To identify environmental carcinogens there is a need for inexpensive and reliable short-term tests that can be used to predict the carcinogenic potential of any ...
Carcinogenesls vol.10 no.8 pp. 1445-1455, 1989
Carcinogens induce intrachromosomal recombination in yeast
Robert H-Scnlest!1*2, R.Danid Gietz1*2, R.D.Mehta3 and P.J.Hastings4 'Department of Biology, University of Rochester, Rochester, NY 14627, GeneBioMed, Inc., PO Box 18121, Rochester, NY 14618, USA, 3Prairie Biological Research, Ltd., 10515-36A Ave, Edmonton, Alberta T6J 2H7 and 4Department of Genetics, University of Alberta, Edmonton, Alberta T6G 2C6, Canada 2
To identify environmental carcinogens there is a need for inexpensive and reliable short-term tests that can be used to predict the carcinogenic potential of any given substance with high accuracy. The Ames assay, which is based on the induction of mutations in Salmonella typhimurium, is the most extensively used short-term test but certain human or animal carcinogens exist that are persistently undetectable as mutagens with the Ames assay or with other short-term tests. There is a need for a short-term test to detect those carcinogens that are missed by the Ames assay. Carcinogenesis is in many cases associated with genome rearrangement. Because of this association a system screening for intrachromosomal recombination that results in genome rearrangement has been constructed for potential use as a short-term test in the yeast Saccharomyces cerevisiae. Evaluation of this recombination system shows that it is readily inducible by a variety of mutagenic as well as nonmutagenk carcinogens, including carcinogens that are not detectable by the Ames assay or by various other short-term tests, such as safrole, urethane, ethionine, auramine, methylene chloride, carbon tetrachloride, cadmium sulfate, aniline, dimethylhydrazine, aminotriazole, acetamide, thiourea and DDE. The present report shows the data for these as well as for additional agents, their response profiles with different concentrations of the agents and the protocol for the DEL system.
Introduction At present the production and use of new chemicals that may pose a potential hazard to human health is regulated mainly based on long-term animal bioassays (1). Long-term animal bioassays cost >$1 million for each chemical and take 2 - 4 years to perform. Short-term bioassays on the other hand cost only $1000-10 000, with the microbial tests being priced at the lower end of this range (1) and they may produce results within 1 week or sooner. Therefore a prescreen of chemicals in a short-term test would be highly desirable, especially if it would be possible to predict the carcinogenic potential of any given substance with reasonably high accuracy. A large number of data establish that animal and human carcinogens without apparent genotoxic activity exist. These
•Abbreviations: DEL, deletion assay; ICR, interchromosomal recombination; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; NQO, 4-nitroquinoline Akixide; TPA, 12-0-tetradecanoylphorbol-13-acetate; DDE, 2,2-6u(4-chlorophenyl)-l,l-dichloroethylene; DMSO, methyl sulfoxide.
carcinogens are difficult or impossible to detect by short-term tests (2). In fact, the sensitivity of die Ames test, which is based on the induction of mutations in Salmonella typhimurium, and which is currently the most widely used short-term test, has been recendy reported by Zeiger et at. (3) to be 54% and by Tennant et al. (4) to be only 45 %, using the results of updated databases from long-term tests. The overwhelming majority of chemicals in our environment have not been tested for their potential to induce cancer. More chemicals have been tested with short-term tests, but this is die basis for die concern that there may be a considerable number of carcinogens in our environment that are not detected by die conventional short-term tests. Long-term tests are inherently unsuitable to solve tiiis problem in die near future. On the orner hand, short-term tests, if their sensitivity could be increased, may be used to solve this problem. A model calculation has been proposed based on hypothetical values for die social costs of nonidentified carcinogens in our environment and the assumption that 2% of the new chemicals may turn out to be carcinogens, in which case an increase in the accuracy of short-term tests of only 1% from 90 to 91% would be worth > $10 000 per chemical tested (1). Furthermore, since models for carcinogenesis of 'nongenotoxic carcinogens' are largely missing, a significant regulatory dilemma in dieir assessment of risk to humans exists (5). Not only because of economic benefits but also because of social concerns, our society has an obligation to remedy this situation especially since it becomes ever more severe in a time of growing chemical production. There is evidence in the current literature that substantial genome rearrangements are associated with cancer (6-10). Furthermore, deletions have been implicated in carcinogenesis caused by mutations in recessive oncogenes in cases such as retinoblastoma or colon cancer (11,12). Because of the association of genome rearrangement widi cancer, a system selecting for intrachromosomal recombination which results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae (13,14) (Figure 1). A plasmid with an internal fragment of die HIS3 gene has been integrated at the HIS3 locus yielding an integrative disruption of the HIS3 gene. This resulted in two copies of the HIS3 gene, each having one terminal deletion. This construct reverts to HIS3+ by recombination of the two his3 deletion alleles, which is in 99% of the cases associated with loss of the integrated plasmid (Figure 1) (14). Because plasmid excision and sister chromatid exchange were found not to be involved in reversion of die his3 disruption, die most likely alternative mechanism seems to be sister chromatid conversion, or in a broader sense intrachromosomal recombination (14). This recombination event deletes 6 kb of DNA, which comprises die entire integrated plasmid. This system has been termed deletion assay (DEL*). DEL is under different genetic control than interchromosomal recombination (ICR) and meiotic recombination. It has been shown diat mutations in die DNA repair genes RAD1 and RAD52 each affect the DEL recombination (15). The RAD52 gene product is involved in double-strand break repair, in ICR and 1445
R.H.Schlestl el al. HIS3
Yeast chromosome
Integration Selection for LEU+
his3
\7
LEU2
pBR322
his3
Selection forHIS*
his3
SJ LEU2
pBR322
his3
pBR322 y
his3
Fig. 1. Plasmid pRS6, which contains an internal fragment of the HIS3 gene, was cut within the internal his3 fragment and integrated into the genome at the HIS3 locus (a). This creates a duplication of the his3 gene in which one allele is deleted for its 3' end and the other for its 5' end (b). The two alleles share — 400 bp of homology and thus can recombine with each other to revert to the H1S3+ allele. As shown previously, H1S3+ recombinants do not arise by plasmid excision or by unequal sister chromatid exchange (14). In these studies the frequency of plasmid excision was determined by putting a yeast origin of replication sequence onto the integrating plasmid. Excised plasmids could be recovered but were 100-fold less frequent than the frequency of HIS"1" formation, which suggests that plasmid excision does not occur in the majority of recombinants. Reciprocal products expected to result from sister chromatid exchange were analyzed by Southern blotting. The pattern characteristic for sister chromatid exchange was not found in any of 25 events examined. Thus, as a likely alternative mechanism conversion between sister chromatids (c) is suggested which results in deletion of the integrated plasmid on one chromatid (d). After segregation of the two chromatids the HIS+ recombinant should show a HIS + leu~ phenotype which is found in 99% of all HIS"1" recombinants (14). Thus as a possible model it is proposed that a double-strand break initates the recombination event which is extended by an exonuclease to a gap. This gap is repaired by gene conversion from the sister chromatid as donor (14).
in meiotic recombination (16). The RAD1 gene product is involved in excision repair and has been shown to have no effect on ICR or meiotic recombination (15). It has further been shown that in a radl rad52 double mutant strain these two mutations act synergistically, reducing DEL recombination to a level far below the effect of each single mutant, suggesting that the radl and rad52 genes work in different recombination pathways (15). Since DEL is UV inducible in the radl mutant but not in the rad52 mutant the RAD52 pathway seems to be mainly responsible for the induction. These data suggest that the mechanism of DEL 1446
recombination differs from that of ICR and meiotic recombination. It has been shown that a variety of carcinogens that are not detectable by the Ames assay or many other tests are detectable with DEL (17). In the present report we show the comprehensive data for these chemicals as well as for eight additional agents, including physical agents and several mutagens without apparent carcinogenic activity of which three out of four do not induce DEL. Also shown are the response profiles with different concentrations of the agents and the protocol for DEL.
Carcinogens induce intracfaromosomal recombination in yeast
Materials and methods Strains and media The diploid strain RSI 12 (MATa/a ura3-52/ura3-52 Ieu2-3,1I2/Ieu2-A98 trp5-27/TRP5 arg4-3/ARG4 ade2-40/odc2-101 Uvl-92/lLVl HIS3 •••• pRS6/his3-A20O LYS2/lys2-SOI) (15) was used and contains the DEL system on one homolog (HIS3 •• pRS6) and a deletion of the entire open reading frame of HIS3 on the other homolog (his3-A200, 18). YPAD and synthetic minimal media (SC) were prepared similarly as described previously (19). YPAD: yeast extract 1%, peptone 2%, dextrose 2%, adenine sulfate 30 mg/1, agar 2% in distilled water. SC: yeast nitrogen base (Difco Laboratories, Detroit, MI) 0.67%, dextrose 2%, agar 2%, plus the following amino acids and bases per liter of distilled water: 20 mg each of L-tryptophan, L-histidine—HC1, L-arginine—HCI, L-methionine, L-isoleucine, L-tyrosine, L-lysine-HCl, adenine sulfate, uracil and 30 mg of L-leucine, 350 mg of L-tnreonine and 75 mg of L-valine. Omission media: SC medium lacking one of the nutritional supplements, e.g. SC-HIS, lacking histidine; SC-ADE, lacking adenine. Determination of the DEL recombination rates and interchromosomal recombination frequencies Plflsmid pRS6 containing an internal fragment of the HIS3 gene has been integrated at the genomic H1S3 site. This resulted in two copies of the his3 gene, one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end (Figure 1) (14). In cells of strain RS112 carrying the genomic integration of the plasmid pRS6, - 9 9 % of HIS3~ recombinants lose the LEW. gene (Figure 1) (14). Therefore the cultures used to select for HIS3+ recombinants were pregrown on medium lacking leucine and after treatment were plated onto medium lacking histidine. Thus growth and accumulation of recombinants does not occur in the pre-culture, and therefore the HJS3+ frequency is a measure of the recombination rate which results in high reproducibility of spontaneous rates. Strain RSI 12 is also heteroallelic for ade2-40 and ade2-101 so that ICR between homologs can be measured. The influence of various agents on the frequency of deletions (DEL) and ICR was determined as described in (17). Single colonies were picked from YPAD medium, inoculated into 5-25 ml of SC-LEU medium and grown for 24 h at 30°C under constant shaking. Cells were counted and the cell density was adjusted to 2 x 10* cells/ml in fresh SC-LEU medium. The medium containing the cells was distributed in aliquots of 5 ml each into disposable 15-ml tubes. The agent to be tested was added, the tubes sealed and the cells were incubated for 17 h at 30°C under constant shaking. Cells were pelleted in a clinical tabletop centrifuge. The cells for 4-nitroquinoline W-oxide (NQO), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) treatment were washed once with a 5% solution of sodium thiosulfate to inactivate the agent and a second time with sterile distilled water; for all other chemicals the cells were washed twice with sterile distilled water. Thereafter cells were resuspended in 0 . 5 - 1 ml of sterile distilled water, transferred to a glass tube and sonicated to disperse any clumps. Cells were counted and appropriate numbers were plated onto SC medium to determine the number of survivors, onto SC-HIS medium to score for DEL events and onto SC-ADE medium to determine the frequency of ICR events. The cells did not contain any clumps or tetrads. Colonies were counted after 2 - 3 days of incubation at 30°C. All data given reflect the total number of recombination events per number of cells capable of forming colonies (survivors) on non-selective medium. Data derived from less than five colonies were not included. A minimum increase of 2-fold over the spontaneous frequency in a dose-dependent manner has been regarded as evidence for inducibility. Each experiment was repeated at least three times and the results obtained were highly reproducible, two plates were used for each system and each concentration. The same qualitative result was obtained with other diploid as well as haploid strains containing the DEL system. Irradiation of cells Irradiation of cells was carried out with log-phase cells harvested at ~ 5 x 10* cells/ml. UV irradiation was carried out as described in Prakash and Prakash (20). For 7-irradiation cells were suspended in sterile distilled water at different concentrations and irradiated with a ^ ? o source at a dose rate of — 9 krad/min. Cells were then plated onto the respective medium for survival or selection for recombinants. Chemicals MMS and EMS were purchased from VWR Scientific and purified by vacuum distillation at 10—15 mmHg pressure before use. NQO, mechJorethamine, 1,2-dibromoethane, epichlorohydrin, aflatoxin Bl, sym-dimethylhydrazine dihydrochloride, ethidium bromide, 4-aminoantipyrine, sodium azide, 5-bromouracil, 12-O-tetradecanoylpborbol 13-acetate (TPA), mezerein, diethylstilbestrol, safrole, ethkmine, auramine O, cadmium chloride, cadmium sulfate, aniline, 3-amino-l,2,4-triazole, acetamide, thioacetamide, thiourea and 2-aminopurine were obtained from Sigma Chemical Company. Formaldehyde, methylene
chloride, carbon tetrachloride and acetone were obtained from J.T.Baker Chemical Company. Urethane, 2,2-Aii(4-chlorophenyl)-l,l-dichlorocthylene (DDE), 2-imidazolidinethione, hydroxybmine hydrochloride and methyl sulfoxide (DMSO) were purchased from Aldrich Chemical Company.
Results
To determine whether DEL is inducible by carcinogens, a diploid strain, RS 112, was used which contains the DEL system on one homolog and a deletion of the entire open reading frame of HIS3 on the other. Therefore there is no homology between the duplicated his3 deletion alleles and its homolog so that all recombination events have to occur intrachromosomally between the two his3 deletion alleles. One major advantage of DEL is that it permits selection against the recombination events in the pre-culture. Figure 1 shows that concomitantly with gain of the HIS3 function the LEU2 gene is lost, which is true for 99% of all recombinants (14). This allows for selection against recombinants on medium lacking leucine preceding selection for the recombinants on medium lacking histidine. Therefore the recombination frequency reflects the recombination rate and the variation in parallel cultures is minimal (14,15). Strain RSI 12 is also heteroallelic for ade2 so that interchromosomal recombination (ICR) between homologs can be measured and the two recombination systems can be compared in their performance to detect carcinogens. As mutagenic carcinogens,UV (21) and -y-irradiation (21,22), MMS (23,24), EMS (23,24), NQO (23), nitrogen mustard (23,24), epichlorohydrine (24,25,26) and aflatoxin B, (23,24) were used and gave strong inductions with DEL (HIS + ; 20- to 220-fold, Table I) as well as with ICR (ADE + ), as has been noted previously (27). All experiments were carried out in the absence of S9 and thus aflatoxin 1^ has been activated by yeast. Ethylene dibromide is a carcinogen (24,28), it is weakly positive with the Ames assay (23) and induces DEL as well as ICR (Table I). Dimethylhydrazine is a carcinogen (24,29) and has been reported negative with the Ames assay by McCann et al. (23) but was weakly positive with one strain in another study (25); it increases DEL 5-fold, but ICR < 2-fold (Table I). The carcinogens formaldehyde, safrole, ethionine, urethane, auramine, carbon tetrachloride, cadmium chloride, cadmium sulfate, aniline, aminotriazole, acetamide, thioacetamide, thiourea, DDE and ethylenethiourea have been chosen because they are negative with all five strains currently used in the Ames assay (23,25,28), yet they have been shown to be animal carcinogens (see below). Repair-deficient Escherichia colt cells are more sensitive to the carcinogens formaldehyde, carbon tetrachloride, cadmium chloride, cadmium sulfate and aniline than are the isogenic wild-type E.coli cells, suggesting that these agents may cause DNA damage (25). The structure of at least the carcinogens methylene chloride, carbon tetrachloride and DDE does not classify them as potential carcinogens (28) and they are therefore included in this category. Formaldehyde causes nasal cancer in rats (24,29,30) and has been proposed to act as tumor promoter (30). Table II shows that formaldehyde produces an increase in DEL of ~ 20-fold; it also induces ICR to a much lesser extent. Safrole produces liver cancer in mice and rats (24,26,29). Safrole results in an increase in DEL of 13-fold without a corresponding increase in ICR (Table H). Most interestingly, safrole is not only negative in the Ames assay, but is also negative in the rat liver foci assay and the sister chromatid exchange and chromosomal aberration assays widi mammalian cells (29). Ethionine is a liver carcinogen (26) and is, in addition to the Ames assay, negative in the sister chromatid exchange and chromosomal aberration assays with mammalian 1447
R.H.Scfaiestl et at.
cells (29). Ethionine induces DEL in yeast ~ 5-fold without resulting in increased ICR frequencies (see also 27,31) (Table II). Urethane is very effective in producing lung as well as skin rumors in mice, rats and hamsters (24,26,29). Urethane induces the DEL assay — 5-fold at high doses without increasing the ICR frequency (Table II). Auramine induces liver tumors and local sarcomas in mice and rats (24) and is a suspected human carcinogen (24,32); it shows a very steep loss in viability over a short concentration range and increases the frequency of DEL events but fails to increase ICR. Methylene chloride or
dichloromethane is a non-mutagenic carcinogen (28,30,32) and induces cancer at multiple organ sites in mice and rats (28). However, the structure of methylene chloride does not classify it as a potential carcinogen (28). As shown in Table II, methylene chloride induces DEL in a dose-dependent manner ~ 4-fold without inducing ICR. Carbon tetrachloride is a liver carcinogen (24,29,30) and has been proposed to act as tumor promoter (30). Carbon tetrachloride induces DEL starting at a concentration of 4 mg/ml (Table II). Cadmium cloride and cadmium sulfate induce cancer at multiple organ sites in mice and rats (24,32) and a study
TaWe I. Carcinogens that are detectable with the Ames assay UV irradiation UV fluence (J/m2) Survivors (%) HIS+/104 cells ADE+/10 5 cells
0 100 3.6 (145) 0.28 (11)
10 96 11.9 3.5
y-ray exposure •y-ray (krad) Survivors (%) HIS+/104 cells ADE+/10 5 cells
0 100 3.6 (145) 0.28 (11)
10 84 18.5 23
Methyl methanesulfonate (MMS) Cone, (/ig/ml) No. of generations Survivors (%) HIS+/104 cells ADE+/10 5 cells
0 4.4 100 1.39 (87) 0.32 (20)
13 4.3 100 3.9 1.9
Ethyl methanesulfonate (EMS) Cone. 0
