Formation of Methylhydrazine from Acetaldehyde /V-Methyl-A/- Formylhydrazone, a Component of Gyromitra Esculenta ^

[CANCER RESEARCH 37, 3458-3460, September 1977]

Brief Communication Formation of Methylhydrazine from Acetaldehyde /V-Methyl-A/- formylhydrazone, a Component of Gyromitra esculenta ^

Donald Nagel, L. Wallcave, Bela Toth, and Robert Kupper

The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105

SUMMARY recently been confirmed at levels of 0.3% gyromitrin and 0.05% MFH in the dried mushrooms (14). Gyromitrin, acetaldehyde /V-methyl-N-formylhydrazone, An earlier study indicated that, MH, a known tumorigen is a toxin present in edible wild mushroom Gyromitra escu producing lung tumors in mice (17) and malignant histiocy- lenta. At 37Â°under different acidic conditions (pH 1 to 3), tomas and tumors of the cecum in hamsters (20), was mimicking the milieu of human stomach, gyromitrin is con formed from either gyromitrin or MFH by treatment with verted to methylhydrazine, a known tumor inducer in mice strong acid (11). The present study was undertaken to de and hamsters, through an intermediate, /v-methyl-/V-formyl- termine whether MH could be formed under pH conditions hydrazine. In addition, methylhydrazine is formed in the simulating the milieu of the human stomach and to attempt mouse stomach after p.o. administration of gyromitrin. an in vivo demonstration of its formation from gyromitrin in These findings imply that consumption of G. esculenta the mouse stomach. could present a carcinogenic, as well as an acutely toxic, health hazard. MATERIALS AND METHODS

INTRODUCTION MH was purchased from Eastman Kodak Co., Rochester, N. Y. MFH was prepared by a modification of the original Gyromitra esculenta, false morel (a wild edible mush method (9) from MH and methyl formate in ethanol at -15Â°. room), grows in sandy soil under conifer trees in Europe, Gyromitrin was prepared from MFH and acetaldehyde in North America, and elsewhere (12, 13). It is collected and diethyl ether, as previously described (9). eaten on a considerable scale in Michigan, Idaho, Colo 1-Methyl-1[(2-nitrophenyl)methylene]diazane (2-nitro- rado, and North Carolina. Gourment shops in the United benzaldehyde methylhydrazone) was prepared by refluxing States also stock a variety canned in Europe (15). 3.0 g of nitrobenzaldehyde and 1.0 g of MH in 50 ml of G. esulenta is widely consumed, and over 500 poisonings methanol for 2 hr. Evaporation of the solvent left a red oil, resulting from its ingestion are documented in the literature which crystallized on standing in the cold. Recrystallization (4). Seventy-four of the poisoning were fatal (2, 5, 6). Fatal from ether-hexane gave bright orange-yellow needles, m.p. poisoning is obviously infrequent on a percentage basis, 39-40Â°[from the literature, 37-39Â°(8)]. The spectral charac and this is undoubtedly a result of the almost universal teristics (IR, UV, PMR, CMR) of these compounds were practice of boiling the mushroom a long time in a large consistent with the assigned structures. quantity of water and then discarding the liquid (15). Never PMR spectra were determined in D.,0 or CDCI;, solutions theless, fatalities have been reported in people who ate this on a Varian Model HA-100 nuclear magnetic resonance mushroom after cooking it and discarding the juice (7, 21, spectrometer, CMR spectra were determined on a Varian 22). Model CFT-20 spectrometer, IR spectra were determined on In G. esculenta the toxin was originally identified as hel- a Beckman Model IR-9 spectrophotometer, and UV spectra velic acid, but this was later found to be a mixture of fatty were determined on a Gary Model 14 spectrophotometer. acids (1). Recently, in a series of papers (9-11 ), 2 hydrazine GLC determinations of gyromitrin were performed on a derivatives (a hydrazone, acetaldehyde A/-methyl-/V-for- Beckman Model GC-45 Chromatograph with the use of 2-m mylhydrazone, which was termed gyromitrin, and a hydra- x 2-mm glass column of Chromosorb 103 (Supelco, Inc., zide, MFH2) were identified as toxins in this fungus. The Bellefonte, Pa.) (column temperature, 160Â°;helium flow latter structure was postulated as a hydrolysis product of rate, 20 ml/min). Under these conditions the retention gyromitrin. The presence of these hydrazine derivatives has time of gyromitrin was 17.0 min, and that of MFH was 15.7 min. GLC determinations of 1-methyl-2-[(2-nitro-

' Research supported by USPHS Contract 1 CP33278 from the National phenyl)methylene]diazene were performed on a Varian Cancer Institute, NIH. Model 3700 gas Chromatograph with the use of a 2-m x 2- 2 The abbreviations used are: MFH, A/-methyl-W-formylhydrazine; MH, mm glass column packed with 8% SE-30 on 100/120 mesh methylhydrazine; PMR, proton magnetic resonance; CMR, carbon magnetic Chromasorb W AW-DMCS (column temperature, 160Â°;he resonance; GLC, gas-liquid chromatography or Chromatograph. Received August 23, 1976; accepted June 21, 1977. lium flow, 20 ml/min). Under these conditions the retention

3458 CANCER RESEARCH VOL. 37

time of the hydrazone was 7.25 min and that of 2-nitrobenz- the blanks, there did not appear to be a correlation between aldehyde was 1.4 min. the time and the amount of MH present. The kinetic studies were conducted at 37Â°in potassium Product studies conducted in vitro at pH 1 indicated that chloride-hydrochloric acid buffer (pH 2) or potassium hy gyromitrin was converted to MFH, which in turn was hydrol- drogen phthalate-hydrochloric acid buffer (pH 2.5 and 3.0). ized to MH, as illustrated in Chart 1. The products were Aliquots of the gyromitrin-buffer mixture were taken at characterized by GLC, PMR, and CMR with the aid of au specified intervals, neutralized to pH 8 with 1.5 N sodium thentic synthetic compounds. No other products were no hydroxide, and injected on the GLC. The rate of reaction ticed. was monitored by integration of the gyromitrin and MFH At pH 2 at 37Â°,gyromitrin at an initial concentration of 1.0 peak areas. x 10~3M was hydrolyzed to MH with a half-life of 122 min, as The molar concentrations of gyromitrin and MFH were illustrated in Chart 2. At pH 2.5 the half-life for the formation calculated with the response from standard samples. The of MH was 10.5 hr, whereas at pH 3.0 only 9.0% MH was amount of MH formed at time f, [MHt], was calculated with produced in 72 hr. the formula:

[MH,] = [Gâ€ž]-[Gt] - [MFH,] DISCUSSION

where [Gâ€ž]isthe initial concentration of gyromitrin. These studies showed that MH was produced by acid For determination of the extent of MH formation from hydrolysis from gyromitrin through MFH. As would be ex gyromitrin in the mouse stomach, female Swiss mice from pected from the chemistry of hydrazones and hydrazides, the Eppley Institute colony, 10 weeks old, were fasted for 24 the rate of hydrolysis was strongly pH dependent (16). hr and given 4.0 mg of gyromitrin in 0.5 ml of distilled water MH formation was monitored by measuring the disap intragastrically. After the specified times, the animals were pearance of gyromitrin and MFH rather than the formation killed, the abdominal wall was incised, and the esophagus of MH, since the GLC of MH directly from aqueous solutions was ligated. The stomach was removed and slit, and the gave poorly reproducible results due to decomposition dur contents were gently scraped out. The inner surface was ing chromatography. washed with distilled water, and a combined volume of For determination of MH in the in vivo mouse experi about 4 ml was collected. After determination of the pH of ments, it was, of course, essential to measure MH in a more the stomach contents, 0.14 ml of 5.0 M potassium acetate Table 1 was added, followed by sufficient 5 M HCI (usually 0.04 MH in mouse stomach after p.o. administration of 4.0 mg of ml) to give a pH of 5.3 to 5.7. The buffered stomach sus gyromitrin pension was centrifuged, and the supernatant was trans Time(min)0153045120240MH"(/*g)0.42.55.511.05.06.08.010.512.06.06.57.0StomachpHND"2.42.45.62.93.52.22.24.24.83.6 ferred to a 2nd graduated centrifuge tube. The precipi tate was rinsed with about 1 ml of water. The combined volume was diluted, if necessary, to 5.0 ml, and 1.0 ml of a 0.6% solution of 2-nitrobenzaldehyde in methanol was added. The stoppered tube was kept in a bath at 50Â°for 30 min in the dark. One ml of carbon disulfide was added to the cooled mixture, and the contents were vigorously shaken for 1 min. After centrifugaron to break an emulsion, 3.0 /Â¿I of organic phase were used for GLC determination of 1- methyl-2-[(2-nitrophenyl)methylene]diazane. A calibration curve was prepared by adding measured quantities of MH, from 2.0 to 21.0 Â¿tg,to5.0 ml of buffer at pH 5.5 and treating " Amount of MH found in 4 mg of gyromitrin in 0.5 ml of water. the solution with 2-nitrobenzaldehyde, as described. Simi Average of 3 determinations. Each value represents a single ani mal. larly, blanks were prepared from the reaction of 4.0 mg of * ND, not determined. gyromitrin in 5.0 ml of buffer with 2-nitrobenzaldehyde. At an electrometer setting of 4 x 10~" /ua, full-scale recorder CH, .CH, deflection was obtained with about 30 /^.gof hydrazine. The GYROMITRIN solution of the hydrazone in carbon disulfide was stable in HC the dark for 24 hr.

RESULTS CH3 -NH, N-METHYL-N-FORMYLHYDRAZINE MH at levels from 6- to 30-fold greater than are found in HC H gyromitrin blanks was measured in the mouse stomach 0 after administration of the latter compound. These values are shown in Table 1. The pH of the stomach contents varied considerably among animals (2.2 to 5.6). Although CMHNH METHYLHYDRAZINE the amount of MH present was considerably higher than in Chart 1. Hydrolysis pathway for formation of MH from gyromitrin.

SEPTEMBER 1977 3459

lOOr- Recently, it has been shown in this laboratory that the convulsive, toxic, and lethal effects of several hydrazines 90 that induce tumors in mice, including MH (18, 20), were prevented by administering pyridozine hydrochloride (19). 80 At present studies are under way to determine the effect of ^ 70 this vitamin on the tumorigenicity of these compounds. M g 60 O ti, = 122min REFERENCES X 50

1. Boehm, R., and KÃ¼lz,E. Ãœberden giftigen Bestandtheil der essbaren | 40 Morchel (Helvetia esculenta). Arch. Exptl. Pathol. Pharmakol.. 79: 403- LÃ¼ 414, 1885. J 30 2. Dearness, J. Gyromitra Poisoning. Mycologia, 76: 199. 1924. 3. Dee, L. A. Gas Chromatographie Determination of Aqueous Trace Hydra 20 zines and Methylhydrazine as Corresponding Pyrazoles. Anal. Chem., 43:1416-1419,1971. 4. Franke, S., Freimuth, U., and List, P. H. ÃœberdieGiftigkeit der FÃ¼hjahrs- l 0 lorchel Gyromitra (Helve/la)esculenta. Arch. Toxicol., 22: 293-332,1967. 5. Giusti, G. V., and Carnevale, A. A Case of Fatal Poisoning by Gyromitra l J_ J_ l j esculenta. Arch. Toxicol.,33: 49-54, 1974. 0 40 80 120 160 200 240 280 320 6. Hendricks, H. V. Poisoning by False Morel (Gyromitra esculenta). J. Am. TIME (min) Med. Assoc., 774: 1625, 1940. Chart 2. Rate of decomposition of gyromitrin in pH 2 buffer at 37Â°atan 7. Herzog, G. Zur pathologisch-anatomischen Kenntnis von Pilzvergiftun initial concentration of 1.0 x 10~3 M. Each point represents 3 individual gen. Muench. Med. Wochschr, 64: 1366-1367, 1917 determinations. Half-life was determined from the graph. 8. Heugebert, F. C., and Willems, J. F. A Synthetic Approach to 1,3,4- Thiodiazolidine-2-thiones. Tetrahedron, 22: 913-923, 1966. 9. List, P. H., and Luft, P. Gyromitrin, das Gift der FrÃ¼hjahrlorchel,Gyromi direct manner. A previously reported GLC method in which tra (Helvetia) esculenta. Tetrahedron Letters, 1893-1894, 1967. MH was reacted with 2,4-pentanedione to give a pyrazole (3) 10. List, P. H., and Luft, P. Gyromitrin. das Gift der FrÃ¼jahrlorchel.Arch. Pharm., 301: 294-305, 1968. was not usable because of inadequate sensitivity and partial 11. List, P. H., and Luft, P. Nachweis und Gehaltsbestimmung von Gyromi- interference from gyromitrin. The method developed in this trin in frischen Lorchein. Arch. Pharm., 302: 143-146, 1969. 12. Miller, O. K. Mushrooms of North America, pp. 215-218. New York: E. P. study was more sensitive, was free from all interferences, Dutton and Co., Inc., 1969. and gave excellent linearity between peak height and MH 13. Savonius, M. Mushrooms and Fungi, pp. 36-37. New York: Crown Pubi., concentration. Inc., 1973. 14. Schmidlin-MÃ©szÃ¡ros,J.Gyromitrin in Trockenlorcheln (Gyromitra escu The formation of MH in the mouse stomach was probably lenta sice.). Mitt. Gebiete Lebensm. Hyg., 65: 453-465, 1974. a result of the acid hydrolysis of gyromitrin, since enzymatic 15. Simmons, D. M. The Mushroom Toxins. Del. Med. J., 43: 177-187,1971. 16. Smith, P. A. S. Open Chain Nitrogen Compounds, Vol. 2. pp. 153-154. metabolism was unlikely. No correlations between mea New York: W. A. Benjamin, Inc., 1966. sured stomach pH and the amount of MH formed or be 17. Toth, B. Hydrazine, Methylhydrazine and Methylhydrazine Sulfate Carci- tween time and MH were observed. Since stomach pH was nogenesis in Swiss Mice, Failure of Ammonium Hydroxide to Interfere in the Development of Tumors. Intern. J. Cancer, 9: 109-118, 1972. probably not constant with time and since nothing was 18. Toth, B. Synthetic and Naturally Occurring Hydrazines as Possible Can known about the rates of elimination of any of the equilib cer Causative Agents. Cancer Res., 35: 3693-3697, 1975. 19. Toth, B., and Erickson, J. Reversal of the Toxicity of hydrazine Ana rium components or about their possible absorption, quan logues by Pyridoxine Hydrochloride. Toxicology, 7: 31-36, 1977. titative interpretations would be speculative. Nevertheless, 20. Toth, B., and Shimizu, H. Methylhydrazine Tumorigenesis in Syrian inasmuch as these studies indicate that MH would probably Golden Hamsters and the Morphology of Malignant Histiocytomas. Can cer Res., 33: 2744-2753, 1973. be formed in the human stomach after consumption of G. 21. Umber, F. Vorsicht beim Morchelgenuss. Deut. Med. Wochschr., 42: esculenta, these mushrooms should be considered to have 627, 1916. a carcinogenic, as well as an acutely toxic, potential. 22. Welsmann, L. Lorchelvergiftung. Z. Pilzkunde, 73: 119-120, 1934.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. Formation of Methylhydrazine from Acetaldehyde N-Methyl-N -formylhydrazone, a Component of Gyromitra esculenta

Donald Nagel, L. Wallcave, Bela Toth, et al.

Cancer Res 1977;37:3458-3460.

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