Skip to main content
SpringerLink
Log in

Interference of acetoxyalkyl-nitrosamines with limb bud differentiation in organ culture

  • Original Investigations
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

α-acetoxynitrosamines have been used as model compounds to study biological activity of N-nitrosamines. After hydrolytic cleavage they yield an “active intermediate” (the hydroxynitrosamine) which presumably also arises as metabolite of N-nitrosamines. We tested eight N-nitrosamines mono-substituted at theα-carbon with an acetoxy group for their teratogenic potential in a mouse limb bud culture system. The following results were obtained:

  1. 1.

    The methyl compound (MOAc-MNA) — a derivative of dimethynitrosamine — proved to be the strongest teratogen in this group of chemicals.

  2. 2.

    The tertiary-butyl derivative — releasing a carbonium ion of low chemical reactivity — possessed the lowest activity in our test system.

  3. 3.

    The primaryα-acetates with unbranched side chains (POAc-MNA or BOAc-MNA) were more active than the corresponding derivatives with branched side chains.

  4. 4.

    The secondaryα-acetate (EOAc-ENA) was clearly less active than the primaryα-acetate (EOAc-MNA).

  5. 5.

    A teratogenic potential could also be demonstrated in the organ culture system with the cyclic derivative: N-nitroso-α-acetoxy-pyrrolidine (NAPYR).

  6. 6.

    With the use of limb buds from 12-day-old mouse embryos the explants showed the highest susceptibility to the teratogens on the first day of culture. No effect could be produced if the substances were added to the culture medium at the third day of culture or later.

  7. 7.

    When initiating the culture with limb buds from 11-day-old mouse embryos the concentrations needed to induce typical effects were lower than those tested with 12-day-old explants.

  8. 8.

    Typical and pronounced impairment with morphogenetic differentiation could be induced by MOAc-MNA if the substance was present in the medium for less than 60 min — the shortest period tested was 15 min.

  9. 9.

    A different abnormality pattern could be induced with the various substances tested. This may partly be explained by a quite different stability of the compounds in the test system.

  10. 10.

    N-Nitrosamines must be expected to be highly teratogenic if they can be “activated” in embryonic tissues. Such an activation into potent electrophilic agents does not occur in rodents under normal conditions. It will be interesting to study if such activity can be “induced” in embryonic tissues of rodents or if it is already present in embryonic tissues of primates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DMNA:

Dimethyl-nitrosamine

MOAc-MNA:

N-Methyl-N-(α-acetoxy)methyl-nitrosamine

EOAc-MNA:

N-Ethyl-N-(α-acetoxy)methyl-nitrosamine

POAc-MNA:

N-Pro-pyl-N-(α-acetoxy)methyl-nitrosamine

BOAc-MNA:

N-Butyl-N-(α-acetoxy)methyl-nitrosamine

i-POAc-MNA:

N-Isopropyl-N-(α-acetoxy)methyl-nitrosamine

t-BOAc-MNA:

N-tert-Butyl-N-(α-acetoxy)methyl-nitrosamine

EOAc-ENA:

N-Ethyl-N-(α-acetoxy)ethyl-nitrosamine

NAPYR:

N-Nitroso-α-acetoxy-pyrrolidine

References

  • Appel KE, Frank N, Wiessler M (1981) Metabolism of nitrosoacetoxymethyl-methylamine in liver microsomes. Biochem Pharmacol 30:2767–2772

    PubMed  Google Scholar 

  • Barrach H-J, Baumann I, Neubert D (1978) The applicability of in vitro systems for the evaluation of the significance of pharmacokinetic parameters for the induction of an embryo-toxic effet. In: Neubert D, Merker H-J, Nau H, Langman J (eds) Role of pharmacokinetics in prenatal and perinatal toxicology. Thieme, Stuttgart, pp 323–336

    Google Scholar 

  • Barrach H-J, Neubert D (1980) Significance of organ culture techniques for evaluation of prenatal toxicity. Arch Toxicol 45:161–187

    PubMed  Google Scholar 

  • Bartsch H, Margison GP, Malaveille C, Camus AM, Brun G, Margison JM (1977) Some aspects of metabolic activation of chemical carcinogens in relation to their organ specificity. Arch Toxicol 39:51–63

    PubMed  Google Scholar 

  • Berman JJ, Rice JM, Wenk ML, Roller PP (1979) Intestinal tumors induced by a single intraperitoneal injection of methyl(acetoxymethyl)nitrosamine in three strains of rats. Cancer Res 39:1462–1466

    PubMed  Google Scholar 

  • Blankenburg G (1981) Limb bud organ cultures; Method description. In: Neubert D, Merker H-J (eds) Culture techniques, de Gruyter, Berlin, pp 590–593

    Google Scholar 

  • Blankenburg G, Bluth U, Neubert D (1981) On the significance of ascorbate and of cysteine on differentiation of limb buds in organ culture. In: Neubert D, Merker H-J (eds) Culture techniques, de Gruyter, Berlin, pp 197–206

    Google Scholar 

  • Bochert G (1975) Comparative studies on the formation of methylated bases in DNA of adult and fetal mouse tissues by dimethylnitrosamine in vivo. In: Neubert D, Merker H-J (eds) New approaches to the evaluation of abnormal embryonic development. Thieme, Stuttgart, pp 554–572

    Google Scholar 

  • Bochert G, Platzek T, Wiessler M (1981) Comparison of effects on limb development in vivo and in vitro using methyl(acetoxymethyl)nitrosamine. In: Neubert D, Merker H-J (eds) Culture techniques. de Gruyter, Berlin, pp 223–235

    Google Scholar 

  • Camus A-M, Wiessler M, Malaveille C, Bartsch H (1978) High mutagenicity of N-(α-acyloxy)alkylnitrosamines in S. typhimurium: model compounds for metabolically activated N,N-dialkylnitrosamines. Mutat Res 49:187–194

    PubMed  Google Scholar 

  • Chahoud I, Kwasigroch TE (1977) Controlled breeding of laboratory animals. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, pp 78–91

    Google Scholar 

  • Druckrey HR, Preussmann R, Ivankovic S, Schmähl D (1967) Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitrosoverbindungen an BD-Ratten. Z Krebsforsch 69:103–201

    PubMed  Google Scholar 

  • Eisenbrand G, Janzowski C, Preussmann R (1977) Analysis, formation and occurrence of volatile and non-volatile N-nitroso compounds: recent results. In: Tinbergen BJ, Krol B (eds) Proc. 2. Internatl. Symp. on Nitrite in Meat Products, Zeist, 1976. Wageningen, Centre for Agricultural Publishing and Documentation, pp 155–169

    Google Scholar 

  • Frank N, Janzowski C, Wiessler M (1980) Stability of nitrosoacetoxymethyl-methylnitrosamine in in vitro systems and in vivo and its excretion by the rat organism. Biochem Pharmacol 29:383–387

    PubMed  Google Scholar 

  • Habs M, Schmähl D, Wiessler M (1978) Carcinogenicity of acetoxymethyl-methyl-nitrosamine after subcutaneous, intraveneous and intrarectal application in rats. Z Krebsforsch 91:217–221

    Google Scholar 

  • Hecht SS, Chi-Hong BC, McCoy GD, Hoffmann D (1979)α-hydroxylation of N-nitrosopyrrolidine and N′-nitrosonornicotine by human liver microsomes. Cancer Lett 8:35–41

    PubMed  Google Scholar 

  • Hecker LI (1980) In vitro metabolism of N-nitrosopyrrolidine by rat lung subcellular fractions:α-hydroxylation in a non-target tissue. Chem-Biol Interact 30:57–65

    PubMed  Google Scholar 

  • Huang G-F, Mochizuki M, Anjo T, Okada M (1981) Mutagenicity of N-alkyl-N(α-acetoxyal-kyl)nitrosamines in V79 Chinese hamster cells in relation to alkylating activity. Gan 72:531–538

    PubMed  Google Scholar 

  • IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Some N-Nitroso Compounds. World Health Organization. vol 17. Lyon (1978)

  • Ivankovic S, Schmähl D, Zeller WJ (1974) N-Demethylierung des Carcinogens Dimethylnitrosamin durch embryonales menschliches Gewebe. Z Krebsforsch 81:269–272

    Google Scholar 

  • Johansson-Brittebo E, Tjälve H (1979) Studies on the distribution and metabolism of14C-dimethylnitrosamine in foetal and young mice. Acta Pharmacol Toxicol 45:73–80

    Google Scholar 

  • Kleihues P, Doerjer G, Keefer LK, Rice JM, Roller PP, Hodgson RM (1979) Correlation of DNA methylation by methyl(acetoxymethyl)nitrosamine with organ-specific carcinogenicity in rats. Cancer Res 39:5136–5140

    PubMed  Google Scholar 

  • Lessmöllmann U, Neubert D, Merker H-J (1975) Mammalian limb buds differentiation in vitro as a test system for the evaluation of embryotoxic effects. In: Neubert D, Merker H-J (eds) New approaches to the evaluation of abnormal embryonic development. Thieme, Stuttgart, pp 99–114

    Google Scholar 

  • Maekawa A, Ogiu T, Onodera H, Furuta K, Matsuoka C, Mochizuki M, Anjo T, Okada M, Odashima S (1982) Carcinogenicity of N-alkyl-N-(acetoxymethyl)Nitrosamines after subcutaneous injections in F-344 rats. J Cancer Res Clin Oncol 104:13–21

    PubMed  Google Scholar 

  • Magee PN, Barnes JM (1956) The production of malignant primary hepatic tumours in the rats by feeding dimethylnitrosamine. Br J Cancer 10:114–122

    PubMed  Google Scholar 

  • Mochizuki M, Suzuki E, Anjo T, Wakabayashi Y, Okada M (1979) Mutagenic and DNA-damaging effects of N-alkyl-N-(α-acetoxyalkyl)nitrosamines, models for metabolically activated N,N-dialkylnitrosamines, Gan 70:663–670

    PubMed  Google Scholar 

  • Mochizuki M, Anjo T, Okada M (1980) Isolation and characterization of N-alkyl-N-(hydroxymethyl)-nitrosamines from N-alkyl-N-(hydroperoxymethyl)nitrosamines by deoxygenation. Tetrahedron Lett 21:3693–3696

    Google Scholar 

  • Napalkov NP, Alexandrov VA (1968) On the effects of blastomogenic substances on the organism during embryogenesis. Z Krebsforsch 71:32–50

    PubMed  Google Scholar 

  • Neubert D, Barrach H-J (1977a) Significance of in vitro techniques for the evaluation of embryotoxic effects. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, pp 202–209

    Google Scholar 

  • Neubert D, Barrach H-J (1977b) Organotropic effects and dose-response relationships in teratology. In: Neubert D, Merker H-J, Kwasigroch TE (eds) Methods in prenatal toxicology. Thieme, Stuttgart, pp 405–412

    Google Scholar 

  • Neubert D, Barrach H-J, Merker H-J (1980) Drug-induced damage to the embryo or fetus. In: Grundmann E (ed) Current topics in pathology, vol 69. Springer, Berlin Heidelberg New York, pp 241–331

    Google Scholar 

  • Neubert D, Bluth U (1980) Limb bud organ cultures from mouse embryos after apparent induction of monooxygenases in utero. Effects of cyclophosphamide, Dimethylnitrosamine and some Thalidomide-derivatives. In: Neubert D, Merker H-J (eds) Culture techniques, de Gruyter, Berlin, pp 175–195

    Google Scholar 

  • Platzek T, Bochert G, Rahm U (1982) Embryotoxicity induced by alkylating agents. Teratogenicity of acetoxymethyl-methyl nitrosamine: Dose-response relationship, application route dependency and phase specificity. Arch Toxicol 51:1–25

    Google Scholar 

  • Pool BL, Wiessler M (1981) Investigations on the mutagenicity of primary and secondaryα-acetoxynitrosamines with Salmonella typhimurium: activation and deactivation of structurally related compounds by S-9. Carcinogenesis 2:991–997

    PubMed  Google Scholar 

  • Preussmann R, Schmähl D, Eisenbrand G (1977) Carcinogenicity of N-Nitrosopyrrolidine: Dose-response study in rats. Z Krebsforsch 90:161–166

    Google Scholar 

  • Roller PP, Shimp DR, Keefer LK (1975) Synthesis and solvolysis of methyl-(acetoxymethyl)nitrosamine. Solution chemistry of the presumed carcinogenic metabolite of dimethylnitrosamine. Tetrahedron Lett 25:2065–2068

    Google Scholar 

  • Stahlmann R, Bluth U, Neubert D (1981) Effects of some “indirectly” alkylating agents on differentiation of limb buds in organ culture. In: Neubert D, Merker H-J (eds) Culture techniques, de Gruyter, Berlin, pp 207–222

    Google Scholar 

  • Ward JM, Rice JM, Roller PP, Wenk ML (1977) Natural history of intestinal neoplasms induced in rats by a single injection of methyl(acetoxymethyl)nitrosamine. Cancer Res 37:3046–3052

    PubMed  Google Scholar 

  • Wiessler M (1975) Chemie der Nitrosamine II. Syntheseα-funktioneller Dimethylnitrosamine. Tetrahedron Lett 30:2575–2578

    Google Scholar 

  • Wiessler M, Schmähl D (1976) Zur carcinogenen Wirkung von N-Nitrosoverbindungen. 5. Mitteilung: Acetoxymethyl-Methyl-Nitrosamin. Z Krebsforsch 85:47–49

    Google Scholar 

  • Wiessler M, Habs M, Schmähl D (1978) Zur carcinogenen Wirkung von N-Nitrosoverbindungen. Z Krebsforsch 91:317–322

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Toxikologie und Embryopharmakologie, Freie Universität Berlin, Garystrasse 9, D-1000, Berlin 33

    Ralf Stahlmann, Ursula Bluth & Diether Neubert

  2. Deutsches Krebsforschungszentrum, Institut für Toxikologie und Chemotherapie, D-6900, Heidelberg, Federal Republic of Germany

    Manfred Wiessler

Authors
  1. Ralf Stahlmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ursula Bluth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manfred Wiessler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diether Neubert
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stahlmann, R., Bluth, U., Wiessler, M. et al. Interference of acetoxyalkyl-nitrosamines with limb bud differentiation in organ culture. Arch Toxicol 54, 109–129 (1983). https://doi.org/10.1007/BF01261380

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01261380

Key words

Search

Navigation