Detection of sister chromatid exchanges in the newly hatched chick

74 Effect o f selenium on chemically-induced sister chromatid exchanges Selenium exhibits both antitumorigenic [1,2] and antimutagenic [3] properties when used in conjunction with selected chemical carcinogens in animal and bacterial systems. Since exposure to chemical carcinogens generally results in an increased sister chromatid exchange (SCE) frequency, a study of the effect of selenium on SCE production was initiated. Toxicity experiments showed t h a t cultured leukocytes could survive a 96-h exposure to 1.27 X 10 -9_ 1.27 X 10-6M sodium selenite and a 15-h exposure to 1.27 X 10-9--1.59 X 10 -s M sodium selenite. Sodium selenite concentrations ranging from 1.27 X 10 -6 M to 6.36 X 10 -4 M resulted in no change in SCE frequency above control (6--7 SCE/cell), b u t a higher concentration (9.54 X 10 -6 M) caused a significant increase in the average number of SCE's/cell (P < 0.01). Co-exposure of leukocytes to sodium selenite (1.27 X 10 -6 M) and m e t h y l methanesulfonate (1 X 10 -4 M MMS) for the last 15 h of culture significantly reduced the SCE frequency observed when MMS was added alone (P < 0.01). The decreased SCE frequency is consistent with the decreased mutagenicity and tumorigenicity detected with chemical carcinogens in the presence of selenium. (Supported by USPHS Grants GM-00143 and GM-19513.) 1 Jacobs, M.M., B. Jansson, A.C. Griffin, Cancer Letters, (1976) in the press. 2 Schrauzer, G.N., D. Ishmael, Ann. Clin. Lab. Sci., 2 (1974) 441--447. 3 Jacobs, M,M., T.S. Matney, A.C. Griffin, Cancer Letters, submitted for publication.

16 L.C. Altenburg, J.H. Ray and M.M. Jacobs, University of Texas Health Science Center, Medical Genetics Center, and M.D. Anderson Hospital and T u m o r Institute, Houston, Tex. 77030 (U.S.A.)

Toxicity and frequency of sister chromatid exchanges produced by N-hydroxy2-acetylaminofluorene (N-OH-AAF) in human lymphocytes The mutagenicity of 2-acetylaminofluorene (AAF) and its activated metabolite, N-OH-AAF, have been established in the Ames' bacterial tester system. Because of the relatively high mutagenic potency of AAF and N-OH-AAF, a study of the effectiveness of these compounds to produce sister chromatid exchanges (SCE's) in cultured h u m a n l y m p h o c y t e s was undertaken. Initially, a toxicity experiment was performed to determine the o p t i m u m concentration at which cells could be exposed with m i n i m u m viability. The results of this experiment revealed the high toxicity of N-OH-AAF in that exposure of h u m a n cells for 15 h to concentrations greater than 4 X 10 -s M resulted in a complete absence of metaphase spreads and large numbers of dead cells. At 4 X 10 -s M N-OH-AAF, cell viability was marginally adequate to allow SCE frequency measurements. At this concentration, an analysis o f 20 spreads showed a combined mean SCE frequency of 16.3/ce11, whereas the control (no exposure to N-OHAAF) mean was 6.2/cell. Exposure o f cells to lower concentrations gave the following results: 2 X 10 -s M = 13.3/cell; 1 X 10 -s M = 9.6/cell; 4 X 10 -6 M = 7.6/cell; lower than 4 X 10 -6 = same as control (6.2/cell). Compared to other

75 mutagens (e.g. MMS) the toxicity of N-OH-AAF is extremely high, while the number of SCE's produced is relatively low. The lower toxicity and complete absence of SCE's produced by the parent compound (AAF) at 4 X 10 -s M in this system is consistent with the reported lower mutagenicity of AAF relative to N-OH-AAF. (Supported by USPHS Grant GM-19513.) 17 S.E. Bloom and A.D. Kligerman, Cornell University, Ithaca, N.Y. 14853

(U.S.A.) Detection of sister chromatid exchanges in the newly hatched chick The presence of minute amounts of chemical mutagens can be detected using sister chromatid exchange (SCE) analysis. This technique depends upon BrdU incorporation into chromosomes through 2 rounds of replication. We have developed in vivo systems for SCE analysis, including the chick embryo (S.E. Bloom and T.C. Hsu, Chromosoma, 51 (1975) 261), and the adult mudminnow (A.D. Kligerman and S.E. Bloom, Chromosoma, 56 ( 1 9 7 6 ) 1 0 1 ) . To expand our system for detecting SCE to the neonate, the newly hatched chick was selected. Chicks have several advantages as they are easily obtained and handled, and chromosomes from several tissues may be sampled. Furthermore, whereas the in vivo mammalian systems require from 6--14 BrdU injections, as few as 3 injections suffice in the chick. This is achieved by supplying only water (and no feed) to the chicks. The absence of exogenous nutrients results in enhanced BrdU incorporation into the cells. The resorbed yolk sac material is sufficient to keep the chicks healthy for up to 5 days post-hatch. Weak sister chromatid differentiation (SCD) was seen with a single injection of BrdU (0.24 mg/40 g chick) into the yolk sac. To enhance SCD and measure exchange rates due to chemical mutagens, 3 injections of BrdU (each containing 0.08 mg BrdU/g body weight) were given at equal intervals over a 6-hour period. After 30 h total exposure to BrdU, animals were sacrificed and chromosomal preparations made. SCD was revealed in chromosomes from bone marrow, bursa of Fabricius and thymus after staining with 33258 Hoechst. The rate of SCE was 0.86 per metaphase. After EMS treatment (given 1.5 hours after BrdU) the rate was elevated to 6.2 SCE per metaphase. The results of our studies suggest that the chicken, as embryo and neonate, should be very useful for measuring mutagenicity of environmental pollutants as reflected by SCE. 18 A.D. Kligerman and S.E. Bloom, Cornell University, Ithaca, N.Y. 14853

(U.S.A.) Increased rates of sister chromatid exchange in the central m u d m i n n o w following in vivo exposure to alkylating agents Sister chromatid exchange (SCE) analysis is a sensitive method by which chromosome damage and repair can be measured following incorporation of the DNA base analog, 5-bromodeoxyuridine. Most systems currently in use for SCE analysis employ in vitro cell culture methods which inherently have high