1997 9 3 Carcinogenesis T eratogenesis and M utagenesis V o l 9 N o 3 1997 831 1 T O ng 2 1 610041 2 D ivision of R esp irato ry D isease Studies,N IO SH,U SA Am es V 79 H PR T (SCE) 831 ( ) 2, 4 6 (DCA P) :, S9 ; DCA P V 79 SCE (P < 0. 01) DCA P DNA ; g ; S9, 2, 4 ; 2, 4 ; SCE; ; STUD IES ON THE GENOTOX IC ITY OF 83- ITS M AJOR M ETABOL ITE 1 HERB IC ID E AND H eng Zhengchang 1, T O ng 2 1 W est Ch ina U n iversity of M ed ica l S ciences, Cheng d u 610041, 2 D iv ision of R esp ira tory D isease S tud ies, N IOS H, U SA Abstract A com parative studies on m u tagen icity and clastogen icity induced by 8 3-1 (3),,, 1..., (4),,, 4. 0. 5-1% (V gv ) 80, : 0. 5% (V gv ) 80 1% (V gv ) 80, (ς 2 = 0. 58, P > 0. 05),, 1995: 7. : 2.... ( ). 1986: 7578. 3... :, 1995: 306307 546. 4.,.. :, 1983: 419 420 425. 5... :, 1987: 64. 6.. ( ). :, 1991: 215216. 154
herb idice (2, 4- dich lo ro- 6- n itropheno l amm on ium, ) and its m ajo r m etabo lite, 2, 4- dich lo ro - 6- am inopheno l (DCA P), w ere perfo rm ed u sing Am es test and gene fo rw ard m u tation (at H PR T locu s) assay, SCE assay, and m icronucleu s assay (M N ). R esu lts show ed that does no t p roduce a po sitive respon se fo r any endpo in t in the studies w ith o r w ithou t liver S9. How ever, the treatm en t of V 79 cu ltu res w ith DCA P cau sed a sign ifican t in2 crease in SCE s and M N in a do se - dependen t m anneṙ T hese resu lts indicated that DCA P w as a DNA dam aging agen t and likely clastogen ic in V 79 cells; the m etabo lic activation in m amm alian w as needed fo r the geno tox icitygcarcinogen icity of ; liver S9 u sed in vitro cou ld be in sufficien t fo r activation of. Key words2, 4- dich lo ro - 6- n itropheno l amm on ium ; 2, 4- dich lo ro - 6- am inopheno l; SCE;M icronucleu s assay;m etabo lic activation 831, 2, 4 6 3. V 79 D ṙ Chang (2, 4 - dich lo ro - 6 - n itropheno l amm on i2 um, ) 10% 1mM L - 1% (1) : 831 Eagle s, (D 7 D 61. M ) (2) 37 5%CO 2 95100% DNA ; ; 4. DCA P 2, 4 6 DM SO (2, 4 - dich lo ro - 6 - am inopheno l, DCA P) (3), 50, 100, 200 400Λggm l, DCA P 50, 100, 200 300Λggm l, Am es V 79 3h, DCN 2 H PR T PA, S9 (SCE) DCA P 5. 1. V 79 H PR T, > 97% ; DCA P 6T G (T G r ), B radley, > 99% (4), 2, 4, 7 7d,, (M NN G) A ldrich ; 6 (CFE ), (6T G) 5 (B r2 100, 6, 7d ; du ) Sigm a 2. Am es TA 98 TA 100 (M ich igan State U n iversity), V 79, PBS ; 100, 6 ; 7d,, GT r, 2. 5 10 5, 6, 10ΛM 6T G S9 10d,, 155
(M F): 1. S9 M F= g( CFE) Am es 1 TN F M NN G 6. SCE Perry W o lff TA 98 TA 100 (5) 50 DCA P 7. H eddle,, (6), 3000 S9 S9 ( ) Table 1. Results of the Sa lm onellagm icrosoma l a ssays for DCNPA and DCAP Do se Λggp late N um ber of Revertant Co lonies (x s) a TA 98 TA 100 p late- inco b p re- incu c p late- inco b p re- incu c 0 25. 23. 3 20. 71. 9 111. 45. 5 103. 212. 8 4 23. 02. 0 20. 32. 1 116. 77. 5 107. 08. 7 20 24. 22. 3 21. 62. 1 113. 86. 9 101. 614. 8 100 24. 03. 2 18. 61. 5 106. 04. 5 97. 014. 0 500 15. 68. 5 6. 53. 9 60. 417. 4 23. 211. 0 DCA P 0 28. 73. 1 24. 25. 8 117. 816. 2 109. 217. 9 4 28. 33. 1 25. 32. 3 126. 07. 9 108. 08. 7 20 27. 83. 9 24. 07. 3 118. 611. 7 115. 821. 9 100 29. 24. 2 24. 07. 6 121. 416. 2 116. 629. 4 500 19. 63. 8 15. 65. 2 97. 52. 1 5. 52. 1 TN F 0. 4 798. 421. 0 855. 517. 0 M NN G 2. 0 1042180. 0 992. 3203. 0 a: average of 3 p lates, b p late- inco: p late- inco rpo ration test, c p re- incu: p re- incubation tesṫ 2. DCA P, V 79 (1) DCA P, 19. 6% 17. 3%, DCA P 3., DCN 2 PA DCA P V 79 6T G r ( 2), 1Λggm l M NN G 6T G r 30 F igure 1. Relative survival of V 79 cells expo sed to and DCA P. 156
Table 2. Frequenc ies of 6- TG r M utan ts in V79 Trea ted with DCNPA and DCAP Λggm l Co loniesgp late (x s) Survivo rs a 10 5 M F b 10 5 0 2. 71. 9 2. 4 1. 3 50 1. 61. 5 2. 4 1. 0 100 2. 01. 9 2. 2 0. 9 200 2. 21. 6 1. 9 1. 2 400 0. 60. 9 1. 9 0. 3 DCA P 0 2. 21. 9 1. 5 1. 5 50 2. 82. 8 1. 7 1. 7 100 1. 61. 3 2. 0 0. 8 200 2. 82. 4 2. 1 1. 3 300 1. 40. 9 1. 7 0. 8 M NN G 1 72. 519. 3 1. 9 38. 2 a Survivo rs= (num ber of cells p lated) CFE, b M utant F requency 4. SCE DCA P (r= 0. 987, P < 0. 01) ; V 79 SCE 3 (100, 200 300Λg gm l) S CE SCE (P < 0. 01), DCA P SCE Table 3. Frequenc ies of SCE in V79 cells Trea ted with DCNPA and DCAP Λggm l Cells sco red Range SCEs per cell x s 0 50 212 6. 32. 76 50 50 210 6. 52. 31 100 50 312 6. 42. 04 200 50 214 6. 62. 53 400 50 314 6. 52. 64 DCA P 0 50 311 6. 12. 08 50 50 214 6. 12. 25 100 50 316 8. 7 3 2. 64 200 50 430 10. 4 3 4. 80 300 50 547 14. 1 3 9. 16 M NN G 1 50 4182 69. 1 3 15. 07 3 P < 0. 001, compared to so lvent contro l by Student s t test 5. DCA P V 79 ; DCA P (4) SCE 200 300Λggm l, 157
(P < 0. 01), 17. 3 21. 3, 2. 831 831 Table 4. Frequenc ies of M N in V79 Cells Trea ted with DCNPA and DCAP Λggm l M icronucleated Cells To ltal a Cells 0 17 5. 7 50 16 5. 3 100 18 6. 0 200 16 5. 3 400 17 5. 7 DCA P 0 16 5. 3 50 17 5. 7 100 18 6. 0 200 52 17. 3 3 300 64 21. 3 3 M NN G 1 59 19. 8 3 a N um ber of m icronucleated cells in 3000 cells sco red, 3 P < 0. 005, comp ared w ith solvent control D CN PA, S 9 (1) ;, V 79 S CE D CN PA 831 g S 9 ( ) ;,, : S 9, D CN PA 1. D CA P ; D CA P,, D CN PA, D CA P V 79 S CE S CE ( ), D CA P, DN A D CA P S 9 V 79 (V 79) H PR T D CA P, (7) 2, 4, 6 (8) (9), DN A 158, (3) : D CA P, 831 D CN PA D CA P,, D CA P S CE 831 g,, D CN PA 6 3. 831, D CN PA, D CN PA S 9, D CA P S CE,, S 9 D CN PA DN A D CA P, 1.,,,. 831 DN A., 1992; 6 (3) : 206. 2.,,. 831., 1993; 24 (1) : 97. 3.,,,. 831., 1993; 12 (4) : 43.
1997 9 3 Carcinogenesis T eratogenesis and M utagenesis V o l 9 N o 3 1997 250062,A (120m ggkg) B (60m ggkg) 10d, (P < 0. 01),, (P < 0. 01), C (30m ggkg) (P > 0. 05), (P < 0. 05); D (10m ggkg) ;, 1w k, 15 (Cory d a lis B ung eana, TU R C I ) 25,, 4. 13, 6,,,, 1. 120 60 30 10m ggkg, d6-15,,, 5%, 5m ggm l 2. (CTX ) (C - ) 5% 0. 5m lggd, 900501 d0; 6, A B C D 4, 1, 10 ; (C + ) 14m gg kgctx, d11, 5 ; d6, 10, 15 1, 3. 8w k, ; 3040g, 3240g, d18,,, 4. B radley M D, Bhuyan B, F rancis M C, et al. M utagenesis by chem ical agents in V 79 Ch inese ham ster cells: A re2 view and analysis of the literature. A repo rt of The Gene - Tox P rogram. M u tat R es, 1981; 87: 81. 5. Perry P,W o lff S. N ew Giem sa m ethod fo r the differential staining of sister chrom atids. N ature, 1974; 251: 156. 6. H eddle JA, H ite M, Ko rkhard B, et al. T he induction of m icronuclei as a m easure of geno toxicity. A repo rt of the U. S. Environm ental P ro tection A gency Gene- Tox P ro2 gram. M u tat R es, 1983; 123: 61. 7. M o riya M, O h ta T, W atanabe K, et al. Further m uta2 genicity studies on pesticides in bacterial reversion assay system s. M u tat R es, 1983; 116: 185. 8. H awo rth S, L aw lo r T, M o rtelm ans K, et al. Salmonella m utagenicity test results fo r 250 chem icals. E nv iron M utagenesis, 1983; 3: 142. 9. Ho lm e JA, Hom gslo JK, B jo rnstad C, et al. Toxic effects of paracetamo l and related structures in V 79 Chinese ham ster cells. M u tag enesis, 1988; 3: 51. 159