Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Micronucleus incidence and hematological effects in bullfrog tadpoles (Rana catesbeiana) exposed to 2-acetylaminofluorene and 2-aminofluorene


The micronucleus technique used withRana catesbeiana tadpoles is useful for the evaluation of aromatic amine toxicity. When exposed to increasing concentrations of the aromatic amine 2-acetylaminofluorene (2-AAF 1 × 10−8 M to 5 × 10−5 M),R. catesbeiana tadpoles responded with an increasing frequency of micronuclei (MN) in the peripheral blood. Micronuclei increased fourfold with the 5 × 10−5 M 2-AAF exposure. Micronuclei frequency significantly increased with concentrations as low as 5 × 10−8 M. The time from initial mutagen exposure to the time of maximum number of MN present in the peripheral blood occurred at two separate times: 2 d and 14 d after exposure. The occurrence of two MN frequency peaks may be due to the presence of two erythrocyte populations from two hemopoietic sources (liver and kidneys) in young tadpoles.

The occurrence of immature erythrocytes, as well as general degenerative changes [such as fragmentation (schistocytosis)] in the erythocytes and vacuolated cytoplasms suggested 2-AAF may contribute to hemolytic anemia effects. 2-AAF also increased the frequency of rubricytes found in the peripheral blood, but had no significant effect on the hematocrit values of animals exposed to concentrations up to 1 × 10−5 M. The tadpoles may have been producing rubricytes to replace the damaged red blood cells (RBCs). The direct-acting mutagen 2-aminofluorene (2-AF) was considerably more toxic to peripheral RBCs than the indirect-acting 2-AAF. Hematocrit values of the 2-AF (1 × 10−5 M)-exposed tadpoles declined severely (from 22 to 10%) because of a significant increase in osmotic fragility of erythrocytes, resulting in cell lysis.

The general nature of these stress responses indicates that measurement of tadpole blood characteristics, including erythrocyte morphology, may provide a sensitive means of early warning for some water quality changes adverse to aquatic life.

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


  1. Al-Subti K, Hardig J (1990) Micronucleus test in fish for monitoring the genotoxic effects of industrial waste products in the Baltic Sea, Sweden. Comp Biochem Physiol C Comp Pharmacol Toxicol 97(1):179–182

  2. Amphlett GE, Delow GF (1984) Statistical analysis of the micronucleus test. Mutation Res 128:161–166

  3. Andaya AA, Di Giulio RT (1987) Acute toxicities and hematological effects of two substituted naphthoquinones in channel catfish. Arch Environ Contain Toxicol 16:233–238

  4. Brown BA (1984) Hematology: principles and procedures. In: Routine hematology procedures. Lea Febiger, Philadelphia, Chap 2

  5. Broyles RH, Johnson GM, Maples PB, Kindell GR (1981) Two erythroporetic microenvironments and two larval red cell lines in bullfrog tadpoles. Develop Biol 81:299–314

  6. Buckley JA (1977) Heinz body hemolytic anemia in coho salmon (Oncorhynchus kisutch) exposed to chlorinated wastewater. J Fish Res Board Can 34:215–224

  7. Buckley JA, Whitmore CM, Matsuda RI (1976) Changes in blood chemistry and blood cell morphology in coho salmon (Oncorhynchus kisutch) following exposure to sublethal levels of total residual chlorine in municipal wastewater. J Fish Res Board Can 33:776–782

  8. Countryman PI, Heddle JA (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Res 41:321–332

  9. Diggs LW, Sturm D, Bell A (1985) The morphology of human blood cells, 5th ed. Abbott Laboratories, Abbott Park, IL

  10. El Kan E, Reichenbach-Klinke H (1974) Color atlas of the diseases of fish, amphibians, and reptiles. In collaboration with M. Landolt (ed.) TFH Publications, Inc, Ltd, NJ

  11. Fernandez M, Gauthier L, Jaylet A (1989) Use of a newt larvae forin-vivo genotoxicity testing of water results on 19 compounds evaluated by the micronucleus test. Mutagenesis 4(1):17–26

  12. Forman JL, Just JJ (1976) The life span of red blood cells in the amphibian larvae,Rana catesbeiana. Develop Biol 50:537–540

  13. Gauthier L, Levi Y, Jaylet A (1989) Evaluation of the clastogenicity of water treated with sodium hypochlorite or monochloramine using a micronucleus test in newt larvaePleurodeles waltilii. Mutagenesis 4(3):170–173

  14. Grinfeld S, Jaylet A, Siboulet R, Deparis P, Chouroulinkov I (1986) Micronuclei in red blood cells of the newtPleurodeles waltl after treatment with benzo(a)pyrene: dependence on dose, length of exposure, posttreatment time, and uptake of the drug. Environ Mutat 8:41–51

  15. Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW, Salamone MF (1983) The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency gene-tox program. Mutat Res 123:61–118

  16. Heddle JA, Lue CB, Saunders EF, Benz RD (1978) Sensitivity to five mutagens in Fanconi's anemia as measured by the micronucleus method. Cancer Res 38:2983–2988

  17. Hooftman RN, de Raat WK (1982) Induction of nuclear anomalies (micronuclei) in peripheral blood erythrocytes of the eastern mud-minnowUmbra pygmaea by ethyl methanesulfonate. Mutat Res 104:147–152

  18. Hose JE, Cross JN, Smith SG, Diehl D (1987) Elevated circulating erythrocyte micronuclei in fishes from contaminated sites off southern California, USA Mar Environ Res 22(3):167–176

  19. Hose JE, Hannah JB, Puffer HW, Landolt ML (1984) Histologic and skeletal abnormalities in benzo(a)pyrene-treated rainbow trout alevins. Arch Environ Contam Toxicol 3:675–684

  20. Krauter PW, Anderson SL, Harrison FL (1987) Radiation-induced micronuclei in peripheral erythrocytes ofRana catesbeiana: An aquatic animal model for in vivo genotoxicity studies. Environ Mol Mutagen 10:285–296

  21. Larsson A, Lehtinen K, Haux C (1980) Biochemical and hematological effects of a titanium dioxide industrial effluent on fish. Bull Environ Contam Toxicol 25:427–435

  22. MacGregor JT, Wehr CM, Gould DH (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. Environ Mutat 2:509–514

  23. McLeay DJ (1973) Effects of a 12-hr and a 25-day exposure to kraft pulp mill effluent on the blood and tissues of juvenile coho salmon (Oncorhynchus kitsutch). J Fish Res Board Can 30:395–400

  24. Manna GK, Sadhukhan A (1986) Use of cells of gill and kidney of tilapia fish in micronucleus test mnt. Curr Sci 55(10):498–501

  25. Miller EC, Miller JA (1969) Studies of the mechanism of activation of aromatic and amide carcinogens to ultimate carcinogenic electrophile reactants. Ann NY Acad Sci 163:731–750

  26. Miller JA (1970) Carcinogenesis by chemicals: An overview. Cancer Res 30:559–576

  27. Neumann HG (1984) Analysis of hemoglobin as a dose monitor for alkylating and arylating agents. Arch Toxicol 56:1–6

  28. Ohsawa M, Kawai K (1981) Cytological shift in lymphocytes induced by cadmium in mice and rats. Environ Res 24:192–200

  29. Reichenbach-Klinke H, El Kan E (1965) The principal diseases of lower vertebrates. In collaboration with M. Landolt (ed), TFH Publications, Academic Press, London

  30. Schmid W (1976) The micronucleus test for cytogenetic analysis. In: Hollaender A (ed) Chemical mutagens, Vol. 4. Plenum Press, NY, pp 31–53

  31. Sittig M (1985) Handbook of toxic and hazardous chemicals and carcinogens, 2nd ed. Noyes Publication, NJ, pp 455–456

  32. Smith CE (1968) Hematological changes in coho salmon fed a folic acid deficient diet. J Fish Res Board Can 25:125–156

  33. Smith CE, McLain LR, Zavgg WS (1971) Phenylhydrazine-induced anemia in chinook salmon. Toxicol Appl Pharmacol 20:73–81

  34. Taylor AC, Kollros JJ (1946) Stages in the normal development ofR. pipiens larva. Anat Rec 94:723

  35. Te-Hsiu M, Cabrera GL, Kim K (1987) The bullfrog (Rana catesbeiana) tadpole—a potential organism for cytogenetic bioassay for freshwater pollution. 1987 EMS Abstracts, p 65

  36. Thorgeirsson SS, Jollow DJ, Sasame HA, Green J, Mitchell JR (1973) The role of cytochrome P-450 in N-hydroxylation of 2-acetylaminofluorene. Mol Pharmacol 9:398–404

  37. Whitmore CM (1965) A microcytic anemia of juvenile chinook salmon resulting from diets deficient in vitamin E. Fish Comm Oreg, Portland, OR, p 31

Download references

Author information

Correspondence to P. W. Krauter.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Krauter, P.W. Micronucleus incidence and hematological effects in bullfrog tadpoles (Rana catesbeiana) exposed to 2-acetylaminofluorene and 2-aminofluorene. Arch. Environ. Contam. Toxicol. 24, 487–493 (1993). https://doi.org/10.1007/BF01146167

Download citation


  • Aromatic Amine
  • Osmotic Fragility
  • Water Quality Change
  • Micronucleus Frequency
  • Rana Catesbeiana