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methanol impurity in alcohol drinks [ and aspartame ] is turned into neurotoxic formic acid, prevented by folic acid, re Fetal Alcohol Syndrome, BM Kapur, DC Lehotay, PL Carlen at U. Toronto, Alc Clin Exp Res 2007 Dec. plain text: Murray 2008.02.24

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  • Rich Murray
    methanol impurity in alcohol drinks [ and aspartame ] is turned into neurotoxic formic acid, prevented by folic acid, re Fetal Alcohol Syndrome, BM Kapur, DC
    Message 1 of 1 , Feb 24, 2008
      methanol impurity in alcohol drinks [ and aspartame ] is turned into
      neurotoxic formic acid, prevented by folic acid, re Fetal Alcohol Syndrome,
      BM Kapur, DC Lehotay, PL Carlen at U. Toronto, Alc Clin Exp Res 2007 Dec.
      plain text: detailed biochemistry, CL Nie et al. 2007.07.18: Murray
      2008.02.24
      http://rmforall.blogspot.com/2008_02_01_archive.htm
      Sunday, February 24, 2008
      http://groups.yahoo.com/group/aspartameNM/message/1524
      ____________________________________________________


      [ Rich Murray comments: As a medical layman volunteer information
      activist for aspartame and related toxicity issues since January 1999,
      I note with appreciation the remarkable exponential progress on all
      fronts, including a rapidly emerging consensus about the primary
      importance of all toxicity challenges for our world.

      This lengthy review features in detail two quite different, revolutionary
      contributions, from Canada, and England and China.

      It is indicative of our times that the CL Nie et al. study, 2007
      appears in a free, open access journal -- indeed,
      as all life and death information must.

      Following rather vigorously, indeed blindly, the imperatives of
      single-minded, profit-driven capitalist competition -- manipulating
      adroitly research, education, media, citizens, governments -- many
      great global corporations have inevitably created results that
      oppose the common good. Alcohol and tobacco are well known.

      Realistically, any further manipulations can only lead to inevitable
      and even sudden corporate meltdowns, in the context of an
      unfettered, cooperative, democratic global information forum,
      the Internet.

      Now, it is as easy and cheap to compose and instantly post a
      30-page review as 3 pages a decade ago -- and such reviews
      are archived forever in multiple collections, open via global search
      engines to a billion Net citizens.

      Perforce, and increasingly happily, all societal entities will have to
      operate by high and shared voluntary universal standards
      for the common good. ]


      http://www.blackwell-synergy.com/doi/abs/10.1111/j.1530-0277.2007.00541.x

      Alcoholism: Clinical and Experimental Research
      Volume 31 Issue 12 Page 2114-2120, December 2007

      Bhushan M. Kapur, b.kapur@...;
      Arthur C. Vandenbroucke, PhD, FCACB
      Yana Adamchik,
      Denis C. Lehotay, dlehotay@...;
      Peter L. Carlen carlen@...;
      (2007) Formic Acid, a Novel Metabolite of Chronic Ethanol
      Abuse, Causes Neurotoxicity, Which Is Prevented by Folic Acid
      Alcoholism: Clinical and Experimental Research 31 (12), 2114-2120.
      doi:10.1111/j.1530-0277.2007.00541.x

      Abstract

      Background:
      Methanol is endogenously formed in the brain and is present as a
      congener in most alcoholic beverages.

      Because ethanol is preferentially metabolized over methanol (MeOH)
      by alcohol dehydrogenase, it is not surprising that MeOH
      accumulates in the alcohol-abusing population.

      This suggests that the alcohol-drinking population will have higher
      levels of MeOH’s neurotoxic metabolite, formic acid (FA).

      FA elimination is mediated by folic acid.

      Neurotoxicity is a common result of chronic alcoholism.

      This study shows for the first time that FA,
      found in chronic alcoholics, is neurotoxic
      and this toxicity can be mitigated by folic acid administration.

      Objective:
      To determine if FA levels are higher in the alcohol-drinking
      population and to assess its neurotoxicity in organotypic
      hippocampal rat brain slice cultures.

      Methods:
      Serum and CSF FA was measured in samples from both ethanol
      abusing and control patients, who presented to a hospital emergency
      department. [ CSF = Cerebral Spinal Fluid ]

      FA’s neurotoxicity and its reversibility by folic acid were assessed
      using organotypic rat brain hippocampal slice cultures using clinically
      relevant concentrations.

      Results:
      Serum FA levels in the alcoholics
      (mean ± SE: 0.416 +- 0.093 mmol/l, n = 23)
      were significantly higher than in controls
      (mean ± SE: 0.154 +- 0.009 mmol/l, n = 82) (p < 0.0002).

      FA was not detected in the controls’ CSF (n = 20),
      whereas it was >0.15 mmol/l in CSF of 3 of the 4 alcoholic cases.

      Low doses of FA from 1 to 5 mmol/l added for 24, 48 or 72 hours
      to the rat brain slice cultures caused neuronal death as measured by
      propidium iodide staining.

      When folic acid (1 umol/l) was added with the FA,
      neuronal death was prevented. [ umol = micromole ]

      Conclusions:
      Formic acid may be a significant factor in the neurotoxicity of
      ethanol abuse.

      This neurotoxicity can be mitigated by folic acid administration
      at a clinically relevant dose.

      Key Words:
      Formic Acid, Folic Acid, Methanol, Neurotoxicity, Alcoholism.

      From the Department of Clinical Pathology (BMK),
      Sunnybrook Health Science Centre,
      Division of Clinical Pharmacology and Toxicology,
      The Hospital for Sick Children, Toronto, Ontario, Canada;

      St. Michael’s Hospital (ACV), Toronto, Canada;

      Department of Laboratory Medicine and Pathobiology
      (BMK, ACV), Faculty of Medicine,
      University of Toronto, Toronto, Ontario, Canada;

      Departments of
      Medicine (Neurology) and Physiology (YA, PLC),
      Toronto Western Research Institute,
      University of Toronto, Toronto, Ontario, Canada;

      and University of Saskatchewan (DLC), Saskatchewan, Canada.

      Received for publication May 1, 2007;
      accepted September 24, 2007.

      Reprint requests: Dr. Bhushan M. Kapur,
      Department of Clinical Pathology,
      Sunnybrook Health Science Centre,
      2075 Bayview Ave, Toronto, Ontario, M4N 3M5, Canada;
      Fax: 416-813-7562; E-mail: b.kapur@...;

      Copyright 2007 by the Research Society on Alcoholism.
      DOI: 10.1111/j.1530-0277.2007.00541.x
      Alcoholism: Clinical and Experimental Research 2007 Dec.
      Alcohol Clin Exp Res, Vol. 31, No 12, 2007: pp 2114–2120

      NEUROTOXICITY AND BRAIN damage are common
      concomitants findings of chronic alcoholism
      (Carlen and Wilkinson, 1987; Carlen et al., 1981; Harper,
      2007).

      The cause of ethanol-induced neurotoxicity is still unclear.

      We present here a novel hypothesis for neurotoxicity:
      increased formic acid (FA) levels produced from methanol
      (MeOH), whose catabolism is blocked by ethanol.

      Axelrod and Daly (1965) demonstrated the endogenous formation
      of MeOH from S-adenosylmethionine (SAM) in the pituitary
      glands of humans and various other mammalian species.

      Presence of MeOH in the breath of human subjects was
      reported by Ericksen and Kulkarni (1963).

      Most alcoholic beverages also have a small amount of MeOH
      as a congener (Sprung et al., 1988).

      As ethanol (EtOH) has a higher affinity for
      alcohol dehydrogenase (ADH) than MeOH,
      EtOH is preferentially metabolized (Mani et al., 1970).

      As a result, MeOH accumulation from endogenously produced
      MeOH, and/or, that consumed as part of an alcoholic beverage,
      has been reported in concentrations up to 2 mmol/l in heavy
      drinkers (Majchrowicz and Mendelson, 1971).

      Toxicity resulting from MeOH consumption is extensively
      documented in both humans and animals and has been
      attributed to its metabolite, FA (Benton and Calhoun, 1952;
      Roe, 1946, 1955; Wood, 1912; Wood and Buller, 1904).

      The rate of formate oxidation and elimination is dependent on
      adequate levels of hepatic folic acid, particularly hepatic
      tetrahydrofolate (THF)
      (Johlin et al., 1987; Tephly and McMartin, 1974).

      Significantly higher formate levels were obtained when
      folate-deficient animals were exposed to MeOH as compared
      with folate-sufficient animals (Lee et al., 1994;
      McMartin et al., 1975; Noker et al., 1980).

      To understand ethanol’s toxicity, one must consider FA
      produced from MeOH, and its elimination mediated by folic acid.

      We postulate that in the chronically drinking patient,
      we will find higher levels of FA than in the nondrinking population,
      and that formate is neurotoxic.

      We also hypothesize that treatment with folic acid, which is a
      critical factor in the catabolism of FA, can prevent or
      diminish FA neurotoxicity.

      METHODS

      Patient Samples

      During our study period of 4 months, 23 patients whose serum
      showed the presence of both ethanol and trace amounts
      (<2 mmol/l) of MeOH presented themselves to our emergency
      department.

      During the same period 4 patients, who were positive
      for EtOH at admission, were admitted to the hospital.

      During their stay, we received multiple samples as part
      of their clinical follow-up.

      We also received CSF and serum samples from 4 other patients
      who were admitted in the hospital for alcohol abuse during our
      study period.

      All samples were analyzed for EtOH, MeOH, and FA.

      As controls, we analyzed randomly the received serum (n = 82)
      and CSF (n = 20) samples from inpatients, who did not have
      any alcohol present at the time of admission.

      All serum and CSF samples were collected as part of the patients’
      clinical evaluation or follow-up.

      Ethanol and MeOH were analyzed using headspace gas
      chromatographic procedure.

      FA was also analyzed by headspace gas chromatography
      (Abolin et al., 1980).

      Lower limits of detection for EtOH, MeOH, and FA
      were 0.4, 0.8, and 0.13 mmol/l, respectively.

      Organotypic Brain Slice Cultures

      To study the neurotoxicity of FA, 2 sets of experiments using
      organotypic brain slice cultures were performed:

      (i) FA at concentrations of 1, 2 and 5 mmol/l was added to
      organotypic hippocampal rat brain slice cultures
      (n = 7 for each concentration).

      To a second set of rat brain slice cultures, both FA,
      at the above-mentioned concentrations, and 1 umol/l of folic acid
      were also added.

      Control brain slices, with and without folic acid, were also
      processed with the experimental slices.

      The time course and extent of cell death were determined by
      measuring the fluorescence of the viability indicator,
      propidium iodide (PI), at 24, 48 and 72 hours
      after the application of FA alone
      and in combination with folic acid.

      Ensuring stable and reproducible measures of damage following
      FA administration depended critically on the tissue culture
      conditions of the hippocampal slice.

      We noted that cultures which showed evident cell death before
      experimental manipulations were more vulnerable to damage
      from FA.

      Hence, we were careful to use cultures that did not demonstrate
      any apparent cell death.

      The initial control images using PI were taken approximately 1 hour
      before experimental measurements or manipulations.

      Preparation of Organotypic Slice Cultures

      Techniques for culturing brain slices have been described in detail
      by Stoppini et al. (1991).
      Briefly, the brains of 7-day-old male Wistar rats were aseptically
      removed and immersed in ice-cold dissecting medium at pH 7.15
      containing 50% minimum essential medium (MEM)
      with no bicarbonate, 50% calcium and magnesium-free
      Hanks balanced salt solution, 20 mM HEPES and 7.5 g/l d-glucose.
      Hippocampi were dissected and coronal sections, 400-um thick,
      were obtained.
      Slices were transferred to a dish containing dissecting medium,
      at room temperature.
      The slices were then carefully separated
      and transferred to sterile, porous membrane units with 0.4-um
      diameter pores (Millicell-CM).
      The membrane units were placed into 6-well trays,
      each well containing 1 ml of culture medium, which is composed
      of 50% MEM with Earl’s salts, 2 mM l-glutamine, 25%
      Earl’s balanced salt solution, 25% normal horse serum,
      6.5 g/l d-glucose, 20 mM HEPES buffer
      and 50 mg/ml streptomycin–penicillin.
      The pH of the medium was adjusted to 7.2 with HEPES buffer.

      Cultures were kept in a tissue culture incubator for 2 weeks
      at 36.8°C in 5% CO2
      before the beginning of the experiments,
      and fed 2 times a week by a 50% exchange of medium.

      Assessment of Cell Death and Fluorescence Microscopy

      Propidium iodide was applied to each dish at 10 mM,
      30 minutes prior to the toxicity assessment.

      PI fluorescence emission was measured immediately before,
      at 24, 48 and 72 hours after the administration of FA
      with a 4-X objective, using a confocal microscope
      (BioRad, Hercules, CA).
      A rhodamine filter (510 to 590 nm) was used to visualize PI
      fluorescence emission. [ nm = nanometer ]
      Gains and black levels were standardized for each experiment.
      Fluorescence images were acquired and analyzed with the
      Comos and the Confocal Assistant software packages (Bio-Rad).
      Pixel intensity was measured either for the whole slice
      or in selected areas of the hippocampus, CA1, CA3,
      and dentate gyrus (DG), using a standard sized box.

      At the end of each experiment, slices were killed by incubating
      for 48 hours at 4°C in the presence of PI (Fig. 3).

      The final PI fluorescence obtained after this treatment was
      considered to be the fluorescence that closely represents
      100% cellular death.

      Cell death was then expressed as a percentage of the final
      fluorescence minus the background fluorescence
      taken before experiments.

      The statistical comparisons between the control and injured groups
      were performed using the unpaired Student’s t-test.

      Numerical values are expressed in the figures as mean
      and standard error of mean.

      Slices exhibiting PI staining before experiments or those revealing
      any incomplete or absent hippocampal layers were excluded from the
      assessment.

      RESULTS

      Human Subjects

      Serum FA levels were significantly higher in the ethanol
      positive patients when compared with the alcohol negative
      controls (0.42 vs. 0.15 mmol/l; p < 0.0002) (Table 1).

      In all the sequentially received samples from 4 inpatients,
      EtOH declined linearly following zero-order kinetics.

      Figure 1 shows the profile of 1 of these patients.

      Both MeOH and FA levels remained almost constant
      for a considerable period of time
      and appeared to be at equilibrium.

      In the final sample of all these patients,
      MeOH levels had declined to almost 0,
      whereas FA levels had risen exponentially (Table 2, Fig. 1).

      This pattern was consistent in all patients.

      The CSF and serum from 4 different alcohol-abusing patients
      had FA in all 4 serum samples and
      FA in 3 of the 4 CSF samples (Table 3).

      In the CSF of nonethanol drinking control patients,
      EtOH, MeOH, and FA were all
      below the detection limit of the assays.

      Organotypic Hippocampal Brain Slices Cultures Incubated
      With FA

      Formic acid from 1 to 5 mmol/l added for 72 hours caused
      neuronal death as measured by PI staining (Figs 2 and 4).

      A dose–response relationship was also observed (Fig. 2)
      (p < 0.01).

      When 1 uM folic acid was added to these slice cultures
      along with the FA, neuronal death, secondary to FA,
      was prevented (p = NS as compared
      with control slice cultures with folic acid).

      The effect was more pronounced at
      48 hours than at 24 hours,
      when compared with controls slice cultures with no folic acid.

      Figure 3 (controls) shows with PI staining that there was
      minimal cell death after 72 hours in cultures.

      Figure 4 illustrates the dose and time response of FA neurotoxicity,
      which affected the CA1 neuronal layer more
      than the dentate granule cell layer.

      It also shows the neuro-protective effect of 1 uM folic acid.


      Table 1. Formic Acid Levels in Alcohol Positive
      and Randomly Collected Samples
      ------------------------------- n --- Mean +- SE (mmol/l)

      Control serum from
      nonalcoholics---------------- 82 --- 0.154 --- 0.009

      Alcohol-positive serum ------ 23 --- 0.416 --- 0.093
      p < 0.0002


      Table 2. Examples of Formic Acid Profile
      in Ethanol-Positive Patients [ serum ]

      [ To simplify, the highest levels were in Patient no. 4:
      whose admission serum samples
      and last serum samples at 30 h had:

      Methanol --- 1.1 and ND mmol/l

      Formic acid 0.25 and 1.95 mmol/l

      ND = not detected

      So, there is no data about the specific levels of
      Formic acid in vulnerable tissues, like brain and eye.

      However, it is clear that 30 hours after alcohol use,
      all ethanol and methanol are gone from the blood serum,
      while formic acid can be as high as 2 mmol/l. ]


      Table 3. CSF and Serum
      [ in 4 patients admitted for alcohol abuse.

      At admission for alcohol abuse,
      Patient no. 6 had in serum, methanol 1.7 mmol/l
      and in CSF NSQ, Not Sufficient Quantity available for analysis,
      and in serum, formic acid 2.25
      and in CSF 0.7. ]


      Fig. 1. Ethanol, methanol, and formic acid elimination profile
      in an alcoholic during his stay in the hospital.


      Fig. 2. Rat brain hippocampal slice cultures.
      Control = no formic or folic acid;
      control folic acid = 1 lmol folic acid only.
      Data represented are means ± standard error of 7 slice cultures.
      *p < 0.01 when compared with
      controls at the corresponding time.
      p = NS when 2 mmol formic + 1 umol folic acid
      compared with control folic acid.


      Fig. 3. Hippocampal slice cultures.
      Intact controls: images at 24, 48 (B),
      and 72 hours (C) and
      killed slice cultures (D, 48 hours at 4 deg C).
      (propidium iodide stains dead cells white).


      Fig. 4. Hippocampal slice cultures:
      Damage by formic acid is both dose and time dependent
      and protection by folic acid (1 umol).
      This figure shows effect of dose, time
      and the neuro-protective effect provided by folic acid.
      Hippocampal slice cultures treated
      with 1, 2 and 5 mmol⁄ L of formic acid in
      the presence and absence of folic acid (1 umol).
      Images at 48 hours with
      and without folic acid. (A, B)
      Formic acid, 1 mmol; (C, D)
      formic acid, 2 mmol; (E, F)
      formic acid, 5 mmol;
      protected slices, B, D, and F.
      (Propidium iodide stains dead cells white).


      DISCUSSION

      There are at least 2 sources of MeOH:
      endogenous production of MeOH (Axelrod and Daly, 1965;
      Ericksen and Kulkarni, 1963; Gilg et al., 1987;
      Iffland and Staak, 1990; Jones and Lowinger, 1988;
      Majchrowicz and Mendelson, 1971; Roine et al., 1989;
      Sarkola and Eriksson, 2001; Sprung et al., 1988),

      and its presence as a congener in most alcoholic beverages
      (Sprung et al., 1988).

      MeOH concentrations between 4 and 4500 mg/l can be
      present in various alcoholic beverages (Sprung et al., 1988).

      Majchrowicz and Mendelson (1971) in an elegant experiment,
      showed a rise in MeOH levels in subjects
      drinking MeOH-free alcohol, thus supporting
      the previous findings of endogenous production of MeOH.

      Endogenous production of MeOH was described again in
      2001 by Sarkola and Eriksson (2001).

      These authors gave 4-methyl pyrazole,
      a competitive inhibitor of ADH,
      to volunteers not exposed to EtOH and observed a significant
      elevation in endogenous EtOH and MeOH plasma levels.

      MeOH levels rose linearly from 20 ± 14 umol/l to 39 ± 22 umol/l.

      It took 195 minutes for EtOH levels to reach their peak (from
      <5 umol/l to 30 ± 20 umol/l) concentrations as compared
      with 420 minutes for MeOH,
      suggesting gradual accumulation of MeOH
      and preferential elimination of EtOH.

      Altered pharmacokinetic behavior of MeOH in the presence of
      EtOH has been demonstrated by various authors
      (Lesch et al., 1990; Martensson et al., 1988).

      As a result of continuous drinking
      and the preferential metabolism of EtOH,
      MeOH levels will rise in chronic drinkers
      (Gilg et al., 1987; Iffland and Staak, 1990;
      Jones and Lowinger, 1988; Majchrowicz and Mendelson, 1971;
      Roine et al., 1989; Sprung et al., 1988).

      MeOH has even been suggested as a marker for alcohol abuse
      (Iffland and Staak, 1990; Roine et al., 1989).

      As MeOH is metabolized to FA, this would suggest
      that there could be a steady increase in FA levels
      to some concentration at which equilibrium is reached.

      It has been suggested that the concentration of MeOH
      remains almost constant until EtOH levels have decreased to
      about 4 mmol/l (Martensson et al., 1988).

      Our data do indeed show this pattern.

      In the 4 patients in whom we had multiple samples,
      initially there was equilibrium between MeOH and FA.

      The frequency of sample collection in all our patients
      was based on the attending physician’s clinical reason.

      As a result, in all the 4 patients and the patient represented in
      Fig. 1, there is a large time gap between the last 2 samples.

      Our patient data (Fig. 1) do suggest that there must have been
      an exponential rise in FA as EtOH approached 4 mmol/l
      (Table 2).

      Our data suggest that in the plasma of an alcohol-drinking person,
      there can be elevated levels of FA (Table 3).

      Two nonfree radical pathways have been proposed for formate
      conversion to carbon dioxide: oxidation through the
      catalase-peroxidative system (Chance, 1950),
      and one-carbon pool.

      Formate enters the one-carbon pool by combining with
      THF to form 10-formyl-THF, a reaction catalyzed
      by 10-formyl-THF synthetase (Johlin et al., 1987).

      This is followed by the oxidation of 10-formyl-THF
      to carbon dioxide mediated
      by 10-formyl THF dehydrogenase (10-FTHFDH).

      Studies have shown that this is the major route of formate
      metabolism (Chiao and Stokstad, 1977; Johlin et al., 1987;
      Makar and Tephly, 1976; Palese and Tephly, 1975)

      and the predominant one in primates (McMartin et al., 1977).

      Formate oxidation to carbon dioxide is dependent upon folic acid
      in rats, monkeys (McMartin et al., 1977; Noker et al., 1980),
      and in humans (liver) (Johlin et al., 1989).

      Although liver is the main source for folate,
      Neymeyer and Tephly (1994) and Neymeyer et al. (1997))
      showed the presence of folate and 10-FTHFDH in the
      retina, optic nerve, and in the various regions of the rat brain.

      Folate was found to be between 3% and 14%
      of that found in the liver.

      The presence of folate and 10-FTHFDH in brain suggests
      that formate can be metabolized in these tissues.

      Folic acid deficiency is a common finding in chronic alcoholics,
      (Eells et al., 2000; Halsted et al., 2002b; Herbert, 1990).

      Chronic alcohol ingestion reduces the intestinal absorption of
      dietary folic acid leading to a decrease in the folate metabolic
      pool (Halsted et al., 2002b).

      A decrease in this pool prolongs the formate blood levels
      by decreasing the rate at which formate combines with THF,
      the first step in its metabolism to carbon dioxide
      and leads to formate-mediated cytotoxicity
      (McMartin et al., 1977).

      Folate deficiency can lead to a decrease in SAM
      (Miller et al., 1994).

      The overall status of the one-carbon pathway is also dependent
      on the levels of methionine and vitamin B6 and B12
      (Bailey and Gregory,1999; Barak et al., 1991;
      Barber et al., 1999; Halsted et al., 2002a; Lucock, 2000;
      Scott et al., 1993).

      In situation of poor folate status, S-adenosylhomocysteine (SAH)
      concentration increases due to the impairment of methyl group
      synthesis and homocysteine re-methylation.

      Inhibition by the resulting product, SAH, suppresses many of the
      (SAM)-dependent methyl transferase reactions
      (Selhub and Miller, 1992; Sokoro, 2007).

      A number of studies have shown that there is enzymatic
      activity in the brain which can metabolize both ethanol and
      acetaldehyde (Brzezinski et al., 1999; Kapoor et al., 2006;
      Roberto et al., 2006; Sun and Sun, 2001; Upadhya et al.,
      2000; Vasiliou et al., 2006; Yadav et al., 2006;
      Zimatkin et al., 2006).

      Vasiliou et al. (2006) suggested that "Although the
      contribution and CYP2E1 and catalase in ethanol oxidation
      may be of little significance, these enzymes appear to play a
      significant role in ethanol metabolism in the brain."

      Patients in whom we had a CSF samples,
      FA was present in 3 of the 4 patient’s CSF.

      Formic acid was present in all the 4 corresponding serum samples.

      The presence of FA in the CSF suggests that either FA crosses
      the blood-brain barrier or is formed in situ from the metabolism
      of water-soluble MeOH that must have crossed
      the blood-brain barrier.

      Carlen et al. (1980) showed profound CSF anion gap metabolic
      acidosis in alcoholic patients.

      Our data showing the presence of FA in CSF may indeed explain
      (Holt and Karty, 2003) the observed acidosis.

      Formate can cause oxidative stress by producing free radicals
      through the Fenton-like reaction (Dikalova et al., 2001;
      Walling, 2007).

      In this reaction, a hydroxyl radical (OH) is
      formed through the Fenton-like reaction, which in turn
      oxidized formate (HCO2),
      forming the carbon dioxide anion radical (CO2).

      The carbon dioxide anion radical then reacts
      with molecular oxygen forming carbon dioxide and
      the cytotoxic reactive oxygen species (ROS)- superoxide radical.


      H2O2 + Fe,2+ --> *OH + Fe,3+ + OH,-

      HCO2,- + *OH --> *CO2,- + H2O

      *CO2,- + O2 --> CO2 + *O2,-


      Chance has shown that formate can be metabolized by the
      catalase-peroxidative system (Chance, 1950).

      When anti-oxidants are depleted, increased ROS are formed
      (Treichel et al., 2004).

      Formic acid-induced cell damage has been attributed
      to the generation of the cytotoxic ROS species.

      FA disrupts mitochondrial electron transport and energy production
      by inhibiting cytochrome oxidase activity (Nicholls, 1975, 1976;
      Sharpe et al., 1982)
      and causes cell death by increased production of cytotoxic ROS
      secondary to the blockade of the electron transport chain
      (Reed and Savage, 1995).

      Formyl group (CHO) is transferred to THF
      resulting in the formation of carbon dioxide and water
      Makar et al., 1990; Medinsky et al., 1997).

      Our organotypic brain slice studies suggest that there is a
      dose and time relationship between FA and neuronal cell death.

      FA levels achieved in the blood of the alcohol drinking
      population can cause neuronal cell death.

      The FA concentrations we used in our studies are representative
      and were achieved in 2 of the 4 patients in whom we had sequential
      samples.

      It is remarkable that neuronal cell death could be prevented
      by folic acid, although the mechanism of this protection is unknown.

      There is a large body of literature relating folic acid deficiency
      to neural tube defect, but, there are no references
      relating low levels of FA to neurotoxicity.

      There are a few studies relating FA and mitochondrial inhibition,
      with MeOH intoxication and retinal damage
      (Seme et al., 1999, 2001).

      Another study demonstrated toxic effects of high concentrations
      of formate in dissociated primary mouse neural cell cultures
      (Dorman et al., 1993).

      The concentration of formate that resulted
      in 50% lactate dehydrogenase leakage after an 8-hour incubation
      was estimated to be 45 mmol/l.

      The total intracellular ATP concentration was significantly
      decreased following either 20 or 40 mmol/l FA
      exposure for 8 hour.

      This is consistent with the hypothesis that FA may inhibit
      mitochondrial function resulting in decreased intracellular ATP
      and formate-induced neurotoxicity.

      Using organotypic hippocampal slices, which preserve neuronal
      circuitry and are easily accessible for experimental manipulations
      (Stoppini et al., 1991),
      our group has previously shown that
      free radical overproduction in hippocampal pyramidal neurons
      during ischemia/reoxygenation
      depended on the activation of glutamate receptors,
      and was associated with elevations of intracellular calcium.

      Mitochondria are thought to be the principal source of
      glutamate-mediated, calcium-dependent free radical production
      in cultured cortical neurons
      (Dugan et al., 1995; Reynolds and Hastings, 1995).

      Although we did not investigate FA levels below 1 mmol/l,
      it is conceivable that a continuous exposure to low,
      but, above normal levels (>0.15 mmol/l), may also be cytotoxic
      and may be part of the pathology of alcohol-related
      organ damage (Jiang et al., 2003)
      including the fetal alcohol spectrum disorder.

      CONCLUSION

      Our studies, for the first time, have shown that MeOH from
      endogenous sources and from congeners present in alcoholic
      beverages can lead to FA concentrations that are neurotoxic.

      Therapeutic intervention with folic acid could be a significant
      treatment modality in preventing FA mediated cytotoxicity,
      especially neurotoxicity, in alcoholics.

      ACKNOWLEDGMENT

      This study was supported by a grant from the CIHR.

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      ____________________________________________________



      folic acid prevents neurotoxicity from formic acid, made by body
      from methanol impurity in alcohol drinks [ also 11 % of aspartame ],
      BM Kapur, PL Carlen, DC Lehotay, AC Vandenbroucke,
      Y Adamchik, U. of Toronto, 2007 Dec., Alcoholism Cl. Exp. Res.:
      Murray 2007.11.27
      http://rmforall.blogspot.com/2007_11_01_archive.htm
      Wednesday, November 27, 2007
      http://groups.yahoo.com/group/aspartameNM/message/1495


      http://www.faslink.org/Formic%20Acid%20Kapur.htm

      Brief Summary:

      Methanol in small amounts is present along with ethanol in beverage
      alcohol.
      [Murray: and about the same amounts from aspartame diet sodas]

      The body's natural enzymes preferentially metabolize ethanol while
      methanol breaks down into highly neurotoxic Formic Acid.

      Use of high levels of Folic Acid was found to inhibit brain damage
      caused by the methanol.

      The use of Folic Acid during pregnancy has been recommended
      for several years to prevent neural tube defects.

      However, this study indicates that even higher levels of Folic Acid
      can be very beneficial to the developing baby, particularly where
      alcohol exposure is a factor.

      Folic Acid is mandated as an additive to all flour sold in Canada.

      The debate has begun on its required addition to all beverage
      alcohol to help mitigate damage caused to both infants and adults.


      Formic Acid in the Drinking patient and the expectant mother
      Dr. Bhushan M. Kapur
      Departments of Laboratory Medicine,
      St. Michael's Hospital , Toronto, Ontario, Canada

      Abstract

      Methanol is produced endogenously in the pituitary glands of humans
      and is present as a congener in almost all alcoholic beverages.

      Ethanol and methanol are both bio-transformed by alcohol
      dehydrogenase; however, ethanol has greater affinity for the enzyme.

      Since ethanol is preferentially metabolized by the enzyme, it is not
      surprising that trace amounts of methanol, most likely originating from
      both sources, have been reported in the blood of people
      who drink alcohol.

      Toxicity resulting from methanol is very well documented
      in both humans and animals and is attributed to its toxic metabolite
      formic acid.

      To understand ethanol toxicity
      and Fetal Alcohol Spectrum Disorders, it is important to consider
      methanol and its metabolite, formic acid, as
      potential contributors to the toxic effects of alcohol.

      Accumulation of methanol suggests that alcohol-drinking
      population should have higher than baseline levels of formic acid.

      Our preliminary studies do indeed show this.

      Chronic low-level exposure to methanol has been suggested to
      impair human visual functions.

      Formic acid is known to be toxic to the optic nerve.

      Ophthalmological abnormalities are a common finding in children
      whose mothers used alcohol during pregnancy.

      Formic acid, a low molecular weight substance, either crosses the
      placenta or may be formed in-situ from the water soluble methanol
      that crosses the placenta.

      Embryo toxicity from formic acid has been reported
      in an animal model.

      To assess neurotoxicity we applied low doses of formic acid
      to rat brain hippocampal slice cultures.

      We observed neuronal death with a time and dose response.

      Formic acid requires folic acid as a cofactor for its elimination.

      Animal studies have shown that when folate levels are low, the
      elimination of formic acid is slower and formate levels are elevated.

      When folic acid was added along with the formic acid
      to the brain slice cultures, neuronal death was prevented.

      Therefore, folate deficient chronic drinkers may be at higher risk of
      organ damage.

      Women who are folic acid deficient and consume alcohol may have
      higher levels of formic acid and should they become pregnant,
      their fetus may be at risk.

      To our knowledge low level chronic exposure to formic acid and its
      relationship to folic acid in men or women who drink alcohol has
      never been studied.

      Our hypothesis is that the continuous exposure to low levels of
      formic acid is toxic to the fetus and may be part of the etiology of
      Fetal Alcohol Spectrum Disorders.
      ____________________________________________________


      http://www.come-over.to/FAS/

      The incidence of Fetal Alcohol Syndrome in America
      is 1.9 cases per 1,000 births (1/500).

      Incidence of babies with disabilities
      resulting from prenatal alcohol exposure: 1/100!
      ____________________________________________________


      http://groups.yahoo.com/group/aspartameNM/message/1067
      eyelid contact dermatitis by formaldehyde from aspartame,
      AM Hill & DV Belsito, Nov 2003: Murray 4.4.4 rmforall [150 KB]

      [ Extracts ]

      McMartin, KE et al 1979, put 3,000 mg/kg methanol in the
      stomachs of small monkeys and, 18 hours later found accumulation
      of formate in liver, kidney, optic nerve, cerebrum, and midbrain
      in 2 of three monkeys.

      Biochemical Pharmcacology 1979: 28; 645-649.
      Lack of a role for formaldehyde in methanol poisoning in the monkey.
      Kenneth E. McMartin, Gladys Martin-Amat, Patricia E. Noker
      and Thomas R. Tephly kmcmar@...;
      The Toxicology Center, Dept. of Pharmacology,
      University of Iowa, Iowa City, Iowa 52242

      K.E. McMartin and T.R. Tephly, authors of many pro-aspartame
      studies, in Biochemical Pharmacology (1979) remarked,
      "It is now generally accepted
      that the toxicity of methanol is due to the formation of toxic
      metabolites, either formaldehyde or formic acid."

      They put damage doses of methanol into the stomachs
      of three monkeys,
      and, using insensitive tests, found no formaldehyde in many tissues --
      except for a single datum in the midbrain,
      1.5 times their detection limit.

      They did report widespread accumulation of formic acid
      in five tissues.

      The use of inadequate tests is common in industry research that is
      funded to claim the safety of profitable toxins.

      Since then, industry scientists have been very wary of doing studies
      on primates, which all too easily show the dangers to humans.

      "Abstract [ not given in PubMed ]:
      [ My briefer comments are in square brackets. ]

      Methanol was administered [ by nasogastric tube ] either to untreated
      cynomolgus monkeys [ 2-3.5 kg ] or to a folate-deficient cynomolgus
      monkey which exhibits exceptional sensitivity to the toxic effects of
      methanol.

      Marked formic acid accumulation in the blood and in body fluids and
      tissues was observed.

      No formaldehyde accumulation was observed in the blood and no
      formaldehyde was detected in the urine, cerebrospinal fluid, vitreous
      humor, liver, kidney, optic nerve, and brain in these monkeys at a
      time when marked metabolic acidosis and other characteristics of
      methanol poisoning were observed.

      Following intravenous infusion into the monkey, formaldehyde was
      rapidly eliminated from the blood with a half-life of about 1.5 min
      and formic acid levels promptly increased in the blood.

      Since formic acid accumulation accounted for the metabolic acidosis
      and since ocular toxicity essentially identical to that produced in
      methanol poisoning has been described after formate treatment,
      the predominant role of formic acid as the major metabolic agent
      for methanol toxicity is certified.

      Also, results suggest that formaldehyde is not a major factor in the
      toxic syndrome produced by methanol in the monkey."

      "It is now generally accepted that the toxicity of methanol is due to
      the formation of toxic metabolites (1,2),
      either formaldehyde or formic acid."

      So, this is an acute toxicity study, with little relevance for chronic
      long-term, low-level exposure.

      Monkeys, like people, are susceptible to methanol toxicity.

      This team cites their six previous methanol in monkey studies,
      from 1975 to 1977.

      The report is difficult to understand, since the three monkeys were
      treated differently, and different assays were used.

      For the methanol sensitive, folate-deficient monkey A, the assay
      used was the chromatropic acid method,
      with a detection limit of .025 mmol/L.

      None of the five tissues showed any formaldehyde with this assay,
      except the midbrain, 0.14 mmol/kg wet weight tissue
      [ units converted from their 0.14 micromole/gm -- just
      1.5 times the detection limit of .09 mmol/kg wet tissue weight
      (given on p. 648).
      [ Since 1 kg of water is 1 L, 1 mmol/kg is equivalent to 1 mmol/L. ]

      Meanwhile, in the methanol sensitive, folate-deficient monkey A,
      the blood formate level rose by 18 hours from 0.18 to 10.02 mEq/L.
      [ I assume that a mEq is equivalent to a mmol -- let me know
      if I'm wrong. ]

      The formate detection limits for the assays were not given
      in this report.

      The formate level in the vitreous humor of the eye of monkey A
      was 7.90 mEq/L.

      It is well known that formate is extremely damaging to the eye.

      For unexplained reasons, formate levels in the five tissues and
      cerebrospinal fluid were not measured in the methanol sensitive,
      folate-deficient monkey A.,
      in the cerebrospinal fluid of monkey B,
      or in the optic nerve of monkey C.

      Formaldehyde was not measured in the optic nerve of Monkey A.

      The kidney formate level for monkey B was 6.33
      and for C was only 0.44,
      with no comment or explanation given.

      The experiment seems arbitrary, capricious, and erratic.

      For monkey A, after 18 hours, the urine formaldehyde level was
      below detection level, while urine formate was 115.80 mEq/L -- so
      much of the formaldehyde had been converted into formic acid,
      another cumulative, potent toxin.

      "In the presence of high formate values and definitive evidence of
      toxicity in methanol-poisoned monkeys, no measurable formaldehyde
      was found in the body tissues that were tested."

      It is reasonable to surmise that more sensitive assays would have found
      formaldehyde and formate bound to and reacted with a variety of cellular
      substances in all tissues -- just as the 1998 Trocho study confirmed.
      (Appendix E)

      Monkeys B and C were normal, not extra vulnerable to methanol,
      and were given 3,000 mg/kg methanol, and samples taken at 18 hr.

      Formaldehyde was detected only in the blood of Monkey B,
      while formate was found in 8 and 10, respectively,
      of the 10 fluid and tissue samples in Monkeys B and C.

      For instance, the lowest value of formate, except for zero-time blood,
      for each monkey was in the midbrain, 2.16 mmol/kg for Monkey B
      (24 times the detection limit for the chromatropic acid method)
      and 1.02 mmol/kg (1.3 times the detection for the dimedon method)
      for Monkey C.

      This shows accumulation of formate in liver, kidney, optic nerve,
      cerebrum, and midbrain.

      "Thus, whereas one can associate formate intimately with ocular
      toxicity in the monkey, no association of formaldehyde with ocular
      toxicity can be made at this time.

      It is not possible to completely eliminate formaldehyde as a toxic
      intermediate because formaldehyde could be formed slowly within
      cells and interfere with normal cellular function without ever obtaining
      levels that were detectable in body fluids..."

      "Acknowledgements-- This research was supported by
      NIH grant GM 19420
      and GM 12675." [not funded by the industry]


      Life Sci 1991; 48(11): 1031-41.
      The toxicity of methanol.
      Tephly TR.
      Department of Pharmacology, University of Iowa, Iowa City 52242.

      "Abstract:
      Methanol toxicity in humans and monkeys is characterized by a latent
      period of many hours followed by a metabolic acidosis
      and ocular toxicity.

      This is not observed in most lower animals.

      The metabolic acidosis and blindness is apparently due to
      formic acid accumulation in humans and monkeys,
      a feature not seen in lower animals.

      The accumulation of formate is due to a deficiency in formate
      metabolism which is, in turn, related, in part,
      to low hepatic tetrahydrofolate (H4 folate).

      An excellent correlation between hepatic H4 folate and
      formate oxidation rates has been shown within and across species.

      Thus, humans and monkeys possess low hepatic H4 folate levels,
      low rates of formate oxidation and accumulation of formate
      after methanol.

      Formate, itself, produces blindness in monkeys in the absence of
      metabolic acidosis.

      In addition to low hepatic H4 folate concentrations, monkeys and
      humans also have low hepatic 10-formyl H4 folate dehydrogenase
      levels, the enzyme which is the ultimate catalyst for conversion of
      formate to carbon dioxide.

      This review presents the basis for the role of folic acid-dependent
      reactions in the regulation of methanol toxicity.
      Publication Types: Review Review, Academic PMID: 1997785"

      p. 1035 "In the past, formaldehyde has often been suggested as the
      methanol metabolite which produces toxicity (34,35).

      Today, a great deal of information is available concerning its lack of
      such a role.

      The presence of elevated formaldehyde levels in body fluids or
      tissues following methanol administration has not been observed.

      No formaldehyde has been detected in blood, urine or tissues
      obtained from methanol-treated animals (36,37) and,
      in methanol-poisoned humans, formaldehyde increases
      have not been observed....

      About 85% of a low dose of 14C-formaldehyde [radioactive label]
      is excreted as pulmonary 14CO2 (49,50)....."

      [ This suggests that 15% of the formaldehyde is indeed retained in
      the body, a very significant result, considering its extreme
      and complex toxicity. ]

      49. W.B. Neely, Biochem. Pharmacol. 13: 1137-1142 (1964).

      50. Xenobiotica 1982 Feb; 12(2): 119-24.
      Formaldehyde metabolism by the rat: a re-appraisal.
      Mashford PM, Jones AR.
      1. The metabolism of [14C]formaldehyde has been investigated
      in the male Sprague-Dawley rat.
      It is extensively oxidized to CO2 and formate,
      which is excreted in the urine.
      2. Two radioactive compounds isolated from the urine of rats dosed
      with [14C] formaldehyde have been identified as
      N-(hydroxymethyl)urea and
      N,N'-bis-(hydroxymethyl)urea, and shown to be urinary artefacts.
      3. Previous studies of the metabolism of formaldehyde by rats have
      been re-appraised.
      Differences in the rate of oxidation of formaldehyde in various strains
      of rats result in the excretion of different urinary metabolites and, in
      some cases, formaldehyde.
      Excretion of formaldehyde leads to the formation of several artefacts
      depending on the components present in the urine. PMID: 6806997
      ____________________________________________________


      new details on how formaldehyde and formic acid from methanol are
      neurotoxic: Chun Lai Nie, Rong Giao He, et al, PLoS ONE 2(7):
      e629 2007.07.18 Chinese Academy of Sciences, Beijing:
      Murray 2007.09.01
      http://groups.yahoo.com/group/aspartameNM/message/1470

      " Recent studies have shown that neurodegeneration
      is closely related to misfolding and aggregation of neuronal tau. "

      " The significant protein tau aggregation induced by formaldehyde
      and the severe toxicity of the aggregated tau to neural cells may
      suggest that toxicity of methanol and formaldehyde ingestion
      is related to tau misfolding and aggregation. "

      " Neuronal tau is an important protein in promoting and stabilizing
      the microtubule system involved in cellular transport and neuronal
      morphogenesis. "

      " Both formaldehyde and acetaldehyde can go through the
      blood-brain barrier and cause some lesions to CNS,
      especially our visual system [38].

      Clinically, the lethal dose of formaldehyde for human beings is
      about 0.08% in the circulation [39].

      We have shown in the present study that formaldehyde can
      significantly induce tau aggregation and polymerization at
      concentrations even lower than 0.08%,
      the clinical dose of toxicosis. "

      " Formaldehyde exposure leads to formation of DNA/protein
      crosslinks, a major mechanism of DNA damage.

      The DNA/protein crosslinks have been used as a measure
      of dose in drug delivery [20].

      Formaldehyde, as a crosslinking agent, also reacts with
      thiol and amino groups, leading to protein polymerization [21], [22].

      Furthermore, methanol ingestion is an important public health
      concern because of the selective actions of its toxic metabolites,
      formaldehyde and formic acid, on the retina, the optic nerves
      and the central nervous system (CNS) [23].

      Illicit consumption of industrial methylated spirits can cause severe
      and even fatal illness [24].

      In the liver and retina, methanol is oxidized by alcohol
      dehydrogenase, resulting in formaldehyde.

      In semicarbazide-sensitive amine oxidase (SSAO)-mediated
      pathogenesis of Alzheimer's disease, formaldehyde interacts
      with B-amyloids and produces irreversibly cross-linked neurotoxic
      amyloid-like complexes [21], [22], [25].

      We have examined the role of formaldehyde in misfolding
      of protein tau [26].

      In particular, we investigated the toxicity of formaldehyde-induced
      tau aggregates on human neuroblastoma cells (SH-SY5Y cell line)
      and rat hippocampal cells [27].

      The results showed that low concentrations (0.01 - 0.1%) of
      formaldehyde are sufficient to induce formation of amyloid-like tau
      aggregates, which can induce apoptosis of both SH-SY5Y
      and hippocampal cells.

      This may be significant to understand the mechanism of chronic
      damage caused by methanol toxicity
      and formaldehyde stress [18], [28].

      However, we have still not known the mechanism of protein tau
      aggregation in the presence of formaldehyde at low concentrations.

      The present study concerns the characteristic of misfolding and
      polymerization of extracellular and intracellular neuronal tau induced
      by formaldehyde at low concentrations. "

      http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17637844
      http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000629
      free full text

      Formaldehyde at Low Concentration Induces Protein Tau
      into Globular Amyloid-Like Aggregates In Vitro and In Vivo
      PLoS ONE. 2007 Jul 18; 2(7): e629.
      doi:10.1371/journal.pone.0000629
      Chun Lai Nie 1,
      Yan Wei 1,
      Xinyong Chen 2,
      Yan Ying Liu 1,
      Wen Dui 1,
      Ying Liu 1,
      Martyn C. Davies 2, Martyn.Davies@...;
      Saul J.B. Tendler 2, Saul.Tendler@...;
      Rong Giao He 1* herq@...;

      1 State Key Laboratory of Brain and Cognitive Science,
      Institute of Biophysics, Graduate School,
      Chinese Academy of Sciences, Chaoyang District, Beijing, China,

      2 Laboratory of Biophysics and Surface Analysis,
      School of Pharmacy, The University of Nottingham,
      Nottingham, United Kingdom

      Received: March 5, 2007; Accepted: June 13, 2007;
      Published: July 18, 2007

      Copyright: © 2007 Nie et al.
      This is an open-access article distributed under
      the terms of the Creative Commons Attribution License, which
      permits unrestricted use, distribution, and reproduction in any
      medium, provided the original author and source are credited.

      * To whom correspondence should be addressed.
      E-mail: herq@...;

      Abstract

      Recent studies have shown that neurodegeneration is closely
      related to misfolding and aggregation of neuronal tau.

      Our previous results show that neuronal tau aggregates in
      formaldehyde solution and that aggregated tau induces apoptosis
      of SH-SY5Y and hippocampal cells.

      In the present study, based on atomic force microscopy (AFM)
      observation, we have found that formaldehyde at low concentrations
      induces tau polymerization whilst acetaldehyde does not.

      Neuronal tau misfolds and aggregates into globular-like polymers
      in 0.01 - 0.1% formaldehyde solutions.

      Apart from globular-like aggregation, no fibril-like polymerization
      was observed when the protein was incubated with formaldehyde
      for 15 days.

      SDS-PAGE results also exhibit tau polymerizing in the presence
      of formaldehyde.

      Under the same experimental conditions, polymerization of bovine
      serum albumin (BSA) or a-synuclein was not markedly detected.

      Kinetic study shows that tau significantly misfolds and polymerizes
      in 60 minutes in 0.1% formaldehyde solution.

      However, presence of 10% methanol prevents protein tau from
      polymerization.

      This suggests that formaldehyde polymerization is involved in tau
      aggregation.

      Such aggregation process is probably linked to the tau's special
      "worm-like" structure, which leaves the e-amino groups of Lys
      and thiol groups of Cys exposed to the exterior.

      Such a structure can easily bond to formaldehyde molecules
      in vitro and in vivo.

      Polymerizing of formaldehyde itself results in aggregation of
      protein tau.

      Immunocytochemistry and thioflavin S staining of both endogenous
      and exogenous tau in the presence of formaldehyde at low
      concentrations in the cell culture have shown that formaldehyde can
      induce tau into amyloid-like aggregates in vivo during apoptosis.

      The significant protein tau aggregation induced by formaldehyde
      and the severe toxicity of the aggregated tau to neural cells may
      suggest that toxicity of methanol and formaldehyde ingestion is
      related to tau misfolding and aggregation.

      Funding: This project was supported by NSFB (06J11),
      the NSFC (Nos. 90206041, 30570536 and 30621004)
      and 973-Project (2006CB500703 and 2006CB911003).

      Competing interests: The authors have declared that no competing
      interests exist.

      Academic Editor: Christophe Herman, Baylor College of Medicine,
      United States of America

      Introduction

      Neuronal tau is an important protein in promoting and stabilizing the
      microtubule system involved in cellular transport
      and neuronal morphogenesis.

      The tau molecule can be subdivided into an amino-terminal domain
      that projects from the microtubule surface and a carboxy-terminal
      microtubule-binding domain.

      The discovery that incubation of bacterially expressed human tau
      with sulphated glycosaminoglycans leads to bulk assembly of tau
      filaments [1], making it possible to obtain structural information [2].

      By using circular dichroism measurement, Schweer et al. have found
      that protein tau lacks secondary structures and is considered in a
      "worm-like" conformation with a high flexibility [3].

      Therefore, the side-chains of amino acids such as Lys, Cys, Thr
      and Ser are mostly exposed and vulnerable to chemical modification.

      Recently, many laboratories have found that misfolding and
      aggregation of protein tau are involved in neurodegeneration
      [2], [4] - [6].

      Protein tau has been found as the major component of paired
      helical filaments in neurofibrillary tangles in the brains of Alzheimer's
      patients, where abnormal hyper-phosphorylation induces tau to
      misfold and form the paired helical filaments,
      depositing in the cytoplasm of neurons [7] - [10].

      Recently, a great deal of evidence has demonstrated that oxidation
      and glycation stresses are key causal factors of neuronal degenerative
      diseases [11] - [13].

      Both of them inevitably produce a variety of unsaturated carbonyls
      as intermediates, like malondialdehyde and 4-hydroxynonenal,
      which usually cause carbonyl-amino crosslinking and lead to
      accumulation of irreversible changes (like lipofuscin) related to
      various neurodegenerative diseases in particular [14] - [16].

      Such carbonyl stress-related reactions (carbonylation) can form
      unstable and reversible 1:1 amino-carbonyl (Shiff's base)
      compounds at an early stage of protein modification [16], [17].

      Carbonylation binds and blocks a-/e- amino groups,
      and results in changes in charge and conformation of a protein.

      In order to investigate the relationship between carbonylation and
      protein tau misfolding, the basic and simplest carbonyl compound
      formaldehyde [18] has come into our attention.

      Formaldehyde is a common environmental agent found in paint, cloth,
      exhaust gas and many other medicinal and industrial products [19].

      Formaldehyde exposure leads to formation of DNA/protein
      crosslinks, a major mechanism of DNA damage.

      The DNA/protein crosslinks have been used as a measure of dose
      in drug delivery [20].

      Formaldehyde, as a crosslinking agent, also reacts with thiol and
      amino groups, leading to protein polymerization [21], [22].

      Furthermore, methanol ingestion is an important public health
      concern because of the selective actions of its toxic metabolites,
      formaldehyde and formic acid, on the retina, the optic nerves
      and the central nervous system (CNS) [23].

      Illicit consumption of industrial methylated spirits can cause severe
      and even fatal illness [24].

      In the liver and retina, methanol is oxidized by alcohol
      dehydrogenase, resulting in formaldehyde.

      In semicarbazide-sensitive amine oxidase (SSAO)-mediated
      pathogenesis of Alzheimer's disease, formaldehyde interacts
      with B-amyloids and produces irreversibly cross-linked neurotoxic
      amyloid-like complexes [21], [22], [25].

      We have examined the role of formaldehyde
      in misfolding of protein tau [26].

      In particular, we investigated the toxicity of formaldehyde-induced
      tau aggregates on human neuroblastoma cells (SH-SY5Y cell line)
      and rat hippocampal cells [27].

      The results showed that low concentrations (0.01 - 0.1%) of
      formaldehyde are sufficient to induce formation of amyloid-like tau
      aggregates, which can induce apoptosis of both SH-SY5Y
      and hippocampal cells.

      This may be significant to understand the mechanism of chronic
      damage caused by methanol toxicity
      and formaldehyde stress [18], [28].

      However, we have still not known the mechanism of protein tau
      aggregation in the presence of formaldehyde at low concentrations.

      The present study concerns the characteristic of misfolding and
      polymerization of extracellular and intracellular neuronal tau induced
      by formaldehyde at low concentrations.....

      Discussion

      Clinical lethal dose of formaldehyde

      Why did we investigate tau misfolding in the presence of
      formaldehyde at low concentrations (0.01 - 0.1%)?

      Methanol and ethanol are metabolized to formaldehyde and
      acetaldehyde respectively in our hepatocytes
      and some neural cells [36], [37].

      Both formaldehyde and acetaldehyde can go through the
      blood-brain barrier and cause some lesions to CNS,
      especially our visual system [38].

      Clinically, the lethal dose of formaldehyde for human beings is
      about 0.08% in the circulation [39].

      We have shown in the present study that formaldehyde can
      significantly induce tau aggregation and polymerization at
      concentrations even lower than 0.08%,
      the clinical dose of toxicosis.

      The same low concentration of formaldehyde did not induce
      polymerization of BSA though theoretically it will cause any
      protein to polymerize if the concentration is high enough.

      On the other hand, although it is known that acetaldehyde is
      acutely toxic and would covalently bind to proteins and other
      macromolecules [40], in our AFM and <br/><br/>(Message over 64 KB, truncated)
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