased DNA
repair;
it is also associated with a striking impairment of the capacity of the
liver to utilize oxygen.

Moreover, acetaldehyde promotes GSH depletion, free-radical-mediated
toxicity, and lipid peroxidation.

In addition, acetaldehyde affects hepatic collagen synthesis;
both in vivo (in our baboon model of alcoholic cirrhosis)
and in vitro (in cultured myofibroblasts and lipocytes);
ethanol and its metabolite acetaldehyde were found to increase collagen
accumulation and mRNA levels for collagen.

This new understanding may eventually improve therapy with drugs and
nutrients.

Encouraging results have been obtained with some "super" nutrients.

On the one hand, SAMe, the active form of methionine, was found to
attenuate the ethanol-induced depletion in SAMe and GSH and associated
mitochondrial lesions.

On the other hand, phosphatidylcholine,
purified from polyunsaturated lecithin,
was discovered to oppose the ethanol-induced fibrosis by decreasing the
activation of lipocytes to transitional cells,
and possibly also by stimulating collagenase activity,
an effect for which dilinoleoylphosphatidylcholine,
its major phospholipid species, was found to be responsible.
PMID: 8337602

Clin Pharmacokinet. 1987 Nov; 13(5): 273-92.
Clinical pharmacokinetics of ethanol.
Holford NH.
Department of Pharmacology and Clinical Pharmacology, School of
Medicine, University of Auckland.

The pharmacokinetics of ethanol after typical doses are described by a
1-compartment model with concentration-dependent elimination.

The volume of distribution estimated from blood concentrations
is about 37 L/70 kg.

Protein binding of ethanol has not been reported.

Elimination is principally by metabolism in the liver with small
amounts excreted in the breath (0.7%), urine (0.3%), and sweat (0.1%).

Metabolism occurs, principally by alcohol dehydrogenase,
in the liver to acetaldehyde.

Models of ethanol input and absorption are crucial to the description
and understanding of the effects of ethanol dose on bioavailability.

Little attention has been paid to evaluation of potential models.

First-pass extraction of ethanol is predicted to be dependent on
hepatic blood flow and ethanol absorption rate,
with a typical extraction ratio of 0.2.

The overall elimination process can be described by a capacity-limited
model similar to the Michaelis-Menten model for enzyme kinetics.

The maximum rate of elimination of ethanol (elimination capacity or
Vmax is 8.5 g/h/70 kg.

This would be equivalent to a blood ethanol disappearance rate of 230
mg/L/h if metabolism took place at its maximum rate.

The elimination rate is half of the elimination capacity at a
peripheral blood ethanol concentration (Km) of about 80 mg/L.

Ethanol is readily detectable in expired air.

The usual blood:expired air ratio is 2300:1
and breath clearance at rest is 0.16 L/h.

The renal clearance of ethanol is 0.06 L/h
and sweat clearance is 0.02 L/h.

The use of a zero-order model of ethanol elimination has been
widespread although the limitations of this model have been known for a
long time.

Much of the published work on ethanol pharmacokinetics must be regarded
with suspicion because of this assumption.
PMID: 3319346

Wien Klin Wochenschr. 1988 Apr 29; 100(9): 282-8.
[Methanol--an up-to-now neglected constituent of all alcoholic
beverages. A new biochemical approach to the problem of chronic
alcoholism]
[Article in German]
Sprung R,
Bonte W,
Lesch OM.
Institut fur Rechtsmedizin, Universitat Gottingen.

Alcoholism is usually understood as ethanolism.

There is some evidence that its oxidation product acetaldehyde may
condense with endogenous amines to form
tetrahydroisoquinoline (TIQ) and - tetrahydro-beta-carboline (THBC)
alkaloids which ultimately might be responsible for addiction.

In most animal experiments pure ethanol solutions were fed,
but chronic alcoholics prefer normal alcoholic beverages,
and it is widely ignored that all these beverages without exception
also contain methanol.

Its metabolite formaldehyde
is a much more potent reaction partner for TIQ and THBC formation than
acetaldehyde.

As our findings in chronic alcoholics proved that these persons in
contrast to healthy subjects are able to oxidize methanol despite high
ethanol levels, there must be a continuous leakage of formaldehyde.

And it seems possible that methanol plays a more significant role in
the pathophysiology and possibly the etiology of chronic alcoholism
than ethanol.
PMID: 3291400

Wien Klin Wochenschr. 1991; 103(22): 684-9.
[Methanol metabolism in chronic alcoholism]
[Article in German]
Soyka M,
Gilg T,
von Meyer L,
Ora I.
Psychiatrische Klinik, Universitat Munchen.

Serum methanol concentrations (SMC) exceeding 10 mg/l are highly
suggestive of long-term alcohol intoxication
and can be considered as marker for chronic alcohol abuse.

Endogenously formed or consumed methanol is almost exclusively
metabolized by alcohol dehydrogenase.

As long as blood alcohol concentrations exceed 0.2-0.5 g/l
methanol cannot be metabolized and accumulates.

In a prospective study on 78 patients admitted for alcohol
detoxification, elevated SMC up to 78 mg/l were found,
with a mean SMC of 29.4 mg/l.

No correlation was demonstrated between SMC and severity of the alcohol
withdrawal syndrome.

Further clinical, forensic and biochemical aspects of methanol
metabolism are discussed.
PMID: 1776249
*******************************************************

http://groups.yahoo.com/group/aspartameNM/message/1340
aspartame groups and books: updated research review of 2004.07.16:
Murray 2006.05.11

http://groups.yahoo.com/group/aspartameNM/message/1371
Russell L. Blaylock, MD discusses MSG, aspartame, excitotoxins with
Mike Adams: Murray 2006.09.27

"Of course, everyone chooses, as a natural priority,
to actively find, quickly share, and positively act upon the facts
about healthy and safe food, drink, and environment."

Rich Murray, MA  Room For All  rmforall@comcast.net
505-501-2298  1943 Otowi Road   Santa Fe, New Mexico 87505

http://groups.yahoo.com/group/aspartameNM/messages
group with 77 members, 1,373 posts in a public, searchable archive
http://RMForAll.blogspot.com

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

http://groups.yahoo.com/group/aspartameNM/message/1279
all three aspartame metabolites harm human erythrocyte [red blood cell]
membrane enzyme activity, KH Schulpis et al, two studies in 2005,
Athens, Greece, 2005.12.14: 2004 research review, RL Blaylock:
Murray 2006.01.14

http://groups.yahoo.com/group/aspartameNM/message/1271
combining aspartame and quinoline yellow, or MSG and brilliant blue,
harms nerve cells, eminent C. Vyvyan Howard et al, 2005
education.guardian.co.uk, Felicity Lawrence: Murray 2005.12.21

http://groups.yahoo.com/group/aspartameNM/message/1349
NIH NLM ToxNet HSDB Hazardous Substances Data Bank
inadequate re aspartame (methanol, formaldehyde, formic acid):
Murray 2006.08.19

http://toxnet.nlm.nih.gov/cgi-bin/sis/search/f?./temp/~HwoSfJ:1
HSDB  Hazardous Substances Data Bank: Aspartame

ASPARTAME   CASRN: 22839-47-0
METHANOL  CASRN: 67-56-1
FORMALDEHYDE   CASRN: 50-00-0
FORMIC ACID  CASRN: 64-18-6

http://groups.yahoo.com/group/aspartameNM/message/1307
formaldehyde from 11% methanol part of aspartame or from red wine
causes same toxicity (hangover) harm: Murray 2006.05.24

Dark wines and liquors, as well as aspartame, provide
similar levels of methanol, above 120 mg daily, for
long-term heavy users, 2 L daily, about 6 cans.

Within hours, methanol is inevitably largely turned into formaldehyde,
and thence largely into formic acid --  the major causes of the dreaded
symptoms of "next morning" hangover.

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, 37 mg,
is 18.5 times the USA EPA limit for daily formaldehyde in
drinking water, 2.0 mg in 2 L average daily drinking water.

Any unsuspected source of methanol, which the body always quickly
and largely turns into formaldehyde and then formic acid, must be
monitored, especially for high responsibility occupations, often with
night shifts, such as pilots and nuclear reactor operators.

http://www.HolisticMed.com/aspartame    mgold@holisticmed.com
Aspartame Toxicity Information Center    Mark D. Gold
12 East Side Drive #2-18 Concord, NH 03301     603-225-2100

http://www.holisticmed.com/aspartame/abuse/methanol.html
"Scientific Abuse in Aspartame Research"

http://groups.yahoo.com/group/aspartameNM/message/1052
DMDC: Dimethyl dicarbonate 200mg/L in drinks adds methanol 98 mg/L
( becomes formaldehyde in body ):  EU Scientific Committee on Foods
2001.07.12:  Murray 2004.01.22
*******************************************************