Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Parkinson's disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson's disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson's disease by energizing the cells and increasing their viability.

Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis.

Near-infrared light via light-emitting diode treatment has documented therapeutic effects on neurons functionally inactivated by tetrodotoxin or methanol intoxication. Light-emitting diode pretreatment also reduced potassium cyanide-induced cell death, but the mode of death via the apoptotic or necrotic pathway was unclear. The current study tested our hypothesis that light-emitting diode rescues neurons from apoptotic cell death. Primary neuronal cultures from postnatal rat visual cortex were pretreated with light-emitting diode for 10 min at a total energy density of 30 J/cm2 before exposing to potassium cyanide for 28 h. With 100 or 300 microM potassium cyanide, neurons died mainly via the apoptotic pathway, as confirmed by electron microscopy, Hoechst 33258, single-stranded DNA, Bax, and active caspase-3. In the presence of caspase inhibitor I, the percentage of apoptotic cells in 300microM potassium cyanide was significantly decreased. Light-emitting diode pretreatment reduced apoptosis from 36% to 17.9% (100 microM potassium cyanide) and from 58.9% to 39.6% (300 microM potassium cyanide), representing a 50.3% and 32.8% reduction, respectively. Light-emitting diode pretreatment significantly decreased the expression of caspase-3 elicited by potassium cyanide. It also reversed the potassium cyanide-induced increased expression of Bax and decreased expression of Bcl-2 to control levels. Moreover, light-emitting diode decreased the intensity of 5-(and -6) chloromethy-2', 7-dichlorodihydrofluorescein diacetate acetyl ester, a marker of reactive oxygen species, in neurons exposed to 300 microM potassium cyanide. These results indicate that light-emitting diode pretreatment partially protects neurons against cyanide-induced caspase-mediated apoptosis, most likely by decreasing reactive oxygen species production, down-regulating pro-apoptotic proteins and activating anti-apoptotic proteins, as well as increasing energy metabolism in neurons as reported previously.

Increased mitochondrial K(ATP) channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics?

Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K(+) (K(ATP)) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial K(ATP) channel. The K(ATP) channel opener bimakalim (1 micromol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (FIO(2)=0.21) hearts to values (42+/-4% to 67+/-5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (FIO(2)=0.12) hearts (69+/-5% to 72+/-5%). Conversely, the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-hydroxydecanoate (5-HD, 300 micromol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92+/-0.23 micromol ATP. min(-1). mg mitochondrial protein(-1)) were significantly greater than rates in normoxic hearts (2.95+/-0.08 micromol ATP. min(-1). mg mitochondrial protein(-1)). Bimakalim (1 micromol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial K(ATP) channel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-HD (300 micromol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial K(ATP) channels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.

Differential recovery of retinal function after mitochondrial inhibition by methanol intoxication.

PURPOSE: The authors' laboratory has previously documented formate-induced retinal toxicity in a rodent model of methanol intoxication. These studies determined functional, bioenergetic, and structural recovery of the retina after methanol intoxication. METHODS: Rats were intoxicated with methanol, and retinal function was assessed by electroretinography 72 hours after the initial dose of methanol and after a 72-hour recovery period. Retinal energy metabolites, glutathione (GSH) concentrations, and histology were determined at the same time points. RESULTS: Both rod-dominated and UV-cone-mediated electroretinogram responses were profoundly attenuated in methanol-intoxicated rats. In rats allowed to recover from methanol intoxication, there was significant, although incomplete, recovery of rod-dominated retinal function. However, there was no demonstrable improvement in UV-cone-mediated responses. Retinal adenosine triphosphate (ATP), adenosine diphosphate (ADP), and GSH concentrations were significantly reduced after intoxication. Although retinal energy metabolites returned to control values after the recovery period, retinal GSH remained significantly depleted. Histopathologic changes were apparent in the photoreceptors after methanol intoxication, with evidence of inner segment swelling and mitochondrial disruption. In animals allowed to recover from methanol intoxication, there was no evidence of histopathology at the light microscopic level; however, ultrastructural studies revealed subtle photoreceptor mitochondrial alterations. CONCLUSIONS: These findings support the hypothesis that formate inhibits retinal mitochondrial function and increases oxidative stress. They also provide evidence for a differential sensitivity of photoreceptors to the cytotoxic actions of formic acid, with a partial recovery of rod-dominated responses and no recovery of UV-cone-mediated responses.

Methanol poisoning. A rodent model with structural and functional evidence for retinal involvement.

Methanol ingestion can lead to visual impairment, central nervous system dysfunction, or death. The extent of ocular involvement has been difficult to determine because the toxicity is restricted to humans and nonhuman primates due to species differences in methanol metabolism. A rodent model of methanol toxicity recently developed by us was used to evaluate retinal dysfunction in methanol poisoning. Formic acidemia and visual toxic reactions developed in methanol-intoxicated rats. Electroretinographic analysis indicated a significant early deficit in b-wave amplitude followed by a temporally delayed, lesser reduction in a-wave amplitude. Histologic evaluation of the eyes 60 hours after methanol administration revealed generalized retinal edema and vacuolation in the photoreceptors and retinal pigment epithelium. Ultrastructural examination showed swelling and disruption of the mitochondria in photoreceptor inner segments, optic nerve, and the retinal pigment epithelium. These studies document direct retinal involvement in this nonprimate model of methanol toxicity.

Development and characterization of a rodent model of methanol-induced retinal and optic nerve toxicity.

Methanol is an important public health and environmental concern because of the selective actions of its neurotoxic metabolite, formic acid, on the retina, optic nerve and central nervous system. Humans and non-human primates are uniquely sensitive to methanol-induced neurotoxicity as a consequence of the limited capacity of primate species to oxidize and thus detoxify formic acid. The toxic syndrome in primates is characterized by formic acidemia, metabolic acidosis and blindness or serious visual impairment. Nonprimate species are normally resistant to the accumulation of formate and associated metabolic and visual toxicity. We have characterized retinal and optic nerve toxicity in a nonprimate model of methanol toxicity using rats in which folate-dependent formate oxidation has been selectively inhibited, allowing formate to accumulate to toxic concentrations following methanol administration. Methanol-intoxicated rats developed formic acidemia, metabolic acidosis and visual toxicity analogous to the human methanol poisoning syndrome. Visual dysfunction was manifested as reductions in the electroretinogram and the flash-evoked cortical potential which occurred coincident with blood formate accumulation. Histological studies revealed mitochondrial disruption and vacuolation in the retinal pigment epithelium, photoreceptor inner segments and optic nerve. The temporal relationship between methanol administration and the onset and development of ocular toxicity, as well as, the degree of metabolic acidosis and extent of formic acidemia in this rodent model are remarkably similar to that documented in human methanol intoxication. Moreover, the functional and morphologic findings in methanol-intoxicated rats are consistent with the hypothesis that formate acts as a mitochondrial toxin in the retina and optic nerve. The establishment and characterization of this nonprimate animal model of methanol intoxication will facilitate research into the mechanistic aspects of methanol toxicity and the development and testing of treatments for human methanol poisoning.

Effects of intraocular mescaline and LSD on visual-evoked responses in the rat.

The effects of mescaline and LSD on the flash-evoked cortical potential (FEP) were determined in unrestrained rats with chronically-implanted electrodes. Systemic administration of mescaline or LSD significantly attenuated the primary component of the FEP at three stimulus intensities with the greatest effect observed 60-90 minutes following drug administration. The magnitude and specificity of the effects of these agents on the primary response suggest that they produce deficits in conduction through the retino-geniculato-cortical system. The serotonin receptor antagonists, cyproheptadine and methysergide, antagonized the mescaline-induced depression of the FEP in accordance with neurochemical and behavioral evidence that mescaline acts as a partial agonist on serotonin receptors. Topical or intraocular administration of atropine antagonized the actions of systemically-administered mescaline. In addition, intraocular administration of mescaline or LSD attenuated the FEP indicative of an action of these hallucinogens on visual processing in the retina which is modulated by muscarinic receptor activity.

Methanol-induced visual toxicity in the rat.

Human methanol poisoning is characterized by formic acidemia, metabolic acidosis and blindness or serious visual impairment. Nonprimate species are ordinarily resistant to the accumulation of formate and the associated metabolic and visual toxicity. A nonprimate model of methanol-induced visual toxicity was developed using rats treated with subanesthetic concentrations of nitrous oxide to inhibit the oxidation of methanol's toxic metabolite, formic acid. Methanol-intoxicated rats developed formic acidemia, metabolic acidosis and visual toxicity within 36 hr of methanol administration analogous to the human methanol poisoning syndrome. Visual dysfunction was measured as reductions in the flash-evoked cortical potential and electroretinogram, which occurred coincident with blood formate accumulation. Alterations in the electroretinogram occurred at formate concentrations lower than those associated with other visual changes and provide functional evidence of direct retinal toxicity in methanol poisoning.

Pyrethroid insecticides evoke neurotransmitter release from rabbit striatal slices.

The effects of the synthetic pyrethroid insecticide fenvalerate ([R,S]-alpha-cyano-3-phenoxybenzyl[R,S]-2-(4-chlorophenyl)-3- methylbutyrate) on neurotransmitter release in rabbit brain slices were investigated. Fenvalerate evoked a calcium-dependent release of [3H]dopamine and [3H]acetylcholine from rabbit striatal slices that was concentration-dependent and specific for the toxic stereoisomer of the insecticide. The release of [3H]dopamine and [3H]acetylcholine by fenvalerate was modulated by D2 dopamine receptor activation and antagonized completely by the sodium channel blocker, tetrodotoxin. These findings are consistent with an action of fenvalerate on the voltage-dependent sodium channels of the presynaptic membrane resulting in membrane depolarization, and the release of dopamine and acetylcholine by a calcium-dependent exocytotic process. In contrast to results obtained in striatal slices, fenvalerate did not elicit the release of [3H]norepinephrine or [3H]acetylcholine from rabbit hippocampal slices indicative of regional differences in sensitivity to type II pyrethroid actions.

Identification, development, and regional distribution of ribonucleotide reductase in adult rat brain.

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [14C]cytidine diphosphate (CDP) to [14C]deoxycytidine diphosphate with subsequent hydrolysis and separation of the product ([14C]deoxycytidine) from substrate ([14C]cytidine) by Dowex-1-borate ion-exchange chromatography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 +/- 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 +/- 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 +/- 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 +/- 0.62 units/mg protein (n = 3) in newborn animals to 0.06 +/- 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the Km for CDP. The Km for CDP in brain of newborn and adult rats was 80-90 microM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.

Determination of ribonucleosides, deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.

Purine and pyrimidine base and nucleoside concentrations in human cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were determined in adult human lumbar (CSF) and plasma by reversed-phase high performance liquid chromatography (HPLC). Guanine, thymine, cytosine and uracil were not detectable (less than 0.1 microM) in human CSF or plasma. Adenine was detectable in plasma (0.3 microM) but was not found in CSF (less than 0.2 microM). Hypoxanthine and xanthine levels in CSF were each approximately 2.5 microM. Plasma levels of hypoxanthine and xanthine were considerably lower (0.4-0.6 microM). Purine and pyrimidine ribonucleosides in human CSF were less than or equal to 0.2 microM with the exception of uridine which was present at concentrations of 2-3 microM. Although low concentrations of thymidine and deoxyuridine (0.2 microM) were present in human plasma, purine and pyrimidine deoxyribonucleosides were less than 0.1 microM in human lumbar CSF.

Methanol toxicity: treatment with folic acid and 5-formyl tetrahydrofolic acid.

After methanol administration to monkeys, an accumulation of formate in blood occurs coincident with the development of metabolic acidosis and a depletion of blood bicarbonate. Formate metabolism in monkeys depends upon and is regulated by a folate-dependent system; therefore, the effect of folic acid pretreatment and 5-formyl tetrahydrofolic acid administration on methanol toxicity was investigated. Treatment of monkeys with repetitive doses of either sodium folate (48, 24, 12, and 4 hr prior to methanol) or 5-formyl tetrahydrofolic acid (2 mg/kg at 0, 4, 8, 12, and 18 hr after methanol) resulted in a marked decrease in the levels of blood formate and an absence of both metabolic acidosis and depletion of blood bicarbonate following methanol administration. Also, 5-formyl tetrahydrofolic acid reversed methanol toxicity once it was established in the monkey. The results indicate that folate compounds decrease formate accumulation after methanol by stimulating formate oxidation or utilization and suggest a possible use for folates in the treatment of certain cases of human methanol poisoning.

Nucleoside and oxypurine homeostasis in adult rabbit cerebrospinal fluid and plasma.

In adult New Zealand white rabbits, the effects of food deprivation and of massive elevations of plasma uridine or thymidine concentrations on CSF and plasma nucleoside and oxypurine concentrations were studied. Nucleoside and oxypurine levels were determined by high performance liquid chromatography using unequivocal methods of compound identification. After 48 and 96 h of food deprivation, the concentrations of uridine, cytidine, inosine, thymidine, deoxycytidine, deoxyuridine, hypoxanthine, xanthine, and uric acid in CSF and plasma were not different than in controls, except at 96 h, when the plasma uridine concentration was 35% lower (p less than 0.05). After elevation of the plasma and CSF thymidine concentrations to approximately 200 and 100 microM, respectively, with intravenous thymidine for 5 h, there was a large increase in CSF and plasma thymidine to approximately 100 microM and a smaller increase in plasma and CSF deoxyuridine concentrations. After elevation of the plasma and CSF uridine concentrations to 0.6 and 0.2 mM, respectively, there was a large increase in CSF and plasma uracil and a smaller increase in plasma and CSF deoxyuridine concentrations. Elevated plasma concentration of thymidine and uridine significantly decreased the CSF to plasma ratios of deoxyuridine and thymidine; however, only elevated plasma uridine concentrations decreased the CSF to plasma ratio of uridine. These results document the powerful homeostatic mechanisms that regulate the concentrations of the principal nucleosides and oxypurine bases in CSF.

Differences in the neuroexcitatory actions of pyrethroid insecticides and sodium channel-specific neurotoxins in rat and trout brain synaptosomes.

The effects of pyrethroid insecticides and other sodium channel-specific neurotoxins on synaptosomal membrane potential were investigated in rat and trout brain synaptosomes using the membrane-permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+). Concentration-dependent and tetrodotoxin-sensitive decreases in TPP+ accumulation, indicative of membrane depolarization, were produced by veratridine, aconitine, scorpion (Leiurus quinquestriatus) venom, and type I and type II pyrethroids, in both species. Veratridine, aconitine, and Leiurus venom were more potent and efficacious membrane-depolarizing agents in rat synaptosomes than in trout synaptosomes. Type II (deltamethrin, cypermethrin) pyrethroids produced similar depolarizing responses in rat and trout synaptosomes; however, the 1R-cis-alpha R isomer of deltamethrin, which had no effect on membrane potential in rat synaptosomes, depolarized trout synaptosomes. This isomer of deltamethrin was also shown to produce toxicity in trout, but not in rats. The type I pyrethroids, permethrin and NRDC 157, exhibited significantly greater intrinsic activity in trout brain synaptosomes, producing maximal membrane depolarizations that were three times greater than those observed in rat brain synaptosomes. These results provide evidence of species-specific differences in the membrane-depolarizing properties of pyrethroid insecticides and sodium channel-specific neurotoxins. They also suggest that some of the neurotoxin binding domains of the voltage-sensitive sodium channel in trout brain differ from those in mammalian brain. The hypersensitivity of fish to the neurotoxic actions of pyrethroid insecticides may be related to these differences.

Pyrethroid insecticide-induced alterations in mammalian synaptic membrane potential.

The neuroexcitatory actions of two toxicologically distinct classes of pyrethroid insecticides were characterized in rat brain synaptosomes using [3H]tetraphenylphosphonium to measure changes in synaptosomal membrane potential and by measuring the release of [3H]acetylcholine. Both type I (permethrin) and type II (deltamethrin, cypermethrin and fenvalerate) pyrethroids produced a concentration-dependent tetrodotoxin-sensitive membrane depolarization which was stereospecific for the neurotoxic isomer of each pyrethroid. Deltamethrin was the most potent and efficacious pyrethroid in these studies, with an EC50 of 30 nM and a maximal estimated membrane depolarization of 27 mV, followed by cypermethrin, fenvalerate and permethrin. The phenoxybenzyl pyrethroids also increased the spontaneous release of [3H]acetylcholine from rat brain synaptosomes, further supporting a depolarizing action of these insecticides on nerve terminal membranes. Pyrethroid-induced release of [3H]acetylcholine was tetrodotoxin-sensitive and occurred over the same concentration range as membrane depolarization. These data indicate that type I and type II phenoxybenzyl pyrethroids act potently and stereoselectively on the voltage-sensitive sodium channel to increase sodium influx into synaptic terminals producing membrane depolarization and neurotransmitter release. Furthermore, they show that pyrethroid-induced alterations in synaptosomal membrane potential is a sensitive measure of pyrethroid action on the sodium channel and of pyrethroid toxicity.

Mitochondria-mediated cell injury. Symposium overview.

Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.

Formate-induced alterations in retinal function in methanol-intoxicated rats.

Formic acid is the toxic metabolite in methanol poisoning. Permanent visual damage in methanol-intoxicated humans and non-human primates has been associated with prolonged exposures (> 24 hr) to blood formate concentrations in excess of 7 mM; however, little information is available on the toxicity associated with chronic low-level or repeated exposure to methanol. The present studies compared the effects on retinal function and structure of rapidly increasing formate concentrations typical of acute methanol intoxication with low-level plateau formate concentrations more likely to be generated by subacute or chronic methanol exposure. Rats that accumulated formate concentrations of 8-15 mM developed metabolic acidosis, retinal dysfunction, and retinal histopathologic changes. Retinal dysfunction was measured as reductions in the a- and b-waves of the electroretinogram that occurred coincident with blood formate accumulation. Histopathologic studies revealed vacuolation in the retinal pigment epithelium and photoreceptor inner segments. Rats exposed to formate concentrations ranging from 4 to 6 mM for 48 hr showed evidence of retinal dysfunction in the absence of metabolic acidosis and retinal histopathology. These data indicate that formic acid generated from methanol oxidation acts as a direct retinal toxin. Formate-induced retinal dysfunction in methanol-intoxicated rats can be produced by steadily increasing concentrations of formate and importantly can also be produced by prolonged exposure to lower concentrations of formate. Our findings substantiate evidence based on clinical case reports and a small number of epidemiological studies and support the hypothesis that the visual system toxicity produced by acute, subacute, or chronic methanol poisoning share a common mechanism.

Methanol toxicity in the monkey: effects of nitrous oxide and methionine.

Methanol poisoning in monkeys and humans is characterized by the development of formic acidemia, metabolic acidosis and ocular toxicity. Formate, the metabolite associated with the toxicity of methanol, is oxidized to carbon dioxide by a tetrahydrofolate-dependent pathway. Nitrous oxide treatment was used to inhibit the tetrahydrofolate-generating enzyme, 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase, E.C. 2.1.1.13.), to delineate the role of this enzyme in regulating formate oxidation in the monkey. The importance of methionine in the regulation of formate oxidation in the monkey also was evaluated. Nitrous oxide inhibited the oxidation of formate generated from the metabolism of methanol (1 g/kg i.p.) in the monkey, resulting in the development of severe metabolic acidosis and high blood formate levels in these animals compared with air-breathing monkeys administered the same dose of methanol. Treatment of nitrous oxide-exposed monkeys with repetitive doses of methionine (100 mg/kg 10, 12 and 14 hr after methanol) reversed the effects of nitrous oxide on formate oxidation, resulting in a marked decrease in blood formate levels and an increase in the rate of [14C]O2 formation from methanol. Methionine treatment also reversed the development of metabolic acidosis and bicarbonate depletion observed in nitrous oxide-exposed monkeys. These results indicate that hepatic methionine synthetase is important in the regulation of tetrahydrofolate-dependent metabolism in the monkey and that the generation of tetrahydrofolate by this enzyme is a major factor in determining the sensitivity of a species to methanol poisoning.