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J. PROTOZOOL., VOL. 39, NO. 4, JULY-AUGUST 1992

ACKNOWLEDGMENTS W e acknowlege support from the U S . Agency for International Development (DPE-0453-00-4027-00), the U.S. Public Health Service (AT-10645) a n d the United Nations Development Program for Research a n d Training in Tropical Medicine. We thank Kiet Dan Luc for his excellent technical assistance. LITERATURE C I T E D 1. Aikawa, M., Carter, R., Ito, Y. & Nijhout, M. 1984. New observations on gametogenesis, fertilization and zygote formation in Plasmodium gallinaceum. J. Protozool., 31:403-4 13. 2. Canning, E. U. & Sinden, R. E. 1973. The organization of the ookinete and observations on nuclear division in oocysts of Plasmodium berghei. Parasitology. 67:2940. 3. Garnham, P. C. C., Bird, R. G. & Baker, J. R. 1962. Electron microscope studies of motile stages of malaria parasites Ill. The ookinetes of Haemamoeba and Plasmodium. Trans. R. SOC. Trop. Med. HVR.. 56:l 16-120. 4. Huber, M.. Cabib, E. & Miller, L. H. 1991. Malaria parasite chitinase and penetration of the mosquito peritrophic membrane. Proc. Natl. Acad. Sci. USA, 88:2807-2810. 5. Kaushal, D. C., Carter, R., Rener, J., Grotendorst, C. A., Miller, L. H. & Howard, R. J. 1983. Monoclonal antibodies against surface determinants on gametes of Plasmodium gallinaceum block transmission of malaria parasites to mosquitoes. J. Immunol.. 131:2557-2562. 6. McCutchan, T. F., Dame, J. B., Miller, L. H. & Barnwell J. 1984. Evolutionary relatedness of Plasmodium species as determined by the structure of DNA. Science, 225:808-8 1 1. 7. Mehlhorn, H., Peters, W. & Haberkorn, A. 1980. The formation

of ookinetes and oocysts in Plasmodium gallinaceum (Haemosporidia) and considerations on phylogenetic relationships between Haemosporidia, Pirpolasmida and other coccidia. Protistologica, 16: 135-1 54. 8. Meis, J. F. G. M. & Ponnudurai, T. 1987. Ultrastructural studies on the interaction of Plasmodium falciparum ookinetes with the midgut epithelium of Anopheles stephensi mosquitoes. Parasitol. Rex, 73:500508. 9. Meis, J. F. G. M., Pool, G., Gemert, G. J. van, Lensen, A. H. W., Ponnudurai, T. & Meuwissen, J. H. E. T. 1989. Plasmodium falciparum ookinetes migrate intercellularly through Anopheles stephensi midgut epithelium. Parasitol. Rex. 76:13-19. 10. Rudin, W. & Hecker, H. 1989. Lectin-binding sites in the midgut of the mosquitoes Anopheles stephensi Liston and Aedes aegypti L. (Diptera: Culicidae). Parasitol. Res., 75:268-279. 11. Sieber, K. P., Huber, M., Kaslow, D., Banks, S. M., Toni, M., Aikawa. M. & Miller, L. H. 1991. The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes. Exp. Parasitol., 72: 145-1 56. 12. Stohler, H. 1957. Analyse des infektionsverlaufes von Plasinodium gallinaceum im darme von Aedes aegypfi. Arta Trop. (Basel), 14:302-352. 13. Syafruddin, Arakawa, R., Kamimura, K. & Kawamoto, F. 199 1. Penetration of the mosquito midgut wall by the ookinetes cf Plasmodium yoelii nigeriensis. Parasitol. R e x , 77:2 30-2 36. 14. Waters, A. P., Higgins, D. G., & McCutchan, T. F. 199 1. Plasmodiurn.falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proc. Narl. Acad. Sci. USA, 88:31403 144.

Received 10-1-91, 12-30-91; accepted 2-13-92

J. Profozwl.. 39(4). 1992, pp. 453-456 0 1992 by the Society of Prot0200logi~t~

Genotoxic Effects of Linear Alkyl Benzene Sulfonate, Sodium Pentachlorophenate and Dichromate on Tetrahymena pyriformis YUPING WU and YUNFEN SHE” Institulc qf Hydrobiology Academia Sinica. U’uhan. 430072, People> Republic of China

ABSTRACT. DNA in macro- and micronuclei of Tetrahymena pyriformis treated with linear alkyl benzene sulfonate (LAS) and sodium pentachlorophenate (PCP-Na) were determined by microspectrophotometry. The effects on rate of formation of macronuclear DNA extrusion bodies were also studied. We found DNA content of micronuclei in 0.14 ppm LAS and 0.9 ppb PCP-Na was lower than in that of the control, and LAS was able to increase the formation rate of macronuclear DNA extrusion bodies (the formation rate was 54% in 11.3 ppm LAS and 25.6% in 16.7 ppm dichromate). We concluded that 0.14 ppm LAS (below the maximum acceptable toxicant concentration) was genotoxic, whereas 0.014 ppm LAS was not. Dichromate 0.05 ppm and 0.9 ppb PCP-Na, equal to and below the maximum acceptable toxicant concentration, respectively, were potentially genetoxic. Key words. DNA. macronuclear DNA extrusion bodies, micronucleus, microspectrophotometry.

0

VER 1,000 chemicals are introduced in the world each year. These chemicals may not only be acutely toxic but may also have long-term harmful effects. Among these chemicals, insecticides, detergents a n d heavy metals deserve particular attention. In addition t o safe levels a n d mechanisms of toxicities, potential genetic effects are important. T h e detergents used in China are primarily linear alkyl benzene sulfonates (LAS). Judging from global trends, LAS will remain the major detergent, and hence will continue t o accumulate in the environment. I n China sodium pentachlorophenate (PCP-Na). which is widely used as a preservative, insec-

‘ To whom correspondence should be addressed.

ticide, herbicide, and antibiotic, often leads t o significant local pollution. Chromium, an important element i n industry, is causing increasing concern since its discovery as a possible carcinogen. C h r o m i u m can cause skin ulcers, nose inflammations, and genetic malformations. Dichromate is a frequently used laboratory material. Therefore, t h e toxic effects of LAS, PCP-Na and dichromate t o organisms merit more study. Reports on the toxic effects of LAS, PCP-Na and dichromate on Tetrahymena pyriformis have been limited to their effects o n cell growth, motility, a n d other physiological processes [4, 6, 9-1 21; hence, we studied the mechanisms of toxicity and t h e genotoxicity. There a r e a t least five kinds o f genotoxicity: gene mutation, chromosome aberration, primary damage o f D N A , interference with D N A topoisomerases and carcinogenic transformation.

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WU & SHEN-GENOTOXIC EFFECTS ON TETRAHYMENA

Table 1. Effects of LAS and PCP-Na on DNA content in the macro- and micronuclei of T. pyrifomis S1 (mean k SD).' PCP-Na concentration

LAS concentration 11.31 ppm

Nuclei group

0.15 ppm

0.14 ppm

0.9 ppb

Micronuclei Experiment

2,861.90 t345.67 3,494.82 t 5 16.63 P < 0.001

Control Significance (t-test) Macronuclei Experiment

2,961.42 f244.52 3,272,88 1332.20

P < 0.001

11,434 21,872 13,534 k1,731 P < 0.002

Control Significance (t-test)

1,391.72 k223.06 1,789.78 k282.74 P < 0.001

12,490

13,020 +1,886 14,440 ?2,416 P < 0.05

k 1,479

13,037 f2,168 P > 0.05

2.636.83 k302.16 3,276.37 t223.59 P < 0.001 12,498 k2.144 13,363 t2,254

P > 0.05

"The number of measured micro- and macronuclei is 50 and 35, respectively.

MATERIALS AND METHODS Organisms and growth medium. Tetrahymena pyriformis strains SI and BJ4 were grown axenically at 25-28" C in 250ml flasks in a medium consisting of 2% (w/v) polypeptone and 0.1 Yo (w/v) glucose plus 0.1O/o (w/v) yeast extract. Determination of DNA content in micro- and macronuclei. Cells were grown in stationary vessels. Cells were obtained by gentle centrifugation (700 g) and the pellet was washed three times in isotonic Osterhout solution. The cells were recentrifuged and suspended. The chemicals were added at a final concentration of 0.14 ppm LAS, 11.31 ppm LAS, 9 x 10 ppm PCPNa, and 0.15 ppm PCP-Na. Cells cultured in an isotonic medium served as controls. Culture medium was centrifuged off after 5 h and cells were fixed in Carnoy's solution for 0.5 h. Slides were Feulgen stained. DNA was measured spectrophotometrically. The conditions of microspectrophotometry in this test were as follows: 1) Measurement of micronuclei DNA-X-field (steps) 8, Y-field (steps) 8, X-stepsize (pm) 0.5, Y-stepsize (pm) 0.5, the wave oflight 560 nm; 2) Measurement ofmacronuclei DNAX-field (steps) 10, Y-field (steps) 10, X-stepsize (pm) 2, Y-stepsize (pm) 2, the wave of light 560 nm. Measurements of macronuclear DNA extrusion bodies. Cultures of cells (amicronucleate strain BJ4) in the log phase were used and treated as described above. Final concentrations were 0.0 14 ppm LAS, 0.14 ppm LAS, 1.4 ppm LAS, 11.30 ppm LAS, 0.05 ppm dichromate, 5 ppm dichromate, and 16.7 ppm dichromate. The chemicals were removed after 4 h. The cells were recultured for 4 h, then fixed with Carnoy's solution, stained with Feulgen, and counterstained with Fast Green. The macronuclear DNA extrusion bodies (MaEB) were identified under an oil lens using over 2,000 cells for each sample. Because the Tetrahymena pyriformis strain BJ4 is amicronucleate, the micronuclei discovered under an oil lens should be toxicological MaEB.

data). We chose two doses for the former two chemicals; the high dose was near 12h-LCSoand the low dose was near or lower than the maximum acceptable toxicant concentration (MATC). The MATC of LAS is 0.17 ppm [ 5 ] , and that of K2Cr40,is 0.05 ppm according to the National Drinking Water Quality Standard (TJ20-76, a serial number of the legal standard in China). The MATC of PCP-Na is