Document GKR2oBnE0vRMR4b19YwvJr8jV

u/lhq ft 125 Mutation Reteareh, 68 (1979) 126--132 Q Elaevir/North*HolUnd Biomedical Press gOTICI: mUrM. * )> protaotad bj ooyyTlant NON-MUTAGENICITY FOR SALMONELLA OF THE CHLORINATED HYDROCARBONS AROCLOR 1254.1.2.4-TRICHLOROBENZENE MIREY AND KEPONE RITA S. SCHOENY, CARL C. SMITH md JOHN C. LOPER DEC 1 4 '979 Department of Environmental Health and Microbiology, Uniuereity of Cincinnati Medical College, Kettering Laboratory, 3223 Eden Avenue, Cincinnati, Ohio 45267 (U.S.A.) (Received 13 June 1979) (Accepted 18 June 1979) Summary A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-tri chlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlo robenzene were shown to differ from S9 of either control or Aroclor 1254induced rats in the capacity to activate 2-ammoanthracene mutagenesis. Chlorinated hydrocarbons have become widespread contaminants of the environment. Polychlorinated biphenyls (PCB) used as plasticizers and solvents, as dielectric and hydraulic fluids, and as heat-transfer media [3] have been found in human adipose, brain and liver tissue as well as in human milk (16, 24). Highly chlorinated PCBs and the insecticides mirex and kepone show only limited metabolic change and degradation [8,12,16,27,28]. Small molecules such as the chlorinated benzenes are found as major breakdown products of less stable pesticides, e.g., hexachlorobenzene [9,14], and are present in drink ing water [29). All these compounds induce hepatic enzymes and are known to have some deleterious biological effects [1--3,6,9,11,15,21--24]. PCBs have produced toxic responses in humans and are suspect carcinogens in rats [17,24]. Mirex induces tumors in mice [13] and neoplastic liver lesions in rats [30). Kepone was responsible for the acute poisoning of over 100 inhabitants of Hopewell (VA), the site of its manufacture, producing symptoms of neurologic and hepatic damage [ 7 ] and has been shown to be carcinogenic for rats and mice HONS 0029*9 126 [10,25). Among the triehlorobenzenes, which are suspected of causing hepatic porphyria [26), the 1,2,4-trichloro isomer is of interest as a potent inducer of liver enzymes [5). The capacity of PCB for hepatic induction is relied upon routinely in the preparation of microsomes for use in the Salmonella mutagenesis assay system developed by Ames [4]. This study of 4 chlorinated hydrocarbons applied the Salmonella/microsomal assay to accomplish 2 objectives: (1) to evaluate mu* tagenicity; (2) to test whether these substances could cause induction in rate such that liver microsomal preparations would metabolize the parent chemical to detectably mutagenic forms. Materials and methods Reagents. 2-Aminoanthracene (2AA), benzo[a]pyrene, gold label (BAP) and iV-methyl-A/'-nitro-iV-nitrosoguanidine (MNNG) were purchased from Aldrich. 4-Nitroquinoline-l-oxide (NQNO) from Koch-Light Laboratories, Ltd. was the gift of Dr. Tong Man Ong of the National Institute of Environmental Health Sciences. Methyl methanesulfonate (MMS) was purchased from Eastman. Cal* biochem was the supplier for aflatoxin B, (AFB). Supelco, Inc. was the source of the polychlorinated biphenyl mixture Aroclor 1254 (PCB) while 1,2,4-tri chlorobenzene, lot No. 038967 (TCB) was obtained from Aldrich. Commercial grade kepone (decachlorooctahydro-l,3,4-methono-2//-cyclohuta|c.dlpntalert2-one) (KPN) was donated by the Health Effects Research Laboratory, Ne* tional Environmental Research Center, Environmental Protection Agency, Ctrv* cinnati, Ohio. Commercial grade mirex (dodecachlorooctahydrol.3.4-metheno 2H-cyclobuta fc.djpentalene) (MRX) was obtained through the Kettering Lab* oratory, Cincinnati, Ohio. EmuiphorR EK-620, a polyoxyethylated vegetable , oil, was obtained from GAF Corporation. Dimethyl sulfoxide (DMSO) was of distiJled-in-glass quality from Burdick and Jackson Laboratories, Inc. Preparation of S9. Young (150 g) male Sprague-Dawley rata from Charto River Laboratories were maintained 2/cage and provided water and Purina Chow ad libitum. Animals underwent an acclimation period of at least 4 days prior to treatment. Dosing was by intraperitoneai injection of com oil solutions prepared so as to deliver 2 ml/kg, according to the following schedule to groups of 4 animals or as indicated: PCB, (6 rats) 50 mg/kg/da, 5 days; TCB, 50 or 200 mg/kg/da, 4 days or 200 mg/kg/da, 2 days; KPN, 18, 25, 36 or 50 mg/kg/da, 2 days; MRX, 25 mg/kg/da (3 rats) or 100 mg/kg/da for 2 days; com oil 2 ml/ kg/da for 4 days. Doses were administered at 9:00 a.m. except for the TCB group which received one-half the dose in the morning and the second half 12 h later. Animals were fasted for 24 h following the last morning injection and were then killed by decapitation. Additionally 5 animals injected with KPN 36 mg/kg/da for 2 days, then were maintained 9 days as before, starved 24 h and killed. Liver extracts were prepared according to the method described by Ames (4), in a homogenizing buffer consisting of 0.15 M KC1 and 0.05 M Trie-- HC1. For each liver homogenate the supernatant fluid resulting from 20 min si 9000 g was mixed to insure homogeneity and stored os 1-ml aliquots at --70"C. S9s were checked for bacterial contamination and for their ability to activate 2AA, BAP and AFB to bacterial mutagens; equal volumes were pooled by 002930 HONS 127 treatment group on the day of assay. Other constituents of the S9 mix were as described by Ames (4]. Microsomal protein concentrations were determined by the method of Lowry et a!., on pellets from centrifugation of S9s at 100 000 g. Mutagenesis assays. Assays reported here used 4 Salmonella typhimurium strains and procedures described by Ames (4]. The missense mutant TA1535, and the frameshift indicator strains TA1537 and TA1538 are all deficient in excision repair (vuvrfl) and have a minimal cell wall [^7gal, rfa). Strains TA98 and TA100 are TA1538/pKM101 and TA1535/pKM101; the R factor pKMIOl renders these strains particularly sensitive to the actions of certain types of mu tagens [20]. Although initial assays for mutagenicity of chlorinated hydrocar bons used TA1638, this strain was dropped in favor of TA98 which retains much the same mutagen specificity while showing greater sensitivity. TA1538 and TA100 were used in the assay of fixed amounts of 2AA with varying con centrations of S9. As TA100 is less specific in its response than TA1535, both these strains were utilized in mutagenicity testing of the chlorinated hydrocar bons. TCB was delivered in a solvent system composed of EmuIphorR; ethanol (95%); physiological saline; 1:1:5; v/v/v. All other test solutions were in DMSO (0.1 ml/plate). Standard assays consisted of duplicate plates for each test point and for positive controls and 3 plates for spontaneous controls. All assays were repeated at least once. To be scored as mutagenic in these studies a substance must have reverted one or more strains in a dose-dependent fashion producing numbers of colonies at least 2 X those obtained spontaneously. Tox icity was scored on the basis of numbers of colonies significantly below spon taneous levels, by the clearing of the background lawn, or by the appearance of auxotrophic (his*) colonies. Results and discussion Using a fixed concentration of S9, 50 /jl/plate, PCB, mirex, kepone and TCB were assayed far mutagenicity at 7 or 8 concentrations over a 3-log dose range with strains TA1535, TA1537, TA98 and TA100. When tested directly or with either uninduced or PCB-induced S9, PCB and the other 3 test materials were all non-mutagenic. As these 3 substances are also known to induce liver enzymes, S9s were prepared from rats treated by various regimens with each of them. Spontaneous colony counts of the 4 strains were not affected by these S9t (50 jul/plate). All these microsomal preparations were used in activation tests in which each substance was tested with homologously induced S9; addi tionally mirex and kepone were assayed reciprocally, using S9s from the MRX25, MRX100 and KPN25, KPNCO-induced rats. Again no mutagenicity was detected. By our criteria for toxicity all 4 test materials were toxic for Salmonella at high doses in both the absence and presence of S9. These data are summarized in Table 1, while data for the concurrent positive and negative controls arc given in Table 2. The data in Table 2 are from assays using S9s prepared from rats given the highest dose of MRX, KPN or TCB and are repre sentative of the lower treatment groups. As noted, data in Tables 1 and 2 was generated using the respective S9s at 50 jjl/plate. The amount of S9 used per plate can be critical in the detection of i HONS 002931 128 TABLE 1 TOXICITY AND NON-MUTAGEN 1CJTY OF CHLORINATED HYDROCARBON* FOR 4 SALMONELLA STRAIN* Test substance * Number o( doses b tened and rant* (Ul/plate) Toxic C dose (Ul/plate) Mutagentcliv d PCB Mir** Kepone TCB ft <0.5-500) 7 (0.1-100) 7 (0.1-100) ft <102-1.4 X 10*) boo 100 50 <509 non* non* non* non* * Substances tested were PCS: Aroelor 1264: TCB: t .2,4-trlchlorob*nzrne: mirea and lirpon* wrr cammerclsl ftadc. b Tested with (trains TA1535. TA1537, TAd* and TA100 in absence and prtwnrc at SO (SO nUglu*l, prepared aa described in Ui* Methods section of the teat, from uninduced. ECS* and homnlngmaRv* indueed rata. Mint and kepont am also tested uiint STS 1?nm kepone- and mlrea-induerd rata reaped tlveiy. Data were obtained from at least repticate assays. r Based upon evident killing on mutagenesis plates of one or more stains as dracribed In text. d Criteria (or mutagenicity are as described in the lest. some pre-mutagens in the Salmonella/microsomal assay {4}. Accordingly TCB was tested further using the 4 strains of Table 1 with 10 and 100 n\ uninduced and PCB-induced S9/plate, and PCB was assayed with TA98 and TA100 using these concentrations of uninduced and PCB-induced S9s; sufficient S9 was not available for the assay of mirex and kepone in this fashion. These variations had no effect on either toxicity or nonmutagenicity of PCB tested at 0.56--500 Mg/ plate or of TCB tested at 102--2914 Mg/plate. As noted above, none of the teat substances when used as inducers in rats from which the S9s were prepared affected their own in vitro activation to mutagens. Two of these, however, PCB TABLE 2 MUTAGENIC RESPONSE OP STRAINS WITH CONTROL MUTAGENS OR IN TIIK PRESENCE Of LIVER SB PREPARATIONS FROM RATS TREATED WITH CHLORINATED HYDROCARBONS Compound * (amount/plate) SB 50 yl/plate and source Strains ................... ............. --' - TA1535 TAI53T TAB# --.. TAIOO NQNO erystal MNNG crystal ICR 191 crystal MMS 12.9 UK 2AA 9.0 ui OMSO 100.0 u> DMSO 100.0 Ul DMSO 100.0 ul DMSO 100.0 ui DMSO 100.0 ul DMSOino.Oul none non* non* none PCS non* unindueed PCB mlrex kepone TCB Nl) NO ND 75 t 28 21 t 20 16 t S 19 t 13 14 t 1 13 t 1 24i 4 ND * ND 93 i 58 18*14 25 s 27 12 t 7 13 t 7 9t 3 14 13 ** ND ND ND SO9 t 299 23 t 12 30 t 14 30 1 IB 30 t 11 27 t 1ft 37 t 19 ND NO ND 1594 ? 8B7 10*4 ? 417 114 t 2 120 t 4t 122 t 27 114 * 9 127 t 9 147 t 44 n Abbreviations art* aa In test. b All numrrlcsi values sr* means ol revertant rnlonln/plsls standard deviations: a > i. e Crystals were spotted on the overlay o( anil scar plus haeteria. I\*stw* response* tn thss* wswsssmHu live aaaava are tcored aa follows: -- , <1(1 revertant coiontal/piate; , 10--100: 100--900. *. iOO-lOOO: tno numerous to count: ND, not dono. HUMS 002932 TABLE 3 MUTAGENESIS OF 2-AM1NOANTHRACENE IN THE PRESENCE OF LIVER S9 PREPARATION FROM RATS TREATED WITH CHLORINATED HYDRO CARBONS Stnis * Source of S9 * Revcrtant eoioaies/pUt* b 2-AaunoanihraceAC <5 pc/pUte) SB/pUt (d) 10 2S SO Without 2 amino* inthnctnt S9/plate (jii) 100 ISO iso TA1539 TA100 uoMuflM PCS TCB 200 TCB 400 TCB SOO uninduced PCI TCB 200 TCB400 TCB 800 771 t <00 3383 1320 1000 258 t 81 789 2 315 2651 3798 l 1297 1 8051 983 * 8 753 347 366 361 1919x 3321 i 25652 1487 i 1644 t 56 949 706 400 371 1694 l 1636 4694 1 1595 3206 1 139 3064 2 1601 2554 * 937 4239 * 2222 2435 i 1043 3627 4 454 2580 t 1551 2648 2 1329 3679 t 1793 2453 * 1306 3570 * 549 2515 * 1866 4029 * 2401 3615 1 698 899 1 425 4072 i 504 2283 t nil 3429 t 3360 3216 756 1269 * 835 5220 i 1404 2745 1223 4339 i 3360 4150 : 505 410 1 178 2006 i 994 2318 : 1983 924 t 452 4633 i 1200 1127 1 564 2975 i 1485 1928 2 1334 2728 i 1718 3055 1 224 i 1705 1 1552 i 935 i 563 72 502 327 281 4407 1302 663 i 315 1925 t 1065 2077 2 1525 2234 i 593 28 2 28 1 20 2 24 2 34 2 12 9 6 4 4 140 s 13 227 2 102 145! 31 116 2 11 95 2 16 4 Direct muuitn controls were a* follows; TA1538. ICR-191 crystal ++; TA100. MMS 12.9 pg/plate. 1373 eolonies/plate. 0 Numbers are means t standard deviation*, n 3 i except for TA100 revertant colomes/pUte using 50pi of umnduced 59 where = 3. NO. not done. c PCB, Arodor 1254. 50 mg/kg/da. 5 days. TCB 200. TCB, 50 mg/kg/da. 4 days: TCB 400, TCB. 200 mg/kg/da. 2 days; TCB 600. TCB. 200 mg/kg/da. 4 days- HONS 0 0 2 9 3 3 ll 130 uugcnlc activation of 2-aminoanthrarrna. Mitmtemii SB fraction* were prepared from liver* of rut* treated with com oil (unlndueed) 01 a* deanttoed in ttoe text. 8-AminoonUuraceae was used at & vt/Plitt. The S9 concentration* tested are indicated a* wi/plate from uninduced (). TCB (0), or TCB 2UU (A) Induced reta. Data pointa are the meana of Platt eownta pmenied In Table 3 lor TMUt and TMOO. and TCB were able to influence the mutagenicity of 2AA (5 Mg/plate) as tested in strains TA1538 and TA1Q0, by virtue of their capacities as hepatic enzyme inducers. S9 extracts from rats treated with PCB and TCB were used at 6 com centrations from 5 to 150 pl/plate in the activation of the fixed amount of 2AA. As can be seen from Table 3 increasing the concentration of uninduced S9 promoted increasingly greater activation of 2AA (see also Fig. 1). By com parison the PCB-induced S9 was most effective at low concentration and its activation of this premutagen decreased sharply at the higher concentrations. TCB-induced S9 produced an intermediate effect. That is. numbers of revertants on experimental plates increased less sharply with increasing TCB-induced S9 concentration than with PCB-induced S9, but did not decrease as much after the maximum point had been reached as was observed using PCB-induced S9. These relative effects for uninduced. PCB. and TCB200 S9 are presented in Fig. 1. TCB was administered for induction over a 4-fold dose range in 3 dosing regimens. The treatment schedules involving 4 days (TCB 200 and TCB 800) resulted in S9s which activated 2AA (5 pg/plate) in a similar dose-dependent fashion. The TCB treatment for 2 days (TCB 400) appeared to be less effective in this regard (Table 3). PCB, mirex and kepone produce neoplastic lesions (10,13,17,25,30). and for PCB and mirex there is independent evidence for limited metabolism in test animals (12,27,28]. Nevertheless, our data show no indication of activation of these compounds to mutagens for TA1535, TA1537, TA98 and TA100. This is HONS 002934 131 true using liver microsomal extracts prepared from control rats as well as from rats which were effectively induced by treatment with PCB. Moreover, although we have shown mirex and TCB induction to alter the activation of a known pre-corunogcn, treatment with TCB, mirex or kepone did not result in SO preparations capable of in vitro activation of the homologous compounds. Other chlorinated hydrocarbons, including the animal carcinogens ODE and dieldrin, have escaped detection in the Salmonella/microsomal assay [19]. As has been suggested by McCann and Ames, it may be that lack of response by the Salmonella strains in such cases is due to the failure of in vitro liver prepa rations to effect the necessary dechlorination. Acknowledgements This work was supported by U.S. Public Health Service research grant AI 11516 from the National Institute of Allergy and Infectious Diseases (JCL) with ancillary support from research grant R804202 of the U.S. Environmental Pro* tection Agency. We are grateful to Patty Gallagher and Ted Lum for technical assistance. References 1 Abatort, KA.. and J.D. Yarbrough. Tha in vivo |fel of mirex on soluble hepatic intymti In Uir rat, Pril. Mloehein. Phyilol.,6 (1976) 192--199. 2 Alvar**, A.P., A. fItchbaln, K E. Anderson and A. Kappa*. Alteration* in drug metabolism in worker* mpoeed to polychlorinated biphanyia. CUn. 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