Carcinogenicity tests of acetoxymethylphenylnitrosamine and benzenediazonium tetrafluoroborate in Syrian hamsters

Cancer Letters, 15 (1982) 289-300 ElsevierlNorth-Holland Scientific Publishers Ltd.

289

CARCINOGENICITY TESTS OF ACETOXYMETHYLPHENYLNITROSAMINE AND BENZENEDIAZONIUM TETRAFLUOROBORATE IN SYRIAN HAMSTERS

BARRY GOLD and SHAHROKH SALMASI The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105 (U.S.A.) (Received 29 September 1981) (Revised version received 20 December 1981) (Accepted 21 December 1981)

SUMMARY

The metabolic activation of the esophageal carcinogen methylphenylnitrosamine (MPhN) via a-hydroxylation to hydroxymethylphenylnitrosamine (HO-MPhN) should afford benzenediazonium ion (BDI) as the ultimate electrophilic metabolite. To determine if this proposed activation pathway is accurate., BDI, as its tetrafluoroborate (BF,) salt, was tested by chronic subcutaneous injection and gavage in Syrian golden hamsters. Acetoxymethylphenylnitrosamine (AMPhN), which is rapidly hydrolyzed to HO-MPhN in vivo, was similarly tested by S.C. injection. AMPhN was weakly carcinogenic, while BDI-BF., did not induce a significant tumor incidence by subcutaneous administration. When orally administered, BDI was inactive. Both AMPhN and BDI-BF, were mutagenic only in Salmonella typhimurium strain TA1537 without enzymic activation. The parent nitrosamine, MPhN was also mutagenic in TA1537, but only with enzymic activation. The mechanistic and ehvironmental significance of these results are discussed.

INTRODUCTION

MPhN is a strong carcinogen in the rat when administered orally and induces tumors of the esophagus, pharynx and forestomach [l--4]. The lung is the primary target tissue in the mouse [5]. Since the currently accepted theory of nitrosamine activation involves an a-hydroxylation, i.e. dealkylation, BDI should be the ‘ultimate’ carcinogenic form of MPhN (see Fig. 1) [6,7]. Recently, Kroeger-Koepke and Michejda have demonstrated that demethylation of MPhN occurs in vitro in the presence of microsomal and so-called ‘pH 5 enzymes’ [ 81, further indicating that BDI 0304-3835/82/0000-0000/$02.75 o 1982 Elsevier/North-Holland

Scientific Publishers Ltd.

290 Ph-N(NOL-CH,OCOCH, AMPhN

esterase

t

e"Zyme

Ph-N(NO)-CH,

*

MPhN

Ph-N(NOl-CH,OH HO-MPhN

I

-CH,O

-OH4

Ph-N;

covalent

Ph-N=N-OH

binding

to cellular macromolecules

Fig. 1. Pathway

for the electrophilic

activation

of MPhN.

forms during the metabolism of this nitrosamine. AMPhN, a protected derivative of hydroxymethylphenylnitrosamine (HO-MPhN), the proximate carcinogenic form (see Fig. l), should be a directly acting carcinogen, because it will be rapidly hydrolyzed to HO-MPhN by endogenous esterases

[91. The biological activity of arenediazonium ions has been demonstrated by their mutagenicity in the Ames system. The insecticide p-dimethylaminobenzenediazonium sodium sulfonate salt shows dose-dependent mutagenicity in S. typhimurium TA 1537 and 1538 [lo], as does the heterocyclic diazonium ion, 4-diazoimidazole-5-carboxamide in TA 100

[Ill. Besides being of mechanistic interest, the carcinogenicity of arenediazonium ions may be environmentally significant because they are used specifically in the commercial synthesis of azo dyes [ 121 and generally as intermediates in other organic chemical syntheses [ 131. They have also been reported in the commercial mushroom Aguricus bisporus [14], although it is not clear if the 4-hydroxymethylbenzenediazonium ion, trapped as a derivative, is continually being generated in the mushroom or if it is stabilized by an, as yet unidentified, counter ion. The presence of this diazonium compound in the commercial mushroom has been recently re-confirmed (A. Ross, pers. comm.). Its carcinogenicity in the mouse has also recently

291

been reported [ 151, and the results show it to induce tumors of the skin and subcutis when injected S.C. However, control mice S.C. treated with NaBF4 developed subcutaneous tumors (6% incidence) at the injection site, making it impossible to rule out a synergistic effect between the diazonium ion and the BF, counter-ion in this species. Thus, because of mechanistic and environmental interest, we report herein the bioassay of AMPhN and BDI-tetrafluoroborate (BF,) by subcutaneous injection and BDI-BF4 by gavage, in Syrian golden hamsters. The hamster has been chosen for these studies because this species has been a useful animal model in evaluating other nitrosamines and activated nitrosamines administered subcutaneously [ 16-191. Mutagenicity studies are also discussed. MATERIALS

AND METHODS

Chemicals BDI-BF, was synthesized by standard procedures. The product was crystallized from acetone-ether to yield analytically pure material, which was stored in vacua at -20°C. AMPhN was prepared by a modification of the method of Saavedra [ 201. N-Phenylglycine (15.1 g, 0.1 mol) was dissolved in 5% HCl(500 ml) and treated with solid sodium nitrite (24.15 g, 0.35 mol) over a period of 2 h. The solid that formed was collected by filtration, washed with Hz0 and dried in vacua. This crude product afforded pale brown needles upon crystallization from diethyl ether; yield 14.5 g (81%); m.p. 97.5-99°C; UV(MeOH) 365 nm (sh, E = 200); NMR (C2HC13) 6 4.67 (s, 2H, CH,-N) and 7.48 (s, 5H, aryl). Elem. anal.: calc’d. for CsH,NZ03: C, 53.33%; H, 4.44%; N, 15.56%; found: C, 53.15%; H, 4.26%; N, 15.40%. The N-nitroso-N-phenylglycine (3.21 g, 17.1 mmol) was dissolved in 50 ml glacial acetic acid, in which potassium acetate (16.4 g, 191 mmol) was suspended. The mixture was cooled to 10°C and vigorously stirred under Nz atmosphere. Lead (IV) tetraacetate (11.5 g, 23.4 mmol) was slowly added in portions over 2.5 h. The reaction mixture was then filtered through Celite, the filtrate diluted with cold HZ0 (50 ml) and extracted with CH&l, (3 X 100 ml). The combined CH,C& extracts were washed with chilled 1% NaHC03 until all acetic acid that had been extracted into the organic layer was neutralized. The CHzC!lz solution was then washed with cold brine and dried over Na,SO,. After filtering through charcoal and concentrating in vacua, a brown residue was obtained, which was purified by dry column chromatography on silica gel (Woelm, activity 111/30 mm) using ether/hexane (1 : 1, v/v) to afford 700 mg (21%) of a pale yellow oil which was homogeneous by TLC (0.25 mm silica gel; ether/hexane, 1 : 1) and GLC (3% SE-30, column temperature 100°C); UV (MeOH) sh 368 nm (destroyed by addition of 5% methanolic KOH); IR (CH,C&) 3060(s), 3000(s), 2370(m), 2320(m), 1755(vs), 1500(m), 1425(m) and

292

1270 cm-’ (vs); NMR (C2HC13) 6 2.04 (s, lH, CH,-C=O of anti-isomer), 2.08 (s, lH, CH,-C=O of syn-isomer), 5.74 (s, 2H, CH2--N of syn-isomer), 6.43 (s, lH, CH*--N of anti-isomer) and 7.48 (m, 5H, aryl of syn- and anti-isomers). The ratio of syn to anti forms was 2 : 1 by NMR. This compound in the presence of either hog liver esterase (Sigma Type II) or alkali could be rapidly coupled to 1-naphthol to yield 2-phenylazo-lnaphthol, the same product obtained when authentic BDI-BF, is coupled to l-naphthol. Animals Randomly bred, 8-week-old Syrian golden hamsters from the Eppley Colony were housed by sex in plastic cages in groups of 5. Animals were fed Wayne pelleted diet (Allied Mills, Chicago, IL) and given water ad libitum. The animals were kept under standard conditions (21 f 5°C 55 + 5% humidity) and weighed once weekly. The acute toxicities (LDsO) of AMPhN (KC.) and BDI-BF4 (s.c. and orally) were determined by the method of Weil [ 211, using mortality within an 8day observation period. Each dose group and control group consisted of 15 females and 15 males. The animals were injected S.C. once per week with the prescribed dose of AMPhN in olive oil or BDI-BF,, in 0.9% saline (see Tables 1 and 2). For control purposes, only solvent was injected and for the BDI-BF, bioassay an additional control group received sodium tetrafluoroborate (NaBF,) in saline (Table 2). To test BDI-BF., by oral administration the compound was gavaged in distilled water once per week. For control, sodium tetrafluoroborate in distilled water was gavaged weekly (Table 3). All hamsters either died naturally or were killed when moribund. Animals were completely necropsied, organs were fixed in 10% buffered formalin, bones decalcified in D-Calcifier (Lemer Labs, Stamford, CN) and tissues embedded in Paraplast. Step sections were prepared from nasal cavities, larynx, trachea with stem bronchi, lungs, pancreas, urinary bladder with rectum and vagina or urinary bladder with Cowper’s gland and prostate. The paraplast sections were stained with hematoxylin and eosin, and when necessary, with phosphotungstic acid -hematoxylin, van Gieson’s and periodic acid -Schiff. Mutagenicity of AMPhN, BDI-BF4 and MPhN The mutagenicity of AMPhN, BDI-BF4 and MPhN was determined using a modification of the Ames liquid incubation assay [22] reported by Yahagi et al. [ 231. Solutions of AMPhN or MPhN in acetone and BDI-BF, in pH 7.4 buffer were made up just prior to use. The preparation of S-9 from rat liver was performed essentially as reported by Ames et al. [22], and 7.5 mg protein was added per plate. RESULTS

The LDso of AMPhN in Syrian golden hamsters

was determined

by S.C.

12.50 12.50 6.25 6.25 3.13 3.13 1.56 1.56 -

F M F M F M F M F M

AMPhN AMPhN AMPhN AMPhN AMPhN AMPhN AMPhN AMPhN Control Control

31.7 34.8 37.3 41.3 37.1 32.2 36.0 45.3 40.6 56.3

f f f f f f f * f f

20.9 14.5 17.3 17.9 12.0 11.8 8.4 11.2 12.8 26.0

Age at death (mean weeks * S.D.)

-

4 4 4 4 For For For For life life life life

Treatment period (weeks)

15 15 15 15 15 15

15 16 15 15

0

12 15 14 15 14 13

10 14 11 13

20

4 65 11 7 11

6 7 8 10

40

0 01 1 2 9

1 0 0 2

60

0 00 0 0 4

0 0 0 0

80

No. of hamsters at week

OF AMPhN TO SYRIAN GOLDEN HAMSTERS

0 00 0 0 0

0 0 0 0

100

lb (3@) 3b (58b’c) (27b,41e,35bqd) t lb 0 0 0

0 0 0 0

No. of animals with tumors at injection site (age at death)

f

D(l),Q(l) N4hE(1U’(1) ~____.

None A(3),T(l) A(l),N(l) E(1) (A(l), P(1) A(l),N(l) A(2),B(4),E(2)

Other tumor sites (no.)g

aSubcutaneously injected in olive oil. bMalignant fibrous histiocytoma. ‘Fibrosarcoma. dPulmonary metastases. eBenign histiocytoma. ‘Level of significance relation to control: P < 0.04. ‘A, adrenal gland-cortical adenoma; B, Cowper’s gland-adenoma; C, pancreas-islet cell adenoma; D, forestomach-papilloma; E, parathyroid-gland adenoma; F, uterus-leiomyoma; G, liver-cholangioma; H, liver-hemangioma; I, liver-Kuppfer cell sarcoma; J, malignant lymphoma; K, rhabdomyosarcoma; L, ovarythecoma; M, gall bladder-adenocarcinoma; N, harderian gland-adenoma; 0, mesenchymoma-malignant; P, thyroid gland-adenoma; Q, pancreas-ductular adenoma; R, Osteosarcoma; S, thyroid gland-carcinoma; T, leukemia; U, prostate glandadenoma; V, spleen hemangioma; W, brain germinoma; X, kidney hamartoma.

Dosea (mg/kg/week)

Sex

ADMINISTRATION

Compound

SUBCUTANEOUS

TABLE 1

F M F M

F M F M

F M F M F M

BDI-BF, BDI-BF, BDI-BF, BDI-BF.

BDI-BF, BDI-BF, BDI-BF, BDI-BF,

BDI-BF, BDI-BF, NaBF! NaBF, Control Control

1.19 1.19 19.0 19.0 -

4.75 4.75 2.38 2.38

19.00 19.00 9.50 9.50

Dose* (mg/kg/ week)

-

58.0 62.4 51.7 57.3 40.6 56.3

52.4 51.0 53.1 61.1

35.1 24.0 37.7 61.0

t f * * * f

f f f f

f f f f

,,,,

/,)

,,

,,,,

F

,*

8.8 13.4 14.0 20.5 12.8 26.0

18.1 13.4 12.9 17.4

-

For For For For

*

life life life life

10 10 For life For life

10 10 10 10

Treatment period (weeks)

,,

15 15 15 15 15 15

15 15 15 15

15 15 16 15

0

,,

15 15 15 15 147 13

13 15 15 15

10 6 13 15

20

11

14 14 11 10

11 13 12 13

8 3 6 12

40

,,

,,,

,,

7 9 4 8 20 9

5 6 5 8

10 0 2 7

60

,,,

4

0 1 1 2

0 0 0 3

0 1 4

80

No. of hamsters at week

,,

0 0 0 0 00 0

0 0 0 0

0 0 0 0

100

OF BDI-BF, TO SYRIAN GOLDEN HAMSTERS

19.4 14.2 20.8 23.5

Age at death (mean weeks * S.D.)

%ubcutaneously injected 0.9% saline. bMalignant schwannoma. ‘Benign schwannoma. dSodium tetrafluoroborate. eSee Table 1.

Sex

Compound

SUBCUTANEOUS ADMINISTRATION

TABLE 2

0

/,

,,I

0 2b ( 71b,84C) 0 0

0 0 2 (50b, 66b) 1 (84b)

0 0 0 0

8,

,,8I,I

No. of animals with tumors at injection site (age at death)

E(1),P(1),Q(1) -WMV),D(2) W),W),J(W(1) -‘V4W(1UW),Q(1) A(4LB(4W(l) P(lLQU) B(4LWLH(1),J(1) D(1),Q(l) A(4UW),P(1)

None C(l),D(l),N(l) A(4),B(l),C(l) D(2),E(l),S(l) P(1) B(4)

J(l),O(l)

Other tumor sites (no.)’

35.0

35.0

17.5 17.5 8.75 8.75 21.2 21.2 ( -

F

M

F M F M F M F

M

BDTF

BDTF

BDTF BDTF BDTF BDTF NaBDTFb NaBDTF Control

Control

f f f * * f f

15.8 27.1 18.0 19.8 21.4 18.8 23.3

63.7 * 23.8

48.9 60.4 49.1 47.1 40.6 45.1 59.8

58.7 f 31.2

55.4 f 20.7

-

For For For For For For -

life life life life life life

For life

For life

15

15 15 15 15 15 15 15

15

15

0

15

15 13 12 14 14 14 14

12

14

20

14

9 12 7 9 10 9 11

11

11

40

11

5 10 2 2 4 4 7

9

8

60

5

1 3 1 1 0 1 3

4

2

80

No. of hamsters at week

Treatment period

Age at death (mean weeks f S.D.)

TETRAFLUOROBORATE

OF BENZENEDIAZONIUM

‘Intragastrically administered in distilled water. bSodium tetrafluoroborate. ‘See Table 1.

-

Dose* (mg/kg/ week)

Sex

Compound

ORAL ADMINISTRATION

TABLE 3

0

0 0 0 0 0 0 0

0

0

100

1

1 2 0 0 1 1 0

1

1

No. of forestomach tumor-bearing animals (age at death)

Other tumor sites (no.)”

(BDTF) TO SYRIAN GOLDEN HAMSTERS

296

injection in olive oil vehicle to be 134.0 mg/kg and 116.6 mg/kg in females and males, respectively. Animals were injected weekly S.C. with AMPhN in olive oil at 12.5, 6.25 and 3.13 mg/kg, which correspond to l/10, l/20 and l/40 of the average acute LDsO (Table 1). At the 2 higher doses, animals developed serious sores at the injection site and therefore treatment was terminated after 4 weeks for these 2 dose groups. A lower dose of 1.56 mg/kg was then initiated. The results of this experiment appear in Table 1. It should be pointed out that the life spans of both control and treated animals were poor (Tables 1 -3) and the total number of animals at risk was low. The acute LDSO of BDI-BF4 in hamsters by S.C. injection in 0.9% saline was 166.2 mg/kg and 219.2 mg/kg in females and males, respectively. For chronic testing, freshly prepared solutions of BDI-BF4 were injected S.C. weekly at 19, 9.5 and 4.75 mg/kg. One control group received S.C. injections of saline solution and another 11.5 mg/kg of sodium tetrafluoroborate in 0.9% saline, a dose corresponding to the molar equivalent of the highest dose (19 mg/kg) of BDI-BF4 used (Table 2). At these 3 doses of BDI-BF,, the hamsters rapidly developed serious abscesses and open sores at the injection sites and treatment was halted after 10 weeks. An additional 2 doses at 2.38 mg/kg and 1.19 mg/kg were started and these animals were treated for life. The carcinogenicity results are shown in Table 2. BDI-BF4 was less toxic by intragastric administration, with the acute LDso being 353.7 mg/kg in both females and males. Animals were gavaged weekly with BDI-BF, dissolved in distilled water at doses of 35.0, 17.5 and 8.75 mg/kg. Sodium tetrafluoroborate in distilled water was gavaged for control purposes at a dose of 21.2 mg/kg which corresponded to the molar equivalent of the highest oral dose of BDI-BF4. No significant qualitative or quantitative difference in tumors was found between treated and control animals (Table 3). Mutagenicity Evidence for the mutagenicity of AMPhN, BDI-BF4 and MPhN was obtained in S. typhimurium TA1.537. AMPh induced revertants in a dosedependent manner from 515.5 nmol to 64.6 nmol (Table 4). BDI-BF4 also showed mutagenic activity, although it was toxic above 260.4 nmol level (Table 4). MPhN was also mutagenic in TA1537 but only in the presence of an activating enzyme system. All 3 compounds were inactive in TA1535, with or without S-9 activation. DISCUSSION

As anticipated, AMPhN induced a statistically significant number of injection site tumors (P < 0.04), although the tumor incidence was unexpectedly low (Table 1). Even more surprising is that BDI-BF4 did not afford a statistically significant number of tumors upon S.C. injection.

291

TABLE 4 MUTAGENICITY

OF AMPhN, BDI-BF, AND MPhN

Compounda

Amount (nmol/plate)

His’ revertants/

AcetoneC Buff erd AMPhNe

0 0 515.5 386.6 257.8 128.9 64.4 260.4 130.2 65.1 32.6 1838.2 735.3 275.9

14 * 3 14 f 5 211 f 56 123*32 91 +_22 48 f 4 28 f 4 7 f 3 (toxic) 91 f 5 55 f 3 39 * 10 65 f 11 57 f 18 617 * 79

BDI-BFge

MPhNf 2-Aminoanthracenef

plateb

aAMPhN added in acetone; BDI-BF, added in pH 7.4 phosphate buffer. bSalmonello typhimurium TA1537 (see Methods). ‘10 ~1 used in control. d10 ~1 used in control. eResults are without S-9 fraction. f Results are with S-9 fraction (7.5 mg protein/plate).

The relatively weak carcinogenic activity of AMPhN and inactivity of BDIBF* may be a result of the nature of BDI, the ‘ultimate’ carcinogenic form for both compounds. Although BDI could arylate cellular macromolecules by a heterolytic or homolytic mechanism [ 241, formation of azo coupling products to protein and nucleic acid probably predominates [ 25--271, An alternative explanation is that poor survival time in both treated and untreated hamsters (Tables l-3) could result in a low tumor incidence if both AMPhN and BDI-BF4 required a long tumor induction period. Because the tumors observed are not particularly fatal, no correlation can be made between survival time and tumor latency, and thus it is not clear what effect the poor survival rates had on tumor incidence. In contrast to AMPhN, l-acetoxypropylpropylnitrosamine induced a 90% sarcoma incidence at the injection site with an average latency period (time at which tumorbearing animals died) of 30 weeks when administered subcutaneously to Syrian golden hamsters [ 161. Mutagenicity testing of AMPhN, BDI-BF4 and MPhN showed all 3 compound5 to be active in S. typhimurium TA1537, the latter compound only with enzyme activation (Table 4). While other diazonium ions have manifested mutagenic activity in TA1537 [lO,ll], which carries a frameshift mutation, no dialkyl-nitrosamine or ol-acetoxynitrosamine has been reported to be active in this tester strain [ 28-341. a-Acetoxynitrosamines and the

298

corresponding parent nitrosamines are mutagenic in TA1535 and TA98, which carry point mutations. The only reported exception is another methylarylnitrosamine, 2-nitrosomethylaminopyridine, which is active in TAlOO [35]. We assume that the anomalous behavior of AMPhN and MPhN is due to the formation of covalently bound products by an azo coupling reaction, in which the nitrogens are retained. In addition to being structurally different from alkylation adducts, in which the nitrogens are lost, arenediazonium ions attack different sites on cellular macromolecules than do the reaction intermediates responsible for alkylation of DNA by dialkylnitrosamines. In light of the evidence above, which is consistent with the activation pathway in Fig. 1, we can better understand why MPhN and other asymmetrical nitrosamines (e.g methylbenzyl-, methylarnyl-, methylcyclohexylnitrosamine) [l] mainly induce esophageal tumors in rats. Post-activation (cu-hydroxylation) factors, including the site and structure of covalently bound adducts, chemical stability of adducts and specificity and rates of enzyme repair do not appear to play a role in the observed organ specificity, since MPhN reacts with cellular macromolecules by a different mechanism than do these other nitrosamines [25-271. It is far more logical to conclude, as Hodgson, Wiessler and Kleihues have proposed, that the induction of esophageal tumors by certain nitrosamines in the rat may be explained by the preferential bioactivation of the carcinogen in the target organ [ 361 prior to covalent binding. In conclusion BDI, produced by the e-hydroxylation of MPhN [37], the hydrolyses of AMPhN or the ionization of BDI-BFI, appears to be a directly acting carcinogen, although it is less potent than anticipated. Oral ingestion of BDI-BF4 was innocuous. This latter result agrees with the failure of 4-hydroxymethylbenzenediazonium ion to induce tumors by repeated oral ingestion in the mouse (B. Toth and D. Nagel, pers. comm.), further indicating that orally ingested arenediazonium ions which may be present in mushrooms [14] may not be a factor in human cancer. ACKNOWLEDGEMENTS

We would like to thank Dr. K. Patil for help in the statistical analyses, Dr. E. Rogan and Ms. B. Walker for Ames studies, Ms. K. Stepan and Mr. L. Hines for technical assistance and Ms. M. Susman for editorial help. This work was funded by Public Health Service Contracts CP33278 and CP05627 from the Division of Cancer Cause and Prevention of the National Cancer Institute. REFERENCES 1 Druckrey, H., Preussmann, R., Ivankovic, S. and Schmiihl, D. (1967) Organotrope carcinogene Wirkung bei 65 verschiedenen N-nitroso-Verbindungen an BD-Ratten. Z. Krebsforsch., 69, 103-201. 2 Goodall, C.M., Lijinsky, W., Tomatis, L. and Wenyon, C.E.M. (1970) Toxicity and

299

3

4 5

6

7 8

9

10

11

12 13 14 15

16

17 18 19

20 21 22

oncogenicity of nitrosomethylaniline and nitrosomethylcyclohexylamine. Toxicol. Appl. Pharmacol., 17, 426-432. Boyland, E., Roe, F.J.C., Gorrod, J.W. and Mitchley, B.C.V. (1964) The carcinogenicity of nitrosoanabasine, a possible constituent of tobacco smoke. Br. J. Cancer, 18, 265-270. Napalkov, N. and Pozhariaski, K.M. (1969) Morphogenesis of experimental tumors of the esophagus. J. Natl. Cancer Inst., 42, 922-Q40. Greenblatt, M., Mirvish, S.S. and So, B.T. (1971) Nitrosamine studies: induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. Natl. Cancer Inst., 46, 1029-1034. Druckrey, H., Steinhoff, D., Preussmann, R. and Ivankovic, S. (1963) Krebsforschung durch einmalige Dosis von Methylnitrosoharnstoff und verschiedenen Dialkylnitrosaminen. Naturwissenschaften, 24, 735-736. Magee, P.N. and Barnes, J.M. (1967) Carcinogenic nitroso compounds. Adv. Cancer Res., 10, 163-246. Kroeger-Koepke, M.B. and Michejda, C.J. (1979) Evidence for several demethylase enzymes in the oxidation of dimethylnitrosamine and phenylmethylnitrosamine by rat liver fractions. Cancer Res., 39, 1587-1591. Kleihues, P., Doerjer, G., Keefer, L.K., Rice, J.M., Roller, P.P. and Hodgson, R.M. (1979) Correlation of DNA methylation by methyl(acetoxymethyl)nitrosamine with organ-specific carcinogenicity in rata. Cancer Res., 39, 5136-5140. Kada, L., Moriya, M. and Shirasu, Y. (1974) Screening of pesticides for DNA interactions by “REC-ASSAY” and mutagenesis testing and frameshift mutagens detected. Mutat. Res., 26, 243-248. Lower, Jr., G.M., Lanphear, S.P., Johnson, B.M. and Byran, G.T. (1977) Aryl and heterocyclic diazo compounds as potential environmental electrophiles. J. Toxicol. Environ. Health., 2, 1095-1107. Zolinger, H. (1973) Reactivity and stability of arenediazonium ions. Act. Chem. Res., 6, 335-341. Zollinger, H. (1961) Azo and Diazo Chemistry, Aliphatic and Aromatic Compounds. Wiley Interscience, New York. Levenberg, B. (1962) An aromatic diazonium compound in the mushroom Agaricus bisporus. B&him. Biophys. Acta, 63, 212-214. Toth, B., Patil, K. and Hwan-Soo, J. (1981) Carcinogenesis of 4-(hydroxymethyl)benzenediazonium ion (tetrafluoroborate) of Agaricuo bisporus. Cancer Res., 41, 2444-2449. Althoff, J., Grandjean, C., Pour, P. and Gold, B. (1977) Local and systemic effects of 1-acetoxypropylpropylnitrosamine in Syrian golden hamsters. Z. Krebsforsch., 90,127-140. Althoff, J., Grandjean, C., Gold, B. and Runge, R. (1977) Carcinogenicity of loxopropylnitrosamine (N-nitroso-N-propyl-proprionamide) in Syrian hamsters. Z. Krebsforsch., 90, 221-225. Althoff, J., Grandjean, C. and Gold, B. (1977) Diallylnitrosamine: a potent respiratory carcinogen in Syrian golden hamsters: brief communication. J. Natl. Cancer Inst., 59,1569-1571. Gold, B., Salmasi, S., Linder, W. and Althoff, A. (1981) Biological and chemical studies involving methyl-t-butylnitrosamine, a non-carcinogenic nitrosamine. Carcinogenesis, 2, 529-532. Saavedra, J.E. (1978) Oxidative decarboxylation of nitrosoamino acids: a synthetic approach to cyclic a-acetoxynitrosamines. Tetrahedron Lett., 1923-1926. Weil, C. (1952) Tables for convenient calculation of median effective dose (LD,, or ED,,) and instructions in their use. Biometrics, 8, 249-263. Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods of detecting carcinogens and mutagens with the Salmonella/mammalian microsomes mutagenicity test. Mutat. Res., 31, 347-364.

300 23 Yagi, T., Nagao, M., Seino, V., Matsushima, T., Sugimura, T. and Okada, M. (1977) Mutagenicities of N-nitrosamines on Salmonella. Mutat. Res., 48, 121-130. 24 Zollinger, H. (1978) Nitrogen as leaving group: dediazoniations of aromatic diazonium ions. Angew. Chem., Int. Ed., 17, 141-150. 25 Moudrianakis, EN. and Beer, M. (1965) Determination of base sequence in nucleic acids with the electron microscope. II. The reaction of a guanine-selective marker with the mononucleotides. B&him. Biophys. Acta, 95, 23-39. 26 Moudrianakis, E.N. and Beer, M. (1965) Base sequence determination in nucleic acids with the electron microscope, III. Chemistry and microscopy of guaninelabeled DNA. Biochemistry, 53, 564-571. 27 Chin, A., Hung, M.-H. and Stock, L.M. (1981) Reactions of benzenediazonium ions with adenine and its derivatives. J. Org. Chem., 46, 2203-2207. 28 Okada, M., Suzuki, E., Anjo, T. and Mochizuki, M. (1975) Mutagenicity of o-acetoxydialkylnitrosamines: model compounds for an ultimate carcinogen. Gann, 66, 457458. 29 Bartsch, H., Camus, A. and Malaveille, C. (1976) Comparative mutagenicity of Nnitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions. Mutat. Res., 37, 149-162. 30 Bartsch, H., Malaveille, C. and Montesano, R. (1976) In vitro metabolism and microsome-mediated mutagenicity of dialkylnitrosamines in rat, hamster and mouse tissues. Cancer Res., 35, 644+51. 31 Tannenbaum, S.R., Kraft, P., Baldwin, J. and Branz, S. (1977) The mutagenicity of methylbenzylnitrosamine and its ol-acetoxy derivatives. Cancer Letters, 2, 305310. 32 Camus, A.M., Wiessler, M., Malaveille, C. and Bartsch, H. (1978) High mutagenicity of N-(cu-acyloxy)-alkyl-N-alkylnitrosamines in S. typhimuriumr model compounds for metabolically activated N,N-dialkylnitrosamines. Mutat. Res., 49, 187-194. 33 Jacobson, M.K., Barton, R.A., Yoder, S.S., Singh, S. and Lyle, R.E. (1979) Mutagenicity of the non-carcinogenic dibenzylnitrosamine and an alpha-acetoxy derivative. Cancer Letters, 6, 83-87. 34 Masataka, M., Suzuki, E., Anjo, T., Wakabayashi, Y. and Okada, M. (1979) Mutagenic and DNA-damaging effects of N-alkyl-N-(cu-acetoxyalkyl)nitrosamines, models for metabolically activated N,N-dialkylnitrosamines. Gann, 70, 663470. 35 Preussmann, R., Habs, M. and Pool, B.L. (1979) Carcinogenicity and mutagenicity testing of three isomeric N-nitroso-N-methylaminopyridines in rats. J. Natl. Cancer Inst., 62, 153-156. 36 Hodgson, R.M., Wiessler, M. and Kleihues, P. (1980) Preferential methylation of target organ DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine. Carcinogenesis, 1, 861-866. 37 Druckrey, H. (1975) Chemical carcinogenesis on N-nitroso derivatives. Gann, 17, 107-132.