Formate-Induced Alterations in Retinal Function in Methanol-Intoxicated Rats

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

140, 58–69 (1996)

0197

Formate-Induced Alterations in Retinal Function in Methanol-Intoxicated Rats1 JANIS T. EELLS,*,2 MICHELE M. SALZMAN,* MICHAEL F. LEWANDOWSKI,†

AND

TIMOTHY G. MURRAY†,3

*Department of Pharmacology and Toxicology and †Department of Ophthalmology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226 Received November 7, 1995; accepted April 11, 1996

Methanol is an important public health and environmental concern because of its selective neurotoxic actions of the human visual system. Blindness or serious visual impairment is a well-known effect of human methanol poisoning; however, the mechanisms responsible for the toxic actions of this agent are not understood (Tephly and McMartin, 1984; Eells, 1992). Methanol is a widely used industrial solvent and is being developed as an alternative fuel and energy source throughout the world (Posner, 1975; Gray and Alson, 1985; Marshall, 1989; Kavet and Nauss, 1990). The increased use and availability of methanol will further expand the potential for accidental poisoning and underscores the importance of understanding the mechanisms responsible for its toxicity. Methanol has been recognized as a human visual neurotoxin for more than a century and the clinical features of acute human methanol toxicity have been extensively documented (Wood and Buller, 1904; Benton and Calhoun, 1952; Roe, 1955). The acute effects of methanol toxicity appear after an asymptomatic latent period of about 24 hr and consist of formic acidemia, uncompensated metabolic acidosis, visual toxicity, coma, and, in extreme cases, death. Visual disturbances generally develop between 18 and 48 hr after methanol ingestion and range from mild photophobia and misty or blurred vision to markedly reduced visual acuity and complete blindness. Susceptibility among individuals to the acute effects of methanol is highly variable and the minimum lethal dose is considered to be between 300 mg/ kg and 1 g/kg (Roe, 1982; Jacobson and McMartin, 1986; Eells, 1992). The minimum dose causing permanent visual defects is unknown, although blindness has been reported after ingestion of as little as 4 ml of methanol (Bennett et al., 1953; Tong, 1982). Methanol toxicity is primarily attributable to its metabolite, formic acid. Formic acid is the toxic metabolite responsible for the metabolic acidosis observed in methanol-intoxicated humans (McMartin et al., 1980; Jacobsen and McMartin, 1986) and nonhuman primates (McMartin et al., 1975, 1977; Eells et al., 1983) and for the ocular toxicity produced in nonhuman primates (Martin-Amat et al., 1977, 1978;

Formate-Induced Alterations in Retinal Function in MethanolIntoxicated Rats. EELLS, J. T., SALZMAN, M. M., LEWANDOWSKI, M. F., AND MURRAY, T. G. (1996). Toxicol. Appl. Pharmacol. 140, 58–69. Formic acid is the toxic metabolite in methanol poisoning. Permanent visual damage in methanol-intoxicated humans and nonhuman primates has been associated with prolonged exposures (ú24 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. q 1996 Academic Press, Inc.

1 This research was presented in part at the 1993 Annual Meeting of the Society of Toxicology, New Orleans, LA and at the 1994 Annual Meeting of the Association for Research in Vision and Ophthalmology, Sarasota, FL. 2 To whom reprint requests should be addressed. 3 Present address: Bascom Palmer Eye Institute, Miami, FL.

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0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Hayreh et al., 1980). Formic acid is also believed to be the toxic metabolite responsible for the ocular toxicity seen in methanol-poisoned humans (Sharpe et al., 1982; Jacobson and McMartin, 1986; Murray et al., 1995). Permanent visual damage in methanol-intoxicated humans (Jacobsen and McMartin, 1986) and nonhuman primates (Hayreh et al., 1977, 1980) has been associated with prolonged exposures (usually greater than 24 hr) to blood formate concentrations in excess of 7 mM. However, very little information is available on the health effects associated with chronic low-level or repeated exposure to methanol. On the basis of clinical case reports and a small number of epidemiological studies, it has been suggested that prolonged exposures to methanol concentrations above 260 mg/m3 impair human visual function (Frederick, 1984; Kingsley and Hirsch, 1954; Andrews, 1987). Although it is possible that acute and chronic effects may share common mechanisms of action, dose–effect and time course relationships between blood and tissue formate concentrations and the onset and development of visual toxicity remain to be established. Our understanding of the pathogenesis of methanol poisoning has, until recently, been limited by the lack of animal models that mimic the human poisoning syndrome. Humans and nonhuman primates are uniquely sensitive to methanolinduced neurotoxicity as a consequence of the limited capacity of primate species to oxidize and thus detoxify formic acid (Makar et al., 1968; Eells et al., 1983; Eells, 1992). We have developed a nonprimate model of methanol toxicity using rats in which formate oxidation has been selectively inhibited by treatment with nitrous oxide (Eells et al., 1981, 1983, 1996; Eells, 1991; Murray et al., 1991). Subanesthetic concentrations of nitrous oxide inactivate the enzyme methionine synthase (Deacon et al., 1980) reducing the production of tetrahydrofolate, the cosubstrate for formate oxidation, thus allowing formate to accumulate to toxic concentrations following methanol administration (Eells et al., 1981). We have previously shown that nitrous oxide exposure selectively depletes hepatic tetrahydrofolate and renders rats susceptible to formic acidemia and metabolic acidosis following methanol administration (Eells et al., 1981, 1982). Recent studies in our laboratory have established this rodent model of methanol-induced visual toxicity and have documented abnormalities in the electroretinogram (ERG) and histopathologic changes in the neural retina and optic nerve in methanol-intoxicated rats (Eells, 1991; Murray et al., 1991; Eells et al., 1996). The present studies were undertaken to determine if prolonged exposure to blood formate concentrations lower than those generally associated with methanol poisoning could produce retinal toxicity in this animal model. These studies compared the effects on retinal function and structure of rapidly increasing formate concentrations typical of acute methanol intoxication with low-level plateau formate con-

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centrations more likely to be generated by subacute or chronic methanol exposure. The work reported here documents evidence of retinal dysfunction in the absence of metabolic acidosis and overt histopathologic changes in the retina in rats exposed to formate concentrations ranging from 4 to 6 mM for 48 hr. METHODS Materials. Methanol (HPLC grade) obtained from Sigma Chemical Co. (St. Louis, MO) was diluted in sterile saline and administered as a 20% w/ v solution. Sodium pentobarbital was purchased from Steris Laboratories (Phoenix, AZ) and tiletamine HCl/zolazepam HCl (1:1) (Telazol) was obtained from A. H. Robbins Co. (Richmond, VA). Phenylephrine HCl, 2.5% (Neosynephrine) was acquired from Winthrop Pharmaceuticals (New York, NY). All other chemicals were reagent grade or better. Animals. Male Long–Evans rats (Harlan Sprague–Dawley, Madison, WI), which weighed 250–300 g, were used throughout these experiments. All animals were supplied food and water ad libitum and maintained on a 12-hr light/dark schedule in a temperature- and humidity-controlled environment. Animals were handled in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Methanol-intoxication protocol. Rats were placed in a Plexiglas chamber (22 1 55 1 22 cm) and exposed to a mixture of N2O/O2 (1:1; flow rate, 2 liters/min) for 4 hr prior to the administration of methanol or saline. N2O/O2 exposure was continued throughout the course of the experiment. Two methanol dosing regimens were employed. In the first regimen (high formate), methanol (20% w/v methanol in saline) was administered by intraperitoneal injection at a dose of 4 g/kg followed by supplemental doses of 2 g/kg at 12-hr intervals. In the second regimen (low formate), methanol was administered at a dose of 4 g/kg followed by supplemental doses of 1 g/kg at 12-hr intervals. These dosage regimens were designed to maintain blood formate concentrations ranging from 8 to 15 mM (high formate) or from 4 to 6 mM (low formate) for 30–40 hr. Control groups were exposed to N2O and received saline (N2O control) or were not exposed to N2O and received methanol (methanol control). Rats were periodically removed from the exposure chamber for electrophysiological measurements and to obtain blood samples. Blood samples for formate analysis and blood gas measurements were obtained from the tail or orbital sinus (under Telazol anesthesia, 10 mg/kg) after the electrophysiological recording sessions. Electrophysiologic measurements. ERGs were recorded from lightly anesthetized rats using circular silver wire electrodes (Murray et al., 1991). Animals were anesthetized prior to ERG recordings with pentobarbital sodium at 10 mg/kg and 0.5% tetracaine hydrochloride was used as a topical anesthetic followed by pupillary dilation with 2.5% phenylephrine hydrochloride (Neosynephrine) and 1% tropicamide (Mydriacyl). Two baseline recordings were obtained from each rat and experimental ERGs were recorded at 24, 48, and 60 hr after methanol administration. Animals were dark adapted for 30 min and the averaged ERG was recorded using a Nicolet system (CA1000 Flash DC200) in response to four flashes (3.2 1 103 lx), 10 msec in duration presented at 0.03 Hz. Amplitude measurements were made in microvolts from the peaks of the negative a wave or the positive b wave of the ERG. Treatment effects were calculated as percentage of the control (mean of baseline measurements) amplitude and averaged across animals (Eells, 1991). Histopathologic analysis. Animals were anesthetized with sodium pentobarbital (60 mg/kg) and perfused (intracardiac) with phosphate-buffered 2.5% glutaraldehyde/2.5% formaldehyde, pH 7.4. Eyes were enucleated and immersed in the above fixative for 72 hr. The anterior segment and vitreous were removed, then full-thickness pieces of eye wall were dissected from the posterior pole, including the optic nerve. Tissues were postfixed

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in phosphate-buffered 2% OsO4 , dehydrated in a graded ethanol series, and embedded in epoxy resin. Thick sections (1 m) for light microscopy were stained with toluidine blue. Blood gas analysis and blood and tissue formate analysis. Blood gases and blood pH were measured on orbital sinus blood samples using a blood gas analyzer (Radiometer, ABL2). Bicarbonate values were calculated from pH and pCO2 values using the Henderson–Hasselbach equation. For tissue formate determination, rats were euthanized by decapitation 60 hr after the initial dose of methanol. The brain and eyes were quickly removed and the retina, optic nerve, and brain regions were dissected on ice. Tissues were weighed and a 25% w/v homogenate was prepared in 0.1 M sodium phosphate buffer (pH 7.4). Protein-free supernatants for formate analysis were prepared by the sequential addition of 100 ml of 7.5% ZnSO4 (7 H2O) and 0.4 N NaOH to 150 ml of tissue homogenate followed by centrifugation at 35,000g for 5 min (Eells, 1991). Formate concentrations were determined on orbital sinus or tail blood samples (McMartin et al., 1975) and tissue samples using the fluorometric assay of Makar and Tephly (1982). Statistical analysis. Statistical comparison of group means were made by using a group Student t test if only one comparison was made between two groups. In all cases in which several comparisons were required, oneway analysis of variance with repeated measures was performed. This was followed by a Dunnett’s test procedure for multiple comparisons with a control (Winer, 1972). In all cases, the minimum level of significance was taken as p õ 0.05.

RESULTS

Blood formate, pH, and bicarbonate concentrations in methanol-intoxicated rats. The administration of methanol to N2O-exposed rats has been shown to result in the accumulation of formate in the blood as a consequence of the inhibition of formate oxidation (Eells et al., 1981; Eells, 1991; Murray et al., 1991). In the present studies, two methanol intoxication regimens were utilized to produce and maintain blood formate concentrations ranging from 8 to 15 mM (highformate group) or 4 to 6 mM (low-formate group) for 30– 40 hr. Blood formate concentrations in these two treatment groups are shown in Fig. 1. Blood formate concentrations in N2O-exposed rats administered methanol under the highformate dosage regimen increased from 0.8 to 7 mM within 12 hr of methanol administration and continued to increase linearly for 60 hr (Fig. 1). Sixty hours after methanol administration, blood formate concentrations were 15 { 3 mM in this treatment group. Rats in the high-formate treatment group also developed uncompensated metabolic acidosis concomitant with formic acidemia (Table 1). Sixty hours after methanol administration, blood bicarbonate values in these animals had declined from 25.7 to 7.7 mEq/liter and blood pH had declined to 6.92 from a normal pretreatment value of 7.36. A different pattern of formate accumulation was observed in the low-formate treatment group. In these animals, blood formate concentrations increased from basal values of 0.5 to 4 mM within 12 hr of the initial dose of methanol and plateaued at 4–6 mM for the following 48 hr. No alterations from control values in blood bicarbonate or blood pH were observed in the low-formate treatment group. Saline-treated rats exposed to N2O (N2O control) and metha-

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FIG. 1. Blood formate concentrations in methanol-intoxicated and methanol control rats. Methanol-intoxicated rats were exposed to a mixture of N2O/O2 (1:1) for 4 hr prior to methanol administration [4 g/kg at zero time followed by 2 g/kg (high-formate treatment group) or 1 g/kg (lowformate treatment group) at 12-hr intervals] and exposure to the gas mixture was continued throughout the experiment. Methanol control (control) rats were exposed to room air and administered methanol (4 g/kg at zero time followed by 2 g/kg at 12-hr intervals). Blood formate concentrations were determined prior to methanol administration and at 12-hr intervals following methanol administration for 60 hr. Shown are the mean values { SE from six rats in each experimental treatment group. Significant differences from zero-time measurements are indicated with asterisks (repeated measurement analysis of variance with Dunnett’s test; p õ 0.05). Blood formate concentrations in untreated control and N2O control rats [exposed to N2O/O2 (1:1) and administered saline on the same injection schedule] did not exceed 1.0 mM (data not shown).

nol-treated rats exposed to room air (methanol control) also exhibited no significant differences in blood formate, blood pH, or blood bicarbonate concentrations relative to untreated rats (Table 1). Tissue-specific formate disposition in methanol-intoxicated rats. Formate concentrations in ocular and brain tissues were determined 60 hr after the initial dose of methanol in both high-formate and low-formate treatment groups. Similar profiles of formate disposition were observed in the central nervous system and in ocular tissues of rats that accumulated high or low concentrations of blood formate (Fig. 2). The highest concentrations of formate in both groups of methanol-intoxicated rats were measured in the vitreous humor, retina, and blood. Formate concentrations in brain regions and optic nerve were significantly lower than those measured in the vitreous humor or retina. The lowest concentrations of formate were measured in the optic nerve and were 15–20% of the levels measured in the retina. Formate concentrations in brain regions were approximately 50% lower than those measured in the retina and showed no regional variation. Concentrations of formate in the blood, vitreous humor, retina, and brain regions in untreated control, N2O control, and methanol control rats did not exceed 1.6 mmol/ml or 1.0 mmol/g.

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TABLE 1 Blood Formate, pH, and Bicarbonate Concentrations in Methanol-Intoxicated Rats Blood formate (mM)

Blood bicarbonate (mEq/liter)

0.8 { 0.1 0.5 { 0.1 1.6 { 0.5

24.6 { 1.3 25.6 { 1.4 24.3 { 0.7

7.36 { 0.03 7.34 { 0.03 7.22 { 0.04

5.9 { 0.6* 16.1 { 0.7*

24.5 { 2.6 7.7 { 1.2*

7.32 { 0.01 6.90 { 0.06*

Treatment Untreated control N2O control Methanol control Methanol intoxicated Low formate High formate

Blood pH

Note. Methanol (4 g/kg, ip) was administered to rats at zero time and supplemental injections of 2 g/kg (high-formate treatment group; methanol control group) or 1 g/kg (low-formate treatment group) were given 12, 24, 36, and 48 hr after the initial dose. Methanol-intoxicated rats were exposed to a mixture of N2O/O2 (1:1) for 4 hr prior to methanol administration and exposure to the gas mixture was continued throughout the experiment. Methanol control rats were exposed to room air. N2O control rats were exposed to N2O/O2 (1:1) and administered saline on the same injection schedule. Blood formate concentrations and blood gas measurements were determined 60 hr after the initial dose of methanol or saline. Shown are the mean values { SE of blood formate concentrations, blood bicarbonate concentrations, and blood pH determined in six rats in the low-formate treatment group and in four rats in each of the other treatment groups. * Significant difference from untreated control measurements (Student’s t test; p õ 0.05).

Disruption of retinal function in methanol-intoxicated rats. The ERG was measured to investigate the direct effects of methanol intoxication on retinal function. The averaged ERG (Fig. 3, zero time) consisted of a negative a-wave (90–200 mV; 15 msec) followed by a positive b-wave (200– 700 mV; 40 msec). The a-wave of the ERG reflects the hyperpolarization of the photoreceptors and the b-wave is generated by Mu¨ller glial cells in response to an increase in extracellular potassium released by the bipolar cells (Dowling, 1987). Comparison of ERG recordings obtained under control conditions (prior to methanol administration, zero time) and 24, 48, and 60 hr after methanol illustrate the time-dependent and formate concentration-dependent attenuation of the ERG in one animal representative of the lowformate treatment group and the virtual elimination of the ERG in one animal representative of the high-formate treatment group (Fig. 3). In the high-formate treatment group, reductions in the b-wave of the ERG were evident as early as 24 hr after methanol administration and the b-wave was profoundly attenuated or abolished by 48–60 hr (Fig. 4). Significant reductions in a-wave amplitudes were also apparent in the high-formate treatment group (Fig. 4). These were less pronounced than the b-wave reductions. Reductions in both the a-wave and the b-wave of the ERG were also observed in rats in which blood formate levels plateaued at 4– 6 mM (Figs. 3 and 4). In these animals, significant reductions

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in a-wave and b-wave amplitudes were measured 60 hr after the initial dose of methanol. No significant alterations were observed in either the a- or b-wave amplitude in N2O control (Murray et al., 1991) or methanol control animals. Correlation between formic acid accumulation and ERG alterations. Reductions in ERG a-wave and b-wave amplitudes occurred coincident with the linear increase in blood formate concentrations in the high-formate treatment group of methanol-intoxicated rats. The data in Fig. 5 show the relationship between the amplitude of the a-wave or the bwave of the ERG and the corresponding concentration of formate in the blood in the high-formate treatment group of methanol-intoxicated rats. A highly significant negative correlation was demonstrated between blood formate concentration and these parameters of retinal function. These findings are indicative of a concentration-dependent disruption of retinal function by formate under conditions in which blood levels of formate are steadily increasing. Threshold

FIG. 2. Formate disposition in the eye and central nervous system of methanol-intoxicated rats. Formate concentrations in ocular and brain tissues following methanol intoxication were determined 60 hr after the initial dose of methanol in rats exposed to either low blood formate (low formate) or high blood formate (high formate) concentrations. Shown are the mean values { SE from six rats in each experimental treatment group. Significant differences from formate concentrations measured in blood for each treatment group are denoted with asterisks (Student’s t test; p õ 0.05). No regional differences in formate concentrations were apparent in the brain. Concentrations of formate in the vitreous humor, retina, and brain regions in untreated control, methanol control, or N2O control animals did not exceed 1.6 mmol/ml or 1.0 mmol/g.

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FIG. 3. Electroretinogram (ERG) alterations in methanol-intoxicated rats exposed to high and low formate concentrations. ERGs were recorded from lightly anesthetized rats using circular silver wire electrodes. ERG recordings were obtained prior to methanol administration (zero time) and 24, 48, and 60 hr after the initial dose of methanol. Baseline ERG amplitudes were comparable in methanol-intoxicated rats exposed to low formate concentrations (low formate: a-wave Å 181 { 19 mV; b-wave Å 524 { 55 mV; n Å 6) or high formate concentrations (high formate: a-wave Å 176 { 22 mV; b-wave Å 529 { 58 mV; n Å 6). ERG analysis revealed reductions in the amplitude of the a-wave and the b-wave of the ERG in methanol-intoxicated rats that accumulated low concentrations of blood formate (low formate) and high concentrations of blood formate (high formate). Shown are the ERG recordings from one animal representative of each treatment group.

concentrations for formate-induced ERG reductions predicted from these curves range from 3 to 5 mM with more pronounced reductions apparent in b-wave amplitudes than in a-wave amplitudes. Blood formate concentrations plateaued at 4–6 mM in the low-formate treatment group of methanol-intoxicated rats. In these animals, significant reductions in ERG a-wave and b-wave amplitude were not observed until 60 hr after methanol administration, indicative of a time-dependent retinotoxic action of formate at lower concentrations. Effects of high-formate and low-formate exposure on retinal structure. The effects of high-formate and low-formate exposure on retinal histology in methanol-intoxicated rats were assessed by light microscopy. Figure 6 shows light micrographs of the retina from a methanol control rat (methanol treated, but not exposed to N2O) (Fig. 6A), a lowformate-exposed (60-hr blood formate: 5.2 mM) (Fig. 6B), and a high-formate-exposed (60-hr blood formate: 15.2 mM) (Fig. 6C) experimental animal. Tissues were prepared 60 hr after the initial dose of methanol. Relative to controls, retinas from methanol-intoxicated animals exposed to blood formate concentrations increasing from 7 to 15 mM over the course of 48 hr showed prominent vacuolation in the photoreceptors near the junction of the inner and outer segments, with accumulations of densely stained material in the inner segments

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near the outer limiting membrane. The outer nuclear layer appeared mildly edematous. Vacuoles were also present in the bases of retinal pigment epithelial cells. In contrast, no histopathologic changes were apparent at the light microscopic level in the methanol-intoxicated rats exposed to formate concentrations of 4–6 mM for 48 hr (Fig. 6B). Retinal histology in all animals from this treatment group was indistinguishable from that observed in untreated control, methanol control, and N2O control animals. DISCUSSION

Methanol poisoning in humans and nonhuman primates is characterized by formic acidemia, metabolic acidosis, and blindness or serious visual impairment. Permanent visual damage in methanol-poisoned humans (Jacobson and McMartin, 1986) and in methanol- and formate-intoxicated primates (Hayreh et al., 1980) occurs following prolonged exposures (usually greater than 20 hr) to blood formate concentrations in excess of 7 mM. Our laboratory has previously reported retinal dysfunction and histopathologic changes in the retina and optic nerve associated with exposure to blood formate concentrations ranging from 8 to 20 mM for 20–30 hr in methanol-intoxicated rats (Eells, 1991; Murray et al., 1991) and in a fatal human case of methanol poisoning (Eells

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al., 1988; Murray et al., 1996; Hayreh et al., 1980), suggesting that the ERG provides an extremely sensitive measure of formate-induced retinotoxicity. The ERG is a global measure of retinal function in response to light (Armington, 1986; Dowling, 1987). The awave of the ERG reflects the hyperpolarization of the photoreceptors. The b-wave of the ERG is generated by depolarization of the Mu¨ller glial cells and reflects the conduction of impulses from photoreceptors through the bipolar cells to the ganglion cells (Armington, 1986; Dowling, 1987). ERG analysis in methanol-intoxicated rats revealed two distinct patterns of attenuation of retinal function. In rats exposed to low blood formate concentrations, significant reductions in a- and b-wave amplitudes were not observed until 60 hr after methanol administration, indicative of a time-dependent retinotoxic action of formate at low concentrations. In contrast, in rats in which formate concentrations were stead-

FIG. 4. Amplitude of the a-wave and b-wave of the ERG as a function of time after methanol administration. ERG recordings were obtained prior to methanol administration (zero time) and 24, 48, and 60 hr after the initial dose of methanol in methanol-treated control rats (control) and in methanolintoxicated rats that accumulated low blood formate (low formate) and high blood formate (high formate) concentrations. Data are expressed as percentage of mean baseline (zero time) values of ERG a-wave or b-wave amplitude. Shown are the mean values { SE from six rats in each experimental treatment group. Significant decreases in amplitude from zero-time measurements are denoted with asterisks (repeated measurement analysis of variance with Dunnett’s test; p õ 0.05).

et al., 1991; Murray et al., 1996). However, there is very little information on the effects of prolonged low-level formate exposure on visual function. The present studies document retinal dysfunction in animals subacutely exposed to low concentrations (4–6 mM) of formate. Significant reductions in both the a-wave and the b-wave of the electroretinogram were observed following 48 hr of formate exposure in these animals. Moreover, these alterations in retinal function occurred in the absence of metabolic acidosis, characteristic of methanol intoxication, and the absence of retinal histopathology. Formate-induced reductions in the ERG occurred at formate concentrations lower than those required to diminish the flash-evoked cortical potential in methanol-intoxicated rats (Eells, 1991) and lower than those associated with retinal and optic disc edema, pupillary dilatation, or histopathologic alterations in the retina or optic nerves in humans or nonhuman primates (McMartin et al., 1980; Jacobsen et

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FIG. 5. Correlation of ERG a-wave and b-wave amplitude with blood formate concentrations in methanol-intoxicated rats. ERG a-wave and bwave amplitudes and blood formate concentrations were determined prior to methanol administration and at 24, 48, and 60 hr after the initial dose of methanol in methanol-intoxicated rats that accumulated high blood formate concentrations (high-formate treatment group). Each point represents the value (mean { SE) for the ERG a-wave or b-wave amplitude (expressed as percentage of zero-time measurements) and corresponding blood formate concentrations (mean { SE; mM) measured in six methanol-intoxicated rats at these time points. The correlation coefficient is indicated and is highly significant (p õ 0.01).

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FIG. 6. Effect of methanol intoxication on retinal histology. Light micrographs of retinal tissue prepared 60 hr after the initial dose of methanol from a methanol-treated control rat (A), a low-formate-exposed rat (B), and a high-formate-exposed rat (C). Histopathologic changes were apparent only in retinas from methanol-intoxicated rats exposed to high concentrations of formate. The arrows indicate prominent vacuoles in the region between the inner and outer segments of photoreceptor cells and in the bases of the retinal pigment epithelial cells (toluidine blue, 1210).

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FIG. 6 —Continued

ily increasing, there was a significant negative correlation between blood formate concentrations and ERG a-wave and b-wave amplitudes indicative of a concentration-dependent retinotoxic action of formate under these conditions. Threshold concentrations for formate-induced ERG reductions predicted from these curves range from 3 to 5 mM. This predicted threshold concentration range for formate-induced retinal dysfunction is consistent with previous studies from our laboratory documenting reversible b-wave attenuation at comparable formate concentrations (Eells, 1991) and is also consistent with the ERG b-wave reductions reported by Lee et al. (1994b) in methanol-intoxicated folate-deficient rats. In addition, the negative correlation between ERG b-wave amplitude and rising formate concentrations obtained in anesthetized rats in the present study is strikingly similar to that reported by our laboratory in awake rats implanted with scleral electrodes, indicative of a negligible anesthetic effect on the ERG. The present studies also show a significant negative correlation between blood formate concentrations and a-wave amplitude. The b-wave was more profoundly attenuated than the a-wave following exposure to either high or low formate concentrations. The b-wave has also been reported to be proportionally more diminished than the awave in methanol-intoxicated primates (Ingmansson, 1983) and in human studies investigating ERG responses in chronic abusers of methanol (Ruedemann, 1961).

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Selective attenuation of the b-wave, in the absence of awave reductions, has been reported in folate-deficient rats given single oral doses of methanol ranging from 1.5 to 3.5 g/kg or by inhalation exposure (2000 ppm for 20 hr/day for 3 days) (Lee et al., 1994b). Formic acid accumulation is comparable in both methanol-sensitive rodent models; therefore, the differences in the ERG responses observed in N2O-treated tetrahydrofolate-deficient and dietary folate-depleted rats are most likely due to the differences in stimulation and recording parameters. Flash intensity was substantially greater in the studies by Lee and colleagues (4.5 1 107 compared to 3.2 1 103 lx in our studies), producing a more intense visual stimulation and potentially eliminating the a-wave decrements recorded at the lower stimulus intensities employed in our studies. Electroretinographic studies are currently being conducted in our laboratory to define the time course and stimulus–response relationships in methanol-intoxicated rats over an extended range of stimulus intensities. The marked reductions of the ERG observed in methanolintoxicated rats indicate that retinal function is directly disrupted by formate. The ERG alterations produced in methanol-intoxicated rats in this study as well as those reported in methanol-intoxicated humans (Ruedemann, 1961; Murray et al., 1996) and nonhuman primates (Ingmansson, 1983) are indicative of a direct retinotoxic action of formate that is initially manifested as a disruption of Mu¨ller glial cell or

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bipolar cell function and later as a disruption of photoreceptor function. Electrophysiologic studies by Garner and colleagues (Garner and Lee, 1994; Garner et al., 1995a) in methanol-intoxicated (folate-deficient) rats further support a direct toxic effect of formate on Muller glial cell function and provide evidence that formate does not disrupt retinal synaptic transmission or bipolar cell function. Formate-induced retinal dysfunction can be produced within 48 hr by steadily increasing concentrations of formate and importantly can also be produced by prolonged exposure to lower concentrations of formate. Human studies have shown that prolonged exposure to methanol vapor concentrations exceeding 260 mg/m3 can produce headache and blurred vision, suggesting that visual system function may be disrupted by prolonged exposure to low concentrations of formate (Frederick et al., 1984; Kingsley and Hirsch, 1954; Andrews et al., 1987). In addition, Lee (1989) has presented evidence of ERG b-wave reductions in folate-deficient rats exposed to 800 ppm of methanol vapor for 90 days. Our findings substantiate these clinical and experimental observations and support the hypothesis that the visual system toxicity produced by acute, subacute, or chronic methanol poisoning share a common mechanism. Formate has been hypothesized to produce retinal and optic nerve toxicity by disrupting mitochondrial energy production (Martin-Amat et al., 1977, 1978; Sharpe et al., 1982; Eells, 1991). Formate has been shown in vitro to inhibit the activity of cytochrome oxidase, a vital component of the mitochondrial electron transport chain involved in ATP synthesis (Nicholls, 1975, 1976; Erecinska and Wilson, 1980). Inhibition occurs subsequent to the binding of formic acid with the ferric heme iron of cytochrome oxidase (Keyhani and Keyhani, 1980) and the apparent inhibition constant is between 5 and 30 mM (Nicholls, 1975, 1976). Furthermore, as pH decreases, cytochrome oxidase inhibition increases, indicating that the active inhibitor is undissociated formic acid, which can cross the inner mitochondrial membrane (Nicholls, 1976). The concentrations of formate present in the retina and vitreous humor of both treatment groups of methanol-intoxicated rats fall within this range, as do the concentrations of formate measured in the blood, vitreous humor, and CSF of methanol-poisoned humans and monkeys (McMartin et al., 1980; Sejersted et al., 1983; Martin-Amat et al., 1978; Eells, 1991). The retina is dependent on oxidative and glycolytic energy metabolism (Winkler, 1981; Ames et al., 1992; Steinberg, 1987). Inhibition of cytochrome oxidase activity by formate would be expected to profoundly disrupt retinal oxidative energy metabolism leading to neuronal dysfunction and neuronal damage. Under normal conditions, mitochondrial electron transport is coupled to oxidative phosphorylation (Siesjo, 1992). A reduction in the efficiency of the electron transport chain would reduce the synthesis of ATP, which

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is essential for active ion pumping to maintain the resting membrane potential, fast axoplasmic transport, and the synthesis of macromolecules and neurotransmitters. Maintenance of ionic homeostasis constitutes the major energyconsuming function of neurons, and in retinal photoreceptor cells cation pumping associated with the dark current accounts for nearly half of the oxygen consumption of the retina (Siesjo, 1992; Ames et al., 1992). Studies showing a reduction in retinal cytochrome oxidase activity (Eells et al., 1995) and retinal ATP concentrations (Eells et al., 1995; Garner et al., 1995a) in methanol-intoxicated rats strongly support the hypothesis that formate acts as a metabolic poison in the retina. Inhibition of aerobic respiration is also known to stimulate anaerobic glycolysis, resulting in increased lactate production and intra- and extracellular acidosis (Siesjo, 1992). Metabolic and lactic acidosis are both hallmark features of severe human methanol intoxication (Koivusalo, 1970; Jacobson and McMartin, 1986; Erlanson et al., 1965). The uncompensated metabolic acidosis produced in rats exposed to high concentrations of formic acid is likely to have accelerated the disruption of retinal energy metabolism because the undissociated formic acid is the active inhibitor of cytochrome oxidase. The pronounced attenuation of the ERG and histopathologic changes observed in the retinas of these animals agree with this interpretation. However, retinal dysfunction was produced in the absence of metabolic acidosis in rats exposed to low formate concentrations for a similar period of time, indicating that acidosis is not essential to the manifestation of formate toxicity. Inhibition of mitochondrial function in the retina and optic nerve is also consistent with the functional and morphologic findings in experimental and clinical methanol intoxication. The b-wave of the ERG has been shown to be extremely sensitive to conditions that interfere with retinal energy metabolism and is reduced or abolished following brief ischemia or the administration of metabolic poisons (Bresnick, 1989; Armington, 1986; Dowling, 1987), consistent with our findings in methanol-intoxicated rats (Eells, 1991; Murray et al., 1991; Eells et al., 1996) and in human methanol intoxication (Ruedemann, 1961; Eells et al., 1991; Murray et al., 1996). The most striking ultrastructural alteration observed in the retinas of methanol-intoxicated rats (Murray et al., 1991) and humans (Eells et al., 1991; Murray et al., 1996) exposed to high formate concentrations was vacuolation and mitochondrial swelling in inner segments of the photoreceptor cells and in the retinal pigment epithelium. Both these retinal cell types are highly metabolically active and a disruption of ion pumping and ionic homeostasis secondary to inhibition of cytochrome oxidase activity would be anticipated to produce such morphologic alterations (Ames et al., 1992; Steinberg, 1987; Tessier-Lavigne, 1991). Similar morphologic alterations have been reported in the

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retinas of patients with mitochondrial diseases that inhibit electron transport (McKechnie et al., 1985; Runge et al., 1986; McKelvie et al., 1991) and in certain forms of lightinduced retinal degeneration in which inactivation of cytochrome oxidase is postulated to play a role in the pathology (Lawwill, 1982; Rapp et al., 1990; Pautler et al., 1990). The apparent selective vulnerability of the retina and optic nerve to the neurotoxic actions of formate in methanol poisoning has been the subject of considerable speculation (Sharpe et al., 1982; Roe, 1955; Potts, 1955; Jacobson and McMartin, 1986). Although methanol intoxication is known to disrupt brain function and severe intoxication results in coma and death, the most common permanent sequela of methanol intoxication is blindness (Benton and Calhoun, 1952; Roe, 1955). Our studies indicate that one component of this selectivity may relate to the differences in the distribution of formate in the eye and the brain. Formate concentrations measured in the vitreous humor and retinas of methanol-intoxicated rats were equivalent to or greater than corresponding blood formate concentrations. In contrast, the concentrations of formate in the optic nerve and in the brain were significantly lower than blood formate concentrations. Similar distribution patterns have also been observed in methanol-intoxicated monkeys (J. Eells and T. Tephly, unpublished observations). These data suggest that the toxic actions of methanol on the visual system may be a consequence of the selective accumulation of formate in the vitreous humor and the retina compared with other regions of the central nervous system. The differential disposition of formate may be indicative of tissue-specific differences in formate and/or methanol oxidation. Preliminary in vitro studies in our laboratory indicate that formate oxidation occurs at a lower rate in the retina than in the brain (J. Eells et al., unpublished observations). Studies by Garner et al. (1995b) also suggest that the intraretinal metabolism of methanol to formate contributes significantly to formate-mediated retinal toxicity. A second predisposing factor may be the high degree of oxidative energy metabolism in the retina, especially at the inner segments of the photoreceptors (Ames et al., 1992; Wong-Riley, 1989; Steinberg, 1987). More metabolically active neurons respond earlier and to a greater extent to functional insult than less active neurons (Wong-Riley, 1989). In conclusion, our 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 poison-

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ing share a common mechanism. This mechanism involves the inhibition of retinal mitochondrial function and energy production by formic acid. ACKNOWLEDGMENTS The project described was supported by Grant ES06648 from the National Institute of Environmental Health Sciences, NIH, and by Grant EYO1931 from the National Eye Institute, NIH. The excellent technical assistance of Anna Fekete is appreciated.

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