Carcinogenesfa vol.13 no. 11 pp. 1957-1960, 1992
A study of tobacco carcinogenesis XLVIII. carcinogenicity of W-nitrosonornicotine in mink (Mustela vison)
Nils Koppang1"2, Abraham Rivenson3, Albrecht Reith4, Hans K.Dahle2, 0ystein Evensen1 and Dietrich Hoffmann3*5 'National Veterinary Institute, N-OO33 Oslo 1, 2Norwegian College of Veterinary Medicine, N-0033 Oslo 1, Norway, 'American Health Foundation, Valhalla, NY 10595, USA and Norwegian Radium Hospital and Institute for Cancer Research, 0310 Oslo 3, Norway To whom requests for reprints should be sent
During tobacco processing and smoking, nicotine and nornicotine give rise to W-nitrosonornicotine (NNN), a highly abundant, strong carcinogen. NNN is known to exert carcinogenic activity in mice, rats and hamsters. Major target organs for NNN carcinogenicity in the rat are the esophagus and the nasal mucosa, and in the Syrian golden hamster trachea and nasal mucosa. In comparison with the rat, the mink (Mustela vison) has a markedly expanded nasal mucosa. Therefore, we explored in this study whether the mink could serve as a non-rodent model for nasal carcinogenesis using NNN as the carcinogen. Twenty random-bred mink, beginning at the age of 3 weeks, received twice weekly s.c. injections of NNN, a total dose of 11.9 mM per animal over a 38 week period. All of the 19 mink at risk developed malignant tumors of both the respiratory and the olfactory region of the nose within 3.5 years. In most animals the malignant rumors, primarily esthesioneuroepithelioma, invaded the brain. Remarkably, NNN induced no other tumors in the mink. None of the control animals developed nasal tumors nor tumors at other sites during the 3.5 years of the assay. The historical data from the farm did not reveal any spontaneous occurrence of nasal tumors in mink at any age. This study supports the concept that NNN is a proven carcinogen for multiple species of mammals and that the mink can serve as a non-rodent, non-inbred animal model for nasal carcinogenesis, especially since NNN induces only tumors in the nasal cavity in this species and not at other sites, as it does in mice, rats and hamsters.
Materials and methods Chemicals NNN was synthesized according to an earlier published method (8). Its purity was >99% according to GC, HPLC and MS (9). NNN is stable in an aqueous solution when stored at room temperature in the dark. Animals The random-bred mink were offspring from our breeding unit, which has an almost zero tumor incidence (at the Norwegian College of Veterinary Medicine's Research Farm for Fur-bearing Animals, Heggedal, Norway). For the first 7 weeks of their lives the cubs were nursed by their mothers. Subsequently, they were housed individually and fed a standard diet as reported earlier (7); water was offered ad libitum. At the age of 3 weeks the cubs weighed — 50 g; at the age of 65 days average weights were — 1000 g for the males and 750 g for the females. Males reach their maximal weight of -2300 g after 180 days, the maximal average weight for females, - 1200 g, is reached after 150 days (Figure 2) (10). The average life-span of these mink is assumed to be 10 years.
Introduction W-Nitrosonornicotine (NNN*) is the most abundant tobaccospecific A/-nitrosamine in tobacco and its smoke. It is formed from nicotine and nornicotine during tobacco processing and during smoking (1). NNN is a potent carcinogen that induces lung adenoma and adenocarcinoma in mice, benign and malignant tumors in the nasal cavity and esophagus of rats, and papilloma and carcinoma in the nasal cavity and trachea of Syrian golden hamsters (2). Autoradiographic studies with mice and rats, using 14 C-labeled and tritiated NNN have shown the highest degree of labeling in the olfactory region of the nose, especially in cells of the Bowman's gland (3). This phenomenon is apparently related to (i) the capacity of the cells in the olfactory region to 'Abbreviations NNN, A^-nitrosonornicotine; NDMA, W-nitiosodiiriethyiamine; OE, olfactory esthesioneuroepithelioma.
Fig. 1. Arborescent turbinate bone forming a labyrinth covered by respiratory mucosa of the nasal cavity of minks. The corresponding surface of a rat turbinate would be about the size shown in the framed area.
1957
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
5
metabolize and bind the active NNN metabolites, and (ii) the erectile vascular network which becomes engaged and provides the nasal mucosa with larger quantities of NNN for a longer period of time (4). To advance the study of tobacco-specific nitrosamine carcinogenesis further we decided to apply NNN to larger, non-rodent animals. We turned to the mink which is bred and raised in industrial settings and is readily available for research. As a subject for nasal carcinogenesis, in comparison with rats, mink have the advantage of a markedly expanded nasal mucosa by virtue of the luxuriant arborization of the turbinate bones (Figure 1). The carcinogens so far tested in mink are A'-nitrosodimethylamine (NDMA) and aflatoxin (5-7). The assay described in this paper was done with 20 mink which were 3 weeks old at the onset of the injections with NNN solution. The nitrosamine was given twice weekly for 38 weeks [total dose 11.9 mM of NNN (2.1 g)]. After an additional 144 weeks of observation the assay was terminated.
N.Koppang et al. Males
2400 2000 1600 -
i
if
Well
£
Females
/
1200 -
. — •
800 400 •
/
30
60
•
•
90 120 150 180 210 Age(days)
Fig. 2 . Weight curves of male and female mink maintained at the Norwegian College of Veterinary Medicine's Research Farm for Fur-bearing Animals, Heggedal, Norway.
lOOum
Table I. Mink exposed to N N N beginning at the age of 3 weeks No.
Sex
Died/killed* weeks after first injection
Remarks about terminal stage and macroscopic changes at autopsy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
M F F F F M F F F F F F F F F M M M M M
45 48 65 92 93 107 109 130 136 143 152 152 158 156 182 182 182 182 182 182
had liver failure (safran liver), no tumors moribund; killed, tumor in nose and forebrain died; cachetic, tumor in nose and forebrain moribund; killed, tumor in nose and forebrain moribund; killed, tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain moribund; killed, tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain anorexic and sick, tumor in nose and forebrain normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose
Fig. 3 . Tip of turbinate branch with image of respiratory squamous metaplasia (thick arrow) and adenosquamous papillary proliferation. Nasal respiratory epithelium marked by thin arrows.
^^m^%^^m^
'Animals 1 — 14 were moribund and killed; animals 15—20 were killed upon termination of the bioassay.
Bioassay Based on availability seven male and 13 female mink constituted the experimental group; four females were controls. At the age of 3 weeks the cubs began to receive s.c. injections of aqueous solutions of N N N close behind the scapula, twice weekly for 38 weeks. The first injection was only 1.5 mg N N N per cub, this dose was increased to 30 mg at week 8 (the mean for the first 5 weeks in this assay was 15 mg N N N per injection), whereafter the doses injected were constant for each mink. Given twice weekly for 38 weeks, the total dose amounted to 2130 mg N N N per animal; the same dose was given to females and males. The dose per kg body weight for males is therefore only about half of that given to the females. A complete autopsy of each mink was performed. The head, trachea, lung, esophagus, heart, liver, pancreas and kidney were fixed in neutral buffered formalin (pH 7.4—7.7). To search for tumors of the nasal cavity and forebrain, the head of each mink was skinned; mandibula, muscles and the dorsal part of the skull were removed before fixation in formalin for —2 days; then the brain was removed and inspected while the nose and skull were decalcified. The decalcified
1958
Fig. 4. Olfactory esthesioneuroepithelioma (OE) invading the brain. Perivascular round-cells inflammatory infiltrate at the border line between tumor and nervous substance. piece was cut into 3—4 mm thick slices, beginning at the cranial part of the nose and ending with the 7th piece which contained the rest of the forebrain that had not been removed with the brain. All pieces obtained were embedded in paraffin, processed and stained as reported earlier (5).
Results None of the mink showed any macroscopic changes upon external inspection. However, after removing the brain, tumorous tissues were recognized in the forebrain. In some of the animals such
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
•
0
F!g. 6. OE brain invasion, palisading near Wood vessels. At this level (arrows) the cells take an organoid arrangement, mimicking normal olfactory mucosa with neurons (clearer, nucleolated nuclei) and sustaining cells (darker nuclei). Many cells have a carrot or tadpole shape with one long end anchored on a basement-like membrane (arrows).
tumors were > 1 cm in diameter, often with small cysts. In the nose, tumor tissues were first seen after decalcification when the nose was sliced into pieces. Table I summarizes the results of the bioassay in mink in which a total dose of 11.9 mM of NNN (2.1 g) was given s.c. in 38 subdoses to 20 animals beginning at the age of 3 weeks. With the exception of one male, which died of liver insufficiency (safran liver), all of the remaining 19 mink developed tumors of the nasal cavity; of the 13 females, eight died, the other five were killed because of illness. None of the four control animals died of tumors or of other diseases; they were killed upon termination of the bioassay at an age of 185 weeks. Among the male mink at risk one died suddenly due to deep invasion of the nasal tumor into the forebrain; the remaining five males looked healthy when they were killed upon termination of the assay after 182 weeks. All of them were found to have nasal cavity tumors. The female mink exhibited tumors of the nasal mucosa at an earlier time; eight died, the other five had to be killed because of the extent of tumor growth. The average induction time for nasal tumors among females was 128 ± 23 weeks after the first NNN application. No tumors (nasal or other) were observed in the control animals, nor, for that matter, in other mink of our colony.
Fig. 7. Rosetting around vascular connective bands. Many cells have a foothold on some kind of membrane. These are neuroepithelial stem cells with some commitment towards olfactory differentiation.
Fig. 8. Flexner-Uke rosette (R) in olfactory tumor. 'Fleurette' arrangement. One of the cells is in prophase. A pycnotic nucleus is in the center. Wur^f*
^vv
• p*^h
— -II—
t
_
- _—
T-F—hffcVi n r a
A
v^« I **^±s>&* *+
»«* *^^1 A n *r*
*r*
i n
#«4^h
I ••>•• I ^ •
Fig. 9. Groups of poorly differentiated, neuroblast-like cells in olfactory tumor. Numerous mitoses.
The lesions induced in the treated animals showed various degrees of development from metaplastic and hyperplastic changes to malignant tumors aggressively invading the brain. All of the animals with tumors had multicentric neoplasms along the 1959
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
Fig. 5. OE invading brain; extensive palisading and resetting.
N.Koppang et al.
Fig. 10. Homer Wright rosette in olfactory tumor (R). 'Flower' arrangement.
nasal mucosa; both respiratory and olfactory parts were affected. In the respiratory part, we could see large benign tumors in various stages of development as squamous papillomas or adenosquamous tumors (Figure 3); the olfactory part also generated pleomorphic neoplasms from the Bowman's glands or from metaplastic olfactory cells. However, these were always outpaced by a more virulent tumor, the olfactory esthesioneuroepithelioma (OE), which secondarily invaded the brain. Most of these tumors display Flexner and Wright rosettes, palisades and lobulation, or just sheets of cells, medullary-like, still containing the two types of nuclei: neuronal (larger, vesicular, nucleolated) and neuroglial (smaller, more compact, darker). The typical elongated carrot-like and tadpole-like cells were abundantly present (Figures 4 - 1 0 ) . Discussion The induction of nasal cavity tumors in mink treated with NNN confirms that the tumorigenic activity of this compound is not limited to rodents. In fact, the mink seemed more sensitive than the rat in terms of nasal carcinogenesis, if we compare the period between injections and the appearance of tumors in relation to the normal life-span of the species. The mink also had an increased physiopathologic sensitivity to NNN, similar to that for NDMA and aflatoxin. We know that the mink is far more sensitive to NDMA and aflatoxin than are the common laboratory animals. The LD^ for NDMA in mink is 7 mg/kg body wt, in rats it is 40 mg/kg body wt (5,11). In the NDMA experiment with mink, a dose-related response is demonstrated in addition to organ specificity (5,7). The total absence of tumors in other organs is especially notable. Of the 63 N-nitrosamines known to induce nasal tumors in laboratory animals all induce benign and/or malignant tumors at other sites in the rat, primarily in the esophagus and liver, and in the Syrian golden hamster primarily in the liver, trachea and lung (12,13). The lack of tumors at other sites makes the treatment of mink with NNN especially suitable for model studies in nasal carcinogenesis. The expanded nasal mucosa of the mink makes this animal highly susceptible to nasal carcinogens. The shorter latency of the tumor development in the females is probably due to the significantly higher dose of NNN given to them during the application period (9.5 versus 5.7 mM NNN/kg given to each male); thus, we see, in fact, a dose-related response. All masses invading the brain had the distinctive characteristics 1960
Acknowledgements The synthesis of NNN and the study of its histopathological effects at the American Health Foundation were both supported by grant CA-29580 from the US National Cancer Institute.
References 1. Bmnnemann,K.D. and Hoffmann,D. (1991) Analytical studies on A'-nitrosamines in tobacco and tobacco smoke. Recent Adv. Tobacco Sd., 17, 71-112. 2. Hecht.S.S. and Hoffmann,D. (1989) The relevance of tobacco-specific nitrosamines to human cancer, Cancer Surv., 8, 273—294. 3. Tj&lve.H. (1991) The tissue distribution and the tissue specificity of bioactivation of some tobacco-specific and some other Af-nitrosamines. CRC Crit. Rev. ToxicoL, 21, 265-294. 4. Rivenson,A., Hecht,S.S. and Hoffrnann.D. (1991) Carcinogenicity of tobaccospecific A'-nitrosamines (TSNA): the role of the vascular network in the selection of the target organs. CRC Crit. Rev. Taricol., 21, 255-264. 5. Koppang.N. and Rimeslatten.H. (1976) Toxic and carcinogenic effects of nitrosodimethylamine in mink. In Walker.E.A., Bogovslri.P. and Griciute.L. (eds), Environmental N-Nitroso Compounds. Analysis and Formation. IARC Sd. Publ, 14, 443-452. 6. Koppang.N. and Helgebostad,A. (1972) Aflatoxinforgiftning hos mink. (Aflatoxin intoxication in mink.) Nord. Vet.-Med., 24, 213-219. 7. Koppang.N. and Helgebostad.A. (1987) Vascular changes and liver tumors induced in mink by high levels of nitrite in feed. In Bartsch,H., O'Neill,I. and Schulte-Hermann.R. (eds), The Relevance of N-Nitroso Compounds to Human Cancer: Exposures and Mechanisms, IARC Sd. Publ., 84, 256-260. 8. Hu,M.W., Bondinell.W.E. and Hoffmann.D. (1974) Chemical studies on tobacco smoke. XXH1. Synthesis of carbon-14 labeled myosmine, nornicotine and Af-nitrosonomicotine. /. Labeled Compd, 10, 7 9 - 8 8 . 9. Hecht.S., Chen.C.B., Dong,M., Omaf.R.M. and Hoffmann.D. (1977) Chemical studies on tobacco smoke. LI. Studies on non-volatile nitrosamines in tobacco. Beitr. Tabakforsch., 9, 1 —6. 10. Enggaard Hansen.N., Finne.L., Skrede.A. and Tauson,A.-H. (1991) Enegiforsyninger hos mink og raev. (Energy consumption by mink and fox.) Nordiske Jordbrukssforskeres Forening, JNF-Utredning Kobenhavn, p. 15. 11. Druckrey.H., Preussmann.R., Ivankovic.S. and Schmfihl.D. (1967) Organotrope carcinogene Wirkungen bei 65 verschiedenen A'-NitrosoVerbindungen an BD-Ratten. (Organotropic carcinogenicity of 65 different A'-nitroso compounds in BD rats.) Z. Krebsforsch., 69, 103-201. 12. Preussmann.R. and Stewart.B.W. (1984) A'-Nitroso carcinogens. In Searle.C.E. (ed.), Chemical Carcinogens. 2nd edn. Am. Chem. Soc Monogr., 182, pp. 643-828. 13. Lijinsky.W. (1987) Structure relations in carcinogenesis by A'-nitroso compounds. Cancer Metastasis Rev., 6, 301-356. Received on June 12, 1992; revised on July 29, 1992; accepted on August 5, 1992
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
of esthesioneuroepitheliomas which, as is the case in rats, become malignant very early, invading the brain, and therefore precluding the development and/or malignant conversion of tumors at other sites of the nasal mucosa. This bioassay demonstrates that NNN is a strong carcinogen in the mink. At a dose of 11.9 mM per animal NNN induces malignant tumors in the nasal cavity of all of the males and females within one-third of the life-span of the mink. In mice, rats and hamsters, NNN and other nitrosamines are known to elicit not only nasal rumors but also tumors at other sites of the aerodigestive tract. The absence of such tumors in the mink appears to make this animal a unique model for the study of early diagnosis, prevention and therapy of nasal cancer. The larger area of the nasal cavity of the mink as compared to rodents makes this model also especially suitable for studies on the metabolism, binding and molecular biology of nasal carcinogens.
A study of tobacco carcinogenesis XLVIII. carcinogenicity of W-nitrosonornicotine in mink (Mustela vison)
Nils Koppang1"2, Abraham Rivenson3, Albrecht Reith4, Hans K.Dahle2, 0ystein Evensen1 and Dietrich Hoffmann3*5 'National Veterinary Institute, N-OO33 Oslo 1, 2Norwegian College of Veterinary Medicine, N-0033 Oslo 1, Norway, 'American Health Foundation, Valhalla, NY 10595, USA and Norwegian Radium Hospital and Institute for Cancer Research, 0310 Oslo 3, Norway To whom requests for reprints should be sent
During tobacco processing and smoking, nicotine and nornicotine give rise to W-nitrosonornicotine (NNN), a highly abundant, strong carcinogen. NNN is known to exert carcinogenic activity in mice, rats and hamsters. Major target organs for NNN carcinogenicity in the rat are the esophagus and the nasal mucosa, and in the Syrian golden hamster trachea and nasal mucosa. In comparison with the rat, the mink (Mustela vison) has a markedly expanded nasal mucosa. Therefore, we explored in this study whether the mink could serve as a non-rodent model for nasal carcinogenesis using NNN as the carcinogen. Twenty random-bred mink, beginning at the age of 3 weeks, received twice weekly s.c. injections of NNN, a total dose of 11.9 mM per animal over a 38 week period. All of the 19 mink at risk developed malignant tumors of both the respiratory and the olfactory region of the nose within 3.5 years. In most animals the malignant rumors, primarily esthesioneuroepithelioma, invaded the brain. Remarkably, NNN induced no other tumors in the mink. None of the control animals developed nasal tumors nor tumors at other sites during the 3.5 years of the assay. The historical data from the farm did not reveal any spontaneous occurrence of nasal tumors in mink at any age. This study supports the concept that NNN is a proven carcinogen for multiple species of mammals and that the mink can serve as a non-rodent, non-inbred animal model for nasal carcinogenesis, especially since NNN induces only tumors in the nasal cavity in this species and not at other sites, as it does in mice, rats and hamsters.
Materials and methods Chemicals NNN was synthesized according to an earlier published method (8). Its purity was >99% according to GC, HPLC and MS (9). NNN is stable in an aqueous solution when stored at room temperature in the dark. Animals The random-bred mink were offspring from our breeding unit, which has an almost zero tumor incidence (at the Norwegian College of Veterinary Medicine's Research Farm for Fur-bearing Animals, Heggedal, Norway). For the first 7 weeks of their lives the cubs were nursed by their mothers. Subsequently, they were housed individually and fed a standard diet as reported earlier (7); water was offered ad libitum. At the age of 3 weeks the cubs weighed — 50 g; at the age of 65 days average weights were — 1000 g for the males and 750 g for the females. Males reach their maximal weight of -2300 g after 180 days, the maximal average weight for females, - 1200 g, is reached after 150 days (Figure 2) (10). The average life-span of these mink is assumed to be 10 years.
Introduction W-Nitrosonornicotine (NNN*) is the most abundant tobaccospecific A/-nitrosamine in tobacco and its smoke. It is formed from nicotine and nornicotine during tobacco processing and during smoking (1). NNN is a potent carcinogen that induces lung adenoma and adenocarcinoma in mice, benign and malignant tumors in the nasal cavity and esophagus of rats, and papilloma and carcinoma in the nasal cavity and trachea of Syrian golden hamsters (2). Autoradiographic studies with mice and rats, using 14 C-labeled and tritiated NNN have shown the highest degree of labeling in the olfactory region of the nose, especially in cells of the Bowman's gland (3). This phenomenon is apparently related to (i) the capacity of the cells in the olfactory region to 'Abbreviations NNN, A^-nitrosonornicotine; NDMA, W-nitiosodiiriethyiamine; OE, olfactory esthesioneuroepithelioma.
Fig. 1. Arborescent turbinate bone forming a labyrinth covered by respiratory mucosa of the nasal cavity of minks. The corresponding surface of a rat turbinate would be about the size shown in the framed area.
1957
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
5
metabolize and bind the active NNN metabolites, and (ii) the erectile vascular network which becomes engaged and provides the nasal mucosa with larger quantities of NNN for a longer period of time (4). To advance the study of tobacco-specific nitrosamine carcinogenesis further we decided to apply NNN to larger, non-rodent animals. We turned to the mink which is bred and raised in industrial settings and is readily available for research. As a subject for nasal carcinogenesis, in comparison with rats, mink have the advantage of a markedly expanded nasal mucosa by virtue of the luxuriant arborization of the turbinate bones (Figure 1). The carcinogens so far tested in mink are A'-nitrosodimethylamine (NDMA) and aflatoxin (5-7). The assay described in this paper was done with 20 mink which were 3 weeks old at the onset of the injections with NNN solution. The nitrosamine was given twice weekly for 38 weeks [total dose 11.9 mM of NNN (2.1 g)]. After an additional 144 weeks of observation the assay was terminated.
N.Koppang et al. Males
2400 2000 1600 -
i
if
Well
£
Females
/
1200 -
. — •
800 400 •
/
30
60
•
•
90 120 150 180 210 Age(days)
Fig. 2 . Weight curves of male and female mink maintained at the Norwegian College of Veterinary Medicine's Research Farm for Fur-bearing Animals, Heggedal, Norway.
lOOum
Table I. Mink exposed to N N N beginning at the age of 3 weeks No.
Sex
Died/killed* weeks after first injection
Remarks about terminal stage and macroscopic changes at autopsy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
M F F F F M F F F F F F F F F M M M M M
45 48 65 92 93 107 109 130 136 143 152 152 158 156 182 182 182 182 182 182
had liver failure (safran liver), no tumors moribund; killed, tumor in nose and forebrain died; cachetic, tumor in nose and forebrain moribund; killed, tumor in nose and forebrain moribund; killed, tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain moribund; killed, tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain died; tumor in nose and forebrain anorexic and sick, tumor in nose and forebrain normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose normal; killed, tumor in the nose
Fig. 3 . Tip of turbinate branch with image of respiratory squamous metaplasia (thick arrow) and adenosquamous papillary proliferation. Nasal respiratory epithelium marked by thin arrows.
^^m^%^^m^
'Animals 1 — 14 were moribund and killed; animals 15—20 were killed upon termination of the bioassay.
Bioassay Based on availability seven male and 13 female mink constituted the experimental group; four females were controls. At the age of 3 weeks the cubs began to receive s.c. injections of aqueous solutions of N N N close behind the scapula, twice weekly for 38 weeks. The first injection was only 1.5 mg N N N per cub, this dose was increased to 30 mg at week 8 (the mean for the first 5 weeks in this assay was 15 mg N N N per injection), whereafter the doses injected were constant for each mink. Given twice weekly for 38 weeks, the total dose amounted to 2130 mg N N N per animal; the same dose was given to females and males. The dose per kg body weight for males is therefore only about half of that given to the females. A complete autopsy of each mink was performed. The head, trachea, lung, esophagus, heart, liver, pancreas and kidney were fixed in neutral buffered formalin (pH 7.4—7.7). To search for tumors of the nasal cavity and forebrain, the head of each mink was skinned; mandibula, muscles and the dorsal part of the skull were removed before fixation in formalin for —2 days; then the brain was removed and inspected while the nose and skull were decalcified. The decalcified
1958
Fig. 4. Olfactory esthesioneuroepithelioma (OE) invading the brain. Perivascular round-cells inflammatory infiltrate at the border line between tumor and nervous substance. piece was cut into 3—4 mm thick slices, beginning at the cranial part of the nose and ending with the 7th piece which contained the rest of the forebrain that had not been removed with the brain. All pieces obtained were embedded in paraffin, processed and stained as reported earlier (5).
Results None of the mink showed any macroscopic changes upon external inspection. However, after removing the brain, tumorous tissues were recognized in the forebrain. In some of the animals such
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
•
0
F!g. 6. OE brain invasion, palisading near Wood vessels. At this level (arrows) the cells take an organoid arrangement, mimicking normal olfactory mucosa with neurons (clearer, nucleolated nuclei) and sustaining cells (darker nuclei). Many cells have a carrot or tadpole shape with one long end anchored on a basement-like membrane (arrows).
tumors were > 1 cm in diameter, often with small cysts. In the nose, tumor tissues were first seen after decalcification when the nose was sliced into pieces. Table I summarizes the results of the bioassay in mink in which a total dose of 11.9 mM of NNN (2.1 g) was given s.c. in 38 subdoses to 20 animals beginning at the age of 3 weeks. With the exception of one male, which died of liver insufficiency (safran liver), all of the remaining 19 mink developed tumors of the nasal cavity; of the 13 females, eight died, the other five were killed because of illness. None of the four control animals died of tumors or of other diseases; they were killed upon termination of the bioassay at an age of 185 weeks. Among the male mink at risk one died suddenly due to deep invasion of the nasal tumor into the forebrain; the remaining five males looked healthy when they were killed upon termination of the assay after 182 weeks. All of them were found to have nasal cavity tumors. The female mink exhibited tumors of the nasal mucosa at an earlier time; eight died, the other five had to be killed because of the extent of tumor growth. The average induction time for nasal tumors among females was 128 ± 23 weeks after the first NNN application. No tumors (nasal or other) were observed in the control animals, nor, for that matter, in other mink of our colony.
Fig. 7. Rosetting around vascular connective bands. Many cells have a foothold on some kind of membrane. These are neuroepithelial stem cells with some commitment towards olfactory differentiation.
Fig. 8. Flexner-Uke rosette (R) in olfactory tumor. 'Fleurette' arrangement. One of the cells is in prophase. A pycnotic nucleus is in the center. Wur^f*
^vv
• p*^h
— -II—
t
_
- _—
T-F—hffcVi n r a
A
v^« I **^±s>&* *+
»«* *^^1 A n *r*
*r*
i n
#«4^h
I ••>•• I ^ •
Fig. 9. Groups of poorly differentiated, neuroblast-like cells in olfactory tumor. Numerous mitoses.
The lesions induced in the treated animals showed various degrees of development from metaplastic and hyperplastic changes to malignant tumors aggressively invading the brain. All of the animals with tumors had multicentric neoplasms along the 1959
Downloaded from http://carcin.oxfordjournals.org/ at The University of British Colombia Library on June 19, 2015
Fig. 5. OE invading brain; extensive palisading and resetting.
N.Koppang et al.
Fig. 10. Homer Wright rosette in olfactory tumor (R). 'Flower' arrangement.
nasal mucosa; both respiratory and olfactory parts were affected. In the respiratory part, we could see large benign tumors in various stages of development as squamous papillomas or adenosquamous tumors (Figure 3); the olfactory part also generated pleomorphic neoplasms from the Bowman's glands or from metaplastic olfactory cells. However, these were always outpaced by a more virulent tumor, the olfactory esthesioneuroepithelioma (OE), which secondarily invaded the brain. Most of these tumors display Flexner and Wright rosettes, palisades and lobulation, or just sheets of cells, medullary-like, still containing the two types of nuclei: neuronal (larger, vesicular, nucleolated) and neuroglial (smaller, more compact, darker). The typical elongated carrot-like and tadpole-like cells were abundantly present (Figures 4 - 1 0 ) . Discussion The induction of nasal cavity tumors in mink treated with NNN confirms that the tumorigenic activity of this compound is not limited to rodents. In fact, the mink seemed more sensitive than the rat in terms of nasal carcinogenesis, if we compare the period between injections and the appearance of tumors in relation to the normal life-span of the species. The mink also had an increased physiopathologic sensitivity to NNN, similar to that for NDMA and aflatoxin. We know that the mink is far more sensitive to NDMA and aflatoxin than are the common laboratory animals. The LD^ for NDMA in mink is 7 mg/kg body wt, in rats it is 40 mg/kg body wt (5,11). In the NDMA experiment with mink, a dose-related response is demonstrated in addition to organ specificity (5,7). The total absence of tumors in other organs is especially notable. Of the 63 N-nitrosamines known to induce nasal tumors in laboratory animals all induce benign and/or malignant tumors at other sites in the rat, primarily in the esophagus and liver, and in the Syrian golden hamster primarily in the liver, trachea and lung (12,13). The lack of tumors at other sites makes the treatment of mink with NNN especially suitable for model studies in nasal carcinogenesis. The expanded nasal mucosa of the mink makes this animal highly susceptible to nasal carcinogens. The shorter latency of the tumor development in the females is probably due to the significantly higher dose of NNN given to them during the application period (9.5 versus 5.7 mM NNN/kg given to each male); thus, we see, in fact, a dose-related response. All masses invading the brain had the distinctive characteristics 1960
Acknowledgements The synthesis of NNN and the study of its histopathological effects at the American Health Foundation were both supported by grant CA-29580 from the US National Cancer Institute.
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of esthesioneuroepitheliomas which, as is the case in rats, become malignant very early, invading the brain, and therefore precluding the development and/or malignant conversion of tumors at other sites of the nasal mucosa. This bioassay demonstrates that NNN is a strong carcinogen in the mink. At a dose of 11.9 mM per animal NNN induces malignant tumors in the nasal cavity of all of the males and females within one-third of the life-span of the mink. In mice, rats and hamsters, NNN and other nitrosamines are known to elicit not only nasal rumors but also tumors at other sites of the aerodigestive tract. The absence of such tumors in the mink appears to make this animal a unique model for the study of early diagnosis, prevention and therapy of nasal cancer. The larger area of the nasal cavity of the mink as compared to rodents makes this model also especially suitable for studies on the metabolism, binding and molecular biology of nasal carcinogens.
