Document 3erYqv14EN2ovaDZJqwdeELdy

R&S 112676 BIO-MEDICAL. RESEARCH DOCUMENT DESCRIPTION FORM 63 68 69 76 Duplicate . in all cards: --> 1i uuuosres year as-1961- File number [Right justify [Numeric only] Author(s), as Last Name FS (No Punctuation) and coden for journal as JAMA preceeded by one blank space 1 20 21 40 4160 Z Al/M &*//. A-fi s. ---f-T---K---s-t /O' *//-s--S----- J. 77 78 Sub-Index Code 61 62 Title of Report; end with space-hyphen-hyphen-space. Follow with Index Terms, separated from each other with comma-space. Avoid other punctuation; do not abbreviate. 61 62 21 22 23 24 Source (Journal, Vol., Number, Pages, Date) 12 Pg\/~/)rrfcr Brief Summary 12 10 SUMMARY: ///rsrsis) 61 62 zx J. / /f?? 32 61 62 61 62 63 64 0000385 STUDIES OH THE MUTAGENICITY OF VINYL CHLORIDE METABOLITES .AND RELATED CHEMICALS A. D. Lajnnbach, S. Lee,.J. Wong, -and. U.. H. Streips Department of Microbiology and Immunology University of Louisville School of Medicine Department of Chemistry ' Louisville, Kentucky 402Q1 I. INTRODUCTION The studies by Viola et al (26) and Maltcni et al (1?) established the carcinogenic potential of vinyl chloride monomer. The detection of angiosarcoma In industrial workers exposed to polyvinyl chloride suggested e causal relationship between this chemical and the development of hepatic abnormalities (4,12). Hefner et al (7) have delineated the metabolic' fate of Inhaled vinyl chloride in rats and proposed that the epoxide, chlarooxirane, and chloroacetaldehyde were the carcinogenic intermediates. Their hypothesis has been supported by the work of several laboratories (1,14,16,19, Elmore, Wong, Laumbach and Streips, submitted for publication) using bacterial strains as mutagenic indicators. In-this communication we present additional data concerning the mutagenicity and the potential mechanisms of action of several vinyl chloride metabolites. Including the previously unreported chloroacetadehyde monomer hydrate, chloroacetaldehyde dimer hydrate, and chloroacetaldehyde trimer. Epichlorohydrin, a mutagenlc/carclnogenic (21,25) methylene homolog of chlorooxlrane was also examined. H. PROCEDURES AND MATERIALS USED A. Bacterial Strains The bacterial strains utilized in these studies are presented In Table I. The Bacillus subtills strains were all maintained on AK agar (BHL). Salmonella typhimurium cultures were obtained from B. N. Ames (l) and were stored on Nutrient Agar (Difco) plus 5g NaCl per liter. R&S 112678 w. . ',1 iW* V - . *di --4b.-- * B. Mutagenicity Assays i The Indirect assay utilized repair deficient strains of B. subtllis. The procedure was a modification of the "rec-assay" describe? by Kada et al (9). Cells were grown overnight in Nutrient Broth (Difco) at 17C in a rotary incubator shaker, then diluted tenfold in phosphate buffer (pH 7.0). The suspended cultures were streaked onto Nutrient Agar plates (Difco). ..Filter paper discs (6 mm) were saturated with the chemical solutions to be examined, then were placed onto the agar plates nert to the streaked bacterial cultures. Following incubation at 37C overnight the plates were examined and the lethality and mutagenic potential of the teat chemicals were assessed by comparing inhibition zones between the B. subtllis 168 wild type, a repair^-capable strain and the various DNA repair-deficient strains. In all these studies 4-nitroquinoline-l-ortde (4NQ0) was used as the positive mutagenic control.' Direct mutagenicity assays utilized the S. typhimurium tester strains described by Ames (1). .The chemicals were examined by the methods of vr^Cnnn et al (16).. The cultures were grown In Nutrient Broth plus 0.5* NaCl overnight in a rotary incubator shaker at -yiC. ' A mixture of the tesrfc chemical (0.1 ml) in dimethyl sulfoxide (DM50) and 2 ad of soft agar (0.62 agar, 0.62 NaCl, 0.5 mM biotin, and 0.5 mil histidine) was added to 0.1 ml of the bacterial culture. The solutions were mixed thoroughly and overlaid onto Trtn-tmat plates CVogel-Bonner E medium (27), 1.52 agar, and 22 glucoseControl samples were prepared by emitting the teat chemicals For the positive mutagenesis control, 4NQ0 was added to the mixtures in place of the test chemicals. All plates were incubated for 48 hr at 27C prior to the enumeration of revertant colonies. " C.. Chemical Compounds The chemical compounds utilized in these studies were prepared, purified, and analyzed by previously reported techniques (ELmare, Wong, Laumbach, and Streips, submitted for publication).- D. Preparation of DMA Transforming DNA was isolated from B. subtllis cultures by the method described by Young and Wilson (29T* : in some of the experiments the cultures were pretreated for 15 min either with chloroacetaldehyde (16 nil) or epichlorohydrin (16 mil) prior to the extraction procedure. In alternate experiments S-9 liver homogenate mix was added to the compounds prior to addition to bacteria. .The S-9 liver homogenate contains per ml, 0.3 ml of the S-9 fraction, 8 nil MgCl2> 33 mM KC1, 5 nil glucose--6-P, 4 mM NADP, and 100 nil sodium phosphate (pH 7.4)* The DNA concentration in ail lysates were assayed by the method of Richards (20). E. Treatment of DNA In vitro with Chemicals A sample of B. subtllis transforming DMA (0.9 ml) la standard noHne citrate (ssc) (0.15 M NaCl-0.015 M trisodium citrate, pH 7.0) was combined with O.k ml chloroacetaldehyde (1.0 M in DMS0) or 0.1 ml 156 .: -V . p -n p epichlorohydrin (1.0 U in E&ISO). The mixture was allowed to react for 1 hr with occasional shaking. Following this treatment the treated DMA. was dialyzed at OC against three 500 ml changes of SSC for 24 hrs. In alternate experiments the DNA-chendcal mixtures were placed in a dialysis bag and immersed in the S-9 liver homogenate mix. These samples were dialyzed in SSC as above. F.. Competent Cultures for Transformation Assays The procedures for the development of competence were similar to . those described (23). B. aubtilis cells were grown in a modified SpdLzlzen's medium \CiJj.) (.29) for 90 min at J7C after cessation of logarithmic, growth in a rotary incubator, shaker. The cells were then diluted tenfold into QH1 medium (29) and incubated for an additional 60 min at J7C in the shaker. At this time the culture has attained maximum competence. '* G,, Transformation Procedures A sample (0.1 ml) of extracted, treated or untreated DNA was added to 0.8 ml of the competent cultures and incubated at J7C. for 30 min in the shaker. The reaction was terminated by the addition of 0.1 ml of deoxyribonuclease (500 vg/ml, Worthington Biochem. Corp.) for 15 min at 37C. The cells were plated on appropriate selective minimal, media and incubated at 370 for .43 hrs. III. BESOLTS A summary of preliminary mutagenesis screening experiments with potential vinyl chloride monomer metabolites and related compounds is presented in Table U. It Is evident that chlarooxirane -and chloroacetaldehyde are the ultimate mutagens is this system. These results agree with the published data (3,16). In addition, this table describes the mutagenicity of the other chemical forms of chloroacetaldehyde, not- ably a monomer hydrate, a dimer hydrate, and a trimer. The hydrate and dimer hydrate forms have been shorn to form an equilibrium mixture by > the spontaneous rearrangement of chloroacetaldehyde under physiological conditions (Elmore, Wong, Laumbach, and Strelps, submitted for publication),and these hydrate forms must he regarded as potential metabolites of consequence. Purified dimer hydrate and trimer were synthesized under laboratory conditions. Neither acetaldehyde, chloroacetic acid, nor chloroethanol showed a significant level of mutagenicity in thes assays. Other Investigators have reported the mutagenicity of chloroethanol, however, either high concentrations or activation with microsomal enzymes was required for activity (3j16). Out results agree with those of KcCaim et al (16). These experiments suggest a molecular relationship involving the proximity of the chloride group to the aldehyde moiety for mutagenic activity. In this regard we are currently examining structurally analogous ketones, substituted with various halogens. Epichlorohydrin (l-ehloro-2,3 epoxypropane) was also mutagenic in screens using the Salmonella tester strain TA100. 157 \ 37 CO to O) o00 Further experiments examined the effect of proposed metabolites on several different DMA repair deficient strains of B. subtills. Chlorooxirane and the different forms of chloroacetaldehyde were all found to specifically inhibit the growth of strain VC-1, which lacks recombination repair (17) (Table III). Epichlorohydrin was capable of moderate reactivity only in the presence of the S-9 fraction. Quantitative mutagenesis assays with Salmonella strain TA100, an indicator for base-pair substitution mutations, revealed that chloro acetaldehyde monomer had the highest mutagenic capacity of all the reactive metabolites (Table IV). The monomer-dimer hydrates, dimer ' hydrate, and trimer show progressively decreasing mutagenic, efficiency as evidenced by the higher chemical, concentration required for eliciting maximum reversion. All forms of chloroacetaldehyde were very toxic, thus the mutagenic response of, each compound was limited to a narrow range of concentrations. However, epichlorohydrin, a weak mutagen by comparison, has a broad mutagenic spectrum and a corresponding low toxicity. Since the mutagenic. activity of the compounds constituted strong evidence that DNA was a primary target of attack, we examined the inter action of chloroacetaldehyde end epichlorohydrin with transforming DMA. It is known that the biological activity of transforming DMA can be altered by exposure to physical and chemical agents (8,22). Previous studies have shown that chloroacetaldehyde can hind to DMA in vitro (ll). Accordingly;, transforming DMA Isolated from B. subtills 168W was treated with either chloroacetaldehyde or epichlorohydrin as described In Materials and Methods. The treated DMA was examined in transformation assays utilizing several different auxotrophic strains of B. subtills as the. recipients. Data presented in Table V reveals that in vitro treatment of DNA with either compound has little or no apparent efTect on the biological activity of this DMA in transformation. Since both chloroacetaldehyde and epichlorohydrin demonstrated mutagenic activity in the Salmonella IA100 strain, we examined the effect . of these two compounds on B. subtjlis DNA in vivo. Transforming DNA was Isolated from B. subtills followingT 15 a2n exposure to the mutagenic chemicals. The* DMA concentration was calculated from these samples, and levels equivalent to those used in the in vitro assays were added to competent cultures. The results of these transformation assays are shown in Table VI. Two major effects are evident with chloroacetaldehyde in vivo treated DMA. First, there was a major depression of the biological activity in the transforming DNA. Secondly, the depression showed genetic marker specificity. Moreover, the DMA segments containing genetic markers which have previously been shown to be associated to macromolecular structures such as the cell membrane.-(6,24,28) or the cell wall (Streips, Doyle, Sueoka, Brown, and Fan, submitted for publication) were selectively protected from attack by chloroacetaldehyde and epichlorohydrin. The activity of epichlorohydrin was less In these experiments, however, the patterns of specific marker inactivation are quite similar. The addition of the S-9 mix to the chemicals prior to additidn.to the cells, did-not cause significant alteration in tronsformationefficiency (results not shown). In some samples there was an effect on the transforming DMA by DfiSO, therefore all. transformation values were corrected to account for this parameter. 1S8 1I 7* * i.i' '4 Th< 1) We I oxirane, chloroac the pre' have she correct: .acetaldf DMA onl; oT the r apparent mutagen! To such as bothjttj or cellv identifi mediated in micro the viny standing blocking Rec caused b Sal, monel the Capa erpress able metaboli are rela evoke th chemical repair, types of has been groups ( chemical repair tt The chemical. Recocbin sense it we can p; replica: chloride replica t. has teen IV. DISCUSSION The major findings reported in this manuscript can be summarized: 1) We have confirmed the mutagenicity of chloroacetaldehyde end chlorocoirane, and extended it to include the additional potential metabolites, chloroacetaldehyde monomer hydrate, dimer hydrate and trimer, as veil as the previously unreported chlorooxirane homolog, epichlorohydrin. 2) Ve have shown that recombination repair appears to be the mechanism for the correction of vinyl chloride metabolite elicited damage. 3) Chloro acetaldehyde causes & decrease in the biological activity of transforming DMA only if the cells are treated with the mutagen prior to the extraction of the DNA. In vitro studies shoved no effeot. 4) Epichlorohydrin apparently diTTora markedly from the vinyl chloride metabolites in mutagenic activity. Io understand the mutagenic potential of an environmental carcinogen, such as vinyl chloride and related chemicals,it is necessary to determine both its metabolic fate end probable mechanism of action for alteration of cellular1 processes-. This-report,, as veil. as. others, .(3,14,16).has . identified the potential active metabolites In vinyl chloride monomer mediated carcinogenesis, furthermore, on the basis of a aeries of studies in microbial systems, ve can postulate probable mechanisms of action of the vinyl chloride monomer metabolites and related chemicals. Under standing of these mechanisms is necessary for the development of possible blocking agents to the carcinogenic activity. Recombination repair appears to be induced to correct DNA lesions caused by vinyl chloride monomer metabolites and epichlorohydrin. Salmonella strainJCAlOO which lacks excision repair (uvr~), yet retains ^he capacity for recombination repair is capable of recovery can Brpress mutation following exposure. Furthermore, experiments with Several repair-deficient 5. subtilia mutants demonstrate that only the recombination repair mutant is specifically sensitive to the active metabolites, whereas the excision .repair mutants and the wild type strain are relatively unaffected. The nature of the lesions may specifically evoke the recombination repair mechanism (10), or, alternatively, the chemical reactivity of the metabolites may directly suppress other repair. It is known that recombination repair is inducible, while other types of repair are mostly constitutive (5). Since chloroacetaldehyde has been shown to specifically Interact with proteins containing -SH groups (J. Hoffman, personal communication}. It is possible that the chemical could inactivate the constitutive repair enzymes leaving the repair to an inducible system. The requirement for recombination repair of damage Induced by these chemicals suggests the potential route of mutagenesis in bacteria. Recombination repair has been shown to be error prone (16). In this sense it resembles postreplication repair in maamallan cells (13). Thus, we can postulate that the analogous error prone repair pathway, postreplication repair, may function in. mammalian cells in response to vinyl chloride metabolite elicited damage. A relationship between postreplication repair caused errors and somatic mutation and carcinogenesis has been suggested in patients with the skin disease, xeroderma pig mentosum (13). 159 The increased inhibitory activity of the chloroacetaldehyde diner and trimer forms for the other repair-deficient B. subtills strains (Table III) may have been nonspecific killing of the cells, since all the strains other than MC-l showed identical levels of inhibition. The necessity for metabolic activation of epichlorohydrin could reflect either a lack, of permeability of the nanactivated compound or the requirement of a metabolite of this compound as the true mutagenic species. ,. . Neither chloroacetaldehyde nor epichlorohydrin seemed to affect transforming DNA in vitro (Table V). Although several Investigators have reported that CAA specifically modifies bases and. causes mismatched base pairs (2,11), this reaction in vitro does not seem to affect the biological activity of the DUa.` In" contrast, DMA which was isolated from cells treated with, either chloroacetaldehyde or. epichlorohydrin (in vivo. Table VI) was severely affected.- The overall biological activity of the transforming DMA is depressed, and It appears that the regions of the genome which are not protected by either the cell membrane or cell wall are most susceptible to attack and inactivation. It has also been postulated both in Escherichia, coll and B. subtills that the replication 73 ; origin, and wp'iinat-lon fork are all outer surface bound. P (IS,24). Thus, these would be protected regions from chloroacetaldehyde W attack and the nonreplicating DNA in the cytoplasm would be most _j. susceptible. In this connection, recent experiments in our laboratory -* (Laumbach, Lee, Wong, and Strelps, manuscript in preparation) have NJ shows that, chloroacetaldehyde causes enhanced mutation levels in cultures 00 with nonreplicating genomes. This may imply that chloroacetaldehyde to could be active in mammalian cells during growth stages where little DNA synthesis occurs. The?mode of action of epichlorohydrin, a known carcinogen (25), differs from that of the vinyl chloride monomer metabolites. Although epichlorohydrin causes similar base substitution mutations in Salmonella tester strain TA 100, it Is a comparatively weaker alkylating agent based on quantitative assay. Epichlorohydrin also; exhibits a lower toxicity level than vinyl chloride monomer metabolites, thus epichloro hydrin can demonstrate mutagenic activity through a wider range of concentrations. In addition, our laboratory has preliminary evidence that epichlorohydrin produces higher levels of mutation in Salmonella cultures which are actively replicating DNA than in cultures which have been arrested in DMA replication (Laumbach, Lee, Wong, and Strelps, manuscript in preparation). The different activity apectra between chlorooxlrane end its homolog epichlorohydrin points out the necessity for a multifaceted study of carcinogens. V. SUMMAET Our laboratories have utilized strains of B. subtills and Salmonella typhimurlum to investigate the mutagenicity 0/ vinyl chloride metabolites and related compounds. The major findings reported in this manuscript are: 1) Confirmation of mutagenicity of chloroacetaldehyde and chlorooxlrane. 2) Description of mutagenicity of additional potential metabolites of vinyl chloride, chloroacetaldehyde monomer hydrate, dimer hydrate, and trimer, as well as the mutagenic 160 / i- ** --. JT carcinogenic chlorooxirane homolog, epichlorohydiin. 3} Recombination repair is postulated to be the mechanism for correcting vinyl chloride metabolite elicited damage. 4) Chloroacetaldehyde affects the transformation activity of DMA. only.if cells are treated with the mutagen prior to the extraction of the DMA,. In vitro the fhumiuni had no, effect. 5) Epiehlarohydrln differs from vinyl chloride, metabolites in mode of action. YI. ACKNOWLEDGEMENTS. Ve wish to thank Vary A. Kirmaraan for her extremely able technical assistance. Ve are grateful to Dr. Jerald Hoffman for maUng available preliminary results and to Dr. B. M. Ames for providing the Salmonella tester strains. This work was supported by a grant from the B. F. Goodrich Company to the Cancer Center at the University of Louisville, School of Medicine. 161 R&S 112684 ef1- Table U JArtagenic Activity Assayed by Bacterial Test Systems Compounds Indirect Screen Direct -Test4 B. aubtllla S. typhimariw "Repair-Assay" *"Stralh "TaIOO Ac taldehyde .Ohloroace-Uc, Acid . ... Chloroethanol Vlnylidene Chloride Vinyl Chloride Chlorooxlrane Chloroacetaldsbyde_ jnffliftMvl *" IT ' Vnloroacetaldehyde (monomer-dimer hydrates) Chloroacetaldebyde (dimer hydrate) Chloroacetaldehyde (trlmer) Eplchlorohydrln MRb NR . NR NR C M-f +4 4 4- NR NR ...... NR NR NR +d 444 44 4 4 4* "Experiments performed in absence of liver homogenatemediated activation. ^NR no reaction detected c Reactive Moderately reactive *+++ Very reactive 163 n .a>; 3 Surfv 'o5ivr*-:^s = J.-V * V ' :--V; V-V** 9892 H S*U Compounds Table 111' ' "Repair-Assay" with Bacillus subtills Strains _______________________________________ J___________________________________________________________________________________________: Molar Concentration Growth Inhibition in Millimeters6 168WT MC-1 tlcr-9 FB-13 (hcr+, reo+) (hcr+, rec~) (hor~, rec+) (uvr+, rec+) Chloroacetaldehyde (monomer) Chloroaoetald ehyde {monomer-dimer hydrate) Chloroacetaldehyde (dimer hydrate) Chloroacetaldehyde (trimer) Chlorooxirane Epichlorohydrin Epichlorohydrin (plus liver homogenate)0 0.10 0.115 0.097 0.096 0.26 0.997 0.997 2.0 NIb 2.0 7.0 : nx' NI. NI 28.0 23.0 10.0 15.0 10.0 HI 3.0 aAveragc Inhibition calculated from multiple experiments. bNo inhibition detected, ^jOOO x g supernatant (S-9) + HADPH generating system. 4.0 NI 2.0 6.0 ; | ; NI ' NI NX 3.0, NI 2.0 7.0 NI ' NI ' 1 NI 164 X k&';* fMEiV.s-- m e<n/t Li.'"; ' ^>vr,*i*,*r-3-*-*'`Si :V?y-A Table IV Quantitative Mutagenicity Assay by Salmonella TA100 Reversion 1 Compound 1 Concentration in Soft Agar Layer n*4/Platea Average Humber Revertants/Plate^ Chioroaoetaldehyde (monomer) Chloroacetaldehyde (monomer-dimer hydrate) Chloroacetaldehyde (dimer hydrate) Chloroacetaldehyde (trimer) Epichlorohydrin 0.0004 0.054 0.490 0.240 4.746 'f highest effective non-toxle concentration for reversion. ^Spontaneous background revertants subtracted. 265 977 311 ' 159 2856 I l 8892U S?U Table V Effect of Chloroacetaldehyde and Epichlorohydrin of Transforming DNA In vl.tro Recipient Strains c Relative Transformation Efficiency1 metBlO leu-8 cy3A . hisAl ura-1 trpC2 lys-8 purB6 Epichlorohydrin treated DNA BUL 714 RUB 783 BUL 709 Oo>V BR 151 .92 .-9? .97 1.16 .91 .60 .98 .77 .99 .95 1.02 .85 1.48 1.07 .62 Chloroacetaldehyde treated*-DNA > BUL 714. 1.43 .92 ' .55 .91 RUB 783 BUL 709 BR 151 .93 1.45 .86 1.33 .75 .89 .90 .75 NDb .77 Relative transformation efficiency calculated: number of transformants with treated DMA DiJot determined. number of transformants with untreated DNA Conditions for competence and transformation as described In Materials and Methods. Table VI EFFECT OF CHLOROACETALDEHYDE AND EPICHLOROHYDRIN ON TRANSFORMING DNA IN VIVO RelntlvA TpnnqfrtT)mo+.: ad ftn amnvd EFFECT OF CKLOROACETALDEHYDE AND EPICHLORQHYDRIN ON TRANSFORMING DNA IN VIVO Recipient Strains'5 Relative Transformation Efficiency8' motBlO leu-8 cysA hlsAl ura-1 trpC2 lys-3 ` purBl6 Chloroacetaldehyde in vivo treated DNA i| . BUL 714 RUB 783 BUL 709 BR 151 .34 .09 .08 .36 .33 .10 35 . .53 * .07 .32 .34 .37 .13 .11 .10 Epichlorohydrin in vivo treated DNA . \ BUL 714 " RUB 783 BUL 709 BR 151 .55 .17 ?5 .46 .52 .59 .15 .15 .38 .50 .36 .37 .11 .19 ND Relative transformation efficiency calculated! number transformanta with treated DNA . number transformants with untreated DkA "Conditions for competence and transformation as described in Materials and Methods. cNot determined. R&S 112690 LITERATURE CITED 1. Ames, B. N., Lee, F. D., and Durston,'V. E. An Improved Bacterial Test System For Detection And Classification Of Mutagens And " Carcinogens. Proc. Nat. Acad. 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Impaired Transfoxaability Of Bacillus subtilis Mutant Sensitive To Mitomycin 0 And Ultraviolet RaeEation. J. Mol. Biol., 15: 440-454, 1966. 18. Olsen, V. L., Heidrich, H. G., Hannig, K., and Hofahneider, P. H. Deoxyribonucleic Add-Envelope Complexes Isolated From Escherichia coll By Free-Flow Electrophoresis: Biochemical And Electron Microscope Characterization. J. Bacterlol., 118: 646-653, 1974. 19. Rannug, U., Johansson, A., Ramel, C., and Wachtmeister, C. A. The Mutagenicity Of Vinyl Chloride After Metabolic Activation. AMBIO, 3: 194-197, 1974. 20. Richards, G. Modifications Of The Diphenylandne Reaction Giving Increased Sensitivity And Simplicity In The Estimation Of DNA. Anal. Biochem., 57: 369-376, 1974. 21. Strauss, B.y*and Okubo, S. Protein Synthesis And The Induction Of Mutations In Escherichia coll By Alkylating Agents. J. Bacteriol.. 79: 464-473, 2$So. 22. Strauss, B., Reiter, H., and Searashi, T. Recovery From Ultra violet And Alkylating Agent-Induced Damage In Bacillus subtilis. Rad. Res. Supp., 6: 201-211, 1966. 23. Streipa, U. N., and Young, F. E. Transformation In Bacillus subtills Using Excreted DNA. Molec. Gen. Genetics, 133: 47-55, T97 . 24. Sueoka, N., end Quinn, W. Membrane Attachment Of The Chromoseme Replication Origin In Bacillus subtilis. Cold Spring Harbor Syiira. Quant. Biol., 33: 695-705, .1968" - 25. Van Duuren, B. L. On The Possible Mechanism Of Carcinogenic Action Of Vinyl Chloride. Aim. N. Y. Acad. Sci., 246 : 258-267, 1975. 26. Viola, P. L., Bigotti, A., and Caputo, A. Oncogenic Response Of Rat SfcLn, Lungs And Bones To Vinyl Chloride. Cancer Res., 31: 516- 522, 1971. ~ 169 rrrts * '`A** . Iv\--J-. . * xjVi-' -; ' 1 "* M ?*'* " * *= 'iSiV-v -4** -r ' 27. Vogel, H. J., and Benner, D. M. Acetylornithinase Of Escherichia coll: Partial Purification And Some Properties. J. Biol. Chem., ggT 97-106, 1956. 28. Yamagudin, K., and Yoshihawa, H. Association Of The Replication Terminus Of The Bacillus suhtllls Chromosome To The Cell Membrane. J. Bacterid., 124; 1030-1033, 1975. 29. Young, F. E., and Wilson, G. A. Bacillus subtills. In; Handbook Of Genetics. Ed.: Robert C. King, Plenum Pressi New York, 1: 69- 114, 1974. " R&S 112692 170