Bra;>i (1976), 99, 183-192
DEGENERATION IN CENTRAL AND PERIPHERAL NERVOUS SYSTEMS PRODUCED BY PURE n-HEXANE: AN EXPERIMENTAL STUDY by HERBERT H. SCHAUMBURG and PETER S. SPENCER
INTRODUCTION
THE industrial solvent n-hexane (CH3CH2CH2CH2CH2CH3) has been implicated as the principal agent responsible for the development of peripheral neuropathy in a variety of industrial workers and in individuals inhaling glue vapours for their euphoric properties (Yamada, 1964; Wada, Okanoto and Takagi, 1965; Sobue, Yamamura, Ando, Iida and Takayanagi, 1968; Yamamura, 1969; Iida, Yamamura and Sobue, 1969; Inoue, Takeuchi, Takeuchi, Yamada, Suzuki, Matsushita, Miyagaki, Maeda and Matsumoto, 1970; Herskowitz, Ishii and Schaumburg, 1971; Gonzalez and Downey, 1972; Matsumura, Inoue and Ohnishi, 1972; Gaultier, Rancurel, Pivaet and Efthymiou, 1973; Goto, Matsumura, Inoue, Murai, Shida, Santa and Kuroiwa, 1974; Shirabe, Tsuda, Terao and Araki, 1974; Korobkin, Asbury, Sumner and Nielsen, 1975). Each outbreak of peripheral neuropathy involved individuals who were exposed to mixed solvents of which n-hexane was only one major component, the others including compounds such as toluene, ethyl acetate and acetone. Because of the importance of n-hexane to industry, the present study systematically investigates the chronic effects of the pure compound on experimental animals, and reports for the first time the development of distinctive damage both in the central and in the peripheral nervous systems. MATERIAL AND METHODS Eleven adult Sprague-Dawley rats were used for this study. All were intoxicated with 99-98 per cent pure n-hexane. Three 400 g animals received a variety of increasing doses of n-hexane by subcutaneous injection, five days a week, for up to thirty-five weeks. The dosages were 650 mg/kg for a total period of ninety days and 2 g/kg for a further period of thirty-nine days (total = 98-7 g/kg). 650 mg/kg for ninety days followed by 980 mg/kg for ninety days (total = 102-8 g/kg), and 550 mg/kg for sixty-five days,
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
(From the Saul R. Korey Department of Neurology and the Department of Pathology (Neuropathology), Albert Einstein College of Medicine, The Bronx, New York, USA)
184
H. H. SCHAUMBURG AND P. S. SPENCER
11 g/kg for ninety days, plus 1-6 g/kg for ninety days (total = 198-8 g/kg). A second group of eight 200 g animals were exposed continuously to air containing 400-600 ppm /i-hexane for periods up to one hundred and sixty-two days. The n-hexane was volatilized by bubbling air through a flask containing 400 c.c of the compound at room temperature, and the animals were exposed in a 25-3 litre chamber made of wood and perspex. The atmospheric concentration of n-hexane in the chamber was monitored by a Bendix Gastec(R» gas sampler system and, on one occasion, by gas chromatography. Animals were examined periodically for neurological signs. After the period of intoxication, animals were anesthetized with sodium barbitone containing heparin, and perfused with 4 per cent paraformaldehyde followed by 5 per cent glutaraldehyde, each in a phosphate buffer (pH 7-4). Tissue was sampled as follows: from up to eight sites along the sciatic, tibial and plantar nerves in the hindlimb, the median nerve in the forelimb, lumbar and cervical spinal cord, medulla oblongata, pons, mesencephalon, optic nerves, olfactory bulb, frontal cortex, hypothalamus, posterior pituitary and cerebellum. In addition, gastrocnemius, soleus, interosseus and lumbrical muscles were sampled from the hindlimb.
RESULTS
Animals intoxicated by inhalation developed insidiously an unsteady, waddling gait after forty-five to sixty-nine days of exposure. Further exposure resulted in a progressive, symmetrical, distal hindlimb weakness with foot-drop (fig. 1).
FIG. 1. Animal with severe hindlimb weakness and foot-drop after one hundred and thirty days of inhalation exposure.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Tissue was postfixed in 2 per cent Dalton chrome osmium, dehydrated, immersed in propylene oxide and infiltrated with epoxy resin. Single nerve fibres were teased apart from tibial and plantar nerves and examined by light microscopy. Transverse 1 fjm sections, cut from embedded tissues, were stained with toluidine blue and examined by light microscopy. Thin sections of selected areas were stained with uranyl acetate and lead citrate, and examined by electronmicroscopy.
NEUROTOXIC ACTION OF /i-HEXANE
185
Severely affected animals also developed distal weakness of the upper extremities. The 3 animals intoxicated by repetitive subcutaneous injection exhibited no evidence of clinical neuropathy.
The first detectable abnormality in both the peripheral and central nervous systems was local axonal dilatation accompanied by a striking localized fibre swelling (fig. 2). Teased fibre preparations of peripheral nerves showed that the axonal swellings were tapered, discontinuous and had a scattered distribution
FIG. 2. Tibial nerve above popliteal fossa from animal intoxicated by inhalation for one hundred and sixty-two days. Note swollen axons (upper right) and perivascular oedema (lower right). Transverse 1 ^m epon section stained with toluidine blue, x 320.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Pathological changes were detected in the peripheral nervous system in 2 of the 3 animals intoxicated by subcutaneous injection, and in the peripheral and central nervous systems of the 4 animals which were exposed for more than forty-two days in the inhalation chamber. There was a consistent distribution of abnormality of the peripheral and central nervous systems. Degeneration of peripheral myelinated fibres was most prominent in the tibial nerve branches bound for the gastrocnemius and soleus muscles. By contrast, abnormalities were relatively unusual in the intramuscular nerves within these muscles, adjacent levels of the tibial, sural and peroneal nerves, and distally in plantar nerves, interosseous and lumbrical muscles.
186
H. H. S C H A U M B U R G AND P. S. SPENCER
(fig. 3). The myelin sheath overlying the axonal swellings was often abnormally thin. Nerve fibres from animals with neuropathy were in a more advanced stage of degeneration and were often composed of a chain of myelin ovoids. Perivascular oedema was present within the endoneurium of the most heavily affected peripheral nerve fascicles and there were occasional vacuoles within the capillary endothelium. Inspection of the central nervous system (CNS) revealed degeneration of myelinated fibres in the ventromedial and peripheral ventrolateral tracts of the lumbosacral spinal cord, the gracile nuclei, the ventromedial medulla adjacent to the hypoglossal nerves (fig. 4), the perimeter of the ventrolateral quadrants of the medulla, the inferior cerebellar peduncles and in the white matter of the cerebellar vermis.
DISCUSSION
We have demonstrated degeneration of the central and peripheral nervous systems in rats exposed to pure w-hexane administered either by continuous inhalation or by daily subcutaneous injections. This finding demonstrates that n-hexane is a neurotoxin and adds strong support to the previous contentions thatrc-hexanewas the agent responsible for outbreaks of peripheral neuropathy both in industry and in individuals inhaling glue vapours (Yamada, 1964; Wada et al., 1965; Sobue et al., 1968; Yamamura, 1969; Iida et al, 1969; Inoue et al.,
FIG. 3. Single myelinated fibres dissected from posterior tibial nerve of animal intoxicated by inhalation for one hundred and sixty-two days. Note multiple paranodal and intemodal axonal swellings and distal paranodal demyelination (arrow). Fixed with buffered glutaraldehyde and osmium tetroxide, teased in epoxy resin, x 140. FIG. 4. Ventral medulla adjacent to exit of hypoglossal nerve from rat after forty-nine days of inhalation exposure. Swollen myelinated axons (a) are apparent. Transverse 1 fim epon section stained with toluidine blue. x510. FIG. 5. Myelinated axon containing abnormally large numbers of neurofilaments. Electronmicrograph of transverse thin section of the tibial nerve in thigh of rat intoxicated by inhalation for forty-nine days, x 12000.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Electronmicroscope examination of the swollen regions of central and peripheral myelinated axons revealed densely packed, whorled masses of 10 nm neurofilaments (fig. 5). Groups of mitochondria and neurotubules were displaced to the periphery of the axon or were segregated into bundles. Adjacent, unswollen regions of these axons appeared normal although there was an abnormal frequency of adaxonal Schwann cell/axon profiles. These are believed to be evidence of phagocytosis by Schwann cells of abnormal axonal organelles (Spencer and Thomas, 1974). Unmyelinated fibres occasionally appeared abnormal with prominent accumulations of neurofilaments in one or two axons.
187 NEUROTOXIC ACTION OF n-HEXANE
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
188
H. H. S C H A U M B U R G A N D P. S. SPENCER
1970; Herskowitz et al., 1971; Gonzalez and Downey, 1972; Matsumura et al., 1972; Gaultier et al., 1973; Goto et al., 1974; Shirabe et al., 1974; Korobkin et al., 1975).
The clinical findings of distal, symmetrical weakness and the predominantly distal distribution of the changes in the peripheral nerve fibres conform to the pattern of axonal degeneration seen in dying-back disease (Cavanagh, 1964). In contrast to the distribution of changes commonly associated with dying-back neuropathies, where the most distal tips of the largest and longest axons are most vulnerable, n-hexane affects the proximal nerve fibres of the calf muscles before it affects the distal regions of the much longer nerve fibres supplying the hindfeet. A similar distribution of giant axonal swellings has been described in rats intoxicated with 2,5-hexanedione, a major metabolite of the neurotoxic industrial solvent MBK (Spencer and Schaumburg, 1975a). That «-hexane (CH3CH2CH2CH2CH2CH3) is metabolized to 2,5-hexanedione (CH3CO CH2CH2CO CH3) before exerting its neurotoxic effect, as has been suggested for MBK (CH3CO CH2CH2CH2CH3) (Spencer and Schaumburg, 1975a) is a possibility under investigation {see Addendum). A cardinal feature of murine n-hexane neuropathy was the widespread axonal degeneration found in the central nervous system. Similar giant axonal changes have been described in experimental MBK and 2,5-hexanedione neuropathies
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Giant axonal swelling was a consistent early feature of the degenerative process in selected areas of the peripheral nervous system, and appeared to result from focal accumulations of large numbers of 10 nm neurofilaments. This axonal abnormality was not described in previous studies of experimental n-hexane intoxication (Kurita, 1967; Miyagaki, 1967; Truhaut, Laget, Piat, Phu-Lich, Dutertre-Catella and Huyen, 1973) but was seen in nerve biopsies from patients inhaling glue vapours containing n-hexane mixtures (Goto et al., 1974; Korobkin et al., 1975). While the basic mechanism responsible for these accumulations is not known, similar multifocal giant axonal changes have been described in the peripheral nervous system in clinical and experimental methyl n-butyl ketone (MBK) neuropathy (Mendell et al., 1974; Allen, Mendell, Billmaier, Fontaine and O'Neill, 1975; Spencer, Schaumburg, Raleigh and Terhaar, 1975), carbon disulphide intoxication (Haltia, Linnoila, Seppalainen and Palo, 1974), experimental 2,5-hexanedione intoxication (Spencer and Schaumburg, 19756) and genetically determined human giant axonal neuropathy (Asbury, Gale, Cox, Baringer and Berg, 1972; Carpenter, Karpati, Andermann and Gold, 1974; Igisu, Ohta, Tabira, Hosokawa, Goto and Kuroiwa, 1975). Multifocal accumulations of neurofilaments accompanied by less striking axonal swellings have also been described and illustrated in experimental acrylamide neuropathy (Schaumburg, Wisniewski and Spencer, 1974; Spencer and Schaumburg, 19746).
NEUROTOXIC ACTION OF n-HEXANE
189
Current interest in the toxic properties of n-hexane is related to attempts to regulate air pollutants in industry and in the environment. The latest available statistics state that 386 542 000 lb of w-hexane and other C6 hydrocarbons were produced in the USA in 1973 from distillation of crude petroleum (US International Trade Commission, 1973). n-Hexane is used with other C6 hexacarbons to provide an excellent and inexpensive industrial solvent. The British and US Threshold Limit Value (TLV) of 500 ppm was calculated from the results of several acute toxicity trials with man and experimental animals. Studies in man found dizziness and giddiness after ten minutes of exposure to 5 000 ppm w-hexane and none of these symptoms after a similar exposure to 2 000 ppm. Nausea, headache and irritation of eyes and throat have been reported at 1 400-1 500 ppm and no irritation reported at 500 ppm in unacclimated subjects. The 500 ppm limit was set to provide freedom from these acute, reversible central nervous effects {see American Conference of Governmental Industrial Hygienists, 1971). The present study demonstrates that rats exposed to the human TLV for prolonged periods develop structural damage in the CNS. Although continuous intoxication of animals was used in this study, the results emphasize the importance of chronic toxicity testing and the need to
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
(Spencer et al., 1975; Spencer and Schaumburg, 19756). That such changes occur concomitantly with degeneration of peripheral nerves in toxic neuropathies is a characteristic finding and represents the expression of dying-back axonal disease in the CNS. There is every reason to believe a priori that similar degeneration.of axons in the CNS would accompany human neuropathies produced by toxic chemicals, including n-hexane. However, the dysfunction of peripheral nerve in affected individuals probably masks the signs which accompany degeneration of the CNS, a clinical paradox which sometimes occurs in patients with conditions such as spinocerebellar degeneration or motor neuron disease. Individuals who developed neuropathy following prolonged exposure to n-hexane have a stereotyped clinical picture of distal areflexia, flaccid weakness and hypaesthesia. After removal from the toxic environment, they eventually recover from the neuropathy presumably because of efficacious regeneration of the damaged peripheral nerves. Since equivalent recovery from lesions within the CNS is unlikely to occur, one might anticipate the appearance of signs associated with damage to the dorsal columns (permanent sensory loss), spinocerebellar tracts (ataxia) and descending motor tracts (spasticity). This consideration might explain the development of hyperactive deep tendon reflexes and a 'spastic catch' in the lower limbs of a patient after a seventeen-month period of recovery from glue-sniffing neuropathy, where the agent responsible was probably n-hexane (Korobkin et al., 1975). In addition, the clinical evidence of central nervous dysfunction in human non-toxic giant axonal neuropathy (Igisu et al., 1975) and in human and experimental acrylamide intoxication (Schaumburg et al., 1974; Spencer and Schaumburg, 1974a; Igisu et al., 1975) may reflect a distribution of central axonal disease similar to that found in this experimental study.
190
H. H. SCHAUMBURG A N D P. S. SPENCER
lower the TLV forrc-hexane,as has been done in Japan where the current TLV is 100 ppm. SUMMARY
ADDENDUM G. D. DiVincenzo, C. J. Kaplan and J. Dedinas (Toxicology and Applied Pharmacology. In press) have demonstrated 2,5-hexanedione and other metabolites in serum of guinea-pigs after intraperitoneal administration of a single dose of n-hexane.
ACKNOWLEDGEMENTS This work was supported by USPHS grants NS 08952, OH 00535 and NS 03356. Dr. Spencer is the recipient of a Joseph P. Kennedy, Jun., Fellowship in the Neurosciences.
REFERENCES ALLEN, N., MENDELL, J. R., BILLMAIER, D. J., FONTAINE, R. E., and O'NEILL, J. (1975) Toxic
polyneuropathy due to methyl n-butyl ketone. Archives of Neurology, Chicago, 32, 209-213. AMERICAN CONFERENCE OF GOVERNMENTAL INDUSTRIAL HYGIENISTS (1971) Documentation of Threshold
Limit Values for Substances in Workroom Air. 3rd edition: A.C.G.H.I., p. 126. ASBURY, A. K., GALE, M. K., COX, S. C , BARINGER, J. R., and BERG, B. O. (1972) Giant axonal
neuropathy: A unique case with segmental neurofilamentous masses. Acta Neuropathologica, 20, 237-247. CARPENTER, S., KARPATI, G., ANDERMANN, F., and GOLD, M. B. (1974) Giant axonal neuropathy.
Archives of Neurology, Chicago, 31, 312-316. CAVANAGH, J. B. (1964) The significance of the 'dying-back' process in experimental and human neurological disease. International Review of Experimental Pathology, 3, 219-267. GAULTTER, M., RANCUREL, G., PIVAET, C , and EFTHYMIOU, M.-L. (1973) Polyn6vrites et hydrocarbures
aliphatiques. European Journal of Toxicology, 6, 294-296.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Rats intoxicated with pure «-hexane, either by repetitive subcutaneous injection or by continuous inhalation, developed clinical and/or pathological evidence of peripheral neuropathy. Animals intoxicated by inhalation (400-600 ppm) developed clinical signs after forty-five days and displayed giant axonal swellings and fibre degeneration both in the central and peripheral nervous systems. The changes were most striking in tibial nerves supplying calf muscles and in selected areas of the cerebellum, medulla and spinal cord. In contrast to the usual picture associated with dying-back disease, the distal regions of proximal nerve fibres supplying calf muscles degenerated before equivalent regions of longer fibres supplying the hindfeet. The relevance of the central nervous changes to individuals recovering from toxic neuropathies and the need for a reduction of the present Threshold Limit Value (500 ppm) for human exposure are discussed.
NEUROTOXIC ACTION OF n-HEXANE
191
GONZALEZ, E., and DOWNEY, J. (1972) Polyneuropathy in a glue sniffer. Archives of Physical Medicine, 53, 333-337. GOTO, I., MATSUMURA, M., INOUE, N., MURAI, Y., SHIDA, K., SANTA, T., and KUROIWA, Y. (1974)
Toxic polyneuropathy due to glue sniffing. Journal of Neurology, Neurosurgery and Psychiatry, 37, 848-853. HALTIA, M., LINNOILA, I., SEPPALAINEN, A. M., and PALO, J. (1974) Experimental carbon disulphide
poisoning: morphological and neurophysiological studies. In: Proceedings VII International Congress of Neuropathology. Edited by St. Kornyey, St. Tauskand and G. Gosztonyi. Amsterdam: Excerpta Medica. HERSKOWITZ, A., ISHn, N., and SCHAUMBURG, H. (1971) n-Hexane neuropathy: A syndrome occurring as a result of industrial exposure. New England Journal of Medicine, 285, 82-85. IGISU, H., GOTO, I., KAWAMURA, Y., KATO, M., lzum,
K., and KUROIWA, Y. (1975) Acrylamide
encephaloneuropathy due to well water pollution. Journal of Neurology, Neurosurgery and Psychiatry, 38, 581-584. OHTA, M., TABIRA, T., HOSOKAWA, S., GOTO, I., and KUROIWA, Y. (1975) Giant axonal
INOUE, T., TAKEUCHI, Y., TAKEUCM, S., YAMADA, S., SUZUKI, H., MATSUSHITA, T., MIYAGAKI, H.,
MAEDA, K., and MATSUMOTO, T. (1970) A health survey on vinyl sandal manufacturers with high incidence of n-hexane intoxication. Japanese Journal of Industrial Health, 12, 73-84. KOROBKIN, R., ASBURY, A. K., SUMNER, A. J., and NIELSEN, S. L. (1975) Glue-sniffing neuropathy.
Archives of Neurology, Chicago, 32, 158-162. KURTTA, H. (1967) Experimental studies of n-hexane to albino rats. Japanese Journal of Industrial Health, 9, 672-677. MATSUMURA, M., INOUE, N., and OHNISHI, A. (1972) Toxic polyneuropathy due to glue sniffing. Clinical Neurology, 12, 290-296. MENDELL, J. R., SAIDA, K., GANANSIA, M. F., JACKSON, D. B., WEISS, H., GARDIER, R. W., CHRISMAN, C ,
ALLEN, N., COURI, D., O'NEILL, J., MASKA, B., and HETLAND, L. (1974) Toxic polyneuropathy
produced by methyl n-butyl ketone. Science, 185, 787-789. MIYAGAKI, H. (1967) An electrophysiological study on the toxicity of n-hexane on the peripheral nerve. Japanese Journal of Industrial Health, 9, 660-671. SCHAUMBURG, H. H., WISNIEWSKL, H. M., and SPENCER, P. S. (1974) Ultrastructural studies of the
dying-back process. 1. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication. Journal of Neuropathology and Experimental Neurology, 33, 260-284. SHIRABE, T., TSUDA, T., TERAO, A., and ARAKI, S. (1974) Toxic polyneuropathy due to glue-sniffing.
Journal of the Neurological Sciences, 21, 101-113. SOBUE, I., YAMAMURA, Y., ANDO, K., IIDA, M., and TAKAYANAGI, T. (1968) n-Hexane polyneuropathy.
Outbreak among vinyl sandal manufacturers. Clinical Neurology, 8, 393-403. SPENCER, P. S., and SCHAUMBURG, H. H. (1974a) A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure. Canadian Journal of Neurological Sciences, 1, 143-150. (19746) A review of acrylamide neurotoxicity. Part II. Experimental animal neurotoxicity and pathologic mechanisms. Canadian Journal of Neurological Sciences, 1, 151-169. (1975a) Nervous system dying-back disease produced by 2,5-hexanedione. Transactions of the American Neurological Association, 100, 148-151. (19756) Experimental neuropathy produced by 2,5-hexanedione—a major metabolite of the neurotoxic industrial solvent methyl n-butyl ketone. Journal of Neurology, Neurosurgery and Psychiatry, 38, 771-775. RALEIGH, R. L., and TERHAAR, C.J. (1975) Nervous system degeneration produced by the industrial solvent methyl n-butyl ketone. Archives of Neurology, Chicago, 32, 219-222.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
neuropathy: A clinical entity affecting the central as well as the peripheral nervous system. Neurology, Minneapolis, 25, 717-721. IIDA, M., YAMAMURA, Y., and SOBUE, I. (1969) Electromyographic findings and conduction velocity in n-hexane polyneuropathy. Electromyography, 9, 247-261.
192
H. H. SCHAUMBURG A N D P. S. SPENCER
SPENCER, P. S., and THOMAS, P. K. (1974) Ultrastructural studies of the dying-back process: II. The sequestration and removal by Schwann cells and oligodendrocytes of organelles from normal and diseased axons. Journal of Neurocytology, 3, 763-783. TRUHAUT, R., LAGET, P., PIAT, G., PHU-LICH, N., DUTERTRE-CATELLA, H., and HUYEN, V. N. (1973)
Premiers resultats electrophysiologiques apres intoxications experimentales par l'hexane et par l'heptane techniques chez le rat blanc. Archives de maladies professionelles de midecine du travail et de stcuritt sociale, 34, 417-426. UNITED STATES INTERNATIONAL TRADE COMMISSION (1973) Synthetic Organic Chemicals (Publication
No. 728). Washington: US Government Printing Office. WADA, Y., OKANOTO, S., and TAKAGI, S. (1965) Intoxication polyneuropathy following exposure to n-hexane. Clinical Neurology, 5, 591-597. YAMADA, A. (1964) An occurrence of polyneuritis by n-hexane in the polyethylene laminating plants. Japanese Journal of Industrial Health, 6, 192-197. YAMAMURA, Y. (1969) n-Hexane polyneuropathy. Folia Psychiatria Neurologica Japonica, 23, 45-57. Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
{Received September 16, 1975)
DEGENERATION IN CENTRAL AND PERIPHERAL NERVOUS SYSTEMS PRODUCED BY PURE n-HEXANE: AN EXPERIMENTAL STUDY by HERBERT H. SCHAUMBURG and PETER S. SPENCER
INTRODUCTION
THE industrial solvent n-hexane (CH3CH2CH2CH2CH2CH3) has been implicated as the principal agent responsible for the development of peripheral neuropathy in a variety of industrial workers and in individuals inhaling glue vapours for their euphoric properties (Yamada, 1964; Wada, Okanoto and Takagi, 1965; Sobue, Yamamura, Ando, Iida and Takayanagi, 1968; Yamamura, 1969; Iida, Yamamura and Sobue, 1969; Inoue, Takeuchi, Takeuchi, Yamada, Suzuki, Matsushita, Miyagaki, Maeda and Matsumoto, 1970; Herskowitz, Ishii and Schaumburg, 1971; Gonzalez and Downey, 1972; Matsumura, Inoue and Ohnishi, 1972; Gaultier, Rancurel, Pivaet and Efthymiou, 1973; Goto, Matsumura, Inoue, Murai, Shida, Santa and Kuroiwa, 1974; Shirabe, Tsuda, Terao and Araki, 1974; Korobkin, Asbury, Sumner and Nielsen, 1975). Each outbreak of peripheral neuropathy involved individuals who were exposed to mixed solvents of which n-hexane was only one major component, the others including compounds such as toluene, ethyl acetate and acetone. Because of the importance of n-hexane to industry, the present study systematically investigates the chronic effects of the pure compound on experimental animals, and reports for the first time the development of distinctive damage both in the central and in the peripheral nervous systems. MATERIAL AND METHODS Eleven adult Sprague-Dawley rats were used for this study. All were intoxicated with 99-98 per cent pure n-hexane. Three 400 g animals received a variety of increasing doses of n-hexane by subcutaneous injection, five days a week, for up to thirty-five weeks. The dosages were 650 mg/kg for a total period of ninety days and 2 g/kg for a further period of thirty-nine days (total = 98-7 g/kg). 650 mg/kg for ninety days followed by 980 mg/kg for ninety days (total = 102-8 g/kg), and 550 mg/kg for sixty-five days,
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
(From the Saul R. Korey Department of Neurology and the Department of Pathology (Neuropathology), Albert Einstein College of Medicine, The Bronx, New York, USA)
184
H. H. SCHAUMBURG AND P. S. SPENCER
11 g/kg for ninety days, plus 1-6 g/kg for ninety days (total = 198-8 g/kg). A second group of eight 200 g animals were exposed continuously to air containing 400-600 ppm /i-hexane for periods up to one hundred and sixty-two days. The n-hexane was volatilized by bubbling air through a flask containing 400 c.c of the compound at room temperature, and the animals were exposed in a 25-3 litre chamber made of wood and perspex. The atmospheric concentration of n-hexane in the chamber was monitored by a Bendix Gastec(R» gas sampler system and, on one occasion, by gas chromatography. Animals were examined periodically for neurological signs. After the period of intoxication, animals were anesthetized with sodium barbitone containing heparin, and perfused with 4 per cent paraformaldehyde followed by 5 per cent glutaraldehyde, each in a phosphate buffer (pH 7-4). Tissue was sampled as follows: from up to eight sites along the sciatic, tibial and plantar nerves in the hindlimb, the median nerve in the forelimb, lumbar and cervical spinal cord, medulla oblongata, pons, mesencephalon, optic nerves, olfactory bulb, frontal cortex, hypothalamus, posterior pituitary and cerebellum. In addition, gastrocnemius, soleus, interosseus and lumbrical muscles were sampled from the hindlimb.
RESULTS
Animals intoxicated by inhalation developed insidiously an unsteady, waddling gait after forty-five to sixty-nine days of exposure. Further exposure resulted in a progressive, symmetrical, distal hindlimb weakness with foot-drop (fig. 1).
FIG. 1. Animal with severe hindlimb weakness and foot-drop after one hundred and thirty days of inhalation exposure.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Tissue was postfixed in 2 per cent Dalton chrome osmium, dehydrated, immersed in propylene oxide and infiltrated with epoxy resin. Single nerve fibres were teased apart from tibial and plantar nerves and examined by light microscopy. Transverse 1 fjm sections, cut from embedded tissues, were stained with toluidine blue and examined by light microscopy. Thin sections of selected areas were stained with uranyl acetate and lead citrate, and examined by electronmicroscopy.
NEUROTOXIC ACTION OF /i-HEXANE
185
Severely affected animals also developed distal weakness of the upper extremities. The 3 animals intoxicated by repetitive subcutaneous injection exhibited no evidence of clinical neuropathy.
The first detectable abnormality in both the peripheral and central nervous systems was local axonal dilatation accompanied by a striking localized fibre swelling (fig. 2). Teased fibre preparations of peripheral nerves showed that the axonal swellings were tapered, discontinuous and had a scattered distribution
FIG. 2. Tibial nerve above popliteal fossa from animal intoxicated by inhalation for one hundred and sixty-two days. Note swollen axons (upper right) and perivascular oedema (lower right). Transverse 1 ^m epon section stained with toluidine blue, x 320.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Pathological changes were detected in the peripheral nervous system in 2 of the 3 animals intoxicated by subcutaneous injection, and in the peripheral and central nervous systems of the 4 animals which were exposed for more than forty-two days in the inhalation chamber. There was a consistent distribution of abnormality of the peripheral and central nervous systems. Degeneration of peripheral myelinated fibres was most prominent in the tibial nerve branches bound for the gastrocnemius and soleus muscles. By contrast, abnormalities were relatively unusual in the intramuscular nerves within these muscles, adjacent levels of the tibial, sural and peroneal nerves, and distally in plantar nerves, interosseous and lumbrical muscles.
186
H. H. S C H A U M B U R G AND P. S. SPENCER
(fig. 3). The myelin sheath overlying the axonal swellings was often abnormally thin. Nerve fibres from animals with neuropathy were in a more advanced stage of degeneration and were often composed of a chain of myelin ovoids. Perivascular oedema was present within the endoneurium of the most heavily affected peripheral nerve fascicles and there were occasional vacuoles within the capillary endothelium. Inspection of the central nervous system (CNS) revealed degeneration of myelinated fibres in the ventromedial and peripheral ventrolateral tracts of the lumbosacral spinal cord, the gracile nuclei, the ventromedial medulla adjacent to the hypoglossal nerves (fig. 4), the perimeter of the ventrolateral quadrants of the medulla, the inferior cerebellar peduncles and in the white matter of the cerebellar vermis.
DISCUSSION
We have demonstrated degeneration of the central and peripheral nervous systems in rats exposed to pure w-hexane administered either by continuous inhalation or by daily subcutaneous injections. This finding demonstrates that n-hexane is a neurotoxin and adds strong support to the previous contentions thatrc-hexanewas the agent responsible for outbreaks of peripheral neuropathy both in industry and in individuals inhaling glue vapours (Yamada, 1964; Wada et al., 1965; Sobue et al., 1968; Yamamura, 1969; Iida et al, 1969; Inoue et al.,
FIG. 3. Single myelinated fibres dissected from posterior tibial nerve of animal intoxicated by inhalation for one hundred and sixty-two days. Note multiple paranodal and intemodal axonal swellings and distal paranodal demyelination (arrow). Fixed with buffered glutaraldehyde and osmium tetroxide, teased in epoxy resin, x 140. FIG. 4. Ventral medulla adjacent to exit of hypoglossal nerve from rat after forty-nine days of inhalation exposure. Swollen myelinated axons (a) are apparent. Transverse 1 fim epon section stained with toluidine blue. x510. FIG. 5. Myelinated axon containing abnormally large numbers of neurofilaments. Electronmicrograph of transverse thin section of the tibial nerve in thigh of rat intoxicated by inhalation for forty-nine days, x 12000.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Electronmicroscope examination of the swollen regions of central and peripheral myelinated axons revealed densely packed, whorled masses of 10 nm neurofilaments (fig. 5). Groups of mitochondria and neurotubules were displaced to the periphery of the axon or were segregated into bundles. Adjacent, unswollen regions of these axons appeared normal although there was an abnormal frequency of adaxonal Schwann cell/axon profiles. These are believed to be evidence of phagocytosis by Schwann cells of abnormal axonal organelles (Spencer and Thomas, 1974). Unmyelinated fibres occasionally appeared abnormal with prominent accumulations of neurofilaments in one or two axons.
187 NEUROTOXIC ACTION OF n-HEXANE
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
188
H. H. S C H A U M B U R G A N D P. S. SPENCER
1970; Herskowitz et al., 1971; Gonzalez and Downey, 1972; Matsumura et al., 1972; Gaultier et al., 1973; Goto et al., 1974; Shirabe et al., 1974; Korobkin et al., 1975).
The clinical findings of distal, symmetrical weakness and the predominantly distal distribution of the changes in the peripheral nerve fibres conform to the pattern of axonal degeneration seen in dying-back disease (Cavanagh, 1964). In contrast to the distribution of changes commonly associated with dying-back neuropathies, where the most distal tips of the largest and longest axons are most vulnerable, n-hexane affects the proximal nerve fibres of the calf muscles before it affects the distal regions of the much longer nerve fibres supplying the hindfeet. A similar distribution of giant axonal swellings has been described in rats intoxicated with 2,5-hexanedione, a major metabolite of the neurotoxic industrial solvent MBK (Spencer and Schaumburg, 1975a). That «-hexane (CH3CH2CH2CH2CH2CH3) is metabolized to 2,5-hexanedione (CH3CO CH2CH2CO CH3) before exerting its neurotoxic effect, as has been suggested for MBK (CH3CO CH2CH2CH2CH3) (Spencer and Schaumburg, 1975a) is a possibility under investigation {see Addendum). A cardinal feature of murine n-hexane neuropathy was the widespread axonal degeneration found in the central nervous system. Similar giant axonal changes have been described in experimental MBK and 2,5-hexanedione neuropathies
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Giant axonal swelling was a consistent early feature of the degenerative process in selected areas of the peripheral nervous system, and appeared to result from focal accumulations of large numbers of 10 nm neurofilaments. This axonal abnormality was not described in previous studies of experimental n-hexane intoxication (Kurita, 1967; Miyagaki, 1967; Truhaut, Laget, Piat, Phu-Lich, Dutertre-Catella and Huyen, 1973) but was seen in nerve biopsies from patients inhaling glue vapours containing n-hexane mixtures (Goto et al., 1974; Korobkin et al., 1975). While the basic mechanism responsible for these accumulations is not known, similar multifocal giant axonal changes have been described in the peripheral nervous system in clinical and experimental methyl n-butyl ketone (MBK) neuropathy (Mendell et al., 1974; Allen, Mendell, Billmaier, Fontaine and O'Neill, 1975; Spencer, Schaumburg, Raleigh and Terhaar, 1975), carbon disulphide intoxication (Haltia, Linnoila, Seppalainen and Palo, 1974), experimental 2,5-hexanedione intoxication (Spencer and Schaumburg, 19756) and genetically determined human giant axonal neuropathy (Asbury, Gale, Cox, Baringer and Berg, 1972; Carpenter, Karpati, Andermann and Gold, 1974; Igisu, Ohta, Tabira, Hosokawa, Goto and Kuroiwa, 1975). Multifocal accumulations of neurofilaments accompanied by less striking axonal swellings have also been described and illustrated in experimental acrylamide neuropathy (Schaumburg, Wisniewski and Spencer, 1974; Spencer and Schaumburg, 19746).
NEUROTOXIC ACTION OF n-HEXANE
189
Current interest in the toxic properties of n-hexane is related to attempts to regulate air pollutants in industry and in the environment. The latest available statistics state that 386 542 000 lb of w-hexane and other C6 hydrocarbons were produced in the USA in 1973 from distillation of crude petroleum (US International Trade Commission, 1973). n-Hexane is used with other C6 hexacarbons to provide an excellent and inexpensive industrial solvent. The British and US Threshold Limit Value (TLV) of 500 ppm was calculated from the results of several acute toxicity trials with man and experimental animals. Studies in man found dizziness and giddiness after ten minutes of exposure to 5 000 ppm w-hexane and none of these symptoms after a similar exposure to 2 000 ppm. Nausea, headache and irritation of eyes and throat have been reported at 1 400-1 500 ppm and no irritation reported at 500 ppm in unacclimated subjects. The 500 ppm limit was set to provide freedom from these acute, reversible central nervous effects {see American Conference of Governmental Industrial Hygienists, 1971). The present study demonstrates that rats exposed to the human TLV for prolonged periods develop structural damage in the CNS. Although continuous intoxication of animals was used in this study, the results emphasize the importance of chronic toxicity testing and the need to
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
(Spencer et al., 1975; Spencer and Schaumburg, 19756). That such changes occur concomitantly with degeneration of peripheral nerves in toxic neuropathies is a characteristic finding and represents the expression of dying-back axonal disease in the CNS. There is every reason to believe a priori that similar degeneration.of axons in the CNS would accompany human neuropathies produced by toxic chemicals, including n-hexane. However, the dysfunction of peripheral nerve in affected individuals probably masks the signs which accompany degeneration of the CNS, a clinical paradox which sometimes occurs in patients with conditions such as spinocerebellar degeneration or motor neuron disease. Individuals who developed neuropathy following prolonged exposure to n-hexane have a stereotyped clinical picture of distal areflexia, flaccid weakness and hypaesthesia. After removal from the toxic environment, they eventually recover from the neuropathy presumably because of efficacious regeneration of the damaged peripheral nerves. Since equivalent recovery from lesions within the CNS is unlikely to occur, one might anticipate the appearance of signs associated with damage to the dorsal columns (permanent sensory loss), spinocerebellar tracts (ataxia) and descending motor tracts (spasticity). This consideration might explain the development of hyperactive deep tendon reflexes and a 'spastic catch' in the lower limbs of a patient after a seventeen-month period of recovery from glue-sniffing neuropathy, where the agent responsible was probably n-hexane (Korobkin et al., 1975). In addition, the clinical evidence of central nervous dysfunction in human non-toxic giant axonal neuropathy (Igisu et al., 1975) and in human and experimental acrylamide intoxication (Schaumburg et al., 1974; Spencer and Schaumburg, 1974a; Igisu et al., 1975) may reflect a distribution of central axonal disease similar to that found in this experimental study.
190
H. H. SCHAUMBURG A N D P. S. SPENCER
lower the TLV forrc-hexane,as has been done in Japan where the current TLV is 100 ppm. SUMMARY
ADDENDUM G. D. DiVincenzo, C. J. Kaplan and J. Dedinas (Toxicology and Applied Pharmacology. In press) have demonstrated 2,5-hexanedione and other metabolites in serum of guinea-pigs after intraperitoneal administration of a single dose of n-hexane.
ACKNOWLEDGEMENTS This work was supported by USPHS grants NS 08952, OH 00535 and NS 03356. Dr. Spencer is the recipient of a Joseph P. Kennedy, Jun., Fellowship in the Neurosciences.
REFERENCES ALLEN, N., MENDELL, J. R., BILLMAIER, D. J., FONTAINE, R. E., and O'NEILL, J. (1975) Toxic
polyneuropathy due to methyl n-butyl ketone. Archives of Neurology, Chicago, 32, 209-213. AMERICAN CONFERENCE OF GOVERNMENTAL INDUSTRIAL HYGIENISTS (1971) Documentation of Threshold
Limit Values for Substances in Workroom Air. 3rd edition: A.C.G.H.I., p. 126. ASBURY, A. K., GALE, M. K., COX, S. C , BARINGER, J. R., and BERG, B. O. (1972) Giant axonal
neuropathy: A unique case with segmental neurofilamentous masses. Acta Neuropathologica, 20, 237-247. CARPENTER, S., KARPATI, G., ANDERMANN, F., and GOLD, M. B. (1974) Giant axonal neuropathy.
Archives of Neurology, Chicago, 31, 312-316. CAVANAGH, J. B. (1964) The significance of the 'dying-back' process in experimental and human neurological disease. International Review of Experimental Pathology, 3, 219-267. GAULTTER, M., RANCUREL, G., PIVAET, C , and EFTHYMIOU, M.-L. (1973) Polyn6vrites et hydrocarbures
aliphatiques. European Journal of Toxicology, 6, 294-296.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
Rats intoxicated with pure «-hexane, either by repetitive subcutaneous injection or by continuous inhalation, developed clinical and/or pathological evidence of peripheral neuropathy. Animals intoxicated by inhalation (400-600 ppm) developed clinical signs after forty-five days and displayed giant axonal swellings and fibre degeneration both in the central and peripheral nervous systems. The changes were most striking in tibial nerves supplying calf muscles and in selected areas of the cerebellum, medulla and spinal cord. In contrast to the usual picture associated with dying-back disease, the distal regions of proximal nerve fibres supplying calf muscles degenerated before equivalent regions of longer fibres supplying the hindfeet. The relevance of the central nervous changes to individuals recovering from toxic neuropathies and the need for a reduction of the present Threshold Limit Value (500 ppm) for human exposure are discussed.
NEUROTOXIC ACTION OF n-HEXANE
191
GONZALEZ, E., and DOWNEY, J. (1972) Polyneuropathy in a glue sniffer. Archives of Physical Medicine, 53, 333-337. GOTO, I., MATSUMURA, M., INOUE, N., MURAI, Y., SHIDA, K., SANTA, T., and KUROIWA, Y. (1974)
Toxic polyneuropathy due to glue sniffing. Journal of Neurology, Neurosurgery and Psychiatry, 37, 848-853. HALTIA, M., LINNOILA, I., SEPPALAINEN, A. M., and PALO, J. (1974) Experimental carbon disulphide
poisoning: morphological and neurophysiological studies. In: Proceedings VII International Congress of Neuropathology. Edited by St. Kornyey, St. Tauskand and G. Gosztonyi. Amsterdam: Excerpta Medica. HERSKOWITZ, A., ISHn, N., and SCHAUMBURG, H. (1971) n-Hexane neuropathy: A syndrome occurring as a result of industrial exposure. New England Journal of Medicine, 285, 82-85. IGISU, H., GOTO, I., KAWAMURA, Y., KATO, M., lzum,
K., and KUROIWA, Y. (1975) Acrylamide
encephaloneuropathy due to well water pollution. Journal of Neurology, Neurosurgery and Psychiatry, 38, 581-584. OHTA, M., TABIRA, T., HOSOKAWA, S., GOTO, I., and KUROIWA, Y. (1975) Giant axonal
INOUE, T., TAKEUCHI, Y., TAKEUCM, S., YAMADA, S., SUZUKI, H., MATSUSHITA, T., MIYAGAKI, H.,
MAEDA, K., and MATSUMOTO, T. (1970) A health survey on vinyl sandal manufacturers with high incidence of n-hexane intoxication. Japanese Journal of Industrial Health, 12, 73-84. KOROBKIN, R., ASBURY, A. K., SUMNER, A. J., and NIELSEN, S. L. (1975) Glue-sniffing neuropathy.
Archives of Neurology, Chicago, 32, 158-162. KURTTA, H. (1967) Experimental studies of n-hexane to albino rats. Japanese Journal of Industrial Health, 9, 672-677. MATSUMURA, M., INOUE, N., and OHNISHI, A. (1972) Toxic polyneuropathy due to glue sniffing. Clinical Neurology, 12, 290-296. MENDELL, J. R., SAIDA, K., GANANSIA, M. F., JACKSON, D. B., WEISS, H., GARDIER, R. W., CHRISMAN, C ,
ALLEN, N., COURI, D., O'NEILL, J., MASKA, B., and HETLAND, L. (1974) Toxic polyneuropathy
produced by methyl n-butyl ketone. Science, 185, 787-789. MIYAGAKI, H. (1967) An electrophysiological study on the toxicity of n-hexane on the peripheral nerve. Japanese Journal of Industrial Health, 9, 660-671. SCHAUMBURG, H. H., WISNIEWSKL, H. M., and SPENCER, P. S. (1974) Ultrastructural studies of the
dying-back process. 1. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication. Journal of Neuropathology and Experimental Neurology, 33, 260-284. SHIRABE, T., TSUDA, T., TERAO, A., and ARAKI, S. (1974) Toxic polyneuropathy due to glue-sniffing.
Journal of the Neurological Sciences, 21, 101-113. SOBUE, I., YAMAMURA, Y., ANDO, K., IIDA, M., and TAKAYANAGI, T. (1968) n-Hexane polyneuropathy.
Outbreak among vinyl sandal manufacturers. Clinical Neurology, 8, 393-403. SPENCER, P. S., and SCHAUMBURG, H. H. (1974a) A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure. Canadian Journal of Neurological Sciences, 1, 143-150. (19746) A review of acrylamide neurotoxicity. Part II. Experimental animal neurotoxicity and pathologic mechanisms. Canadian Journal of Neurological Sciences, 1, 151-169. (1975a) Nervous system dying-back disease produced by 2,5-hexanedione. Transactions of the American Neurological Association, 100, 148-151. (19756) Experimental neuropathy produced by 2,5-hexanedione—a major metabolite of the neurotoxic industrial solvent methyl n-butyl ketone. Journal of Neurology, Neurosurgery and Psychiatry, 38, 771-775. RALEIGH, R. L., and TERHAAR, C.J. (1975) Nervous system degeneration produced by the industrial solvent methyl n-butyl ketone. Archives of Neurology, Chicago, 32, 219-222.
Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
neuropathy: A clinical entity affecting the central as well as the peripheral nervous system. Neurology, Minneapolis, 25, 717-721. IIDA, M., YAMAMURA, Y., and SOBUE, I. (1969) Electromyographic findings and conduction velocity in n-hexane polyneuropathy. Electromyography, 9, 247-261.
192
H. H. SCHAUMBURG A N D P. S. SPENCER
SPENCER, P. S., and THOMAS, P. K. (1974) Ultrastructural studies of the dying-back process: II. The sequestration and removal by Schwann cells and oligodendrocytes of organelles from normal and diseased axons. Journal of Neurocytology, 3, 763-783. TRUHAUT, R., LAGET, P., PIAT, G., PHU-LICH, N., DUTERTRE-CATELLA, H., and HUYEN, V. N. (1973)
Premiers resultats electrophysiologiques apres intoxications experimentales par l'hexane et par l'heptane techniques chez le rat blanc. Archives de maladies professionelles de midecine du travail et de stcuritt sociale, 34, 417-426. UNITED STATES INTERNATIONAL TRADE COMMISSION (1973) Synthetic Organic Chemicals (Publication
No. 728). Washington: US Government Printing Office. WADA, Y., OKANOTO, S., and TAKAGI, S. (1965) Intoxication polyneuropathy following exposure to n-hexane. Clinical Neurology, 5, 591-597. YAMADA, A. (1964) An occurrence of polyneuritis by n-hexane in the polyethylene laminating plants. Japanese Journal of Industrial Health, 6, 192-197. YAMAMURA, Y. (1969) n-Hexane polyneuropathy. Folia Psychiatria Neurologica Japonica, 23, 45-57. Downloaded from http://brain.oxfordjournals.org/ by guest on April 8, 2016
{Received September 16, 1975)
