haun 1952; Duke-Elder & Scott 1971; McMartin et al. 1980;. Roe 1982; Sharpe ... formaldehyde toxicity in methanol poisoning (Potts & John- son 1952; Cooper ...
C Pharmacology & Toxicology 2001, 89, 74–78. Printed in Denmark . All rights reserved
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Ocular Changes after Intravitreal Injection of Methanol, Formaldehyde, or Formate in Rabbits Yoriko Hayasaka, Seiji Hayasaka and Yasunori Nagaki Department of Ophthalmology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930–0194, Japan (Received November 15, 2000; Accepted May 2, 2001) Abstract: We evaluated the effects of intravitreal injection of methanol, formaldehyde, or formate on rabbit eyes. One hundred ml of 1% methanol, 1% or 0.1% formaldehyde, or 1% formate was injected in the vitreous cavity of the right eyes of rabbits. The eyes were examined by biomicroscopy and ophthalmoscopy weekly. One month after injection, the eyes were enucleated and examined histologically. One week after treatment the animals that received 0.1% formaldehyde showed retinal vessel dilation, and the rabbits that received 1% formaldehyde showed mild posterior subcapsular cataract and retinal vessel dilation and haemorrhages. One month after treatment, the animals that received 0.1% or 1% formaldehyde developed mild posterior subcapsular cataract and retinal lesions. Animals that received 1% methanol or 1% formate showed nearly normal optical media and fundi. Histologically disorganized retina and optic nerve were seen in eyes that received 0.1% or 1% formaldehyde. Eyes that received 1% methanol or 1% formate appeared histologically normal. Our findings indicate that intravitreal injection of formaldehyde causes retinal and optic nerve damage, while methanol and formate are not or less toxic to ocular tissues.
Methanol poisoning causes acute visual impairment, optic atrophy, coma, and sometimes death in man (Benton & Calhaun 1952; Duke-Elder & Scott 1971; McMartin et al. 1980; Roe 1982; Sharpe et al. 1982; Sejersted et al. 1983; Chen et al. 1985; Naeser 1988; Matsui et al. 1991; Hantson et al. 1999; Onder et al. 1999; Sharma et al. 1999). Ocular toxicity is thought to be due to the formation of toxic metabolites, formaldehyde or formate. Earlier investigators reported on formaldehyde toxicity in methanol poisoning (Potts & Johnson 1952; Cooper & Kini 1962; Kini et al. 1962). Conversely, recent researchers have proposed formate-induced ocular toxicity (Martin-Amat et al. 1978; McMartin et al. 1979; McMartin et al. 1980; Roe 1982; Sejersted et al. 1983; Seme et al. 1999). The mechanism by which methanol causes visual impairment is not yet clearly understood. Hantson et al. (1999) reported that in human methanol poisoning, reversible retinal dysfunction (tested by electroretinography) and optic neuropathy (examined by funduscopy and visual evoked potential) were correlated. In monkeys, alterations in the optic nerve heads were found after oral administration of methanol (Baumbach et al. 1977) or intravenous infusion of formate (Martin-Amat et al. 1978). However, no alteration of the retina was found in the monkeys (Baumbach et al. 1977; Martin-Amat et al. 1978). In folate-reduced rats, oral administration of methanol caused loss of b-wave on electroretinogram, but formate infusion Author for correspondence: Yoriko Hayasaka, M.D., Department of Ophthalmology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan (fax π81 76 436 0146, e-mail ophthal/ms.toyama-mpu.ac.jp).
did not alter the electroretinogram (Garner et al. 1995). Furthermore, a trace of formaldehyde was found in the vitreous of a methanol-poisoned patient (Benton & Calhaun 1952). To our knowledge, only a few histologic studies of intravitreal injection of formaldehyde and formate in animals have been reported namely Kini et al. (1962) who reported that 24 hr after intravitreal injection of formaldehyde, histological disorganization of the retina was produced in rabbit. Optic nerve atrophy is usually seen from 4 to 6 weeks after methanol poisoning in human (Roe 1982). We examined the effects of intravitreal injection of methanol, formaldehyde, or formate on ocular tissues in rabbits for one month.
Materials and Methods Animals. Twenty albino male rabbits, weighing 2.1–2.3 kg, were housed and maintained on a 12 hr light/dark cycle, with food and water available ad libitum. The experiment was performed according to the Guide for Care and Use of Laboratory Animals (Department of Health, Education and Welfare publication, NIH 80–23). Intravitreal injection. All animals were confirmed to have normal optical media and fundi prior to initiation of the study. Animals were divided into 5 groups: each group contained 4 rabbits. Methanol, formaldehyde, and formate were purchased from Wako Pure Chemicals (Tokyo) and were diluted in 25 mM sodium phosphate buffer (pH 7.4) – 0.8% NaCl solution. After the animals were anaesthetized with intravenous pentobarbital, 25 mg/kg, 0.5% phenylephrine and 0.4% oxybuprocaine were topically instilled in the right eye. Then, 100 ml of 1% methanol, 0.1% or 1% formaldehyde, or 1% formate was injected through the sclera 4 mm from the limbus into the central vitreous cavity of the right eye using a 27-gauge needle. The right eyes of the control animals received the vehicle,
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FORMALDEHYDE TOXICITY 0.1 ml, served as. After intravitreal injection, 0.3% ofloxacin was topically instilled. Systemic, biomicroscopic, and ophthalmoscopic examination. One day, two days, one week, two weeks, and one month after intravitreal injection, systemic conditions of the animals were inspected. After topical instillation of 0.5% phenylephrine, the eyes were examined by biomicroscopy and ophthalmoscopy. Histological study. One month after intravitreal injection, the animals were killed with an overdose of pentobarbital. The right eyeballs treated with the chemicals or vehicle were enucleated and were fixed in 10% formaldehyde for 5 min. Two windows formed by a 5 mm circumferential incision were made 4 mm from the limbus and fixed in 10% formaldehyde for 1 hr. A circumferential incision was made at the equator, and the posterior eye-cup was fixed in 10% formaldehyde for 6 hr. The tissues including the retina, choroid, and sclera temporal to the optic disc and the optic nerve behind the lamina cribrosa were dissected and fixed. Then, the tissues were processed and stained with haematoxylin and eosin for light microscopic examination.
Results No remarkable alterations in systemic conditions of the animals treated with the chemicals were noted, nor signs of pain and discomfort. The treated eyes of animals within the groups showed the same biomicroscopic and ophthaoscolmoscopic findings. No alterations were noted in the untreated eyes. One and two days after injection, mild postoperative inflammation was observed in the eyes that received the chemicals. One week after the injection, the animals treated with intravitreal injection of the vehicle, 1%
methanol, or 1% formate showed nearly normal optical media and fundi. Animals that received 0.1% formaldehyde showed retinal vessel dilation. Animals that received 1% formaldehyde showed mild posterior subcapsular cataract, retinal vessel dilation, and retinal haemorrhages. One month after injection, animals treated with vehicle (fig. 1a), 1% methanol (fig. 1b), or 1% formate (fig. 1e) showed the nearly normal optical media and fundi. Animals that recived 0.1% (fig. 1c) or 1% (fig. 1d) formaldehyde showed mild posterior subcapsular cataract, vessel dilation, and juxtapapillary retinal haemorrhages. Histopathologic study of eyes that received vehicle (fig. 2a), 1% methanol (fig. 2b), or 1% formate (fig. 2e) showed nearly normal retina. The ganglion cell layer and outer nuclear layer were disorganized in eyes that received 0.1% formaldehyde (fig. 2c). Markedly disorganized retina was observed in eyes that received 1% formaldehyde (fig. 2d). The optic nerve of eyes that received vehicle (fig. 3a), 1% methanol (fig. 3b), or 1% formate (fig. 3e) appeared to be nearly normal. The optic nerve of eyes that received 0.1% (fig. 3c) or 1% (fig. 3d) formaldehyde showed vacuolizations.
Discussion The most substantial effects observed in the present study were mild posterior subcapsular cataract, retinal lesions, and vacuolization of the optic nerve. It is possible that this
Fig. 1. Fundus photographs of eyes that received intravitreal injection of 100 ml of the chemical. a) vehicle. b) 1% methanol. c) 0.1% formaldehyde: Retinal vessel dilation and juxtapapillary haemorrhage (arrow) are seen. d) 1% formaldehyde: Retilnal vessel dilation and juxtapapillary haemorrhage (arrow) are visible. e) 1% formate: The fundus appears normal.
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Fig. 2. The retinas of eyes that received intravitreal injection of 100 ml of the chemical. a) vehicle. b) 1% methanol. c) 0.1% formaldehyde: The ganglion cell layer and inner nuclear layer are disorganized. d) 1% formaldehyde: The retinal structure is markedly disorganized. e) 1% formate: The retinal structure appears almost normal. INL, inner nuclear layer; ONL, outer nuclear layer (haematoxylin and eosin staining, original magnification ¿100).
type of effects usually appears as visual disturbances in the absence of pain. Development of methanol toxicity is reportedly different in species (Roe 1982): optic nerve atrophy occurs in man, while monkey, dog, rabbit, and rat are ordinarily resistant to visual impairment. Methanol-intoxicated monkeys showed optic disc swelling but no alteration in the retina (Baumbach et al. 1977). Nevertheless, methanol-induced retinotoxicity developed in folate-reduced rats (Garner & Lee 1994; Lee et al. 1994). It is likely that methanol-induced ocular lesions may develop in experimental animals under certain conditions. We therefore used rabbits in the present study. McMartin et al. (1979) reported that formate plays a predominant role as the major metabolic agent for methanol toxicity, using chromatropic acid method to assay formaldehyde in the biologic sample. Their assay method may be inadequate for formaldehyde determination in biologic samples, according to Matsumoto et al. (1990). McMartin et al. (1979) and Matsumoto et al. (1990) suspected that the inability to detect formaldehyde after methanol administration was probably due to its rapid metabolism to formate in the liver as well as in the blood. Not all methanol is necessarily metabolized in the liver. Metabolism of methanol in the rat retina has been suggested (Garner et al. 1995). Moore & Lee (1987) reported that methanol was metabolised to formaldehyde by azide-sensitive catalase in the eye. Therefore, it is possible that a small amount of
formaldehyde may be formed from methanol in vivo in human and rabbit retinas. In the present study, we injected 100 ml of 1% methanol, 0.1 or 1% formaldehyde, or 1% formate into the vitreous body. The weight of the rabbit’s vitreous body was about 1.4 g (1.4 ml). Therefore, the concentrations of 100 ml of 1% methanol, 0.1% and 1% formaldehyde, and 1% formate in the vitreous were calculated to be about 700 mg/ml (22 mM) methanol, 70 mg/ml (2.3 mM) and 700 mg/ml (23 mM) formaldehyde, and 700 mg/ml (15 mM) formate, respectively, in the present study. Jaanus & Bartlett (1984) reported that the amounts responsible for acute ocular symptoms varied from 4–500 ml of 40% methanol in man; which levels are equivalent to about 260 to 32,000 mg/ml of blood when the same doses of methanol are dissolved in 5,000 ml of blood. The high concentration of methanol (1,730 mg/ ml) in the post mortem vitreous body of a patient with methanol intoxication was also shown (Chen et al. 1985). Therefore, it is likely that the intravitreal concentration of methanol (700 mg/ml) in the present study may be within the range of the blood and tissue fluid levels in acute human intoxication. Kini et al. (1962) reported that intravitreal injection of formaldehyde (10 mM in the vitreous body) inhibited the incorporation of 32P into retinal phospholipids, and that those (20 mM in the vitreous body) caused degeneration of the ganglion cell layer after 24 hr. In our present study, the histologically disorganized retina and optic nerve were
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Fig. 3. Cross-sections of optic nerves of eyes that received intravitreal injection of 100 ml of the chemical. a) vehicle. b) 1% methanol. c) 0.1% formaldehyde: Vacuolizations are seen. d) 1% formaldehyde: Many vacuolizations are visible. e) 1% formate: The tissue structure is reserved (haematoxylin and eosin staining; original magnification ¿400).
shown one month after intravitreal injection of 0.1% or 1% formaldehyde (2.3 mM or 23 mM in the vitreous body). Formaldehyde is known to be an extremely reactive molecule, forming readily reversible adducts with nucleophiles in biological systems (Heck & Casanova-Schmitz 1984). It is likely that even a small amount of formaldehyde may be toxic in the retina. In the present study, 700 mg/ml of methanol (22 mM) and formate (15 mM) did not induce ocular lesion, while 70 mg/ml (2.3 mM) of formaldehyde in the vitreous body produced retinal and optic nerve lesions. It is likely that a small amount of formaldehyde may be formed in the ocular tissues and may be toxic, although formate accumulation may play a major role in methanol poisoning. References Baumbach, G. L., P. A. Cancilla, G. Martin-Amat, T. R. Tephly, K. E. McMartin, A. B. Makar & S. S. Hayreh: Methyl alcohol poisoning. IV. Alterations of the morphological findings of the retina and optic nerve. Arch. Ophthalmol. 1977, 95, 1859–1865. Benton, C. D. Jr. & F. P. Calhaun Jr.: The ocular effects of methyl alcohol poisoning. Report of a catastrophe involving three hundred and twenty persons. Trans. Acad. Ophthalmol. 1952, 56, 875–883. Chen, N. B., E. R. Donoghu & M. I. Schaffer: Methanol intoxication: Distribution in post mortem tissues and fluids including vitreous humor. J. Forens. Sci. 1985, 30, 213–216. Cooper, J. R. & M. M. Kini: Biochemical aspects of methanol poisoning. Biochem. Pharmacol. 1962, 11, 405–416. Dukeee-Elder, S. & G. I. Scott: Methyl alcohol. In System of ophthalmology, vol 12. Ed.: S. Duke-Elder. Henry Kimpton, London, 1971, pp. 159–166.
Garner, C. D. & Lee, E. W. 1994: Evaluation of methanol-induced retinotoxicity using oscillatory potential analysis. Toxicology 1994, 93, 113–124. Garner, C. D., E. W. Lee, T. S. Terzo & R. T. Louis-Ferdinand: Role of retinal metabolism in methanol-induced retinal toxicity. J. Toxicol. Environ. Health 1995, 44, 43–56. Hantson, P., M. de Tourtchaninoff, G. Simons, P. Mahieu, A. Boschi, C. Beguin & J. M. Guerit: Evoked potentials of visual function after methanol poisoning. Crit. Care Med. 1999, 27, 2707– 2715. Heck, H. & M. Casanova-Schmitz: Biochemical toxicology of formaldehyde. Rev. Biochem. Toxicol. 1984, 6, 155–189. Jaanus, S. D. & J. D. Bartlett: Adverse ocular effects of systemic drug therapy. In Clinical ocular pharmacology. Eds.: J. D. Bartlett & S. S. Jaanus. Butterworth, Boston, 1984, pp. 917–939. Kini, M. M., D. W. King Jr. & J. R. Cooper: Biochemistry of methanol poisoning. V. Histological and biochemical correlates of effects of methanol and its metabolites on the rabbit retina. J. Neurochem. 1962, 9, 119–124. Lee, E. W., C. D. Garner & T. S. Terzo: A rat model manifesting methanol-induced visual dysfunction suitable for both acute and long-term exposure studies. Toxicol. Appl. Pharmacol. 1994, 128, 199–206. Martin-Amat, G., K. E. McMartin, M. S. Hayreh & T. R. Tephly: Methanol poisoning. Ocular toxicity produced by formate. Toxicol. Appl. Pharmacol. 1978, 45, 201–208. Matsui, H., S. Noda, S., Hayasaka & T. Setogawa: A case of methanol intoxication presented glaucomatous cupping on the eleventh day. Jap. Rev. Clin. Ophthalmol 1991, 85, 2129–2131. Matsumoto, K., F. Moriya & R. Nanikawa: The movement of blood formaldehyde in methanol intoxication. II. The movement of blood formaldehyde and its metabolism in rabbit. Jap. J. Legal. Med. 1990, 44, 205–211. McMartin, K. E., G. Martin-Amat, P. E. Noker & T. R. Tephly: Lack of a role for formaldehyde in methanol poisoning in the monkey. Biochem. Pharmacol. 1979, 28, 645–649.
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Roe, O.: Species differences in methanol poisoning. CRC Crit. Rev. Toxicol. 1982, 10, 275–286. Sejersted, O. M., D. Jacobsen, S. Ovrebo & H. Jansen: Formate concentrations in plasma from patients poisoned with methanol. Acta Med. Scand. 1983, 213, 105–110. Seme, M. T., P. Summerfelt, M. M. Henry, J. Neitz & J. T. Eells: Formate-induced inhibition of photoreceptor function in methanol intoxication. J. Pharmacol. Exp. Therap. 1999, 289, 361– 370. Sharma, M., N. J. Volpe & E. B. Dreyer: Methanol-induced optic nerve cupping. Arch. Ophthalmol. 1999, 117, 286. Sharpe, J. A., M. Hostovsky, J. M. Bilbao & N. B. Rewcastle: Methanol optic neuropathy: A histopathological study. Neurology 1982, 32, 1093–1100.
