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

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1 Carcinogenesis T eratogenesis and M utagenesis V o l 9 N o T O ng 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 , 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 (3),,, 1..., (4),,, % (V gv ) 80, : 0. 5% (V gv ) 80 1% (V gv ) 80, (ς 2 = 0. 58, P > 0. 05),, 1995: 7. : ( ). 1986: :, 1995: ,.. :, 1983: :, 1987: ( ). :, 1991:

2 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, V 79 D ṙ Chang (2, 4 - dich lo ro n itropheno l amm on i2 um, ) 10% 1mM L - 1% (1) : 831 Eagle s, (D 7 D 61. M ) (2) 37 5%CO % DNA ; ; 4. DCA P 2, 4 6 DM SO (2, 4 - dich lo ro am inopheno l, DCA P) (3), 50, 100, Λggm l, DCA P 50, 100, Λggm l, Am es V 79 3h, DCN 2 H PR T PA, S9 (SCE) DCA P 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, , 6, 10ΛM 6T G S9 10d,, 155

3 (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 DCA P TN F M NN G 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

4 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 DCA P M NN G a Survivo rs= (num ber of cells p lated) CFE, b M utant F requency 4. SCE DCA P (r= , P < 0. 01) ; V 79 SCE 3 (100, Λg 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 DCA P M NN G P < , compared to so lvent contro l by Student s t test 5. DCA P V 79 ; DCA P (4) SCE Λggm l, 157

5 (P < 0. 01), , 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 DCA P M NN G a N um ber of m icronucleated cells in 3000 cells sco red, 3 P < , 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 , 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) : ,,. 831., 1993; 24 (1) : ,,,. 831., 1993; 12 (4) : 43.

6 Carcinogenesis T eratogenesis and M utagenesis V o l 9 N o ,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,,,, m ggkg, d6-15,,, 5%, 5m ggm l 2. (CTX ) (C - ) 5% 0. 5m lggd, 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: 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: 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: 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: 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: 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: