[CANCER RESEARCH 41, 2444-2449, June 1981] 0008-5472/81 /0041-0000 $02.00 Carcinogenesis of 4-(Hydroxymethyl)benzenediazonium Ion (Tetrafluoroborate) of Agaricus bisporus1
Bela Toth, Kasinath Patii, and Hwan-Soo Jae
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68105
ABSTRACT MATERIALS AND METHODS
4-(Hydroxymethyl)benzenediazonium tetrafluoroborate was Swiss albino mice from the colony randomly bred by us since administered as 26 weekly s.c. injections of 50 /ig/g body 1951 were used. They were housed in plastic cages with weight to randomly bred Swiss mice. In addition, as a solvent granular cellulose bedding, separated according to sex in control, sodium tetrafluoroborate was given as 26 weekly s.c. groups of 5, and given Wayne Lab-Blox diet in regular pellets injections at 25 fig/g body weight in 0.9% NaCI solution to (Allied Mills, Inc., Chicago, III.) and tap water ad libitum. another group of mice. The 4-(hydroxymethyl)benzenedi- The chemicals used were HMBD (Chart 1) (M.W. 221.96; azonium tetrofluoroborate treatment induced tumors in the m.p. 61-62°; purity, 95%) and STB (M.W. 103.79; m.p. 384° subcutis and skin in incidences of 20 and 12%, respectively; with decomposition; Alfa Division, Danvers, Mass.). HMBD was while in the solvent sodium tetrafluoroborate-injected mice, the synthesized in this laboratory in the following way. corresponding tumor incidences were 6 and 0%, respectively. A 1.75-g (1.5 mmol) sample of nitrosyl tetrafluoroborate was Histopathologically, the tumors were classified as a fibroma, suspended in 15 ml of ethyl acetate maintained at 0°. The fibrosarcomas, rhabdomyosarcomas, and an angiosarcoma in solution was cooled to —20°,and a solution of p-aminobenzyl the subcutis and also as squamous cell papillomas and carci alcohol in 15 ml of ethyl acetate was added in one portion. The nomas of the skin. mixture was stirred for 1 hr and filtered (yield, 2.0 g; purity, 4-(Hydroxymethyl)benzenediazonium ion is an ingredient of 90%; m.p. 51-52°). Recrystallization produced white crystals the cultivated mushroom of commerce Agaricus bisporus. (m.p. 61-62°).
C7H7BF4N2O Calculated: C 37.84, H 3.15, N 12.61 INTRODUCTION Found: C 38.13, H 3.20, N 12.49 Evidence for the occurrence of 4-(hydroxymethyl)ben- 1H-nuclear magnetic resonance (D2O): «58.53 (d, J = 8.3 zenediazonium ion in basal-stalk sections of the commonly Hz, 2H); 7.90 (d, J = 8.3 Hz, 2H); 4.89 (s, 2H); 4.66 (broad s, cultivated mushroom of commerce Agaricus bisporus (Figs. 1 1H). IR (KBr): N = N 2275/cm. UV (H2O): e27425,000. and 2) was provided as early as 1962 (9). Also, the possibility A toxicity study was performed for 35 days before the was raised that this diazonium ion may be formed enzymatically chronic experiment. Five dose levels of HMBD such as 200, from agaritine, a constituent of this fungus (9). In addition, the 150, 100, 50, and 10 fig/g body weight were administered in presence of an enzyme y-glutamyltransferase (y-glutamyltran- 0.01 ml 0.9% NaCI solution as single s.c. injections to Swiss speptidase) was demonstrated, which catalyzes the hydrolysis mice. By taking into account 4 parameters (survival rates, body of agaritine to L-glutamate and 4-hydroxymethylphenylhydra- weights, dose of chemical, and histological changes), the 50- zine(7, 10). /xg/g body weight dose was found to be suitable for the chronic In earlier experiments, 4-methylphenylhydrazine, a water- treatment. This toxicity technique was developed in this labo soluble substance related to the aforementioned series of ratory (15). compounds, induced statistically significant incidence of soft- The chronic experimental group and the controls were as tissue tumors at injection sites when given by repeated s.c. follows. injection in mice (22, 28). From this finding, it was postulated Group 1. This consisted of 50 female and 50 male Swiss that a corresponding diazonium ion may have been formed mice, 6 weeks (43 days) old at the beginning of the experiment. locally and was ultimately responsible for cancer induction. They received 26 injections s.c. at weekly intervals of 50 jug This hypothesis was particularly promising, since none of the HMBD in 0.01 ml 0.9% NaCI solution per g body weight. other known carcinogenic water-soluble hydrazines had in Group 2. This consisted of 50 female and 50 male mice, 6 duced tumors at application sites (17, 18). weeks (44 days) old at the beginning of the experiment. They The present study thus demonstrates the tumorigenicity of HMBD2 administered by repeated weekly s.c. injections to received 26 injections s.c. at weekly intervals of 25 fig STB (this corresponds to an equimolar dose of the tetrafluoroborate Swiss albino mice. anión) in 0.01 ml 0.9% NaCI solution per g body weight. The experimental and control animals were carefully checked and weighed at weekly intervals, and the gross patho ' This study was supported by National Institute of Environmental Health logical changes were recorded. Complete necropsies were Sciences Contract NO1-CP05629 and was presented in part at the 71st Annual performed in all animals. All organs were examined macro- Meeting of the American Association for Cancer Research. San Diego, Calif., May 31, 1980(1). scopically and fixed in 10% buffered formalin. Histological 2 The abbreviations used are: HMBD. 4- 2444 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Carcinogenesis of a Diazonium Salt turbinais, at least 4 lobes of the lungs of each mouse, and Finally, in 2 instances, rhabdomyosarcomas were observed those organs with gross pathological changes. Sections from which were composed of multinucleated giant cells and spindle these tissues were stained with hematoxylin and eosin. cells with the typical striations. Histopathologically, these le sions were similar to those described in humans (5). Tumors of Skin. Of the HMBD-treated females, 9 (18%) RESULTS developed such tumors at injection sites. Of these, 4 mice had The survival rates after weaning are recorded in Table 1. As squamous cell papillomas, 4 mice had squamous cell carcino can be seen from the data, the treatment shortened substan mas, and a mouse developed a squamous cell papilloma and tially the survival when compared with the solvent-injected a squamous cell carcinoma. In the HMBD-treated males, 3 controls. (6%) developed 3 skin tumors at the injection sites. Of these, The number, percentages of animals with tumors, and their 2 mice had squamous cell papillomas, and one mouse had a ages at death (latent periods) are summarized in Table 2. The squamous cell carcinoma. 2 most important neoplasms are described in detail below. No skin tumors were observed in the STB-injected mice of Tumors of s.c. Tissue. Of the HMBD-treated females, 11 both sexes. (22%) developed 13 such neoplasms at injection sites. Of Macroscopically, tumors of the skin exhibited localized over these, 10 mice had 11 fibrosarcomas, and one mouse devel growth of the epidermis capped often by keratinized material. oped a fibrosarcoma and an angiosarcoma. In the HMBD- They ranged from 3 to 15 mm in diameter (Fig. 6). These treated males, 9 (18%) developed 10 tumors at injection sites. tumors were classified into benign and malignant varieties. In These neoplasms consisted of 5 fibrosarcomas in 5 mice, 2 the squamous cell papillomas, the epithelial and fibrous com rhabdomyosarcomas in 2 mice, one angiosarcoma in one ponents were present in various proportions and were char mouse, and one fibrosarcoma and one angiosarcoma in one acterized by the papillary pattern of the proliferating, folded, mouse. and thickened epidermis (Fig. 7). In the squamous cell carci In the females given STB injections, 3 mice (6%) developed nomas, the irregular masses of the typical malignant epidermal tumors on the back, all classified as fibrosarcomas; while in cells descended downwards deep into the dermis (Fig. 8). the males given STB injections, 3 animals (6%) developed s.c. Macroscopically, these skin tumors were similar to those de tissue neoplasms, of which one was classified as a fibroma and scribed by other investigators in mice (3). the remaining 2 as fibrosarcomas. Other Tumors. A few other types of tumors were found also The connective tissue tumors were observed on the back in the treated groups shown in Table 2. Since they occurred in and on the side of the chest, exhibiting hard consistency and low incidences, their appearance cannot be attributed to the various shapes and sizes up to 60 mm in diameter. Sometimes treatment. the skin of the tumor was ulcerated (Fig. 3). The majority of these soft-tissue tumors were composed of the typical elon DISCUSSION gated spindle-shaped cells arranged in an interlacing fashion characteristic of fibrosarcomas (Fig. 4). The malignant tumor The current study demonstrates that HMBD, given as 26 also invaded the liver (Fig. 5). In a single instance, a benign weekly s.c. injections on a SO-jig/g body weight basis, induced variety of the connective tissue tumor, a fibroma was observed. tumors of the subcutis and skin in Swiss mice. The s.c. tissue It consisted of the cells of fibrous tissue with varying amounts tumor incidence rose from 6 to 22% (p < 0.046) in females of collagen and reticulin fibers. In a few instances, angiosar- and from 6 to 18% in males, whereas the skin tumor incidence comas were seen. They exhibited the characteristic vascular increased from 0 to 18% (p < 0.0056) in females and from 0 spaces and clefts lined by the neoplastic endothelial cells. to 6% in males, compared with that in the solvent-injected controls. Statistical analysis was carried out by using Fisher's CH2-OH exact test for 2 x 2 tables (1 ). Light microscopic examination of the lesions revealed the characteristic appearance of fi broma, fibrosarcomas, rhabdomyosarcomas, and angiosarco- mas in the subcutis and squamous cell papillomas and carci nomas of skin at injection sites. 4-(Hydroxymethyl)benzenediazonium ion, a naturally occur ring compound, is the first chemical of this class shown to be carcinogenic. The various diazonium salts are also commonly used intermediates in the industrial synthesis of azo dyes (2, 6, Chart 1. Chemical structure of HMBD. 12, 14, 29) and may be formed in the stomach as a result of Table 1 Treatment and survival rate of HMBD-treated and STB-injected Swiss mice survivorsGroup12TreatmentHMBD, No. of no.5050SO50SexFMFM10wk5050505020wk4948504830wk4543504640wk3124484250wk1212483960wk15423270wkwk1133 80wk 90wk 100 wk1 10 wk120 wt,26 50 ng/g body weekly s.c. injec tionsSTB, 26weekly25 fig/g body wt, 725 24 8 s.c. injectionsInitial 12 8 52 JUNE 1981 2445 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. B. Toth et al. Table 2 Treatment and tumor distribution in HMBD-treated and STB-injected Swiss mice ofGroup Animals with tumors (wk)% at death (wk)Av.51Range23-76Otherat death Treatment1 tiveNo.50 no. Sex Av.22 %41-76 No. tissues3 30)a HMBD, 50 fig/g bodyEffec F 11SubcutisAge 51Range 9 18SkinAge malignant lymphomas (13,26, wt, 26 weekly s.c. in 2 adenomas of lungs (35, 76) jections 1 angioma in liver (59) 1 polypoid adenoma of cecum (54) 1 adenocarcinoma of gall bladder (31) 50 18 47 37-56 53 51-56 4 adenomas of lungs (40, 42, 48, 71) 1 adenoma and adenocarcinoma of lungs (60) 1 malignant lymphoma (29) 1 polypoid adenoma of cecum (43) STB, 25(ig/g body wt. 50 78 59-88 9 adenomas of lungs (58, 60, 72. 72, 26 weekly s.c. injec 77, 84, 88, 88, 106) tions 2 adenocarcinomas of lungs (81 , 84) 1 adenoma and adenocarcinoma of lungs (71) 5 malignant lymphomas (34, 53, 71 , 79, 84) 2 adenocarcinomas of breasts (88, 100) 2 granulosa cell tumors (79, 107) 1 angioma in ovary (83) 1 adenocarcinoma of gl. stomach (72) 1 adenocarcinoma of cecum (80) 1 adenocarcinoma of ovary (89) 1 angiosarcoma, abdominal (60) 50 68 57-81 6 adenomas of lungs (71 , 71 , 74, 77, 109, 110) 3 adenocarcinomas of lungs (68, 72, 91) 2 adenoma and adenocarcinoma of lungs (78, 100) 3 malignant lymphomas (75, 93. 1 10) 1 angiosarcoma. abdominal (71) 1 angioma in liver (1 09) 1 angiosarcoma in liver (72) 1 papilloma of bladder (110) 1 adenocarcinoma of prostate (100) * Numbers in parentheses, age at death given in weeks. ingestion of primary aromatic amines and nitrite (11 ). It is also and now the tetrafluoroborate form of 4-(hydroxymethyl)- theorized that enzymatic a-hydroxylation is a distinct possibility benzenediazonium ion of the cultivated mushroom of com in the case of dialkyl- and alkylarylnitrosamines. Accordingly, merce, A. bisporus, induced a series of tumors in mice (20, for instance, the carcinogenic methylphenylnitrosamine should 23, 24). Therefore, it seems advisable to continue and to act through the phenyldiazonium ion as the "ultimate" carcin expand the research in these environmentally important fields ogen (4). of interest. The 4-(hydroxymethyl)benzenediazonium ion was detected To date, 3 diazonium ions have been demonstrated to be in A. bisporus in 1962 (9). This compound was also shown to mutagenic in bacterial systems. The first was 4-dimethylami- be present in an aqueous extract of this fungus at pH 3, at a nobenzenediazonium ¡on(sodium sulfonate salt), which has level of 0.6 ppm in this laboratory. Furthermore, in vitro studies been used as a pesticide and showed a dose-dependent mu- carried out here also indicated that this ion is generated from tagenicity in Salmonella typhimurium TA 1537 and TA 1538 agaritine by an enzyme system present in A. bisporus acting in (8). Subsequently, a heterocyclic diazonium ion, 4-diazoimi- situ.3 dazole-5-carboxamide was demonstrated to be highly muta The present study is also part of our long-range objective to genic in the Ames system using TA 100 strain (11 ). Finally, the reveal the carcinogenic potencies and mode of action of chem presently studied mushroom ingredient, HMBD, was proven to ical ingredients of edible mushrooms. To date, 3 compound be mutagenic recently in this laboratory in the TA 1537 strain ingredients of one of the false morel mushrooms Gyromitra (13). esculenta, i.e., methylhydrazine, W-methyl-W-formylhydrazine, and acetaldehyde methylformylhydrazone, induced a variety of REFERENCES tumors in mice and hamsters (16, 19, 21, 25-27). In addition, 1. Armitage, P. Statistical Methods in Medical Research, pp. 136-138. Oxford, the A/'-acetyl derivative of 4-hydroxymethylphenylhydrazine England: Blackwell Scientific Publications, Ltd., 1971. 2. Butler, R. N. The diazotization of heterocyclic primary amines. Chem. Rev.. ' A. Ross, unpublished observations. 75: 241-257, 1975. 2446 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Carcinogenesis of a Diazonium Salt 3. Delia Porta, G., Terracini, B., Dämmert, K., and Shubik, P. Histopathology 17. Toth, B. Synthetic and naturally occurring hydrazines as possible cancer of tumors induced in mice treated with polyoxyethylene sorbitan monostear- causing agents. Cancer Res., 35: 3693-3697, 1975. ate. J. Nati. Cancer Inst., 25: 573-605, 1960. 18. Toth, B. Actual new cancer-causing hydrazines, hydrazides, and hydra- 4. Druckrey, H., Preussmann, R., Schmähl, D., and Ivankovic, S. Organotrope zones. J. Cancer Res. Clin. Oncol., 97: 97-108, 1980. carcinogene Wirkungen bei 65 verschiedenen N-nitroso-Verbindungen an 19. Toth, B. Carcinogenesis by gyromitrin of Gyromitra esculenta. Fed. Proc., BD-Ratten. Z. Krebsforsch., 69: 103-201, 1967. 39. 884, 1980. 5. Evans, R. W. Mistological Appearances of Tumors, Ed. 2. Baltimore: The 20. Toth, B. Carcinogenesis by mushroom hydrazines. Additional investigations Williams & Wilkins Co., 1968. with naturally occurring mycotoxins. In: Biology of the Cancer Cell, Proc. V. 6. Fuser, L. F. Experiments in Organic Chemistry, Ed. 3. Boston: D. C. Heath Meeting of Europ. Assoc. Cancer Res., Vienna, Austria, pp. 45-50. 1980. and Co., 1957. 21. Toth, B.. and Nagel, D. Tumors induced in mice by W-methyl-W-formylhydra- 7. Gigliotti, H. Studies on the y-Glutamyltransferase and Arylhydrazine Oxidase zine of the false morel Gyromitra esculenta. J. Nati. Cancer Inst., 60: 201- Activities of Agaricus bisporus. Ph.D. Dissertation. Ann Arbor, Mich.: The 204, 1978. University of Michigan, 1963. 22. Toth, B., and Nagel, D. Studies on the tumorigenic potential of 4-substituted 8. Kada, T., Moriya, M., and Shirasu, Y. Screening of pesticides for DMA phenylhydrazines by subcutaneous route. J. Toxicol. Environ. Health, in interactions by "Recassay" and mutagenesis testing, and frameshift muta- press, 1981. gens detected. Mutât.Res., 26: 243-248, 1974. 23. Toth, B., Nagel, D., Patii, K., Erickson, J., and Antonson, K. Tumor induction 9. Levenberg, B. An aromatic diazonium compound in the mushroom Agaricus with the N'-acetyl derivative of 4-hydroxymethylphenylhydrazine a metabo bisporus. Biochim. Biophys. Acta, 63. 212-214, 1962. lite of agaritine of Agaricus bisporus. Cancer Res.. 38. 177-180, 1978. 10. Levenberg, B. Isolation and structure of agaritine, a y-glutamyl-substituted 24. Toth, B., Nagel, D., and Ross, A. Occurrence and the carcinogenic action of arylhydrazine derivative from Agaricacae. J. Biol. Chem., 239: 2267-2273, 4- JUNE 1981 2447 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Fig. 3. An ulcerated growth in the subcutis on the right side of the back and chest in a HMBD-treated female mouse. Gross photograph, x 2. Fig. 4. Fibrosarcoma in subcutis. Composed of spindle-shaped cells arranged in bands. Same tumor as shown in Fig. 3. H & E, x 200. Fig. 5. Metastasis of fibrosarcoma in liver. A large area in the liver is obliterated by the malignant fibroblasts. Same tumor as shown in Fig 4 H & E, x 200. 2448 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. //jf ÃS?8^'^^^£^'¿'fjp^fV^-' ty¿&^*S%£'''-.- - '--'ì^^f-^^v-- "'•••*•'"•'•%'*-"•»i Fig. 6. Growths of skin on the back. Some of the lesions are keratinizing and protruding over the surface. A male mouse treated with HMBD. Gross photograph. ">C9 Fig. 7. Squamous cell papilloma of skin. It exhibits a thin stroma and the highly thickened and folded epidermis covered with a varying amount of keratin. Female mouse treated with HMBD. H & E, x 40. Fig. 8. Squamous cell carcinoma of skin. The irregular masses of atypical epidermal cells are distinct, and the invasion of the lower part of dermis is evident. Same animals as shown in Fig. 6. H & E, x 80. 2449 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Carcinogenesis of 4-(Hydroxymethyl)benzenediazonium Ion (Tetrafluoroborate) of Agaricus bisporus Bela Toth, Kasinath Patil and Hwan-Soo Jae Cancer Res 1981;41:2444-2449. Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/6/2444 E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected]. Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/41/6/2444. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. 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