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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

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  • Rich Murray
    aspartame rat brain toxicity re cytochrome P450 enzymes, expecially CYP2E1, Vences-Mejia A, Espinosa-Aguirre JJ et al, 2006 Aug, Hum Exp Toxicol: relevant
    Message 1 of 1 , Sep 29, 2006
      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 of 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@...

      ISRAEL PÉREZ LÓPEZ,

      JAVIER J. ESPINOSA AGUIRRE, jjea@...
      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.

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      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@...
      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@...
      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@...
      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@...
      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@...

      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@...

      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@...

      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@...
      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@...
      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
      *******************************************************
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