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Effect of Cadmium Exposure on Background and anti-5-Methylchrysene-1,2-dihydrodiol 3,4-Epoxide-Induced Mutagenesis in the supF Gene of pS189 in Human Ad293 Cells R. Rita Misra,*,† John E. Page,‡ George T. Smith,† Michael P. Waalkes,† and Anthony Dipple‡ Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, Division of Basic Sciences, National Cancer Institute, and Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received October 8, 1997
Cadmium is a toxic environmental contaminant that is carcinogenic in humans and rodents. Although cadmium has proven to be mutagenic in a variety of assay systems, exactly how cadmium achieves gentoxicity is poorly understood. To define the mechanism(s) underlying the mutagenicity and comutagenicity of cadmium, human Ad293 cells were exposed to subtoxic doses of the metal and transfected with untreated or anti-5-methylchrysene-3,4-dihydrodiol 1,2-epoxide (5-MCDE)-treated pS189 shuttle vector. Alterations in the frequency, types, and distribution of mutations were subsequently assessed in the supF gene of pS189 that was replicated in Ad293 cells and screened in indicator bacteria. Doses of 0.5 and 1 µM CdCl2 increased the mutation frequency of untreated pS189 by approximately 4- and 8-fold, respectively, with no apparent effect on the types of mutations generated. In contrast, hostcell exposure to cadmium had little or no effect on the frequency, types, or distribution of mutations generated with 5-MCDE-treated pS189. These results indicate that cadmium increases mutagenesis of untreated pS189 by affecting a process that is not involved in mutagenesis of the 5-MCDE-treated vector. Although it is not clear exactly how host-cell exposure to cadmium increases background mutagenesis, presumably, the mutagenic effect does not involve cadmium interaction with the cellular machinery used to replicate past bulky DNA lesions.
Introduction Cadmium (Cd)1 is a toxic environmental contaminant that is considered to be carcinogenic to humans and animals (1). In humans, pulmonary carcinogenesis has been associated with occupational exposure to Cd in dusts and aerosols produced during the smelting and refining of metal ores, during electroplating and welding, and during the manufacture of pigments, plastic stabilizers, and nickel-cadmium batteries. Other sources of human exposure include cigarette smoke and contaminated food, water, and air (1). In general, the route and duration of exposure ultimately determine which organs become targets of Cd toxicity. Acute exposures have been associated with damage to the testes, liver, and lungs, and chronic exposures can lead to emphysema, obstructive airway disease, permanent renal dysfunction, bone disorders, and immunosuppression (2). In experimental animals, Cd has proven to be carcinogenic by all routes of exposure * To whom correspondence should be directed at NCI-FCRDC, Bldg. 538, Rm. 205E, Frederick, MD 21702. † Inorganic Carcinogenesis Section. ‡ Chemistry of Carcinogenesis Laboratory. 1 Abbreviations: Cd, cadmium; 5-MCDE, 5-methylchrysene-3,4dihydrodiol 1,2-epoxide; X-gal, 5-bromo-4-chloro-3-indolyl β-D-galactoside; IPTG, isopropyl β-thiogalactoside; bp, base pair; ROS, reactive oxygen species.
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tested. At particular target sites however, sensitivity to the carcinogenic and toxic effects of Cd often depends on the species, strain, age, and sex of the animal (1). In rodents, Cd has been associated with injection site tumors, as well as tumors of the lung, prostate, liver, testes, and hematopoietic system (3). In many cases, tissue specificity appears to be mediated by the presence of metallothioneins which are small, cysteine-rich, metalstorage proteins that sequester, and may thereby inactivate, free Cd ions (4). Although Cd exhibits limited mutagenic potential in standard bacterial assays (5), it is clearly mutagenic in mammalian cells (6-8). At least three different hypotheses regarding this metal's mode of action currently exist: (1) Cd may interact directly with chromatin to induce strand breaks, cross-links, or conformational changes in the DNA (5, 9); (2) Cd may act indirectly, by affecting proteins involved in transcription, DNA replication, or DNA repair (9-11); or (3) Cd may increase intracellular hydrogen peroxide levels (e.g., by depleting antioxidant levels) and thereby catalyze iron/coppermediated redox reactions which produce free radicals that can break or cross-link the genetic material (5). Alternatively, Cd-induced increases in hydrogen peroxide production could trigger lipid peroxidation which, in turn, could generate mutagenic adducts in DNA (12).
This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 02/10/1998
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In the present study, a modification of the shuttlevector mutagenicity assay developed by Seidman (13) was used to assess the mutagenic and comutagenic potential of Cd in mammalian cells. It is our hope that such information may provide further insight into the mechanism(s) underlying Cd carcinogenicity. Particular advantages of this assay system include the fact that the pS189 vector used to monitor mutagenicity can be exposed to a known mutagen prior to transfection, thereby eliminating any direct effects of that mutagen on host-cell replication or repair. According to our experimental scheme, human Ad293 cells were exposed to subtoxic doses of Cd and transfected with untreated or anti-5-methylchrysene-3,4-dihydrodiol 1,2-epoxide (5MCDE)-treated vector. Alterations in the frequency, types, and distribution of mutations were subsequently assessed in the supF gene of pS189 that replicated in Ad293 cells and had been screened in indicator bacteria. In these experiments, host-cell exposure to Cd resulted in increased mutagenesis of untreated pS189 but had little or no effect on mutagenesis of 5-MCDE-treated vector.
Materials and Methods Caution: Cd has been classified by IARC as a group I carcinogen, and 5-MCDE is a potent mutagen and carcinogen; therefore, all precautions should be taken to prevent exposure. Materials. Annhydrous cadmium chloride was purchased from Sigma Chemical Co. (St. Louis, MO). 5-Bromo-4-chloro3-indolyl β-D-galactoside (X-gal) and isopropyl β-thiogalactoside (IPTG) were purchased from Amersham/United States Biochemical (Arlington Heights, IL). 5-MCDE was a gift from Dr. R. G. Harvey (University of Chicago, Chicago, IL) and was synthesized as described (14). The plasmid pS189 (13), human embryonic adenovirus-transformed kidney cells (Ad293), and Escherichia coli strain MBM7070 were gifts from Dr. M. Seidman (Codon Pharmaceuticals, Gaithersburg, MD). Cell Cultures. Ad293 cells were grown at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Assay of Cytotoxicity. Chemically induced inhibition of cell growth was monitored as described previously (15). Growing Ad293 cells were seeded at densities of 100, 250, and 500 cells/ 100-mm dish. CdCl2 was dissolved in distilled, deionized water and filter-sterilized, and various concentrations were delivered in fresh, complete medium 16 h later. At 24 h after Cd administration the metal was removed by medium replacement. The cells were fed once over the course of the next 7 days, and colonies were visualized with Giemsa stain. Three individual dishes of each seeding density were assayed per treatment, and mean values were used to estimate viability (the plating efficiency of the Cd-exposed cells divided by the plating efficiency of the unexposed cells, multiplied by 100). Treatment of pS189 with Racemic anti-5-MCDE. pS189 (36 µg) in 10 mM Tris-HCl, 1 mM EDTA (TE), pH 7.6 (324 µL), was treated for 1.5 h at 37 °C with various amounts (0.12 or 0.18 µg) of racemic anti-5-MCDE delivered in 36 µL of acetone. Excess hydrocarbon was subsequently removed by organic solvent extraction (16). Transfection of Human Cells. Growing Ad293 cells were plated at 5 × 105 cells/100-mm dish. At 68 h after plating, the cells received fresh media containing 0, 0.5, or 1 µM CdCl2. Exactly 24 h later, the Ad293 cells were transfected with untreated or 5-MCDE-treated vector (10 µg/dish) using a calcium phosphate coprecipitation technique (17). At 48 h after the transfection, plasmid DNA was isolated and purified using an alkaline lysis extraction procedure that had been adapted from a previously described method (18). In the modified purification procedure, low-molecular-weight DNA was digested
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Figure 1. Effect of Cd on the colony-forming ability of Ad293 cells. Cells were seeded at 100, 250, and 500 cells/100-mm dish and exposed to 0, 1, 5, 10, or 50 µM CdCl2 for 24 h, and colonies were allowed to grow for 1 week before counting. Three dishes of each seeding density were assayed per treatment, and mean values were used to estimate viability. The data shown represent results from cultures seeded at 100 cells/dish. Similar results were obtained at the higher seeding densities. with proteinase K, RNase A, and dpnI restriction endonuclease to remove residual protein, RNA, and unreplicated/ methylated DNA, respectively. Purified pS189 was dissolved in 50 µL of TE buffer, pH 7.6, and stored at -20 °C until used for bacterial transformation. Bacterial Transformation and Mutant Selection. MBM7070 cells were transformed with pS189 that had been recovered from the Ad293 cells. The transformation was accomplished by electroporation as previously described (19). Luria-Bertani agar dishes (100 mm) supplemented with ampicillin (50 µg/mL), X-gal (2 mg/dish), and IPTG (5 mg/dish) were used for mutant selection (bacteria harboring pS189 with a wildtype supF gene give rise to blue colonies on such plates, whereas those containing a mutated supF gene give rise to white or paleblue colonies). White and pale-blue colonies were isolated and restreaked to confirm the selection, and mutant plasmids were extracted using a Wizard miniprep kit (Promega, Madison, WI). Analysis of Mutants. The supF genes of pS189 plasmids isolated from white or pale-blue colonies were sequenced using an ABI PRISM Ready Reaction Dye Terminator sequencing kit (Perkin-Elmer Corp., Foster City, CA), a model 480 DNA thermal cycler (Perkin-Elmer), and a model 377 ABI PRISM DNA sequencer (Perkin-Elmer). Statistical Analysis. Chi-square analysis was used to determine if the mutation frequencies of Cd-exposed cells were significantly different from appropriate controls (p < 0.1 was considered significant). Mutational “hot spots” were identified using the Poisson distribution to calculate the expected number of randomly distributed mutations at each base pair (bp) in the supF gene.
Results Cd-Induced Cytotoxicity in Ad293 Cells. A standard cell survival assay was used to establish a subtoxic dose range for CdCl2 exposure of Ad293 cells. Based on the results presented in Figure 1, concentrations of 0.5 and 1 µM Cd were used in subsequent mutagenesis studies. Effect of Cd on Mutation Frequencies Measured in the supF Gene of Untreated and 5-MCDETreated pS189. In the absence of Cd, the background mutation frequency measured in the supF gene of pS189
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Table 1. Types of Mutations Induced by Cd in the supF Gene of Untreated pS189 in Human Cells Cd dose transbase insertions/ mutation (µM) formants substitutions deletions frequency × 104 0 0.5 1.0
26 572 32 541 30 317
1a 3b 5d
0 3c 6e
0.38 1.5 3.0
a Mutant was 164 G:C f T:A (number refers to the position in the gene). b Mutants were (i) 152 G:C f C:G, (ii) 155 G:C f C:G plus 157 A:T f G:C. c Mutants were (i) 3-bp insertion at position 131, (ii) 2-bp deletion/5-bp insertion at position 130, (iii) large deletion (>65 bp) directly upstream of position 165. d Mutants were (i) 155 G:C f T:A, (ii) 163 G:C f C:G, (iii) 133 G:C f C:G plus 169 G:C f T:A plus 172 G:C f A:T. e Mutants were (i) 1-bp deletion at position 167, (ii) 3-bp deletion at position 151, (iii) 5-bp deletion at position 105, (iv) 5-bp deletion at position 105, (v) 6-bp deletion at position 164, (vi) large deletion (>200 bp) directly upstream of position 224.
that had replicated in Ad293 cells was 0.38/104 bacterial transformants (Table 1). This frequency is similar to values reported previously by Bigger et al. (0.49/104 and 0.15/104) using the same shuttle-vector assay (16, 20). Exposure of Ad293 cells to subtoxic doses of 0.5 and 1 µM CdCl2 increased the mutation frequency of untreated vector in a dose-related manner, by approximately 4- and 8-fold, respectively (Table 1). At these doses, Cd had no measurable effect on transformant yield. The Cd-related increases in mutation frequency of untreated vector were statistically significant (p ) 0.08 and 0.01, for 0.5 and 1 µM Cd, respectively), indicating that Cd is mutagenic in this assay. In the absence of Cd, the mutation frequencies measured in the supF gene of pS189 treated with 0.12 and 0.18 µg of anti-5-MCDE were 15 × 10-4 and 19 × 10-4, respectively. These values were substantially greater (∼32- and ∼50-fold, respectively) than those observed for untreated vector and are consistent with those reported previously (ref 20, Figure 2). In addition, no dramatic differences in the mutation frequencies of 5-MCDEtreated pS189 were found when the host cells were exposed to Cd (in all cases, p > 0.1). The 5-MCDErelated increases in mutation frequency were 106, 93, and 102 mutants/104 transformants/µg of 5-MCDE at 0, 0.5, and 1 µM CdCl2 (Figure 2). Again, these Cd doses had no demonstrable effect on transformant yield. Thus, in contrast to our results with untreated pS189, exposure of Ad293 cells to 0.5 or 1 µM CdCl2 had no apparent effect on the mutation frequency of 5-MCDE-treated vector. Effect of Cd on the Types of Mutations Generated in the supF Gene of Untreated and 5-MCDETreated pS189. Analysis of the types of mutations generated in these experiments was undertaken in order to determine if Cd may affect mutagenesis in a manner not readily apparent from analyses of overall mutation frequency. This analysis involved sequencing all of the mutants obtained using untreated pS189 and approximately 25 mutants from each of the six remaining treatment groups (i.e., from each dose of 5-MCDE at 0, 0.5, and 1 µM CdCl2). Comparison of the types of mutations obtained using untreated pS189 in the presence and absence of Cd revealed Cd-related increases in both base substitutions and insertions/deletions (Table 1). Although the number of mutants analyzed in the present study was extremely limited, comparison of our results with previous analyses of “background” mutagenesis using the same experimental system (16, 20, 21) revealed no Cd-related differences in the ratios of the
Figure 2. Lack of effect of Cd on mutation frequencies measured in the supF gene of 5-MCDE-treated pS189 in human cells. Ad293 cells were exposed to 0 (top panel), 0.5 (middle panel), or 1 (bottom panel) µM Cd. Replicated plasmids were recovered 48 h later and used to transform MBM7070 bacterial cells. Mutation frequencies were determined by screening an average of 28 000 transformants/treatment group. In all cases, the estimated mutation frequency fell within 90% confidence limits for the proportion as determined by transformant sample size (39). Associations between mutation frequency and 5-MCDE dose were analyzed by linear least-squares regression. A reasonable fit (r > 0.9) indicated that the relationship was consistent throughout the range of doses tested. The slope of each line served as a measure of the increase in mutation frequency per µg of 5-MCDE.
types of mutations generated. These results suggest that Cd acts by enhancing at least one of the processes involved in background mutagenesis. As expected, analysis of the types of mutations generated during replication of anti-5-MCDE-treated pS189 in Ad293 cells that were not exposed to Cd revealed the nearly exclusive induction of base substitutions, the vast
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Table 2. Types of Mutations Generated in the supF Gene of 5-MCDE-Treated pS189 in Human Cellsa Cd dose (µM)b base change transversions G:C f T:A G:C f C:G A:T f T:A A:T f C:G transitions G:C f A:T A:T f G:C total
0c
0.5d
1.0e
32 (60%) 16 (30%) 1 (2%) 0
29 (59%) 12 (25%) 1 (2%) 2 (4%)
30 (60%) 13 (26%) 0 2 (4%)
4 (8%) 0 53
2 (4%) 3 (6%) 49
5 (10%) 0 50
a Mutants were pooled from experiments using pS189 treated with either 0.12 or 0.18 µg of 5-MCDE. b Values represent number of mutants of each type (percent total mutations). c Three mutants had two base substitutions; one mutant had a large deletion (68 bp) at position 61. d Six mutants had two base substitutions; one mutant had a 4-bp deletion at position 119; one mutant had a 7-bp duplication at position 125. e Three mutants had two base substitutions; one mutant had a base substitution plus a 1-bp deletion at position 116; one mutant had a 1-bp deletion at position 172; one mutant had a 13-bp deletion at position 156; one mutant had a large deletion (135 bp) at position 171.
majority of which occurred at G:C base pairs (Table 2). Such results are similar to those previously reported for 5-MCDE (20) and are consistent with the finding that this diol epoxide reacts principally with guanine bases in DNA (22). Exposure of the Ad293 cells to Cd resulted in slight alterations in the relative amounts of G:C f C:G and A:T f C:G transversions obtained from the 5-MCDE-treated vector (Table 2), but such differences were statistically insignificant (23). Thus, the types of mutations generated from the 5-MCDE-treated template were essentially the same whether the Ad293 cells were exposed to Cd or not. Effect of Cd on the Mutational Spectra of 5-MCDE in the supF Gene of pS189. Although Cd exposure of the mammalian host cells had no apparent effect on the types of mutations generated using 5-MCDEtreated pS189, analysis of the distribution of mutations was undertaken to determine if Cd affected mutagenesis of 5-MCDE-treated pS189 at specific sites in the supF gene. Such information can sometimes be useful in detecting differences in the pattern of DNA damage and/ or repair (24, 25). In the present study, the Poisson distribution was used to identify mutational “hot spots” on 5-MCDE-treated template (“hot spots” were defined as sites where the observed number of mutagenic events exceeded the expected number by a factor of 5 or more). Hot spots occurred at positions 123, 133, 155, 156, and 164 in the supF gene of 5-MCDE-treated pS189 that had replicated in Cd-exposed Ad293 cells and at positions 127 and 133 in vectors recovered from unexposed host cells (Figure 3). With a larger collection of mutants, Bigger et al. (20) detected mutational hot spots at positions 123, 127, 133, 159, and 168 in the supF gene of 5-MCDEtreated pS189. Although the hot spot at position 127 obtained in the absence of Cd and the hot spots at positions 155, 156, and 164 obtained in the presence of Cd suggest that Cd does affect mutagenesis of 5-MCDEtreated pS189 at particular sites within the supF gene, such effects are hardly dramatic; careful examination of the data presented in Figure 3 reveals no substantial Cdrelated difference in the relative number of mutations at sites 155 and 156, and several mutants occurred at position 127 in the presence of Cd and at position 164 in
the absence of Cd. These findings, together with those from analyses of the frequency and types of mutations generated from pS189 in the presence or absence of Cd, indicate that Cd increases background mutagenesis through a process that plays no substantial role in mutagenesis of 5-MCDE-treated vector.
Discussion In this investigation, host-cell exposure to subtoxic doses of Cd resulted in increased mutagenesis of untreated pS189 with no apparent alteration in the types of mutations observed. In addition, Cd exposure of Ad293 cells had little or no effect on the frequency, types, or distribution of mutations generated with 5-MCDEtreated vector. These results indicate that Cd increases background mutagenesis by affecting a process that is not involved in mutagenesis of 5-MCDE-treated DNA. Although it is not clear exactly how host-cell exposure to Cd increases mutagenesis of untreated pS189, presumably, Cd does not act by inhibiting any of the processes involved in replicating past bulky lesions on the shuttle vector. Our finding that Cd exposure of Ad293 cells increases background mutagenesis of pS189 is consistent with demonstrations of Cd mutagenesis in the hprt gene of various hamster cells (6, 8). In both assay systems, increases in mutagenicity most likely result from Cdinduced DNA damage, inhibition of DNA repair, or decreases in the fidelity of DNA replication. In vitro, Cd ions can bind to bases and phosphates in DNA (9); however, such binding does not result in DNA strand breaks, cross-links, or adducts (5). Interaction between Cd and some unidentified nuclear proteins and cofactors appears to be a prerequisite for Cd-induced production of DNA lesions (5). In mammalian cells, highdose Cd exposure (>20 µM) has been linked to the induction of DNA strand breaks (6, 26, 27) and chromosomal aberrations (28). Amelioration of Cd-induced DNA damage with various antioxidants (28) and/or superoxide dismutase (6) further suggests that reactive oxygen species (ROS) mediate such effects. At high doses, Cd probably acts by reducing intracellular concentrations of antioxidants (29) or by stimulating lipid peroxidation (12). In the present report, exposure of Ad293 cells to subtoxic doses of Cd substantially increased the incidence of base substitutions and insertions/deletions generated using untreated pS189. There was no obvious Cd-related effect, however, on the types of mutations that occurred. Several different laboratories have shown that endogenously generated ROS induce primarily large deletions, G:C f T:A transversions, and G:C f A:T transitions in mammalian cells (30, 31). Since the spectrum of mutations within a particular genetic locus should be similar regardless of which metal ion is used to generate endogenous ROS (32), our findings suggest that Cd mutagenicity is not mediated by ROS. In a recent study characterizing Cd mutagenesis in the hprt gene of hamster cells, exposure to 1 µM Cd was associated with a mutation frequency 20 times higher than background levels (8). In the same report, coexposure to D-mannitol protected against Cd-induced increases in cytotoxicity and mutation frequency, whereas coexposure to nontoxic doses of a catalase inhibitor
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Figure 3. Effect of Cd on the distribution of 5-MCDE-induced point mutations in the supF gene of pS189 in human cells. (A) Mutants were pooled from Ad293 cells that were transfected with pS189 treated with either 0.12 or 0.18 µg of 5-MCDE. (B) Mutants were pooled from Ad293 cells that were exposed to 0.5 or 1 µM Cd and transfected with pS189 that received 0.12 or 0.18 µg of 5-MCDE. Asterisks indicate “hot spots” of mutagenesis; 9 indicates a deletion.
potentiated such effects. Although these results were taken as evidence that ROS mediate the cytotoxic and mutagenic effects of the metal, subsequent analysis of the mutants generated revealed higher frequencies of G:C f C:G and T:A f A:T transversions in the Cd-treated cells. In fact, nearly 50% of Cd-induced base substitutions in the hprt gene occurred at T:A base pairs (8). In other words, these investigators also found no preferential increase in mutations that are characteristic of ROS exposure. Relevant to the possibility that Cd mutagenesis results from an inhibition of DNA repair, we found that Cd had little or no effect on mutagenesis of 5-MCDE-treated vector. Since exposure to 5-MCDE produces bulky adducts in DNA, our results suggest that Cd has no effect on nucleotide excision repair. To what extent damaged pS189 is repaired in Ad293 cells is as yet poorly defined; therefore, our findings do not necessarily preclude the possibility that Cd may affect repair in other experimental systems, especially those which involve chromosomal DNA. It is possible, however, that the Cd-related increases in mutagenesis of untreated vector were due to inhibited repair of “spontaneous” DNA lesions. Evidence for an effect of Cd on DNA repair has been provided by a number of different laboratories: Scicchitano and Pegg (33) reported that Cd can inhibit the ability of O6methylguanine methyltransferase to remove promutagenic adducts from DNA in vitro; Hartwig and Beyersmann (34) demonstrated that Cd can reduce the incidence of repair-associated chromosomal breaks in mammalian cells; and most recently, Dally and Hartwig (35) found that low-dose Cd exposure can inhibit removal
of oxidative DNA damage (most likely 8-hydroxyguanosine) from human cells. Others have shown that Cd-related increases in the incidence of chemically induced chromosomal aberrations depend upon the repair capacity of the particular cell line (36), and results from a recent study using human cell-free extracts and untreated or UV-treated plasmids suggest that Cd can inhibit lesion recognition and/or the strand incision/ displacement step of nucleotide excision repair (37). Although Cd had no obvious effect on the replication of 5-MCDE-treated plasmid in our experiments, it is conceivable that a reduction in the fidelity of replication of untreated pS189 may be responsible for the Cd-related increases in background mutagenesis. The fact that we saw no Cd-related effect on the types of mutations generated using untreated vector supports the idea that Cd may act by increasing the “spontaneous” error rate of at least one of the polymerases involved in replicating untreated pS189. Similarly, a lack of effect on an errorprone polymerase involved in replicating past bulky DNA lesions could explain why mutagenesis of 5-MCDEtreated vector appeared to be unaffected by host-cell exposure to Cd. At least five different polymerases are known to be involved in the replication of mammalian DNA (38). Further studies will be necessary in order to elucidate which, if any, of these is affected by Cd.
Acknowledgment. The authors thank Dr. Gary Pauly and Dr. Jeanne Cahill for helpful advice and discussions regarding this work. This research was sponsored in part by the National Cancer Institute, DHHS, under contract by ABL.
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References (1) IARC. (1993) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Beryllium, Cadmium, Mercury and Exposures in the Glass Manufacturing Industry, Vol. 58, pp 119-237, IARC, Lyon, France. (2) Friberg, L., Elinder, C.-G., and Kjellstrom, T. (1986) In Cadmium and Health: A Toxicological and Epidemiological Appraisal (Friberg, L., Elinder, C.-G., Kjellstrom, T., and Nordberg, G. F., Eds.) Vol. II, pp 1-246, CRC Press, Boca Raton, FL. (3) Waalkes, M. P., Infante, P., and Huff, J. (1994) The scientific fallacy of route specificity of carcinogenesis with particular reference to cadmium. Regul. Toxicol. Pharmacol. 20, 119-121. (4) Cherian, M. G., Howell, S. B., Imura, N., Klaassen, C. D., Koropatnick, J., Lazo, J. S., and Waalkes, M. P. (1994) Role of metallothionein in carcinogenesis. Toxicol. Appl. Pharmacol. 126, 1-5. (5) Rossman, T. G., Roy, N. K., and Lin, W. (1992) In Cadmium in the Human Environment: Toxicity and Carcinogenicity (Nordberg, G. F., Herber, R. F. M., and Alessio, L., Eds.) pp 367-375, IARC, Lyon, France. (6) Ochi, T., and Ohsawa, M. (1983) Induction of 6-thioguanineresistant mutants and single-strand scission of DNA by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 111, 6978. (7) Biggart, N. W., and Murphy, E. C., Jr. (1988) Analysis of metalinduced mutations altering the expression or structure of a retroviral gene in a mammalian cell line. Mutat. Res. 198, 115129. (8) Yang, J.-L., Chao, J.-I., and Lin, J.-G. (1996) Reactive oxygen species may participate in the mutagenicity and mutational spectrum of cadmium in Chinese hamster ovary-K1 cells. Chem. Res. Toxicol. 9, 1360-1367. (9) Jacobson, K. B., and Turner, J. E. (1980) The interaction of cadmium and certain other metal ions with proteins and nucleic acids. Toxicology 16, 1-37. (10) Sunderman, F. W., Jr., and Barber, A. M. (1988) Finger-loops, oncogenes, and metals. Ann. Clin. Lab. Sci. 18, 267-288. (11) Hechtenberg, S., Schafer, T., Benters, J., and Beyersmann, D. (1996) Effects of cadmium in cellular calcium and proto-oncogene expression. Ann. Clin. Lab. Sci. 26, 512-521. (12) Stacey, N. H., Cantilena, L. R., Jr., and Klassen, C. D. (1980) Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 53, 470-480. (13) Seidman, M. (1989) The development of transient SV40 based shuttle vectors for mutagenesis studies: Problems and solutions. Mutat. Res. 220, 55-60. (14) Harvey, R. G., Pataki, J., and Lee, H. (1986) Synthesis of the dihydrodiol and diol epoxide metabolites of chrysene and 5-methylchrysene. J. Org. Chem. 51, 1407-1412. (15) McCormick, J. J., and Maher, V. M. (1981) In DNA Repair: A Laboratory Manual of Research Procedures (Friedberg, E. C., and Hanawalt, P. C., Eds.) Vol. 1B, pp 501-521, Marcel Dekker, New York. (16) Bigger, C. A. H., Strandberg, J., Yagi, H., Jerina, D. M., and Dipple, A. (1989) Mutagenic specificity of a potent carcinogen, benzo[c]phenanthrene (4R,3S)-dihydrodiol (2S,1R)-epoxide, which reacts with adenine and guanine in DNA. Proc. Natl. Acad. Sci. U.S.A. 86, 2291-2295. (17) Graham, F. L., and Van der Eb, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467. (18) Birnboim, H. C., and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523. (19) Routledge, M. N., Wink, D. A., Keefer, L. K., and Dipple, A. (1993) Mutations induced by saturated aqueous nitric oxide in the pSP189 supF gene in human Ad293 and E. coli MBM7070 cells. Carcinogenesis 14, 1251-1254. (20) Bigger, C. A. H., Flickinger, D. J., Strandberg, J., Pataki, J., Harvey, R. G., and Dipple, A. (1990) Mutational specificity of the
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
(38)
(39)
anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene. Carcinogenesis 11, 2263-2265. Bigger, C. A. H., St. John, J., Yagi, H., Jerina, D. M., and Dipple, A. (1992) Mutagenic specificities of four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides. Proc. Natl. Acad. Sci. U.S.A. 89, 368-372. Reardon, D. B., Prakash, A. S., Hilton, B. D., Roman, J. M., Pataki, J., Harvey, R. G., and Dipple, A. (1987) Characterization of 5-methylchrysene-1,2-dihydrodiol-3,4-epoxide DNA adducts. Carcinogenesis 8, 1317-1322. Adams, W. T., and Skopek, T. R. (1987) Statistical test for the comparison of samples from mutational spectra. J. Mol. Biol. 194, 391-396. Levy, D. D., Groopman, J. D., Lim, S. E., Seidman, M. M., and Kraemer, K. H. (1992) Sequence specificity of aflatoxin B1-induced mutations in a plamid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52, 5668-5673. Tornaletti, S., and Pfeifer, G. (1994) Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 263, 1436-1438. Ochi, T., Ishiguro, T., and Ohsawa, M. (1983) Participation of active oxygen species in the induction of DNA single-strand scissions by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 122, 169-175. Misra, R. R., Smith, G. T., and Waalkes, M. P. (1998) Evaluation of the direct genotoxic potential of cadmium in four different cultured rodent cell lines. Toxicology, in press. Ochi, T., and Ohsawa, M. (1985) Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells. Mutat. Res. 143, 137142. Ochi, T., Takahashi, K., and Ohsawa, M. (1987) Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction. Mutat. Res. 180, 257-266. Gille, J. J. P., van Berkel, C. G. M., and Joenje, H. (1994) Mutagenicity of metabolic oxygen radicals in mammalian cell cultures. Carcinogenesis 15, 2695-2699. Reid, T. M., Feig, D. I., and Loeb, L. A. (1994) Mutagenesis by metal-induced oxygen radicals. Environ. Health Perspect. 102 (Suppl. 3), 57-61. Rodriguez, H., Holmquist, G. P., D’Agostino, R., Jr., Keller, J., and Akman, S. A. (1997) Metal-ion dependent hydrogen peroxideinduced DNA damage is more sequence specific than metal specific. Cancer Res. 57, 2394-2403. Scicchitano, D. A., and Pegg, A. E. (1987) Inhibition of O6alkylguanine-DNA-alkyltransferase by metals. Mutat. Res. 192, 207-210. Hartwig, A., and Beyersmann, D. (1989) Comutagenicity and inhibition of DNA repair by metal ions in mammalian cells. Biol. Trace Element Res. 21, 359-365. Dally, H., and Hartwig, A. (1997) Induction and repair of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 18, 1021-1026. Yamada, H., Miyahara, T., and Sasaki, Y. F. (1993) Inorganic cadmium increases the frequency of chemically induced chromosome aberrations in cultured mammalian cells. Mutat. Res. 302, 137-145. Calsou, P., Frit, P., Bozzato, C., and Salles, B. (1996) Negative interference of metal (II) ions with nucleotide excision repair in human cell-free extracts. Carcinogenesis 17, 2779-2782. Wood, R. D., and Shivji, M. K. K. (1997) Which DNA polymerases are used for DNA-repair in eukaryotes? Carcinogenesis 18, 605610. Snedecor, G. W., and Cochran, W. G. (1967) Statistical Methods, 6th ed., pp 210-211, Iowa State University Press, Ames, IA.
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