Document K62ZeO2EeNZy2Qn33d3ee4pjo

i tion of pUtinum 11 compounds 307. smcnt of mutation induced by m1 molecule*, in: T.A. Connor* Chemotherapy, Splinter, New n-PttNHjJjCl;, An Anti-tumor d mutagenesis: dependence on -77)93-103. itnehia cofi, B*ct*riol. Rev., 40 t 5 i i Mutation Research, 77 (19S0) 55--63 Elsevicr/North-Holland Biomedical Press 55 CYTOCHROME P450 MEDIATED GENETIC ACTIVITY AND CYTOTOXICITY OF SEVEN HALOGENATED ALIPHATIC HYDROCARBONS IN Sacebaromyces cerevisiae '* DAVID F. CALLEN , C. ROLAND WOLF and RICHARD M. PHILPOT 1 1 School of Biological Sciences, Flinders University of S.A., Bedford Park, South Australia, 5042 (Australia) and 2 National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Pork, NC 27709 (U.S.A.) (Received 28 May 1979) (Revision received 7 August 1979) (Accepted 14 August 1979) Summary Cells of Sacebaromyces cerevisiae, harvested from log-phase cultures, contain cytochrome P-450 and are capable of metabolizing promutagens to genetically active products. The activities of 7 halogenated aliphatic hydrocarbons in the yeast system have been investigated. All of the compounds tested (methylene chloride, halothane, chloroform, carbon tetrachloride, trichloroethylene, tetrachloroethylene and s-tetrachloroethane) induced mitotic gene convertants and recombinants and, to a lesser extent, gene revertants when incubated with logphase cells of the yeast strain D7. An examination of the difference spectra observed upon the addition of carbon tetrachloride, halothane and trichloro ethylene to whole-cell or microsomal suspensions of yeast suggested that cyto chrome P-450 mediated the metabolism of the hydrocarbons tested to cyto toxic and genetically active compounds. Several halogenated aliphatic hydrocarbons are known to be carcinogenic. Epidemiological studies have shown that vinyl chloride is a carcinogen in humans [2]. Recent studies by the National Cancer Institute (U.S.A.) have established that chloroform, carbon tetrachloride, trichloroethylene and several other chlorinated hydrocarbons are carcinogenic [23]. These, compounds generally induce tumours in the livers of mice but are only marginally carcino genic in rats. It has been reported that operating room personnel have an increased risk of cancer [15] which may be due to exposure to anesthetic gases such as halothane (CFjCHClBr). The toxicity and potential carcinogenicity of the hal genated aliphatic hydrocarbons have been related to their metabolic activati n by the hepatic SI. 0397 56 microsomal monooxygenase system. Metabolism to free-radical intermediates has been suggested for some halogenated alkanes [3]. The hepatic microsomal monooxygenase system has been incorporated into in vitro systems which are used for the determination of mutagenicity [12]. Such systems have established that most carcinogenic compounds are also mutagenic e.g. [13]. Some of the halogenoalkanes tested in these systems have been shown to be mutagenic [4,7]; however, tests with carbon tetrachloride [13,22], chloroform [18,22], and halothane [1,18,22] have consistently given negative results. Several of the halogenated alkenes tested, including vinyl chloride [2,8] and trichloroethylene [8,16], have also been shown to be muta genic [8,11,16]. Most in vitro test systems for mutagenicity require mammalian hepatic microsomal fractions as a source of monooxygenase activity. In such systems active metabolites are generated outside of the indicator organism and may react before entering the cell and reaching the DNA and thereby lead to false negative results. Yeast cells harvested from cultures grown on medium containing glucose contain a cytochrome P-450-dependent monooxygenase system [10] and are capable of activating a variety of promutagens [5,6]. Therefore, yeast cells can be used as an indicator organism for promutagens without the need for mamma lian monooxygenase systems. Some halogenated hydrocarbons are metabolized to products which interact with ferrous cytochrome P-450 to give characteristic spectra [3,14,19--21]. In order to obtain evidence that halogenated hydrocarbons enter yeast cells and are metabolized, the spectra formed on the addition of 3 of these compounds to suspensions of whole cells and microsomal preparations were recorded. Materials and methods Strain. D7 is a multipurpose diploid in which the frequencies of gene conver sion at the trpS locus, mitotic recombination at the ade2 locus, and gene con version at the Uul locus can be measured [27]. Mitotic gene conversion can be .induced by a variety of mutagens and mutagen specificity is absent at these loci [25]. The D7 diploid used in these studies was constructed from the parental haploid strains. D7.1528B (aade2-40, trpSb. ilvl-92) and D7.9A (a, ade2-ll9, trpSa, ilvl-92), which were kindly supplied by Dr. F.K. Zimmermann. This D7 strain had a cytochrome P-450 concentration of approximately 13 nmoles/g dry weight in cells harvested from log-phase cultures grown with glucose as carbon source. The strain D5 [26] was used for spectral studies because of its high cytochrome P-450 concentration (up to 38 nmoles/g dry weight). Experi ments [5 and unpublished] have shown that these two strains can activate a variety of promutagens to genetically active products. Media. MIN medium consistent of 0.6% Difco yeast nitrogen base without amino acids and 2% glucose supplemented with 5 mg/1 of adenine, tryptophan (10 mg/1), or isoleucine (60 mg/1) as necessary. Log-phase cultures were grown on complete medium (2% peptone, 1% yeast extract and 2% glucose). Treatment conditions. Cells were harvested from log-phase cultures growing on medium containing glucose as carbon source and resuspended in 0.1 M ph s- SL 034298 ee-radical intermediates been incorporated into i of mutagenicity [12]. ic compounds are also ed in these systems have ith carbon tetrachloride I have consistently given tested, including vinyl been shown to be muta- : \ ] j 1 lire mammalian hepatic ictivity. In such systems ator organism and may md thereby lead to false ! | j I iium containing glucose i ase system [10] and are i Therefore, yeast cells can i )ut the need for mamma- ; i oducts which interact t:tra [3,14,19--21]. In ons enter yeast ceils and of 3 of these compounds .ons were recorded. equencies of gene converide2 locus, and gene con ic gene conversion can be city is absent at these loci ructed from the parental and D7.9A (a, ade2-119, K. Zimmermann. This D7 proximately 13 nmoles/g ;s grown with glucose as tral studies because of its iles/g dry weight). Experitwo strains can activate a ast nitrogen base without ;/l of adenine, tryptophan ohase cultures were grown nd 2% glucose), ^^-phase cultures growing ^Hispended in 0.1 M phos 57 phate buffer, pH 7.0, at a cell concentration of approx. 3 X 107/ml. The test compound -was added directly to a 3-ml aliquot of cell suspension and incu bated in a screw-capped 50-ml glass centrifuge tube at 37 for varying times. Treatments were terminated by the addition of 40 ml of ice-cold buffer. The samples were then centrifuged and the sedimented cells resuspended in 3 ml of buffer. These cell suspensions were then plated on appropriate media to allow for estimation of genetic activity. Assessment of genetic activity. For D7, 0.1-ml aliquots of each treatment were spread on each of 5 plates of MIN + adenine + tryptophan to allow estimation of the ilvl reversion frequency, and on each of 5 plates of MIN + adenine + isoleucine to allow estimation of the trp5 conversion frequency. Each treatment was diluted 104-fold and 0.1-ml aliquots spread on each of 20 plates of MIN medium containing all 3 supplements to allow estimation of cell survival. Survivors and trp5 convertants were scored after incubation of the plates for 2 days and ilvl revertants after incubation for 5 days at 30. The plates were stored at 4 for 7 days and the mitotic recombinants scored, both on the 20 plates containing all 3 supplements and on the 5 MIN + adenine + isoleucine plates. The genetic alterations at the ade2 locus were grouped into two classes: twin spots (red-pink and red-pink-white colonies, which arise from mitotic recombination) and other genetically altered colonies (red, pink, redwhite, and pink-white). Data are given for one of two duplicate experiments from which similar results were obtained in all cases. Spectral analysis.' Cells of the D5 strain were harvested from log-phase cul tures grown on complete medium containing glucose and were resuspended in phosphate buffer, pH 7.0, at 5 X 10 to 109 cells/ml. The cell suspension was divided between two cuvettes (3 ml) and the spectra recorded before and after the addition of the test ligand to the sample cuvette. Microsomal fractions were prepared as described previously [5]. All spectra were recorded with an Aminco DW2A spectrophotometer. Chemicals. Halothane (Ayerst Labs, Inc., New York, NY) and tetrachloroethylene (Eastman Kodak Co., Rochester, NY) contained thymol, 0.01% as stabilizer. Methylene chloride, trichloroethylene, and s-tetrachloroethane were purchased from Fisher, Fair Lawn, NH; chloroform from J.T. Baker, Phillipsburg, NJ and carbon tetrachloride from Mallinckrodt, St. Louis, MO. Results Preliminary experiments established that 4-h treatment of log-phase cells of the strain D4 (used to monitor gene conversion, see ref. 5) or strain D7 with a particular dose of the various halogenated hydrocarbons resulted in increased frequencies of gene conversion per cell survivor (data not shown). However, only marginal increases in the observed numbers of convertant colonies was evident in the treatments as compared with the controls. Such a result could be attributed to differential survival of the various cell phenotypes rather than any genetic effect of the treatment. In subsequent experiments positive genetic effects (as assessed by at least a doubling in the observed numbers of conver tant or mitotic recombinant colonies as compared with the control) were evident when log-phase cells of the strain D7 were treated for 1 h (Table 1). SL 034299 58 TABLE 1 EFFECT OF VARIOUS HALOGENATED HYDROCARBONS ON STRAIN D7 OF YEAST CtUi were harvested from iof-phase cultures of the strain 07 frown on medium with flucose as carbon source. Treatments were incubated at 37C for 1 h. Compound Concentration (mM) Methylene chloride Halothane Chloroform Carbon tetrachloride Trichloroethylene Tetrachloroethyiene syn-Tetrachloroethane 0 104 157 209 0 47 63 79 0 21 41 54 0 21 28 34 0 15 22 0 4.9 6.6 8.2 0 3.1 5.2 7.3 Survival Total number of colonies counted % 1467 1128 617 1 1201 1002 364 0 1423 1302 982 84 1454 1252 1120 152 1201 802 3 1546 1301 891 0 1201 . 1127 426 1 100 77 42 <0.1 100 83 30 <0.1 100 91 69 6 100 86 77 10 100 67 0.3 100 84 58 <0.1 100 94 35 0.1 trp 5 locus Total number of convertants counted 258 314 660 171 355 583 246 274 450 278 285 331 350 506 171 610 221 251 742 171 213 741 c 2 locus' Convertants/ T *1 number 10s survivors o win spots j c nted J i.8 2.8 10.7 ; i !1 - 1.4 3.5 16.0 1.7 2.1 4.6 33.1 2.0 2.6 3.1 61.7 1.4 7.6 1 1 1.4 1.9 8.3 2' 1.4 1.9 17.4 1< < : 5 5 N.D..Tnot determined. The total number of colonies in the different classes represent total counts of colonies from 5 plates in the case of survivals conversion and revertant*frequency estimations. Mitotic recombination was estimated from count* i ini celt* fKW When strain D4 was similarly treated, i.e. for 1 h, only marginal increases in the frequencies of gene conversion, were evident. When cells of the strain D7, harvested from log-phase cultures, were treated with increasing concentrations of each of the haiogenated hydrocarbons there were decreased cell survival and increased frequencies of gene conversion, mitotic recombination and, to a lesser extent, reversion. Although the toxicity of the 7 compounds differed considerably, there were no apparent differences among the genetic activities of the compounds when comparisons were made at similar levels of cell survival, A nonlinear correlation between cell survival and the concentration of the hydrocarbons in the incubation mixtures was apparent (Table 1). In ordei yeast cell: carbon tet or microso When c; with sodiu observed (! shift^wai whoqjpl run and a order to o SL 034300 59 IN D7 OF YEAST medium with glucose ti carbon trph locus Total number of convertants counted 258 314 660 171 355 583 246 274 450 278 285 331 350 506 171 610 221 251 742 171 213 741 ? ;d :ocm% Convertanta/ 10s survivors i Tc of CO 1.8 2.8 10.7 !: jr number xti spots :*d 1.4 3.5 i6.o 1.7 2.1 4.6 33.1 2.0 2.6 3.1 61.7 1.4 7.6 1.4 1-9 8.3 j ; i j 1 i i) i !i 2 1.4 1.9 17.4 1i Mitotic recombwams/104 survivors 3.1 1.9 44.9 3.5 17.0 22.9 1.6 1.7 4-1 44.8 1.6 5.3 5.8 40.1 2.5 36.0 3.3 5,3 52.6 2.5 2.0 56.5 Total number of genetically altered colonies counted 21 20 53 Total frequency of genetically altered colonies/ 103 survivors 3.3 3.9 14.0 13 3.3 29 6.1 43 16.4 6 1.0 11 1.9 43 8.9 47 52.7 11 1.7 19 3.4 16 3.1 65 33.3 13 3.3 53 12.0 18 3.0 27 4.7 102 26.8 13 3.3 19 3.9 60 18.8 ilvl locus Total number of revertants counted Reveilanls/ 10* survivors 39 50 36 43 63 29 61 46 81 50 38 41 57 11 43 59 45 49 N.D. 43 26 64 2.7 4.4 5.8 3.6 6.3 8.0 4.3 3.5 8.2 60.0 2.6 3.3 5.1 7.2 3.6 7.4 2.9 3.8 N.D. 3.6 2.3 15.0 runts of colonies from S plates in tottc recombination was estimated from counts of colonies (rowing on a total of 30 plates, 20 plates containint medium on which all survivin( cells grew and 10 Plates containing medium on which only frp5 convertants grew, / marginal increases in the lase cultures, were treated nated hydrocarbons there icies of gene conversion, ion. Although the toxicity re no apparent differences comparisons were made at i between cell survival and ion mixtures was apparent In order to obtain evidence that these halogenated hydrocarbons entered the yeast cells and were metabolized the spectra obtained on the addition of carbon tetrachloride, halothane or trichloroethylene to whole-cell suspensions or microsomal preparations were recorded (Figs. 1 and 2). When carbon tetrachloride was added to microsomal preparations reduced with sodium dithionite, an absorption peak with a maximum at 460 nm was observed (Fig. 1). The formation of the peak was time-dependent and a gradual shift towards 450 nm was observed. When carbon tetrachloride was added to whole-cell suspensions, two peaks were observed, one with a maximum at 450 nm and a second at 505 nm (Fig. 2). Sodium dithionite was not required in order to obtain these spectra. The formation of both peaks was time-depen- SL 034301 60 Waveltngih (nm) Fit. 1. Difference spectre obtained on the addition of carbon tetrachloride and htlothanc to microtomes prepared from the yeast strain D5, Microtomes were prepared from log-phase cells of the strain D5 harvested from medium containing glucose at carbon source. The concentration of cytochrome P-450 from the carbon monotide difference spectrum was 0.66 AA (450--490 nm)/ml, ----------- . difference spectrum of dithiomte-reduced microtomes 15 min after the addition of carbon tetrachloride, 10 mM, to the sample cuvette.---------- --. difference spectrum of dithionite-rcduced microtomes 6 min after the addition of halothane. 10 m.M, to the sample cuvette.............. , baseline. dent. The peak at 505 nm was still observed after the test cuvette had been bubbled with carbon monoxide. On the addition of carbon monoxide to the cuvette, prior to the addition of carbon tetrachloride, no peak at 505 nm was observed. Similar experiments with halothane resulted in the formation of a peak with a maximum at 450 nm in microsomal preparations (Fig. 1) and with peaks at 437 and 505 nm in suspensions of whole cells (Fig. 2). The addition of tri chloroethylene to whole-cell suspensions gave peaks with maxima at 450 and 505 nm. The peak at 450 nm appeared to reach a maximum after approx. Fig. 3. Difference spectra obtained on the addition of carbon tetrachloride, trichloroethylene and halo thane to cell suspensions of^ the yeast strain D5. Cells of the strain DS were harvested from log-phase cul tures grown on medium containing glucose as carbon source. The concentration of cytochrome P-450 from the carbon monoxide difference spectrum was 0.86 AA (450--490 nm)/ml. " -- , difference spectrum 8 min after addition of trichloroethylene, 15 mM. to the sample cuvette.-------------- , difference spectrum 15 min after the addition of halothane, 12 mM, to the sample cuvette. -- * *, difference spec trum 15 min after the addition of carbon tetrachloride, 10 mM, to the sample cuvette.............. , baseline. 5 min. After thi action was obs preparations aftt Discussion There was an carbons when im D7 for 4 h. Ho; increased frequer gests that these c to be expressed . rapid in the stra suspensions of th P-450 concentrau D4 strain. The o'; cells and test cor toxic properties metabolites, or a c When treatmen log-phase cells of mitotic recombir. recombinanj^ras of induced W re has been reported of some halogenc alkenes to be epo. yeast to these two It has been suge trichloroethylene j impurity is epichi metabolic activatio these genetic effec; active to log-phase Bronzetti, . Zeiger Several lines of e metabolized by a c to products which pounds. Experimen' chloroform were ge with yeast cells har\ galactose (such cells hydrocarbons were evidenced by the sp suspensions of whok observed, however, i preparations [14,19,: The addition of t SL 034302 nd halolhane to microsomts >hase cells of the strain D5 ration of cytochrome P-450 nm)/ml. -----------. difference ion tetrachloride. 10 mM. to -mcrosomes 6 min after the test cuvette had been m monoxide to the 'eak at 505 nm was mation of a peak with 1) and with peaks at . The addition of tri ll maxima at 450 and lximum after approx. - trichloroethylene end haioharveittd from log-ph^se cul* (ration of cytochrome P-4 50 > nm)/mL -----------1 different* cuvette,------------ , difference ette. difference spec* baseline. 61 5 min. After this period an additional peak at 423 nm was observed. No inter action was observed on the addition of sodium dithionite to microsomal preparations after incubation with trichloroethylene and NADPH. Discussion There was an observed marginal genetic effect of these 7 halogenated hydro carbons when incubated with log-phase cells of strain D4 for 1 or 4 h and strain D7 for 4 h. However, treatment of strain D7 for 1 h resulted in significant increased frequencies of mitotic gene conversion and recombination. This sug gests that these compounds need metabolic activation for their genetic activity to be expressed and that the metabolism of these compounds may be more rapid in the strain D7. This latter result may not be unexpected since cell suspensions of this strain, harvested from log-phase cultures, had a cytochrome P-450 concentration up to 5 times higher than a similar cell suspension of the D4 strain. The observation of a marginal genetic activity when incubations of cells and test compounds were continued for 4 h could be the result of the toxic properties of these compounds masking the genetic effects of their metabolites, or a destruction of the metabolic system. When treatment times were 1 h all 7 hydrocarbons tested were activated by log-phase cells of the strain D7, to products which induced gene conversion, mitotic recombination and gene reversion. The frequency of ade2 mitotic recombinants was the most sensitive indicator of genetic damage; frequencies of induced ilvl reversion were low. Reversion of the ilvl gene in D7 diploids has been reported to be mutagen specific [27]. Although the active metabolites of some halogenoalkanes are though to be free radicals [17] and those of alkenes to be epoxides [3], no difference was evident in the response of the yeast to these two classes of hydrocarbons. It has_been suggested [9] that the carcinogenic and mutagenic properties of. trichloroethylene are due to the presence of "impurities. However, the major impurity is epichl6rbhydrinj~a direct-acting mutagen wKich^ does not need metabolic Tcfivafloh.^Therefore, itjs`jun]ikely ~that~this~is an_expianation for these genetic effects observed in yeast since trichloroethylene is_not genetically active to log-phase cultures of the strain D4 or stationary cultures of D7 (G., Bronzetti, E. Zeiger and D. Frezza, personal communication). ^ Several lines of evidence strongly indicate that the hydrocarbons tested are metabolized by a cytochrome P-4 50-dependent yeast monooxygenase system to products which are both mutagenic and more toxic than the parent com pounds. Experiments (data not shown) indicate that methylene chloride and chloroform were genetically inactive and significantly less toxic when tested with yeast cells harvested from log-phase cultures grown in medium containing galactose (such cells have a low level of cytochrome P-450 [5]). Halogenated hydrocarbons were also shown to interact with yeast cytochrome P-450 as evidenced by the spectra obtained on the addition of several compounds to suspensions of whole yeast cells or yeast microsomal preparations. The spectra observed, however, were often different from those observed with mammalian preparations [14,19,20,24]. The addition of trichloroethylene to suspensions of whole cells resulted in SL 034303 62 the formation of a spectrum which was similar to that observed in mammalian microsomes. It has been suggested that the spectrum, with a peak at 450 nm, results from the metabolism of trichloroethylene to carbon monoxide [19]. The peak of absorption at 505 nm which formed on the addition of either halothane or carbon tetrachloride, to suspensions of whole yeast cells remains unexplained. It is concluded that the halogenated hydrocarbons tested are metabolized to genetically active and cytotoxic products by a yeast cytochrome P-450-dependent monooxygenase system. Halothane, chloroform, carbon tetrachloride and tetrachloroethylene have been reported to be inactive in test systems which require the use of a mammalian monooxygenase system [13,18,22] even though they appear to be carcinogenic [23]. Thus, the yeast system described appears to be more sensitive than some other in vitro test systems in the detec tion of the activity of some compounds. The higher sensitivity of this yeastactivation system may result from the production of active metabolites in much closer proximity to the nucleus than is possible with liver S9 in vitro activation systems. The use of this yeast-activation system may be advanta geous where the active metabolites are particularly unstable and reactive; as is likely for those of the halogenated hydrocarbons. References At 1 Baden. J.M.. M. Bnnketnhoff. R.S. Wharton, B.A. Hiff, V.F, Simmon and R.I. Mail*. Mutagenicity of volatile antithetic*: Hajothane, Anesthesiology, 45 (1976) 311-318. 2 Bamch, H., and R. Montcsano. Mutagenic and carcinogenic effects of vinyl chloride. Mutation Ret., 32 (1975) 93-114. 3 Bonic. G,, and D. Henichler, Chemical reactivity, biotransformation and toxicity of polychlorinated aliphatic compound*. CRC Critical Rev. Toxicol.. 4 (197$) 395--410. 4 Brem. H.. A.B. Stein and H.S. Rosenkranz, The mutagenicity and DNA-modifying effect of haloalkane*. Cancer Re*.. 34 (1974) 2576-2579. 5 Callen. D.F., and R.M. Philpot. Cytochrome P-450 and the activation of promutagen* in Saccharomycct eerrvuiar, Mutation Re*.. 45 (1977) 309--324. 6 Callen, D.F.. C.R. Wolf and R.M. Philpot. Cumene hydroperoxide and yeaxt cytochrome P-150: spectral interaction* and effect on the genetic activity of promutagen*. Biochem. Biophyi. Res. Commun.. 83 (1978) 14--20. 7 Fishbein, L., Industrial mutagens and potential mutagens, 1. Halogenated aliphatic derivatives. Muta tion Re*.. 32 (1976) 267-308. 8 Greim, H.. G. Bonte, Z. Radwan, D. Reichert and D. Henschler. Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylene* as a function of metabolic oxirane formation, Biochem. Pharmacol., 24 (1975) 2013--2017. 9. Henschler. D.. E. Eder. T. Neudecker and M. Mctzler, Carcinogenicity of trichloroethylene: fact or artifact? Arch. Toxicol., 37 (1977) 233--236. 10 Kawaguchi, K., K. Iihidste and K. Tagawa. Nonmitochondrial electron transfer system in anaerobi cally grown yeast cells, J. Biochem.. 74 (1973) 817--826. 11 de Lorenzo. F,, S. DeglTnnoconti, A. Ruocco, L. Silcngo and R. Cortese. Mutagenicity of pesticides containing 1,3-dichloropropene. Cancer Res.. 37 (1977) 1915--1917. 12 Mailing. H.V.. Oimethylnitrosamine: formation of mutagenic compounds by interaction with mouse liver microtomes. Mutation Rea.. 13 (1971) 425--429. 13 McCann. J.. E. Choi. E. Yamasaki and 8.N. Ames. Detection of carcinogens aa mutagens in the Salmo nella/microtome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135--5139. 14 Mansuy, D,, W. Nastainciyk and V. Ullrich, The mechanism of halothana binding to microsomal cyto chrome, Naunyn^chmiedeberg's Arch. Pharmacol., 285 (1974) 315--324. 15 Report of'an Ad Hoc committee on the effect of trace aneslhetici on the health of operating room personnel. Anesthesiology, 41 (1974) 321--340. 16 Shahin. M.M.. and R.C. von Borstal, Mutagenic and lethal effects of a-benxene hexachloride, dibutylphthalate and trichloroethylene in Saecharomycet eerrvitiae, Muution Ret., 48 (1977) 173--180. 17 Slat 18 Stu nm, 19 Ta> mic Cor 20 Ueh pro. 1--1 21 Uehl OIUl 22 Uehl lc- r. 23 Weis; <19* 24 Wolf halo? 25 Zm- 32726 Zirs- cros27 Zittit- mtot 386. Si 34304 % ^^tbserved in mammalian - . with a peak at 450 nm, carbon monoxide [19], ie addition of either halohole veast cells remains tested are metabolized to cytochrome P-450-depen* , carbon tetrachloride and ve in test systems which system [13,18.221 even he yeast system described test systems in the detec* r sensitivity of this yeastof active metabolites in ble with liver S9 in vitro system may be advantamstable and reactive; as is on and R.l. Mazze, Mutagenicity of 5. -s of vinyl chloride. Mutation Res., d toxicity of polychlorinsted id D,\ A-modifying effect of haio- .vation of promutagens in Socehe- ,ide and yeast cytochrome P-450: nutagens, Biochem. Biophys. Res. genated aliphatic derivatives, Muta- Mutagenicity in vitro and potential bolic oxirane formation, Biochem. nicity of trichloroethylene: fact or ectron transfer system in anaerobi- Cortese, Mutagenicity of pesticides 7. pounds by interaction with mouse rcinogens as mutagens in the Salmo:. (U.S.A.), 72 (1975) 5135-5139. othane binding to microsomal eyto5-324. cs on the health of operating room of o-benzene hexachloridc. dibutyl* tion Res.. 48 (1977) 173-180. 63 Slater. T.FFree radical mechanisms in tissue injury. Pion Press. London. 1972. Sturrock, J., Lack of mutagenic effect of halothane or chloroform on cultured cells using the xzaguanine test system. Br. J. Anaesth.. 49 (1977; 207--210. Taylor, P.S.. W, Xastainctyk and V. LUr.ch, Conversion of trichloroethylene to carbon monoxide by microsomal cytochrome P-450, in: V, Pinch, 1. Roots. A. Hildebrandt. R.W. Estabrook and A.H. Conney, (Eds,), Microsomes and Drug Oxidations. Pergamon, Oxford, 1977, pp. 615--621. Uchleke. H,, K.H. Hellmer and S. TabareUi, Binding of 1 ^C-carbon tetrachloride to microsomal proteins in Mtro and formation of chloroform by reduced liver microsomes. Xenobiotica, 3 (1973) 1--11. Uehleke. H-. S. Tabarelli-Poplawski. G. Bonse and D. Henschler, Spectral evidence for 2.2,3-trichlooxirane formation during microsomal trichloroethylene oxidation. Arch. Toxicol.. 37 (1917) 95--105. Uehleke, H,, T. Wemer. H. Greim and M. Kramer. Metabolic activation of haloalkanes and tests in vitro Tor mutagenicity. Xenobiotica, 7 (1977) 393--400. Weisburger. E.K.. Carcinogenicity studies on halogenated hydrocarbons. Environ. Hlth. Perspect., 21 (1977) 7--16. Wolf, C.R., D. Mansuy, W. Nastaincryk. G. Deutschmann and V. Ullrich, The reduction of polyhalogenated methanes by liver microsomal cytochrome P-450. Mol. Pharmacol.. 13 (1977) 698--705. Zimmerman, F.K.. Induction of mitotic gene conversion by mutagens. Mutation Res., 11 (1971) 327-337. Zimmerman, F.K.. A yeast strain for visual screening for the two reciprocal products of mitotic crossing-over. Mutation Res.. 21 (1973) 263--269. Zimmerman, FJC.. R. Kern and H. Raser.berger. A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation. Mutation Res., 28 (1975) 381-- 388. SL 034305